0

1|M o M – D a i r y a n d D a i r y P r o d u c t s
2|M o M – D a i r y a n d D a i r y P r o d u c t s
 List of Contributors

Name Organization
Dr. Rajan Sharma ICAR-National Dairy Research Institute, Karnal
Dr. Rajesh R Nair CALF, National Dairy Development Board, Anand
Dr. AK Dikshit Retd. From Division of Agricultural Chemicals, Indian Agricultural
Research Institute (IARI), Pusa, New Delhi

Dr. Alok Kumar Srivastava CSIR-Central Food Technological Research Institute (CFTRI),
Mysuru
Dr. Lalitha Ramakrishna Gowda Retd. From CSIR-Central Food Technological Research Institute
(CFTRI), Mysuru
Dr. Ajit Dua Punjab Biotechnology Incubator, Department of Science and
Technology and Environment, Govt. of Punjab), Mohali
Dr. Jagan Mohan Rao Retd. From CSIR-Central Food Technological Research Institute
(CFTRI), Mysuru
Dr. Kiran N Bhilegaonkar ICAR-Indian Veterinary Research Institute Regional Station, Pune
Dr. Raju Khan CSIR-Advanced Materials & Processes Research Institute, Bhopal
Dr. Mohana Krishna Reddy Mudiam CSIR- Indian Institute of Chemical Technology, Hyderabad
Dr. Prem Saran Tirumalai Department of Botany, Dayalbagh Educational Institute,
Dayalbagh, Agra
Dr. Richa Singh ICAR- National Dairy Research Institute, Karnal
Dr. Rajiv Chawla CALF, National Dairy Development Board, Anand
[Link] Bharatbhai Darji CALF, National Dairy Development Board, Anand
Dr. Harinder Singh Oberoi Food Safety and Standards Authority of India
[Link] Kr Yadav Food Safety and Standards Authority of India
Dr. Dinesh Kumar Food Safety and Standards Authority of India
Ms. Kanika Aggarwal Food Safety and Standards Authority of India
Mr. Pravesh Shanker Chaubey Food Safety and Standards Authority of India
Ms. Sunaina Verma Food Safety and Standards Authority of India

3|M o M – D a i r y a n d D a i r y P r o d u c t s
 TABLE OF CONTENTS

S. No. METHOD NO. METHOD PAGE NO.

A LIQUID MILK
1. FSSAI 01.001:2022 Preparation of Milk Sample 10
2. FSSAI 01.002:2022 Detection of Sucrose (cane sugar) in Milk 11 – 12
3. FSSAI 01.003:2022 Detection of Starch in Milk 13 – 15
4. FSSAI 01.004:2022 Detection of Urea in Milk 16 – 17
5. FSSAI 01.005:2022 Quantitative Estimation of Total Urea Content in Milk 18 – 21
6. FSSAI 01.006:2022 Detection of Ammonium Salts in Milk 22 – 23
7. FSSAI 01.007:2022 Detection of Sulphates in Milk 24 – 25
8. FSSAI 01.008:2022 Detection of Glucose in Milk 26 – 27
9. FSSAI 01.009:2022 Detection of Sodium Chloride in Milk 28 – 29
10. FSSAI 01.010:2022 Detection of Presence of Foreign Fat in Milk 30 – 32
11. FSSAI 01.011:2022 Detection of Nitrate in Milk 33 – 34
12. FSSAI 01.012:2022 Detection of Neutralizers in Milk 35
13. FSSAI 01.013:2022 Detection of Hypochlorite and Chloramines in Milk 36 – 38
14. FSSAI 01.014:2022 Detection of Quaternary Ammonium Compounds in Milk 39 – 40
15. FSSAI 01.015:2022 Detection of Anionic Detergents in Milk 41 – 42
16. FSSAI 01.016:2022 Detection of Formaldehyde in Milk 43 – 45
17. FSSAI 01.017:2022 Detection of Hydrogen Peroxide in Milk 46 – 48
18. FSSAI 01.018:2022 Detection of Boric acid and Borates in Milk 49 – 50
19. FSSAI 01.019:2022 Detection of Salicylic acid in Milk 51 – 52
20. FSSAI 01.020:2022 Detection of Maltodextrin in Milk 53 – 54
21. FSSAI 01.021:2022 Detection of Alkaline Phosphatase in Milk 55 – 58
22. FSSAI 01.022:2022 Checking Efficiency of Sterilization in Liquid Milk 59 – 60
23. FSSAI 01.023:2022 Detection of Skimmed Milk Powder Addition in Species 61 – 62
Identified Milk and Mixed Milk
24. FSSAI 01.024:2022 Determination of Fat Content in Milk 63 – 68
25. FSSAI 01.025:2022 Determination of Solids-Not-Fat (Gravimetric method) 69 – 70
26. FSSAI 01.026:2022 Determination of Total Nitrogen in Milk by Kjeldahl 71 – 85
Method
27. FSSAI 01.027:2022 Determination of True Protein in Milk 86 – 88

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28. FSSAI 01.028:2022 Determination of Non-Protein Nitrogen (NPN) in Milk 89– 91
B CREAM
29. FSSAI 01.029:2022 Preparation of Sample of Cream 92
30. FSSAI 01.030:2022 Determination of Fat in Cream 93 – 95
31. FSSAI 01.031:2022 Detection of Presence of Thickeners in Cream 96 – 98

C DAHI AND YOGHURT

32. FSSAI 01.032:2022 Preparation of Sample of Dahi and Yoghurt 99
33. FSSAI 01.033:2022 Determination of Fat in Dahi and Yoghurt 100 – 101
34. FSSAI 01.034:2022 Determination of Milk Solids-Not-Fat (SNF) in Dahi and 102 – 105
Yoghurt
35. FSSAI 01.035:2022 Determination of Titratable Acidity in Dahi and Yoghurt 106 – 107
36. FSSAI 01.036:2022 Determination of Protein in Dahi and Yoghurt 108 – 109
37. FSSAI 01.037:2022 Detection of Starch in Dahi 110

D CHEESE AND CHEESE PRODUCTS

38. FSSAI 01.038:2022 Preparation of Sample of Cheese and Cheese Products 111
39. FSSAI 01.039:2022 Determination of Moisture in Cheese 112 – 113
40. FSSAI 01.040:2022 Determination of Fat (on dry matter basis) in Cheese by 114 – 115
Reference Method
41. FSSAI 01.041:2022 Determination of Fat (on dry matter basis) in Cheese by 116 – 118
Gerber Method

E CHANNA/PANEER
42. FSSAI 01.042:2022 Preparation of Sample of Channa/Paneer 119
43. FSSAI 01.043:2022 Determination of Moisture in Channa/Paneer 120
44. FSSAI 01.044:2022 Determination of Fat (on dry matter basis) in Channa/Paneer 121 – 122

F ICE CREAM AND RELATED PRODUCTS

45. FSSAI 01.045:2022 Preparation of Sample of Ice cream, Kulfi, Milk Ices, Milk 123– 124
Lollies and Frozen Dessert/Confection
46. FSSAI 01.046:2022 Determination of Total Solids in the Ice Cream 125 – 126
47. FSSAI 01.047:2022 Determination of Weight per Unit Volume or Over-run in 127 – 128
Ice Cream
48. FSSAI 01.048:2022 Determination of Milk Fat in Ice cream 129 – 130

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49. FSSAI 01.049:2022 Method for Determination of Protein in Ice cream 131 – 132

G CONDENSED / EVAPORATED MILK

50. FSSAI 01.050:2022 Preparation of Sample of Condensed/Evaporated Milk 133
51. FSSAI 01.051:2022 Determination of Fat in Condensed/Evaporated Milk 134 – 135
52. FSSAI 01.052:2022 Determination of Sucrose Content in 136 – 148
Condensed/Evaporated Milk
53. FSSAI 01.053:2022 Determination of Milk Solids in Condensed/Evaporated 149 – 152
Milk
54. FSSAI 01.054:2022 Determination of Milk Protein in Milk Solids-not-Fat of 153 – 154
Condensed/Evaporated Milk

H DRIED MILK PRODUCTS

55. FSSAI 01.055:2022 Preparation of Sample for Dried Milk Products 155
56. FSSAI 01.056:2022 Determination of Moisture in Dried Milk Products 156 – 158
57. FSSAI 01.057:2022 Determination of Fat in Dried Milk Products by Reference 159 – 160
Method
58. FSSAI 01.058:2022 Determination of Fat in Whole Milk Powder by Gerber 161 – 163
method
59. FSSAI 01.059:2022 Determination of Titratable Acidity in Dried Milk Products 164 – 166
60. FSSAI 01.060:2022 Determination of Titratable Acidity in Dairy Whitener 167 – 168
61. FSSAI 01.061:2022 Determination of Added Sugar in Dairy Whitener 169 – 172
62. FSSAI 01.062:2022 Determination of Milk Protein (Milk Solids-Not-Fat Basis) 173 – 174
in Dried Milk Products
63. FSSAI 01.063:2022 Determination of Milk Protein in Foods for Infant Nutrition 175 – 176
and Processed Cereal Based Complementary Foods
64. FSSAI 01.064:2022 Determination of Total Ash in Dried Milk Products 177 – 179
65. FSSAI 01.065:2022 Determination of Acid Insoluble Ash in Dried Milk Products 180 – 182
66. FSSAI 01.066:2022 Determination of Crude Fibre Content in Milk Cereal Based 183 – 188
Complementary Food and Processed Cereal Based
Complementary Foods
67. FSSAI 01.067:2022 Determination of Total Carbohydrates in Milk Cereal Based 189 – 190
Complementary Food and Processed Cereal Based
Complementary Food
68. FSSAI 01.068:2022 Determination of Insolubility Index in Dried Milk Products 191 – 197
69. FSSAI 01.069:2022 Determination of Solubility Percentage in Foods for Infant 198 – 200
Nutrition

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70. FSSAI 01.070:2022 Determination of Scorched Particles in Dried Milk Products 201 – 202

I KHOA
71. FSSAI 01.071:2022 Preparation of Sample of Khoa 203
72. FSSAI 01.072:2022 Determination of Total Solids in Khoa 204
73. FSSAI 01.073:2022 Determination of Fat content on Dry Matter Basis in Khoa 205 – 207
74. FSSAI 01.074:2022 Detection of Starch in Khoa 208 – 209
75. FSSAI 01.075:2022 Detection of Sucrose in Khoa 210 – 211
76. FSSAI 01.076:2022 Determination of Titratable Acidity in Khoa 212 – 213
77. FSSAI 01.077:2022 Determination of Total Ash in Khoa 214 – 215
78. FSSAI 01.078:2022 Estimation of Reichert Meissl Value, Polenske Value and 216– 217
Butyro-refractometer Reading of Fat Extracted from Khoa

J BUTTER
79. FSSAI 01.079:2022 Preparation of Sample of Butter 218
80. FSSAI 01.080:2022 Determination of Moisture in Butter 219 – 220
81. FSSAI 01.081:2022 Determination of Fat and Curd (Milk solids not Fat) in 221 – 222
Butter
82. FSSAI 01.082:2022 Determination of Fat in Butter 223 – 225
83. FSSAI 01.083:2022 Determination of Salt Content in Butter 226 – 229
84. FSSAI 01.084:2022 Estimation of Reichert Meissl Value and Butyro- 230 – 231
refractometer Reading of Fat Extracted from Butter

K MILK FAT PRODUCTS

85. FSSAI 01.085:2022 Preparation of Sample of Milk Fat Products 232
86. FSSAI 01.086:2022 Determination of Moisture in Milk Fat Products 233 – 234
87. FSSAI 01.087:2022 Determination of Fat in Milk Fat Products 235 – 236
88. FSSAI 01.088:2022 Determination of Butyro-Refractometer Reading in Milk Fat 237 – 240
Products
89. FSSAI 01.089:2022 Determination of Free Fatty Acids in Milk Fat Products 241 – 242
90. FSSAI 01.090:2022 Determination of Reichert-Meissl and Polenske Value in 243 – 248
Milk Fat Products
91. FSSAI 01.091:2022 Test for Detection of Vanaspati in Milk Fat Products 249 – 250
(Baudouin Test)
92. FSSAI 01.092:2022 Test for Detection of Mineral Oil in Milk Fat Products 251 – 252
(Holde’s test)

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93. FSSAI 01.093:2022 Test for Determination of fatty acid composition in Milk Fat 253 – 260
Products by gas-liquid-chromatography (GLC)
94. FSSAI 01.094:2022 Determination of Peroxide Value in Milk Fat Products 261 – 263
95. FSSAI 01.095:2022 Determination of Iodine Value in Milk Fat Products 264 – 267
96. FSSAI 01.096:2022 Determination of Saponification Value in Milk Fat Products 268 – 270
97. FSSAI 01.097:2022 Detection of adulteration of vegetable oil in Milk Fat 271 – 274
Products by Reversed Phase- High performance liquid
chromatography (RP-HPLC)

L CHAKKA AND SHRIKHAND

98. FSSAI 01.098:2022 Preparation of Sample of Chakka and Shrikhand 275
99. FSSAI 01.099:2022 Determination of Total Solids in Chakka and Shrikhand 276
100. FSSAI 01.100:2022 Determination of Fat (on dry matter basis) in Chakka and 277 – 278
Shrikhand
101. FSSAI 01.101:2022 Determination of Milk Protein (on dry matter basis) in 279 – 280
Chakka and Shrikhand
102. FSSAI 01.102:2022 Determination of Titratable Acidity in Chakka and 281 – 282
Shrikhand
103. FSSAI 01.103:2022 Determination of Total Ash (on dry matter basis) in Chakka 283 – 284
and Shrikhand
104. FSSAI 01.104:2022 Determination of Sucrose Content in Shrikhand 285 - 286

M WHEY POWDER
105. FSSAI 01.105:2022 Preparation of Sample of Whey Powder and Acid Whey 287
Powder
106. FSSAI 01.106:2022 Determination of Moisture in Whey Powder and Acid Whey 288
Powder
107. FSSAI 01.107:2022 Determination of Fat in Whey Powder and Acid Whey 289
Powder
108. FSSAI 01.108:2022 Determination of Milk Protein in Whey Powder and Acid 290 – 291
Whey Powder
109. FSSAI 01.109:2022 Determination of Total Ash (on dry matter basis) in Whey 292 – 293
Powder and Acid Whey Powder
110. FSSAI 01.110:2022 Determination of pH in Whey Powder and Acid Whey 294
Powder
111. FSSAI 01.111:2022 Determination of Lactose in Whey Powder and Acid Whey 295 – 298
Powder

N EDIBLE CASEIN PRODUCTS

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112. FSSAI 01.112:2022 Preparation of Sample of Edible Casein Products 299
113. FSSAI 01.113:2022 Determination of Moisture in Edible Casein Products 300 – 301
114. FSSAI 01.114:2022 Determination of Fat in Edible Casein Products 302 – 304
115. FSSAI 01.115:2022 Determination of Protein in Edible Casein Products 305 – 306
116. FSSAI 01.116:2022 Determination of Casein in Protein in Edible Casein 307 – 309
Products
117. FSSAI 01.117:2022 Determination of Lactose Content in Edible Casein Products 310– 313
(by Photometric Method)
118. FSSAI 01.118:2022 Determination of Fixed Ash (ash including P2O5) Content in 314 – 316
Edible Casein Products
119. FSSAI 01.119:2022 Determination of Free Acidity in Edible Casein Products 317 – 318
120. FSSAI 01.120:2022 Determination of pH in Edible Casein Products 319 – 320

O COLOSTRUM
121. FSSAI 01.121:2022 Preparation of Sample of colostrum 321
122. FSSAI 01.122:2022 Determination of Moisture Content in Colostrum 322 – 323
123. FSSAI 01.123:2022 Determination of Fat (Gravimetric method) in Colostrum 324 – 325
124. FSSAI 01.124:2022 Determination of Milk Protein in Colostrum 326 - 327

125. FSSAI 01.125:2022 Determination of Melamine in Milk, Milk Products and 328 – 331
Infant formula

ANNEXURES
126. FSSAI 01.126:2022 Preparation of 0.1 N Standard Solution Of Sodium 332 – 334
Hydroxide
127. FSSAI 01.127:2022 Preparation of 0.1 N Standard Solution of Hydrochloric 335 – 338
Acid
128. FSSAI 01.128:2022 Preparation of 0.1 N Standard Solution of Silver Nitrate 339 – 341

Note: The test methods given in the manual are standardized/ validated/ taken from national
or international methods or recognized specifications, however it would be the responsibility of
the respective testing laboratory to verify the performance of these methods onsite and ensure
that it gives proper results before putting these methods in to use.

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 A. LIQUID MILK

Preparation of Milk Sample

Method No. FSSAI 01.001:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of liquid milk (buffalo milk,
cow milk, goat milk, sheep milk, camel milk, mixed milk, standardized
milk, full cream milk, recombined milk, toned milk, double toned milk, and
skimmed milk).
Materials and reagents 1. Beakers of various capacity.
2. Water bath.
3. Thermometer.
Preparation of reagents Ammonia solution (Specific Gravity 0.91).
Sample preparation 1. Samples are received after few days of drawl and contains preservative
(0.4% formalin).
2. Warm the sample to 37-40 ℃ by transferring it to the beaker and
keeping it in a water bath maintained at 40-45 ℃.
3. Stir slowly for proper homogenization , mix sample thoroughly by
pouring back into the bottle, mixing to dislodge any residual fat sticking
to the sides and pour it back in the beaker.
4. During mixing do not shake the bottle vigorously.
5. Allow the sample to come to room temperature (26-28 ℃) and withdraw
immediately for analysis.
6. If small clots or lumps are observed in the sample which cannot be
dispersed, a few drops of ammonia solution may be used during
homogenization.
If even after homogenization the sample shows lumps or clots or droplets
Inference (Qualitative of oil are visible suggestive of curdling /splitting of milk, the sample should
analysis) be deemed unfit for analysis and rejected.
Reference IS 1479 (Part II):1961 (Reaffirmed year 2018). Methods of test for Dairy
Industry -Chemical Analysis of Milk. Bureau of Indian Standards, New
Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

10 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Sucrose (cane sugar) in Milk

Method No. FSSAI 01.002:2022 Revision No. & Date 0.0

Scope This method is for the detection of sucrose in milk by Seliwanoff’s test
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Resorcinol: Avoid using for a prolonged period, on a broken skin. For
external use only; avoid contact with eyes, mouth, and
nose. Caution should be exercised in patients with any allergy,
children, elderly, during pregnancy and breastfeeding.
Principle For the detection of sucrose in milk, Seliwanoff’s test is used which is a
test for detection of ketoses such as fructose. Sucrose gives a positive test
with Seliwanoff’s reagent as it is a disaccharide consisting of fructose and
glucose. The dilute hydrochloric acid is used in preparation of Seliwanoff’s
reagent (resorcinol). The boiling helps in dehydrating the sugar; further
keto group more actively attacks resorcinol in comparison to aldehyde
group. In case of sucrose, acid leads to its hydrolysis into constituent
monosaccharides i.e. glucose and fructose. 5-hydroxymethylfurfural is
produced from fructose; a keto sugar. The dehydration product 5-
hydroxymethylfurfural condenses with resorcinol forming cherry red
colour. Test can be done directly on milk or whey obtained from milk
sample.
Apparatus/Instruments 1. Test tube.
2. Water bath.
3. Whatman No. 1 filter paper.
4. Analytical balance (readability 0.01 g).
Materials and Reagents 1. Resorcinol.
2. Concentrated hydrochloric acid (Approximately 11.6 N).
Preparation of reagents 1. Dilute hydrochloric acid: Take 30 mL concentrated hydrochloric acid
and dilute it to 100 mL with distilled water.
2. Resorcinol solution (0.05%): The reagent is prepared by dissolving
0.05 g of resorcinol in 100 mL dilute hydrochloric acid.

11 | M o M – D a i r y a n d D a i r y P r o d u c t s
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Using milk as a medium:
1. Take 3 mL milk and 5 mL resorcinol solution in a test tube.
2. Keep the contents in a boiling water bath for 6 min.
3. Cool the tubes immediately after heating under tap water to retard the
rate of reaction, which if not done would narrow the colour difference
between negative and positive samples.
4. Observe for colour development.

Using whey as a medium:

1. Take 3 mL whey and 5 mL resorcinol solution in a test tube (quantity
of both whey and reagent can be doubled proportionately to get
sufficient filtrate.)
2. Mix and filter the contents using Whatman No. 1 filter paper.
3. Keep the filtrate in boiling water bath for 4 min.
4. Cool the tubes immediately after heating under tap water to retard the
rate of reaction, which if not done would narrow the colour difference
between negative and positive samples.
5. Observe for colour development.
Inference (Qualitative Development of red colour indicates adulteration of sucrose in milk. The
analysis) intensity of red colour increases with increase in the sucrose content in the
milk, whereas pure milk remains light in colour. The limit of detection is:
0.1 g/ 100 mL milk (when test is performed in milk)
0.06 g/ 100 mL milk (when test is performed in whey)
In case sample is preserved with formaldehyde, adulterated milk will show
light pink colour for both milk and whey as a medium of analysis.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 26-28. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. IS 1479 (Part II): 1961 (Reaffirmed Year: 2018). Methods of test for
Dairy Industry – Chemical Analysis of Milk. Bureau of Indian
Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

12 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Starch in Milk

Method No. FSSAI 01.003:2022 Revision No. & Date 0.0

Scope This method is for the detection of starch in milk by Iodine test
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
2. Iodine: It is not compatible with combustibles, strong bases, halogens
and ethanol. It reacts violently or explosively with acetylene,
acetaldehyde, metal hydrides and metal carbides.
3. Glacial acetic acid: Use with adequate ventilation. Keep away from
heat. Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin
and eyes. Keep away from incompatibles such as oxidizing agents,
reducing agents, metals, acids, alkalis.
4. Citric acid: May cause gastrointestinal irritation with nausea, vomiting
and diarrhea. Excessive intake of citric acid may cause erosion of the
teeth. Causes respiratory tract irritation. Repeated exposure may cause
sensitization dermatitis.
Principle Starch in milk is detected by iodine test, where blue colour develops on
addition of iodine in starch solution. The development of blue colour is due
to complex formation between iodine and amylose component of starch.
The other component, amylopectin, gives a red-purple colour which is
much less intense than the amylose. The acidic condition in the reagent
mixture accentuates the blue colour, whereas alkali reduces its intensity,
the blue colour disappears above a pH of about 9.5. Heating the solution
containing starch-iodine complex also destroys the colour although
reversibly. Test can be done directly on milk or whey obtained from milk
sample after coagulation with citric acid.
Apparatus/Instruments 1. Test tube.
2. Conical flask.
3. Water bath.
4. Whatman No. 1 filter paper.
5. Analytical balance (readability 0.01 g).
Materials and Reagents 1. Potassium iodide.
2. Iodine crystals.

13 | M o M – D a i r y a n d D a i r y P r o d u c t s
 3. Citric acid monohydrate.
4. Glacial acetic acid.
Preparation of reagents 1. Iodine solution: Dissolve 2.5 g potassium iodide and 1 g of pure iodine
crystals in 100 mL distilled water. Prepare iodine solution at least a day
before as iodine dissolves slowly.
2. Acetic acid (10%): Dissolve 10 mL glacial acetic acid in distilled water
and make up the volume to 100 mL.
3. Citric acid solution (5%): Dissolve 5 g citric acid monohydrate in
distilled water and make up the volume to 100 mL with distilled water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Using milk as a medium:
1. Take 3 mL milk in test tube; bring the milk to boil on a direct flame or
on a boiling water bath.
2. Cool the test tube to room temperature (25±3 oC) under tap water.
3. Add a drop of 10% acetic acid in the test tube, and 0.2 mL of iodine
solution.
4. Mix the content and observe colour.
Using whey as a medium:
1. Take 20 mL milk in a conical flask, bring the milk to boil on a direct
flame or on a boiling water bath.
2. Add 5% citric drop wise till visible coagulation (approximate
consumption of citric acid required would be 1.5-2 mL).
3. Filter the content using Whatman No. 1 filter paper; let the filtrate cool
to room temperature.
4. Take 3 mL filtrate in another test tube and add 0.1 mL of iodine
solution.
5. Mix the contents and observe colour.
Inference (Qualitative Blue/dark blue colour formation indicates adulteration of milk with starch,
analysis) whereas pure milk remains yellow due to colour of iodine. The limit of
detection is:
0.02 g/ 100 mL milk (when test is performed in milk)
0.01 g /100 mL milk (when test is performed in whey)
In case sample is preserved with formaldehyde, adulterated milk will show
same blue colour for milk and dark blue colour for whey.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 31-32. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. IS 1479 (Part I):2016. Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.

14 | M o M – D a i r y a n d D a i r y P r o d u c t s
 3. Moulay, S. (2013). Molecular iodine/polymer complexes. Journal of
Polymer Engineering, 33(5), 389-443.
Approved by Scientific Panel on Methods of Sampling and Analysis

15 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Urea in Milk

Method No. FSSAI 01.004:2022 Revision No. & Date 0.0

Scope This method is for the detection of urea in milk by p-
dimethylaminobenzaldehyde (DMAB) test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Methanol: It is a highly flammable liquid and vapour. Keep away from
heat, sparks, open flames, hot surfaces. Perform all operations under a
fume hood.
2. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
3. p-Dimethylaminobenzaldehyde: May be harmful if absorbed through
skin. May cause skin irritation. May cause irritation in eyes. May be
harmful if swallowed Good general ventilation should be sufficient to
control worker exposure to airborne contaminants.
Principle This method is based on the principle that urea forms a yellow-coloured
complex with p-dimethylaminobenzaldehyde (DMAB) reagent in low
acidic alcoholic solution at room temperature (25 ± 3 °C). The intensity of
colour can be measured at 440 nm. The colour developed is in proportion
to urea content in the sample.
Apparatus/Instruments 1. Analytical balance (Readability 0.01 g).
2. Test tube.
Materials and Reagents 1. p-Dimethylaminobenzaldehyde reagent (DMAB).
2. Methanol.
3. Concentrated hydrochloric acid (Approximately 11.6 N).
Preparation of reagents p-Dimethylaminobenzaldehyde reagent (DMAB): The reagent is prepared
by dissolving 1.6 g of p-dimethylaminobenzaldehyde in methanol
subsequently adding 10 mL of concentrated hydrochloric acid and making
volume to 100 mL with methanol.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis 1. Take 5 mL milk in a test tube, add 5 mL DMAB reagent.
2. Mix the contents and observe for colour.

16 | M o M – D a i r y a n d D a i r y P r o d u c t s
Inference (Qualitative Formation of distinct yellow colour indicates the presence of added urea in
analysis) milk sample, whereas pure milk shows light yellow colour due to natural
urea. The limit of detection is 0.2 g/100 mL milk.
This method is a preliminary method for screening purpose. Positive
samples are required to be confirmed by quantitative method (Method No.
FSSAI 01.005:2022).
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 23-24. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. IS 1479 (Part I):2016. Methods of test for Dairy Industry, Part – I Rapid
examination of Milk. Bureau of Indian Standards, New Delhi.
3. Bector, B.S., Ram, M. & Singhal, O.P. (1998). Rapid platform test for
the detection /determination of added urea in milk. Indian Dairyman,
50(4): 59-62
Approved by Scientific Panel on Methods of Sampling and Analysis

17 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Quantitative Estimation of Total Urea Content in Milk

Method No. FSSAI 01.005:2022 Revision No. & Date 0.0

Scope
This method is for the quantitative estimation of total urea in milk by
dimethylaminobenzaldehyde (DMAB) method.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
2. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas. Urea:
Avoid contact and inhalation. Do not get in eyes, on skin, on clothing.
Wash thoroughly after handling. Keep in a tightly closed container,
stored in a cooled, dry, ventilated area. Urea reacts with calcium
hypochlorite or sodium hypochlorite to form the explosive nitrogen
trichloride. It is incompatible with sodium nitrite, gallium perchlorate,
strong oxidizing agents (permanganate, dichromate, nitrate, chlorine).
3. p-Dimethylaminobenzaldehyde: Good general ventilation should be
sufficient to control worker exposure to airborne contaminants.
4. Trichloroacetic acid: Trichloroacetic acid is a corrosive chemical and
contact can severely irritate and burn the skin and eyes with possible
eye damage. Trichloroacetic acid can irritate the nose and throat.
Trichloroacetic acid can irritate the lungs causing coughing and/or
shortness of breath.
5. Potassium dihydrogen orthophosphate: Avoid contact with skin and
eyes. Hygiene measures: Wash hands and other exposed areas with
mild soap and water before eating, drinking or smoking and when
leaving work. Storage conditions: Store in a well-ventilated place. Keep
container tightly closed.

18 | M o M – D a i r y a n d D a i r y P r o d u c t s
 6. Dipotassium monohydrogen orthophosphate: On eye and skin contact,
rinse and wash off immediately with plenty of water, also under the
eyelids, for at least 15 min. Obtain medical attention. On inhalation,
move to fresh air; If breathing is difficult, provide oxygen.
Principle
This method is based on the principle that urea forms a yellow-coloured
complex with p-dimethylaminobenzaldehyde (DMAB) reagent in low
acidic alcoholic solution at room temperature (25 ± 3 oC). The intensity of
colour is measured at 425 nm.
Apparatus/Instruments 1. Spectrophotometer: Instrument with maximum band width 1 nm at
425 nm and 1 cm path length.
2. Whatman filter paper: Grade 42.
3. Funnels.
4. Test tubes.
5. Volumetric flasks: 25 mL, 100 mL and 1 L.
6. Analytical balance (readability 0.01 g).
Materials and reagents 1. p-Dimethylaminobenzaldehyde (DMAB).
2. Ethyl alcohol.
3. Concentrated hydrochloric acid (Approximately 11.6 N).
4. Trichloroacetic acid (TCA).
5. Urea.
6. Anhydrous potassium dihydrogen orthophosphate.
7. Anhydrous dipotassium monohydrogen orthophosphate.
Preparation of reagents 1. p-Dimethylaminobenzaldehyde (DMAB) solution: Dissolve 1.6 g
DMAB in 100 mL ethyl alcohol and add 10 mL concentrated
hydrochloric acid. The reagent is stable for 1 month. Prepare new
standard curve with each new batch of reagent.
2. Phosphate buffer pH 7.0 (0.05 M): Dissolve 3.154 g anhydrous
potassium dihydrogen orthophosphate (KH2PO4) and 4.672 g
anhydrous dipotassium monohydrogen orthophosphate (K2HPO4)
separately in 100 mL of distilled water. Combine both the solutions and
dilute to 1 L with water.
3. Trichloroacetic acid (TCA) 24%, w/v (freshly prepared): 24.0 g TCA
is dissolved in distilled water and the volume is made up to 100 mL.
4. Diluting reagent: Equal volumes of 24% TCA and phosphate buffer (pH
7.0) are mixed to make the diluting reagent.
5. Urea Standard Solution (1 mg/mL): Weigh 25 mg of urea and dissolve
in phosphate buffer of pH 7.0 and make up the volume to 25 mL with
phosphate buffer. Store at less than 24 ℃. The reagent is stable for 1
week.

19 | M o M – D a i r y a n d D a i r y P r o d u c t s
Sample preparation
Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis: a) Preparation of standard curve
1. Take 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5 mL of urea standard solution in
20 x 150 mm (25) mL test tube and make up the volume to 5 mL by
adding 4.9, 4.8, 4.6, 4.4, 4.2, 4.0 and 3.5 mL diluting reagent. The
concentration of the solutions will be 20, 40, 80, 120, 160, 200 and 300
μg/mL. Add 5 mL DMAB solution to each to develop the yellow
colour. Prepare reagent blank of 5 mL diluting reagent and 5 mL
DMAB solution.
2. Shake tubes thoroughly and let stand for 10 min. Read absorbance in 1
cm cell at 425 nm with reagent blank at zero.
3. Plot absorbance against the concentration of urea; plot should be
straight line (R2>0.99)
b) Extraction
1. 10 mL of milk sample is mixed with 10 mL of TCA to precipitate the
proteins and filtered using Whatman 42 filter paper.
2. 5 mL of filtrate is then treated with 5 mL of DMAB reagent to develop
the colour.
3. Blank is prepared by taking 5 mL of diluting reagent and treating it with
5 mL of DMAB reagent.
c) Checking of Optical Density
1. The optical density of the yellow color is measured at 425 nm.
Calculation with units of From standard curve, the amount of urea in milk is calculated.
expressions Read from the graph the concentration of urea (μg) corresponding to
absorbance of sample.
Consider the concentration from the standard curve for urea is =Y µg
So, 1 mL of filtrate from sample has = Y μg urea
10 mL of filtrate (milk+ TCA) has = Y * 10 μg urea
i. e. 5 mL milk has = Y * 10 μg urea
𝑌 ∗10 ∗ 100
100 mL of milk sample has = μg urea per 100 mL of milk
5
𝑌
= mg urea per 100 mL of milk
5
Reference 1. IS 1479 (Part I):2016. Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.
2. Bector, B.S., Ram, M. & Singhal, O.P. (1998). Rapid platform test for
the detection /determination of added urea in milk. Indian Dairyman,
50(4): 59-62

20 | M o M – D a i r y a n d D a i r y P r o d u c t s
Approved by Scientific Panel on Methods of Sampling and Analysis

21 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Ammonium Salts in Milk

Method No. FSSAI 01.006:2022 Revision No. & Date 0.0

Scope This method is for the detection of ammonium salts in milk by Nessler’s
test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Mercuric chloride: Reacts violently with strong oxidizers. Avoid
formation of dust, perform all operations under a fume hood.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
3. Potassium iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
4. Citric acid: May cause gastrointestinal irritation with nausea, vomiting
and diarrhea. Excessive intake of citric acid may cause erosion of the
teeth. Causes respiratory tract irritation. Repeated exposure may cause
sensitization dermatitis.
Principle Salts like ammonium sulfate, ammonium nitrate and ammonium
dihydrogen orthophosphate are added in milk to raise the SNF. Ammonium
sulphate is also a nitrogen containing molecule, which if used as adulterant
can also raise nitrogen content if determined by Kjeldahl method. Nessler’s
test is one of the classical methods for qualitative and quantitative analysis
of ammonia and ammonium ions. Nessler’s reagent is an alkaline solution
of potassium mercuric iodide (K2HgI4). On reaction with ammonium ion
Nessler’s reagent produces a yellowish brown colour. The intensity of the
colour is directly proportional to the amount of ammonia/ammonium ion
present.
Apparatus/Instruments 1. Test tube.
2. Conical flask.
3. Water bath.
4. Whatman No. 1 filter paper.
5. Analytical balance (readability 0.01 g).
Materials and Reagents 1. Mercuric chloride.
2. Sodium hydroxide.

22 | M o M – D a i r y a n d D a i r y P r o d u c t s
 3. Potassium iodide.
4. Citric acid.
Preparation of reagents 1. Nessler’s reagent: Dissolve the following chemicals separately.
a) 8.0 g of mercuric chloride in 150 mL distilled water.
b) 60.0 g of sodium hydroxide in 150 mL distilled water.
c) 16.0 g of potassium iodide in 150 mL distilled water.
Add reagent ‘a’ to reagent ‘b’ and mix well. To this mixture, add
reagent ‘c’, mix and dilute the contents to 500 mL. Leave this solution
undisturbed and decant the clear upper layer of the solution. Store in a
stoppered amber glass bottle.
Note: Alternatively, commercially available (readymade) Nessler’s
reagent can also be used.
2. Citric acid solution (5%): Dissolve 5 g citric acid monohydrate in
distilled water and make up the volume to 100 mL with distilled water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis 1. Take 20 mL milk in a conical flask.
2. Warm the milk to 70-80 °C either on direct flame or water bath.
3. Add 5% citric acid solution drop wise in the milk with gentle stirring
till visible coagulation occurs.
4. Flask, if stirred vigorously will result in fine curd particles and which
may impact colour observation.
5. Filter the content using Whatman No. 1 filter paper.
6. Take 5 mL of filtrate into a test tube, add 0.4 mL of Nessler's reagent.
Observe for colour without shaking the test tube.
Inference (Qualitative Milk adulterated with ammonium salts shows instant development of
analysis) orange colour, whereas pale yellow colour indicates unadulterated milk. In
case sample is preserved with formaldehyde, adulterated milk will show
muddy brown colour. Limit of detection is 0.02 g/100 mL milk.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 25-26. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health
and Family Welfare, Government of India, New Delhi.
3. Sharma, R.; Rajput, Y.S. & Naik, N.L. (2012). Detection of adulterants
in milk – a laboratory manual. NDRI Publication No. 88/2012, NDRI,
Karnal, page 49-51).
Approved by Scientific Panel on Methods of Sampling and Analysis

23 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Sulphates in Milk

Method No. FSSAI 01.007:2022 Revision No. & Date 0.0

Scope This method is for the detection of sulphates in milk by barium chloride
test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Barium chloride: Wash thoroughly after handling. Wash thoroughly
after handling. Use with adequate ventilation. Minimize dust
generation and accumulation. Avoid contact with eyes, skin, and
clothing. Do not ingest or inhale.
2. Lactic acid: Ensure adequate ventilation. Wear personal protective
equipment. Do not get in eyes, on skin, or on clothing. Avoid ingestion
and inhalation.
Principle This method is based on the reaction of barium chloride with sulphates.
The solution of barium chloride reacts with sulphate in milk (protein free
filtrate) to give barium sulphate. Using this test, presence of added sulphate
like ammonium sulphate, sodium sulphate, zinc sulphate, magnesium
sulphate, etc. to milk can be confirmed by observing milky-white
precipitates.
Apparatus/Instruments 1. Test tube.
2. Conical flask.
3. Water bath.
4. Whatman No. 42 filter paper.
5. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Barium chloride.
2. Lactic acid (commercially available 85% assay).
Preparation of reagents 1. Barium chloride solution (5%): Dissolve 5.0 g barium chloride in
distilled water and make up the final volume to 100 mL.
2. Lactic acid solution (5%): Take 5 mL of commercially available lactic
acid in 100 mL volumetric flask and make up the volume with distilled
water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation
Method of analysis 1. Take 20 mL milk in a conical flask/test tube bring it to boil on direct
flame, add 2 mL of 5% lactic acid in hot milk and filter the contents
using Whatman No. 42 filter paper.

24 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Take 2 mL filtrate in a separate test tube, add 0.2 mL of 5% barium
chloride and observe for turbidity development.
Inference (Qualitative Formation of turbidity after addition of barium chloride solution indicates
analysis) adulteration of milk with sulphate. The limit of detection is 0.015 g/100
mL milk.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 36-37. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. Singh, P., & Gandhi, N. (2015). Milk preservatives and adulterants:
processing, regulatory and safety issues. Food Reviews International,
31(3), 236-261.
3. Sharma, R.; Rajput, Y.S. & Naik, N.L. (2012). Detection of adulterants
in milk – a laboratory manual. NDRI Publication No. 88/2012, NDRI,
Karnal, page 20-21).
Approved by Scientific Panel on Methods of Sampling and Analysis

25 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Glucose in Milk

Method No. FSSAI 01.008:2022 Revision No. & Date 0.0

Scope This method is for the detection of glucose in milk by Barfoed’s test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Copper acetate: Wash thoroughly after handling. Wash thoroughly after
handling. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation. Store in a cool, dry, well-ventilated area away
from incompatible substances.
2. Lactic acid: Ensure adequate ventilation. Wear personal protective
equipment. Do not get in eyes, on skin, or on clothing. Avoid ingestion
and inhalation.
Principle This test is based on the reaction of glucose (a mononsachharide) with
Barfoed’s reagent. By means of the Barfoed’s reaction, it is possible to
differentiate reducing monosaccharaides from reducing disaccharides
(lactose in case of milk) as monosaccharaides can reduce copper fast
enough in comparison to disaccharides. The formation of green precipitate
is a positive test for reducing monosaccharides.
Apparatus/Instruments 1. Test tube.
2. Water bath.
3. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Copper acetate.
2. Lactic acid (commercially available 85% assay).
Preparation of reagents Barfoed’s reagent: Dissolve 13.3 g of copper acetate in distilled water,
subsequently add 2.0 mL of lactic acid and make up the total volume to
200 mL.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis 1. Take 1 mL milk in a test tube, add 2 mL Barfoed’s reagent.
2. Keep the test tube in boiling water bath for 6 min.
3. Cool the test tube to room temperature (25 ± 3 oC) under tap water.
4. Observe for colour development.
Inference (Qualitative Development of green colour indicates presence of glucose in milk. The
analysis) limit of detection is 0.1 g/100 mL milk. In case sample is preserved with
formaldehyde, adulterated milk will give bluish-green colour.
Reference Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-

26 | M o M – D a i r y a n d D a i r y P r o d u c t s
 [Link], Anand, Page 28-29. Published by Director of Research,
Anand Agricultural University (AAU), Anand (Gujarat).
Approved by Scientific Panel on Methods of Sampling and Analysis

27 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Sodium Chloride in Milk

Method No. FSSAI 01.009:2022 Revision No. & Date 0.0

Scope This method is for the detection of sodium chloride in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Silver nitrate: It is corrosive. It is not combustible, but it is a strong
oxidizer that enhances the combustion of other substances.
2. Potassium chromate: It is a strong oxidizing agent. It is a carcinogen
and mutagen and should be handled with extreme caution.
Principle The presence of extraneously added sodium chloride in milk can be
detected by silver nitrate and potassium chromate reagent. In presence of
sodium chloride in milk, the silver nitrate does not react with potassium
chromate to form brown precipitate of silver chromate.
Apparatus/Instruments 1. Glass test tube.
2. 100 mL volumetric flask.
3. Analytical balance (Readability 0.01 g).
Materials and reagents 1. Silver nitrate.
2. Potassium chromate.
Preparation of reagents 1. Silver nitrate (AgNO3) solution: 0.1 N, aqueous Dissolve 1.6987 g of
AgNO3 and make up the volume to 100 mL with distilled water.
Standardize the prepared silver nitrate with sodium chloride.
2. Potassium chromate (K2CrO4) solution: 10% (w/v) aqueous.: Dissolve
10 g of potassium chromate in distilled water and make up the total
volume to 100 mL.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis 1. Take 5.0 mL of milk sample and add 2.0 mL of 0.1 N silver nitrate
solution
2. Mix the contents thoroughly and add 0.5 mL of 10% potassium
chromate solution and observe the colour.
Inference (Qualitative The appearance of yellow colour indicates presence of added chloride in
analysis) milk, whereas chocolate brown precipitate indicates the absence of added
chloride in milk
Reference 1. Pearson’s Composition and Analysis of Foods, 9th edn, 1991 –
Modified Mohr method, page 14.
2. FSSAI Order. (2018). Revised procedure for detection of sodium
chloride in milk. File No. 1-90/FSSAI/SP (MS&A)/2009 dated 21st
March, 2018. Food Safety Standards Authority of India, New Delhi.

28 | M o M – D a i r y a n d D a i r y P r o d u c t s
Approved by Scientific Panel on Methods of Sampling and Analysis

29 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Method for Detection of Presence of Foreign Fat in Milk

Method No. FSSAI 01.010:2022 Revision No. & Date 0.0

Scope This method is for the detection of presence of foreign fat in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Iso-amyl alcohol: It is a highly flammable liquid. Keep away from
heat/sparks/open flames/hot surfaces. Perform all operations under a
fume hood.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
Principle In the following test, milk fat is isolated from given milk sample and is
subjected to butyro-refractometer (BR) reading. Since, most of vegetable
fat/oils have higher B.R. compared to milk fat, any increase in B.R. reading
above reference value of milk fat indicates adulteration of milk with
vegetable/refined oil. For isolation of milk fat from milk, modified Gerber
butyrometer can be used where both ends of the butyrometer are open.
Stem side opening of the butyrometer (which is generally closed) is closed
with a good quality removable silicon stopper. After the milk fat test,
silicon stopper is removed and milk fat is removed with the help of a
syringe and same is subjected to BR at 40 ℃. Since Gerber sulphuric acid
causes some hydrolysis of fatty acids/triglycerides, the B.R is multiplied
by a factor to obtain corrected B.R. The fat in suspected milk sample can
also be isolated by solvent extraction method in which case correction of
B.R. is not required.
Apparatus/Instruments 1. Modified Gerber Butyrometer (with both ends open)
2. Pipette: 10.75 ± 0.03 mL for milk at 27° C.
3. Automatic measure or tilt measure for dispensing Gerber sulphuric acid:
10 ± 0.25 mL at 27° C.
4. Automatic measure or tilt measure for dispensing iso-amyl alcohol: 1
± 0.05 mL at 27° C.

30 | M o M – D a i r y a n d D a i r y P r o d u c t s
 5. Butyro-refractometer (B.R.).
6. Centrifuge.
7. Analytical balance (Readability 0.01 g).
Materials and reagents 1. Concentrated sulphuric acid (Approximately 36.8 N).
2. Iso amyl alcohol (furfural free). It should have density between 0.807
to 0.809 g/mL at 27 ℃.
3. Diethyl ether.
Preparation of reagents 1. Gerber Sulphuric acid: Take required volume of water in a pyrex flask
(generally 100 mL of water is required for 900 mL of concentrated
sulphuric acid) kept in a basin of ice-cold water. Carefully add the
concentrated sulphuric acid in small quantities at a time keeping the
container sufficiently cold and mix gently. After cooling the flaks,
check the specific gravity of Gerber acid with hydrometer and if
necessary, adjust the Gerber acid to the correct specific gravity with
addition of water or acid taking same precautions as before till specific
gravity is in the range of 1.807 to 1.812 g/mL at 27 ℃ (or 1.815 to
1.820 g/mL at 20 ℃). Store the prepared acid in a glass stoppered bottle
to avoid absorption of water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation
Method of analysis Modified Gerber Method:
1. Isolate the fat from milk by Gerber method using specially designed
milk butyrometer, which is open at both ends; close the stem side
opening with a good quality acid resistant silicon stopper.
2. Add 10 mL of Gerber sulphuric acid, 10.75 mL milk and 1 mL iso-
amyl alcohol; close the neck side with lock stopper; mix the contents
and centrifuge at 1100 rpm (approximately 300 g) for 5 min to get a
clear fat column.
3. Remove the silicon stopper from the stem side and take out the fat from
the stem of the butyrometer using a capillary or a syringe.
4. For taking BR. reading of the milk fat, clean the prism of the butyro-
refractometer with diethyl ether and allow the ether to evaporate to
dryness.
5. Maintain the temperature of the prism at 40 ℃ by circulating water
using a thermostatically controlled water-bath or thermostatically
controlled automatic butyro- refractometer.
6. Calibrate the butyro-refractometer by applying standard liquid solution
of known BR reading.
7. Again, clean the prism with diethyl ether; apply 1-2 drops of clear,
extracted fat between the prism.
8. Wait for 2 min before taking the reading so that the sample should
attain temperature of 40 ℃.
9. A correction of 0.55 is added to the observed B.R. reading for each

31 | M o M – D a i r y a n d D a i r y P r o d u c t s
 degree above 40 ℃ or subtracted for each degree below 40 ℃ to get
corrected B.R. reading of the sample.
Solvent Extraction Method:
1. Extract fat from the milk sample by Rose-Gottlieb method.
2. The B.R. reading at 40 ℃ of the extracted fat and interpret the results.
Cream Extraction Method:
1. Separate the milk fat from the milk sample by centrifugation (5000 g,
10 min, 4 ℃).
2. Carefully remove the cream plug and collect the cream in an aluminium
dish.
3. Heat the dish over burner till ghee formation takes place.
4. Filter the ghee residues and take the BR reading of ghee and interpret
the results.
Note:
(a) If fat is extracted using modified Gerber method, correction factor is
required.
(b) Result can be interpreted directly from the B.R. reading if fat is
extracted using other method. Further, check for the presence of
extraneous fat can be done by checking the fatty acid profile of the
extracted fat by GLC.
Calculation with units of Calculate the corrected B.R. reading of isolated fat as follows:
expression
Corrected B.R. = Observed B.R. × 1.08 (correction factor is required only
in case fat is extracted with modified Gerber method)
Inference (Qualitative If the B.R. reading differs from the prescribed limit specified for states /
analysis) Union Territory (as indicated in Clause 2.1.8 of FSS (Food Products
Standards and Food Additives), 2011) presence of foreign fat in the milk
may be suspected.
Reference 1. IS 1479 (Part I):2016. Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.
2. Arora, K.L.; Lal, D.; Seth, R. & Ram, J. (1996). Platform test for
detection of refined mustard oil adulteration in milk. Indian J. Dairy
Sci., 49 (10): 721-723.
3. Lal, D.; Seth, R.; Arora, K.L. & Ram, J. (1998). Detection of vegetable
oils in milk. Indian Dairyman, 50 (7): 17-18.
Approved by Scientific Panel on Methods of Sampling and Analysis

32 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Nitrate in Milk

Method No. FSSAI 01.011:2022 Revision No. & Date 0.0

Scope This method is for the detection of nitrate in milk by diphenylamine test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Glacial acetic acid: Use with adequate ventilation. Keep away from
heat. Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin
and eyes. Keep away from incompatibles such as oxidizing agents,
reducing agents, metals, acids, alkalis.
3. Diphenylamine: Avoid contact with skin and eyes. Do not breathe
vapours. Provide good ventilation in process area to prevent formation
of vapours.
Principle The test consists of adding a solution of diphenylamine prepared in
sulphuric acid to milk. In the presence of nitrates, diphenylamine is
oxidized, giving a blue coloration. Hence, development of blue colour
indicates the presence of nitrate.
Apparatus/Instruments 1. Test tube.
2. Conical flask.
3. Water bath.
4. Whatman No. 1 filter paper.
5. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Diphenylamine.
2. Concentrated sulphuric acid (Approximately 36.8 N).
3. Glacial acetic acid.
Preparation of reagents 1. Diphenylamine solution: The reagent is prepared by dissolving 0.085 g
diphenylamine in 50 mL distilled water and gradually 450 mL of
concentrated sulphuric acid is added with constant stirring. During
preparation of reagent the content is kept cool by dipping in cold water.
Diphenyl amine solution should be prepared freshly and shall be
colourless.

33 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Acetic acid (10%): Take 10 mL glacial acetic acid in 100 mL
volumetric flask and make up the volume with distilled water.
Method of analysis 1. Take 20 mL milk in a conical flask; warm the milk to 70-80 C either
on direct flame or boiling water bath.
2. Add 10% acetic acid drop wise in the milk with gentle stirring till
visible coagulation.
3. Filter the contents using Whatman No. 1 filter paper.
4. Take 2 mL diphenylamine solution in a test tube, add 1 mL filtrate in
test tube containing diphenylamine solution.
5. Observe for ring formation at the junction of two solutions.
Inference (Qualitative Formation of blue ring at the junction of two solutions indicates
analysis) adulteration of milk with nitrate or surface water. The limit of detection is
0.002 g/100 mL milk.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 34-36. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. Sharma, R.; Rajput, Y.S. & Naik, N.L. (2012). Detection of adulterants
in milk – a laboratory manual. NDRI Publication No. 88/2012, NDRI,
Karnal, page 47-48.
Approved by Scientific Panel on Methods of Sampling and Analysis

34 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Neutralizers in Milk

Method No. FSSAI 01.012:2022 Revision No. & Date 0.0

Scope This method is for the detection of neutralizers in milk by phenol red test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
Principle This reaction is based on the pH of milk, wherein phenol red is used as a
pH indicator. It shows colour transition from yellow to red over the pH
range 6.8 to 8.2. At pH value greater than 8.2 phenol red turns a bright pink.
Neutralization of milk causes increase in the pH which can be detected
using this indicator.
Apparatus/Instruments 1. Analytical balance (Readability 0.01 g)
2. Test tube
Materials and Reagents 1. Phenol red
2. Ethanol
Preparation of reagents 1. Phenol red solution: Dissolve 0.05 g of phenol red in 20 mL ethanol
and make up the volume up to 100 mL using distilled water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation
Method of analysis 1. Take 4 mL milk in a test tube, add 1 mL phenol red solution.
2. Mix the contents and observe for colour.
Inference (Qualitative Development of pinkish orange colour indicates neutralizer adulterated
analysis) milk, whereas unadulterated sample remains yellow. The limit of detection
is as follows:
Sodium hydroxide: 0.04 g/100 mL milk
Na2CO3: 0.08 g/ 100 mL milk
NaHCO3: 0.2 g/ 100 mL milk
In case sample is preserved with formaldehyde, adulterated milk will show
pinkish orange colour with reduced intensity.
Reference Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 42. Published by Director of Research,
Anand Agricultural University (AAU), Anand (Gujarat).
Approved by Scientific Panel on Methods of Sampling and Analysis

35 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Method for Detection of Hypochlorite and Chloramines in Milk

Method No. FSSAI 01.013:2022 Revision No. & Date 0.0

Scope This method is for detection of hypochlorite and chloramines in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Stannous chloride: It is non-combustible; substance itself does not burn
but may decompose upon heating to produce corrosive and/or toxic
fumes. Thermal decomposition can lead to release of irritating gases or
vapours.
2. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
3. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
Principle In this test, yellowish fluorescence is produced due to the presence of
chlorate (potassium or sodium chlorate) in the hypochlorite solution and is
directly proportional to the amount of chlorate present. Stannous chloride
in the reaction acts as reducing agent.
Apparatus/Instruments 1. Centrifuge.
2. Tubes for centrifuge: 12.5 mL capacity.
3. UV lamp.
4. Analytical balance (Readability 0.01 g).
Materials and reagents 1. Stannous chloride (SnCl2.2H2O).
2. Potassium iodide.
3. Concentrated hydrochloric acid (Approximately 11.6 N).

36 | M o M – D a i r y a n d D a i r y P r o d u c t s
 4. Starch.
5. Concentrated sulphuric acid (Approximately 36.8 N).
Preparation of reagents 1. Stannous chloride (SnCl2.2H2O) solution: 0.025%, (w/v) in 73.5%
sulphuric acid (prepared by mixing three volumes of concentrated
sulphuric acid and one volume of distilled water).
2. Potassium iodide (KI) solution: Prepare fresh by dissolving 7 g of KI
in 100 mL water.
3. Dilute hydrochloric acid: To 200 mL of water, add 100 mL
concentrated hydrochloric acid.
4. Starch solution: Boil 1 g starch in 100 mL water. Cool before using.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Method 1. Detection of Hypochlorite:
1. Cool about 3 mL of milk sample in a test tube in a freezing mixture of
ice and salt to 2-5 ℃.
2. In another test tube, take an equal volume of the stannous chloride
solution and similarly cool and add to milk. Shake the tube whilst in
freezing mixture and hold for 3 min.
3. Place the mixture in a centrifuge tube and centrifuge for 3 min at 2500
rpm (approximately 1012 g).
4. A yellow-green colour is produced in the presence of hypochlorite.
Alternatively, after centrifuging, examine the tube in ultraviolet light
for the presence of any yellow fluorescence.
Method 2. Detection of Hypochlorite and Chloramines:
1. To 5 mL of sample in a test tube add 1.5 mL of potassium iodide
solution, mix thoroughly by shaking and note the colour of milk.
2. If unaltered, add 4 mL of dilute hydrochloric acid, mix thoroughly with
a glass rod flattened at one end and note the colour of curd.
3. Subsequently, place the tube in a water bath previously heated to 85 ℃
and allow it to remain for 10 min. The curd will rise to the surface. Cool
the tube rapidly by placing in cold water. Note the colour of the curd
and the liquid.
4. Next add 0.5 to 1.0 mL of starch solution to the liquid below curd and
note the colour.
Inference (Qualitative Table.1. Reactions with various tests to detect residual chlorine in
analysis) milk.

Test Concentration of available chlorine

No 1:1000 1:2000 1:5000 1:10000 1:25000 1:50000

37 | M o M – D a i r y a n d D a i r y P r o d u c t s
 A Yellowish Deep Pale - - -
brown yellow yellow
B Yellowish Deep Light - - -
brown yellow yellow
C Yellowish Deep Yellow Yellow Pale Yellowis
brown yellow yellow h
D Blue purple Blue Blue, Dark Red Pale red-
purple dark purple purple purple
purple
* Indicates the step number followed in Method 2 of the above procedure.
Note: The method is not reliable in the presence of more than 2.5 ppm of
copper.
Reference 1. IS 1479 (Part I): 2016. Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.
st
2. AOAC 922.08. 21 Edn. (2019). Hypochlorites and Chloramines in
milk. AOAC International, USA.
Approved by Scientific Panel on Methods of Sampling and Analysis

38 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Quaternary Ammonium Compounds in Milk

Method No. FSSAI 01.014:2022 Revision No. & Date 0.0

Scope This method is for the detection of Quaternary Ammonium Compounds
(QAC) in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Tetrachloroethane: It is a colorless, volatile, non-flammable, liquid,
chlorinated hydrocarbon with an ether-like odor that may emit toxic
fumes of phosgene when exposed to sunlight or flames.
3. Acetone: It is a flammable liquid and dangerous fire hazard. It reacts
with strong oxidizing agents (such as perchlorates, chlorine, bromine,
fluorine) to form explosive peroxides. Perform all operations under a
fume hood.
4. Citric acid: May cause gastrointestinal irritation with nausea, vomiting
and diarrhea. Excessive intake of citric acid may cause erosion of the
teeth. Causes respiratory tract irritation. Repeated exposure may cause
sensitization dermatitis.
Principle The amount of QAC can be approximately determined by titration with a
standard anionic detergent solution. QAC may be present in milk due to
some residual detergent solution remaining after bottle washing. The
following method detects about 5 mg / kg in milk. .
Apparatus/Instruments 1. 100 mL volumetric flask.
2. Centrifuge tube.
3. Analytical balance (Readability 0.01 g).
Materials/reagents 1. Eosin.
2. Acetone.
3. Tetrachloroethane.
4. Citric acid.
5. Sodium hydroxide.
Preparation of reagents 1. Indicator solution: Prepare a stock solution by dissolving 0.05 g eosin
in 100 mL acetone. Shake 10 mL of stock solution with 90 mL of
tetrachloroethane and 1 g citric acid and filter before use.

39 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Sodium hydroxide solution (50%): Dissolve 50 g sodium hydroxide in
100 mL water.
3. Buffer: Dissolve 25 g citric acid in 100 mL water and adjust to pH 3.5
with 50% sodium hydroxide solution (approximately 15 mL required).
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis 1. To a centrifuge tube add 1 mL milk, 5 mL water, 1 mL indicator
solution and 0.2 mL buffer and shake hard for 10 sec.
2. Centrifuge for 5 min at 3200 rpm (approximately 1660 g).
3. Observe the colour in bottom layer.
Inference (Qualitative QAC present in the sample is indicated by the formation of red or pink
analysis) colouration. In bottom layer If the colour is deep pink or red, the amount
of QAC can be approximately determined by titration with a standard
anionic detergent solution. Samples containing about 1 mg/kg of QAC
show a faint pink colour.
Reference Pearson’s Composition and Analysis of Foods 9thedn 1991, page 548.

Approved by Scientific Panel on Methods of Sampling and Analysis

40 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Anionic Detergents in Milk

Method No. FSSAI 01.015:2022 Revision No. & Date 0.0

Scope This method is for the detection of detergents in milk by methylene blue
test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Methanol: It is a highly flammable liquid and vapour. Keep away from
heat, sparks, open flames, hot surfaces. Perform all operations under a
fume hood.
2. Chloroform: Decomposes slowly on exposure to light and air to release
toxic and corrosive vapours like phosgene, chlorine etc. It reacts
strongly with strong oxidizers and forms toxic vapours. Keep away
from heat and strong oxidizing agents.
Principle Detergents may be intentionally added in milk to emulsify the added
vegetable oil in milk. Detergents are also believed to be part of the so
called “synthetic milk” formulation. Detergents can be anionic, cationic
and non-ionic, among which anionic types are the most commonly used.
Methylene blue is cationic dye which forms complex with anionic
detergents. It is normally water-soluble compound; however, it shows
affinity for anionic detergents, if they are present. In this method,
detergents are first extracted in chloroform and then methylene blue
solution is added. In presence of detergent, blue colour is developed in
chloroform layer of the sample, whereas blue colour is observed in milk
layer in control (pure milk). Chloroform is heavier (density 1.49 g/mL) than
milk (density 1.030 g/mL), and hence settles at the bottom. This implies
that observation of blue colour in the bottom layer indicates presence of
detergents.
Apparatus/Instruments 1. Test tube.
2. Whatman No. 1 filter paper.
3. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Methanol.
2. Methylene blue.
3. Chloroform.
Preparation of reagents 1. Methylene blue solution: 12.5 mg methylene blue is dissolved in 100
mL of distilled water. Protect the solution from direct sunlight.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.

41 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Take 2.5 mL of suspected milk sample in a test tube and add 7.5 mL
methanol, filter the contents through Whatman No. 1 filter paper.
2. Take 2 mL filtrate in a test tube, add 2 mL of methylene blue solution
and shake well.
3. Subsequently, add 4 mL chloroform and shake well again. Allow the
chloroform layer to separate. Compare the colour extracted in the
chloroform layer in suspected milk with that for pure milk.
Inference (Qualitative If the methylene blue colour extracted from a suspected sample into the
analysis) chloroform layer is darker than that extracted from pure milk sample, it
indicates the presence of detergent in milk. The limit of detection is 0.02 g/
100 mL milk.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 22-23. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. Rajput, Y.S.; Sharma, R. & Kaur, S. (2006) A kit for detection of
detergent in milk. Indian Patent Office File No. 1970/Del/2006. Indian
Patent Grant No. 264661 dated 14.01.2015.
Approved by Scientific Panel on Methods of Sampling and Analysis

42 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Formaldehyde in Milk

Method No. FSSAI 01.016:2022 Revision No. & Date 0.0

Scope This method is for the detection of presence of formaldehyde in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
3. Ferric chloride: It is a corrosive chemical. Substance itself does not
burn but may decompose upon heating to produce corrosive and/or
toxic fumes. It can react with metals to form flammable and potentially
explosive hydrogen gas.
Principle Formaldehyde (HCHO) is the simplest aldehyde. Exposure to
formaldehyde is reported to be extremely harmful to human health.
Formaldehyde manifests toxicity in humans, acute exposure can cause
pain, dermatitis, nausea, arrhythmia, coma and renal failure. Chronic
exposure increases the risk of cancer. Formaldehyde is permitted as
preservative only for analytical purposes in milk and milk product sample;
though its presence marketable dairy products is illegal. Two methods can
be used for the detection of presence of formaldehyde in milk.
In the Hehner test for detection of formaldehyde in milk, concentrated
sulphuric acid and ferric chloride is used. The test is an aldehyde-oxidation
reaction of an aromatic amine. Ferric chloride acts as oxidising agent for
formaldehyde. The formaldehyde reaction depends on the presence of the
tryptophan in the protein molecule. The violet colour develops as a result
of the reaction of oxidised formaldehyde with tryptophan. The intensity of
the reaction with different proteins varies in direct proportion to the amount
of tryptophan present in the protein molecule.

43 | M o M – D a i r y a n d D a i r y P r o d u c t s
 In Leech test for detection of formaldehyde, concentrated hydrochloric acid
is used in place of sulphuric acid (which is used in Hehner test). The
contents are heated to enhance oxidation and resultant colour development.
Apparatus/Instruments 1. Test tube.
2. Water bath.
3. Hot plate or burner.
4. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Ferric chloride.
2. Concentrated sulphuric acid (Approximately 36.8 N).
3. Concentrated hydrochloric acid (Approximately 11.6 N).
Preparation of reagents 1. Ferric chloride (10%): Take 10 g of ferric chloride in 100 mL
volumetric flask and make up the volume with distilled water.
2. Sulphuric acid (80%): Add 80 mL concentrated sulphuric acid into 20
mL distilled water.
3. Hydrochloric acid containing ferric chloride: The reagent is prepared
by adding 1 mL of 10% ferric chloride solution in a 500 mL volumetric
flask and making up the volume with concentrated hydrochloric acid.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation
Method of analysis Hehner test:
1. Take 5 mL milk sample in a test tube, add 5 mL distilled water and 0.1
mL of 10% FeCl3 solution.
2. Mix the contents and add 10 mL sulphuric acid (80%) from the side of
the test tube.
Leech Test:
Using milk as a medium:
1. Take 5 mL milk in a test tube, add 3 mL hydrochloric acid containing
ferric chloride.
2. Heat on direct flame for 1 min and observe for colour.
Using whey as a medium:
1. Take 5 mL milk in a test tube, add 3 mL hydrochloric acid containing
ferric chloride (amount of milk and reagent can be increased
proportionately to get sufficient filtrate).
2. Filter the content using Whatman No. 1 filter paper.
3. Take filtrate in a test tube and heat on a direct flame for 1 min.
4. Observe the colour.
Inference (Qualitative Hehner test: Violet ring at the junction of two layers indicates presence of
analysis) formaldehyde. The limit of detection is 0.0005 mL formalin/100 mL milk.
Leech Test: Appearance of violet colour indicates presence of
formaldehyde in milk. The limit of detection is:
0.002 mL formalin/100 mL milk (when test is performed in milk)
0.0005 mL formalin/100 mL milk (when test is performed in whey)

44 | M o M – D a i r y a n d D a i r y P r o d u c t s
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 39-40. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. Kamps, J. J., Hopkinson, R. J., Schofield, C. J., & Claridge, T. D.
(2019). How formaldehyde reacts with amino acids. Communications
Chemistry, 2(1), 1-14.
3. Pearson’s Composition and Analysis of foods, 9th edition, 1991 page
90.
4. IS 1479 (Part II): 1961 (Reaffirmed Year: 2018). Methods of test for
Dairy Industry – Chemical Analysis of Milk. Bureau of Indian
Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

45 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Hydrogen Peroxide in Milk

Method No. FSSAI 01.017:2022 Revision No. & Date 0.0

Scope This method is for the detection of presence of hydrogen peroxide in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Vanadium pentoxide: Should be handled as a carcinogen with extreme
caution. It may react violently with chlorine trifluoride, lithium and
peroxyformic acid. It is incompatible with strong acids like
hydrochloric acid, sulphuric acid, nitric acid.
2. Para-phenylenediamine: Ensure there is adequate ventilation while
working with this chemical. Avoid formation of dust and breathing
vapours, mist or gas.
3. Potassium iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
4. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
Principle Three different methods have been indicated for detection of presence of
hydrogen peroxide. In the first method, appearance of pink or red color
after addition of vanadium pentoxide reagent in milk samples indicates the
presence of hydrogen peroxide in milk. In the second method, para-
phenylenediamine gives blue colour in presence of hydrogen peroxide in
milk. In the third method, hydrogen peroxide present in milk sample
liberates iodine from potassium iodide which in presence of starch gives
blue colour.
Apparatus/ 1. 100 mL volumetric flask.
Instruments 2. Analytical balance (Readability 0.01 g).
Materials and 1. Vanadium pentoxide.
Reagents 2. Para-phenylenediamine.
3. Potassium iodide.
4. Starch.
5. Concentrated sulphuric acid (Approximately 36.8 N).
Preparation of reagents 1. Vanadium pentoxide solution: Dissolve 1 g of vanadium pentoxide

46 | M o M – D a i r y a n d D a i r y P r o d u c t s
 (V2O5) in 100 mL dilute sulphuric acid (6 mL concentrated sulphuric
acid diluted to 100 mL).
2. Para-phenylenediamine solution: Weigh 2.0 g of para-
phenylenediamine and dissolve it in distilled water to obtain 100 mL
solution i.e. 2% aqueous solution, w/v. Dissolution of para-
phenylenediamine in water is difficult and requires thorough mixing.
The solution will appear pale yellow.
3. Potassium iodide solution: Weigh 20 g of potassium iodide and
dissolve it in distilled water to obtain a 100 mL solution.
4. Starch solution: Take 1 g starch powder and dissolve it in distilled water
by heating and make up the volume to 100 mL.
5. Potassium iodide–Starch reagent: Mix equal volumes of 20%
potassium iodide solution and 1% starch solution.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Vanadium pentoxide test:
1. Add 10 to 20 drops of vanadium pentoxide reagent in 10 mL milk
sample and mix. Note the colour of the sample.
Para-phenylenediamine test
1. Add about 2 mL of milk in a test tube; add equal volume of raw milk.
Add two drops of 2% of para-phenylenediamine reagent and mix well.
2. Observe the color of the solution in the tube.
Using Potassium iodide and Starch
1. Take 1 mL of milk sample in a test tube, add 1 mL of the potassium
iodide-starch reagent and mix well.
2. Observe the color of the solution in the tube.
Inference (Qualitative Vanadium pentoxide test: Appearance of pink or red colour indicates the
analysis) presence of hydrogen peroxide in milk.
Para-phenylenediamine test: The milk sample adulterated with H2O2
develops blue colouration, whereas pure milk samples remain white in
colour.
Using potassium iodide and starch: The milk sample adulterated with
H2O2 develops blue colouration, whereas pure milk samples remain white
in colour.
st
Reference
1. AOAC 957.08. 21 Edn. (2019). Official Method- Hydrogen Peroxide
in milk. AOAC International, USA.
2. IS 1479 (Part I) - 2016 Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.
3. Sharma, R.; Rajput, Y.S. & Naik, N.L. (2012). Detection of adulterants
in milk – a laboratory manual. NDRI Publication No. 88/2012, NDRI,
Karnal, page 10.
Approved by Scientific Panel on Methods of Sampling and Analysis

47 | M o M – D a i r y a n d D a i r y P r o d u c t s
48 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Boric acid and Borates in Milk

Method No. FSSAI 01.018:2022 Revision No. & Date 0.0

Scope This method is to test the presence of boric acid and borates in milk sample.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
Mmonium hydroxide: Ammonium hydroxide causes skin irritation;
contact can lead to severe irritation and burns. If inhaled, mild exposure
can cause nose irritation (sneezing, coughing). Exposure to high
concentrations must be avoided
2. Caustic soda: Protect skin and eyes when working with these alkalis as
solids or concentrated solutions. While making solution add pellets to
water and not vice versa.
Principle Appearance of characteristic red colour on the turmeric paper indicates the
presence of boric acid or borax.
Apparatus/ Instruments 1. 250 mL Erlenmeyer flask.
2. Porcelain dish.
3. Whatman filter paper Grade 2.
4. Analytical balance (Readability 0.01 g).
Materials and reagents 1. Turmeric Paper.
2. Concentrated hydrochloric acid (Approximately 11.6 N).
3. Ammonium hydroxide (specific gravity 0.88).
4. Lime water or caustic soda.
5. Ethanol (80%).
Preparation of reagents 1. Preparation of Turmeric Paper: Weigh 1.5 to 2.0 g of turmeric powder
in 250 mL Erlenmeyer flask and add 100 mL 80% (v/v) ethanol. Shake
for 5 min and filter. Collect the filtrate in a flat bottom dish. Dip Whatman
filter paper Grade 2 in the clear filtrate. Remove the paper and hang to
dry in air. After 1 h, cut the paper into 6 × 1 cm strips and store in tightly
stoppered bottle protected from light.

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Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Method 1:
1. Take 20 mL of milk in a porcelain dish and add 1.4 mL of concentrated
hydrochloric acid and mix it thoroughly.
2. Dip a strip of turmeric paper in the acidified milk. Appearance of
characteristic red colour on the turmeric paper indicates the presence of
boric acid or borax (Na2B4O7.10H2O).
Method 2.
1. Take 25 mL of milk sample in a porcelain dish. Make the sample
alkaline with lime water or caustic soda and evaporate it to dryness on
water bath. Ignite the dry residue in the dish by heating over low flame
until the organic matter is completely charred.
2. Cool the charred residue, re-digest with 15 mL distilled water and add
concentrated hydrochloric acid, drop by drop until the ignited residue
is dissolved; then add 1 mL of concentrated hydrochloric acid in excess.
Immerse a strip of turmeric paper in the solution and dry it in air.
3. Observe the colour change.
Inference (Qualitative 1. Method 1: Appearance of characteristic red colour on the turmeric
analysis) paper indicates the presence of boric acid or borax (Na2B4O7.10H2O).
The red colour changes to dark blue green on adding ammonium
hydroxide, but reappears on re-acidification with hydrochloric acid.
2. Method 2: Appearance of characteristic red colour indicates the
presence of boric acid or borax. On addition of ammonium hydroxide,
the red turmeric paper turns deep bluish-green in colour. The red colour
reappears on re-acidification with hydrochloric acid.
Reference IS 1479 (Part I): 2016 Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Salicylic acid in Milk

Method No. FSSAI 01.019:2022 Revision No. & Date 0.0

Scope This method is to test the presence of salicylic acid in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
3. Ferric chloride: It is a corrosive chemical. Substance itself does not
burn but may decompose upon heating to produce corrosive and/or
toxic fumes. It can react with metals to form flammable and potentially
explosive hydrogen gas.
Principle A violet colour outcome by adding ferric chloride solution indicates
presence of salicylic acid.
Apparatus/Instrument 1. Separating funnel.
2. Centrifuge.
3. Analytical balance (Readability 0.01 g).
4. Steam bath
5. Whatman No. 1 filter paper.
Materials and reagent 1. Concentrated hydrochloric acid (Approximately 11.6 N).
2. Diethyl Ether.
3. Ferric Chloride.
4. Ammonium hydroxide (specific gravity 0.88).
Preparation of reagents 1. Dilute hydrochloric acid: dilute one part of acid with three parts of
distilled water)
2. Neutral ferric chloride solution, 0.5% (v/v): Dissolve 0.5 g of ferric
chloride in distilled water and make up the volume to 100 mL with
distilled water. Add ammonimum hydroxide solution dropwise to the
prepared solution till brown colour precipitates appear. Filter the
solution and use the clear filtrate.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.

51 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Take 100 mL of milk in a separating funnel, add 5 mL of dilute
hydrochloric. Shake until curdled and filter.
2. Extract the filterate with 50 to 100 mL diethyl ether and wash the ether
layer with two 5 mL portions of water.
3. Evaporate the ether on steam bath and add one drop of ferric chloride
solution.
4. Observe the colour change.
Inference (Qualitative A violet colour indicates presence of salicylic acid.
analysis)
st
Reference
1. A.O.A.C 975.30. 21 Edn. (2019). Official method Salicylic acid in
Food and Beverages. AOAC International, USA.
2. IS 1479 (Part II): 1961 (Reaffirmed Year: 2018). Methods of test for
Dairy Industry – Chemical Analysis of Milk. Bureau of Indian
Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Maltodextrin in Milk

Method No. FSSAI 01.020:2022 Revision No. & Date 0.0

Scope This method is for the detection of maltodextrin in milk by Iodine test.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Iodine: It is not compatible with combustibles, strong bases, halogens
and ethanol. It reacts violently or explosively with acetylene,
acetaldehyde, metal hydrides and metal carbides.
2. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
3. Citric acid: May cause gastrointestinal irritation with nausea, vomiting
and diarrhea. Excessive intake of citric acid may cause erosion of the
teeth. Causes respiratory tract irritation. Repeated exposure may cause
sensitization dermatitis.
Principle This test is based on the reactionof Maltodextrin with iodine. Similar to
starch, maltodextrin also forms complex with iodine. The dextrins having
chain length of 8 DP (degree of polymerization) to 15 DP forms complex
with iodine giving yellow-brown to brown to reddish purple colour as chain
length increases (Thoma and French, 1960). While, the chain length more
than 45 DP gives blue colour.
Apparatus/Instruments 1. Test tube.
2. Conical flask.
3. Water bath.
4. Whatman No. 1 filter paper.
5. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Potassium iodide.
2. Iodine crystals.
3. Citric acid monohydrate.
Preparation of reagents 1. Iodine solution (1%): Dissolve 2.5 g potassium iodide in 100 mL
distilled water, then add 1 g pure iodine crystals. Prepare iodine
solution at least a day before as iodine dissolves slowly.
2. Citric acid (5%): Dissolve 5 g citric acid monohydrate in distilled water
and make up the volume to 100 mL.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.

53 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Take 20 mL milk in a conical flask, warm the milk to 70-80 C either
on direct flame or boiling water bath.
2. Add 5% citric acid solution drop wise in the milk with gentle stirring
until clear coagulation occurs (approximate consumption of citric acid
required would be 1.5-2 mL).
3. Flask, if stirred vigorously will result in fine curd particles which may
impact colour observation.
4. Filter the contents using Whatman No. 1 filter paper.
5. Take 5 mL filtrate in a test tube, add 0.25 mL of 1% iodine solution.
6. Mix the contents and observe for colour development.
Inference (Qualitative Development of red-brown colour indicates adulteration of milk with
analysis) maltodextrin, whereas pure milk remains yellow in colour. In case sample
is preserved with formaldehyde, adulterated milk will show same red
brown colour. Limit of detection is 0.1g/ 100 mL milk.
Reference 1. Aparnathi, K.D.; Shaikh, A.I. & Patel, S.I. (2020). Qualitative Tests for
Detection of Common Adulterants in Milk. AAU Publication No. RES-
[Link], Anand, Page 30-31. Published by Director of
Research, Anand Agricultural University (AAU), Anand (Gujarat).
2. Thoma, J.A., & French, D. (1960). The Starch-Iodine-Iodide
Interaction. Part I. Spectrophotometric Investigations1. Journal of the
American Chemical Society, 82(16), 4144-4147.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Alkaline Phosphatase in Milk

Method No. FSSAI 01.021:2022 Revision No. & Date 0.0

Scope This method is for the detection of alkaline phosphatase in milk. It may
be noted that the test is applicable to recently pasteurized milk as it has
been reported that denatured alkaline phosphatase is activated during
storage of pasteurized milk.
Caution Follow all safety procedures while handling and disposing solutions.
Wear laboratory apron, shoes, safety goggles and mask while working
with chemicals. Refer to MSDS (Material Safety Data Sheets) for specific
information.
1. Sodium Carbonate: If improperly handled, or in the event of
accidental exposure, sodium carbonate can pose several hazards to
people’s health and safety. Inhalation of this chemical can lead to
adverse effects such as respiratory tract irritation, coughing, shortness
of breath, and pulmonary edema.
2. Sodium bicarbonate: It might increase blood pressure. People who
already have high blood pressure should avoid sodium bicarbonate.
Low potassium levels in the blood: Sodium bicarbonate might lower
potassium blood levels. People who already have low levels of
potassium should avoid sodium bicarbonate.
3. Disodium para nitrophenyl phosphate: Eye and skin Contact - Rinse
immediately with plenty of water for at least 15 min., also under the
eyelids. Inhalation- Remove to fresh air. Ingestion - Clean mouth with
water and drink afterwards plenty of water. Get medical attention if
symptoms occur.
4. Para-nitrophenol: Contact may severely irritate skin and eyes.
Poisonous by ingestion and moderately toxic by skin contact.
5. Trichloroacetic acid: It is a corrosive chemical and contact can
severely irritate and burn the skin and eyes with possible eye damage.
Breathing trichloroacetic acid can irritate the nose and throat.
Breathing trichloroacetic acid can irritate the lungs causing coughing
and/or shortness of breath.
6. Sodium hydroxide: It is very corrosive. It can cause irritation to the
eyes, skin, and mucous membrane; an allergic reaction; eye and skin
burns; and temporary loss of hair. Workers may be harmed from
exposure to sodium hydroxide. The level of harm depends upon the
dose, duration, and work being done
Principle This test is a routine quality control test for assessing the efficiency of
pasteurization of milk. The principle of this method is that, disodium
para-nitrophenyl phosphate which is used as a substrate for alkaline
phosphatase, liberates a yellow-coloured compound para-nitrophenol,

55 | M o M – D a i r y a n d D a i r y P r o d u c t s
 under the alkaline conditions in the presence of alkaline phosphatase.
This liberated yellow colored product is measured spectrophotometrically
at 405 nm. p-Nitrophenol when present in the acidic medium remains in
an undissociated colorless form, while it readily gets converted into its
quinoid structure under alkaline conditions to form a yellow-coloured
product.
Apparatus/Instruments 1. Test tube.
2. Pipettes.
3. Volumetric flask.
4. Water bath.
5. Whatman No. 1 filter paper.
6. Funnel.
7. Tripod stand.
8. UV-Visible Spectrophotometer.
9. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Sodium carbonate.
2. Sodium bicarbonate.
3. Disodium para nitrophenyl phosphate.
4. Para-nitrophenol.
5. Trichloroacetic acid.
6. Sodium hydroxide.
Preparation of reagents 1. Carbonate-bicarbonate buffer (pH 10.0): Dissolve 3.5 g sodium
carbonate (Na2CO3) and 1.5 g sodium bicarbonate (NaHCO3) in 1 L
of distilled water and adjust the pH to 10.
2. Buffer substrate: Dissolve 0.15 g of disodium p-nitrophenyl
phosphate in the carbonate-bicarbonate buffer and make the volume
to 100 mL with the buffer. Store the solution at 4 °C and protect from
light. This solution is stable for one week.
3. Trichloroacetic acid (TCA): Weigh 28 g of trichloroacetic acid and
dissolve in distilled water and make up the volume to 100 mL using
distilled water in a volumetric flask.
4. Sodium hydroxide: 14% solution in distilled water.
Regents for Standard Curve Preparation
1. Stock p-nitrophenol (PNP) solution: Dissolve 100 mg of PNP in 100
mL distilled water.
Working PNP solution: Dissolve 1.0 mL stock solution in 100 mL of
distilled water
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Measurement in milk sample
1. Take 3.5 mL of carbonate-bicarbonate buffer in a test-tube and add 5
mL buffer substrate, keep the tube in a water bath for 5 min at 37 °C.

56 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. After the above-mentioned pre-incubation, add 0.5 mL of milk
(previously diluted 1:10 with distilled water) and mix the contents. A
corresponding blank is carried out using boiled milk (inactivated
enzyme)
3. Incubate the tubes for 30 min at 37 °C.
4. Add 1 mL of TCA to the tubes and mix the contents properly.
5. Filter the content of the test tube through filter paper (Whatman no.
1).
6. To 5 mL of filtrate in a test tube, add 0.5 mL of sodium hydroxide.
Centrifuge, if necessary.
7. Measure the absorbance of developed color in a spectrophotometer at
405 nm.
Preparation of standard curve
1. Prepare solution for different concentration (0-50 g) p-nitrophenol
by serially diluting the working solution.
2. Add 0.5 mL of sodium hydroxide.
3. Measure the absorbance of in a spectrophotometer at 405 nm and
calculate the enzyme concentration from the standard curve
Calculation with units of The concentration of p-nitrophenol in the sample is calculated using the
expression standard curve.
Calculations:

1. Preparation of graph of PNP Concentration v/s Absorbance

1 mL stock PNP solution = 1 mg PNP = 1000 g PNP
Dissolve 1.0 mL stock solution = 1.0 mg PNP in 100 mL H2O
100 mL working PNP solution = 1000 g PNP
1 mL working PNP solution = 10 g PNP
0.1 mL working PNP solution = 1.0 g PNP

Observation table for standard curve:

Test Working H2 O 14% Sodium Conc. Absorba

tube PNP soln. (mL) hydroxide PNP (g) nce at
(mL) (mL) 405 nm
Blank 0.0 5.0 0.5 0.0
1 0.5 4.5 0.5 5.0
2 1.0 4.0 0.5 10
3 1.5 3.5 0.5 15
4 2.0 3.0 0.5 20
5 2.5 2.5 0.5 25

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 6 3.0 2.0 0.5 30
7 3.5 1.5 0.5 35
8 4.0 1.0 0.5 40
9 4.5 0.5 0.5 45
10 5.0 0.0 0.5 50

2. Express the results in terms of enzyme units. One unit of alkaline

phosphatase is defined as the preparation liberating 1 g p-
nitrophenol (PNP) in 30 min at 37 °C. The concentration of p-
nitrophenol in the sample can be calculated using the standard curve.

All the samples giving readings in excess of 10 µg shall be recorded as

unsatisfactory.
Inference (Qualitative Pasteurized milk giving readings less than 10 µg of p-nitrophenol shall
analysis) be recorded as negative for alkaline phosphatase.
Precautions The sample of milk should be examined as soon as possible after arrival
at the laboratory. If not examined immediately, store between 3 to 5 °C
until examined but not for more than 48 hours. The milk sample must be
brought to room temperature (25±3 oC) before being tested.
Reference 1. IS 8479 (Part I): 1977. Method for determination of Phosphatase
activity in milk and milk products. Routine method. Bureau of Indian
Standards, New Delhi.
2. Aschaffenburg, R., & Mullen, J. E. C. (1949). 381. A rapid and simple
phosphatase test for milk. Journal of Dairy Research, 16(1), 58-67.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Checking Efficiency of Sterilization in Liquid Milk

Method No. FSSAI 01.022:2022 Revision No. & Date 0.0

Scope This method is for checking the efficiency of sterilization in liquid milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonium sulphate: May explode if mixed with oxidizers, such as
potassium nitrate, potassium nitrite, and potassium chlorate. Ensure
there is adequate ventilation while working.
Principle The turbidity test depends upon the denaturation of proteins of milk,
especially albumin after sterilization. When solutions of inorganic salts or
acids are added, albumin separates with the casein. The sample after
treatment with ammonium sulphate is filtered, and heating of the filtrate
shows turbidity due to the presence of albumin on account of insufficient
heat treatment. If milk has been sterilized properly, all albumin will have
been precipitated and no turbidity will be produced. The test is not suitable
for UHT milk.
Apparatus/Instruments 1. Conical flask 50mL.
2. Cylinder 25 mL.
3. Test tubes 150 X 16 mm.
4. Funnels, 6 cm diameter.
5. Beaker, 400 mL.
6. Whatman No. 12 or equivalent, 12.5 cm folded filter paper.
7. Analytical balance (Readability 0.01 g).
Materials and reagents Ammonium sulphate.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis 1. Pipette 20 mL of milk in a 50 mL conical flask, add 4.0±0.1 g of
ammonium sulphate.
2. Shake the flask till the ammonium sulphate is completely dissolved.
3. Allow the mixture to settle for 5 min; filter through a folded filter paper
in a test tube.
4. Keep about 5 mL of the above filtrate in a boiling water bath for 5 min.
5. Cool the tube in a beaker of cold water and examine the contents for
turbidity by moving the tube in front of an electric light shaded from
the eyes of the observer.

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Inference (Qualitative The milk is considered sterilized when the filtrate shows no turbidity.
analysis)
Reference 1. IS 4238:2000 Sterilized and Ultra High Temperature Sterilized Milk —
Specification. Bureau of Indian Standards, New Delhi.
2. F.A.O Manual of Food Quality Control 14 / 8 page 26.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Skimmed Milk Powder Addition in Species Identified
Milk and Mixed Milk

Method No. FSSAI 01.023:2022 Revision No. & Date 0.0

Scope This method is for the detection of skimmed milk powder addition in
natural milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Glacial acetic acid: Use with adequate ventilation. Keep away from
heat. Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin
and eyes. Keep away from incompatibles such as oxidizing agents,
reducing agents, metals, acids, alkalis.
2. Phosphomolybdic acid: The product causes burns of eyes, skin and
mucous membranes. It is an oxidizer, so contact with
combustible/organic material may cause fire.
Principle The method is based on the fact that the coagulum obtained from
reconstituted skim milk powder by addition of acetic acid, gives intense
blue colour on boiling with phosphomolybdic acid due to certain reducing
groups present in the proteins of milk powder which are able to cause
reduction of molybdenum blue resulting in formation of blue colour.
Apparatus/Instruments 1. Centrifuge tubes.
2. Test tubes.
3. Centrifuge.
4. Waterbath.
5. Analytical balance (Readability 0.01 g).
Materials and Reagents 1. Acetic acid.
2. Phosphomolybdic acid.
Preparation of reagents 1. Acetic acid (4%): Take 4 mL of acetic acid in 100 mL volumetric flask
and make up the final volume to 100 mL with distilled water.
2. Phosphomolybdic acid (1%): Dissolve 1 g of phosphomolybdic acid in
distilled water and make up the final volume to 100 mL with distilled
water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation
Method of analysis 1. Take 50 ml of milk in a centrifuge tube. Place the tube in the centrifuge
and balance it properly.

61 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Centrifuge at 5000 rpm for 15 minutes. Remove the cream layer and
take out 5 ml of skim milk, carefully in a test tube.
3. Add 2 ml of 4% acetic acid for coagulation and wash the curd using
distilled water. To the curd then add 2 ml of 1% phosphomolybdic
acid.
4. Mix the contents thoroughly and heat in a water bath at boiling
temperature for 5 minutes and then cool.
5. Observe the colour of the solution in the tube.
Inference (Qualitative The curd obtained from pure milk shall be greenish in colour whereas the
analysis) curd of sample containing skimmed milk powder shall be bluish in colour.
The intensity of bluish colour depends on the amount of the skim milk
powder present in the sample
Reference 1. Murthi, T. N. (1985). Colorimetric determination of skim milk powder
in normal milk. Journal of food science and technology, 22(3), 207-
208.
2. FSSAI manual of methods of analysis of foods: milk and milk
products. (2016). Food Safety Standards Authority of India, Ministry
of Health and Family Welfare, Government of India, New Delhi.
3. Sharma, R.; Rajput, Y.S. & Naik, N.L. (2012). Detection of adulterants
in milk – a laboratory manual. NDRI Publication No. 88/2012, NDRI,
Karnal, page 20-21.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat Content in Milk

Method No. FSSAI 01.024:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
5. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
6. Amyl alcohol: It is a highly flammable liquid. Keep away from
heat/sparks/open flames/hot surfaces. Perform all operations under a
fume hood.
Principle Fat content in liquid milk sample can be estimated either by Gerber method
(volumetric method) or Rose-Gottlieb method (gravimeteric method).
The Gerber method is suitable as a routine or screening test. It is an
empirical method and reproducible results can be obtained if procedure is
followed correctly. The milk is mixed with sulphuric acid and iso-amyl
alcohol in a special Gerber tube, permitting dissolution of the protein and
release of fat. The tube is centrifuged and the fat rising into the calibrated
part of the tube is measured as a percentage of the fat content of the milk
sample. This method can be used for routine purposes.

63 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Rose-Gottlieb Method is a gravimetric method in which fat globule
membrane of milk fat globules is ruptured by addition of ammonia to milk
sample to liberate the fat. The liberated fat is extracted using combination
of solvents viz., diethyl ether and petroleum ether. The solvents are
evaporated and obtained fat is dried and weighed. This method is
considered suitable for reference purposes. Strict adherence to details is
essential in order to obtain reliable results.
Apparatus/Instruments 1. Gerber butyrometer, 6, 8 and 10 percent (ISI marked).
2. Pipette: 10.75 ± 0.03 mL for milk at 27° C.
3. Automatic measure or tilt measure for dispensing Gerber sulphuric
acid: 10 ± 0.25 mL at 27° C.
4. Automatic measure or tilt measure for dispensing iso-amyl alcohol: 1
± 0.05 mL at 27° C.
5. Lock stoppers for butyrometer.
6. Lock stopper key.
7. Water-bath: The water-bath shall be made of a suitable material (e.g.
stainless steel). It shall be capable of being maintained at 65±2 ℃ and
shall be of sufficient depth as to support the butyrometer in vertical
position with their scale completely immersed. The bath shall be fitted
with horizontal perforated plates to hold the butyrometer and shall also
carry a suitable thermometer.
8. Gerber Centrifuge. The centrifuge may be hand-driven or electric
driven. The centrifuge shall be capable of producing within 2 min when
fully loaded, a relative centrifugal acceleration of 350 ± 50 gn at the
outer end of the butyrometer stopper. This acceleration is produced by
centrifuges with the following effective radius (horizontal distance
between the centre of the centrifuge spindle and the outer end of the
butyrometer stopper) if operated at the speed indicated against each:
Effective Radius Revolution Per Min (± 70
(mm) rev/min)
240 1140
245 1130
250 1120
255 1110
260 1100
265 1090
270 1080
275 1070
300 1020
325 980

64 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Note: The relative centrifugal acceleration (gn) produced in a centrifuge is
given by the following formula:
1.12 X 10 -6 r n2
where r = effective horizontal radius in mm, and
n = speed in revolutions per min
9. Mojonnier fat extraction flask or any other suitable extraction tube (as
per IS specification).
10. Cork or stopper of synthetic rubber unaffected by usual fat solvents.
11. 100 mL flat bottom flask with G/G joint or stainless steel or aluminium
dishes of 5.5 cm height and 9 cm diameter or glass bowl.
12. Hot air oven (forced air type), capable of being controlled at 102±2 ℃.
Materials and reagents 1. Ammonia solution (Sp. Gr. 0.91).
2. Ethyl alcohol (95%).
3. Diethyl ether, peroxide-free.
4. Petroleum ether, boiling range 40-60 ℃.
5. Concentrated Sulphuric acid (Approximately 36.8 N).
6. Iso amyl alcohol (furfural free). It should have density between
0.807 to 0.809 g/mL at 27 ℃.
Preparation of Reagents 1. Gerber Sulphuric acid: Take required volume of water in a Pyrex flask
(generally 100 mL of water is required for 900 mL of concentrated
sulphuric acid) kept in a basin of ice-cold water. Carefully add the
commercial sulphuric acid in small quantities at a time keeping the
container sufficiently cold and mix gently. After cooling the flasks,
check the specific gravity of Gerber acid with hydrometer and if
necessary, adjust the Gerber acid to the correct specific gravity with
addition of water or acid taking same precautions as before till specific
gravity is in the range of 1.807 to 1.812 g/mL at 27 ℃ (or 1.815 to
1.820 g/mL at 20 ℃). Store the prepared acid in a glass stoppered bottle
to avoid absorption of water.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Method 1: Gerber Method
1. Transfer 10 mL of sulphuric acid into a butyrometer tube, preferably
by use of an automatic dispenser, without wetting the neck of the tube.
Mix the milk sample gently but thoroughly and fill the milk pipette
above the graduation line.
2. Wipe the outside of the pipette and allow the milk level to fall so that
the top of meniscus is level with the mark.
3. Run the milk into the butyrometer tube along the side wall without
wetting the neck, leave to drain for three sec and touch the pipette’s tip
once against the base of the neck of the butyrometer tube.
4. Add 1 mL of amyl alcohol, close with a lock stopper, shake until

65 | M o M – D a i r y a n d D a i r y P r o d u c t s
 homogeneous, inverting it for complete admixture of the acid.
5. Keep in water bath at 65±2 ℃ for at least 3 min and not less than 10
min taking care to have casein particles if any to dissolve fully, and
centrifuge for 5 mins at 1100 rpm (approximately 300 g).
6. The tubes should be put in centrifuge, so as to conform to radial
symmetry, and as evenly spaced as possible, in order to protect bearings
of the centrifuge.
7. Allow the centrifuge to come to rest, remove the butyrometer tubes
and place in water bath at 65±2 ℃ for at least 3 min and not less than
10 min.
8. Read the percentage of fat after adjusting the height in the tube as
necessary by movements of the lock stopper with the key.
9. Note the scale reading corresponding to the lowest point of the fat
meniscus and the surface of separation of the fat and acid.
10. When readings are being taken, hold the butyrometer with the
graduated portion vertical, keep the point being read in level with the
eye, and then read the butyrometer to the nearest half of the smallest
scale division.
In homogenised milk, fat separates with more difficulty and centrifuging
more than once may be required. Below procedure should be followed for
homogenized milk:
1. In case of homogenized milk, obtain the second value of fat content
by repeating centrifugation. If the second value does not exceed the first
value by more than half a smallest scale division of the butyrometer,
the second value shall be recorded as the fat content of the milk.
2. If the second value exceeds the first value by more than half a smallest
scale division, repeat the centrifugation and obtain a third value for the
fat content. If the third value does not exceed the second value by more
than half a smallest scale division, the third value shall be recorded as
the fat content of the milk.
3. If the third value exceeds the second value by more than half a smallest
scale division, repeat the centrifugation and obtain fourth value for the
fat content. The fourth value shall be recorded as the fat content of the
milk, but if this value exceeds the third value by more than half a
smallest scale division, it should be regarded as of doubtful accuracy.
4. If even after the several centrifuging’s, the fat is turbid or dark in colour
or if there is white or black material at the bottom of the fat column the
value for fat content would not be accurate.
5. Holding the tubes too long at 65 ℃ or above, results in esterification of
the amyl alcohol with a consequent increase in the volume of the fat
layer.
6. In case of old samples, if necessary, the concentration of sulphuric acid

66 | M o M – D a i r y a n d D a i r y P r o d u c t s
 may be increased from 90-91% to 92-93% to felicitate better
dissolution.
Note: The butyrometer must always be emptied without delay and the
highly acidic waste disposed of appropriately. The tubes may be cleaned
with chromic acid.
Method 2: Rose-Gottlieb Method
1. Weigh accurately about 10 g of sample (liquid milk) in beaker.
2. Add 1.25 mL of ammonia sp. gr. 0.91 (or an equivalent volume of a
more concentrated ammonia solution may be used), mix and shake
thoroughly.
3. Transfer the content to extraction tubes.
4. Add 10 mL ethyl alcohol and mix again.
5. Add 25 mL of diethyl ether (peroxide free) stopper and shake
vigorously for about a min, then add 25 mL petroleum ether (boiling
range 40-60 ℃ and shake again vigorously for about half a min. Let it
stand until the upper ethereal layer has separated completely and is
clear (alternatively use low r.p.m. Mojonnier centrifuge).
6. If there is a tendency to form emulsion, a little alcohol may be added to
help separation of the layers. Decant off the clear ethereal layer into a
suitable vessel (flask, glass bowl, aluminium dish, etc.).
7. Wash the delivery end of the extraction tube with a little ether and add
the washings to the flask.
8. Repeat twice extraction of the liquid remaining in the extraction tube
using 15 mL of each solvent every time, add the ethereal extract to the
same container and evaporate off completely.
9. Dry the flask in an air oven at 102 ± 2 ℃ for 2 h, cool in a desiccator
and weigh.
10. Heat the flask again in the oven for 30 min. Cool in a desiccator and
weigh. Repeat the process of heating and cooling and weighing until
the difference between two successive weights does not exceed 1 mg.
11. Wash out the fat from the flask with petroleum ether carefully leaving
any insoluble residue in the flask.
12. Dry the flask in the oven and reweigh. The difference in weights
represents the weight of fat extracted from the milk.
13. Correct weight of extracted fat by blank determination on reagents
used. Difference between duplicate determinations obtained
simultaneously by the same analyst should not be more than 0.07 g fat
/100g product.
Calculation with units of Method 1: Gerber Method: See the method of analysis.
expression
Method 2: Rose-Gottlieb Method:

67 | M o M – D a i r y a n d D a i r y P r o d u c t s
 (𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤
Where,
M1 is the mass, in g, of the fat-collecting flask and extracted matter after
drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference 1. F.A.O. Manual of Food Quality Control, 14/8, page 8.
2. IS 1479 (Part II): 1961 (Reaffirmed Year: 2018). Methods of test for
Dairy Industry – Chemical Analysis of Milk. Bureau of Indian
Standards, New Delhi.
3. AOAC 905.02. 21st Edn. (2019). Official method –Fat in milk. AOAC
International, USA.
4. IS 1224: 1977 (Reaffirmed year 2018). Determination of fat by Gerber
Method. Part 1 - Milk. Bureau of Indian Standards, New Delhi.
5. ISO 1211: 2010. Milk — Determination of fat content — Gravimetric
method (Reference method). International Organization for
Standardization, Geneva.
6. Pearson’s Composition and analysis of foods, 9th edn., 1991 page 538.
Approved by Scientific Panel on Methods of Sampling and Analysis

68 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Solids-Not-Fat in Milk

Method No. FSSAI 01.025:2022 Revision No. & Date 0.0

Scope This method is for the determination of Solids-Not-Fat (SNF) in milk
samples by gravimetric method.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.
1. Use thermal protection gloves, tongs and protective eyewear while
handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle In this procedure, first of all total solids in the given sample are estimated.
Fat content (estimated by FSSAI 01.024:2022) is subtracted from total
solids to calculate at SNF content in the sample.
For estimating total solids, a known quantity of milk is dried on a boiling
water bath. Subsequently, the sample is dried in hot air oven at 102 ± 2 ℃
and from the weight of the residue, the total solids content in milk is
determined.
Apparatus/Instruments 1. Analytical Balance (Readability 0.0001 g).
2. Desiccator provided with an efficient desiccant (for example freshly
dried silica gel with a hydrometric indicator).
3. Boiling water bath provided with openings of adjustable size.
4. Hot air oven (forced air type), capable of being controlled at 102 ± 2
℃.
5. Flat bottomed dishes of height 20-25 mm, diameter 50-75 mm and
made of appropriate material (stainless steel, nickel or aluminium)
provided with well fitted readily removable lids.
6. Water bath capable of being maintained at 35 - 40 ℃.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Determination of Total Solids:
1. Transfer sample to a beaker, warm slowly to 35°-40 ℃ on a water bath
with careful mixing to incorporate any cream adhering to the sample.
Cool the sample quickly to room temperature (25 ± 3 oC).
2. Heat a dish with its lid alongside in the drying oven maintained at 102
± 2 ℃ for at least 1 h. Place the lid on the dish and immediately transfer
to a desiccator.
3. Allow to cool to room temperature (at least 30 min) and weigh to the
nearest 0.1 mg.
4. Add 5 mL of prepared sample, place the lid on the dish and weigh again.

69 | M o M – D a i r y a n d D a i r y P r o d u c t s
 5. Place the dish without the lid on a vigorously boiling water bath in such
a way that the bottom of the dish is directly heated by the steam.
6. Continue heating till most of the water is removed.
7. Remove the dish from the water bath, wipe the underside and place it
in the oven alongside the lid and dry it in the oven maintained at 102 ±
2 ℃ for 2 h; place the lid and transfer to the desiccator.
8. Allow the dish to cool and weigh to the nearest 0.1 mg.
9. Again, heat the dish with its lid alongside in the oven maintained at 102
± 2 ℃ for 1 h. Place the lid on the dish and immediately transfer to the
desiccator.
10. Allow to cool and weigh again. Repeat the operation again until the
difference in the two consecutive weighing does not exceed 1 mg.
Record the lowest mass
Determination of fat:
Refer method for determination of fat in milk (FSSAI 01.024:2022).
Calculation with units of 𝑀2 − M
expression Total Solids Content (%) = 𝑥 100
𝑀1 − M
Where,

M is the mass in g of empty dish along with lid;

M1 is the initial mass in g of dish with lid and test portion;
M2 is mass in g of dish with lid and dried test portion
Round the value obtained to nearest 0.01 % (m/m)
SNF Content (%) = Total Solid (%) - Fat (%)
Reference IS 12333 - 2017/ ISO 6731: 2010. Milk, Cream and Evaporated milk. –
Determination of total Solids Content -reference method. Bureau of Indian
Standards, News Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

70 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Total Nitrogen in Milk
by Kjeldahl Method
Method No. FSSAI 01.026:2022 Revision No. & Date 0.0
Scope This method describes the determination of total nitrogen in milk by
Kjeldahl method.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
3. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to
release of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle In this manual, the three commonly used variants of Kjeldahl method have
been presented.
Method 1. Macro Kjeldahl Method by ISO procedure (ISO 8968-1:2014)
Method 2. Block Digestion/ Steam Distillation method (ISO 8968-2:2001)
Method 3. Macro Kjeldahl Method by BIS procedure [IS 7219: 1973 (RA
2015)]

71 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Any of the above method can be employed in the laboratory depending on
the availability of the equipment in the laboratory.
The method involves three major steps. In the first step, the protein is
digested using concentrated sulphuric acid in presence of a catalyst
(potassium sulphate/copper sulphate). In this step, all the organic material
is oxidized except nitrogen, which is converted to ammonium sulphate (Eq
1). In the second step, the digest is neutralized with alkali to liberate
ammonia (Eq 2). The ammonia distilled is collected in boric acid. In the
third step, the collected ammonia in boric acid is titrated with standard
hydrochloric acid (Eq 3 & 4) in the presence of a methyl red-bromocresol
green indicator until the green distillate changes from colourless to pink
(methyl red, methylene blue indicator can also be used).
Reaction
K2SO4, CuSO4, H2SO4
Protein (NH4)2SO4 Eq 1
Heat

(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O Eq 2

NH3 + H3BO3 NH4+.H2BO4- Eq 3

NH4+.H2BO-4+ HCl NH4Cl + H3BO3 Eq 4

(Green) (Pink at pH < 4.8)
The quantity of acid required for titration is equivalent to the concentration
of ammonia in the distillate and to the nitrogen content of the original
protein containing sample The total amount of protein is calculated using
the conversion factor of 6.38.
The basic principle of the Method 3. Macro Kjeldahl Method by BIS
procedure [IS 7219: 1973 (RA 2015)] is also same as indicated above.
However, in this variant of the method, liberated ammonia is quantitatively
distilled into a measured volume of standard hydrochloric acid or sulphuric
acid.
Apparatus/Instruments Method 1. Macro Kjeldahl Method by ISO procedure (ISO 8968-
1:2014)
1. Kjeldahl flasks: Kjeldahl, hard, moderately thick, well-annealed glass,
500 or 800 mL capacity.
2. Distillation flask: Same Kjeldahl flask, fitted with rubber stopper
through which lower end of sufficient rubber bulb or trap passes to

72 | M o M – D a i r y a n d D a i r y P r o d u c t s
 prevent mechanical carryover of sodium hydroxide during distillation.
Connect upper end of the bulb to condenser tube by rubber tubing. Use
graduated 500 mL Erlenmeyer titration flask to collect distillate. Trap
outlet of condenser in manner to ensure complete absorption of
ammonia distilled into boric acid solution.
3. Digestion apparatus, to hold the Kjeldahl flasks in an inclined position
approximately 45 ℃ along with electric heater or gas burners that do
not heat the flasks above the level of their contents, and with a fume
extraction system. The heater source should be adjustable to determine
the maximum heater setting to be used during digestion. Preheat the
heat source at the heater setting for evaluation. In the case of a gas
heater, the preheat period shall be 10 min and for an electric heater the
preheat period shall be 30 min. Determine the heater setting that brings
250 mL of water (including 5-10 boiling aids) with an initial
temperature of 25 ℃ to a rolling boil in 5-6 min for each type of heaters.
This is the maximum heater setting to be used during digestion.
4. Conical or Erlenmeyer flask: 500 mL capacity, graduated at every 200
mL.
5. Burette: 50 mL capacity, graduated at least at every 0.1 mL.
6. Boiling aid, having a mesh size 10. Use plain, high purity amphoteric
alundum granules. Do not reuse the aids. Glass beads of approximately
5 mm diameter are also used, but they do not promote as efficient
boiling as the alundum granules and more foaming problems will be
encountered during digestion with glass beads.
7. Measuring cylinders: 50, 100 and 500 mL capacities, graduated.
Method 2. Block Digestion/ Steam Distillation method (ISO 8968-
2:2001)
1. Digestion block: Aluminium alloy block or equivalent apparatus, fitted
with an adjustable temperature control device for measuring block
temperature.
2. Digestion tubes: 250 mL capacity, suitable for use with digestion flask.
3. Exhaust manifold: Suitable for use with the digestion tubes.
4. Centrifugal scrubber apparatus or filter pump or aspirator: Constructed
of acid resistant material and for use with main water supply.
5. Measuring cylinder: 25, 50 and 100 mL capacities, graduated.
6. Distillation unit: Manual /semi-automatic/automatic, for steam
distillation, suited to accept the 250 mL digestion tubes and 500 mL
conical flasks.
7. Conical or Erlenmeyer flask: 500 mL capacity, graduated at 200 mL.

73 | M o M – D a i r y a n d D a i r y P r o d u c t s
 8. Burette: 50 mL capacity, graduated at least at every 0.1 mL.
Method 3. Macro Kjeldahl Method by BIS procedure [IS 7219: 1973
(RA 2015)]
Digestion flask: Use 500 to 800 mL Kjeldahl flasks. Conduct digestion
over a heating device adjusted to bring 250 mL water at 25 ℃ to rolling
boil in approximately 5 min. To test heaters, preheat for 10 min, if gas, or
for 30 min if electric. Add 3 to 4 boiling chips or glass beads to prevent
superheating.
Distillation: Fit the flask with a rubber stopper through which passes the
lower end of an efficient scrubber trap or bulb to prevent mechanical carry
over of alkali during distillation. Connect the upper end of the trap to a
condenser by rubber or glass tubing. Immerse the trap outlet of the
condenser in such a way as to ensure complete absorption of ammonia
distilled over into acid in a 500 mL Erlenmeyer flask (see Fig. 1).

Fig 1: Distillation assembly (IS 7219:1973)

Materials and Reagents Use all the reagents of AR Grade quality, unless otherwise specified, and
use distilled or demineralized water only.
For Method 1. Macro Kjeldahl Method by ISO procedure (ISO 8968-
1:2014) and Method 2. Block Digestion/ Steam Distillation method
(ISO 8968-2:2001)
1. Potassium sulfate (K2SO4): Nitrogen free or low in nitrogen content.
2. Copper (II) sulfate solution
3. Concentrated sulphuric acid: (Approximately 36.8 N) nitrogen free
4. Sodium hydroxide solution, 50%, m/m (low in nitrogen))
5. Methyl red

74 | M o M – D a i r y a n d D a i r y P r o d u c t s
 6. Bromocresol green
7. Ethanol
8. Boric acid solution (H3BO3)
9. Standard hydrochloric acid solution: 0.1 ± 0.0005 N.
10. Ammonium sulfate (NH4)2SO4
11. Tryptophan (C11H12N2O2) or Lysine hydrochloride (C6H15ClN2O2):
Minimum assay 99%, do not dry these reagents in an oven before use.
12. Sucrose with a nitrogen content of not more than 0.002% (m/m). Do
not dry in an oven before use.
Method 3. Macro Kjeldahl Method by BIS procedure [IS 7219: 1973
(RA 2015)]
1. Concentrated sulphuric acid (Approximately 36.8 N), nitrogen-free.
2. Mercuric oxide or Metallic Mercury or Copper (II) Sulphate
Pentahydrate - nitrogen-free.
3. Potassium sulphate or anhydrous sodium sulphate - nitrogen-free.
4. Zinc Granules
5. Sulphide or thiosulphate Solution - Dissolve 40 g potassium sulphide
or 80 g hydrated sodium thiosulphate in 1 L distilled water.
6. Sodium hydroxide -pellets, flakes or solution; nitrogen free.
7. Hydrochloric or sulphuric acid, standard Solution - 0.1 or 0.5 N.
Standardize against primary standard and against sodium hydroxide
standard solution
8. Sodium hydroxide standard Solution - 0.1 N. Standardize against
primary standard (potassium hydrogen phthalate) and against standard
acid solution.
9. Methyl Red Indicator
Preparation of reagents For Method 1. Macro Kjeldahl Method by ISO procedure (ISO 8968-
1:2014) and Method 2. Block Digestion/ Steam Distillation method
(ISO 8968-2:2001)
1. Copper (II) sulfate solution: Dissolve 5.0 g of copper (II) sulfate
pentahydrate (CuSO4.5H2O) in water and make up the final volume to
100 mL in a 100 mL volumetric flask.
2. Sodium hydroxide solution, 50%, m/m (low in nitrogen): Dissolve 50
g sodium hydroxide pellets in water and finally make to 100 mL.
3. Indicator solution: Dissolve 0.1 g of methyl red in 95% (v/v) ethanol
and dilute to 50 mL with ethanol. Dissolve 0.5 g of bromocresol green
in 95% (v/v) ethanol and dilute to 250 mL with ethanol. Mix 1 part of
methyl red solution with 5 parts of bromocresol green solution or
combine all of both solutions.
4. Boric acid solution (H3BO3): Dissolve 40 g of boric acid in hot water,
allow the solution to cool and dilute to 1 L. Add 3 mL of methyl red-

75 | M o M – D a i r y a n d D a i r y P r o d u c t s
 bromocresol indicator solution, mix and store the solution in
borosilicate glass bottle. The solution will be light orange in colour.
Protect the solution from light and sources of ammonia fume during
storage.
5. Standard hydrochloric acid solution: 0.1 ± 0.0005 N.
6. Ammonium sulfate [(NH4)2SO4]: Minimum assay 99.9% on dried
material. Immediately before use dry the ammonium sulfate at 102 ± 2
℃ for not less than 2 h. Cool to room temperature (25 ± 3 oC) in a
desiccator.
7. Tryptophan or lysine hydrochloride: Minimum assay 99%, do not dry
these reagents in an oven before use.
8. Sucrose with a nitrogen content of not more than 0.002% (m/m). Do
not dry in an oven before use.
Method 3. Macro Kjeldahl Method by BIS procedure [IS 7219: 1973
(RA 2015)]
1. Sulphide or Thiosulphate Solution - Dissolve 40 g potassium sulphide
or 80 g hydrated sodium thiosulphate in 1 L distilled water.
2. Sodium hydroxide solution; nitrogen free. For solution, dissolve about
450 g solid sodium hydroxide in distilled water, cool, and dilute to 1 L.
The specific gravity should be at least 1.36 at 20 °C.
3. Hydrochloric or sulphuric Acid, Standard Solution - 0.1 or 0.5 N.
standardize against sodium hydroxide standard solution
4. Sodium hydroxide standard solution - 0.1 N. standardize against
primary standard (potassium hydrogen phthalate) and used to
standardise acid solution.
5. Methyl Red Indicator - Dissolve 1 g methyl red in 200 mL alcohol.
Sample preparation Refer method FSSAI 01.001:2022for sample preparation.
Method of analysis Method 1. Macro Kjeldahl Method by ISO procedure (ISO 8968-
1:2014)
Test Portion and Pre-treatment:
1. Add to the clean and dry Kjeldahl flask, 5-10 boiling aids, 15 g K2SO4,
1.0 mL of the copper sulfate solution, approximately 5±0.1 g of
prepared milk sample (or milk product sample containing equivalent
amount of protein), weighed to the nearest 0.1 mg, and add 25 mL of
concentrated sulphuric acid.
2. Use the 25 mL acid to also wash down any copper sulfate solution,
K2SO4 or milk left on the neck of the flask. Gently mix the contents of
the Kjeldahl flask.
Digestion:

76 | M o M – D a i r y a n d D a i r y P r o d u c t s
1. Turn on the fume extraction system of the digestion apparatus prior to
beginning the digestion.
2. Heat the Kjeldahl flask and its contents on the digestion apparatus using
a heater setting low enough such that charred digest does not foam up
the neck of the Kjeldahl flask.
3. Digest at this heat-setting for at least 20 min or until white fumes appear
in the flask. Increase the heater setting to half way to the maximum
setting as determined previously (See Digestion apparatus) and
continue the heating period for 15 min. At the end of 15 min period,
increase the heat to maximum setting.
4. After the digest clears (clear with light blue-green colour), continue
boiling for 1 h to 1.5 h at maximum setting. The total digestion time
will be between 1.8-2.25 h.
Note: To determine the specific boiling time required for analysis
conditions in a particular laboratory using a particular set of apparatus, for
milk analysis, select a high-protein, high-fat milk sample and determine its
protein content using different boil times (1 h-1.5 h) after clearing. The
mean protein result increases with increasing boil time, becomes consistent
and then decreases when boil time is too long. Select the boil time that
yields the maximum protein result.
1. At the end of digestion, the digest shall be clear and free of undigested
material. Allow the acid digest to cool to room temperature (25 ± 3 oC)
over a period of approximately 25 min.
2. If the flasks are left on hot burners to cool, it will take longer to reach
room temperature (25 ± 3 oC). The cooled digest should be liquid or
liquid with a few small crystals at the bottom of the flask at the end of
25 min cooling period.
3. Do not leave the undiluted digest in the flask overnight. The undiluted
digest may crystallize during this period and it will be very difficult to
get that back into the solution to avoid this situation.
Note: Excessive crystallization after 25 min is the result of undue acid loss
during digestion and can result in low test values. Undue acid loss is caused
by excessive fume aspiration or an excessively long digestion time caused
by an incorrect maximum burner setting.
1. After the digest is cooled to room temperature (25 ± 3 oC), add 300 mL
of water to 500 mL Kjeldahl flask or 400 mL of water when using 800
mL Kjeldahl flask. Use the water to wash down the neck of the flask
too.
2. Mix the contents thoroughly ensuring that any crystals which separate
out are dissolved. Add 5-10 boiling aids.

77 | M o M – D a i r y a n d D a i r y P r o d u c t s
3. Allow the mixture to cool again to room temperature (25 ± 3 oC) prior
to the distillation. Diluted digests may be stoppered and held for
distillation at a later time.
Distillation:
1. Turn on the condenser water for the distillation apparatus.
2. Add 75 mL of 50% (m/m) sodium hydroxide solution to the diluted
digest by carefully pouring the solution down the inclined neck of the
Kjeldahl flask, so as to form a clear layer at the bottom of the bulb of
the flask. There should be a clean interface between the two solutions.
3. Immediately after the addition of sodium hydroxide solution to the
Kjeldahl flask, connect it to the distillation apparatus, the tip of whose
condenser outlet tube is immersed in 50 mL of boric acid solution with
indicator contained in a 500 mL Erlenmeyer flask.
4. Vigorously swirl the Kjeldahl flask to mix its contents thoroughly until
no separate layers of solution are visible in the flask any more. Set the
flask down on the burner.
5. Turn on the burner to a setting high enough to boil the mixture; continue
distillation until irregular boiling (bumping) starts and then
immediately disconnect the Kjeldahl flask and turn off the burner. Turn
off the condenser water.
6. The distillation rate shall be such that approximately 150 mL distillate
is collected when irregular boiling (bumping) starts and the volume of
the contents of the conical flask will be approximately 200 mL.
7. If the volume of distillate collected is less than 150 mL, then it is likely
that less than 300 mL of water is added to dilute the digest. The
efficiency of the condenser shall be such that the temperature of the
contents of conical flask does not exceed 35 ℃ during distillation.
Titration:
1. Titrate the boric acid receiving solution with standard hydrochloric acid
solution (0.1 N) to the first trace of pink colour.
2. Take the burette reading to at least the nearest 0.05 mL. A lighted stir
plate may aid visualization of the end point.
Blank Test: Simultaneously carry out a blank test by following the
procedure as described above taking all the reagents and replacing the milk
sample with 5 mL water and about 0.85 g of sucrose.
Note:
1. The purpose of sucrose in a blank or a recovery standard is to act as
organic material to consume an amount of sulphuric acid during
digestion that is roughly equivalent to a test portion. If the amount of

78 | M o M – D a i r y a n d D a i r y P r o d u c t s
 residual free sulphuric acid at the end of digestion is too low, the
recovery of nitrogen by both recovery tests (See Section. Nitrogen
recovery test) will be low. If the amount of residual acid present at the
end of the digestion is sufficient to retain all the nitrogen, but the
temperature and time conditions during digestion were not sufficient to
release all the nitrogen from a sample, then the nitrogen recovery will
be acceptable.
2. The amount of titrant used in the blank should always be greater than
0.00 mL. Blanks within the same laboratory should be consistent across
time. If the blank is already pink before the beginning of titration,
something is wrong. Usually, in such cases, the conical flasks are not
clean or water from the air that may condense on the outside of the
condenser apparatus has dripped down into the collection flask to cause
the contamination.

Nitrogen Recovery Test

1. The accuracy of the procedure should be checked regularly by means
of following recovery tests, carried out in accordance with procedure
as in the preceding steps
2. Check that no loss of nitrogen occurs by using a test portion of 0.12 g
of ammonium sulfate along with 0.85 g of sucrose. Add all other
reagents (except milk sample) as stated in Step A. Digest and distill
under same conditions as for a milk sample
3. The percentage of nitrogen recovered shall be between 99.0 and
100.0% for the given apparatus.
4. In the case recoveries of nitrogen exceed 100%, ammonium sulfate is
only useful to determine whether nitrogen loss has occurred or the
normality of titrant is lower than the stated value. For recoveries less
than 99%, the loss could be in the digestion or distillation step.
5. It is possible to use a mixture of ammonium sulfate and small amount
of sulphuric acid (the amount of residual remaining at the end of
digestion) in a Kjeldahl flask. Dilute it with the normal value of water,
add the normal amount of sodium hydroxide solution and distill.
6. If the nitrogen recovery is still low by the same amount, the loss of
nitrogen is in the distillation apparatus and not in that of the digestion.
The probable cause might be a leaky tubing in a traditional system or
the tips of the condensers not submerged under the surface of boric acid
solution early in the distillation. The apparatus should pass this test
before going on to check recoveries by the procedure described below.
7. Check the efficiency of digestion procedure by using 0.16 g of lysine
hydrochloride or 0.18 g of tryptophan along with 0.67 g of sucrose.

79 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Add all other reagents (except milk sample). Digest and distill under
same conditions as for a milk sample. At least 98% of the nitrogen shall
be recovered. If the recovery is lower than 98% after having a 99 -
100% recovery on ammonium sulfate, then the temperature or time of
digestion is insufficient or there is undigested sample material (i.e.,
char) on the inside of the Kjeldahl flask.
8. The final evaluation of performance is best done by participation in a
proficiency testing system, where within and between laboratories
statistical parameters are computed based on analysis of milk samples.
9. Lower results in either of the recovery tests (or higher than 100% in
case of ammonium sulfate) will indicate failures in the procedure
and/or inaccurate concentration of the standard hydrochloric acid
solution.
Method 2. Block Digestion/ Steam Distillation method (SO 8968-
2:2001)
Test Portion and Pre-treatment:
1. Add to the clean and dry digestion tube, 12 g potassium sulphate, 1.0
mL of the copper sulfate solution, approximately 5 ± 0.1 g of prepared
milk sample, weighed to the nearest 0.1 mg, and add 20 mL of
concentrated sulphuric acid.
2. Use the sulphuric acid also to wash down and copper sulfate solution,
K2SO4 or milk left on the upper walls of the digestion tube. Gently mix
the contents of the tube.
Note: The amount of acid used in the block digestors is less than that used
in the Macro Kjeldahl method because the volumes of acid greater than 20
mL in the block digestion systems gives excessive foaming problem during
digestion and variable results. Users of block digestors must note that
maintaining sufficient residual sulphuric acid at the end of digestion needs
more attention by the analyst in the block digestors than in Macro Kjeldahl
method. Excessive acid loss due to over aspiration of fumes is more of
concern in block digestors than Macro Kjeldahl method.
Digestion
1. Set the digestion block at a low initial temperature so as to control
foaming (approximately a temperature between 180-230 ℃). Transfer
the tube to the digestion block and place the exhaust manifold which is
itself connected to a centrifugal scrubber of similar device in the top of
the tube.

80 | M o M – D a i r y a n d D a i r y P r o d u c t s
2. The suction rate of the centrifugal scrubber or similar device shall be
just sufficient to remove fumes. The complete apparatus may need to
be kept inside a fume hood.
3. Digest the test portion for 30 min or until white fumes develop, then
increase the temperature of digestion block to a temperature between
410-430 ℃ and continue digestion of the test portion until the digest is
clear.
Note: It may be necessary to increase the temperature gradually over a
period of approximately 20 min to control foaming. In any event, do not let
foam rise higher than 4-5 cm below the surface of exhaust manifold
inserted into the top of the digestion tube.
1. After the digest clears (clear with light blue-green colour) continue
digestion at a temperature of between 410-430 ℃ for at least 1 h.
2. During this period the sulphuric acid must be boiling. If visible boiling
of the clear liquid is not apparent as bubbles forming at the surface of
the hot liquid around the perimeter of the tube, then the temperature of
the block may be too low. The total digestion time will be between
1.75-2.5 h.
3. To determine the specific boiling time required for analysis conditions
in a particular laboratory using a particular set of apparatus, select a
high-protein, high-fat milk sample and determine its protein content
using different boil times (1-1.5 h) after clearing.
4. The mean protein content increases with increase in boil time,
becomes consistent and then decreases when boil time is too long.
Select the boil time that yields the maximum protein results.
5. At the end of digestion, the digest should be clear and free of undigested
material. Remove the tube from the block with the exhaust manifold in
place.
6. Allow to cool to room temperature (25 ± 3 oC) over a period of
approximately 25 min. The cooled digest should be liquid or liquid with
a few small crystals at the bottom of the tube.
7. Do not leave the undiluted digest in the tube overnight. The undiluted
digest may solidify and it will be very difficult to get that back into the
solution with water.
Note: Excessive crystallization after 25 min is the result of undue acid loss
during digestion and can result in low test values. Undue acid loss is caused
by excessive fume aspiration or an excessively long digestion time caused
by digestion for too long a period at a temperature below the maximum
temperature of the analysis. To reduce acid loss, reduce the rate of fume
aspiration.

81 | M o M – D a i r y a n d D a i r y P r o d u c t s
1. After the digest is cooled to room temperature (25 ± 3 oC), remove the
exhaust manifold and carefully add 85 mL of water to each tube.
2. Swirl to mix while ensuring that any crystals which separate out are
dissolved.
3. Allow the contents of the tube to cool again to room temperature (25 ±
3 oC).
Distillation
1. Turn on the condenser water for the distillation apparatus.
2. Attach the digestion tube containing the diluted digest to the distillation
unit.
3. Place a conical flask containing 50 mL of the boric acid solution under
the outlet of the condenser, in such a way that the delivery tube is below
the surface of the boric acid solution.
4. Adjust the distillation unit to dispense 55 mL of sodium hydroxide
solution.
Note: Where 40% (m/m) sodium hydroxide solution is used; the dispensed
volume should be adjusted to 65 mL. If the automatic delivery of sodium
hydroxide solution is extremely variable due to the partial plugging of the
delivery tubing for the sodium hydroxide, then large variability in duplicate
results will occur.
1. As per the manufacturer’s instructions, operate the distillation unit in
such a way as to steam distill the ammonia liberated by addition of
sodium hydroxide solution, collecting the distillate in the boric acid
solution.
2. Continue with the distillation process until at least 150 mL of distillate
has been collected. Remove the conical flask from the distillation unit.
Titration
1. Titrate the boric acid receiving solution with standard hydrochloric acid
solution (0.1 N) to the first trace of pink colour.
2. Take the burette reading to at least the nearest 0.05 mL. A lighted stir
plate may aid visualization of the end point.
Blank Test
Simultaneously carry out a blank test by following the procedure as
described above taking all the reagents and replacing the milk sample with
5 mL water and about 0.85 g of sucrose. Please see the Note mentioned in
Method 1.
Nitrogen Recovery Test - Please see the details mentioned in Method 1.

82 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method 3. Macro Kjeldahl Method by BIS procedure [IS 7219: 1973
(RA 2015)]
Digestion
1. Accurately weigh 0.7 to 2.2 g of the sample into the digestion flask.
2. Add 0.7 g mercury oxide or 0.65 g mercury or 0.45 g of copper (II)
sulphate pentahydrate and 15 g powdered potassium sulphate or
anhydrous sodium sulphate, and 25 mL sulphuric acid.
3. Ratio of salt to acid (m/v) should be approximately 1: 1 at the end of
digestion for proper temperature control.
4. Digestion may be incomplete at a lower ratio and nitrogen may be lost
at a higher ratio.
5. Each gram of fat consumes 10 mL and each gram of carbohydrate 4
mL sulphuric acid during digestion.
6. Place the flask in an inclined position on a heater and heat gently until
foaming ceases.
7. A small amount of paraffin or silicon antifoam may be added to reduce
foaming. Boil vigorously until the solution becomes clear and then
continue boiling it for 1 to 2 h.
Distillation –
1. Cool, add about 200 mL distilled water, and in order to avoid complex
formation, add 25 mL of the sulphide or thiosulphate solution when
mercury or mercuric oxide is used as catalyst. Mix to precipitate the
mercury.
2. Add a few zinc granules to prevent bumping, incline flask, and add
without agitation 25 g of sodium hydroxide as solid or equivalent as
solution, to make solution strongly alkaline (the thiosulphate or
sulphide solution may be mixed with the sodium hydroxide solution
before addition to the flask).
3. Immediately connect flask to distillation bulb or trap on condenser,
and, with tip of the condenser immersed in a measured quantity
standard hydrochloric acid (usually 50 mL, 0.5 N or an appropriate
quantity of 0.1 N) in the receiver, rotate flask to mix the contents
thoroughly; then heat immediately until all ammonia has distilled over
(at least 150 mL distillate).
4. Lower the receiver before stopping distillation and wash tip of
condenser with distilled water. Back-titrate excess acid with standard
0.1 N sodium hydroxide, using methyl red as indicator. Correct for
blank determination in reagents.
Blank
Conduct determinations using all reagents and 2 g of sugar.

83 | M o M – D a i r y a n d D a i r y P r o d u c t s
Calculation with units of For Method 1. Macro Kjeldahl Method by ISO procedure (ISO 8968-
expression 1:2014) and Method 2. Block Digestion/ Steam Distillation method
(ISO 8968-2:2001)
Calculate the nitrogen content, expressed as a percentage by mass, by the
following formula:
1.4007 x (Vs − VB )x N
Wn =
W
Wn = nitrogen content of sample, expressed as a percentage by mass;
VS = volume in mL of the standard hydrochloric acid used for sample;
VB = volume in mL of the standard hydrochloric acid used for blank test;
N = Normality of the standard hydrochloric acid expressed to four decimal
places;
W = mass of test portion in g, expressed to nearest 0.1 mg.
Express the nitrogen content to four decimal places.
The crude protein content, expressed as percentage by mass is obtained by
multiplying the nitrogen content by 6.38. Express the crude protein results
to three decimal places.
Method 3. Macro Kjeldahl Method by BIS procedure [IS 7219: 1973
(RA 2015)]
Nitrogen content (N) in g= (a - 0.2b) - (c - 0.2d) x 0.007
Where,
a = volume in mL 0.5 N acid measured for main distillation,
b = volume in mL 0.1 N alkali used for back-titrating a,
c = volume in mL 0.5 N acid measured for blank distillation, and
d = volume in mL 0.1 N alkali used for back-titrating c.
OR
Nitrogen content (N) in g= (A - B) - (C - D) x 0.0014
Where,
A = volume in mL 0.1 N acid measured for main distillation,
B = volume in mL 0.1 N alkali used for back-titrating A,
C = volume in mL 0.1 N acid measured for blank distillation, and
D = volume in mL 0.1 N alkali used for back-titrating C.
Calculation of Total Protein
𝑁 𝑥 100 𝑥 6.38
Protein, percent by mass =
𝑊
Where,
N = mass of nitrogen content in g of original sample,
W = mass of sample in g.

84 | M o M – D a i r y a n d D a i r y P r o d u c t s
Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content
(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.
2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content
(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.
3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of
protein in foods and feeds. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

85 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of True Protein in Milk

Method No. FSSAI 01.027:2022 Revision No. & Date 0.0

Scope This method describes the determination of true protein in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Trichloroacetic acid: Trichloroacetic acid is a corrosive chemical and
contact can severely irritate and burn the skin and eyes with possible
eye damage. Trichloroacetic acid can irritate the nose and throat.
Trichloroacetic acid can irritate the lungs causing coughing and/or
shortness of breath.
Principle The method is used for analysis of true protein content by estimating the
nitrogen content obtained only from proteins and not from non-protein
nitrogen (NPN). In this method, the protein in milk sample is precipitated
by the addition of TCA such that the final concentration of TCA in the
mixture is approximately 12%. The precipitated proteins are filtered and
the filtrate will contain the NPN contents. The nitrogen content of the
precipitated protein is then determined by Kjeldahl method which
represents true protein nitrogen content of milk
Apparatus/Instruments Apart from the apparatus listed in Kjeldahl method for the determination
of nitrogen content in milk (Refer method FSSAI 01.026:2022), the
following apparatus required are as below:
1. Conical flask: 25 mL capacity.
2. Pipettes: 10, 20 mL capacities.
3. Filter funnel: 75 mm diameter.
4. Whatman filter paper: Grade 1 (nitrogen free).
5. Beaker: 50 mL capacity.
Materials and Reagents 1. Trichloroacetic acid (TCA)
2. Other reagents as already described in Kjeldahl method for the
determination of total nitrogen content in milk.
Preparation of reagents 1. Trichloroacetic acid (TCA) solution (15%, w/v): Dissolve 15.0 g of
trichloroacetic acid (CCl3COOH) in water and dilute to 100 mL in a
volumetric flask.
Note: Do not use other concentrations of TCA and volumes of solutions
than those specified in this procedure. Other concentrations or volumes of
solutions will change the performance of the method with respect to its
mean value and performance characteristics.

86 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Other reagents as already described in Kjeldahl method for the
determination of total nitrogen content in milk.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Test Portion
1. Weigh approximately 5 ± 0.1 g, to the nearest 0.1 mg, of the prepared
milk sample into a Kjeldahl flask.
2. Immediately add 5.0 ±0.1 mL of water to the Kjeldahl flask, rinsing
any milk on the neck of the flask to the bottom of it.
Precipitation and Filtration
1. Add 40 ± 0.5 mL of TCA solution to the flask and swirl to mix the
contents. Let the flask stand for approximately 5 min to allow the
precipitate to settle.
2. Pour the mixture from the Kjeldahl flask through a Whatman filter
paper Grade 1 and collect the entire filtrate in a clean, dry conical flask.
Some of the precipitate will remain in the Kjeldahl flask and some will
be collected on the filter paper. It is not necessary to remove all of the
precipitate from the flask.
3. Immediately after pouring the mixture (and so as not to allow any
precipitate to dry on the neck of Kjeldahl flask), add, by means of a
pipette, 10 mL of TCA solution.
4. Use the acid solution to also rinse any precipitate from the neck of the
flask down on to the bottom, swirl to mix the contents.
5. Pour the mixture from the flask through the same filter paper, adding
the filtrate to that collected previously.
6. Immediately rinse the neck of the flask with a further 10 mL of TCA
solution again.
7. Swirl to mix the contents and again pour the mixture from the flask
through the same filter paper, adding the filtrate to that collected
previously. This filtrate shall be clear and free of particulate matter.
Note: At this point, the filtrate is no longer needed and may be discarded
in an appropriate manner.
8. Wearing gloves, carefully remove the filter paper from the filter funnel
and fold the filter paper to enclose the precipitate.
9. If any precipitate remains on either the inner or outer lip of the Kjeldahl
flask, wipe with the folded filter paper so that any precipitate adheres
to the paper and then drop the filter paper into the Kjeldahl flask.
Digestion and Distillation
1. Add the appropriate amount of boiling aids, K2SO4, copper (II) sulfate
solution and concentrated sulphuric acid as specified in the Macro
Kjeldahl method for the determination of total nitrogen content in milk.

87 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Determine the nitrogen content of the precipitate and filter paper by the
Macro Kjeldahl method.
Titration
1. Titrate the boric acid receiving solution with standard hydrochloric acid
solution (0.1 N) to the first trace of pink colour.
2. Take the burette reading to at least the nearest 0.05 mL. A lighted stir
plate may aid visualization of the end point.
Blank Test
1. Carry out a blank test as described above. Replace the test portion by a
filter paper washed with TCA solution and record the blank value.
Calculation with units of The true protein nitrogen content, expressed as percentage by mass is
expression calculated by the following formula:
1.4007 x (Vs − VB )x N
WPN =
Wt
Where,
WPN = true protein nitrogen content of the sample, expressed as a
percentage by mass;
VS = volume in mL of the standard hydrochloric acid used for sample;
VB = volume in mL of the standard hydrochloric acid used for blank test;
N = Normality of the standard hydrochloric acid expressed to four decimal
places;
Wt = mass of the test portion in g, expressed to nearest 0.1 mg.
Express the results to four decimal places.
The true protein content is obtained by multiplying the true protein
nitrogen content by 6.38. Express the test result to three decimal places.
Reference ISO 8968-5/IDF 020-5:2001 - Milk - Determination of nitrogen content -
Part 5: Determination of protein-nitrogen content. International
Organization for Standardization, Geneva, Switzerland.
Approved by Scientific Panel on Methods of Sampling and Analysis

88 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Non-Protein Nitrogen (NPN) in Milk

Method No. FSSAI 01.028:2022 Revision No. & Date 0.0

Scope This method describes the determination of non-protein nitrogen in milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Trichloroacetic acid: Trichloroacetic acid is a corrosive chemical and
contact can severely irritate and burn the skin and eyes with possible
eye damage. Trichloroacetic acid can irritate the nose and throat.
Trichloroacetic acid can irritate the lungs causing coughing and/or
shortness of breath.
Principle In this method, proteins are precipitated from milk by the addition of
trichloroacetic acid solution. Final concentration of trichloroacetic acid in
the mixture is about 12%. Precipitated milk proteins are removed by
filtration. Filtrate contains NPN components of milk. Nitrogen content of
filtrate is determined using Kjeldahl method.
Apparatus/Instruments Apart from the apparatus listed in Macro Kjeldahl method for the
determination of nitrogen content in milk (Refer method FSSAI
01.026:2022) , the following apparatus required are as below.
1. Conical flask: 25 mL capacity.
2. Pipettes: 10, 20 mL capacities.
3. Filter funnel: 75 mm diameter.
4. Whatman filter paper: Grade 1 (nitrogen free).
5. Beaker: 50 mL capacity.
Materials and Reagents 1. Trichloroacetic acid (TCA) solution
2. Concentrated Hydrochloric acid (Approximately 11.6 N)
3. Other reagents as already described in Macro Kjeldahl method for the
determination of total nitrogen content in milk.
Preparation of reagents 1. Trichloroacetic acid (TCA) solution (15%, w/v, aq): Dissolve 15.0 g of
trichloroacetic acid (CCl3COOH) in water and dilute to 100 mL in a
volumetric flask.

89 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Note: Do not use other concentrations of TCA and volumes of solutions
than those specified in this procedure. Other concentrations or volumes
of solutions will change the performance of the method with respect to
its mean value and performance characteristics.
2. Standard hydrochloric acid solution: 0.01 ± 0.0001 N: Dilute 0.0872
mL of concentrated hydrochloric acid to 100 mL with distilled water.
Standardize the prepared solution with 0.01 N sodium carbonate
solution.
Sample preparation Refer method FSSAI 01.001:2022 for sample preparation.
Method of analysis Test portion Preparation: Pipette 10.0 ± 0.1 mL of the prepared test
sample into a pre-weighed conical flask. Re-weigh the flask and its
contents recording the weights to the nearest 0.1 mg.
Precipitation and Filtration
1. Add 40 mL of TCA solution to the conical flask. Weigh the flask and
its contents again to the nearest 0.1 mg. Swirl to mix, let the flask stand
for approximately 5 min to allow the precipitate to settle.
2. Filter the contents of the flask through a Whatman filter paper Grade 1
and collect the entire filtrate in a clean, dry conical flask. The filtrate
shall be clear and free of particulate matter. If it is not, repeat the
process of precipitation of filtration with a new test portion. If duplicate
tests are to be done, separate precipitations and filtrations should be
carried out.
3. Swirl the filtrate to ensure the complete mixing. Pipette 20 mL of the
filtrate into a 50 mL beaker and weigh. Pour the filtrate from the beaker
into a Kjeldahl flask containing the appropriate amount of boiling aids,
K2SO4, copper (II) sulfate solution and concentrated sulphuric acid as
specified in the Macro Kjeldahl method for the determination of total
nitrogen content in milk. Immediately re-weigh the empty beaker.
Digestion and Distillation:
1. Continue with the digestion and distillation procedure as described in
method for the determination of total nitrogen content in milk Macro
Kjeldahl method.
Titration:
1. Titrate the boric acid receiving solution with standard hydrochloric acid
(0.01 N) to the first trace of pink colour. Take the burette reading to at
least the nearest 0.05 mL. A lighted stir plate may aid visualization of
the end point.
Blank Test:

90 | M o M – D a i r y a n d D a i r y P r o d u c t s
 1. Simultaneously carry out a blank test. Digest, distil and titrate a blank
comprising about 0.1 g of sucrose and 16 ± 0.5 mL of TCA solution
according to the procedure as given above.
Calculation with units of Calculate the nitrogen content, expressed as a percentage by mass using the
expression following formula:
1.4007 x (Vs − VB ) x N
Wn =
Wf x Wm /(Wt − 0.065 Wm )
Where,
Wn = nitrogen (NPN) content of sample, expressed as a percentage by
mass;
VS = volume in mL of the standard hydrochloric acid used for sample;
VB = volume in mL of the standard hydrochloric acid used for blank test;
N = Normality of the standard hydrochloric acid expressed to four decimal
places;
Wm = mass of test portion in g, expressed to nearest 0.1 mg.
Wf = mass of 20 mL filtrate in g, expressed to nearest 0.1 mg.
Wt = mass of the test portion plus 40 mL of TCA solution in g, expressed
to nearest 0.1 mg.
Note: The factor 0.065 in the denominator assumes that milk contains
about 3.5 % fat and 3.0% true protein (thus 0.035 + 0.030). The factor may
need to be adjusted for other liquid dairy products e.g. concentrated or
fractionated skim or whole milk products.
Express the nitrogen content to four decimal places.
Reference ISO 8968-4/IDF 20-4:2001 - Milk - Determination of nitrogen content -
Part 4: Determination of non-protein-nitrogen content. International
Organization for Standardization, Geneva, Switzerland.

Approved by Scientific Panel on Methods of Sampling and Analysis

91 | M o M – D a i r y a n d D a i r y P r o d u c t s
 B. CR EAM

Sample Preparation for Cream

Method No. FSSAI 01.029:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of cream and similar kind of
products such as reconstituted cream, recombined cream, prepared cream
(pre-packaged liquid cream, whipping cream, cream packed under
pressure, whipped cream, fermented/cultured/sour cream, acidified cream)
and malai.
Sample preparation 1. The preparation of cream sample depends upon its physical condition.
If, at room temperature (25 ± 3 oC), the cream is thin to pour easily,
mix by repeated inversion of the container.
2. If the cream is too thick, stir gently, taking care that the top and the
bottom layers get well mixed.
3. It may not be possible to mix by gentle stirring if the cream is very
thick and the fat is partly separated, or if on stirring, the cream becomes
thick or fat separates. Under these circumstances, warm the cream
sample to temperature between 30 to 40 ℃ in water-bath and, while
cooling it to room temperature (25 ± 3 oC), shake the container gently
or stir the contents at intervals.
4. Keep the container covered as much as possible to avoid loss of
moisture by evaporation.
5. If the cream sample shows any abnormality, it should be recorded. If
satisfactory mixing cannot be achieved, the sample should not be
tested.
6. In the laboratory, the exposure of cream sample to temperature below
freezing point should be avoided.
7. Cream samples should be protected from light, heat, contaminating
odours and be kept in a cool place, at a storage temperature of 0 to 5 ℃.
Note: Sour cream should not be warmed but should be thoroughly stirred.
Reference IS 3509: 1966 (Reaffirmed year 2018). Method of sampling and test for
cream. Bureau of Indian Standards, New Delhi.
IS 12333: 2017/ ISO 6731: 2010. Methods for determination of total solids
content in milk, cream, and evaporated milk- reference method. Bureau of
Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat in Cream

Method No. FSSAI 01.030:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in cream and similar kind of
products such as reconstituted cream, recombined cream, prepared cream
(pre-packaged liquid cream, whipping cream, cream packed under
pressure, whipped cream, fermented/cultured/sour cream, acidified cream)
and malai.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
2. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
5. Sulphuric acid: Not flammable but highly reactive. It reacts violently
with water with evolution of heat; can react with organic materials
explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
6. Iso amyl alcohol: It is a highly flammable liquid. Keep away from
heat/sparks/open flames/hot surfaces. Perform all operations under a
fume hood.
Principle Fat content in cream sample can be estimated either by Gerber Method or
Rose-Gottlieb method.
The Gerber method is suitable as a routine or screening test. It is an
empirical method and reproducible results can be obtained if procedure is
followed correctly. The cream sample is mixed with sulphuric acid and iso-
amyl alcohol in a special Gerber tube, permitting dissolution of the protein

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 and release of fat. The tube is centrifuged and the fat rising into the
calibrated part of the tube is measured as a percentage of the fat content of
the cream sample. This method can be used for routine purposes.
Rose-Gottlieb Method is a gravimetric method in which fat globule
membrane of milk fat globules is ruptured by addition of ammonia to cream
sample to liberate the fat. The liberated fat is extracted using combination
of solvents viz., diethyl ether and petroleum ether. The solvents are
evaporated and obtained fat is dried and weighed. This method is
considered suitable for reference purposes. Strict adherence to details is
essential in order to obtain reliable results.
Apparatus/Instruments Refer apparatus requirement stated for milk (FSSAI 01.024:2022), along
with following additional requirement.
1. Analytical Balance (Readability 0.0001 g).
2. Cream butyrometer; 70 percent (ISI marked).
Materials and reagents Refer requirement stated for analysis of fat content in milk (FSSAI
Preparation of Reagents 01.024:2022)
Sample preparation Refer method FSSAI 01.029:2022 for sample preparation.
Method of analysis Method 1: Gerber Method:
1. Transfer 10 mL of Gerber sulphuric acid into cream butyrometer using
automatic measure, taking care not to wet the neck of the butyrometer
with the sulphuric acid.
2. Weigh 5 ± 0.01 g of sample into the butyrometer without soiling the
neck, using any suitable form of support for the butyrometer on the
balance.
3. Add about 6 mL of the hot water (70 ℃) to the butyrometer; add 1 mL
of amyl alcohol, preferably by use of an automatic dispenser.
4. Adjust the level of the contents to about 5 mm below the shoulder by
further additions of hot water.
5. In no circumstances shall the amyl alcohol be added to the
butyrometer before the cream.
6. Close with a lock stopper, shake until homogeneous, inverting it for
complete admixture of the acid. Keep in a water bath at 65 ± 2 ℃ for
at least 3 min and not less than 10 min taking care to have casein
particles if any to dissolve fully, and centrifuge for 5 min at 1100 rpm
(approximately 300 g).
7. The tubes should be put in centrifuge, so as to conform to radial
symmetry, and as evenly spaced as possible, in order to protect
bearings of the centrifuge. Allow the centrifuge to come to rest.
8. Remove the butyrometer tubes and place in water bath at 65 ± 2 ℃ for
at least 3 min and not less than 10 min.

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 9. Read the percentage of fat after adjusting the height in the tube as
necessary by movements of the lock stopper with the key.
10. Note the scale reading corresponding to the lowest point of the fat
meniscus and the surface of separation of the fat and acid.
11. When readings are being taken hold the butyrometer with the
graduated portion vertical, keep the point being read in level with the
eye, and then read the butyrometer to the nearest half of the smallest
scale division.
Method 2: Rose-Gottlieb Method:
1. Weigh accurately 1-2 g (depending on fat percentage) of cream into
a 50 mL beaker, add 9 mL of 0.5% sodium chloride solution to
disperse and transfer to fat extraction apparatus.
2. Add 1 mL of ammonia sp. gr. 0.91 (or an equivalent volume of a more
concentrated ammonia solution may be used), mix and shake
thoroughly.
3. Proceed for the extraction of the fat by Rose-Gottlieb method as
prescribed for milk (Method No FSSAI 01.024:2022) starting with
the step indication addition of 10 mL ethyl alcohol onwards.
Calculation with units of Method 1: Gerber Method: See the method of analysis.
expression
Method 2: Rose-Gottlieb Method:

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤
Where,
M1 is the mass, in g, of the fat-collecting flask and extracted matter after
drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference 1. IS 3509: 1966 (Reaffirmed year 2018): Method of sampling and test for
cream. Bureau of Indian Standards, New Delhi.
2. IS 1224 (Part II): 1977 (Reaffirmed Year 2018). Determination of fat
by the Gerber method: Milk products. Bureau of Indian Standards, New
Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Presence of Thickeners in Cream

Method No. FSSAI 01.031:2022 Revision No. & Date 0.0

Scope This method is for the detection of presence of thickeners in cream and
similar kind of products such as reconstituted cream, recombined cream,
prepared cream (pre-packaged liquid cream, whipping cream, cream
packed under pressure, whipped cream, fermented/cultured/sour cream,
acidified cream) and malai.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
3. Iodine: It is not compatible with combustibles, strong bases, halogens
and ethanol. It reacts violently or explosively with acetylene,
acetaldehyde, metal hydrides and metal carbides.
4. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
5. Mercury: Avoid all unprotected contact with this product. Avoid
generating vapour or mists. It forms toxic chemicals on contact with
ammonia, oxidizing agents, metals (aluminium) etc.
6. Concentrated nitric acid: It is a corrosive chemical. It is highly reactive
and a dangerous explosion hazard. It reacts violently and explosively
with most metals and powdered metals (antimony, bismuth etc.),
alkaline earth metals (magnesium, calcium etc.), and metal hydrides to
form flammable and explosive hydrogen gas.
7. Picric acid: If necessary, apply water to the powder (misting it if
possible). This will reduce dust formation and make it safe to handle.
Picric acid forms sensitive salts with, and is therefore incompatible with
alkalis, transition metal oxides and alkaline salts, and heavy metal
oxides.
Principle Addition of iodine solution in milk samples gives the appearance blue

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 colour indicating the presence of starch, which disappears when sample is
boiled and reappears on cooling. Gelatine is also used as a thickener in
cream. It is detected by Stokes’ test, in which the sample is mixed with
water and Stokes reagent and filtered. A saturated solution of picric acid is
then added to the filtrate and the colour development is observed. Yellow
precipitate is produced in the presence of gelatine, and cloudiness is
observed when gelatine is present in smaller amounts.
Apparatus 1. Test tube.
2. 250 mL volumetric flask.
3. Whatman filter paper.
4. Analytical Balance (Readability 0.01 g).
Chemicals 1. Iodine.
2. Ethanol (98%).
3. Sodium hydroxide.
4. Sodium carbonate.
5. Mercury.
6. Concentrated nitric acid (Approximately 15.8 N).
7. Picric acid.
Preparation of reagents 1. Iodine solution: Dissolve 2.6 g of iodine and 3 g of potassium iodide in
sufficient quantity of water and make up to 200 mL.
2. Stokes’ reagent: Dissolve mercury in twice its weight of concentrated
nitric acid and dilute to 25 times the volume with water.
Sample preparation Refer method FSSAI 01.029:2022 for sample preparation.
Method of analysis Detection of Starch:
1. Take about 5 mL of cream in a test tube, bring to boiling condition and
allow the test tube to cool to (25 ± 3 C).
2. Add 1-2 drops of iodine solution to the test tube.
3. Observe the colour.
Detection of Gelatin in Cream:
1. Gelatin may be detected by Stokes’ test.
2. Mix together 10 mL cream, 20 mL water and 20 mL of Stokes reagent.
3. To the filtrate add an equal volume of saturated picric acid solution.
4. Yellow precipitate is produced in presence of considerable amount of
gelatin, smaller amounts are indicated by cloudiness.
Inference (Qualitative 1. Detection of starch: Development of blue colour indicates presence of
analysis) starch which disappears when sample is boiled and reappears on
cooling. The limit of detection of method is 0.02%.
2. Detection of Gelatin in Cream: Yellow precipitate is produced in
presence of considerable amount of gelatin, smaller amounts are
indicated by cloudiness.

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Reference 1. IS 1479 (Part I): 2016. Methods of test for Dairy Industry – Rapid
Examination of Milk. Bureau of Indian Standards, New Delhi.
2. AOAC 920.106 (Modified Method). 21st Edn. (2019). Official Method,
Starch in Confectionery, last para. AOAC International, USA.
3. Pearson’s Composition and Analysis of foods, 9th Edn. 1991 page 559.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 C. DAHI AND YOGHURT

Preparation of Sample of Dahi and Yoghurt

Method No. FSSAI 01.032:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of plain dahi, yoghurt, flavoured
dahi, partly skimmed yoghurt, flavoured partly skimmed dahi, skimmed
yoghurt and flavoured skimmed dahi.
Apparatus/Instruments 1. Spatula / Spoon.
2. Mortar and pestle.
Sample preparation For plain dahi, yoghurt, partly skimmed yoghurt, skimmed yoghurt
etc
1. Bring the sample to room temperature (preferably 25 °C).
2. Mix the sample carefully by means of spatula or spoon using a rotary
motion which passes from the lower layers to the surface layers of the
sample so as to displace and mix them well.
3. To get a representative sample, particularly in case of a thick-set
product, it may be necessary to pour out the whole of the contents in a
mortar and mix it thoroughly until the mass is homogeneous.
4. Aliquots are weighed out from this well-mixed sample.
For products containing non-dairy ingredients such as fruits and
vegetables and their products thereof, cereals and cereal products,
coconut and coconut products, honey, chocolate, nuts, coffee, spices,
condiments, culinary herbs etc.
1. Bring the sample to room temperature (preferably 25 °C).
2. Homogenize it using an appropriate device, in order to facilitate the
grinding and dispersion of fruits etc.
3. Aliquots are weighed out from this well-mixed sample.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat in Dahi and Yoghurt

Method No. FSSAI 01.033:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in plain Dahi, yoghurt, flavoured
dahi, partly skimmed yoghurt, flavoured partly skimmed dahi, skimmed
yoghurt and flavoured skimmed dahi.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with adequate
ventilation. Although ammonia is not flammable, it may ignite and burn
with explosive force. It reacts violently with halogens (such as fluorine,
bromine, and chlorine), acids (such as hydrochloric acid, hydrogen
fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes. Perform
all operations in a fume hood.
5. Amyl alcohol: It is a highly flammable liquid. Keep away from
heat/sparks/open flames/hot surfaces. Perform all operations under a
fume hood.
Principle Fat content of Dahi or yoghurt is estimated by Rose-Gottlieb procedure. In
this gravimetric method, fat globule membrane of milk fat globules is
ruptured by addition of ammonia to sample to liberate the fat. The liberated
fat is extracted using combination of solvents viz., diethyl ether and
petroleum ether. The solvents are evaporated and obtained fat is dried and
weighed. This method is considered suitable for reference purposes. Strict
adherence to details is essential in order to obtain reliable results.
Apparatus/Instruments Refer requirement stated for analysis of fat content in milk (FSSAI
Materials and reagents 01.024:2022).
Preparation of reagents
Sample preparation Refer method FSSAI 01.032:2022 for sample preparation.
Method of analysis Rose-Gottlieb Method:
Refer method stated for analysis of fat content in milk (Method No FSSAI
01.024:2022).

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Calculation with units of Refer calculation stated for analysis of fat content in milk (Method No
expression FSSAI 01.024:2022).
Reference IS 1479 (Part II): 1961 (Reaffirmed Year 2018). Methods of test for Dairy
Industry – Chemical Analysis of Milk. Bureau of Indian Standards, New
Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Milk Solids-Not-Fat (SNF) in Dahi and Yoghurt

Method No. FSSAI 01.034:2022 Revision No. & Date 0.0

Scope This method is for the determination of milk solids-not-fat in plain dahi and
yoghurt.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Zinc oxide: Avoid contact with skin and eyes. Avoid formation of dust
and aerosols. Provide appropriate exhaust ventilation at places where
dust is formed.
Principle In this method, total solids in given sample are estimated. Fat content is
subtracted from total solids to calculate at SNF content in the sample. In
cultured dairy products such as Dahi, total solids are determined after
neutralization of developed acidity with alkali. In the following procedure
(IDF 151: 1991), the water is evaporated from a test portion of the sample
in the presence of zinc oxide at a temperature of 100 ± 2 ℃ in drying oven.
Lactic acid (determined by titration method) content is determined
separately in the sample and is added to the moisture determined in the
drying step to compensate for the loss of water as a result of neutralizing the
sample by means of zinc oxide.
Apparatus/Instruments 1. Shallow flat-bottom dishes of aluminium, nickel, stainless steel.
2. Analytical Balance (Readability 0.0001 g).
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Hot air oven (forced air type), capable of being controlled at 100 ± 2
℃.
5. Flat bottom dishes of height 20 to 25 mm, diameter 50 to 75 mm, and
made of appropriate material (for example stainless steel, nickel or
aluminium), provided with well-fitting, readily removable lids.
6. Boiling water-bath.
7. Spoon or spatula.
8. Homogenizer for homogenizing fruit yogurts.

102 | M o M – D a i r y a n d D a i r y P r o d u c t s
 9. Porcelain or silica, 7-8 cm diameter, about 1.5 cm in height and
provided with easily removable but closely fitting lids.
Materials and reagents 1. Phenolphthalein indicator.
2. Sodium hydroxide.
3. Zinc oxide.
4. Ethanol.
Preparation of reagents 1. Standard sodium hydroxide solution – 0.1 N
2. Phenolphthalein indicator solution: Dissolve 1g of phenolphthalein in
100 mL of ethanol (95%, v/v). Add 0.1N Sodium hydroxide solutions
until one drop gives a faint pink coloration. Dilute with distilled water
to 200 mL.
Sample preparation Refer method FSSAI 01.032:2022 for sample preparation.
Method of analysis Determination of total solids
Method 1: Sodium hydroxide method
1. Heat the clean, dry, empty dish and lid in oven maintained at 100 ± 2 ℃
for 1 h, cool in a desiccator and weigh.
2. Quickly weigh to the nearest 0.1 mg, 4-5 g of prepared sample of curd
or dahi, replace the lid and weigh again.
3. Add 1-2 drops of phenolphthalein solution to the sample in the dish and
neutralise with 0.1 N sodium hydroxide solution to a faint pink colour.
4. Note the volume of 0.1 N sodium hydroxide required to neutralise the
sample.
5. Place the dish without lid on a boiling water bath until the water is
removed from the sample. Wipe the under-surface of the dish and place
in the oven maintained at 100 ± 2 ℃, for 3 h.
6. Remove the dish along with the lid and cool in a desiccator and weigh.
Continue heating and re-weighing at hourly intervals until the difference
between successive weighing do not vary by more than 0.5 mg.
7. Deduct half weight of the 0.1 N sodium hydroxide added to neutralize
the sample from the residue after drying and calculate total solids as for
whole milk.
Method 2: Zinc oxide method
1. Bring the sample to temperature of 20-25 ℃. Mix the sample carefully
by means of spatula using a rotatory motion which passes from the lower
layers to the surface layer of the sample so as to displace and mix them
well.
2. Heat an open dish containing approximately 2 g of zinc oxide, together
with lid and a stirring rod for 1 h in the oven maintained at 100 ± 2 ℃.
3. Transfer the dish along with lid and stirring rod to the desiccator, allow
it to cool to room temperature (at least 45 min) and weigh the dish with
the stirring rod and lid to the nearest 0.1mg.

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 4. Move the zinc oxide to one side of the prepared dish by tilting. Place on
the clear space approximately 1.0 g of prepared test sample, replace the
lid on the dish with the stirring rod on top and weigh the dish to the
nearest 0.1 mg.
5. Add 5 mL of water to the test portion and thoroughly mix the diluted test
portion and zinc oxide. Spread the mixture evenly over the bottom
surface of the dish. Heat the dish on the boiling water bath and continue
heating for approximately 30 min with frequent mixing of the contents
of the dish during the early stages of the drying so as to obtain maximum
evaporation of liquid.
6. Remove the dish from the water bath and wipe its base to remove any
water. Leave the stirring rod in the dish and then place it together with
lid by its side, in the hot air oven (100 ± 2 ℃) for 3 h.
7. After drying, cover the dish with its lid and immediately transfer to the
desiccator. Allow the dish and contents to cool in the desiccator to room
temperature (at least 45 min) and weigh to the nearest 0.1 mg.
8. Again, heat the dish and contents together with its lid for a further 1h,
cover the dish and transfer to the desiccator. Allow to cool and weigh to
the nearest 0.1 mg. repeat the process of reheating and weighing until
the difference in mass between two successive weighing does not exceed
1 mg. Record the lowest weight.
9. In order to compensate for the loss of water as a result of neutralizing
the yoghurt by means of zinc oxide, determine the titratable acidity
(expressed as g of lactic acid per 100 g of product) of the sample.
Determination of fat content:
Refer to method for determination of fat content in dahi and yoghurt
(Method No FSSAI 01.033:2022)
Determination of sucrose content (if added in flavoured product)
Refer to method stated for analysis of sucrose content in condensed milk
(FSSAI 01.052:2022)
Calculation with units of
Sodium hydroxide method:
expression
𝑁 × 𝑇.𝑉 × 40
a=
1000 × 2
100 (𝑀2 −𝑎)
Total solids percent (%) (m/m) =
𝑀1
Where,
N = Normality of Sodium hydroxide
T. V = Titre value

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 M2 = Weight in g of residue left after drying

M1= Weight in g of the prepared sample taken

Zinc Oxide method:

The total solids contents, expressed as percentage by mass, is equal to:
𝑀2 − M
= [( ) × 100] + 0.1 𝑎
𝑀1 − M
Where,
M= is the mass, in g of the dish (including zinc oxide), lid and stirring rod.
M1 = is the mass, in g, of the dish (including zinc oxide), lid, stirring rod
and test portion.
M2 = is the mass, in g, of the dish, lid stirring rod and dried test portion
(including zinc oxide)
a = is the mass, in g, of lactic acid as obtained in Step I. it is equal to 0.1 g
per g of lactic acid content.

For plain dahi/ yoghurt

Milk SNF Content (%) = Total solids (%) - Fat (%)

For Flavoured product

Milk SNF Content (%) = Total milk solid (%) - Fat (%) – Added sugar (%)
Reference 1. IS 12333: 2017/ ISO 6731: 2010 (Modified). Milk, Cream and
Evaporated Milk -Determination of Total Solid Content – Reference
method. Bureau of Indian Standards, New Delhi.
2. IDF. (2005). IDF Standard 151, Yoghurt. Determination of total solid
content, International Dairy Federation: Brussels.
3. Pearson’s Composition and Analysis of Foods, 9th Edn. Page 570.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Titratable Acidity in Dahi and Yoghurt

Method No. FSSAI 01.035:2022 Revision No. & Date 0.0

Scope This method is for the determination of titratable acidity in dahi.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
Principle A known quantity of sample is neutralized with standardized sodium
hydroxide solution with phenolphthalein indicator. The amount of sodium
hydroxide required is a function of the amount of natural buffering
substances present in the product, and of developed or added acid or alkaline
substances.
Materials and reagents 1. Sodium hydroxide.
2. Phenolphthalein indicator.
3. Analytical Balance (Readability 0.01 g).
Preparation of reagents 1. Standard sodium hydroxide solution – 0.1 N.
2. Phenolphthalein indicator: Dissolve 1.0 g of phenolphthalein in 100 mL
of 95% ethanol, add 0.1 N Sodium hydroxide solution until one drop
gives a faint pink colouration. Dilute with distilled water to 200 mL.
Sample preparation Refer method FSSAI 01.032:2022 for sample preparation.
Method of analysis 1. Weigh accurately about 10 g of the prepared dahi/yoghurt sample in a
suitable dish or basin.
2. Add 30 mL of warm water, add 1 mL of phenolphthalein indicator.
3. Shake well and titrate against standard NaOH solution (0.1N).
4. Complete the titration in 20 sec. Keep a blank by taking 10 g of prepared
dahi/yoghurt sample diluted with 30 mL of water in another dish for
comparison of colour.
Calculation with unis of 9 𝐴𝑁
Titratable acidity (as lactic acid)percent by mass =
expression 𝑤
Where,

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 A = Volume of standard Sodium hydroxide required for titration
N = Normality of Standard Sodium hydroxide solution
w = weight of the sample taken for test in g
Reference IS 1166: 1986 (Reaffirmed year 2018). Specifications for condensed milk,
partly skimmed condensed milk (Second Revision). Bureau of Indian
Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Protein in Dahi and Yoghurt

Method No. FSSAI 01.036:2022 Revision No. & Date 0.0

Scope This method is for the determination of protein in dahi and yoghurt. The
protein content is determined from the organic Nitrogen content by Kjeldahl
method.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
Principle Refer the method for determination of protein by Kjeldahl method in milk
Apparatus/Instruments and milk product (FSSAI 01.026/027:2022)
Material and reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.032:2022 for sample preparation.
Method of analysis 1. Weigh quickly about 4-5 g of the prepared dahi/yoghurt sample and
transfer to a 500 or 800 mL Kjeldahl flask/digestion tube taking care to
see that no portion of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk products
(FSSAI 01.026/027:2022).
Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.026/027:2022)
Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content
(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.
2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content
(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.

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 3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of
protein in foods and feeds. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Starch in Dahi

Method No. FSSAI 01.037:2022 Revision No. & Date 0.0

Scope This method is for the detection of starch in dahi.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
2. Iodine: It is not compatible with combustibles, strong bases, halogens
and ethanol. It reacts violently or explosively with acetylene,
acetaldehyde, metal hydrides and metal carbides.
3. Glacial acetic acid: Use with adequate ventilation. Keep away from heat.
Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin and
eyes. Keep away from incompatibles such as oxidizing agents, reducing
agents, metals, acids, [Link] acid: May cause gastrointestinal
irritation with nausea, vomiting and diarrhea. Excessive intake of citric
acid may cause erosion of the teeth. Causes respiratory tract irritation.
Repeated exposure may cause sensitization dermatitis.

Principle Addition of iodine solution in dahi samples gives the appearance blue colour
indicating the presence of starch which disappears when sample is boiled and
reappears on cooling.
Apparatus/Instruments Refer requirement stated for detection of starch in milk (FSSAI
Materials and reagents 01.003:2022).
Preparation of reagents
Sample preparation Refer method FSSAI 01.032:2022 for sample preparation.
Method of analysis Refer method prescribed for detection of starch in milk (FSSAI 01.003:2022)
Inference (Qualitative Appearance of blue colouration after addition of iodine solution indicates the
analysis) presence of starch, which disappears when the sample is boiled and reappears
on cooling.
Reference IS 1479 (Part I): 2016. Methods of test for Dairy Industry-Rapid examination
of Milk. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 D. CHEESE AND CHEESE PRODUCTS

Preparation of Sample of Cheese and Cheese Products

Method No. FSSAI 01.038:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of cheese (all varieties).
Sample preparation 1. Prior to analysis, remove the rind or smear or mouldy surface layer of
the cheese, in such a way as to provide a sample representative of the
cheese as it is usually consumed.
2. Grind or grate the sample by means of an appropriate device; mix the
ground or grated mass quickly, and if possible, grind or grate a second
time, and again mix thoroughly.
3. If the sample cannot be ground or grated, mix it thoroughly by intensive
stirring and kneading.
4. Transfer the test sample to an air-tight container to await analysis, which
should be carried as soon as possible after grinding.
5. If delay is unavoidable, take all precautions to ensure proper
preservation of the sample, and to prevent condensation of moisture on
the inside surface of the container.
6. The storage should be at 10 to 12 ℃.
7. All sample preparation should be carried out in a manner which
minimizes moisture loss. Such moisture loss will have the effect of
increasing the apparent fat content.
Reference IS 12758: 2005 (Reaffirmed year 2019)/ I.S.O 1735: 2014. Cheese and
Processed cheese products- Determination of Fat content by gravimetric
method – reference method. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Moisture in Cheese

Method No. FSSAI 01.039:2022 Revision No. & Date 0.0

Scope This method is for the determination of moisture in cheese (all varieties).
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.

Principle The moisture content of cheese is the loss in mass, expressed as a percentage
by mass when the product is heated in an air oven at 102 ± 2 ℃ to constant
mass.
Apparatus/Instruments 1. Flat-bottom dishes with lid: Dishes of nickel, aluminium or of other
suitable metal not affected by boiling water, 70 to 80 mm in diameter
and not more than 25 mm deep, provided with short glass stirring rod
having a widened flat end. The dishes shall have lids which fit well and
can readily be removed.
2. Hot air oven (forced air type), capable of being controlled at 102 ± 1 ℃.
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Sand, which passes through 500 μ sieve and is retained by 180 μ sieve.
It shall be prepared by digestion with concentrated hydrochloric acid,
followed by thorough washing with water. It shall then be dried and
ignited till it is dull red.
5. Analytical balance (Readability 0.0001 g).
Sample preparation Refer method FSSAI 01.038:2022 for sample preparation.
Method of analysis 1. Heat the flat-bottomed metal dish containing 20 g of prepared sand and
a stirring rod, in hot air oven for about 1 h. Allow to cool in an efficient
desiccator for 30 to 40 min.
2. Weigh accurately 3 g of the prepared sample of cheese into a flat-
bottomed dish (with a cover) previously dried and weighed containing
about 20 g of prepared sand and a stirring rod.
3. Saturate the sand by careful addition of a few drops of distilled water,
and thoroughly mix the wet sand with the cheese sample by stirring

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 with the glass rod, smoothing out lumps and spreading the mixture over
the bottom of the dish.
4. Place the dish on a boiling water-bath for 20 to 30 min, then wipe the
bottom of the dish. Transfer the dish containing the material, along with
glass rod after uncovering in an oven maintained at 102  1 ℃ for about
4 h.
5. After 4 h replace the lid, transfer the covered dish to the desiccator,
allow it to cool to 25 ± 3 C and weigh it accurately and quickly to the
nearest 0.1 mg.
6. Heat the uncovered dish and lid in the oven at 102 ± 1 ℃ for further 1
h, replace the lid, allow the covered dish to cool to room temperature
(25 ± 3 oC) in the desiccator and weigh it.
7. Repeat the process of drying, cooling and weighing, until the successive
weighings do not differ by more than 0.5 mg. Record the weight.
Calculation with units of M1 - M2
Moisture % by mass = × 100
expression M1 -M
Where,
M is mass in g, of the empty dish along with sand and glass rod;
M1 is initial mass in g of the dish with lid, glass rod, sand and test portion;
M2 is the mass in g of the dish with lid, glass rod, sand and dried test portion
Express the results to the nearest 0.01% (m/m).
Reference IS 2785: 1979 (Reaffirmed year 2016). Specification for Natural cheese
(Hard Variety), Processed Cheese, Processed Cheese Spread and Soft
Cheese. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat (on dry matter basis) in Cheese by Reference
Method
Method No. FSSAI 01.040:2022 Revision No. & Date 0.0
Scope This method is for the determination of fat in cheese (all varieties).
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
2. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
3. Diethyl ether: Store protected from light. It is extremely flammable
and can react explosively when in contact with Cl2, O3, LiAlH4 or
strong oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
5. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
Principle In this method fat is extracted from the sample by digesting the protein
matrix using hydrochloric acid. The liberated fat is extracted using a
combination of solvents viz., diethyl ether and petroleum ether. The solvents
are evaporated and obtained fat is dried and weighed. The addition of
ethanol facilitates the passage of the fat from the aqueous phase to the
solvents. This method is considered suitable for reference purposes. Strict
adherence to details is essential in order to obtain reliable results.
Apparatus/Instruments Refer requirement stated for analysis of fat content in milk (FSSAI
01.024:2022).

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Materials and reagents 1. Refer requirement stated for analysis of fat content in milk (FSSAI
01.024:2022).
2. Concentrated hydrochloric acid (Approximately 11.6 N)
Preparation of reagents Refer requirement stated for analysis of fat content in milk (FSSAI
01.024:2022).
Sample preparation Refer method FSSAI 01.038:2022 for sample preparation.
Method of analysis 1. Weigh accurately 1-2 g of prepared sample in a 100 mL beaker.
2. Add 10 mL of concentrated hydrochloric acid and heat on a Bunsen
burner boil gently, with shaking, either over a flame or in boiling
waterbath, until all solid particles are dissolved. Cool to room
temperature (25 ± 3 oC) in running water. Transfer the contents to the
Mojonnier fat extraction flask.
3. Proceed for the extraction fat by Rose-Gottlieb method as prescribed
for milk from the step indicating addition of 10 mL ethyl alcohol
(Method No FSSAI 01.024:2022).
Determination of moisture
Refer method for determination of moisture in cheese (FSSAI 01.039:2022)
Calculation with units of
(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
expression 𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,
M1 is the mass, in g, of the fat-collecting flask and extracted matter after
drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Total Solids (%) = 100-moisture (%)
Fat (%)
Fat % (on dry matter basis) = 𝑥100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)
Reference IS 2785 -1979 (Reaffirmed 2016). Specification for Natural cheese (Hard
Variety), Processed Cheese, Processed Cheese Spread and Soft Cheese.
Bureau of Indian Standards, New Delhi

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat (on dry matter basis) in Cheese by Gerber
Method
Method No. FSSAI 01.041:2022 Revision No. & Date 0.0
Scope This method is for the determination of fat in cheese (all varieties).
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sulphuric acid: Highly reactive. It reacts violently with water with
evolution of heat; can react with organic materials explosively. Do not
mix with hydrochloric acid. Good general ventilation should be
provided to keep vapour and mist concentrations below the exposure
limits.
2. Amyl alcohol: It is a highly flammable liquid. Keep away from
heat/sparks/open flames/hot surfaces. Perform all operations under a
fume hood.
Principle Cheese is mixed with sulphuric acid and iso-amyl alcohol in a special
Gerber tube, permitting dissolution of the protein and release of fat. The
tubes are centrifuged and the fat rising into the calibrated part of the tube
is measured as a percentage of the fat content of the cheese sample.
Apparatus/Instruments Refer apparatus requirement stated for milk (FSSAI 01.024:2022), along
with following additional requirement.

3. Analytical Balance (Readability 0.0001 g).

4. Cheese butyrometer; 40 percent (ISI marked).

Materials and reagents 1. Concentrated Sulphuric acid (Approximately 36.8 N).

2. Iso amyl alcohol (furfural free). It should have density between 0.807
to 0.809 g/mL at 27 ℃.
Preparation of Reagents Preparation of Gerber Sulphuric acid: Take required volume of water
in a Pyrex flask (generally 100 mL of water is required for 900 mL of
concentrated sulphuric acid) kept in a basin of ice-cold water. Carefully

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 add the commercial sulphuric acid in small quantities at a time keeping
the container sufficiently cold and mix gently. After cooling the flaks,
check the specific gravity of Gerber acid with hydrometer and if
necessary, adjust the Gerber acid to the correct specific gravity with
addition of water or acid taking same precautions as before till specific
gravity is in the range of 1.807 to 1.812 g/mL at 27 ℃ (or 1.815 to 1.820
g/mL at 20 ℃). Store the prepared acid in a glass stoppered bottle to avoid
absorption of water.
Sample preparation Refer method FSSAI 01.038:2022 for sample preparation.
Method of analysis 1. Grate samples of hard cheese; grind samples of soft cheese. Mix
thoroughly.
2. Measure 10 mL of Gerber sulphuric acid into a cheese butyrometer
tube, preferably by use of an automatic dispenser, without wetting the
neck of the tube.
3. Add gently from the wash bottle sufficient warm water (30 to 40 ℃) to
form a layer about 6 mm deep on top of the acid, allowing the water to
flow down the side of the bulb.
4. Weigh 3 ± 0.01 g of sample into the butyrometer without soiling the
neck, using any suitable form of support for the butyrometer on the
balance.
5. Add 1 mL of amyl alcohol, preferably by use of an automatic
dispenser. Add warm water (30 to 40 ℃) from the wash bottle until the
butyrometer is filled to about 5 mm below the shoulder.
6. Close with a lock stopper, shake until homogeneous, inverting it for
complete admixture of the acid. Keep in a water bath at 65 ± 2 ℃ for
at least 3 mins and not less than 10 mins, taking care to have casein
particles if any to dissolve fully, and centrifuge for 5 min. at 1100 rpm
(approximately 300 g).
7. The tubes should be put in centrifuge, so as to conform to radial
symmetry, and as evenly spaced as possible, in order to protect
bearings of the centrifuge.
8. Allow the centrifuge to come to rest. Remove the butyrometer tubes
and place in a water bath at 65 ± 2 ℃ for at least 3 min and not less
than 10 min.
9. Read the percentage of fat after adjusting the height in the tube as
necessary by movements of the lock stopper with the key.
10. Note the scale reading corresponding to the lowest point of the fat

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 meniscus and the surface of separation of the fat and acid.
When readings are being taken hold the butyrometer with the graduated
portion vertical, keep the point being read in level with the eye, and then
read the butyrometer to the nearest half of the smallest scale division.
Determination of Moisture
Refer to method of determination of moisture in cheese (FSSAI
01.039:2022)
Calculation with units of Total solids (%) = 100- moisture (%)
expression
Fat (%)
Fat % (on dry matter basis) = 𝑥100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)

Reference IS1224 (Part II): 1977 (Reaffirmed Year 2018). Determination of fat by the
Gerber method: Milk products. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 E. CHANNA/PANEER

Preparation of Sample of Channa/Paneer

Method No. FSSAI 01.042:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of channa/paneer.
Apparatus/Instruments 1. Spatula / Spoon.
2. Mortar and pestle.
3. Grating device.
Sample preparation 1. Pass Channa sample through 8 mesh sieve three times or grate sample
and mix thoroughly or grind to a uniform mass in a glass pestle and
mortar.
2. Grate the paneer sample quickly through a suitable grater. Mix the grated
sample thoroughly.
3. Transfer the grated sample to an air-tight container prior to analysis,
which should be carried as soon as possible after grinding.
4. Keep sample in an airtight container until the time of analysis. If delay
is unavoidable, take all precautions to ensure proper preservation of the
sample, and to prevent condensation of moisture on the inside surface of
the container.
5. The storage temperature should be below 10 ℃. All sample preparation
should be carried out in a manner which minimizes moisture loss. Such
moisture loss will have the effect of increasing the apparent fat content.
Reference IS 12758: 2005 (Reaffirmed year 2019)/ I.S.O 1735: 2014. Cheese and
Processed Cheese products - Determination of fat content by gravimetric
method - reference method. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Moisture in Channa/Paneer

Method No. FSSAI 01.043:2022 Revision No. & Date 0.0

Scope This method is for the determination of moisture in channa/paneer.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.
1. Use thermal protection gloves, tongs and protective eyewear while
handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle The moisture content of channa/paneer is the loss in mass, expressed as a
percentage by mass when the product is heated in an air oven at 102 ± 2 ℃
to constant mass.
Apparatus/Instruments Refer method of analysis of moisture in cheese (FSSAI 01.039:2022)

Materials and reagents

Preparation of reagents
Sample preparation Refer method FSSAI 01.042:2022 for sample preparation.
Method of analysis Refer method of analysis of moisture in cheese (FSSAI 01.039:2022)
Calculation with units of M1 - M2
Moisture % by mass = × 100
expression M1 -M
Where,
M is mass in g, of the empty dish along with glass rod;
M1 is initial mass in g of the dish with lid, glass rod and test portion;
M2 is the mass in g of the dish with lid, glass rod and dried test portion
Express the results to the nearest 0.01% (m/m).
Reference 1. IS 2785: 1979 (Reaffirmed year 2016). Specification for Natural cheese
(Hard Variety), Processed Cheese, Processed Cheese Spread and Soft
Cheese. Bureau of Indian Standards, New Delhi.
2. IS 10484: 1983 (Reaffirmed year 2019). Specification for Paneer.
Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat (on dry matter basis) in Channa/Paneer

Method No. FSSAI 01.044:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in channa/paneer on dry matter
basis.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
2. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
3. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
Principle In this method using fat is extracted from the sample by digesting the
protein matrix using hydrochloric acid. The liberated fat is extracted using
combination of solvents viz., diethyl ether and petroleum ether. The solvents
are evaporated and obtained fat is dried and weighed. The addition of
ethanol facilitates the passage of the fat from the aqueous phase to the
solvents. This method is considered suitable for reference purposes. Strict
adherence to details is essential in order to obtain reliable results. Total
solids are separately determined and final results of fat content in the sample
is expressed on dry matter basis.
Apparatus/Instruments Refer requirement stated for analysis of fat content in cheese (FSSAI
01.040:2022).
Materials and reagents

Preparation of reagents
Sample preparation Refer method FSSAI 01.042:2022 for sample preparation.

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Method of analysis Refer method of analysis for determination of fat content in cheese (FSSAI
01.040:2022).
Determination of moisture
1. Refer method for determination of moisture in channa/paneer FSSAI
01.043:2022.
Calculation with units of
expression (𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g

Total Solids (%) = 100 - moisture (%)

Fat (%)
Fat % (on dry matter basis) = 𝑥100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)
Reference 1. IS 2785: 1979 (Reaffirmed year 2016). Specification for Natural cheese
(Hard Variety), Processed Cheese, Processed Cheese Spread and Soft
Cheese. Bureau of Indian Standards, New Delhi.
2. IS 10484: 1983 (Reaffirmed year 2019). Specification for Paneer.
Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 F. ICE CREAM AND RELATED PRODUCTS

Sample Preparation for Ice cream, Kulfi, Milk Ices, Milk Lollies and
Frozen Dessert/Confection

Method No. FSSAI 01.045:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of ice cream, kulfi, milk ices,
milk lollies and frozen dessert/confection.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
Sample preparation Preparation of Sample of Ice Cream: For samples taken in small
packages, remove the packaging and place the sample in a clean, dry
container fitted with an airtight closure. For samples taken from bulk or from
large packages, keep them in their sampling containers.
1. Plain ice cream: Allow the sample to soften at room temperature. It is
not advisable to soften the ice-cream sample by heating on water bath
or over flame because melted fat tends to separate and rise to the
surface. After softening of ice-cream, mix it thoroughly by stirring with
spoon or eggbeater or by pouring back and forth between beakers:
2. Ice cream containing fruits, nuts and chocolate ice-cream
containing insoluble particles: Use a mixer capable of comminuting
product to fine, uniform pulp. Use 100 - 200 g of ice-cream sample to
fill the cup of mixer full to about one-third. Melt the product at room
temperature (25 ± 3 oC) or in an incubator at 37 ℃ in a closed container.
Transfer entire contents to the mixer cup and mix until insoluble
particles are finely divided (about 3-5 min for fruit ice-cream and up to
7 min for nut ice-cream). Alternatively, the product may be grounded
in porcelain or glass pestle and mortar. Transfer the mixed ice-cream
sample to a suitable container for convenience in weighing. After
weighing operation, return the remainder of the ice-cream sample to
the refrigerator, preferably at a temperature not exceeding -15 ℃. In
case of ice cream, where the chocolate or similar covering portions
forms a separate layer, it shall be removed and only the ice cream
portion shall be taken for analysis.
 Note: For determination of overrun in ice-cream, the entire ice-cream
carton/cup should be taken as such.
Reference 1. IS 2802: 1964 (Reaffirmed year 2016). Specification for Ice cream.
Bureau of Indian Standards, New Delhi.

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 2. AOAC 969.20. 21st Edn. (2019). Official method. Ice cream and Frozen
Desserts. AOAC International, USA.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Total Solids in Ice Cream

Method No. FSSAI 01.046:2022 Revision No. & Date 0.0

Scope This method is for the determination of total solids in ice cream, kulfi, milk
ices, milk lollies and frozen dessert/confection.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.
1. Use thermal protection gloves, tongs and protective eyewear while
handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling
period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle In this method the sample is repeatedly heated and cooled till the difference
between consecutive weighing agrees within 0.5 mg. Total solids is
calculated from loss in weight observed.
Apparatus/Instruments 1. Flat bottomed dishes of aluminium, nickel or stainless steel not
affected by boiling water, 7-8 cm in diameter and not more than 2.5
cm deep provided with short stirring rods having a widened flat end.
2. Hot air oven (forced air type), capable of being controlled at 102 ± 2 ℃.
3. Analytical balance (Readability 0.0001 g).
Materials and reagents Sand which passes through 500 micron I.S sieve and is retained on 180-
micron sieve. It shall be prepared by digesting with concentrated
hydrochloric acid followed by thorough washing with water. It shall be dried
and ignited to red heat. (Refer clause 5.7 of IS 11622: 1986 for detailed
procedure of preparation of sand)
Preparation of reagents 1. Quartz sand or sea sand, which passes through 500 microns IS sieve
and is retained on 180 microns IS sieve [IS: 460 (Part I) – 1978], and
which passes the suitability test size as follows:
a. Place approximately 20 g of sand in a dish containing a stirring rod.
Heat the open dish and sand, stirring rod and lid in a hot air oven
controlled at 102 ± 2 ℃, for at least 2 h or preferably overnight. Fit the
lid, allow the dish to cool in a desiccator to room temperature (25 ± 3
o
C) and weigh to the nearest 0.1 mg.
b. Moisten the sand with approximately 5 mL of water, mix the sand and
water using the stirring rod and heat the dish and sand, stirring rod and
lid in a hot air oven, controlled at 102 ± 2 ℃ for at least 4 h. Fit the lid,
allow the dish to cool in the desiccator to room temperature (25 ± 3

125 | M o M – D a i r y a n d D a i r y P r o d u c t s
 o
C) and weigh again to the nearest 0.1 mg. The difference between the
two weighing shall not exceed 0.5 mg.
If the requirements given above is not met, the sand may be made suitable
for the determination as follows:
Leave the sand immersed in 25% (m/m) hydrochloric acid solution for 3
days. Stir occasionally, decant off the supernatant liquid as far as possible,
then wash the sand with water until the acid reaction has disappeared.
Calcine (heat) the sand at 550 ℃ for at least 4 h using a muffle furnace.
Repeat the test for the suitability of the sand as described above (the
difference in weights represents the weight of fat extracted from the milk.
Correct weight of extracted fat by blank determination on reagents used).
Sample preparation Refer method FSSAI 01.045:2022 for sample preparation.
Method of analysis 1. Heat the moisture dish containing 20 g of prepared sand and glass
stirring rod in the oven for 1 h; cool and weigh.
2. Weigh accurately about 5 g of sample into the dish. Add a few drops of
water to assist in spreading the sample with glass rod.
3. Place dish on a boiling water bath for about 30 min.
4. Wipe bottom of dish and transfer to the air oven. Dry for about 4 h in
anoven maintained at 102 ± 2 ℃., remove dish to an efficient desiccator,
allow to cool and weigh.
5. Replace dish in oven for a further period of 1 h, transfer to desiccator,
allow it to cool and weigh.
6. Repeat the process of heating and cooling till consecutive weighing
agree within 0.5 mg. Calculate total solids from loss in weight observed.
Calculation with units of 𝑀2 − M
expression Total Solids Content (%) = 𝑥 100
𝑀1 − M
Where,
M is the mass in g of empty dish along with lid and sand and glass rod;
M1 is the initial mass in g of dish with lid, sand, glass rod and test portion
M2 is mass in g of dish with lid, sand and dried test portion
Round the value obtained to nearest 0.01 % (m/m)
Reference 1. IS 2802: 1964 (Reaffirmed year 2016). Specification for Ice cream.
Bureau of Indian Standards, New Delhi.

2. Pearson’s Composition and analysis of foods,9th Edn,1991 page 604.

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 Determination of Weight per Unit Volume or Over-run in Ice Cream

Method No. FSSAI 01.047:2022 Revision No. & Date 0.0

Scope This method is for the determination of weight per unit volume or over-run
in ice cream.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Amyl alcohol: It is a flammable liquid. Keep away from heat, sparks
and open flame. It is not compatible with strong acids (sulphuric acid,
hydrochloric acid) and strong bases (Sodium hydroxide, potassium
hydroxide).
Principle Over-run is usually defined as the volume of ice-cream obtained in excess
of the volume of the mix. It is usually expressed as a percentage. This
increased volume is composed mainly of the air incorporated during the
freezing process. The amount of air which is incorporated depends upon the
composition of mix and the way it is processed. In this test, the volume of
water and alcohol used corresponds with the volume of air originally
contained in the ice-cream and the difference between the sum of these two
and capacity of the flask is equivalent to the volume occupied by the sample.
Apparatus/Instruments 1. Beaker: 400 mL.
2. Volumetric flask: 250 mL.
3. Glass funnel.
4. Analytical balance (Readability 0.0001 g).
Materials and reagents n-Amyl alcohol (sp. gr. 0.817).
Sample preparation Refer method FSSAI 01.045:2022 for sample preparation.
Method of analysis 1. Weigh a unit of ice-cream and from it calculate the weight of ice-cream
per litre. For example, 200 mL of a full carton of ice-cream can be
obtained, the ice-cream carefully removed and the empty dry carton
weighed. The difference in weights between the carton when filled and
when empty is, therefore, the weight of 200 mL of frozen ice-cream.
Five times this weight would then equal the weight of a litre. To
determine the weight of the mix, proceed as below
2. Weigh and record the exact weight of a clean, dry 400 mL beaker. Into
the beaker, weigh exactly 130 g of the frozen ice-cream.
3. Place the beaker in water bath warmed to 49 ℃ and melt.

127 | M o M – D a i r y a n d D a i r y P r o d u c t s
 4. Weigh and record the exact weight of a 250 mL volumetric flask.
5. Using a glass funnel, transfer 130 g of melted ice-cream into the 250 mL
volumetric flask.
6. Add exactly 10 g of n-amyl alcohol to the flask and mix to break the
surface tension of the melted ice-cream and release the incorporated air.
7. 10 g of n-amyl alcohol occupies a volume of 12.24 mL.
8. Cool the flask with contents to 15.5 ℃ using a cold water or ice water
bath.
9. Rinse the beaker containing melted mix with several small rinsing of
water, adding each rinse to the 250 mL flask.
10. Again cool the flask with contents to 15.5 ℃ and using the final rinse
water, bring the volume to 250 mL mark. The bottom of the meniscus
should correspond with the mark when temperature is exactly 15.5 ℃.
Dry the outside of the flask and reweigh.
11. Calculate the weight in g of the contents. Calculate the weight in g of
the water added to the flask.
Calculation with units of Calculate the volume in mL occupied by the sample of ice-cream.
expression Determine the sp. gr. of the mix by dividing its weight (130 g) by the volume
in mL, which it occupied. Determine the weight in g per litre of mix by
multiplying by the specific gravity.
Reference IS 2802: 1964 (Reaffirmed year 2016). Specification for ice-cream. Bureau
of Indian Standards, New Delhi.
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 Determination of Milk Fat in Ice cream

Method No. FSSAI 01.048:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of milk fat in ice cream,
kulfi, milk ices, milk lollies and frozen dessert/confection.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: It is an extremely caustic liquid and gas. Wear skin,
eye and respiratory protection while handling.; Perform all operations
under a fume hood.
2. Ethyl alcohol: It is flammable. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: It is extremely flammable. Avoid breathing fumes.
Perform all operations under a fume hood.
Principle Sample is treated with ammonia and ethyl alcohol; the former to dissolve
the protein and the latter to help precipitate the proteins. Fat is extracted
with diethyl ether and petroleum ether. Mixed ethers are evaporated and the
residue weighed. This method is considered suitable for reference purposes.
Strict adherence to details is essential in order to obtain reliable results. It
may be noted that this method will determine total fat in the product and if
any non-milk fat is present in the sample, the same will also be determined.
Apparatus/Instruments 1. Mojonnier fat extraction flask or any other suitable extraction tube (as
per IS specification).
2. Cork or stopper of synthetic rubber unaffected by usual fat solvents.
3. 100 mL flat bottom flask with G/G joint or stainless steel or aluminium
dishes of 5.5 cm height and 9 cm diameter or glass bowl.
4. Water bath.
5. Analytical balance (Readability 0.0001 g).
Materials and reagents 1. Ammonia solution (Specific gravity 0.91).
2. Ethyl alcohol (95%).
3. Diethyl ether, peroxide-free.
4. Petroleum ether, boiling range 40 - 60 ℃.

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Sample preparation Refer method FSSAI 01.045:2022 for sample preparation.
Method of analysis Rose-Gottlieb Method:
1. Accurately weigh 4-5 g of the thoroughly mixed sample; transfer it
directly into fat extraction flask or Mojonnier tube, using free flowing
pipette.
2. Dilute with water to approximately to 10 mL, working the sample into
the lower chamber and mix by shaking.
3. Add 2 mL ammonia; mix thoroughly, heat in water bath for 20 mins at
60 ℃ with occasional shaking.
4. Cool and proceed extraction of fat by Rose-Gottlieb method as
prescribed for milk from the step indicating addition of 10 mL ethyl
alcohol (Method No FSSAI 01.024:2022).

Calculation with units of

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
expression 𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g

Reference Pearson’s Composition and analysis of foods 9th edn,1991 page 604.

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 Determination of Protein in Ice-cream

Method No. FSSAI 01.049:2022 Revision No. & Date 0.0

Scope This method is for the determination of protein in ice cream, kulfi, milk
ices, milk lollies and frozen dessert/confection. The protein content is
determined from the organic Nitrogen content by Kjeldahl method.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
Principle The various nitrogenous compounds are converted into ammonium
sulphate by boiling with concentrated sulphuric acid. The ammonium
sulphate formed is decomposed with an alkali (Sodium hydroxide) and the
ammonia liberated is absorbed in excess of standard solution of acid and
then back titrated with standard alkali.
Apparatus/Instruments 1. Refer the method for determination of protein by Kjeldahl method in
milk and milk products (Refer method FSSAI 01.026/027:2022)
Material and reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.045:2022 for sample preparation.
Method of analysis 1. Weigh quickly about 5-8 g of the prepared ice-cream sample and
transfer to a 500 or 800 mL Kjeldahl flask/digestion tube taking care to
see that no portion of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk
products (FSSAI 01. 026/027:2022).

Calculation with units of Refer the method for determination of protein in milk and milk products

131 | M o M – D a i r y a n d D a i r y P r o d u c t s
expression (FSSAI 01. 026/027:2022)

Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content

(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.

3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of

protein in foods and feeds. Bureau of Indian Standards, New Delhi.

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 G. CONDENSED/EVAPORATED MILK

Preparation of Sample of Condensed/Evaporated Milk

Method No. FSSAI 01.050:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of condensed/evaporated milk.
Sample preparation 1. Shake the container thoroughly with frequent inversion.
2. Open the container and pour the milk slowly into another container
made of glass or other suitable material provided with an air tight lid,
taking care to incorporate in the sample any fat or other constituents
adhering to the sides of the original container.
3. Stir vigorously and close the container. Alternatively heat the closed
container in a water bath at 40-60 ℃.
4. Remove and shake the container vigorously every 15 min. After 2 h,
remove the container and cool to 20-25 ℃. Remove the lid and mix
thoroughly by stirring the milk with a spoon or spatula.
Note: If the fat separates, correct results cannot be obtained.
Reference 1. IS 12333: 2017/ ISO 6731: 2010. Methods for determination of total
solids content in milk, cream, and evaporated milk- reference method.
Bureau of Indian Standards, New Delhi.
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 Determination of Fat in Condensed/Evaporated Milk

Method No. FSSAI 01.051:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in condensed/evaporated milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable
and can react explosively when in contact with Cl2, O3, LiAlH4 or
strong oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle Refer principle stated for analysis of fat content in milk (FSSAI
01.024:2022).
Apparatus/Instruments Refer requirement stated for analysis of fat content in milk (FSSAI
01.024:2022).
Materials and reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.050:2022 for sample preparation.
Method of analysis Rose-Gottlieb Method
1. Weigh accurately 2-2.5 g in case of condensed milk and 4 to 5 g for
evaporated milk sample into a beaker.
2. Add 1 mL of water and make into a smooth paste by using a glass rod.
3. Add 9.0 mL of additional water and 1.25 mL of ammonium hydroxide
and warm on a steam bath.
4. Transfer to Mojonnier flask, cool and proceed for the extraction fat
by Rose-Gottlieb method as prescribed for milk from the step

134 | M o M – D a i r y a n d D a i r y P r o d u c t s
 indicating addition of 10 mL ethyl alcohol (Method No FSSAI
01.024:2022).

Calculation with units of

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
expression 𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference 1. IS 1166: 1986 (Reaffirmed year 2018). Specification for condensed
milk, partly skimmed condensed milk. Bureau of Indian Standards,
New Delhi.

2. Pearson’s Composition and analysis of foods 9th edn,1991 page 604.

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135 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Sucrose Content in Condensed/Evaporated Milk

Method No. FSSAI 01.052:2022 Revision No. & Date 0.0

Scope This method is for the determination of sucrose content in
condensed/evaporated milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide solution: It is extremely caustic and can cause
severe burns. Protect skin and eyes when working with these alkalis
as solids or concentrated solutions. While making solution add pellets
to water and not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is
extremely corrosive and can cause serious burns when not handled
properly. It reacts violently with water with evolution of heat; can
react with organic materials explosively. Do not mix with
hydrochloric acid. Good general ventilation should be provided to
keep vapour and mist concentrations below the exposure limits.
3. Ammonia solution: It reacts violently with halogens (such as fluorine,
chlorine, bromine), acids (such as hydrochloric acid, hydrogen
bromide, hydrogen fluoride). It is incompatible with cyanide and
oxidizing agents.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
Principle Lane-Eynon (Volumetric) Method: The principle involves clarifying the
sample with the help of zinc acetate and potassium ferrocyanide, inverting
a part of the sample using hydrochloric acid and determining the reducing
sugars content in both inverted and non-inverted parts of the sample. The
sugar content is estimated by determining the volume of the unknown sugar
solution required to completely reduce a measured volume of Fehling's
solution. Invert sugar reduces the copper in Fehling's solution to red
insoluble cuprous oxide.
Polarimetric Method: The method based on Clerget inversion involves
treatment of milk without acid which brings about complete hydrolysis of

136 | M o M – D a i r y a n d D a i r y P r o d u c t s
 sucrose, but almost none of lactose or other sugars. Sugar content is
obtained from change in rotating power of the solution. A clear filtrate of
sample, without mutarotation by lactose is prepared by treating the solution
with ammonia followed by neutralisation and clarification with successive
addition of zinc acetate and potassium ferrocyanide solution. In a part of
the filtrate, the sucrose is hydrolysed by appropriate method and from the
rotation of filtrate, before and after inversion, sucrose content is calculated.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Volumetric flasks: 100, 250, 500 and 1000 mL capacities.
3. Burette: 50 mL, graduated to 0.1 mL.
4. Funnels.
5. Conical flasks: 250 mL capacities.
6. Pipette: 1, 5 and 10 mL capacities.
7. Measuring cylinder: 25 and 50 mL capacities.
8. Whatman filter paper: Grade 1.
9. Watch glass.
10. Hot plate.
11. Polarimeter or saccharimeter.
12. Polarimeter tube: length 200 mm, of exactly calibrated length).
13. Water bath: Regulated at 60 ± 1 ℃.
Materials and reagents 1. Sodium hydroxide.
2. Sucrose.
3. Methylene blue indicator.
4. Fehling's solution.
5. Zinc acetate.
6. Potassium ferrocyanide.
7. Concentrated hydrochloric acid (Approximately 11.6 N).
8. Concentrated ammonia (Specific gravity 0.88).
9. Copper sulphate.
10. Concentrated sulphuric acid (Approximately 36.8 N).
11. Potassium sodium tartrate.

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Preparation of Reagents 1. Sodium hydroxide solution: Approximately 0.1 N. Weigh 4 g of
Sodium hydroxide and dissolve in 100 mL, cooling the flask. Make
up the volume to 1000mL
2. Stock solution of invert sugar (Solution A): Weigh accurately 9.5 g
of pure sucrose on a watch glass and transfer it to a one litre
volumetric flask with 100 mL of water; add 5 mL of concentrated
hydrochloric acid. Allow this to stand for 3 days at 20 to 25 ℃ and
then make up to volume with water (this solution is stable for several
months).
3. Standard solution of invert sugar (Solution B): Neutralise a known
aliquot of the stock solution of invert sugar (Solution A) with sodium
hydroxide solution using litmus paper and dilute with water to a
known volume, so that more than 15 mL but less than 50 mL of it
shall be required to reduce all the copper in the Fehling's solution
taken for titration. Note the concentration of invert sugar in this
solution as mg per 100 mL. Prepare this solution fresh every day.
Note: When 10 mL of Fehling's solution are taken for titration, a standard
invert sugar solution containing 0.12 to 0.30 percent (m/v) of invert sugar
is used. Usually 40 mL of Stock solution (Solution A) is diluted to 100 mL
to get titration reading of around 15 mL when titrated against Fehling
solution.
4. Methylene blue indicator solution: Dissolve 0.2 g of methylene blue
in water and dilute to 100 mL.
5. Fehling's solution (Soxhlet modification): Prepared by mixing
immediately before use, equal volumes of Solution A and Solution B
prepared as described below.
6. Solution A: Dissolve 34.639 g of copper sulphate (CuSO45H2O) in
water add 0.5 mL of concentrated sulphuric acid of sp. gr. 1.84 and
dilute to 500 mL in a volumetric flask. Filter the solution through
prepared asbestos.
7. Solution B: Dissolve 173 g of Rochelle salt (potassium sodium
tartrate –KNaC4H4O6.4H2O) and 50 g of sodium hydroxide analytical
reagent grade in water, dilute to 500 mL in a volumetric flask and
allow the solution to stand for two days. Filter this solution through
prepared asbestos.
Standardization of Fehling's Solution: Pipette accurately 10 mL of each
Fehling's solutions (Solution A and B) into a 250 mL Erlenmeyer flask.
Add about 10 mL of standard solution of invert sugar (Solution B) to the

138 | M o M – D a i r y a n d D a i r y P r o d u c t s
 flask and heat to boiling. Boil for about 15 secs and add rapidly further
portions of the standard solution of invert sugar until only the faintest
perceptible blue colour remains, then add 2-5 drops of methylene blue
solution, continue the heating and addition of Standard solution of invert
sugar (Solution B) drop wise until the titration is complete, which is shown
by the reduction of the dye (the end point of the titration is blue to red. Do
not stir the flask, mixing is achieved by continuous boiling). Note down
the volume of standard solution of invert sugar (Solution B) required to
reduce all the copper. Repeat the titration, adding before heating almost all
of the standard solution of invert sugar (Solution B) needed to reduce all
the copper so that not more than 0.5-1 mL is required later to complete the
titration. Heat the mixture to boiling and boil gently for 2 min, lowering
the flame sufficiently to prevent bumping. Without removing the flask
from the flame add 2-5 drops of methylene blue indicator and complete the
titration within a total boiling time of about 3 min, by small additions of
standard solution of invert sugar (Solution B) to complete decolourization
of the indicator. Note down the volume of standard solution of invert sugar
(Solution B) required to reduce all the copper.
Note 1: The concentration of the sugar solution should be such that the titre
volume should be between 15 and 50 mL and this can be achieved with
0.2 -0.3 g sugar per 100 mL.
8. Zinc acetate solution: Dissolve 21.9 g of crystalline Zinc acetate in
water and add 3 mL of glacial acetic acid; make volume up to 100
mL.
9. Potassium ferrocyanide solution: Dissolve 10.6 g of crystalline
Potassium ferrocyanide and make up to 100 mL with water.
10. Concentrated hydrochloric acid (Approximately 11.6 N)
11. Dilute hydrochloric acid: 6.35 N ± 0.2 (20-22 %); required for
polarimetric method only.
12. Concentrated ammonia solution (Specific gravity 0.88).
13. Dilute ammonia solution: 10 mL of concentrated ammonia solution
diluted to 100 mL with distilled water.
14. Dilute acetic acid solution: 10 mL of acetic acid solution diluted to
100 mL with distilled water.
Sample preparation Refer method FSSAI 01.050:2022 for sample preparation.
Method of analysis Method 1. Lane-Eynon (Volumetric) Method:
Preparation of the Solution:

139 | M o M – D a i r y a n d D a i r y P r o d u c t s
1. Weigh accurately about 40 g of the well-mixed sample and transfer
to 100 mL beaker.
2. Add 50 mL of hot water at 80 to 90 °C. Mix and transfer to a 250
mL measuring flask washing it with successive quantities of distilled
water at 60 ℃, until the volume is 120 to 150 mL.
3. Mix and cool to room temperature (25 ± 3 °C) and add 5 mL of the
dilute ammonia solution. Mix and allow to stand for 15 min. Add 5
mL of dilute acetic acid to neutralize the added ammonia. This can
be confirmed by placing a drop of solution on a blue litmus paper.
The litmus paper should turn red. Mix and add 12.5 mL of zinc
acetate solution followed by 12.5 mL of potassium ferrocyanide
solution; mix again.
4. Make up to 250 mL mark, allow it to settle and filter. Mark this
solution as B-I.
5. Pipette 50 mL of solution B-I into a 100 mL volumetric flask, add 5
mL of concentrated hydrochloric acid and heat in a water bath at 65-
68 ℃ for 5 min rotating the flask for the first 3 min.
6. Cool the solution and neutralize with sodium hydroxide solution.
Mark this solution as A- I, and make up the volume to 100 mL.
7. Dilute the solutions B-I (usually 50 mL of B-I solution is required
to be diluted to 100 mL to obtain titration reading of around 14 mL
when titrated against Fehling solution) and A-I (usually 15 mL of A-
I solution is required to be diluted to 100 mL to obtain titration
reading of around 18 mL when titrated against Fehling solution) so
that the volume of solution required to react with 10 mL Fehling's
solution is between 15- and 50-mL. Mark them B-II and A-II,
respectively.
Incremental Method of Titration
1. Pour the prepared solution into a 50 mL burette (see Note 6). Pipette
10 mL of mixed Fehling's solution into a 250 mL conical flask and
run in from the burette 15 mL of the solution.
2. Without further dilution, heat the contents of the flask over a wire
gauze, and boil (after the liquid has been boiling for about 15 sec, it
will be possible to judge if the copper is almost fully reduced by the
bright red colour imparted to the boiling liquid by the suspended
cuprous oxide. When it is judged that nearly all the copper is reduced,
add 1 mL of methylene blue indicator solution (see Note 2).
3. Continue boiling the contents of the flask for 1-2 min from the
commencement of bubbling (boiling), and then add the prepared

140 | M o M – D a i r y a n d D a i r y P r o d u c t s
 solution in small quantities (1 mL or less at a time), allowing the
liquid to boil for about 10 sec between successive additions, till the
blue colour of the indicator just disappears (see Note 5).
4. In case there appears to be still much unreduced copper, after the
mixture of Fehling's solution with 15 mL of the prepared solution has
been boiling for a 15 sec, add the prepared solution from the burette
in larger increments (more than 1 mL at a time according to
judgement), and allow the mixture to boil for a quarter of a min after
each addition.
5. Repeat the addition of the prepared solution at intervals of 15 sec until
it is considered safe to add a large increment of the prepared solution.
At this stage, continue the boiling for an additional 1-2 min, add 1 mL
of methylene blue indicator solution and complete the titration by
adding the prepared solution in small quantities (less than 1 mL at a
time) (see also Note 3).
Note 2: It is advisable not to add the indicator until the end point has been
reached, because the indicator retains its full colour until the end point is
almost reached and thus gives no warning to the operator to go slowly.
Note 3: When the operator has had a fair amount of experience with the
method, a sufficiently accurate result may often be obtained by a single
estimation by the incremental method of titration, but for the utmost degree
of accuracy of which the method is capable second titration should be
carried out by the standard method of titration.
Standard Method of Titration
1. Pipette 10 mL of Fehling solution into a 250 mL conical flask and run
in from the burette almost the whole of the prepared solution of B-II
required to effect reduction of all the copper so that, if possible, not
more than 1 mL shall be required later to complete the titration.
2. Gently boil the contents of the flasks for 2 min, at the end of 2 min of
boiling add, without interrupting boiling, 1 mL of methylene blue
indicator solution.
3. While the contents of the flask continue to boil, begin to add the
prepared solution (1 or 2 drops at a time), from the burette till the blue
colour of the indicator just disappears (see Note 4).
4. The titration should be completed within 1 min, so that the contents
of the flask boil altogether for 3 min without interruption.
Note 4: The indicator is so sensitive that it is possible to determine the
end point within one drop of the prepared solution in many cases. The
complete decolourization of the methylene blue is usually indicated by
the whole reaction liquid in which the cuprous oxide is continuously
generated up becoming bright red or orange in colour. In case of doubt,
the flame may be removed from the wire gauze for 1 or 2 sec and the

141 | M o M – D a i r y a n d D a i r y P r o d u c t s
 flask held against a sheet of white paper. (A holder of paper, suitably
affixed round the neck of the flask, is very convenient for this purpose
as it can be left round the neck of the flask without risk of over-
balancing it). The top edge of the liquid would appear bluish if the
indicator is not completely decolourized. It is inadvisable to interrupt
the boiling as the indicator undergoes back oxidation rather rapidly
when air is allowed free access into the flask, but there is no danger of
this as long as a continuous stream of steam is issuing from the mouth
of the flask.
Note 5: It should be observed that with both incremental and standard
methods of titration, the flask containing the reaction mixture is left on
the wire gauze over the flame throughout the titration, may be removed
for a few sec to ascertain if the end point is reached.
Note 6: In adding sugar solution to the reaction mixture the burette may
be held in hand over the flask. The burette may be fitted with a small
outlet tube bent twice at right angles, so that the body of the burette can
be kept out of the steam while adding sugar solution. Burette with glass
taps are unsuitable for this work, as the taps become heated by the steam
and are liable to jam.

Method 2. Polarimetric Method:

Preparation of Sample

A. Samples of recently manufactured product in which no appreciable

separation of components may be expected: Open the container, transfer
all material adhering to the lid into the container and thoroughly mix by an
up and down movement of a spoon so that the top layers and the contents
of the lower corners are intermixed. Transfer the contents of can to a jar
with well-fitting lid.
B. Samples of older products and samples in which separation of
components may be expected: Heat in a water bath at about 40 ℃ until
the sample has nearly reached this temperature, open the container and
proceed as described above. When the product is in a can, transfer the
contents to a jar, scrap out all material adhering to the walls and continue
the mixing until the whole mass is homogeneous. Close the jar with a well-
fitting lid.

Control Determination
Run a control determination as described under "Determination" in
duplicate in a mixture of 100 g of milk (or 110 g of skim milk) and of pure
sucrose (a) 18 g for 40 g of condensed milk (thus containing 45% sucrose).

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Calculate the sugar contents by means of the formula given at the end of
this procedure using formula (1) for m, F and P respectively, which are the
quantity of milk weighed and the fat and protein content of this milk, and
in formula (2) for m, the value of 40. The mean of the values found shall
not differ by more than 0.1 percent from the actual 45 percent.

Procedure:
1. Weigh approximately 40 g of the well mixed sample to an accuracy
of 10 mg, into glass beaker.
2. Add 50 mL of hot distilled water (80-90 ℃) and mix well.
3. Transfer the mixture quantitatively to 200 mL volumetric flask,
rinsing the beaker with successive quantities of distilled water at 60
℃, until the total volume is between 120-150 mL.
4. Mix and cool, add 5 mL of ammonia solution, mix again and then
allow to stand for 15 min.
5. Neutralize the ammonia by adding equivalent quantity of acetic acid,
having determined beforehand the exact number of millilitres by
titration of the ammonia solution with bromothymol blue as indicator.
6. Mix and add with gentle mixing, by rotating the tilted flask, 12.5 mL
of zinc acetate solution. In the same manner add 12.5 mL of potassium
ferrocyanide solution.
7. Bring the contents of the flask to 20 ℃ and add water up to 200 mL
mark.
Note: Up to this stage, all additions of water or reagents is made so as to
avoid the formation of air bubbles, and with the same object in view, all
mixing should be done by rotation of the flask rather than by shaking. If air
bubbles are found to be present before diluting to 200 mL, their removal
can be done by temporarily connecting the flask to a vacuum pump and
rotating the flask; close the flask with a dry stopper and mix thoroughly by
swirling. Let it stand for a few min and filter through a dry filter paper,
rejecting first 25 mL of filtrate.

Direct Polarisation: Determine optical rotation of filtrate at 20 ± 2 ℃.

Inversion:
1. Pipette 40 mL of the filtrate obtained above into the 50 mL volumetric
flask and add 6.0 mL of diluted hydrochloric acid.
2. Place the flask in the water bath at 60 ℃ for 15 min, the entire bulb
of the flask being immersed.
3. Mix by the rotary movement during the first 5 min; by this time the
contents of the flask should have attained the temperature of the bath.
4. Cool to 20 ℃ and make up to the 50 mL mark with distilled water at

143 | M o M – D a i r y a n d D a i r y P r o d u c t s
 20 ℃. Mix, allow to stand for 1 h at this temperature.

Invert Polarization: Determine the rotation of the invert solution at 20 ± 2

℃. (When temperature of the liquid in the polarization tube differs by more
than 0.2 ℃ during the measurement, the temperature correction referred to
under Note 2 shall be applied).
Original Reducing sugars % (ORS) in condensed milk =
Calculation with units of
W1 100 1
expression  250    F1
V1 W2 1000
W1  2F 
Total Reducing sugars % (TRS) in Condensed Milk =  25  2 
W2  V2 
Sucrose % = TRS -ORS
Note: If standardization of Fehling solution is carried out by Lactose
Solution (Reagent L) instead of Standard solution of invert sugar (Solution
B) prepared under acidic conditions at room temperature (25±3oC), multiply
the Sucrose % obtained in above formula by a factor 0.95 for calculating
sucrose content.
Where,
W1= weight in mg of sucrose corresponding to 10 mL of Fehling’s solution
W2 = weight in g of the material taken for the determination
F2 = dilution factor for solution A - II from A - I
V2 = volume in mL of solution A - II corresponding to 10 mL of Fehling’s
solution
F1 = dilution factor for the solution B - II from B - I
V1 = volume in mL of solution B - II corresponding to 10 mL of Fehling’s
solution
Derivation of above formula
1. Standardization of Fehling Solution using Standard Reducing Sugar
Let 1 mL of Standard solution of invert sugar (Solution B) contains = M
mg of Total Invert Sugar

2. Calculation of Lactose (original reducing sugar in SCM sample)

estimation
Let V1 = mL of diluted sample filtrate (B1) required for the titration of 10
mL of Fehling’s solution
Let V = mL of the Standard solution of invert sugar (Reagent C) required
for the titration of 10 mL of Fehling’s solution
Now
10 mL of Fehling’s solution = V mL of standard reducing sugar solution

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 = V1 mL of diluted sample filtrate
Thus
V1 mL of diluted sample filtrate ≡ V mL of standard reducing sugar solution
≡ V x M mg of standard reducing sugar
or ≡ W1 mg of standard reducing sugar
W1
Therefore, 100 mL of sample filtrate contains = 100 mg of original
V1
reducing sugar
Since 100 mL of this sample filtrate was obtained from 50 mL of B1
solution
W1 100
Therefore, 250 mL of B1 sample filtrate will contain =   250
V1 50
mg of original reducing sugar
Since 250 mL of sample filtrate was obtained from 40 g (say W2 g) of SCM
Therefore, Lactose (g) present in 100 gm of SCM (say C1) =
W1 100 100 1
  250   g
V1 50 W2 1000

2. Calculation for Total reducing sugar (Lactose + hydrolyzed Sucrose)

in given sample
Let V2 = mL of diluted sample filtrate (A1) required for the titration of 10
mL of Fehling’s solution
Let V = mL of the Standard solution of invert sugar (Solution B) required
for the titration of 10 mL of Fehling’s solution
Now
10 mL of Fehling’s solution = V mL of standard reducing sugar solution
= V2 mL of diluted sample filtrate
Thus
V2 mL of diluted sample filtrate ≡ V mL of standard reducing sugar solution
≡ V x M mg of standard reducing sugar
or ≡ W1 mg of standard reducing sugar
W1
Therefore, 100 mL of sample filtrate (A-II) contains = 100 mg of
V2
total reducing sugar
Since 100 mL of this sample filtrate was obtained from 15 mL of A1
solution
W1 100
Therefore, 100 mL of A-I sample filtrate will contain =  mg
V2 15
of total reducing sugar

145 | M o M – D a i r y a n d D a i r y P r o d u c t s
Since 100 mL of this sample filtrate was obtained from 50 mL of B1
solution
W1 100 100
Therefore, 100 mL of B-I sample filtrate will contain =  
V2 15 50
mg of total reducing sugar
Therefore, 250 mL of B1 sample filtrate will contain =
W1 100 100
   250 mg of total reducing sugar
V2 15 50
Since 250 mL of sample filtrate was obtained from 40 g (say W2 g) of SCM
Therefore, Total reducing sugars (g) present in 100 g of SCM (say C2) =
 W1 100 100 100 1 
    250   
 V2 15 50 W2 1000 

This calculation is on the assumption that 15 of A-1 solution was diluted to

100 mL
Total sucrose (%) in the SCM = C2 –C1 g
Polarimetric Method: Calculate the sucrose content S, percent by mass,
by means of the following formula.
𝑚
𝑈= (1.08 𝐹 + 1.55 𝑃) − −𝐹𝑜𝑟𝑚𝑢𝑙𝑎 1
100

𝐷 − 5⁄4 𝐼 𝑉 − 𝑈 𝑉
𝑆= 𝑥 𝑥 − −𝐹𝑜𝑟𝑚𝑢𝑙𝑎 2
𝑄 𝑉 𝑒𝑥𝑚
Where,
U = Correction in mL for the volume of the precipitate formed during the
clarification
m = Mass in g of the weighed sample
F = Percentage of fat in the sample
P = Percentage of protein (N x 6.38) in the sample
D = Direct polarimeter reading (polarization before inversion)
I = Polarimeter reading after inversion
Q = Inversion division factor, the values of which are given in Note1
V = Volume in mL to which the sample is diluted before filtration; and
e = Length in mm of the polarimeter tube.
Remarks:
1. When exactly 40 g of the condensed milk are weighed and a

146 | M o M – D a i r y a n d D a i r y P r o d u c t s
 polarimeter with sodium light, angular degrees and a 2 dm
polarimeter tube at 20.0 ± 1 °C is used the sucrose content of normal
condensed milk (C = 9) can be calculated from the following formula;
S = (D = 5/4 I) 2.833 - 0.006 I2F-0.08 78P).
2. If the invert polarisation is measured at a temperature other than 20
°C, the figures should be multiplied by: 1 + 0.0037 (T-20) Values of
the inversion division factor (Q) -The following formula give accurate
values for Q, for various sources of light with corrections, where
necessary for concentration and temperature:
3. Sodium light and polarimeter with angular degrees: Q=0.8825 +
0.0006 (C-9) - 0.0033 (T-20).
4. Mercury green light and polarimeter with saccharimeter with
international sugar scale degrees:
Q = 2.549 + 0.0017 (C-9) - 0.0095 (T-20)
In the above formula:
C = Percentage of total sugars in the invert solution as polarised; and
T = Temperature of the invert solution in the polarimetric reading.
Note 1: The percentage of total sugar C in the inverted solution may be
calculated from the direct reading and the change on inversion in the usual
manner, using the usual values for the specific rotations of sucrose, lactose,
and invert sugar. The correction 0.0006 (C-9), etc., is only accurate when
C is approximately 9. For normal condensed milk, this correction can be
neglected C being close to 9.
Note 2: Variation in temperature from 20 ℃ makes little difference in direct
reading, but variation of over 0.2 ℃ in the invert reading necessitates a
correction. The correction 0.0037 (T-20) etc. is accurate only between 18
and 22 ℃.
Repeatability (Polarimetric method): The difference between the results
of two determinations carried out simultaneously or in rapid succession by
the same analyst in the same laboratory shall not exceed 0.3 g of sucrose
per 100 g of condensed milk.
Reference 1. IS 4079: 1967 (Reaffirmed year 2016). Specification for canned
rasogolla. Bureau of Indian Standards, New Delhi.

2. IS 11764: 2005 (Reaffirmed year 2019). Method for determination of

sucrose content by Polarimetric method in condensed milk. Bureau of
Indian Standards, New Delhi.

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Approved by Scientific Panel on Methods of Sampling and Analysis

148 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Milk Solids in Condensed/Evaporated Milk

Method No. FSSAI 01.053:2022 Revision No. & Date 0.0

Scope This method is for the determination of milk solids in
condensed/evaporated milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to
protect your eyes and skin. Concentrated hydrochloric acid is toxic if
inhaled, so avoid breathing it in and always handle it while under a
fume hood. It reacts with strong oxidizing agents to form toxic
chlorine gas.
Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling
period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle In this method, first total solids are determined in the sample. In case of
evaporate milk, total solids calculated in the sample are milk solids (%). In
case of condensed milk, % sucrose is also estimated separately and milk
solids are calculated by subtracting % sucrose from % total solids.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Flat-bottomed dishes: Made of aluminum alloy, nickel, stainless
steel, porcelain, 50 to 75 mm diameter, 20 to 25 mm depth, and
provided with easily removable but closely fitting lids.
3. Boiling water-bath.
4. Water-bath: Maintained at 40 – 60 ℃.
5. Hot air oven (forced air type), capable of being controlled at 102 ± 2

149 | M o M – D a i r y a n d D a i r y P r o d u c t s
 ℃.
6. Desiccator with efficient desiccant (for example freshly dried silica
gel with a hygroscopic indicator).
7. Short glass stirring rods: Flattened at one end and fitting in to the
dish.
Materials and reagents 1. Concentrated hydrochloric acid (Approximately 11.6 N).
2. Quartz sand or sea sand.
Preparation of reagents 1. Quartz sand or sea sand, which passes through 500 microns IS sieve
and is retained on 180 microns IS sieve [IS: 460 (Part I) – 1978], and
which passes the suitability test size as follows:
a) Place approximately 20 g of sand in a dish containing a stirring
rod. Heat the open dish and sand, stirring rod and lid in a hot air
oven controlled at 102 ± 2 ℃, for at least 2 h or preferably
overnight. Fit the lid, allow the dish to cool in a desiccator to
room temperature (25 ± 3 oC) and weigh to the nearest 0.1 mg.
b) Moisten the sand with approximately 5 mL of water, mix the
sand and water using the stirring rod and heat the dish and sand,
stirring rod and lid in a hot air oven, controlled at 102 ± 2 ℃ for
at least 4 h. Fit the lid, allow the dish to cool in the desiccator to
room temperature (25 ± 3 oC) and weigh again to the nearest 0.1
mg. The difference between the two weighing shall not exceed
0.5 mg.

If the requirements given above is not met, the sand may be made suitable
for the determination as follows:
Leave the sand immersed in 25% (m/m) hydrochloric acid solution for 3
days. Stir occasionally, decant off the supernatant liquid as far as possible,
then wash the sand with water until the acid reaction has disappeared.
Calcine (heat) the sand at 550 ℃ for at least 4 h using a muffle furnace.
Repeat the test for the suitability of the sand as described above (the
difference in weights represents the weight of fat extracted from the milk.
Correct weight of extracted fat by blank determination on reagents used).
Sample preparation Refer method FSSAI 01.050:2022 for sample preparation.
Method of analysis 1. Heat a dish containing about 20 g of the sand with its lid alongside and
a stirring rod on top of the lid, in a hot air oven at 102° ± 2 ℃ for about
1 h.

150 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Place the lid (with the stirring rod on the top) on the dish, immediately
transfer the dish to the desiccator and allow to cool for at least 45 min,
and weigh the dish with lid and rod to the nearest 0.1 mg.
3. Tilt the sand to one side of the prepared dish, and place 2.0 g of the
prepared test sample of condensed milk on the clear space, replace the
lid with the stirring rod on top and weigh the dish to the nearest 0.1 mg.
4. Add 5 mL of distilled water to the test portion in the dish and mix with
the stirring rod. Thoroughly mix together the diluted test portion and
the sand, and spread the mixture evenly over the bottom of the dish.
Leave the stirring end of the rod in the mixture with the other end
resting on the rim of the dish.
5. Heat the dish on a boiling water-bath with the bottom of the dish
exposed to steam, for approximately 30 min while stirring the mixture
frequently in the early stages of drying so that the mixture is well
aerated and becomes crumbly.
6. Lay the stirring rod flat inside the dish; dry the bottom of the dish and
heat the dish with its lid alongside, in a hot air over maintained at 102
± 2 ℃ for 4 h.
7. Place the lid on the dish, allow the dish to cool in the desiccator and
weigh to the nearest 0.1 mg. Repeat the operations described above
(heating the dish for 1 h) until the difference in mass between two
successive weighing does not exceed 0.5 mg.
8. Record the lowest mass.
9. In case of sweetened condesned milk, determine the sucrose (Refer
method FSSA.01.052:2022).
Calculation with units of For evaporated milk
expression 𝑀2 − 𝑀
𝑇𝑜𝑡𝑎𝑙 𝑠𝑜𝑙𝑖𝑑𝑠 % 𝑏𝑦 𝑚𝑎𝑠𝑠 = × 100
𝑀1 − 𝑀
Where
M = mass in g, of the dish, lid, sand and stirring rod;
M1 = mass in g, of the dish, lid, stirring rod, sand and test portion; and
M2 = in g, of the dish, lid, stirring rod and dried test portion.
Note: BIS/ISO method does not recommend use of sand. However, use
of sand helps in evenly spreading the samples.
For sweetened condensed milk
Milk solids (%) = Total solids (%) – Sucrose (%)
Reference IS 11622: 1986. Method for determination of total solids content in
condensed milk. Bureau of Indian Standards, New Delhi.

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Approved by Scientific Panel on Methods of Sampling and Analysis

152 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Milk Protein in Milk Solids-not-Fat of
Condensed/Evaporated Milk

Method No. FSSAI 01.054:2022 Revision No. & Date 0.0

Scope This method is for the determination of milk protein in milk solids not fat of
condensed/evaporated milk.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It reacts
violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
3. Ethanol: Extremely flammable. Use effective fume removal device when
heating or evaporating. Keep away from heat, sparks and open flame;
avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts
with strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to release
of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle Refer the method for determination of protein by Kjeldahl method in milk
Apparatus/Instruments and milk products (FSSAI 01.026/027:2022)
Materials and reagents
Preparation of Reagents
Sample preparation Refer method FSSAI 01.050:2022 for sample preparation.

153 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis Determination of Total Nitrogen in Sample
1. Weigh quickly about 1-2 g of the prepared condensed/evaporated milk
sample and transfer to a 500 or 800 mL Kjeldahl flask/digestion tube
taking care to see that no portion of the sample clings to the neck of the
flask/tube.
2. Refer the method for determination of protein in milk and milk products
(FSSAI 01.026/027:2022).

Determination of fat
Refer the method for determination of fat in condensed/evaporated milk
(FSSAI 01.051:2022).

Determination of milk solids

Refer the method for determination of milk solids in condensed/evaporated
milk (FSSAI 01.053:2022).

Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.026/027:2022).

Milk SNF (mSNF) Content (%) = Milk Solids (%) - Fat (%)

Protein (%)𝑥 100

Protein on mSNF basis (%) =
𝑚𝑆𝑁𝐹

Reference AOAC 991.23. 21st Edn. (2019). Official Method 991.23 Protein Nitrogen
content of Milk. AOAC International, USA.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 H. DRIED MILK PRODUCTS

Preparation of Sample for Dried Milk Products

Method No. FSSAI 01.055:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample dried milk products such as
Whole, Skimmed, Partially Skimmed Milk Powder, Cream Powder, Infant
Milk Food, Infant Formula, Milk Cereal Weaning Food, Processed Cereal
Based Weaning Food, Milk Protein Concentrates, Dairy Permeates,
Colostrum Powder, and Dairy Whitener.
Sample preparation 1. Make the sample homogeneous either by mixing or shaking or
alternately rolling and inverting the container.
2. Avoid excessive temperature and humidity when opening sample
container to prevent absorption of moisture. These products are
hygroscopic in nature. Care must be taken while handling these
samples to avoid moisture absorption, which can give erroneous
results.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Moisture in Dried Milk Products

Method No. FSSAI 01.056:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of moisture in dried milk
products such as whole, skimmed, partially skimmed milk powder, cream
powder, infant milk food, infant formula, milk cereal weaningf, processed
cereal based weaning food, milk protein concentrates, dairy permeates,
colostrum powder, dairy whitener, dried ice-cream mix.
This method gives free moisture content of the powder, i.e. it does not
include water of crystallization of alpha lactose monohydrate. The total
moisture, including water of crystallization can be determined by Karl
Fischer titration or toluene distillation. However, sample of commerce are
expected to contain less than 5% of free moisture, therefore oven method
at 100 ± 2 ℃ is adequate for its determination
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.

Principle The sample is dried to constant weight at 102 ± 2 ℃ and the loss in weight
reported as moisture.
Apparatus/Instruments 1. Flat-bottom moisture dishes with covers made of stainless steel, nickel
or aluminium having approximately 50 mm diameter and 25 mm depth.
The dishes shall have lids which fit well and can readily be removed.
2. Hot air oven (forced air type), capable of being controlled at 102 ± 2 ℃.
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Analytical balance (Readability 0.0001 g).
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation. Weigh sample
in / under dehumidifier or humidity shall not be more than 50%.

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Method of analysis 1. Uncover a dish and place the dish and its lid in a hot air oven at 102 ±
2 ℃ for 1 h. Place the lid on the dish, transfer the covered dish from the
hot air oven to the desiccator; allow it to cool to 25 ± 3 C and weigh.
2. Place approximately 1 g of the the test powder in the dish, cover the
dish with the lid and weigh the covered dish accurately and quickly.
3. Uncover the dish and place it along with its lid in the hot air oven
maintained at 102  2 ℃ for 2 h.
4. Replace the lid, transfer the covered dish to the desiccator, allow it to
cool to 25 ± 3 C (for approximately 30-45 min) and weigh it accurately
and quickly.
5. Heat the uncovered dish and lid in the hot air oven at 102 ± 2 ℃ for
further 1 h; replace the lid, allow the covered dish to cool to room
temperature (25 ± 3 °C) in the desiccator and weigh it.
6. Repeat the process of drying, cooling and weighing until the successive
weighing do not differ by more than 0.5 mg. It is usually found that
drying is complete after the first 2 h.

Calculation with units of

M1 - M2
expression Moisture % by mass = × 100
M1 -M
Where,
M is mass in g, of the empty dish along with lid;
M1 is initial mass in g of the dish with lid and test portion;
M2 is the mass in g of the dish with lid and dried test portion

The maximum deviation between duplicate determinations should not

exceed 0.06% by mass of moisture.
Reference 1. IS 16072: 2012 (Reaffirmed year 2018), Determination of moisture
content in milk powder and similar products, Bureau of Indian
Standards: India.
2. ISO 5537/IDF 026:2004. Dried milk - Determination of moisture
content (Reference method). International Organization for
Standardization, Geneva.
3. F.A.O Manuals of Food quality Control 14/ 8 page 30.
Approved by Scientific Panel on Methods of Sampling and Analysis

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158 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Fat in Dried Milk Products by Reference Method

Method No. FSSAI 01.057:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in dried milk products such as
whole, skimmed, partially skimmed milk powder, cream powder, infant
milk food, infant formula, milk cereal weaning food, processed cereal
based weaning food, milk protein concentrates, dairy permeates, colostrum
powder, and dairy whitener.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks
and open flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable
and can react explosively when in contact with Cl2, O3, LiAlH4 or
strong oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle Sample is treated with ammonia and ethyl alcohol; the former to dissolve
the protein and the latter to help precipitate the proteins. Fat is extracted
with diethyl ether and petroleum ether. Mixed ethers are evaporated and
the residue weighed. This method is considered suitable for reference
purposes. Strict adherence to details is essential in order to obtain reliable
results.
Apparatus/Instruments Refer requirement stated for analysis of fat content in milk (FSSAI
Materials and reagents 01.024:2022).
Preparation of reagents
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis Rose-Gottlieb Method
1. Weigh 1 g of the sample in a Mojonnier flask or suitable extraction

159 | M o M – D a i r y a n d D a i r y P r o d u c t s
 flask.
2. Add 10 mL of warm (65 ± 5 ℃) distilled water.
3. Shake to dissolve the powder.
4. Add 2 mL of ammonia solution, shake well and proceed as per the
extraction fat by Rose-Gottlieb method as prescribed for milk from
the step indicating addition of 10 mL ethyl alcohol (Method No
FSSAI 01.024:2022).

Calculation with units of

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
expression 𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference IS 11721: 2013 (Reaffirmed year 2018). Method of sampling and test for
dried milk sample. Bureau of Indian Standards, New Delhi.

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 Determination of Fat in Whole Milk Powder by Gerber Method

Method No. FSSAI 01.058:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of fat in whole milk
powder only.
Caution Follow all safety procedures while handling and disposing solutions.
Wear laboratory apron, shoes, safety goggles and mask while working
with chemicals. Perform work in fume hood while working with solvents.
Refer to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Iso-amyl alcohol: It is a highly flammable liquid. Keep away from
heat/sparks/open flames/hot surfaces. Perform all operations under a
fume hood.
Principle Sample is mixed with sulphuric acid and isoamyl alcohol in a special
Gerber tube, permitting dissolution of the protein and release of fat. The
tubes are centrifuged and the fat rising into the calibrated part of the tube
is measured as a percentage of the fat content of the milk powder sample.
Apparatus/Instruments Refer apparatus requirement stated for milk (FSSAI 01.024:2022), along
with following additional requirement.
1. Analytical Balance (Readability 0.001 g).
2. Milk butyrometer of 10% scale (ISI marked).

Materials and reagents 1. Concentrated sulphuric acid (Approximately 36.8 N).

2. Isoamyl alcohol (furfural free). It should have density between
0.807 to 0.809 g/mL at 27 ℃.
Preparation of Reagents Preparation of Gerber sulphuric acid: Take required volume of water
in a pyrex flask (generally 100 mL of water is required for 900 mL of
concentrated sulphuric acid) kept in a basin of ice-cold water. Carefully
add the concentrated sulphuric acid in small quantities at a time keeping
the container sufficiently cold and mix gently. After cooling the flaks,
check the specific gravity of Gerber acid with hydrometer and if
necessary, adjust the Gerber acid to the correct specific gravity with

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 addition of water or acid, taking same precautions as before till specific
gravity is in the range of 1.807 to 1.812 g/mL at 27 ℃ (or 1.815 to 1.820
g/mL at 20 ℃). Store the prepared acid in a glass stoppered bottle to avoid
absorption of water.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis 1. Mix the sample thoroughly, and weigh 1.69 ± 0.01 g of milk powder
into the counterpoised scoop.
2. Add 10 mL of Gerber sulphuric acid into a butyrometer tube,
preferably by use of an automatic dispenser, without wetting the neck
of the tube.
3. Add gently, from the wash bottle sufficient cold water to form a layer
about 6 mm deep on top of the acid, allowing the water to flow down
the side of the bulb.
4. Insert the narrow end of the stemless funnel into the neck of the
butyrometer. Transfer the contents of the scoop to the funnel,
removing the last particles with a camel hair brush.
5. Tap the funnel gently until most of the powder is in the butyrometer.
Transfer the powder remaining in the funnel to the butyrometer with
the aid of the glass rod and the camel hair brush.
6. Remove the funnel, add 1 mL of isoamyl alcohol, preferably by use
of an automatic dispenser.
7. Add hot water (70 ℃) from the wash bottle until the butyrometer is
filled to about 5 mm below the shoulder, allowing all air entrained in
the powder to escape.
8. Close the neck of the butyrometer firmly with the stopper without
disturbing the contents.
9. When a double ended stopper is used, screw it in until the widest part
is, at least, level with the top of the neck.
10. When a lock stopper is used, insert it until the rim is in contact with
the neck of the butyrometer.
11. Keep in a water bath at 65 ± 2 ℃ for at least 3 min and not less than
10 min, taking care to have casein particles, if any, to dissolve fully,
and centrifuge for 5 min at 1100 rpm (approximately 300 g).
12. The tubes should be put in centrifuge, so as to conform to radial
symmetry, and as evenly spaced as possible, in order to protect

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 bearings of the centrifuge.
13. Allow the centrifuge to come to rest. Remove the butyrometer tubes
and place in water bath at 65 ± 2 ℃ for at least 3 min and not less than
10 min.
14. If after centrifuging, there is not a sharp dividing line between the fat
and the acid, or if the acid layer is not clear, repeat the temperature
adjustment and centrifuging before taking the reading.
15. Read the scale reading of the butyrometer
Calculation with units of Note down the reading of the scale of butyrometer as follows
expression
Scale reading = B – A

where

A is the reading at the bottom of the fat column;

B is the reading at the top of the fat column.

Calculation the fat content in the sample as follows

Fat content (%) = Scale reading x 20/3

Reference IS 1224 (Part II): 1977 (Reaffirmed Year 2018). Determination of fat by
the Gerber method: Milk products. Bureau of Indian Standards, New
Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Titratable Acidity in Dried Milk Products

Method No. FSSAI 01.059:2022 Revision No. & Date 0.0

Scope This method is for the determination of titratable acidity in milk dried milk
products such as whole, skimmed, partially skimmed milk Powder etc.
Caution Follow all safety procedures while handling and disposing solutions.
Wear laboratory apron, shoes, safety goggles and mask while working
with chemicals. Perform work in fume hood while working with solvents.
Refer to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water
and not vice versa.
2. Ethanol: It is extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
Principle In this method, reconstituted milk is prepared by addition of water to a
test portion of dried milk, corresponding accurately to 5 g of solids-not-
fat. It is titrated with 0.1 N sodium hydroxide solution using
phenolphthalein as an indicator and cobalt (II) sulphate as reference
colour solution. The number of milliliters of 0.1 N sodium hydroxide used
in the titration is multiplied by the factor 2, in order to obtain the number
of milliliters in terms of 10 g of solids-not-fat. The amount of sodium
hydroxide solution required is a function of the amount of natural
buffering substances present in the product and of developed or added
acid or alkaline substances.
Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).
2. Burette, 50 mL graduated in 0.1 mL and with an accuracy of 0.05 mL.
3. Pipettes of 2 mL capacity.
4. Measuring cylinders of 50 mL capacity.
5. Conical flasks capacity 100- or 150-mL capacity with ground necks
and ground glass stoppers.
Materials and reagents 1. Sodium hydroxide.
2. Phenolphthalein.
3. Cobalt sulphate heptahydrate.
4. Ethanol.
Preparation of Reagents 1. Sodium hydroxide standard volumetric solution, (NaOH) = 0.1 ±
0.0002 N: Dissolve 0.4 g of sodium hydroxide in water and make up

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 to 100 mL. Standardize the prepared solution with 0.1N standard
oxalic acid solution using phenolphthalein as indicator.
2. Reference colour solution: Dissolve 3 g of cobalt (ll) sulphate
heptahydrate (CoSO4.7H2O) in water and make up to 100 mL.
3. Phenolphthalein solution: Dissolve 2 g of phenolphthalein in 75 mL
of 95% (v/v) ethanol and add 20 mL of water. Add the sodium
hydroxide solution until 1 drop gives a faint pink coloration, and make
up to 100 mL of water.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis 1. Determine the solids-not-fat content of the sample. For this, estimate
the moisture (Refer method FSSAI 01.056:2022) and fat content
(Refer method FSSAI 01.057:2022). The, cacluate the solids-not-fat
by following equation
Solids-not-fat (%) = 100 – fat % - moisture %
2. Weigh (500/a) ± 0.01 g of the test sample into each of two conical
flasks, “a” being the solids-not-fat content of the sample, expressed as
a percentage to two decimal places.
3. Prepare reconstituted milk by adding 50 mL of water at about 20 ℃
to the test portion and agitating vigorously. Allow to stand for about
20 min.
4. Add to one of the conical flasks 2 mL of the reference colour solution
to obtain a colour standard and mix by slight swirling.
5. If a series of determinations on similar products is to be carried out,
this colour standard may be used throughout. However, it should be
discarded after 2 h.
6. Add 2 mL of the phenolphthalein solution to the second conical flask,
and mix by slight swirling.
7. Titrate the contents of the second conical flask, while swirling, by
adding the sodium hydroxide solution from the burette until a faint
pink colour similar to that of the colour standard persists for about 5
sec.
8. The titration should be completed within 45 sec. Record the volume,
in millilitres, of sodium hydroxide solution used, to the nearest 0.05
mL.
Calculation with units of The titratable acidity = 2 x V
expression
Where,

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 V = volume, in millilitres, of the sodium hydroxide solution used for
titration
Reference IS 11766: 1986 (Reaffirmed Year 2018). Method for determination of
titratable acidity in milk powder and similar products (routine method).
Bureau of Indian Standards, New Delhi
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Titratable Acidity in Dairy Whitener

Method No. FSSAI 01.060:2022 Revision No. & Date 0.0

Scope This method is for the determination of titratable acidity in dairy whitener.

Caution Follow all safety procedures while handling and disposing solutions.
Wear laboratory apron, shoes, safety goggles and mask while working
with chemicals. Perform work in fume hood while working with solvents.
Refer to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water
and not vice versa.
2. Ethanol: It is extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
Principle In this method, the dried test portion of dairy whitener is reconstituted
with water and titrated with 0.1 Nsodium hydroxide solution using
phenolphthalein as an indicator and rosaniline acetate solution as
reference colour solution. The amount of sodium hydroxide solution
required is a function of the amount of natural buffering substances
present in the product and of developed or added acid or alkaline
substances.
Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).
2. Burette (50 mL), graduated in 0.1 mL and with an accuracy of 0.05
mL.
3. Pipettes of 2 mL capacity.
4. Measuring cylinders of 50 mL capacity.
5. Conical flasks capacity 100 or 150 mL capacity with ground necks
and ground glass stoppers.
Materials and reagents 1. Sodium hydroxide.
2. Phenolphthalein.
3. Rosaniline acetate.
Preparation of Reagents 1. 0.1 N standard sodium hydroxide solution: Dissolve 0.4 g of sodium
hydroxide in water and make upto 100 mL. Standardize the prepared
solution with 0.1N standard oxalic acid solution using
phenolphthalein as indicator.
2. Phenolphthalein Indicator solution
3. Rosaniline acetate solution (Stock solution) — Dissolve 0.12 g of
rosaniline acetate in approximately 50 mL of rectified spirit [see IS:

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 323-1959 Specification for Rectified Spirit (Revised)] containing 0.5
mL of glacial acetic acid. Make up to 100 mL with rectified spirit.
4. Rosaniline acetate solution (Bench Solution) — Dilute 1 mL of the
stock solution to 500 mL with a mixture of rectified spirit (see IS: 323-
1959) and distilled water in equal proportions by volume.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis 1. Weigh accurately about 1 g of the sample into each of the two
porcelain dishes.
2. Add 10 mL of boiling water to each dish and stir with the flat end of a
glass rod until a perfectly smooth liquid is obtained.
3. Cool to 25 ± 3 C. Use the contents of one dish as a blank by stirring
on 2 mL of bench solution of rosaniline acetate.
4. Add 1 mL of phenolphthalein indicator solution to the other dish
followed by standard sodium hydroxide solution drop by drop from
burette until by comparison the color matches the pink tint of the
blank.
5. Stir vigorously throughout. The time taken for the complete titration
shall not exceed 20 seconds. The titration shall be preferably made in
north light or under illumination from a day light lamp.
Calculation with units of 9 AN
Titratable acidity (as % lactic acid) by mass =
expression M
Where,
A = volume in mL of the standard sodium hydroxide required for titration
N = normality of the standard sodium hydroxide solution, and
M = mass in g of milk powder taken for the test
Reference IS 1165: 2002 (Reaffirmed Year 2013). Milk Powder-Specifications.
Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Added Sugar in Dairy Whitener

Method No. FSSAI 01.061:2022 Revision No. & Date 0.0

Scope This method is for the determination of added sugar in dairy whitener.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).

Principle The principle involves clarifying the sample with the help of zinc acetate
and potassium ferrocyanide, inverting a part of the sample using
hydrochloric acid and determining the reducing sugars content in both
inverted and non-inverted parts of the sample. The sugar content is
estimated by determining the volume of the unknown sugar solution
required to completely reduce a measured volume of Fehling's solution.
Invert sugar reduces the copper in Fehling's solution to red insoluble
cuprous oxide.
Material and Reagents 1. Sodium hydroxide.
2. Sucrose.
3. Methylene blue.
4. Copper sulphate (CuSO4.5H2O).
5. Concentrated sulphuric acid (Approximately 36.8 N).
6. Rochelle’s salt (potassium sodium tartrate –KNaC4H4O6.4H2O).
7. Zinc acetate.
8. Potassium ferrocyanide.

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 9. Concentrated hydrochloric acid (Approximately 11.6 N).
10. Concentrated ammonia (Specific gravity 0.91).
11. Acetic acid.
Preparation of reagents 1. Sodium hydroxide solution: Approximately 0.1 N prepared from
sodium hydroxide, analytical reagent grade.
2. Stock solution of invert sugar: Weigh accurately 9.5 g of pure sucrose
and transfer it to a one litre volumetric flask with 100 mL of water. Add
5 mL of concentrated hydrochloric acid. Allow this to stand for 3 days
at 20 to 25 °C and then make up to volume with water (This solution is
stable for several months).
3. Standard solution of invert sugar: Neutralize 40mL of the stock solution
of invert sugar (Reagent B) with sodium hydroxide solution using
litmus paper and dilute with water to 100 mL volume.
4. Methylene blue indicator solution: Dissolve 0.2 g of methylene blue in
water and dilute to 100 mL.
5. Fehling's solution (Soxhlet modification): Prepared by mixing
immediately before use, equal volumes of Solution A and Solution B,
prepared as described below.
a) Solution A: Dissolve 34.639 g of copper sulphate (CuSO4.5H2O) in
water add 0.5 mL of concentrated sulphuric acid and dilute to 500
mL in a volumetric flask. Filter the solution through prepared
asbestos.
b) Solution B: Dissolve 173 g of Rochelle salt and 50 g of sodium
hydroxide analytical reagent grade in water, dilute to 500 mL in a
volumetric flask and allow the solution to stand for two days, Filter
this solution through prepared asbestos.
6. Standardization of Fehling's solution: Pipette accurately 10 mL of each
Fehling's solutions (solution A and B) into a 250 mL Erlenmeyer flask.
Pour the standard solution of invert sugar in burette and run in from the
burette almost whole of the invert sugar solution required to reduce all
of the copper so that not more than 1 mL is required to complete the
titration. Heat the contents on wire gauze. Gently boil the contents for
2 min. Add about 1 mL of methylene blue indicator solution. While the
contents of the flask continue to boil, begin to add standard invert sugar
solution till the blue colour of the solution disappears. Titration should
be completed within 1 min so that contents of the flask boil altogether
for 3 min without interruption.
7. Zinc acetate solution: Dissolve 21.9 g of crystalline zinc acetate
(Zn(CH₃CO₂)₂·2H₂O ) in water and add 3 mL of glacial acetic acid.
Make up to 100 mL.

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 8. Potassium ferrocyanide solution: Dissolve 10.6 g of crystalline
potassium ferrocyanide and make up to 100 mL with water.
9. Dilute ammonia solution: 10 mL of concentrated ammonia solution
diluted to 100 mL with water.
10. Dilute acetic acid solution: Approximately equivalent to the dilute
ammonia solution in strength.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation
Method of analysis 1. Weigh accurately about 40 g of the well - mixed sample and transfer to
100 mL beaker. Add 50 mL of hot water at 80 to 90 °C. Mix and transfer
to a 250 mL measuring flask washing it with successive quantities of
distilled water at 60 °C, until the volume is 120 to 150 mL.
2. Mix and cool to room temperature (25 ± 3 oC) and add 5 mL of the
dilute ammonia solution. Mix and allow to stand for 15 min.
3. Add the exact equivalent of dilute acetic acid to neutralize the ammonia
added. Mix and add 12.5 mL of zinc acetate solution followed by 12.5
mL of potassium ferrocyanide solution.
4. Mix again. Make up to 250 mL mark. Allow to settle and filter. Mark
this solution B-I.
5. Pipette 50 mL of solution B-I into a 100 mL volumetric flask, add 5 mL
of concentrated hydrochloric acid and heat in a water bath at 65 – 68
°C for 5 min rotating the flask for the first 3 min.
6. Cool the solution and neutralize with sodium hydroxide solution. Mark
this solution A- I. Make up to 100 mL.
7. Dilute the solutions B-I (usually 50 mL of B-I solution is required to be
diluted to 100 mL to obtain titration reading of around 14 mL when
titrated against Fehling solution) and A-I (usually 15 mL of A-I solution
is required to be diluted to 100 mL to obtain titration reading of around
18 mL when titrated against Fehling solution) so that the volume of
solution required to react with 10 mL Fehling's solution is between 15
and 50 mL.
8. Mark them B-II and A-II, respectively.

Standard method of titration

1. Pour the solution (BII) in a 50 mL burette. Pipette 5 mL of Fehling
solution A and 5 mL of Fehling solution B into a 250 mL conical flask
and run into the solution from the burette to effect reduction of all the
copper (blue colour faints)
2. Boil the contents of flask for 2 min. At the end of 2 min of boiling add,
without interrupting boiling one mL of methylene blue indicator
solution. While the contents of the flask continue to boil, add the

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 prepared solution from the burette until the blue colour disappears. The
titration should be completed within 3 min without interruption.
3. Repeat the steps of standard method of titration using solution AII.
Calculation with units of 25∗𝑊1 2𝑓2 𝑓1
expression
Sucrose, percent by weight = [ − ]
𝑊2 𝑉2 𝑉1

Where,
W1= weight in mg of sucrose corresponding to 10 mL of Fehling’s solution
W2 = weight in g of the material taken for the determination
f2 = dilution factor for solution A - II from A - I
V2 = volume in mL of solution A - II corresponding to 10 mL of Fehling’s
solution
f1 = dilution factor for the solution B - II from B - I
V1 = volume in mL of solution B - II corresponding to 10 mL of Fehling’s
solution
Reference Appendix C of IS 4079: 1967 (Reaffirmed year 2016). Bureau of Indian
Standards, New Delhi.

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 Determination of Milk Protein (Milk Solids-Not-Fat Basis) in Dried
Milk Products
Method No. FSSAI 01.062:2022 Revision No. & Date 0.0
Scope This scope is for the determination of protein content in dried milk products
such as whole, skimmed, partially skimmed milk powder, cream powder and
dairy whitener.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
3. Ethanol: Extremely flammable. Use effective fume removal device when
heating or evaporating. Keep away from heat, sparks and open flame;
avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts
with strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to release
of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle Refer the method for determination of protein by Kjeldahl method in milk
Apparatus/Instruments (FSSAI.01.026/027:2022). To calculate the protein on solids-not-fat basis,
Materials and Reagents milk fat and moisture are calculated separately and finally the results are
Preparation of reagents presented on the basis of % milk protein in milk solids-not-fat.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.

173 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Weigh quickly about 0.5 g of the prepared dahi/yoghurt sample and
transfer to a 500 or 800 mL Kjeldahl flask/digestion tube taking care to
see that no portion of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk products.
Determination of fat
Refer method for determination of fat in dried powder (FSSAI
01.057/058:2022)
Determination of moisture
Refer method for determination of moisture in dried powder (FSSAI
01.056:2022)
Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.027:2022)
For milk powder
Total Solids (%) = 100- moisture (%)
Milk Solids not fat = Total Solids (%) – Fat (%)
For Dairy Whitener
Total Solids (%) = 100- moisture (%)
Milk Solids not fat (%) = Total Solids (%) – Sugar (%) - Fat (%)

Protein on milk SNF basis = % Protein X 100

% Milk SNF

Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content

(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.
3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of
protein in foods and feeds. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

174 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Milk Protein in Foods for Infant Nutrition and
Processed Cereal Based Complementary Foods
Method No. FSSAI 01.063:2022 Revision No. & Date 0.0
Scope This scope is for determination of milk protein in infant milk food, infant
formula, milk cereal weaning food, processed cereal based weaning food,
milk protein concentrates, dairy permeates and colostrum powder.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information
Safety of
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
3. Ethanol: Extremely flammable. Use effective fume removal device when
heating or evaporating. Keep away from heat, sparks and open flame;
avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts
with strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to
release of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle Refer the method for determination of protein by Kjeldahl method in milk
Apparatus/Instruments and milk products (FSSAI 01.027:2022)
Materials and Reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.

175 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Weigh quickly about 0.5 g of the prepared sample and transfer to a 500
or 800 mL Kjeldahl flask/digestion tube taking care to see that no portion
of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein by Kjeldahl in milk and
milk products (FSSAI 01.026/027:2022).
Calculation with units of Refer the method for determination of protein by Kjeldahl in milk and milk
expression products (FSSAI 01.026/027:2022).
Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content
(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.
3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of
protein in foods and feeds. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

176 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Total Ash in Dried Milk Products

Method No. FSSAI 01.064:2022 Revision No. & Date 0.0

Scope This method is for the determination of total ash on dry basis in dried milk
products such as whole, skimmed, partially skimmed milk powder, cream
powder, infant milk food, infant formula, milk cereal weaning food,
processed cereal based weaning food, milk protein concentrates, dairy
permeates, colostrum powder, and dairy whitener. This method is also for the
determination of total ash on fat free, moisture free and sugar free basis.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot crucibles.
2. Hot crucibles will heat air within the desiccator and a vacuum may form
on cooling. Remove desiccator’s cover gradually by sliding to one side
to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle Destruction of organic matter by incinerating the sample to a constant mass
at high temperature of (550 ± 20 °C) in Muffle furnace.
Apparatus/Instruments 1. Platinum or silica crucible, about 70 mm diameter and 25 to 50 mm deep.
2. Muffle furnace, capable of being controlled at 550 ± 20 ℃.
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Safety tongs having long arms.
5. Bunsen burner or electric hot plate.
6. Analytical balance (Readability 0.0001g)
7. Thermal protection gloves, capable to resist temperature up to 550 - 600 °C
8. Hot air oven (forced air type), capable of being controlled at 102 ± 2 °C
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis 1. Weigh accurately about 3 g of the dried milk sample in a crucible,
previously dried in a hot air oven and weighed.
2. Heat the crucible gently on a burner or hot plate at first and then strongly

177 | M o M – D a i r y a n d D a i r y P r o d u c t s
 in a muffle furnace at 550  20 °C till grey ash is obtained.
3. Cool the crucible in a desiccator and weigh it.
4. Heat the crucible again at 550  20 °C for 30 min.
5. Cool the crucible in a desiccator and weigh.
6. Repeat this process of heating for 30 min, cooling and weighing until the
difference between two successive weighing is less than 1 mg.
7. Record the lowest mass.
If ash still contains black particles, add 2-3 drops of preheated water at 60°C.
Break the ash and evaporate to dryness at 100-110°C. Re-Ash at 550 °C until
ash is white or slightly grey.

Determination of fat
Refer method for determination of fat in dried powder (FSSAI
01.057/058:2022).
Determination of moisture
Refer method for determination of moisture in dried powder (FSSAI
01.056:2022)
Determination of added sugar (In Dairy Whitener)
Refer method for determination of added sugar in dried powder (FSSAI
01.061:2022)
M2 − 𝑀
Calculation with units of Total ash % by mass = × 100
expression 𝑀1 − 𝑀
Where,
M2 = mass in g, of the crucible with ash
M = mass in g, of the empty crucible
M1 = mass in g, of the crucible with the material taken for the test and

Total ash (% on dry matter basis) = Total ash (%) × 100

(100−Moisture %)

Total ash (% on fat and moisture free basis)

Total ash (%)
= × 100
(100−Moisture(%)−fat(%))

Total ash (% on fat, sugar and moisture free basis)

Total ash (%)
= × 100
(100−Moisture(%)−fat(%)−sugar(%))

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Reference IS 1165: 2002 (Reaffirmed year 2018). Milk Powder – Specifications.
Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Acid Insoluble Ash in Dried Milk Products

Method No. FSSAI 01.065:2022 Revision No. & Date 0.0

Scope This method is for the determination of ash insoluble in hydrochloric acid
(for infant foods and processed cereal based weaning foods and Dairy
Whitener).
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts
with strong oxidizing agents to form toxic chlorine gas.
2. Silver nitrate: It is corrosive. It is not combustible, but it is a strong
oxidizer that enhances the combustion of other substances.

Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot crucibles.
2. Hot crucibles will heat air within the desiccator and a vacuum may form
on cooling. Remove desiccator’s cover gradually by sliding to one side
to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.

Principle The ‘Acid Insoluble Ash’ content is the proportion of the ash that is not
soluble in acid and is not subsequently volatilised upon the incineration of
this acid insoluble residue.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Platinum or silica crucible about 70 mm diameter and 25 to 50 mm
deep.
3. Muffle furnace, capable of being controlled at 550 ± 20 ℃.

180 | M o M – D a i r y a n d D a i r y P r o d u c t s
 4. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
5. Safety tongs having long arms.
6. Bunsen burner or electric hot plate.
7. Boling water bath (100 ± 2 oC)
8. Ashless Whatman filter paper Grade 42.
9. Watch-glass.
10. Hot air oven (forced air type), capable of being controlled at 100 ± 2 ℃.
Materials and reagents 1. Concentrated hydrochloric acid (Approximately 11.6 N).
2. Silver nitrate.
Preparation of reagents 1. Dilute hydrochloric acid (5 N): Dilute 43 mL of concentrated
hydrochloric acid to 100 mL with distilled water.
2. Silver nitrate solution: 0.1 N: Dissolve accurately 1.6987 g of silver
nitrate in 25 mL distilled water and makeup the volume to 100 mL with
distilled water.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis 1. To the ash contained in the crucible (obtained in the previous method),
add 25 mL of dilute hydrochloric acid (5 N), cover it with a watch-glass
and heat on water-bath for 10 min.
2. Allow to cool and filter the contents of the crucible through an ashless
Whatman filter paper Grade 42.
3. Wash the filter paper with water until the washings are free from the
acid [check with AgNO3 solution (0.1 N solution); till the washings do
not form white precipitate with AgNO3 solution] and return the washed
filter paper to the crucible.
4. Keep the filter paper in a hot air oven maintained at 100  2 ℃ for about
3 h.
5. Ignite the crucible in a muffle furnace at 550  20 ℃ for 1 h. Cool the
crucible in a desiccator to room temperature (for approximately 30-45
min) and weigh it.
6. Heat the crucible again at 550  20 ℃ for 30 min, cool in a desiccator
and weigh. Repeat this process of heating for 30 min, cooling and
weighing until the difference between two successive weighing is less
than 1 mg.

181 | M o M – D a i r y a n d D a i r y P r o d u c t s
 7. Record the lowest mass.

Calculation with units of 𝑀2 − 𝑀

Acid insoluble ash, % (m/m) = 𝑥 100
expression 𝑀1 − 𝑀

Where,
M is mass in g of the empty crucible;
M1 is mass in g of the crucible with the material taken for the test;
M2 is mass in g of the crucible with acid insoluble ash
Reference IS 1165 – 2002 (Reaffirmed year 2018). Milk Powder – Specifications.
Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

182 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Crude Fibre Content in Milk Cereal Based
Complementary Food and Processed Cereal Based Complementary
Foods
Method No. FSSAI 01.066:2022 Revision No. & Date 0.0
Scope This method is for the determination of crude fibre content in milk cereal
based complementary food and processed cereal based complementary
foods.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is
extremely corrosive and can cause serious burns when not handled
properly. It reacts violently with water with evolution of heat; can
react with organic materials explosively. Do not mix with
hydrochloric acid. Good general ventilation should be provided to
keep vapour and mist concentrations below the exposure limits.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water
and not vice versa.
3. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to
protect your eyes and skin. Concentrated hydrochloric acid is toxic if
inhaled, so avoid breathing it in and always handle it while under a
fume hood. It reacts with strong oxidizing agents to form toxic
chlorine gas.
5. Petroleum ether: Extremely flammable. Avoid breathing fumes,
perform all operations under a fume hood. Keep away from heat,
sparks and open flame.
Principle In this method, after any necessary grinding and defatting, the sample is
boiled with sulphuric acid solution of standard concentration, the insoluble
residue is then separated and washed. The obtained residue is then boiled
with sodium hydroxide solution of standard concentration; it is then
separated, washed and dried. The residue is then weighed and the loss in
mass on incineration is determined.
Apparatus/Instruments 1. Grinding device which is easy to clean, suited to the nature of the

183 | M o M – D a i r y a n d D a i r y P r o d u c t s
 product and allows grinding of the product without causing undue
heating or significant change in the moisture content.
2. Burette graduated in 0.1 mL and with an accuracy of 0.05 mL.
3. Sieve, of metal wire cloth, aperture size 1 mm, complying with the
requirements of IS0 3310/ 1.
4. Hot air oven (forced air type), capable of being controlled at 130 ± 2
℃.
5. Wide-mouthed vessel, provided with a condenser, for example a flask
having a minimum capacity of 600 mL fitted with a reflux condenser,
or a beaker without spout, of capacity 600 mL, covered by a round-
bottom flask of 500 mL capacity containing 456 mL of cold water or
automatic system.
6. Heating device, for example an electrically heated hotplate fitted with
a magnetic stirrer, capable of maintaining 200 mL of reagents at a
gentle boil.
7. Incineration dish of capacity 25 to 50 mL, resistant to attack under
the test conditions, or a filter crucible, suitable for separation and
incineration of the residue.
8. Muffle furnace, provided with air circulation and temperature
control, suitable for carrying out incineration at 550 ± 25 ℃.
9. Desiccator containing an efficient desiccant (for example freshly
dried silica gel with a hygroscopic indicator).
10. Separating device.
11. Analytical balance (Readability 0.0001 g).
Materials and reagents 1. Concentrated sulphuric acid (Approximately 36.8 N).
2. Sodium hydroxide.
3. Acetone or 95% ethanol, methanol or propan-2-ol.
4. Concentrated hydrochloric acid (Approximately 11.6 N).
5. n-hexane, petroleum ether, diethyl ether or any other solvent.
Preparation of Reagents 1. Sulphuric acid solution: Standard volumetric solution 0.255 ± 0.005
N. Take 12.68 mL of concentrated sulphuric acid and make up to the
volume to 1000 mL in a volumetric flask.
2. Sodium hydroxide solution: Standard volumetric solution, (0.313 ±
0.005 M). Weigh 12.5 g of sodium hydroxide. Dissolve in water and
make up to 1000 mL This solution shall be as free as possible from
carbonates.
3. Acetone or 95% (v/v) ethanol, or methanol, or propan-2-ol.
4. Extraction solvent: Technical grade n-hexane, or light petroleum
(having a boiling range between 40 and 60 ℃), or diethyl ether, or
another solvent or mixture of solvents more suitable for the extraction
of fatty substances from certain products to be analysed.
5. Hydrochloric acid: 0.5 mol/L solution (in the case of products rich in

184 | M o M – D a i r y a n d D a i r y P r o d u c t s
 carbonates). Carefully add 21.6 mL of concentrated hydrochloric acid
to water in a standard flask. Make up the volume to 500 mL with
distilled deionised water.
6. Filter aid.
7. Anti-foam agent (for example an aqueous emulsion containing 30
percent m/m of silicone), if necessary, known to have no effect on the
results.
8. Anti-bumping agent (glass beads), if necessary, resistant to attack
under the test conditions or known to have no effect on the results.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis: Products requiring grinding: Products that do not pass through the test
sieve without leaving a residue, shall be ground. If the results are to be
expressed relative to the product as received, determine beforehand the dry
matter content of the sample by an appropriate method. Grind the
laboratory sample in the grinding device so that the product passes through
the sieve without leaving a residue.
Extraction of fatty substances: If the fatty matter content is less than 1%,
extraction of fatty matter is not required. Extraction is is recommended if
the fat content is between 1 and 10%. If the fatty matter content is more
than 10%, prior extraction is essential. For products containing fatty
substances which cannot be removed directly, the extraction shall be
carried out after the acid treatment.
1. Weigh to the nearest 1 mg, about 3 g of the test sample and which is
presumed to contain more than 1% of crude fibre.
2. In the case of products having moisture content too high for them to
be mixed or ground as received, carry out a preliminary drying of the
product at an appropriate temperature.
3. In this case, weigh the product before the preliminary drying and
again just before preparation of the test sample.
Acid treatment:
1. Transfer the test portion, which may have had the fat and oils and
carbonates removed into the vessel.
2. Add the prescribed quantity of filter aid and the anti-foam agent and
the anti-bumping agent.
3. Measure 200 mL of the sulphuric acid solution, bring it to a
temperature of 95 to 100 ℃ and add it to the contents of the vessel.
4. Fit the condenser, bring it rapidly to the boil (in about 2 min) using
the heating device and continue boiling gently for 30 ± 1 min.
5. Swirl the vessel from time to time so that any particles adhering to
the interior walls are returned to the solution.
6. After the specified boiling period, add about 50 mL of cold water and
separate rapidly the insoluble residue using the separating device

185 | M o M – D a i r y a n d D a i r y P r o d u c t s
 chosen.
7. Wash the vessel with 50 mL portions of hot water (of temperature 95
to 100 ℃) and pour the washings over the insoluble residue remaining
in the separating device.
8. Repeat the washing of the insoluble residue until the filtrate is
substantially neutral to litmus paper.
9. The separation and washing of the insoluble residue shall be
completed in less than 30 min.
Alkaline treatment:
1. Return the washed insoluble residue to the vessel and add the anti-
foam agent and the anti-bumping agent.
2. Measure 200 mL of the sodium hydroxide solution, bring it to a
temperature of 95 to 100 ℃.
3. Fit the condenser and bring rapidly to boil (in about 2 min) using the
heating device, and continue to boil gently for 30 ± 1 min.
4. After the specified boiling period, add about 50 mL of cold water and
rapidly separate the insoluble residue using the separating device
chosen.
5. Wash the residue with 25 mL of the sulphuric acid solution measured
at room temperature (25 ± 3 oC) and then raised to a temperature of
95 to 100 ℃.
6. Wash with water as described in Dry the residue with one of the
reagents; wash with solvent to remove unsaponifiable fatty matter.
According to the filtration ‘technique chosen, collect the entire
residue in the incineration dish or in the filter crucible.
Drying:
1. Dry the incineration dish or the filter crucible with its contents in the
oven at 130 ± 2 °C.
2. Allow to cool to room temperature (25 ± 3 °C) in the desiccator and
quickly weigh to the nearest 0.5 mg.
3. Repeat these operations until the difference between two successive
weighing, following drying in the oven and cooling in the desiccator,
does not exceed 1 mg.
Incineration:
1. After drying, incinerate the dry residue in the muffle furnace at 550
± 25 ℃ to constant mass.
2. Allow to cool to room temperature (25 ± 3 °C) in the desiccator and
weigh again to the nearest 0.5 mg.
Number of determinations: Carry out at least two determinations on the
same test sample.

Blank test: If asbestos is used as a filter aid, carry out a blank test under

186 | M o M – D a i r y a n d D a i r y P r o d u c t s
 the same conditions as described in.
Calculation with units of Crude fibre content relative to product as received:
expression
The crude fibre content, expressed as percentage by mass relative to the
product as received, is given by the following formula:
a) for products not requiring grinding:

100
Crude fibre = [M1 – (M2 + M3)] x
𝑀𝑜

b) for products requiring grinding:

100 100 𝑀𝑠
Crude fibre= [M1 – (M2 + M3)] × × ×
𝑀𝑜 𝑀"𝑠 100

Where,
M0 is mass, in g, of the test portion
M1 is total mass, in g, of the dry residue and its support after drying,
M2 is total mass, in g, of the dry residue and its support after incineration
M3 is difference in mass, in g, observed during the incineration process in
the blank test, taking into account the quantity of filter aid used;
Ms is dry matter content, expressed as a percentage by mass, of the product
as received,
M”s is dry matter content, expressed as a percentage by mass, of the test
sample,

Crude fibre content relative to the dry matter content of the product:
The crude fibre content, expressed as a percentage by mass relative to the
dry matter content of the product, is given by the following formulae:

100 100
Crude fiber= [M1 – (M2 + M3)] x 𝑀𝑜 x 𝑀"𝑠

Where,
M0 is the mass, in g, of the test portion
M1 is the total mass, in g, of the dry residue and its support after drying,
M2 is the total mass, in g, of the dry residue and its support after
incineration,
M3: is the difference in mass, in grams, observed during the incineration
process in the blank test, taking into account the quantity of filter aid used;
M”s: is the dry matter content, expressed as a percentage by mass, of the
test sample,
Case of preliminary drying:

187 | M o M – D a i r y a n d D a i r y P r o d u c t s
 If a preliminary drying was carried out, the crude fibre content, expressed
as a percentage by mass relative to the product as received, is obtained by
multiplying the result calculated according to crude fibre content relative
to the product as received by the ratio:
M5/M4
Where,
M4: is the mass, in g, of the initial moist sample before preliminary drying,
M5: is the mass, in g, of the same sample after the preliminary drying.
Reference IS 10226 (Part 1): 1982 (Reaffirmed Year 2015). Method for
determination of crude fibre content: Part 1 General method. Bureau of
Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

188 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Total Carbohydrates in Milk Cereal Based
Complementary Food and Processed Cereal Based Complementary
Food
Method No. FSSAI 01.067:2022 Revision No. & Date 0.0
Scope This method is for the determination of total carbohydrates in milk cereal
based complementary food and processed cereal based complementary
food.
Caution This method is based on calculation and thus follow the caution listed in
individual method of determination (moisture, fat, protein and ash content).
Principle Total carbohydrate is determined by calculation method. After adding
moisture, fat, protein and ash content; the value obtained is then deducted
from 100.
Apparatus/Instruments This method is based on calculation and thus use the apparatus/instruments
listed in individual method of determination (moisture, fat, protein and ash
content).
Material and Reagents This method is based on calculation and thus use the materials and reagents
listed in individual method of determination (moisture, fat, protein and ash
content).
Preparation of reagents This method is based on calculation and thus use the preparation of
reagents listed in individual method of determination (moisture, fat, protein
and ash content).
Sample preparation This method is based on calculation and thus use the sample preparation
listed in individual method of determination (moisture, fat, protein and ash
content).
Method of analysis 1. Total carbohydrate is determined by subtracting from 100, the
addition of moisture (FSSAI 01.055:2022), fat (FSSAI
01.056/057:2022), protein (FSSAI 01.061:2022) and ash content
(FSSAI 01.063:2022).
2.
Calculation with units of Total carbohydrate including sucrose, dextrose
expression and dextrin, maltose or lactose percent by weight = 100 – (A+B+C+D)
Where A = Percent by mass of moisture
B = Percent by mass of total protein
C = Percent by mass of fat and

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 D = Percent by mass of total ash
Reference IS 1656: 2017 (Reaffirmed year 2018). Milk cereal based Weaning foods
– Specification. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Insolubility Index of Dried Milk Products

Method No. FSSAI 01.068:2022 Revision No. & Date 0.0

Scope This method is for the determination of insolubility index in dried milk
products such as whole, skimmed, partially skimmed milk powder, milk
protein concentrates and dairy whitener.

Caution Wear laboratory apron, shoes, safety goggles and mask while working in
the laboratory.
Principle Water at 24 ℃ (or at 50 ℃ if appropriate) is added to a test portion, which
is reconstituted using a special mixer. After a specified standing period, a
specified volume of the reconstituted milk or milk product is centrifuged
in a graduated tube. The supernatant liquid is removed and the sediment
is re-dispersed after the addition of water at the same temperature as used
for the reconstitution. The mixture is centrifuged and the volume of
sediment (insoluble residue) obtained is recorded.
Apparatus/Instruments 1. Thermometer, capable of measuring a temperature of 24 ℃ or 50
℃ with an error not exceeding ± 0.2 ℃.
2. Water bath, capable of being maintained at 24.0 ± 0.2 ℃ and/or 50
℃ ± 0.2 ℃, in which one or more mixing jars can be placed.
3. Mixing jar made of glass, of capacity 500 mL, as supplied for use
with the mixer.
4. Scoop, with a smooth surface, or sampling paper, black, glazed (of
dimensions 140 mm × 140 mm), for weighing the test portion.
5. Analytical balance (Readability 0.01 g).
6. Measuring cylinder made of plastic material, of capacity 100 mL
± 0.5 mL.
7. Stainless steel brush, suitable for removing any residual test
portion from the scoop or sampling paper.
8. Electric mixer, equivalent to that manufactured for the solubility
index method of the American Dry Milk Institute (ADMI) with the
following characteristics:
a. The 16-bladed impeller (stainless steel) shall have the shape and
diameter shown in Fig. 1 and shall be attached to the shaft of the
mixer so that the “flat” side of the impeller is underneath, as also
shown in Fig. 1. The slope of the blades is upward from right to left;
this is for clockwise rotation (see the Note).

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b. The pitch of the impeller blades shall be 30° and the horizontal
distance between the blades (around the circumference of the
impeller) shall be 8.73 mm, as shown in Fig. 1. With usage of the
impeller, these dimensions can change and hence periodic inspection
and maintenance are essential.
c. When the mixing jar is fitted to the mixer, the length of the mixer
shaft shall be such that the distance from the lowest part of the
impeller to the bottom of the jar is 10 mm ± 2 mm; this means that
for a jar of depth 132 mm the distance from the top of the jar to the
lowest part of the impeller is 122 mm ± 2 mm, and to the plane of the
lowest part of the impeller blades is 115 mm ± 2 mm. The impeller
shall also be located centrally in the jar.
d. When the mixing jar, containing 100 mL of water at 24 ℃ with or
without the addition of an appropriate test portion is fitted to the
mixer and the mixer switched on, the impeller shall come to its
operational fixed rotational frequency of (3600 ± 100) min−1 in less
than 5 s. The direction of rotation of the impeller shall be clockwise
(viewed from above). The rotational frequency of the impeller under
load (as described above) shall be checked periodically with an
electronic tachometer; this is especially necessary with older models
of the mixer, with a non-synchronous motor, whose rotational
frequency has to be adjusted to (3600 ± 100) min−1 by means of a
speed control and a speed indicator (which may not remain accurate).
NOTE: Some mixers for the ADMI method cause the impeller to rotate in
an anticlockwise direction (viewed from above). These mixers require an
impeller whose blades slope upward from left to right so that liquid in the
mixing jar moves in the same way as with a clockwise rotating impeller.
In all respects, including its manner of attachment to the shaft and its
distance from the bottom of the mixing jar, an anticlockwise-rotating
impeller is equivalent to a clockwise-rotating impeller.

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 Fig-1: Mixing Jar and Impeller (Source: IS 12789: 2019)
9. Interval timer, indicating 0 s to 60 s and 0 min to 60 min.
10. Spoon spatula of length approximately 210 mm.
11. Centrifuge tubes of glass, conical, with the shape, dimensions,
graduation scheme, inscriptions and patch of matt surface shown in Fig.
2, and provided with rubber stoppers. The graduation lines, graduation
numbers and the inscription “mL (20 ℃)” shall be marked in a
permanent manner and the graduation lines shall be fine and clean. The
maximum errors (±) in capacity at 20 ℃ shall be as follows: at 0.1 mL:
± 0.05 mL; from 0.1 mL to 1 mL inclusive: ± 0.1 mL; from 1 mL to 2
mL inclusive: ± 0.2 mL; from 2 mL to 5 mL inclusive: ± 0.3; mL from
5 mL to 10 mL: ± 0.5 mL; at 10 mL: ±1 mL. For routine production
control purposes, tubes of different dimensions may be used provided

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 that they comply with limits for maximum error in capacity, as listed
above. In all cases of dispute or where a definitive result is required, the
tubes specified in this sub clause should be used.

Fig-2: Centrifuge tube and Siphon Fitting (Source: IS 12789: 2019)

12. Centrifuge, electric, with speed indicator (min−1 or r/min), with vertical-
loading swing-out cups for accommodating the centrifuge tubes and
capable of producing an acceleration of 160 gn. At the internal bottom
of the tubes and maintaining a temperature of 20 ℃ to 25 ℃ within the
closed centrifuge.
 NOTE — The acceleration, in terms of gn, produced in a centrifuge is
equal to
1.12 rn2 × 10−6
where, r = effective horizontal radius of spinning, in millimeters;
n= rotational frequency, per min.
13. Siphon fitting or suction tube attached to water pump, made of glass
tubing and with upturned tip, for removing supernatant liquid from a
centrifuge tube
14. Stirring rod made of glass, of length 250 mm and diameter 3, 5 mm.
15. Magnifying lens, suitable for aiding the reading of the volume of
sediment.

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Materials and reagents Silicone antifoaming agent.
Preparation of Reagents Silicone Antifoaming Agent (for example an aqueous emulsion
containing 30 percent m/m of silicone): Test the suitability of the silicone
antifoaming agent by carrying out the procedure described in the method
without a test portion. No more than a trace of silicone fluid (≤ 0.01 mL)
should be visible at the bottom of the tube at the end of the procedure.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis 1. According to whether the insolubility index is to be measured at 24
℃ or at 50 ℃, adjust the temperature of the mixing jar to 24.0 ℃ ±
0.2 ℃ or 50.0 ℃ ± 0.2 ℃, respectively, by keeping the jar in the
water bath, with the water level near the top of the jar, for a sufficient
period of time. Weigh, to the nearest 0.01 g, in the scoop or on the
sampling paper, a test portion of:
a) 13.00 g, in the case of dried whole milk, dried partly skimmed milk,
and infant food based on either of these,
b) 10.00 g, in the case of dried skimmed milk and dried buttermilk, or
c) 7.00 g, in the case of dried whey.
2. Remove the mixing jar from the water bath, quickly wipe dry the
outside of the jar and, using the measuring cylinder, add to the jar
100 mL ± 0.5 mL of water at 24.0 ℃ ± 0.2 ℃ or 50.0 ℃ ± 0.2 ℃ as
appropriate.
3. Add 3 drops of the silicone antifoaming agent to the water in the
mixing jar and then transfer the test portion to the jar, using the
brush, if necessary, so that the entire test portion falls onto the
surface of the water.
4. Fit the mixing jar to the mixer, switch on the mixer, and after the
mixer has operated for exactly 90 s, switch it off.
5. If the mixer is of the type with a nonsynchronous motor, speed
control and speed indicator, bring the rotational frequency of the
impeller to 3600 ± 100 min−1 within the first 5 secs of the 90 sec
mixing period.
6. Remove the mixing jar from the mixer (allow a few secs for liquid
on the impeller to drain into the jar) and leave the jar to stand at
laboratory temperature for not less than 5 min and not more than 15
min.
7. Add 3 drops of the silicone antifoaming agent to the mixture in the
mixing jar; thoroughly mix the contents of the jar by stirring (not too
vigorously) for 10 secs with a spoon spatula.
8. Immediately pour sufficient of the mixture into a centrifuge tube to

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 fill the tube up to the 50 mL mark, that is, until the top level is
coincident with the 50 mL mark.
9. Place the centrifuge tube (counterbalanced) in the centrifuge set at
20 ℃ to 25 ℃.
10. Bring the centrifuge as quickly as possible to the rotational
frequency, producing an acceleration of 160 gn at the internal bottom
of the tube, and then spin the tube at this rotational frequency for 5
min.
11. Remove the centrifuge tube from the centrifuge and using a spoon
spatula, remove and discard any top layer of fatty material in the
tube.
12. Hold the centrifuge tube in a vertical position and remove the
supernatant liquid with a siphon fitting or a suction tube until the top
level is coincident with the 15 mL mark, if a roller-dried product is
being tested, or coincident with the 10 mL mark, if a spray-dried
product is being tested, taking care not to disturb the sediment.
13. However, if it is evident that the volume of sediment exceeds 15 mL
or 10 mL, respectively, discontinue the procedure at this stage and
record the insolubility index as “greater than 15 mL” or “greater than
10 mL”, indicating the reconstituting temperature.
14. Otherwise, add water at 24 ℃ or 50 ℃ as appropriate to the
centrifuge tube until the top level is coincident with the 30 mL mark.
15. Completely disperse the sediment with the stirring rod, tap the
bottom of the rod against the inside of the tube to collect adhering
liquid and add more water at the same temperature until the top level
is coincident with the 50 mL mark.
16. Close the centrifuge tube with a rubber stopper; invert the tube
quickly five times so as to mix its contents thoroughly, remove the
stopper (draw the bottom of the topper across the rim of the tube to
collect adhering liquid) and spin the tube in the centrifuge for 5 min
at the required rotational frequency and temperature.
17. It is recommended that the centrifuge tube be placed in the
centrifuge cup so that, when the tube is in the spinning position, the
scale lines are not facing upwards or downwards but are midway
between these two positions. Then, if the top of the sediment is
sloping, the volume of sediment will be easier to estimate.
18. Remove the centrifuge tube from the centrifuge, hold the tube in a
vertical position against a suitable background with the top of the
sediment at eye level and using the magnifying lens, read the volume
of sediment to the nearest 0.05 mL if the volume is less than 0.5 mL
and to 0.1 mL if the volume is more than 0.5 mL.
19. Estimate the volume if the top of the sediment is sloping. If the top

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 of the sediment is uneven, leave the tube in a vertical position for a
few min; the top of the sediment will usually become less uneven
and the volume of sediment can be read more easily. Record the
temperature of the water used for the reconstitution.
NOTE: Viewing the tube against either a light or a dark background, as
preferred, makes the top of the sediment more distinct.
Calculation with units of The insolubility index of the laboratory sample is equal to the volume, in
expression mL, of sediment recorded. Report the result with the temperature of the
water used for reconstitution, for example as follows:
a) 0.10 mL (24 ℃); and
b) 4.1 mL (50 ℃).
Reference IS 12759: 2019. Dried Milk and Dried Milk Products — Determination of
Insolubility Index (First Revision). Bureau of Indian Standards, New
Delhi
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Solubility Percentage in Foods for Infant Nutrition

Method No. FSSAI 01.069:2022 Revision No. & Date 0.0

Scope This method is for the determination of solubility percentage in infant milk
substitutes.
Caution Wear laboratory apron, shoes, safety goggles and mask while working in
the laboratory.
Principle In this method, a definite quantity of powder is reconstituted in water
under specified conditions. Reconstituted sample is centrifuged and the
fat layer is removed. The sediments are again dissolved and total solids
(TS-1) are determined in an aliquot. The sample is again centrifuged and
total solids of an aliquot of supernatant layer (TS-2) is determined.
Difference in total solids of two (TS -1 and TS-2) provide solubility of
powder. The solubility percent is then calculated by taking into account of
initial weight of sample taken.
Apparatus/Instruments 1. Shallow, flat-bottom dishes of aluminium, nickel, stainless steel,
porcelain or silica, 7-8 cm diameter, about 1.5 cm in height and
provided with easily removable but closely fitting lids.
2. 50 mL boiling tube.
3. Water bath (50 ± 1 ℃).
4. Hot air oven (forced air type), capable of being controlled at 100 ±
2 ℃.
5. Centrifuge capable of running at 770 × g.
6. Analytical balance (Readability 0.01 g).
7. Aluminium dish with lid.
8. Refrigerator (4-7 oC).
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation.
Method of analysis Reconstitution of Milk Powder
1. Weigh accurately 4 g of the material into a 50 mL boiling tube, add
32 mL of water at 50 ± 1 ℃; cork the tube and shake for 10 sec.
2. Place the tube in a water-bath maintained at 50 ± 1 ℃ for 5 min.
3. Shake the tube for one min.
Removal of Fat

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 1. Fill the reconstituted milk into a 25 mL centrifuge tube and
centrifuge for 10 min at 2000 rpm with a radius of 17 cm (giving a
force of 770 g).
2. Cool in a refrigerator or in ice until the fat solidifies (taking care
that milk does not freeze).
3. Remove the fat layer with a spoon shaped spatula.
4. Bring the milk to room temperature (27 ± 1 ℃).
5. Break up the deposit with a glass rod, cork the tube and shake
vigorously until the liquid is homogenous.
Determination of Total Solids
1. Transfer about 2 mL of the homogenous liquid to a previously
weighed dry tared aluminium dish (No. 1) (6 cm in diameter and
2.5 cm in height) with a tight-fitting lid.
2. Weigh the dish with the lid on and place it aside.
3. Centrifuge the tube again for 10 min at 2000 rpm with a radius of
17 cm (giving a force of 770 g).
4. Pipette about 2 mL of the upper layer of the supernatant liquid
without disturbing the sediment, into a second aluminium dish (No.
2) of the same type as No. 1.
5. Cover the dish and weigh.
6. Uncover both the dishes and place them side by side on a water-
bath until dry.
7. Place the dishes in an air-oven at 100 ± 2 ℃ for 90 min.
8. Cover and transfer the dishes to a desiccator; cool and weigh the
dish individually.
Calculations with units of 𝑀4 × 𝑀1 ×100
expression Solubility, percent by weight =
𝑀3 × 𝑀2
Where,
M1 is Mass in g of the liquid taken immediately after the removal of fat
in dish No.1
M2 is Mass in g of the supernatant liquid taken in dish No. 2
M3 is Mass in g of total solids in dish No. 1, and
M4 is Mass in g of total solids in dish No. 2
Reference IS 14433: 2007 (Reaffirmed year 2018). Infant Milk Substitutes (Annex
F, Amendment No. 3). Bureau of Indian Standards, New Delhi.

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Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Scorched Particles in Dried Milk Products

Method No. FSSAI 01.070:2022 Revision No. & Date 0.0

Scope This method is for the determination of scorched particles in whole,
skimmed, partially skimmed milk powder, cream powder, milk protein
concentrates, dairy permeates, colostrum powder, and dairy whitener.
Caution Wear laboratory apron, shoes, safety goggles and mask while working in the
laboratory.
Principle In this method, dried milk powder after reconstitution with distilled water is
passed through a cotton filter disc and dried. The resultant disc is compared
with the scorched particle disc test cards.
Apparatus/Instruments 1. Scorched particles standard photo prints for dry milks / scorched
particles discs test cards.
2. Analytical Balance: Torsion or similar type, approximately 500 g
capacity and 0.1 g or better sensitivity.
3. Mixer: Waring blender or similar type. Waring Blender with worn or
bent shaft should be replaced immediately since metal particles may be
produced, given false scorched particles reading.
4. Scorched particles filtering discs: Cotton discs 3.8 cm (1.25’) diameter
or cotton pads mounted on test cards use with the aspirator type tester.
5. Scorched particle tester: Aspirator or pressure type, 3.2 cm (1.125”)
filtering diameter.
6. 250 mL measuring cylinder
Materials and reagents 1. Defoaming agent: Diglycol laurate S or Antifoam B emulsion.
2. Water: should be sediment free and distilled with temperature between
32.5 and 41 ℃.
Sample preparation Refer method FSSAI 01.055:2022 for sample preparation
Method of analysis 1. Measure 250 mL sediment free water and weigh 25 g of non-fat dry
milk or dry butter milk or 32.5 g dry whole milk.
2. Transfer sediment free water and weighed sample to waring blender
jar and add approximately 0.5 mL of diglycol laurate S.
3. Mix for 60 secs in the blender and filter the entire solution through a
standard cotton disc, using an aspirator or pressure type tester.
4. Rinse the mixing container in tester with approximately 50 mL of
sediment free water, also passing this through the cotton disc.

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 5. If re-liquefied sample is allowed to stand before filtering, stir
vigorously just before pouring it into the tester. Do not allow samples
to stand uncovered.
6. Remove the filter disc, place it in a scorched particle disc test card and
dry at 30 to 40 ℃ in a dust free atmosphere.
7. Compare the dry disc, placed on a table and viewed from directly
above with the scorched particle standard photo prints under uniform,
indirect light. Any test falling between two standard discs should be
assigned the higher disc’s letter.
Example: A disc showing more scorched particle than the standard disc A
but less than B should be assigned B, and similarly for the other discs.

NOTE - for illustrative purpose only, a black and white reproduction of

these standard discs is given below. These illustrations shall not be used for
official classification of scorched particle content (see Figure)

Figure. Scorched particle standards for dry milk (Source IS 13500-1992)

Reference: IS 13500: 1992 (Reaffirmed 2018). Spray dried milk powder - Scorched
particles- determination. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 I. KHOA

Preparation of Sample of Khoa

Method No. FSSAI 01.071:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of khoa.
Apparatus/Instruments 1. Grating device.
2. Spatula/Spoon.
3. Airtight container for appropriate size.
Sample preparation 1. Take all precautions to ensure proper preservation of the sample and to
prevent condensation of moisture on the inside surface of the container.
2. The storage temperature should be below 10 ℃. Grind or grate the
laboratory sample of khoa by means of an appropriate device; mix the
ground or grated mass quickly, and if possible, grind or grate a second
time, and again mix thoroughly.
3. If the sample cannot be ground or grated, mix it thoroughly by
intensive stirring and kneading.
4. Transfer the test sample to an air-tight container to await analysis,
which should be carried as soon as possible after grinding.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Total Solids in Khoa

Method No. FSSAI 01.072:2022 Revision No. & Date 0.0

Scope This method is for the determination of total solids in khoa.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle The total solids content of khoa is determined by drying the product in
hot air oven at 102 ± 2 ℃ to constant mass.
Apparatus/Instruments Refer requirement stated for analysis of moisture in cheese (FSSAI
Materials and reagents 01.039:2022).
Preparation of reagents
Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis Refer method prescribed for analysis of moisture in cheese (FSSAI
01.039:2022).
Calculation with units of 𝑀2 − M
expression Total Solids Content (%) = × 100
𝑀1 − M
Where,
M is the mass in g of empty dish along with lid;
M1 is the initial mass in g of dish with lid and test portion
M2 is mass in g of dish with lid and dried test portion
Round the value obtained to nearest 0.01 % (m/m)
Reference IS 2785 -1979 (Reaffirmed year 2016). Specification for Natural cheese
(Hard Variety), Processed Cheese, Processed Cheese Spread and Soft
Cheese. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat Content on Dry Matter Basis in Khoa

Method No. FSSAI 01.073:2022 Revision No. & Date

Scope This method is for the determination of fat content in khoa.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.\
5. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
Principle In this method fat is extracted from the sample using either by digesting the
protein matrix using hydrochloric acid or ammonia. The process of
extraction of fat from khoa depends on whether sugar is present or not. If
sugar is present, fat is extracted with Rose Gottlieb, and if not, then fat is
extracted by acid digestion method. The liberated fat is extracted using a
combination of solvents viz., diethyl ether and petroleum ether. The
solvents are evaporated and obtained fat is dried and weighed. The addition
of ethanol facilitates the passage of the fat from the aqueous phase to the
solvents. The total solids in the sample are determined separately. Using
calculations, fat content on dry matter basis is then calculated.
Apparatus/Instruments Refer requirement stated for analysis of fat content in cheese and dried
Materials and reagents milk (FSSAI 01.040:2022 and FSSAI 01.056:2022).
Preparation of Reagents

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Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis Method 1: If sample shows presence of sugar:
1. Accurately weigh 2-3 g of the sample in a glass beaker, add 5 mL of
warm water and break the lumps with the help of a glass rod.
2. Transfer the contents carefully to a Mojonnier flask or other extraction
flask.
3. Wash the beaker and glass rod with warm water, add 1.25 mL of
ammonia solution
4. Proceed for the extraction of fat by Rose-Gottlieb method as prescribed
for milk from the step indicating addition of 10 mL ethyl alcohol
(Method No FSSAI 01.024:2022).
Method 2: If sample shows absence of sugar:
1. Accurately weigh 2-3 g of sample in a small beaker, add a few drops of
water and rub to a smooth paste by using a glass rod.
2. Add 9 mL of water (the first few drops being used to wash the tip of the
glass rod).
3. Add 10 mL of concentrated hydrochloric acid and boil gently, with
shaking, either over a flame or in boiling waterbath, until all solid
particles are dissolved. Cool to room temperature (25±3oC) in running
water. Transfer the contents to the Mojonnier fat extraction flask.
4. Proceed for the extraction of fat by Rose-Gottlieb method as prescribed
for milk from the step indicating addition of 10 mL ethyl alcohol
(Method No FSSAI 01.024:2022)
Determination of total solids
Refer method of analysis for total solids content in khoa
(FSSAI.01.072.2022).

Calculation with units of

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
expression 𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask

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 M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Total Solids (%) = Refer method of analysis for total solids moisture
content in khoa (FSSAI.01.072.2022)
Fat (%)
Fat % (on dry matter basis) = × 100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)
Reference 1. IS 4883: 1980 (Reaffirmed year 2019). Specification for Khoa.
Bureau of Indian Standards, New Delhi.
2. AOAC 905.02. 21st Edn. (2019). Official method -Fat in milk. AOAC
International, USA.
3. Pearson’s Composition and analysis of foods, 9th edn, 1991 page 538.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Detection of Starch in Khoa

Method No. FSSAI 01.074:2022 Revision No. & Date 0.0

Scope This method is for the determination of starch in khoa.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Iodine: It is not compatible with combustibles, strong bases, halogens
and ethanol. It reacts violently or explosively with acetylene,
acetaldehyde, metal hydrides and metal carbides.
2. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
Principle In this method, a definite quantity of sample is dispersed in hot water and
the diluted sample is treated with iodine solution. The appearance of blue
colour confirms presence of starch in khoa.
Apparatus/Instruments 1. 50 mL volumetric flask.
2. 100 mL volumetric flask.
3. Analytical balance (Readability 0.01 g).
Materials and reagents 1. Iodine.
2. Potassium iodide.
Preparation of Reagents 1. Iodine solution: Dissolve 1 g of iodine and 5 g of potassium iodide in
sufficient quantity of water and make up the volume to 100 mL.
Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis 1. Take 11.0 g of grated khoa sample in a beaker, add 20 mL of hot water
(80 - 90 ℃) and with the help of glass rod, make a paste.
2. Transfer the sample quantitatively to 50 mL volumetric flask and make
the volume to 50 mL. Shake well before use.
3. Take about 5 mL of prepared sample of khoa in a test tube, add 0.2 mL
of iodine solution to the test tube and mix well.
4. Observe the colour.

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Inference (Qualitative Development of blue colour indicates presence of starch and control sample
analysis) remains yellow. The limit of detection is 0.05%.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
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 Detection of Sucrose in Khoa

Method No. FSSAI 01.075:2022 Revision No. & Date 0.0

Scope This method is for the detection of sucrose in khoa.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents.
Refer to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Resorcinol: Avoid using for a prolonged period, on a broken skin. For
external use only; avoid contact with eyes, mouth, and
nose. Caution should be exercised in patients with any allergy,
children, elderly, during pregnancy and breastfeeding.
Principle In this method, a definite quantity of sample is dispersed in hot water and
the diluted sample is treated with resorcinol solution. The appearance of
red colour confirms presence of sugar in khoa.
Apparatus/Instruments 1. 50 mL volumetric flask.
2. Analytical balance (Readability 0.01 g).
Materials and reagents 1. Resorcinol flakes (should be white in colour).
2. Concentrated hydrochloric acid (Approximately 11.6 N).
Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis 1. Preparation of sample: Take 11.0 g of grated khoa sample in a beaker,
add 20 mL of hot water (80-90 ℃) and with the help of glass rod, make
a paste. Transfer the sample to 50 mL volumetric flask and make the
volume to 50 mL. Shake well before use.
2. Take about 5 mL of prepared sample of khoa in a test tube, add 0.5 mL
of concentrated hydrochloric acid followed by addition of 0.005 g of
resorcinol.
3. Mix the contents and place the test tube in boiling water bath for 5 min.
4. Observe the colour.

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Inference (Qualitative Appearance of deep red colour indicates presence of sucrose in sample,
analysis) whereas control samples will remain light pink.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
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 Determination of Titratable Acidity in Khoa

Method No. FSSAI 01.076:2022 Revision No. & Date 0.0

Scope This method is for the determination of titratable acidity in khoa.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
Principle A known quantity of sample is neutralized with standardized sodium
hydroxide solution with phenolphthalein indicator. The amount of sodium
hydroxide required is a function of the amount of natural buffering
substances present in the product, and of developed or added acid or
alkaline substances.
Apparatus/Instruments Analytical balance (Readability 0.01 g).
Materials and reagents 1. Sodium hydroxide.
2. Phenolphthalein indicator.
3. Ethanol.
Preparation of reagents 1. Standard Sodium hydroxide solution – 0.1 N.
2. Phenolphthalein Indicator – Dissolve 1.0 g of phenolphthalein in 100
mL of 95% ethanol. Add 0.1 N Sodium hydroxide solution until one
drop gives a faint pink colouration. Dilute with distilled water to 200
mL.
Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis 1. Weigh accurately about 2 g of the material in a suitable dish or basin,
add 3 mL of hot water and render it to paste; add further 17 mL of hot
water washing off any adherents.
2. Add 1 mL of phenolphthalein indicator, shake well and titrate against
standard Sodium hydroxide solution; complete the titration in 20 sec.

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 3. Keep a blank by taking 2 g of material diluted with 20 mL of water in
another dish for comparison of colour.

Calculation with units of 9 𝐴𝑁

Titratable acidity (as lactic acid)percent by mass =
expression 𝑤
Where,
A = Volume of standard Sodium hydroxide required for titration
N = Normality of Standard Sodium hydroxide solution
w = weight of the sample taken for test in g
Reference IS 4883: 1980 (Reaffirmed year 2019). Specification for Khoa. Bureau of
Indian Standards, New Delhi.
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 Determination of Total Ash in Khoa

Method No. FSSAI 01.077:2022 Revision No. & Date 0.0

Scope This method is for the determination of total ash in khoa.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot crucibles.
2. Hot crucibles will heat air within the desiccator and a vacuum may form
on cooling. Remove desiccator’s cover gradually by sliding to one side
to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle Sample is kept at 550 ± 20 ℃ in a muffle furnace and ignited so that all the
organic matter will be burnt. The leftover inorganic residue is then weighed
and the total ash is determined and expressed as percentage.
Apparatus/Instruments 1. Platinum or silica crucible about 70 mm diameter and 25 to 50 mm
deep.
2. Muffle furnace: Capable of being controlled at 550 ± 20 ℃.
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Safety tongs having long arms.
5. Bunsen burner or electric hot plate.
6. Analytical balance (Readability 0.0001 g).
7. Muffle furnace tongs.
8. Gloves.
Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis 1. Weigh accurately about 3 g of the khoa sample in the crucible,
previously dried in a hot air oven and weighed.
2. Heat the crucible gently on a burner or hot plate at first and then
strongly in a muffle furnace at 550  20 ℃ till grey ash is obtained.
3. Cool the crucible in a desiccator and weigh it; heat the crucible again
at 550  20 ℃ for 30 min.

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 4. Cool the crucible in a desiccator and weigh.
5. Repeat this process of heating for 30 min, cooling and weighing until
the difference between two successive weighing is less than 1 mg.
6. Record the lowest mass.
𝑀2 − 𝑀
Calculation with units of Total ash % by mass = × 100
𝑀1 − 𝑀
expression
Where
M is mass in g, of the empty crucible;
M1 is mass in g, of the crucible with the material taken for the test;
M2 is mass in g, of the crucible with ash.
Reference IS 1165: 2002 (Reaffirmed year 2018). Milk Powder – Specifications.
Bureau of Indian Standards, New Delhi.

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 Estimation of Reichert Meissl Value, Polenske Value and Butyro-
Refractometer Reading of Fat Extracted from Khoa

Method No. FSSAI 01.078:2022 Revision No. & Date 0.0

Scope This method is applicable for determination of Reichert Meissl (RM) value,
Polenske Value and Butyro-Refractometer Reading (BRR) of the fat
extracted from khoa.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle The sample is extracted with light petroleum ether. The solvent is distilled off
and the residue dried and weighed.
Apparatus/Instruments 1. Soxhlet extraction apparatus.
2. Extraction thimbles, free of matter soluble in light petroleum and
having porosity consistent with the requirements.
3. Hot air oven (forced air type), capable of being controlled at 100 ± 3 ℃.
4. Extraction Cups.
5. Analytical balance (Readability 0.001 g).
Materials and Reagents Light petroleum ether, boiling range: 40 to 60 ℃.
Sample preparation Refer method FSSAI 01.071:2022 for sample preparation.
Method of analysis 1. Weigh approximately 15 g of the sample (M1), transfer it to an
extraction thimble and cover with a fat-free cotton wool. The sample
may be taken in quadruplicateto obtain sufficient quantity of fat (12 g
fat may be obtained from 60 g of sample).

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 2. Place the thimble in extraction cup then place the cup in extractor and
pour 150 mL of petroleum ether.
3. Turn on the heating mantle and run the extraction for approximately 4
to 6 h at condensation rate of 5 to 6 drops per second.
4. Collect all petroleum ether in the conical glass flask and evaporate.
5. Place the flasks in hot air oven for approximately two h at 100 ℃.
6. Leave the flasks at 25 ± 3 C to cool.
7. Use the extracted fat for estimation for Reichert Meissl Value &
Polenske value (FSSAI 01.090:2022) and Butyo-refractometer
reading (FSSAI 01.088:2022) as per the method specified for milk fat
products
Calculation with units of Refer calculation as described for Reichert Meissl Value, Polenske value
expression and butyo-refractometer reading in method specified for milk fat products
(FSSAI 01.090:2022 and FSSAI 01.088:2022).
Reference AOAC 948.22 21st Edn. (2019). Fat (Crude) in Nuts and Nut Products.
AOAC International, USA.
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 J. BUTTER

Preparation of Sample of Butter

Method No. FSSAI 01.079:2022 Revision No. & Date 0.0

Scope This method specifies the sample preparation of butter for further physical
and chemical analysis
Apparatus/Instruments 1. Water bath/oven maintained at 37 ± 2 ℃.
2. Airtight container for appropriate size.
Sample preparation 1. Warm the sample in an airtight container with the lid screwed down
tightly or with the glass stopper in an oven or water bath maintained
at 37 ± 2 ℃.
2. Shake vigorously to obtain a homogeneous fluid emulsion, free from
unsoftened pieces.
3. In case the sample does not mix up properly (water separation can be
seen), reject the sample.
Reference 1. IS 3507 – 1966 (Reaffirmed year 2018) Method of sampling and test
for Butter. Bureau of Indian Standards, New Delhi.

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 Determination of Moisture in Butter

Method No. FSSAI 01.080:2022 Revision No. & Date 0.0

Scope This method describes the determination of moisture in butter using.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.
1. Use thermal protection gloves, tongs and protective eyewear while
handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling
period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle The sample is kept in a hot air oven at 100  1 ℃. The moisture content in
butter is determined by the reduction in weight of the sample.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Hot air oven (forced air type), capable of being controlled at 100 ± 1 ℃.
3. Flat bottom moisture dish: Dishes of height at least 25 mm and at least
50 mm in diameter, and made of appropriate material (for example
stainless steel, nickel or aluminium) not affected by boiling water.
4. Glass rods with one end flattened and about 9 cm in length.
5. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
6. Boling water-bath with rings to take dishes of 50 mm diameter.
7. Clay pipe triangles.
Sample preparation Refer method FSSAI 01.079:2022 for sample preparation.
Method of analysis 1. Clean the dish and the glass rod and dry them in the hot air oven
maintained at 100  1 ℃ for at least 1 h.
2. Allow to cool to the room temperature (25 ± 3 °C) in a desiccator and
weigh the dish. Accurately weigh (to the nearest 0.1 mg) into the dish
3 to 4 g of the prepared butter sample.
3. Place the dish on a boiling water-bath supported on a clay pipe triangle
for at least 20 min, stirring at frequent intervals until no moisture can
be seen. Wipe the bottom of the dish and transfer it to the oven
maintained at 100  1 ℃ and keep it for 90 min. Allow the dish to cool
in the desiccator and weigh to the nearest 0.1 mg.
4. Heat the dish again in an oven for 30 min. Repeat the process of heating,
cooling and weighing until the difference between two consecutive
weights does not exceed 0.1 mg. Record the lowest mass and preserve
the residue for the determination of curd.

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 5. Note: As per IDF (IDF 80-1, 2001) procedure, the weight of butter
sample taken for moisture determination is 5 g and drying temperature
is 102 ± 2 ℃ for 1 h.
Calculation with units of M -M
Moisture % by mass = 1 2 × 100
expression M1 -M
Where,
M is mass in g, of the empty dish along with glass rod;
M1 is initial mass in g of the dish, glass rod and test portion;
M2 is the mass in g of the dish, glass rod and dried test portion
Express the results to the nearest 0.01% (m/m).
Reference 1. IS 3507: 1966 (Reaffirmed year 2018). Methods of sampling and test
for Butter. Bureau of Indian Standards, New Delhi.
2. AOAC 920.116. 21st Edn. (2019). Official method Moisture in Butter;
IDF (2001). AOAC International, USA.
3. IDF Standard 80-1. Butter – Determination of moisture, non-fat solids
and fat contents (Part 1): Determination of moisture content (reference
method) International Dairy Federation, Brussels.

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 Determination of Fat and Curd (Milk solids not Fat) in Butter

Method No. FSSAI 01.081:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat and curd (milk solids not fat) in
butter.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle In this method, the fat portion is removed using petroleum ether and residue
is dried for determination of curd content. In case of table butter, it is curd
and salt content, and thus salt content has to be determined separately
(Refer method FSSAI 01.083:2022) for calculating curd content.
Apparatus/Instruments 1. Gooch crucible or sintered funnel - with filter flask and adapter.
2. Glass funnel with folded 12.5 cm Whatman filter paper Grade 1.
3. Flat bottom flask of 250 mL capacity.
4. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
5. Celite 545 (diatomaceous earth).
6. Hot air oven (forced air type), capable of being controlled at 100 ± 1
℃.
7. Conical flask: 250 mL capacity.
8. Glass beads.
Materials and reagents Petroleum ether – boiling range 40-60 °C.
Sample preparation Refer method FSSAI 01.079:2022 for sample preparation.
Method of analysis 1. Prepare a celite mat in a Gooch crucible or sintered funnel. Dry it in a
hot air oven maintained at 100  1 ℃, cool in the desiccator and weigh.
Alternatively, dry, cool and weigh ordinary glass funnel with folded
12.5 cm filter paper.
2. Melt the residue in the moisture dish (obtained after determination of
moisture) and add 25 to 50 mL of petroleum ether and mix well.
3. Fit the crucible to the filter flask or place the funnel with filter paper on
a filter stand.
4. Wet the celite mat or the filter paper with petroleum solvent and decant
the fatty solution from the dish into the celite or the filter paper, leaving
the sediment in the dish.
5. Macerate the sediment twice with 20 to 25 mL of petroleum solvent
and decant again the fatty solution into the asbestos or the filter paper.

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 6. Filter the solution and collect the filtrate in a clean, dried, tared 250 mL
flat bottom flask, containing 1 to 2 glass beads.
7. With the aid of a wash-bottle containing petroleum solvent, wash all
the fat and sediment from the dish into the crucible or the filter paper.
8. Finally, wash the crucible or the filter paper until free from fat,
collecting all the filtrate in the conical flask. Preserve the filtrate for the
determination of fat. Dry the crucible or filter paper in the oven
maintained at 100  1 ℃ for at least 30 min.
9. Note: If only fat is to be determined, transfer all the filtrate to a pre-
dried and weighed fat flaks containing 2-3 glass beads. Rinse the
conical flask with petroleum ether. Evaporate the ether, first on the
water-bath and then in the oven at 102 ± 2 ℃ for 1 h or till the time the
constant weight is obtained. Cool in the desiccator and weigh. Repeat
drying, cooling and weighing until the loss of weight between the
consecutive weighing does not exceed 0.1 mg. Preserve the residue for
the determination of salt.
Calculation with units of Calculate the fat content form the residues obtained by using following
expression formula
% Fat =
(𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑎𝑡 𝑓𝑙𝑎𝑠𝑘+𝑓𝑎𝑡 𝑟𝑒𝑠𝑖𝑑𝑢𝑒𝑠)−𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑓𝑙𝑎𝑠𝑘
× 100
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛
Curd and salt
𝑀1 − 𝑀2
Curd and salt, % by mass (C) = 𝑥 100
𝑀
Where,
M is mass in g, of the sample
M1 is mass in g, of the filter paper with residue;
M2 is mass in g, of the filter paper alone;
Curd in butter
Curd percent by weight is obtained by subtracting the value of salt percent
by weight from the value of C. Refer method FSSAI 01.083:2022 for
determination of salt in butter.

Reference 1. IS 3507: 1966 (Reaffirmed year 2018). Method of sampling and Test
for Butter. Bureau of Indian Standards, New Delhi.
2. Pearsons Composition and Analysis of Foods 9thedn, page 576.
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 Determination of Fat in Butter

Method No. FSSAI 01.082:2022 Revision No. & Date 0.0

Scope This method is for the determination of fat in butter.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
2. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may
ignite and burn with explosive force. It reacts violently with halogens
(such as fluorine, bromine, and chlorine), acids (such as hydrochloric
acid, hydrogen fluoride, hydrogen bromide).
3. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
4. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
5. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
6.
Principle Fat content in butter can be estimated by either Gerber Method or Rose-
Gottlieb method. The Gerber method is suitable as a routine or screening
test. The butter is mixed with sulphuric acid and iso-amyl alcohol in a
special Gerber tube, permitting dissolution of the protein and release of fat.
The tube is centrifuged and the fat rising into the calibrated part of the tube
is measured as a percentage of the fat content of butter. This method can
be used for routine purposes.
Rose-Gottlieb Method is a gravimetric method in which fat globule
membrane of milk fat globules is ruptured by addition of ammonia to milk
sample to liberate the fat. The liberated fat is extracted using combination
of solvents viz., diethyl ether and petroleum ether. The solvents are
evaporated and obtained fat is dried and weighed. This method is

223 | M o M – D a i r y a n d D a i r y P r o d u c t s
 considered suitable for reference purposes. Strict adherence to details is
essential in order to obtain reliable results.
Apparatus/Instruments Refer apparatus requirement stated for milk (FSSAI 01.024:2022), except
Butyrometer required for analysis of butter would be range up to 70% (ISI
marked).
Materials and reagents Refer requirement stated for analysis of fat content in milk (FSSAI
01.024:2022).
Preparation of Reagents Refer requirement stated for analysis of fat content in milk (FSSAI
01.024:2022).
Sample preparation Refer method FSSAI 01.079:2022 for sample preparation.
Method of analysis Method 1: Gerber Method
1. Weigh 2.5 g of sample in a 25 mL beaker and mix it with small portions
of 1:1 sulphuric acid. Transfer the contents into the butyrometer with
the help of a small funnel.
2. Rinse the beaker about six times with small quantities of dilute
sulphuric acid to make sure that all butter has been transferred.
3. Mix 10 mL of water and 10 mL of sulphuric acid in a small beaker; add
between 15 to 20 mL of the mixture to the butyrometer depending on
its size.
4. Add 1 mL of amyl alcohol, preferably by use of an automatic dispenser.
Continue analysis as per method prescribed for analysis of fat in milk
by Gerber method (FSSAI 01.024:2022).

Method 2: Rose-Gottlieb Method

1. Weigh accurately 1 g of butter sample into a 50 mL beaker, add 9 mL
of 0.5% sodium chloride solution to disperse and transfer to fat
extraction apparatus.
2. Add 1 mL of concentrated ammonia and mix well.
3. Proceed for the extraction fat by Rose-Gottlieb method as prescribed
for milk from the step indicating addition of 10 mL ethyl alcohol
(Method No FSSAI 01.024:2022).
Calculation with units of Method 1: Gerber Method: See the method of analysis.
expression
Method 2: Rose-Gottlieb Method:

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤
Where,
M1 is the mass, in g, of the fat-collecting flask and extracted matter after drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after drying

224 | M o M – D a i r y a n d D a i r y P r o d u c t s
 M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference 1. IS 3507: 1966 (Reaffirmed year 2018). Method of sampling and Test
for Butter. Bureau of Indian Standards, New Delhi.

2. IS 3509: 1966. (Reaffirmed year 2018). Method of sampling and test

for cream. Bureau of Indian Standards, New Delhi.
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 Determination of Salt Content in Butter

Method No. FSSAI 01.083:2022 Revision No. & Date 0.0

Scope This method is for the determination of salt content in butter.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Nitric acid: It is a corrosive chemical. It is highly reactive and a
dangerous explosion hazard. It reacts violently and explosively with
most metals and powdered metals (antimony, bismuth etc.), alkaline
earth metals (magnesium, calcium etc.), and metal hydrides to form
flammable and explosive hydrogen gas.
2. Ferric ammonium sulphate: Provide exhaust ventilation or other
engineering controls to keep the airborne concentrations of vapors
below their respective threshold limit value.
3. Nitrobenzene: It is highly flammable; keep away from heat, sparks and
open flame. Nitrobenzene is highly toxic (Threshold Limit Value 5
mg/m3) and readily absorbed through the skin. Prolonged exposure,
avoid breathing fumes/aerosols. Perform all operations under an
effective fume removal device.
4. Silver nitrate: It is corrosive. It is not combustible, but it is a strong
oxidizer that enhances the combustion of other substances.
Principle In this method, salt present in the butter sample is extracted with hot water
from the dried fat-free residue obtained in moisture determination. The
chlorides are precipitated by adding excess of silver nitrate. The unused
silver nitrate is titrated with potassium thiocyanate using ferric ammonium
sulphate indicator.
Reaction
Ag+ (excess) + Cl- AgCl (solid)

Ag+ + SCN- AgSCN (solid)

Fe+3 + SCN- [FeSCN]+2 (Reddish brown)

Apparatus/Instruments 1. Beakers of 100, 250 mL capacity.

2. Volumetric flask of 100 mL, 1 L capacity.
3. Conical flask of 250 mL capacity.
4. Water-bath maintained at 60 to 70 ℃.

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 5. Burette of 50 mL capacity, graduated to 0.1 mL.
6. Pipette capable of delivering 2.0 mL.
7. Measuring cylinder of 100 mL capacity, graduated.
8. Analytical balance (Readability 0.001 g).
Materials and reagents 1. Silver nitrate.
2. Sodium chloride 99.9-100% pure – dried at 140 ℃.
3. Nitric acid (Approximately 15.8 N).
4. Ferric ammonium sulphate.
5. Potassium thiocyanate (KCNS).
6. Potassium chromate.
7. Calcium carbonate: Analytical Grade, free from chloride.
8. Nitrobenzene.
Preparation of Reagents 1. Standard silver nitrate solution: 0.05 N, Dissolve slightly more than
theoretical quantity (8.7 g per 1 L of water) of silver nitrate (equivalent
weight 169.89) in halogen-free water and dilute to volume (1 L) and
standardize against standard 0.05 N sodium chloride.
2. Nitric acid: Approximately 5 N. Take 31 mL of concentrated nitric acid
and make up the volume to 100 mL with distilled water.
3. Ferric ammonium sulphate indicator solution: Dissolve 50 g of ferric
ammonium sulphate [Fe2(SO4)3.(NH4)2SO4.24H2O] in 95 mL of water
containing 5 mL of 5 N nitric acid.
4. Standard potassium thiocyanate (KCNS) solution (Approx. 0.05 N):
Weigh approx. 5.25 g KCNS and dissolve in 1 L water. Allow to stand
overnight and filter, if necessary, to get a clear solution, standardize by
titration against 0.05 N AgNO3 and dilute with requisite volume of
water to get exactly 0.05 N KCNS solution.
5. Potassium chromate indicator (5%, w/v): Dissolve 50 g of potassium
chromate (K2CrO4) in 1 L of water.
6. Calcium carbonate: Analytical Grade, free from chloride.
Sample preparation Refer method FSSAI 01.079:2022 for sample preparation.
Method 1: (Volhard's Method)
Method of analysis
1. Extract the salt from the residue of curd and salt by repeated washing
of the Gooch crucible or filter paper with hot water, or by placing the
crucible or filter paper in a beaker of hot water.
2. Collect the rinsing in a 100 mL volumetric flask, passing the solution
through a filter paper. Allow to cool to room temperature (25 ± 3 oC)
and make up to volume.
3. Take 25 mL water extract into a 250 mL conical flask, and add an
excess (normally 25 to 30 mL) of 0.05 N silver nitrate solution.
4. Acidify with 5N nitric acid; add 2 mL of the indicator solution and 1
mL nitrobenzene. Mix and determine the excess of silver nitrate by

227 | M o M – D a i r y a n d D a i r y P r o d u c t s
 titration with potassium thiocyanate solution until the appearance of an
orange tint, which persists for 15 s.
5. In the same manner, determine the equivalent of 25 mL or the added
amount of silver nitrate as thiocyanate using the same volumes of
reagents and water.

Method 2: (Mohr's Method)

1. Weigh accurately 5 g of butter sample into the 250 mL conical flask.
Carefully add 100 mL of boiling distilled water; mix the contents of the
conical flask. Allow to stand with occasional swirling for 5 to 10 min.
2. After cooling to 50 to 55 ℃ (titration temperature), add 2 mL of
potassium chromate solution; mix by swirling. Add about 0.25 g of
calcium carbonate and mix by swirling.
3. Titrate at 50 to 55 ℃ with standard silver nitrate solution while swirling
continuously, until the brownish colour persists for half a min.
4. Carry out a blank test with all the reagents in the same quantity except
the butter sample. The maximum deviation between duplicate
determinations should not exceed 0.02% of sodium chloride.
Calculation with units of Method 1. (Volhard's Method)
expression 23.38 × 𝑁 × (𝐴−𝐵)
NaCl, % by mass =
𝑀
Where,
N = normality of potassium thiocyanate solution;
A = volume in mL, of potassium thiocyanate in blank titration;
B = volume in mL, of potassium thiocyanate in the sample titration; and
M = mass in g, of the butter sample
Method 2. (Mohr's Method)
5.844 ×𝑁 (𝑉1 − 𝑉2 )
NaCl, % by mass = =
𝑀
Where,
N = normality of silver nitrate solution (0.1N);
V1 = volume in mL, of silver nitrate used in the sample titration;
V2 = volume in mL, of silver nitrate used in the blank titration; and
M = mass in g, of the butter sample.
Reference IS 3507: 1966 (Reaffirmed year 2018). Methods of sampling and test for
butter. Bureau of Indian Standards, New Delhi.

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Approved by Scientific Panel on Methods of Sampling and Analysis

229 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Estimation of Reichert Meissl Value and Butyro-refractometer
Reading of Fat Extracted from Butter

Method No. FSSAI 01.084:2022 Revision No. & Date 0.0

Scope This method is applicable for determination of Reichert Meissl (RM) value,
and Butyro-Refractometer (BR) Reading of the fat extracted from butter.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid. Good
general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle In this method, fat from the butter is extracted by heating the sample on hot
plate/burner followed by filtration. The extracted fat is further used for
determination of RM Value and BR reading.
Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).
2. Flat bottom dish of Stainless steel / nickel / aluminium having 7-8 cm
diameter and depth of approximately 2.5 cm.
3. Hot plate / burner.
Materials and Reagents 1. Light petroleum ether, boiling range: 40 to 60 ℃.
2. Funnel.
3. Ordinary filter paper.
Sample preparation Refer method FSSAI 01.079:2022 for sample preparation.
Method of analysis 1. Take appropriate quantity of butter into a flat bottom dish (20 g butter
may yield 15 g fat).

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 2. Heat the dish on hot plate / burner till the disappearance of foam
indicating removal of moisture.
3. Filter the content with ordinary filter paper.
4. Use the extracted fat for estimation for Reichert Meissl Value (FSSAI
01.090:2022), and Butyo-refractometer reading (FSSAI 01.088:2022)
as per the method specified for milk fat products.
Calculation with units of The fat extracted in different flask can be used further for determination of
expression Reichert Meissl value and BR value as per the methods specified for milk
fat products (FSSAI 01.088:2022 and FSSAI 01.090:2022)
Reference AOAC 948.22 21st Edn. (2019). Fat (Crude) in Nuts and Nut Products.
AOAC International, USA.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 K. MILK FAT PRODUCTS

Preparation of Sample of Milk Fat Products

Method No. FSSAI 01.085:2022 Revision No. & Date 0.0

Scope The method describes the sample preparation procedure for milk fat, ghee,
butter oil, anhydrous milk fat and anhydrous butter oil.
Apparatus/Instruments 1. Water bath.
2. Whatman filter paper- Grade 4.
3. Funnel.
4. Bottles.
5. Moisture determination apparatus.
Sample preparation 1. Mix the milk fat product sample in the container in which it is received
until homogenous.
2. Carry out this operation in a cool place, away from direct sunlight, and
complete it in shortest possible time.
3. In the event of any separation taking place in between, that is, mixing
and commencement of the chemical analysis for moisture
determination, remix the sample.
4. Use this for determining moisture.
5. After moisture determination, place the container or glass bottle
containing milk fat products in a water-bath at a temperature not higher
than 50 ℃ till completely melted.
6. Filter through a dried, fluted open-texture, 15 cm Whatman filter paper
Grade 4 with the help of a hot water funnel, directly into receiving
bottle. The filtered ghee should be bright and clear.
Reference IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test for
Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Moisture in Milk Fat Products

Method No. FSSAI 01.086:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of moisture in milk fat,
ghee, butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.

Principle The moisture content of milk fat product is the loss in mass, expressed as a
percentage by mass when the product is heated in a hot air oven at 105 ± 1
℃ to constant mass.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Moisture dish: Aluminium, nickel or stainless steel, 7-8 cm in diameter,
2-2.5cm deep provided with tight fitting sip on covers.
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Hot air oven (forced air type), capable of being controlled at 105 ± 1 ℃.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis 1. Weigh accurately about 10 g of the sample into a moisture dish which
has been dried previously and weighed.
2. Place it in a hot air oven for 1 hour at 105 ± 1 ℃.
3. Remove the dish from the oven, cool in a desiccator and weigh.
4. Repeat the process by keeping the dish in the oven for half an hour,
cooling and weigh till two successive weighing’s do not exceed 1 mg.
Calculation with units of M -M
Moisture % by mass = 1 2 × 100
expression M1 -M
Where,
M is mass in g, of the empty dish along with glass rod;
M1 is initial mass in g of the dish with lid, glass rod and test portion;

233 | M o M – D a i r y a n d D a i r y P r o d u c t s
 M2 is the mass in g of the dish with lid, glass rod and dried test portion
Express the results to the nearest 0.01% (m/m).
Reference IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test for
Ghee (Butterfat). Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Fat in Milk Fat Products

Method No. FSSAI 01.087:2022 Revision No. & Date 0.0

Scope This method describes the determination of fat in milk fat, ghee, butter oil,
anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may ignite
and burn with explosive force. It reacts violently with halogens (such as
fluorine, bromine, and chlorine), acids (such as hydrochloric acid,
hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle Rose-Gottlieb Method is a gravimetric method in which fat globule
membrane of milk fat globules is ruptured by addition of ammonia to cream
sample to liberate the fat. The liberated fat is extracted using combination
of solvents viz., diethyl ether and petroleum ether. The solvents are
evaporated and obtained fat is dried and weighed. This method is considered
suitable for reference purposes. Strict adherence to details is essential in
order to obtain reliable results.
Apparatus/Instruments Refer requirement stated for determination of fat in milk (FSSAI
Materials and reagents 01.024:2022)
Preparation of reagents
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis 1. Weigh accurately 0.5 g of prepared product into a 50 mL beaker, add 9
mL of 0.5% sodium chloride solution to disperse and transfer to fat
extraction apparatus.
2. Add 1 mL of ammonia sp. gr. 0.91 (or an equivalent volume of a more
concentrated ammonia solution may be used), mix and shake

235 | M o M – D a i r y a n d D a i r y P r o d u c t s
 thoroughly.
3. Proceed for the extraction of the fat by Rose-Gottlieb method as
prescribed for milk (Method No FSSAI 01.024:2022) starting with the
step indication addition of 10 mL ethyl alcohol onwards.
Calculation with units of (𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
expression 𝑤
Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference 1. IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test
for Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
2. IS 1479 (Part II): 1961 (Reaffirmed Year: 2018). Methods of test for
Dairy Industry – Chemical Analysis of Milk. Bureau of Indian
Standards, New Delhi.
3. IS 3509: 1966 (Reaffirmed year 2018). Method of sampling and test
for cream. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Butyro-Refractometer Reading in Milk Fat
Products
Method No. FSSAI 01.088:2022 Revision No. & Date 0.0
Scope This method is used to determine the butyro-refractometer (BR) Reading
and applicable for milk fat, ghee, butter oil, anhydrous milk fat and
anhydrous butter oil.
Caution Wear laboratory apron, shoes, safety goggles and mask while working in
laboratory.
Principle Refractive index is the ratio of the velocity of light in vacuum to the velocity
of light in the sample medium; more generally, it is expressed as the ratio
between the sine of angle of incidence to the sine of the angle of refraction,
when a ray of light of a definite known wavelength (usually 589.3 m the
mean of the D-line of sodium) passes from air into ghee. The refractive
index of ghee may be read on an Abbe refractometer which gives the true
refractive index or on a thermostatically controlled butyro-refractometer,
which reads on an arbitrary scale (BR reading) at constant temperature.
Apparatus/Instruments 1. Precision butyro-refractometer which is thermostatically controlled and
fitted with an accurate thermometer reading from 40 to 50 ℃. Check the
calibration of the instrument as per the manufacturer’s instructions.
2. Device to maintain and control the temperature of prism constant at 40
 1 ℃. Peltier controlled butyro-refractometer which are commercially
available can also be used.
3. Sodium lamp; daylight can also be used if the refractometer has an
achromatic compensator.
4. Standard fluid for checking the accuracy of the instrument. Usually, this
fluid is provided by the manufacturer for calibration of the
refractometer.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis The sample should be rendered optically clear and free from water and
suspended impurities, this can be done by using following procedure:
1. After moisture determination, place the container or glass bottle
containing ghee in a water-bath at a temperature not higher than 50 ℃
till completely melted.
2. Filter through a dried, fluted open-texture 15 cm Whatman filter paper
Grade 4 with the help of a hot water funnel, directly into the receiving
bottle. The filtered ghee should be bright and clear.

237 | M o M – D a i r y a n d D a i r y P r o d u c t s
3. The correctness of the butyro-refractometer shall be tested before
carrying out the test with liquid of known refractive index (at 40 ℃).
4. Open the double prism of the instrument and place a few drops of the fat
sample on prism. The fat sample shall completely fill the space between
the two prisms and shall show no air bubbles; close prisms firmly.
5. Allow the instrument to stand for few min before reading is taken so that
temperature of ghee sample and instrument is same.
6. The reading shall be taken after ghee has been kept in the prism for 2 to
5 min and after it has been ensured that it has attained constant
temperature (40 ℃) by taking two or more readings.
7. B.R. reading of ghee decreases with the rise in temperature and vice
versa. A correction is needed whenever temperature of the butyro-
refractometer is either below or above 40 ℃.
8. Method of measurement is based upon observation of position of border
line on the scale of the instrument. Hold the sector firmly and move
backward or forward until the field of vision is divided into light and
dark portion. Line dividing these portions may not be sharp but a band
of colours.
9. The colours are eliminated by rotating screw head of compensator until
sharp, colourless line is obtained. Read the B.R. of the sample directly
on the scale.
Notes- It should be borne in mind that presence of free fatty acids
considerably lowers the refractive index.
10. In the homologous series of saturated fatty acids from butyric to stearic,
the refractive index rises steeply among the lower members, and flattens
out at higher chain lengths. A double bond elevates the refractive index:
stearic acid has a lower refractive index than oleic, which in turn has a
lower value than linoleic.
11. For conversion of refractive index values into butyro-refractometer
reading and vice versa use the following formula (Rangappa and
Achaya, 1974) or table (BIS, 1966) as follows:
Inter-conversion of refractive index and butyrometer degrees:
Butyrometer reading in degrees = 42.0 + factor (observed refractive index -
1.4538)
The factor to be used varies with the range of refractive index, as shown
below:
Observed refractive
Factor
index range
1.4500 - 1.4515 1400

1.4515 - 1.4530 1410

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 1.4530 - 1.4545 1420

1.4545 - 1.4560 1430

1.4560 - 1.4575 1440

Butyrometer degrees to Refractometer index:

Refractometer index = 1.4538 + factor (Observed B.R. - 42.0)
The factor to be used varies inversely with temperature.

Observed BR reading Factor

range
37.5 - 40.0 0.00072
40.0 - 42.5 0.00071
42.5 - 45.0 0.00070
45.0 - 47.5 0.00069
The refractive index decreases with a rise, and increases with a fall in
temperature. If the temperature is not exactly at 40 ℃, X is added to the
observed reading for each degree above or subtracted for each degree below
40 ℃ pro rata, where,
X for butyro-refractometer = 0.55
X for Abbe refractometer = 0.000365
Normally, the temperature of observation shall not deviate by more than 
2℃
Butyro-refractometer Readings and Indices of Refraction
BR Refractive BR Refractive BR Refractive
Reading Index Reading Index Reading Index
35.0 1.4488 40.5 1.4527 46.0 1.4565
35.5 1.4491 41.0 1.4531 46.5 1.4569
36.0 1.4495 41.5 1.4534 47.0 1.4572
36.5 1.4499 42.0 1.4538 47.5 1.4576
37.0 1.4502 42.5 1.4541 48.0 1.4579
37.5 1.4506 43.0 1.4545 48.5 1.4583
38.0 1.4509 43.5 1.4548 49.0 1.4586
38.5 1.4513 44.0 1.4552 49.5 1.4590
39.0 1.4517 44.5 1.4555 50.0 1.4593
39.5 1.4520 45.0 1.4558

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 40.0 1.4524 45.5 1.4562

Calculation with units of The maximum difference between duplicate determinations shall not exceed
expression 0.0002 units for the refractive index and 0.1 for the butyro-refractometer
reading.
Reference 1. IS 3507: 1966 (Reaffirmed year 2018). Methods of sampling and test
for Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
2. Rangappa, K.S. & Achaya, K.T. (1974). Indian Dairy Products. Asia
Pub. House, Bombay.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Free Fatty Acids in Milk Fat Products

Method No. FSSAI 01.089:2022 Revision No. & Date 0.0

Scope This method is used for the determination of free fatty acids in milk fat,
ghee, butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
3. Potassium hydroxide: It is highly corrosive. It reacts violently with
strong acids. While making solutions add pellets to water and not vice
versa.
Principle The FFA present in milk fat products is estimated by acid-base titration with
alkali (Sodium hydroxide) using phenolphthalein as an indicator and the end
point comes at around pH 8.3 (BIS, 1966) and expressed as % oleic acid
RCOOH + NaOH RCOONa + H2O

Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).

2. Conical flasks of 250 mL capacity.
3. Burette: 50 mL, graduated to 0.1 mL.
4. Pipettes.
Materials and Reagents 1. Ethanol or rectified spirit: 95% (v/v), sp. gr. 0.816, neutral to
phenolphthalein.
2. Sodium hydroxide or potassium hydroxide.
3. Potassium hydrogen phthalate.
4. Phenolphthalein indicator.
Preparation of reagents 1. Sodium hydroxide or potassium hydroxide: 0.1 N aqueous solution
accurately standardized potassium hydrogen phthalate.
2. Phenolphthalein indicator: 1.0% solution in 95 % (v/v) ethanol or
rectified spirit.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.
Method of analysis 1. Weigh 10 g of the milk fat sample in a 250 mL conical flask.

241 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. In another flask, bring 50 mL of ethanol to the boiling point and while
still above 70 ℃, neutralize it with 0.1 N sodium hydroxide using
phenolphthalein (using 0.5 mL).
3. Add the neutralized alcohol to flask containing milk fat sample and mix
the contents of the flask.
4. Bring the mixture to boil, and while it is still hot, titrate with
standardized 0.1 N sodium hydroxide, shaking vigorously during the
titration.
5. The end point of the titration is reached when the addition of a single
drop produces a slight, but a definite colour change persisting for at least
15 s.
Calculation with units of Free fatty acids: is expressed as the percentage of free fatty acids in the
expression sample, calculated as oleic acid, using the following formula:
T
Free fatty acids (as % oleic acid) = x 2.82
w
Where,
T is volume of 0.1 N alkali required for titration in mL;
w is mass in g, of milk fat sample taken.
In case, strength of alkali used in the titration is other than 0.1 N, use
following formula
Free fatty acids (as % oleic acid) =
𝑉 𝑥 28.2 𝑥 𝑁
𝑊
Where,
V is volume of alkali required for titration in mL;
N is the normality of alkali used in the titration
w is mass in g, of milk fat sample taken.

Reference 1. IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test
for Ghee (Butterfat). Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Reichert-Meissl and Polenske Value in Milk Fat
Products
Method No. FSSAI 01.090:2022 Revision No. & Date 0.0
Scope This method is used to determine the Reichert-Meissl and Polenske value in
milk fat, ghee, butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
Principle The fat is saponified using glycerol-potash diluted with water and acidified,
and thereafter steam distilled in a glass apparatus (Polenske distillation
apparatus) at a controlled rate. The condensed and cooled distillate is
filtered; the water-soluble acids which pass through are estimated by
titration with alkali to give the R.M. value, while the water-insoluble acids
collected on the filter paper are dissolved out in alcohol and titrated to give
the Polenske value.
Reichert-Meissl value is the number of mL of 0.1 N aqueous alkali solution
required to neutralize the water-soluble, steam volatile fatty acids distilled
from 5 g of milk fat under the precise conditions specified in the method.
The PV is the number of mL of 0.1 N aqueous alkali solution required to
neutralize the water-insoluble, steam volatile fatty acids distilled from 5 g
of milk fat under the precise conditions specified in the method.
Apparatus/Instruments 1. Graduated cylinders: 25 mL and 100 mL capacities.
2. Pipette: 50 mL capacity.

243 | M o M – D a i r y a n d D a i r y P r o d u c t s
3. The assembly of the apparatus for the distillation:
4. Flat-bottom boiling flask (Polenske): The flask shall be made of heat-
resistance glass and shall conform to the following details:
a. Volume contained to bottom of neck: 310 ± 10 mL
b. Length of neck: 75 ± 5 mm
c. Internal diameter of neck: 21 ± 1 mm
d. Overall height: 160 ± 5 mm
e. Diameter of base: 45 ± 5mm
f. Still-head: The still-head shall be made of glass tubing of wall
thickness 1.25 ± 025 mm, and shall conform to the shape shown
in Figure 5.3, and with the following dimensions:

A 180 ± 5 mm
B 107.5 ± 2.5 mm
C 80 ± 5 mm
D 70 ±5 mm
E 20 ± 2 mm
F 4 ± 1 mm
G (external diameter of bulb) 37.5 ± 2.5 mm
Internal diameter of tubing 8.0 ± 0.5 mm
Acute angle between sloping
60 ± 2°
part of still-head and vertical
A rubber stopper, fitted below the bulb of the longer arm of the still-head,
and used for connecting it to the flask shall have its lower surface 10 mm
above the centre of the side hole of the still-head.
a. Condenser: The condenser shall be made of glass and conform to the
following dimensions:

Overall length 520 ± 5 mm

Length of water jacket 300 ± 5 mm

Length of widened part above water jacket 70 ± 10 mm

Wall thickness of widened part 1.25 ± 0.25 mm

Internal diameter of widened part 20 ± 1 mm

External diameter of inner tube within water 12 ± 0.5 mm
jacket
Wall thickness of inner tube 1.0 ± 0.2 mm

244 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Wall thickness of outer jacket 1.25 ± 0.25 mm

External diameter of water jacket 30 ± 2 mm

b. Receiver: The receiver shall be a flask with two graduation marks on the
neck, one at 100 mL and the other at 110 mL.
c. Asbestos-board: An asbestos-board of 120
mm diameter and 6 mm in thickness, with a
circular hole of about 65 mm in diameter
shall be used to support the flask over the
burner. During distillation, the Polenske
flask shall fit snugly into the hole in the
board to prevent the flame from impinging
on the surface of the flask above the hole. A
new asbestos-board may conveniently be
prepared by bevelling the edge of the hole,
soaking in water, moulding the edge with a
flame, and drying.
d. Gas burner: The burner should be
sufficiently large to allow the distillation to
be completed in the time specified in the
procedure.
The apparatus shall be supported on a retort
Figure. Apparatus for
stand. determination of R.M. Value
of Ghee (IS 3508:1966)
A. Glass Funnel: Of approximate diameter
6 cm.
Materials and Reagents 1. Glycerol 98% (m/m).
2. Sodium hydroxide.
3. Concentrated sulphuric acid (specific gravity 1.84).
4. Ethanol 95% (v/v).
5. Glass beads, approximately 1.5 to 2.0 mm in diameter.
6. Phenolphthalein indicator.
7. Sodium hydroxide.
Preparation of reagents 1. Sodium hydroxide solution (50% (m/m)): Dissolve sodium hydroxide
in an equal weight of water and store the solution in a bottle protected
from carbon dioxide. Use the clear portion free of the solution from

245 | M o M – D a i r y a n d D a i r y P r o d u c t s
 deposit.
2. Dilute sulphuric acid: Use 1 N sulphuric acid (H2SO4) prepared by
adding 27 mL of concentrated sulphuric acid in 1 L distilled water.
Adjust so that 40 mL of 1 N sulphuric acid neutralize 2 mL of the 50%
sodium hydroxide solution.
3. Ethanol: 95% (v/v), neutralized to phenolphthalein immediately before
use, or neutralized denatured spirit.
4. Phenolphthalein indicator: 0.5% solution in 95% (v/v) ethanol or
rectified spirit.
5. Sodium hydroxide solution: Approximately 0.1 N aqueous solution of
Sodium hydroxide of accurately determined strength.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis
1. Weigh 5.00  0.01 g of fat sample into a Polenske flask, add 20 g of
glycerol and 2 mL of 50% Sodium hydroxide solution.
2. Heat the flask over a direct flame using a Bunsen burner, with
continuous mixing, until thw fat, including any drops adhering to the
upper parts of the flask is saponified and the liquid becomes perfectly
clear; avoid overheating during this saponification. Cover the flask with
a watch-glass.
3. Make a blank test without ghee, but using the same quantities of reagents
and following the same procedure, again avoiding overheating; such
overheating would be indicated by darkening of the solution. Measure
93 mL of boiling distilled water, which has been vigorously boiled for
15 min, into a 100 mL graduated cylinder.
4. When the soap is sufficiently cool to permit addition of the water
without loss, but before the soap has solidified, add water, draining the
cylinder for 5 secs, and dissolve the soap. If the solution is not clear
(indicating incomplete saponification), or is darker than yellow
(indicating overheating), repeat the saponification with a fresh sample
of ghee.
5. Add two glass beads, followed by 50 mL of the dilute sulphuric acid,
and connect the flask at once with the distillation apparatus.
6. Heat the flask without boiling its contents, until the insoluble acids are
completely melted, then increase the flame and distil 110 mL in between
19 and 21 min. Keep the water flowing in the condenser at a sufficient
speed to maintain the temperature of the issuing distillate between 18
and 21 ℃.
7. When the distillate reaches the 110 mL mark, remove the flame and

246 | M o M – D a i r y a n d D a i r y P r o d u c t s
 replace the 110-mL flask by a cylinder of about 25 mL capacity, to
collect drainings. Close 110 mL flask with its stopper and without
mixing the contents; place it in water at 15 °C for 10 min so as to
immerse the 110 mL mark. Remove the flask from the water, dry from
outside, and invert the flask carefully, avoiding wetting the stopper with
insoluble acids. Mix the distillate by four or five double inversions,
without violent shaking.
8. Filter through a dry 9 cm open-texture filter paper (Whatman filter paper
Grade 4) which fits snugly into the funnel. Reject the first runnings and
collect 100 mL in a dry volumetric flask; cork the flask and retain the
filtrate for titration.
9. Detach the still-head and wash the condenser with three successive 15
mL portions of cold distilled water, passing each washing separately
through the cylinder, the 110 mL flask, the filter and the funnel, nearly
filling the paper each time and draining each washing before filtering
the next. Discard the washings.
10. Dissolve the insoluble acids by three similar washings of the condenser,
the cylinder, and the filter, with 15 mL of neutralized ethanol, collecting
the solution in the 110 mL flask and draining the ethanol after each
washing. Cork the flask, and retain the solution for titration (for
Polenske value).
11. Reichert-Meissl or soluble volatile acid value: Pour 100 mL of the
filtrate (obtained in step F) containing the soluble volatile fatty acids into
a titration flask, add 0.1 mL of phenolphthalein indicator and titrate with
0.1 N sodium hydroxide solution until the liquid becomes pink.
12. Polenske or insoluble volatile acid value: Titrate the alcoholic solution
of the insoluble volatile acids after addition of 0.25 mL of
phenolphthalein indicator, with the 0.1 N sodium hydroxide solution
until the solution becomes pink.
Calculation with units of Reichert-Meissl value = 11 × (Actual Normality of sodium hydroxide) ×
expression (T1-T2)
Polenske value = 10 × (Actual Normality of sodium hydroxide) × (T1-T2)
Where,
T1 = volume in mL of 0.1 N sodium hydroxide solution used for
sample under step I;
T2 = volume in mL of 0.1 N sodium hydroxide solution used for blank
under step I;

247 | M o M – D a i r y a n d D a i r y P r o d u c t s
 T3 =volume in mL of 0.1 N sodium hydroxide solution used for sample
under step J,
T4 = volume in mL of 0.1 N sodium hydroxide solution used for blank
under step J;
Polenske values, and to a much slighter extent Reichert-Meissl
values, have been found to be low when determined at low barometric
pressures, such as may occur at high altitudes. The following factors may
be applied to values determined at a barometric pressure to convert them to
the values determined at normal pressure.

Correct Reichert Value = (Observed value - 10) log 760

 10
log P

Corrected Polenske Value = Observed value x 760 - 45

P - 45

Where,
P =barometric pressure in mm of mercury at the place and time of
determination.
Reichert-Meissl Value: The maximum deviation between duplicate
determinations shall not exceed 0.5 units.
Polenske Value: The maximum deviations between duplicate
determinations shall not exceed 0.3 units.
Reference IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test for
Ghee (Butterfat). Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

248 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Test for Detection of Vanaspati in Milk Fat Products (Baudouin Test)

Method No. FSSAI 01.091:2022 Revision No. & Date 0.0

Scope This method is applicable for the detection of vanaspati in milk fat, ghee,
butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your
eyes and skin. Concentrated hydrochloric acid is toxic if inhaled, so
avoid breathing it in and always handle it while under a fume hood. It
reacts with strong oxidizing agents to form toxic chlorine gas. Ethanol:
Extremely flammable. Use effective fume removal device when heating
or evaporating. Keep away from heat, sparks and open flame; avoid
breathing vapours.
2. Furfural: Keep away from heat, sparks and open flame. It reacts
violently with strong oxidizers. Perform all operations under an effective
fume removal device.
Principle Addition of sesame (til) oil to vanaspati is mandated by FSSAI. Therefore,
detection of sesame oil in milk fat is used as a marker to detect the
adulteration of milk fat with vanaspati. Sesame oil contains sesamolin and
sesamol, which are used to indirectly determine the presence of vanaspati.
Sesamolin on condensation with furfural produces a pink colour which is
used to detect vanaspati.
Apparatus/Instruments 1. 25 mL measuring cylinder.

Materials and Reagents 1. Concentrated hydrochloric acid (Approximately 11.6 N).

2. Furfural.
3. Ethanol.
Preparation of reagents 1. Furfural solution: 2% solution of furfural, in ethanol, distilled not
earlier than 24 h prior to the test from rectified spirit.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis 1. Take 5 mL of the melted ghee in 25 mL measuring cylinder provided

with a glass stopper, add 5 mL of hydrochloric acid and 0.4 mL of
furfural solution. Insert the glass stopper and shake vigorously for 2 min.

249 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Allow the mixture to separate.
3. Observe the colour in the acid layer.
Inference (Qualitative The development of a pink or red colour in the acid layer indicates presence
analysis) of vanaspati.
Confirm by adding 5 mL of water and shaking again. If the colour in acid
layer persists, vanaspati is present. If the colour disappears, it is absent.
Reference IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test for
Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

250 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Test for Detection of Mineral Oil in Milk Fat Products (Holde’s test)

Method No. FSSAI 01.092:2022 Revision No. & Date 0.0

Scope This method is used to detect the presence of mineral oil in milk fat, ghee,
butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Potassium hydroxide is a highly corrosive chemical and contact
can irritate and burn the skin and eyes
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
Principle Unlike oils and fats, mineral oils are not saponifiable by alkali. This
characteristic behaviour of mineral oils is used as the basis for their
detection in ghee. Known as Holde’s test, the presence of as little as 0.3 %
of mineral oil in ghee is detected by saponifying the fat sample and mixing
the hot soap solution with water. The appearance of turbidity indicates the
presence of mineral oil in the sample (Winton and Winton, 1999).
Apparatus/Instruments Flat-bottom flask, 250 mL capacity, fitted with reflux condenser.
Materials and Reagents 1. Potassium hydroxide.
2. Ethanol.
Preparation of reagents Alcoholic potassium hydroxide (4%): Dissolve 4 g of potassium hydroxide
in approximately 10 mL water and make the final volume to 100 mL with
ethanol (95%, v/v). The solution should be colourless or very pale yellow.
Store this solution in a dark place.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.
Method of analysis 1. Saponify 1 g of the fat with 25 mL of 4% alcoholic potassium hydroxide
for about 1 h.
2. Cool and add 100 mL hot (just boiled) distilled water.
3. Appearance of turbidity indicates the presence of mineral oil.
Inference (Qualitative Appearance of turbidity indicates the presence of mineral oil.
analysis)
Reference 1. Kumar, A.; Lal, D.; Seth.; Sharma. V. (2005) Turbidity test for
detection of liquid paraffin in ghee. Indian Journal of Dairy Science.
58(4): 298
2. Winton, A.L. and Winton, K.B. (1999) Techniques for Food Analysis.

251 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Allied Scientific Publishers, Bikaner, p. 358.
Approved by Scientific Panel on Methods of Sampling and Analysis

252 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Test for Determination of Fatty Acid Composition in Milk Fat
Products by Gas-Liquid-Chromatography (GLC)
Method No. FSSAI 01.093:2022 Revision No. & Date 0.0
Scope This method is for the determination of fatty acid composition in milk fat,
ghee, butter oil, anhydrous milk fat and anhydrous butter oil. by gas-liquid-
chromatography (GLC).
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Methanol: It is a highly flammable liquid and vapour. Keep away from
heat, sparks, open flames, hot surfaces. Perform all operations under a
fume hood.
2. Potassium hydroxide: Avoid formation of dust; avoid breathing dust,
vapours, mist, gas. Use local exhaust ventilation to control airborne
levels below recommended exposure limit.
3. n-hexane: Avoid storage near extreme heat, ignition sources or open
flame. Use respiratory protective device against the effect of
fumes/aerosols. Perform all operations under an effective fume removal
device.
Principle The fatty acid composition of milk fat is defined as mass fraction of
individual fatty acids as free acid expressed as g/100 g of total fatty acids.
Gas liquid chromatography (GLC) has become a useful tool in the
quantitative analysis of the fatty acid composition of natural lipids. This
method (IDF, 1999a, b) involves base-catalyzed methanolysis of the
glycerides in an essentially non- alcoholic solution. After a certain reaction
time, the mixture is neutralized by addition of crystalline NaHSO4 to avoid
saponification of pre-formed esters. Fatty acid methyl esters (FAME) are
separated and determined by capillary gas-liquid-chromatography (GLC).
Quantification of individual FAME in the test sample is done by reference
to a milk fat with known composition.
Apparatus/Instruments 1. Test tube: 16 mm x 100 mm, fitted with PTFE-lined screw cap.
2. Graduated pipettes of 1 mL and 10 mL capacity.
3. Vortex mixer.
4. Centrifuge, as used for the butyrometric determination of milk fat.
5. Gas-liquid chromatography: The instructions given relate to the usual
equipment for gas-liquid chromatography, employing a suitable
injection system, a capillary column and a flame ionization detector
(FID).

253 | M o M – D a i r y a n d D a i r y P r o d u c t s
6. Injector: Injectors of the vaporizing type (split injector or programmed
temperature injector, PTV) that are maintained at least at 220 ℃ (in the
case of a PTV, final injector temperature should be at least 220 ℃) or,
in the case of a cold on-column injector, the temperature should be
maintained some degrees below the boiling point of the solvent.
7. Oven, capable of being temperature-programmed from near ambient
temperature up to 260 ℃.
8. Columns: Columns made of deactivated glass or fused silica with an
inner diameter of 0.20 mm to 0.53 mm, a length of 10 m to 100 m and a
stationary phase film thickness of 0.2 m to 1.0 m. The stationary
phase is of the nitroterephthalic acid-modified or unmodified
polyethylenglycol type (for example Carbowax 20M, Supelcowax 10,
DB-Wax, Stabilwax, HP-20M, Superox II, CP-Wax 52CB, BP-20,
FFAP, SP-1000, DB-FFAP, CP-Wax 58CB).
9. Flame ionization detector capable of being heated to a temperature 20
℃ above the final temperature of the column oven.
10. Carrier gas pneumatics of the oxygen-diffusion proof type. The column
head pressure should be maintained to give a linear carrier gas velocity
of 15 to 25 cm/s for nitrogen, 25 to 35 cm/s for helium, and 35 to 55
cm/s for hydrogen at the initial oven temperature. If a vaporizing split
injector is used, the split vent flow is controlled to give a split-ratio of
1:50 to 1:100.
11. Syringe of the ‘plunger-in-barrel’ type, with maximum capacity of 10
l, graduated to 0.1 l.
12. Computing integrator, capable of baseline indication and reintegration.
Note:
13. Performance specification of the GLC: Prepare a test mixture consisting
of methyl butyrate, methyl stearate and methyl oleate at a concentration
of 0.1 mg/mL each in the fat solvent. Separate this mixture by gas
chromatography using the same operating conditions as applied to milk
fat samples. The maximum of a test substance peak should rise to
approximately three-quarters full scale of the computing integrator.
Largest possible separation of methyl butyrate from the solvent peak and
a resolution R of 1.5 (baseline resolution) between methyl stearate and
methyl oleate should be obtained. R is calculated as:
14. R = 2d/ (W1 + W2)
Where,
a. W1 and W2 are the widths, in mm, of the peaks for methyl
stearate and methyl oleate, measured between the points of
intersection of the tangents at the inflexion points of the curve
with the baseline;

254 | M o M – D a i r y a n d D a i r y P r o d u c t s
 b. d is the distance, in mm, between the respective peak maxima
for methyl stearate and oleate.
15. Operating conditions for GLC: Select operating conditions, that is,
column length and inner diameter, stationary phase film thickness,
initial oven temperature, temperature programme rate(s), final oven
temperature, carrier gas flow rate to fulfill the specifications as
described above in performance specifications of GLC.
Materials and Reagents Use only reagents of recognized analytical grade, unless otherwise
specified, and distilled or demineralized water or water of equivalent purity.
1. n-alkane (n-pentane, n-hexane or n-heptane).
2. Methanol, containing not more than 0.5% (m/m) water.
3. Sodium hydrogen sulfate monohydrate (NaHSO4.H2O).
4. Potassium hydroxide (approximately 2 N methanolic solution
5. Reference fatty acid methyl esters: For identification purposes. The
reference FAME set should be of high purity (> 90%) and should
consist, at least, of the saturated, even numbered, straight chain fame
having 4 to 22 carbon atoms in addition to oleic, linoleic and linolenic
acid methyl ester.
6. Reference milk fat: For quantification purposes use a milk fat with
known fatty acid composition (for example reference material CRM
164, Community Bureau of Reference, Commission of the European
communities, Brussels, Belgium).
7. Fat solvent: An appropriate n-alkane (pentane, hexane, heptane) or
dichloromethane.
8. Carrier gas: Hydrogen, helium or nitrogen, purity at least 99.999%. The
oxygen content should be < 2 ppm.
9. Other gases: Nitrogen and hydrogen, purity at least 99,995%, free from
organic impurities (CnHm < 1 ppm); synthetic air, free from organic
impurities (CnHm < 1 ppm).
10. Transesterification reagent, potassium hydroxide or sodium methoxide,
methanolic solution of approximately 2 mol/l. Dissolve 11.2 g of
potassium hydroxide in 100 mL of methanol and mix well.
Alternatively, dissolve 10.8 g of sodium methoxide in 100 mL of
methanol and mix well. The sodium methoxide methanolic solution may
also be prepared by dissolving 4.6 g of metallic sodium in methanol or
by diluting a commercially available solution of approximately 5.4
mol/l.
Preparation of reagents 11. Potassium hydroxide (approximately 2 N methanolic solution): Dissolve
11.2 g potassium hydroxide in 100 mL methanol. Alternatively, sodium
methoxide, approximately 2 N methalonic solution may be used.

255 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Prepare this solution by dissolving 10.8 g methoxide in 100 mL
methanol.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.
Method of analysis Step A: Preparation of fatty acid methyl esters of milk fat
1. Weigh to the nearest 5 mg, 100 mg of the prepared milk-fat sample in a
test tube.
2. Dissolve the sample in 5 mL solvent (n-alkane) and mix, add 0.2 mL
transesterification reagent, cap the tube and mix the contents vigorously
by means of the vortex mixer for 1 min.
3. After an additional reaction time of 5 min, add 0.5 g solid sodium
sulphate and mix. Centrifuge the sample for 3 min at room temperature
(25±3oC). An aliquot from the clear supernatant is taken for gas
chromatographic analysis.
4. The ester solution may be decanted and stored at refrigeration
temperatures or, preferably, in a deep-freezer for several days.
Precautions have to be taken to avoid losses due to the volatility of milk
fat fatty acid methyl esters.
Notes: Sodium methoxide in methanol may be prepared by dissolving 4.6 g
metallic sodium in methanol. Special precautions must be taken when
handling metallic sodium.
n-acyl lipids (for example sphingolipids) are not properly transesterified by
base-catalyzed methanolysis. These substances occur primarily in lipids of
whey derived products.
Alternative procedure for the preparation of FAME using acid
catalysed transesterification of glycerides: This method is suitable for the
analysis of partially lipolysed milk fat (fat acidity greater than 1 mmol free
fatty acids per 100 g of milk fat). In such a case, the following method for
the preparation of methyl esters of milk fat should be followed. In this
method, the partially hydrolyzed milk fat is converted to FAME by acid-
catalysed transesterificaiton of glycerides bound acids with concomitant
esterification of free fatty acids:
1. Weigh to the nearest 0.10 g, 2.0 g of the prepared ghee sample in an
ampule, add 0.4 mL transesterification reagent. Flame seal the ampule.
2. Heat the ampule for 3 h in an oven maintained at 105 ℃ with intermittent
mixing of the contents. Cool to room temperature (25±3oC) and allow
for complete phase separation.

256 | M o M – D a i r y a n d D a i r y P r o d u c t s
3. An aliquot of the upper phase is taken for further analysis. Dilution of
the esters formed with an appropriate solvent may be necessary.
Notes: Substitution of the ampule with another suitable derivitization vial
or similar dimension is possible, provided leak-tight closure prevents losses
of volatile esters.
Instead of a hot air oven, a boiling water-bath may be used.
Step B: Determination of fatty acid composition by GLC
1. Sample injection: In general, follow the manufacturer’s instructions for
the injector employed. In the case of a vaporizing split injector, the
following guidelines should be obeyed-
2. A 0.5 l to 1.5 l test portion fatty acid methyl ester (FAME) is drawn
up with a micro syringe.
3. Withdraw the sample into the barrel of the syringe.
4. Insert the needle into the heated injector and after a dwell time of 3 secs
to 5 sec, the plunger is depressed rapidly.
5. Immediately thereafter, the syringe needle is removed from the injector.
If sampling is done by the cold on-column technique, the test portion is
diluted sufficiently with solvent (n-alkane; for example, 1:10) and
injected at an oven temperature at or some degrees below the boiling
point of the solvent used.
a. Qualitative analysis:
1. Analyze reference FAME under the same working conditions as used
for the test sample. Note the retention times of the reference substances.
Esters elute in order of increasing number of carbon-atoms and in order
of increasing number of double bonds for a given number of carbon-
atoms, for example methyl palmitate elutes before methyl stearate; fame
with 18 carbon-atoms elute in the order: methyl stearate, methyl oleate,
methyl linoleate, methyl linolenate. Branched chain FAME elute before
the straight chain ester with the same number of c-atoms in the order:
iso-branched before anteiso-branched. The chromatogram given in
Figure 5.10 is a further aid for the tentative identification of milk fat
FAME. Identify the peaks of the test sample by comparison with the
retention data obtained with the reference mixture and by comparison
with the chromatogram in the Figure below.

257 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Figure: Separation of milk fat fatty acid methyl ester by GLC
(Source: IDF 184: 2002)
b. Quantitative analysis
1. Determination of correction factors: Transmethylate the reference milk
fat as described above (See Step A for the preparation of FAME), and
analyze the obtained fame mixture under the same working conditions
as used for the test sample. Terminate recording the chromatogram after
complete elution of behenic acid (C22:0). To estimate the percentage of
a component represented by a peak in the chromatogram, use the method
of normalization, which assumes that all components of the sample are
represented in the chromatogram, so that the sum of the area under the
relevant peaks represents 100% of the sample constituents (100%
elution). Determine the areas of peaks attributable to fame (all peaks in
the chromatogram except the solvent peak, peaks due to addition of
stabilizing agents and those which appear also in blank runs) by using
the computing integrator. Calculate the percentage of the area
represented by the peak of component i by:

Percent area of component Ai (Ai%) =

Ai
x 100
 Ai

258 | M o M – D a i r y a n d D a i r y P r o d u c t s
Where Ai is the area of the peak corresponding to component i, and Ai, is
the sum of the areas under all peaks.
To determine correction factors CFi which are used to convert the
percentages of peak areas into mass percentages of components, the known
mass percentage (mi%) of the component i in the reference milk fat is
divided by the area percentage of the corresponding peak in the calibration
run:

mi% m i x  Ai
Cfi = Ai% =  mi x A i

Where mi is the mass (expressed as free fatty acid) of component i and mi
is the total of the masses (expressed as free fatty acids) of the various
components in the reference milk fat.
Individual CFi values are standardized with respect to the CF value of
methyl palmitate (relative CF’palmitate = 1) to obtain the relative correction
factor of component i (CF’i):
CFi
Cf’i = CF palmitate
ii. Test sample: Analyze the transmethylated test sample and determine
peak areas as described in quantitative analysis of reference fat. Mass
percentages of individual components expressed as g free fatty acid per 100
g of total fatty acids are obtained by:
CF' i x A i x 100
Percent mass of component i = CF' i x A i 
Unidentified sample components (“unknown”) are included in the final
report and a CF’ value of 1 is used for calculations.
Notes:
1. Instead of injecting samples manually, an automatic sample may be
used.
The stationary phase specified in apparatus (specification of column) may
be substituted by other polar phases provide that a similar resolution of
FAME is obtained.
Method 2

259 | M o M – D a i r y a n d D a i r y P r o d u c t s
 (AOAC 996.06 21st Edn. 2019)
Fat and fatty acids are extracted from food by hydrolytic methods (acidic
hydrolysis for most products, alkaline hydrolysis for dairy products, and
combination for cheese). Fat is extracted into ether, then methylated to fatty
acid methyl esters (FAMES) using BF3 in methanol. FAMEs are
quantitatively measured by capillary gas chromatography (GC) with Flame
Ionization Detector (FID). Results of different individual or category of
fatty acids are calculated based upon peak area of individual fatty acids and
combining them respectively. FAME mixture of 37 fatty acids is used for
identification.
Calculation with units of As mentioned in the Method of Analysis
expression
Reference 1. IDF (2002) IDF Standard 182, Milk fat. Preparation of fatty acid methyl
esters. International Dairy Federation, Brussels;
2. IDF (2002) IDF Standard 184, Milkfat. Determination of the fatty acid
composition by gas-liquid chromatography. International Dairy
Federation, Brussels.
3. AOAC 996.06 21st End. 2019 Fatty Acids in Oils and Fats
Approved by Scientific Panel on Methods of Sampling and Analysis

260 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Peroxide Value in Milk Fat Products

Method No. FSSAI 01.094:2022 Revision No. & Date 0.0

Scope This method is applicable for determining the of peroxide value of milk fat,
ghee, butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Chloroform: Inhaling chloroform vapors may cause drowsiness or
dizziness. Use in a well ventilated hood Perform all operations under a
fume hood.
2. Potassium iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
Principle The usual method of assessment of rancidity in ghee is by determination of
peroxide value (PV) which is reported in units of milliequivalents of
peroxide oxygen per kg of fat or mL of 0.002 N sodium thiosulphate per g
of sample. The most common method for PV determination is based on
iodometric titration which measures the iodine produced from potassium
iodide by the peroxides present in a fat or oil.

Apparatus/ 1. 250 mL glass stoppered Erlenmeyer flask.

Instruments
2. Analytical balance (Readability 0.001 g).
Material and Reagents 1. Glacial acetic acid.
2. Chloroform (AR grade).
3. Sodium thiosulphate (>99% purity).
4. Potassium iodide.
5. Sodium thiosulphate.

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Preparation of reagents 1. Acetic acid-chloroform solution - Mix 3 volumes of acetic acid with 2
volumes of chloroform.
2. Potassium iodide solution, saturated – Dissolve excess KI in freshly
boiled water; excess solid must remain; store this solution in dark. Test
daily by adding 0.5 mL to 30 mL CH3COOH-CHCl3 (solution a), then
add 2 drops of 1% starch solution. If solution turns blue requiring more
than 1 drop of 0.1 N sodium thiosulphate to discharge colour, prepare
fresh solution.
3. Standard sodium thiosulphate solution (0.1 N) - Dissolve approximately
24.8 g of sodium thiosulphate crystals (Na2S2O3.5H2O) in distilled water
and make up to 1000 mL.

Standardise this solution by the following procedure:

Weigh accurately about 5.0 g of finely powdered potassium dichromate
which has been previously dried at 105 °C ± 2 °C for one hour, dissolve
it in distilled water and make up to 1 L. For standardization of sodium
thiosulphate, pipette 25 mL of this solution into a 250 mL conical flask.
Add 5 mL of concentrated hydrochloric acid and 15 mL of a 10 percent
potassium iodide solution. Allow to stand in dark for 5 min and titrate
the content with sodium thiosulphate solution using starch as indicator
at the end. End point is change of blue colour to green

N = 25W/49.03 V

Where, N = Normality of the sodium thiosulphate

W = Weight in g of the potassium dichromate
V = Volume in mL of sodium thiosulphate solution required for
titration
For 0.01 N dilute 0.1 N with freshly boiled and cooled water.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis 1. Weigh 5 ± 0.5 g sample in a 250 mL glass stoppered Erlenmeyer flask,
add 30 mL of acetic acid-chloroform solution and swirl to dissolve.
2. Add 0.5 mL of saturated KI solution from Mohr pipette, let it stand with
occasional shaking for 1 min, and add 30 mL water.
3. Slowly titrate with 0.1 N sodium thiosulphate solution with vigorous
shaking until yellow colour is almost gone.
4. Add about 0.5 mL of starch solution and continue titration by shaking
vigorously to release all iodine from chloroform layer until blue just

262 | M o M – D a i r y a n d D a i r y P r o d u c t s
 disappears.
5. If less than 0.5 mL 0.1 N Na2S2O3 is used, repeat determination with
0.01 N Na2S2O3.
6. Conduct blank determination (must be less than 0.1 mL 0.1N Na2S2O3);
subtract from sample titration.
Calculation with units of S x N x 1000
expression Peroxide value (milliequivalents oxygen / kg oil) =
Weight of Sample

Where,
S = mL of Na2S2O3 (blank corrected) used and
N = Normality of Na2S2O3
Reference 1. AOAC 965.33. 21st Edn. (2019). Official method Peroxide Value of Oils
and Fats. AOAC International, USA.
2. IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test
for Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

263 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Iodine Value in Milk Fat Products

Method No. FSSAI 01.095:2022 Revision No. & Date 0.0

Scope This method is applicable for determining the iodine value in milk fat, ghee,
butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your
eyes and skin. Concentrated hydrochloric acid is toxic if inhaled, so
avoid breathing it in and always handle it while under a fume hood. It
reacts with strong oxidizing agents to form toxic chlorine gas.
2. Carbon tetrachloride: Carbon tetrachloride is a carcinogen. Avoid
contact with skin and eyes. Avoid inhalation of vapour or mist. Carry
out all reaction in a fume hood.
3. Potassium Iodide: It is incompatible with strong oxidizing and reducing
agents. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Avoid
ingestion and inhalation.
Principle The iodine value of oil/fat is the number of grams of iodine absorbed by 100
g of the oil/fat, when determined by using Wij’s solution. The iodine value
is a measure of the amount of unsaturation (number of double bonds) in a
fat. The oil / fat sample taken in carbon-tetrachloride is treated with a known
excess of iodine monochloride solution in glacial acetic (Wij’s solution).
The excess of iodine monochloride is treated with potassium iodide and the
liberated iodine is estimated by titration with sodium thiosulfate solution.
Apparatus/Instruments 1. 250 mL stopper conical flask (Iodine flask).
2. Analytical balance (Readability 0.001 g).
Materials and Reagents 1. Potassium dichromate CRM grade.
2. Concentrated hydrochloric acid (Approximately 11.6 N).
3. Glacial acetic acid, free from ethanol.
4. Carbon tetrachloride.

264 | M o M – D a i r y a n d D a i r y P r o d u c t s
 5. Iodine mono-chloride (ICl).
6. Sodium thiosulphate.
7. Mercuric iodide.
8. Potassium iodide (free from potassium iodate).
9. Starch.
Preparation of Reagents 1. Potassium iodide (free from potassium iodate) - 10% solution. Prepare
a freah solution by dissolving 10 g of potassium iodide in 90 ml of water.
2. Starch solution - Mix 5 g of starch and 0.01 g of the mercuric iodide
with 30 mL of cold water and slowly pour it with stirring into one litre
of boiling water; boil for three min. Allow to cool and decant off the
supernatant clear liquid.
3. Wij’s Iodine monochloride solution: Prepare this solution by one of the
following three methods, and store in a glass stoppered bottle in a cool
place, protected from light.
(a) Dissolve 10 mL of iodine monochloride in about 1800 mL of glacial
acetic acid and shake vigorously. Pipette 5 mL of this solution, add 10 mL
of potassium iodide solution and titrate with 0.1 N standard sodium
thiosulphate solution using starch as indicator. Adjust the volume of the
solution till it is approximately 0.2 N
(b) Dissolve 13 g resublimed iodine in 1 litre acetic acid, using gentle heat,
if necessary, and determine the strength by titration with standard sodium
thiosulphate solution. Set aside 50 to 100 ml of the solution and introduce
chrlorine gas into the remainder until the charachterisitc colour change
occurs and the halogen content is nearly double as ascertained again by
titration. If the halogen content has been more than doubled, reduce it by
adding the requisite quantity of the iodine-acetic acid solution. A slight
excess of iodine does no harm, but avoid an excess of chlorine.
Example: If the titration of 20 ml of original iodine-acetic acid solution
requires 22 ml of standard thiosulphate, 20 ml of the finished Wij’s solution
should require between 43 and 44 ml (and not more than 44 ml) of the same
sodium thiosulphate solution.
(c) Dissolve 8 g of iodine trichloride in approximately 450 ml of acetic acid.
Dissolve separately 9 g of iodine in 450 ml acetic acid using heat, if
necessary. Add gradually the iodine solution to the iodine trichloride until
the colour has changed to reddish brown. Add 50 ml more of iodine solution
and dilute the mixture with acetic acid till 10 ml of the mixture is equivalent
to 20 ml of standard sodium thiosulphate solution when the halogen content
is estimated by titration in the presence of an excess of potassium iodide
and water. Heat the solution to 100 °C for 20 min and cool. Prevent access
of water vapour in preparing the solution.

265 | M o M – D a i r y a n d D a i r y P r o d u c t s
 4. Standard sodium thiosulphate solution (0.1 N) - Dissolve
approximately 24.8 g of sodium thiosulphate crystals (Na2S2O3.5H2O)
in distilled water and make up to 1000 mL.

Standardise this solution by the following procedure:

Weigh accurately about 5.0 g of finely powdered potassium dichromate
which has been previously dried at 105±2 °C for one hour, dissolve it
in distilled water and make up to 1 L. For standardization of sodium
thiosulphate, pipette 25 mL of this solution into a 250 mL conical flask.
Add 5 mL of concentrated hydrochloric acid and 15 mL of a 10 percent
potassium iodide solution. Allow to stand in dark for 5 min and titrate
the content with sodium thiosulphate solution using starch as indicator
at the end. End point is change of blue colour to green

N = 25W/49.03 V

Where, N = Normality of the sodium thiosulphate

W = Weight in g of the potassium dichromate
V = Volume in mL of sodium thiosulphate solution required for titration

Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis 1. Weigh accurately 0.40 to 0.45 g of milk fat/ghee, into a 500 mL conical
flask with glass stopper, to which 15 mL of carbon tetrachloride has
been added; mix the contents well.
2. Add 25 mL of Wij's solution using burette and replace the glass stopper.
Swirl for proper mixing and keep the flasks in dark for one h.
3. After standing, add 20 mL of potassium iodide solution, followed by
150 mL of recently boiled and cooled water, rinsing in the stopper also.
4. Titrate liberated iodine with standardized sodium thiosulphate solution,
using starch as indicator at the end until the blue colour formed
disappears after thorough shaking with the stopper on.
5. Conduct blank determinations in the same manner as test sample but
without oil/fat.
6. Slight variations in temperature appreciably affect titre of iodine
solution as chloroform has a high coefficient of expansion. It is thus
necessary that blanks and determinations are made at the same time.

266 | M o M – D a i r y a n d D a i r y P r o d u c t s
Calculation with units of 12.69 (B −S)N
expression Iodine value =
W
Where,
B = volume in mL of standard sodium thiosulphate solution required for
the blank.
S = volume in mL of standard sodium thiosulphate solution required for
the sample.
N = normality of the standard sodium thiosulphate solution.
W = weight in g of the sample.
Reference A.O.A.C. 17th edn, 2000, Official method 920. 159 – Iodine absorption
number of oils and fats.
IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test for
Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

267 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Saponification Value in Milk Fats

Method No. FSSAI 01.096:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of saponification value in
milk fat, ghee, butter oil, anhydrous milk fat and anhydrous butter oil.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Potassium hydroxide is a highly corrosive chemical and contact can
irritate and burn the skin and eyes.
2. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your
eyes and skin. Concentrated hydrochloric acid is toxic if inhaled, so
avoid breathing it in and always handle it while under a fume hood. It
reacts with strong oxidizing agents to form toxic chlorine gas.
Principle The milk fat sample is saponified by refluxing with a known excess of
alcoholic potassium hydroxide solution. The alkali required for
saponification is determined by titration of the excess potassium hydroxide
with standard hydrochloric acid. The saponification value is expressed as
the number of mg of potassium hydroxide required to saponify 1 g of fat.
Apparatus/Instruments 1. 250 mL capacity conical flask with ground glass joints.
2. One m long air condenser, or reflux condenser (65 cm minimum in
length) to fit the 250 mL capacity conical flask.
3. Hot water bath or electric hot plate fitted with thermostat.
4. Analytical balance (Readability 0.001 g).
Materials and Reagents 1. Aldehyde free alcohol: conforming to IS: 323-1959.
2. Potassium hydroxide.
3. Phenolphthalein indicator.
4. Concentrated hydrochloric acid (Approximately 11.6 N).

Preparation of reagents 1. Alcoholic potassium hydroxide solution (0.5 N): Dissolve 35 to 40 g of

potassium hydroxide in 20 mL of distilled water, and add sufficient
aldehyde-free alcohol to make up to 1000 mL. Allow the solution to
stand in a tightly stoppered bottle for 24 h, then quickly decant the clear

268 | M o M – D a i r y a n d D a i r y P r o d u c t s
 supernatant into a suitable, tight container, and standardize the solution
to 0.5 ± 0.005 N and keep in a bottle closed tight with a cork or rubber
stopper.
2. Phenolphthalein indicator solution: Dissolve 1.0 g of phenolphthalein in
100 mL 95% ethanol .
3. Standard hydrochloric acid (0.5 N ± 0.005 N): Dilute 4.31 mL of
concentrated hydrochloric acid to 100 mL with distilled water.
Standardize the prepared solution against 0.5 N sodium carbonate
solution.
4. Neutral ethyl alcohol soluuion: Titrate 95% ethanol with few drops of
sodium hydroxide solution (preferably 0.1 N) in presence of
phenolphthalein indicator until a faint pink colour is obtained that persist
for 15-30 sec.
Sample preparation Refer method FSSAI 01.085:2022 for sample preparation.
Method of analysis 1. Mix the melted sample thoroughly and weigh about 1.5 to 2.0 g of dry
sample into a 250 mL Erlenmeyer flask.
2. Pipette 25 mL of the alcoholic potassium hydroxide solution into the
flask. Add 1 or 2 glass beads..
3. Connect the sample flask with air condensers, keep on the water bath,
boil gently but steadily until saponification is complete, as indicated by
absence of any oily matter and appearance of clear solution. Clarity may
be achieved within 1 h of boiling.
4. After the flask and condenser have cooled wash down the inside of the
condenser with about 10 mL of hot ethyl alcohol neutral to
phenolphthalein.
5. Titrate the excess potassium hydroxide with standardised 0.5 N
hydrochloric acid, using about 0.5 mL phenolphthalein indicator.
6. Make a blank determination upon the same quantity of the potassium
hydroxide solution at the same time under the same conditions..
Calculation with units of 56.1 (B −S)N
expression Saponification Value =
W
Where,
B = Volume in mL of standard hydrochloric acid required for the blank.
S = Volume in mL of standard hydrochloric acid required for the sample

269 | M o M – D a i r y a n d D a i r y P r o d u c t s
 N = Normality of the standard hydrochloric acid
W = Weight in g of the oil/fat taken for the test
56.1 = molecular weight of potassium hydroxide
Reference 1. A.O.A.C 17th edn, 2000, Official method 920.160 Saponification
number of oils and fats /
2. IS 3508: 1966 (Reaffirmed year 2018). Methods of sampling and test
for Ghee (Butterfat). Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

270 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Detection of Adulteration of Milk Fat Products with Vegetable Oils by
Reversed Phase- High Performance Liquid Chromatography (RP-
HPLC)
Method No. FSSAI 01.097:2022 Revision No. & Date 0.0
Scope This method is applicable for the detection of adulteration ghee with
vegetable oils by RP-HPLC. The method is applicable for the detection of
the soybean oil, coconut oil, sunflower oil, groundnut oil and designer
vegetable oil in ghee.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Potassium hydroxide: It is highly corrosive. It reacts violently with
strong acids. While making solutions add pellets to water and not vice
versa.
2. Chloroform: Decomposes slowly on exposure to light and on exposure
to air it releases toxic and corrosive gases/vapours (phosgene, chlorine,
hydrogen chloride). Perform all operations under a fume hood.
3. Methanol: Highly flammable; keep away from heat, sparks and open
flame. Perform all operations under a fume hood.
4. n-hexane: Avoid storage near extreme heat, ignition sources or open
flame. Use respiratory protective device against the effect of
fumes/aerosols. Perform all operations under an effective fume removal
device.
5. Acetonitrile: Do not inhale substance/mixture. Avoid generation of
vapours/aerosols. Keep away from open flames, hot surfaces and
sources of ignition. Keep container tightly closed in a dry and well-
ventilated place (+5 °C to +30 °C).

Principle The RP-HPLC method is based on the detection of cholesterol and β-

sitosterol as markers in the unsaponifiable matter (USM). β- Sitosterol, a
plant sterol serves as an indicator for the adulteration in ghee by vegetable
oils
Apparatus/Instruments 1. Beakers (100 mL, 1000 mL).
2. Volumetric flasks (10 mL,100 mL, 200 mL).
3. Screw capped test tubes (50 mL).
4. Micro centrifuge stands (2 No.).
5. Adjustable micro pipettes (1 mL and 10 mL).
6. Millipore filter paper Nylon (0.22 µm).
7. Analytical balance (Readablity 0.001 g).
8. Shaking water bath.
9. Vortex mixer.
10. Centrifuge.

271 | M o M – D a i r y a n d D a i r y P r o d u c t s
 11. HPLC equipped with Photo Dioade Array (PDA) detector, pumps, auto
sampler, column oven and degasser.
12. Column: Reversed phase C-18, 4.6 × 250 mm ID, 5μ 120 Å particle
size
13. Micro concentrator.
Materials and Reagents 1. β- sitosterol- Purity.
2. Stigmasterol- Purity.
3. Methanolic potassium hydroxide.
4. n-Hexane (HPLC grade).
5. Chloroform.
6. Methanol.
7. Cholesterol.
8. Acetonitrile (HPLC grade).
9. Isopropanol (HPLC grade).
Preparation of reagents Preparation of stock solution of standards
1) Stock solution A (Cholesterol): Prepare 1 mg/mL of stock solution
in chloroform.
2) Stock solution B (Stigmasterol): Prepare 1 mg/mL of stock solution
in chloroform.
3) Stock solution C (β-sitosterol): Prepare 1 mg/mL of stock solution in
chloroform.
4) Prepare diluted standards up to 1 µg/mL of individual and mixture of
(1 to 3) in methanol.

Sample Preparation Refer method FSSAI 01.085:2022 for sample preparation.

Method of analysis Preparation of unsaponifiable matter (USM)
1. Weigh 1 g of fat in a screw capped test tube.
2. Add 25 mL of 5% methanolic potassium hydroxide.
3. Keep the tubes in water bath maintained at 90 ℃ for 50 min with
vigorous shaking at regular intervals.
4. Take out the tubes from water bath and add 5 mL water and 15 mL
hexane.
5. Vortex the mixture for 1min.
6. Centrifuge at 1700 g (around 3000 rpm) for 5 min.
7. Pipette out the upper hexane layer completely and dry (preferably in
micro concentrator) to USM.
8. Dissolve the dried USM in 300 µl of chloroform and add 500 µl
methanol, vortex and mix.
9. Filter the above extract through 0.22 µm nylon filter paper.

HPLC analysis

272 | M o M – D a i r y a n d D a i r y P r o d u c t s
Perform the HPLC analysis on the material obtained above after filtration.
HPLC conditions are mentioned below Run the sample (obtained after
filteration from from 0.22 µm nylon filter paper), reference standards of
cholesterol, stigmasterol and β- sitosterol also on HPLC.

HPLC Condition
Column C-18, 4.6 mm (i.d.) × 250 mm, 5µm
Column Oven Temp 30 ℃
Flow rate 1.5 mL/min
PDA wavelength 205 nm
Injection volume 20 µl
Run time 35 min
Washing solution Acetonitrile: Isopropanol (9:1)

Mobile Phase: Acetonitrile: Isopropanol (9:1), filtered and degassed

The RP-HPLC method resolves cholesterol, stigmasterol and β- sitosterol
at Retention Time (RT values) of 22.8 ±0.2, 25.5 ±0.2, 29.05 ±0.2 min
under prescribed conditions of the method.

Peak identification and Confirmation

1. Identify the peaks in the sample by comparing the retention time with
that of reference standards.
2. Limit of Detection (LOD) would vary with the type of oil adulterated.
3. Method is capable of detecting coconut oil at 5%, refined soybean oil at
1%, groundnut oil at 2% and sunflower oil at 1%.
4. The detection and reporting of any result below than 1% should be done
cautiously.

273 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Calculation with units of
expression
a

Figure: a) HPLC chromatogram of standard cholesterol (Retention time:

17.697 min), Campasterol and stigmasterol (Retention time: 19.933 min),
β-sitosterol (Retention time: 22.780 min). b) HPLC chromatogram of
unsaponifiable matter of pure cow ghee (Rani et al., 2016)
Inference (Qualitative 1. In pure/genuine ghee sample the peak of β- sitosterol must be absent
analysis) 2. The detection of β- sitosterol peak denotes the adulteration of ghee with
vegetable fat.

10 Reference 1. FSSAI Order. (2019). Method for detection of adulteration in ghee

(clarified milk fat) with vegetable oils. File No. 1-90/FSSAI/SP
(MS&A)/2009 dated 25th March, 2018. Food Safety Standards
Authority of India, New Delhi.
2. Rani, A., Sharma, V., Arora, S., & Ghai, D. L. (2016). Comparison of
rapid reversed phase high-performance liquid chromatography (RP-
HPLC) method with rapid reversed phase thin layer chromatography
method for detecting vegetable oils in ghee (clarified milk fat).
International Journal of Food Properties, 19(5), 1154-1162.
Approved by Scientific Panel on Methods of Sampling and Analysis

274 | M o M – D a i r y a n d D a i r y P r o d u c t s
 L. CHAKKA AND SHRIKHAND

Preparation of Sample of Chakka and Shrikhand

Method No. FSSAI 01.098:2022 Revision No. & Date 0.0

Scope This method is used for the preparation of chakka and shrikhand for
analysis.
Apparatus/Instruments Mortar and pestle
Sample preparation 1. To get a representative sample, particularly in case of a thick-set
product, it may be necessary to pour out the whole of the contents in a
mortar and mix it thoroughly until the mass is homogeneous.
2. Aliquots are weighed out from this well-mixed sample.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

275 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Total Solids in Chakka and Shrikhand

Method No. FSSAI 01.099:2022 Revision No. & Date 0.0

Scope This method is used for the determination of total solids in chakka and
shrikhand.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.
1. Use thermal protection gloves, tongs and protective eyewear while
handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle Total solids is determined by drying the product in hot air oven at 102 ± 2
℃ to constant mass.
Apparatus/Instruments Refer method prescribed for analysis of total solids in condensed/evaporated
Materials and reagents milk (FSSAI 01.053:2022).
Preparation of reagents
Sample preparation Refer method FSSAI 01.098:2022 for sample preparation.
Method of analysis Refer method prescribed for analysis of total solids in condensed/evaporated
milk (FSSAI 01.053:2022).
Calculation with units of 𝑀2 − M
expression Total Solids Content (%) = × 100
𝑀1 − M

Where,
M is the mass in g of empty dish along with lid;
M1 is the initial mass in g of dish with lid and test portion
M2 is mass in g of dish with lid and dried test portion
Round the value obtained to nearest 0.01 % (m/m)
Note: BIS/ISO method does not recommend use of sand. However, use of
sand help in even spreading of samples.
Reference IS 11622: 1986. Method for determination of total solids content in
condensed milk. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

Determination of Fat (on dry matter basis) in Chakka and Shrikhand

Method No. FSSAI 01.100:2022 Revision No. & Date 0.0

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Scope This method is used for the determination of fat on dry matter basis in
chakka and shrikhand.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may ignite
and burn with explosive force. It reacts violently with halogens (such as
fluorine, bromine, and chlorine), acids (such as hydrochloric acid,
hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
5.
Principle Refer principle stated for analysis of fat in condensed milk (FSSAI
01.051:2022).
Apparatus/Instruments Refer requirement stated for analysis of fat in condensed milk (FSSAI
Materials and Reagents 01.051:2022).
Preparation of reagents

Sample preparation Refer method FSSAI 01.098:2022 for sample preparation.

Method of analysis Rose-Gottlieb Method
Refer method stated for analysis of fat in condensed milk (FSSAI
01.051:2022).

Determination of Total Solids

Refer method stated for analysis of total solids in chakka and shrikhand
(FSSAI 01.099:2022).

(𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
Calculation with units of 𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
expression 𝑤

277 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Fat (%)
Fat % (on dry matter basis) = × 100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)
Reference 1. IS 9352: 1980 (Reaffirmed year 2019). Specifications for chakka and
shrikhand. Bureau of Indian Standards, New Delhi.

2. IS 1166: 1986 (Reaffirmed year 2018). Specifications for condensed

milk, partly skimmed condensed milk (Second Revision). Bureau of
Indian Standards, New Delhi.

3. Pearson’s Composition and analysis of foods 9th edn,1991 page 604.

Approved by Scientific Panel on Methods of Sampling and Analysis

278 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Milk Protein (on dry matter basis) in Chakka and
Shrikhand
Method No. FSSAI 01.101:2022 Revision No. & Date 0.0
Scope This method is used for the determination of milk protein in chakka and
shrikhand and expressed on dry matter basis
Introduction/ Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer to
MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It reacts
violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and not
vice versa.
3. Ethanol: Extremely flammable. Use effective fume removal device when
heating or evaporating. Keep away from heat, sparks and open flame;
avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts with
strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to release
of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle Refer the method for determination of protein by Kjeldahl method in milk and
milk products (FSSAI 01.026/027:2022).
Apparatus
Material and Reagents The total solids in the product is calculated separately and the % protein is
Preparation of Reagents calculated on dry matter basis

Sample preparation Refer method FSSAI 01.098:2022 for sample preparation.

279 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Weigh quickly about 1-2 g of the prepared sample and transfer to a 500 or
800 mL Kjeldahl flask/digestion tube taking care to see that no portion of
the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk products
(FSSAI 01.026/027:2022).
Determination of Total Solids
Refer method stated for analysis of total solids in chakka and shrikhand
(FSSAI 01.099:2022).

Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.026/027:2022)
Protein (%)
Protein % (on dry matter basis) = 𝑥100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)

Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content

(Part 1): Kjeldahl method. International Organization for Standardization,
Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.

3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of

protein in foods and feeds. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Titratable Acidity of Chakka and Shrikhand

Method No. FSSAI 01.102:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of titratable acidity in
chakka and shrikhand.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
Principle A known quantity of sample is neutralized with standardized sodium
hydroxide solution with phenolphthalein indicator. The amount of sodium
hydroxide required is a function of the amount of natural buffering
substances present in the product, and of developed or added acid or alkaline
substances.
Apparatus/Instruments 1. Analytical balance (Readability 0.01 g).
2. Burette, 50 mL.
3. Porcelain dishes, white hemispherical of approximately 60 ml capacity.
4. Stirring rods, of glass, flattened at one end.
5. Pipettes, 10 mL and 1 mL.
Materials and Reagents 1. Sodium hydroxide.
2. Phenolphthalein indicator.
3. Ethanol.
Preparation of reagents 1. Standard sodium hydroxide solution – 0.1 N.
2. Phenolphthalein Indicator – Dissolve 1.0 g of phenolphthalein in 100 mL
of 95% ethanol. Add 0.1 N Sodium hydroxide solution until one drop
gives a faint pink colouration. Dilute with distilled water to 200 mL.
Sample preparation Refer method FSSAI 01.098:2022 for sample preparation.

281 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Weigh accurately about 10 g of the sample into each of the two porcelain
dishes.
2. Add 30 ml of lukewarm water to each dish and stir with the flat end of a
glass rod until a fine paste is obtained. Use the contents of one dish as a
blank for comparison of colour.
3. Add 1 ml of phenolphthalein indicator solution in the other dish, shake
well and titrate against standard sodium hydroxide solution. Stir
vigorously throughout.
Note: The time taken for the complete titration shall not exceed 20 seconds.
The persistence of slight pinkish tinge for 30 seconds. The titration shall be
preferably made in north light or under illumination from a day light lamp.

Calculation with units of 9 AN

Titratable acidity (as lactic acid)percent by mass = 𝑤
expression
Where,
A = Volume of standard Sodium hydroxide required for titration
N = Normality of standard Sodium hydroxide solution
w = weight of the sample taken for test in g
Reference IS 1166: 1986 (Reaffirmed year 2018). Specifications for condensed milk,
partly skimmed condensed milk (Second Revision). Bureau of Indian
Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

282 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Total Ash (on dry matter basis) in Chakka and
Shrikhand
Method No. FSSAI 01.103:2022 Revision No. & Date 0.0
Scope This method is applicable for the determination of total ash on dry matter basis
in chakka and shrikhand.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot crucibles.
2. Hot crucibles will heat air within the desiccator and a vacuum may form
on cooling. Remove desiccator’s cover gradually by sliding to one side to
prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle Sample is kept at 550 ± 25 ℃ in muffle furnace so that all the organic matter
will be burn left behind the inorganic matter that is ash. Total solids are
estimated separately in the sample (Refer method FSSAI 01.099:2022). Then,
total ash (on dry matter basis) is calucalted in the sample.
Apparatus/Instruments 1. Platinum or silica crucible, about 70 mm diameter and 25 to 50 mm
deep.
2. Muffle furnace, capable of being controlled at 550 ± 25 ℃.
3. Desiccator containing an efficient desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
4. Steam bath
5. Safety tongs having long arms.
6. Bunsen burner or electric hot plate.
7. Analytical balance (Readability 0.0001g)
8. Thermal protection gloves, capable to resist temperature up to 550 - 600
°C
9. Hot air oven (forced air type), capable of being controlled at 102 ± 2
°C
Refer method FSSAI 01.098:2022 for sample preparation.
Sample preparation

Method of analysis 1. Weigh accurately about 3 g of the sample in a crucible, previously dried
in a hot air oven and weighed.

283 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Evaporte the moisture in the sampel by keepting the dish on steam bath.
3. Heat the crucible gently on a burner or hot plate at first and then strongly
in a muffle furnace at 550  25 °C till grey ash is obtained.
4. Cool the crucible in a desiccator and weigh it.
5. Heat the crucible again at 550  25 °C for 30 min.
6. Cool the crucible in a desiccator and weigh.
7. Repeat this process of heating for 30 min, cooling and weighing until the
difference between two successive weighing is less than 1 mg.
8. Record the lowest mass.

Determination of Total Solids

Refer method stated for analysis of total solids in chakka and shrikhand FSSAI
01.099:2022.
M2 − M
Calculation with units of Total ash (% by mass) = × 100
M1 − M
expression
Where,
M = mass in g, of the empty crucible
M1 = mass in g, of the crucible with the material taken for the test; and
M2 = mass in g, of the crucible with ash;
Total ash (%)
Total ash % (on dry matter basis) = 𝑥100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)
Reference IS 1165: 2002 (Reaffirmed year 2018). Milk Powder – Specifications. Bureau
of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

284 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Sucrose Content in Shrikhand

Method No. FSSAI 01.104:2022 Revision No. & Date 0.0

Scope This method is for the determination of sucrose content in shrikhand. Sucrose
content can be determined by any of the following methods: Volumetric
method (Lane - Eynon) and Polarimetric method.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer to
MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide solution: It is extremely caustic and can cause severe
burns. Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It reacts
violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
3. Ammonia solution: It reacts violently with halogens (such as fluorine,
chlorine, bromine), acids (such as hydrochloric acid, hydrogen bromide,
hydrogen fluoride). It is incompatible with cyanide and oxidizing agents.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts with
strong oxidizing agents to form toxic chlorine gas.
Principle Refer method prescribed for analysis of sucrose content in
Lane-Eynon (Volumetric) condensed/evaporated milk (FSSAI 01.052:2022).
Method,
Polarimetric Method
Apparatus/Instruments
Materials and Reagents
Preparation of Reagents
Sample preparation Refer method FSSAI 01.098:2022 for sample preparation.

Method of analysis Refer method prescribed for analysis of sucrose content in condensed milk

285 | M o M – D a i r y a n d D a i r y P r o d u c t s
 (FSSAI 01.052:2022).

Calculation with units of Refer method prescribed for analysis of sucrose content in condensed milk
expression (FSSAI 01.052:2022).

Reference 1. IS 4079 – 1967 (Reaffirmed year 2016). Specification for canned

rasogolla. Bureau of Indian Standards, New Delhi).

2. IS 11764 – 2005 (Reaffirmed year 2019). Method for determination of

sucrose content by polarimetric method in condensed milk. Bureau of
Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

286 | M o M – D a i r y a n d D a i r y P r o d u c t s
 M. WHEY POWDER

Preparation of Sample of Whey Powder and Acid Whey Powder

Method No. FSSAI 01.105:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of whey powder.
Sample preparation 1. Make the sample homogeneous either by mixing or shaking alternately,
and rolling and inverting the container.
2. Avoid excessive temperature and humidity when opening the sample
container to prevent absorption of moisture.
Reference FSSAI manual of methods of analysis of foods: milk and milk products.
(2016). Food Safety Standards Authority of India, Ministry of Health and
Family Welfare, Government of India, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

287 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Moisture in Whey Powder and Acid Whey Powder

Method No. FSSAI 01.106:2022 Revision No. & Date 0.0

Scope This method is used for the determination of moisture in all whey powders.

Caution Follow all safety procedures and wear laboratory apron and safety goggles.
1. Use thermal protection gloves, tongs and protective eyewear while
handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle The sample is dried to constant weight at 102 ± 2 ℃ and the loss in weight
reported as moisture.
Apparatus/Instruments Refer method prescribed for analysis of moisture content in milk powder
Materials and Reagents (FSSAI 01.056:2022). Weigh sample in / under dehumidifier or humidity
Preparation of reagents shall not be more than 50%.
Sample preparation Refer method FSSAI 01.105:2022 for sample preparation.

Method of analysis Refer method prescribed for analysis of moisture content in milk powder
Calculation with units of (FSSAI 01.056:2022).
expression
Reference 1. IS 16072: 2012 (Reaffirmed year 2018). Determination of moisture
content in milk powder and similar products, Bureau of Indian
Standards: India.

2. ISO 5537/IDF 026:2004 - Dried milk - Determination of moisture

content (Reference method). International Organization for
Standardization, Geneva.

3. F.A.O Manuals of Food quality Control 14/ 8 page 30).

Approved by Scientific Panel on Methods of Sampling and Analysis

288 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Fat in Whey Powder and Acid Whey Powder

Method No. FSSAI 01.107:2022 Revision No. & Date 0.0

Scope This method is used for the determination of fat in all whey powders.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may ignite
and burn with explosive force. It reacts violently with halogens (such as
fluorine, bromine, and chlorine), acids (such as hydrochloric acid,
hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle Refer method prescribed for analysis of fat content in milk powder (FSSAI
Apparatus/Instruments 01.057:2022).
Materials and Reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.105:2022 for sample preparation.
Method of analysis Refer method prescribed for analysis of fat content in milk powder (FSSAI
Calculation with units of 01.057:2022).
expression
Reference IS 11721 – 2013 (Reaffirmed year 2018). Method of sampling and test for
dried milk sample. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Milk Protein in Whey Powder and Acid Whey
Powder
Method No. FSSAI 01.108:2022 Revision No. & Date 0.0
Scope This method is used for the determination of protein in all whey powders.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe
burns. Protect skin and eyes when working with these alkalies as
solids or concentrated solutions. While making solution add pellets
to water and not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is
extremely corrosive and can cause serious burns when not handled
properly. It reacts violently with water with evolution of heat; can
react with organic materials explosively. Do not mix with
hydrochloric acid. Good general ventilation should be provided to
keep vapour and mist concentrations below the exposure limits.
3. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to
protect your eyes and skin. Concentrated hydrochloric acid is toxic
if inhaled, so avoid breathing it in and always handle it while under
a fume hood. It reacts with strong oxidizing agents to form toxic
chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to
release of irritating gases and vapours. Minimize dust generation and
accumulation.

Principle Refer the method for determination of protein by Kjeldahl method in milk
Apparatus/Instruments and milk products (FSSAI 01.026/027:2022).
Materials and Reagents
Preparation of Reagents
Sample preparation Refer method FSSAI 01.105:2022 for sample preparation.

290 | M o M – D a i r y a n d D a i r y P r o d u c t s
Method of analysis 1. Weigh quickly about 1-1.3 g of the prepared whey powder sample and
transfer to a 500 or 800 mL Kjeldahl flask/digestion tube taking care to
see that no portion of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk products
(FSSAI 01.026/027:2022).

Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.026/027:2022)
Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content
(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.

3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of

protein in foods and feeds. Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Total Ash (on dry matter basis) in Whey Powder
and Acid Whey Powder

Method No. FSSAI 01.109:2022 Revision No. & Date 0.0

Scope This method is used for the determination of total ash (on dry matter basis)
in whey powder and acid whey powder.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot crucibles.
2. Hot crucibles will heat air within the desiccator and a vacuum may form
on cooling. Remove desiccator’s cover gradually by sliding to one side
to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle The sample is kept at 550 ± 20 ℃ in a muffle furnace so that all the organic
matter will be burnt. The leftover inorganic matter is the total ash.
Apparatus/Instruments Refer method prescribed for analysis of total ash in milk powder (FSSAI
Materials and Reagents 01.064:2022).
The total solids in the product are calculated and the % total ash is calculated
Preparation of reagents on dry matter basis.
Sample preparation Refer method FSSAI 01.105:2022 for sample preparation.

Method of analysis Determination of ash

Refer method prescribed for analysis of total ash in milk powder (FSSAI
01.064:2022).

Determination of moisture
Refer method prescribed for analysis of moisture in whey powder (FSSAI
01.106:2022).

Calculation with units of M2 − M

Total ash (on dry basis), % by mass =
(100 − M0 ) x ( M1 − M)
× 100
expression
Where,
M0 = moisture, % by mass, calculated as per the method for dried milk
M = mass in g, of the empty crucible;
M1 = mass in g, of the crucible with the material taken for the test; and
M2 = mass in g, of the crucible with ash;

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Reference IS 1165 – 2002 (Reaffirmed year 2018). Milk Powder – Specifications.
Bureau of Indian Standards, New Delhi.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of pH in Whey Powder and Acid Whey Powder

Method No. FSSAI 01.110:2022 Revision No. & Date 0.0

Scope This method is used for the determination of pH in whey powder and acid
whey powder.
Caution Wear laboratory apron, shoes, safety goggles and mask while working in
laboratory.
Principle Refer method prescribed for analysis of pH in Casein / Caseinate (FSSAI
Apparatus/Instruments 01.120:2022)
Materials and Reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.105:2022 for sample preparation.
Method of analysis Refer method prescribed for analysis of pH in Casein / Caseinate (FSSAI
01.120:2022).
Calculation with units of Refer method prescribed for analysis of pH in Casein / Caseinate (FSSAI
expression 01.120:2022).
Reference IS 11918: 2017. Method for determination of pH in caseins and caseinates
(Reference Method). Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Lactose in Whey Powder and Acid Whey Powder

Method No. FSSAI 01.111:2022 Revision No. & Date 0.0

Scope This method is used for the determination of lactose in whey powder and
acid whey powder by Lane – Eynon method.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
2. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
3. Copper sulphate: Non-combustible, substance itself does not burn but
may decompose upon heating to produce corrosive and/or toxic fumes.
4. Glacial acetic acid: Use with adequate ventilation. Keep away from heat.
Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin and
eyes. Keep away from incompatibles such as oxidizing agents, reducing
agents, metals, acids, alkalis etc.
Principle Lane and Eynon method is based on the principle of reduction of Fehling’s
solution by reducing sugars (Lactose). Fehling’s solution is a mixture of
copper sulphate and alkaline Rochelle salt (sodium potassium tartarate).
Rochelle salt complexes with the cupric hydroxide formed in alkaline
solution and prevent it from precipitation. Lactose reduces the complexed
cupric hydroxide to red, insoluble cuprous oxide under the experimental
conditions. An oxidation-reduction indicator, usually methylene blue,
detects the end point of the reaction.
Apparatus/Instruments 1. Analytical balance (Readability 0.01 g).
2. Hot plate.
3. Burette (50 mL cap.) with an off-set tip.
4. Volumetric flask, 250 mL.
5. Pipette, 5 mL and 25 mL.

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 6. Conical flask, 250 mL.
7. Funnel (small).
8. Whatman No. 1 filter circles.
Materials and Reagents 1. Copper sulphate.
2. Concentrated sulphuric acid (Approximately 36.8 N).
3. Rochelle salt (sodium potassium tartrate).
4. Sodium hydroxide.
5. Acetic acid.
6. Lactose.
7. Methylene blue.
Preparation of reagents Dilute sulphuric acid (1 N): Dilute 27.17 mL of concentrated sulphuric acid
to 1000 mL with distilled water.
Fehling's Solution: It is freshly prepared by mixing equal volumes of the
following two solutions, termed as Fehling's A and Fehling's B.
1. Fehling's A: Dissolve 34.639 g of copper sulphate in sufficient distilled
water, add a few drops of 1 N sulphuric acid to prevent formation of
cloudy appearance or turbidity (use a minimum quantity of acid). Dilute
the solution with distilled water to 500 mL. If necessary, filter through
prepared asbestos, on Gooch crucible.
2. Fehling's B: Dissolve 173 g Rochelle salt in distilled water. Add to it 50
g of sodium hydroxide and dilute the mixture to 500 mL with distilled
water. Allow to stand in a rubber or polyethene stoppered container for
two days and filter through prepared asbestos.
3. Acetic acid solution: 10% (w/v; aqueous) solution.
4. Methylene blue indicator: 1% (w/v; aqueous) solution.
5. Standard lactose solution: 0.5% (w/v; aqueous) solution: Weigh
accurately 5 g of this dried lactose and dissolve in freshly boiled and
cooled distilled water. Transfer the solution quantitatively to l L
volumetric flask and dilute up to mark with distilled water. Mix well and
store it in a cool place. One milileter of this solution contains 5 mg
(0.005 g) lactose.
Sample preparation Refer method FSSAI 01.105:2022 for sample preparation.
Method of analysis Preparation of sample filtrate
1. Take 2-2.5 g of prepared sample into a 100 mL volumetric flask.
2. Add 30 to 40 mL distilled water and the content of the flask are warmed
to 40 to 45 °C, immediately add 1.5 mL acetic acid (10%). Mix well and
make the final volume with distilled water up to mark and mix again.
Keep the flask undisturbed for 30 min.
3. After 30 min, filter the content of the flask through a Whatman filter
paper Grade 42. Discard the first few milileter of the filtrate and collect
the rest in a clean dry Erlenmeyer flask fitted with stopper.

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B. Standardization of Fehling's solution
Preliminary titration
1. Pipette 5 mL of Fehling's solution A and 5 mL of Fehling's solution B
using two separate pipettes in a 250 mL Erlenmeyer flask.
2. Fill up a burette with the standard lactose solution and connect the
burette end with an offset tube to keep the burette tube out of steam.
3. Heat the content of the flask to boiling over burner or heater and
maintain moderate boiling for 2 min. To prevent bumping add some inert
boiling chips. Add 3 to 4 drops of methylene blue indicator without
removing from the flame.
4. Titrate the content of the flask against standard lactose solution from the
burette until the blue colour disappears and the bright brick-red colour
of precipitated Cu2O appears (at the end point the Cu2O suddenly settles
down giving a clear supernatant).
5. Note the volume of lactose solution required for the standardization of
Fehling's solution
Final titration
1. After this preliminary titration, further titration or titrations should be
carried out, adding practically the whole of the standard lactose solution
volume (one mL less than required as observed in first titration) required
before commencing the heating and continuing the titration as before.
2. The titration must be completed within 3 min from the commencement
of boiling. Let V1 mL be the titre for this experiment.
Note: Carefully note the first disappearance of blue colour. Once missed, it
is difficult to ascertain the end point. Maintain the boiling at a uniform rate
during the titration.
C. Determination of lactose in sample
Here also the titration is taken up to two stages. In the first stage, an
approximate estimation is made, while in the second stage more accurate
result is obtained.
Preliminary titration
1. Pipette 5 mL of Fehling's solution A and 5 mL of Fehling's solution B
using two separate pipettes in a 250 mL Erlenmeyer flask.
2. Fill up a burette with the prepared milk filtrate and connect the burette
end with an offset tube to keep the burette tube out of steam.
3. Add 10 mL of the milk filtrate from the burette into flask containing
Fehling's solution and heat to boiling on wire gauge over burner.
4. Boil for about 15 secs and rapidly add further amount of lactose filtrate
until only faint perceptible blue colour remains. Add 3 - 4 drops of
methylene blue indicator and complete the titration to the first
disappearance of blue colour by adding lactose filtrate drop-wise.
Final Titration

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 Repeat the experiment of Stage 1, but add almost the entire prepared milk
filtrate (one milileter less than required as observed in first titration)
required to reduce all copper, as determined by Stage 1, and complete the
titration as described under standardization of Fehling's solution. Let V2 mL
be the titer for this experiment.
Calculation with units of 0.005 × 𝑉1 × 100
𝐿𝑎𝑐𝑡𝑜𝑠𝑒 (% 𝑏𝑦 𝑚𝑎𝑠𝑠) = × 100
expression 𝑉2 × 𝑊
Where
V1 = volume in mL of standard lactose solution taken to reduce 10 mL of
Fehling's solution; and
V2 = volume in mL of prepared milk filtrate taken to reduce 10 mL of
Fehling's solution.
W = Mass of sample taken.
Reference Sharma, R., Mann, B., Lal, D., Rajput, Y.S. & Lata K. (2013). Laboratory
Manual on Milk Carbohydrates, Minerals and Water-Soluble vitamins,
Published by NDRI.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 N. EDIBLE CASEIN PRODUCTS

Preparation of Sample of Edible Casein Products

Method No. FSSAI 01.112:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of sample of edible acid casein, edible
rennet casein and edible caseinates.
Apparatus/Instruments Sieve (500 μm)
Sample preparation 1. Thoroughly mix the sample by repeatedly shaking and inverting the
container.
2. Transfer about 50 g of the thoroughly mixed sample to the test sieve
(500 μm).
3. If the 50 g portion directly passes or almost completely passes through
the sieve, use it for analysis, otherwise, grind the 50 g portion, using the
grinding device until it passes through the sieve.
4. Immediately transfer the entire sieved sample to an air-tight container of
sufficient capacity and mix thoroughly by repeatedly shaking and
inverting.
5. During these operations take precautions to avoid any change in the
water content of the product. After the test sample has been prepared,
the analysis should be carried out as soon as possible.
Reference IS 11920: 2012 (Reaffirmed year 2018). Caseins ad Caseinates –
Determination of moisture content (reference method). Bureau of Indian
Standards: India.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Moisture in Edible Casein Products

Method No. FSSAI 01.113:2022 Revision No. & Date 0.0

Scope This method is used for the determination of moisture in edible acid casein,
edible rennet casein and edible caseinates.
Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.

Principle
The sample is dried to constant weight at 102  2 ℃ and the loss in weight
reported as moisture. The method described here has been followed from IS
16072: 2012.
Apparatus/Instruments 1. Flat-bottom moisture dishes with cover made of stainless steel, nickel or
aluminium having approximately 50 mm diameter and 25 mm depth.
The dishes shall have lids which fit well and can readily be removed.
2. Hot air oven (forced air type), capable of being controlled at 102 ± 2 ℃.
3. Desiccator containing an efficient desiccant. (for example freshly dried
silica gel with a hygroscopic indicator).
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis 1. Uncover a dish and place the dish and its lid in a hot air oven at 102 ± 2
℃ for 1 h. Place the lid on the dish, transfer the covered dish from the
hot air oven to the desiccator. Allow it to cool to 25 ± 3C and weigh it.
2. Put approximately 2 g of the caseinate sample in the dish, cover the dish
with the lid and weigh the covered dish accurately and quickly.
3. Uncover the dish and put it with its lid in the hot air oven maintained at
102  2 °C for 3 h.

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 4. Replace the lid, transfer the covered dish to the desiccator, allow it to
cool to 25 ± 3 C (for approximately 30-45 min) and weigh it accurately
and quickly.
5. Heat the uncovered dish and lid in the hot air oven at 102 ± 2 ℃ for
further 1 h; replace the lid and allow the covered dish to cool to 25 ± 3
C in the desiccator and weigh it.
6. Repeat the process of drying, cooling and weighing, until the successive
weighing do not differ by more than 0.5 mg. It is usually found that
drying is complete after the first 2 h.
Calculation with units of M1 - M 2
Moisture % by mass = × 100
expression M1 -M
Where,

M is mass in g, of the empty dish with lid;

M1 is initial mass in g of the dish with lid and test portion;

M2 is the mass in g of the dish with lid and dried test portion
Express the results to the nearest 0.01% (m/m).

Note: Commercial automatic moisture determination apparatus can also be

used for determination of moisture in milk powder.
ISO (ISO 5537:2004) method advocates the use of drying oven set at 87°C
(for 5 h) and use of copper tubes.
Reference 1. IS 11920: 2012 (Reaffirmed year 2018). Caseins ad Caseinates –
Determination of moisture content (reference method). Bureau of Indian
Standards: India.

2. ISO 5537/IDF 026:2004. Dried milk - Determination of moisture

content (Reference method). International Organization for
Standardization, Geneva.

Approved by Scientific Panel on Methods of Sampling and Analysis

301 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Fat in Edible Casein Products

Method No. FSSAI 01.114:2022 Revision No. & Date 0.0

Scope This method is used for the determination of fat in edible acid casein, edible
rennet casein and edible caseinates.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Light petroleum ether: Avoid storage near extreme heat, ignition
sources or open flame. Use respiratory protective device against the
effect of fumes/aerosols. Perform all operations under an effective fume
removal device.
Principle In this method, a test portion is digested with hydrochloric acid followed by
addition of ethanol. The acid-ethanolic solution is subsequently extracted
with diethyl ether and light petroleum, followed by removal of solvents by
distillation or evaporation and thereafter the determination of mass of the
substances extracted which are soluble in light petroleum. The following
method is as per IDF guidelines.
Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).
2. Centrifuge, for the centrifugation of fat-extraction flasks or tubes at
500 to 600 rpm to produce a gravitational field of 80 to 90 g at the
outer end of the flasks or tubes (the use of the centrifuge is optional
but recommended).

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 3. Distillation or evaporation apparatus to enable the solvents and ethanol
to be distilled from the fat-collecting flasks or to be evaporated from
beakers and dishes at a temperature not exceeding 100 ℃.
4. Hot air oven (forced air type), capable of being controlled at 102 ± 2 ℃.
5. Boiling water bath or hot plate.
6. Mojonnier type fat-extraction tube; the flask shall be provided with
good quality bark corks or stoppers of other material for e.g. silicon
rubber or polytetrafluoroethylene (PTFE) unaffected by the reagents
used.
7. Fat-collecting vessels for e.g., flat bottom flask of capacity 125 to 250
mL.
8. Measuring cylinder of capacities 5 and 25 mL.
9. Pipettes graduated of capacity 10 mL.
Materials and Reagents 1. Hydrochloric acid (Approximately 11.6 N).
2. Ethanol or ethanol denatured by methanol at least 94% (w/v).
3. Congo red.
4. Diethyl ether, free from peroxides containing no, or not more than 2
g/kg of antioxidants.
5. Light petroleum, having any boiling range between 30 and 60 ℃.
Preparation of reagents 1. Hydrochloric acid solution of ρ20 approximately 1.125 g/mL. Dilute
675 mL of concentrated hydrochloric acid, ρ20 1.18 g/mL, to 1000 mL
with water.
2. Congo red solution: Dissolve 1 g of congo red in water and dilute to
100 mL
3. Mixed solvents; prepared shortly before use by mixing equal volumes
of diethyl ether and the light petroleum ether.
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis 1. Mix and weigh immediately 2 to 3 g of test sample to the nearest 1
mg, directly or by difference, into a fat-extraction flask, or into 100
mL beaker or flask.
2. Add 7.5 to 10 mL of hydrochloric acid solution so as to wash the test
portion into the small bulb of the extraction flask (as per BIS method,
take 5 g of the sample and 10 mL of hydrochloric acid solution.

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 3. Carry out a blank test simultaneously with the determination, using the
same procedure and same reagents, but omitting the test portion.
4. Heat by gently moving the vessel in a boiling water-bath or on a hot
plate, until all the particles are entirely dissolved. Allow the vessel to
stand for 20 to 60 min in the boiling water bath, shaking occasionally
during the initial 15 min, or keep it gently boiling over the flame or on
to the hotplate for 10 min; cool in running water.
5. Proceed for the extraction fat by Rose-Gottlieb method as prescribed
for milk from the step indicating addition of 10 mL ethyl alcohol
(Method No FSSAI 01.024:2022).
Calculation with units of
expression (𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Approved by Scientific Panel on Methods of Sampling and Analysis

304 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Determination of Protein in Edible Casein Products

Method No. FSSAI 01.115:2022 Revision No. & Date 0.0

Scope This method is applicable for the determination of protein in edible acid
casein, edible rennet casein and edible caseinates.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
3. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to
release of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle Refer the method for determination of protein by Kjeldahl method in milk
Apparatus/Instruments and milk products (FSSAI 01.027:2022)
Materials and Reagents
Preparation of reagents
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis 1. Weigh quickly about 1-2 g of the prepared sample and transfer to a
500 or 800 mL Kjeldahl flask/digestion tube taking care to see that no

305 | M o M – D a i r y a n d D a i r y P r o d u c t s
 portion of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk
products (FSSAI 01.026/027:2022).
Determination of moisture
Refer method for determination of moisture in edible casein products
er FSSAI 01.113:2022.
Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.026/027:2022).

Total Solids (%) = 100 - moisture (%)

Protein (%)
Protein % (on dry matter basis) = × 100
𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%)

Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content

(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.

3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of

protein in foods and feeds. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Casein in Protein in Edible Casein Products

Method No. FSSAI 01.116:2022 Revision No. & Date 0.0

Scope This method is for the determination of casein in protein in edible acid
casein, edible rennet casein and edible caseinates.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Glacial acetic acid: Use with adequate ventilation. Keep away from
heat. Do not breathe gas/fumes/ vapor/spray. Avoid contact with skin
and eyes. Keep away from incompatibles such as oxidizing agents,
reducing agents, metals, acids, alkalis.
2. Sodium acetate: Combustion products may include carbon oxides or
other toxic vapours. Thermal decomposition can lead to release of
irritating gas or vapours. Avoid generating dust; fine dust dispersed in
air in sufficient concentrations and in the presence of an ignition source
is a potential dust explosion hazard.
3. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
4. Potassium sulphate: Hazardous decomposition products are formed
under fire like sulphur oxides, potassium oxides. Avoid breathing and
formation of dust. Perform all operations in a well-ventilated area.
5. Copper sulphate: Non-combustible, substance itself does not burn but
may decompose upon heating to produce corrosive and/or toxic fumes.
Principle Casein is precipitated from milk at pH 4.6 using acetic acid and sodium
acetate solutions in Kjeldahl flask. The acidified solution which contains the
non-casein nitrogen components is separated from casein precipitate by
filtration. Nitrogen content of the casein precipitate is determined by
Kjeldahl method and multiplied by 6.38 to obtain casein in protein.
Apparatus/Instruments 1. Kjeldahl apparatus
2. Analytical balance (Readability 0.0001 g)
Materials and Reagents 1. Sodium acetate solution.

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 2. Acetic acid solution.
3. Buffer solution.
4. Sodium acetate trihydrate.
5. Glacial acetic acid.
6. Potassium sulphate.
7. Copper sulphate.
8. Concentrated Sulphuric acid (Approximately 36.8 N).
Preparation of reagents 1. Sodium acetate solution (1 M / litre) using AR sodium acetate or
sodium acetate trihydrate: Transfer 4.10 ± 0.1 g sodium acetate or 6.80
± 0.1 g sodium acetate trihydrate into 50 mL volumetric flask and dilute
to volume with water; prepare fresh weekly.
2. Acetic acid solution (10%) Mix 10 g of acetic acid with 50 mL distilled
water and makeup the volume to 100 mL with distilled water.
3. Buffer solution: Dilute 1 ± 0.1 mL sodium acetate and 1 ± 0.1 mL 10 5
acetic acid to 100 mL with water; prepare fresh.
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis 1. Weigh 1-2 g of sample into the beaker, add 50 mL water and 0 .75 mL
of 10% acetic acid to the flask and swirl gently. Leave the mixture for
10 min.
2. Add 0.75 mL of sodium acetate and swirl gently.
3. Filter the mixture on to a pleated filter paper (Whatman No1, 15 cm)
and collect the filtrate; let it drain completely before the next pour.
4. Add 30 mL of buffer solution to beaker and swirl to mix.
5. Pour the mixture on to the filter paper after the first filtration is
complete and combine the filtrates.
6. Add another 30 mL of the buffer solution and add this filtrate to the
previous 2 filtrates.
7. The filtrate should be clear and free of particulate matter.
8. If particulates appear, recycle the filtrate through the same filter paper
or repeat the test.
9. Remove filter paper; ensure that no precipitate is lost on the filter paper.
10. Drop the filter paper into kjeldahl flask/digestion tube and proceed as
per method for determination of protein in milk and milk products.
Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.027:2022)

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Reference 1. AOAC 998.06. 21st Edn. (2019). Official Method Casein Nitrogen
Content of Milk). AOAC International, USA.

2. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content

(Part 1): Kjeldahl method. International Organization for
Standardization, Geneva.

3. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.

4. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of

protein in foods and feeds. Bureau of Indian Standards, New Delhi.
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Lactose Content in Edible Casein Products (by
Photometric Method)
Method No. FSSAI 01.117:2022 Revision No. & Date 0.0
Scope A photometric method for determination of lactose content in edible acid
casein, edible rennet casein and edible caseinates is specified
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It
reacts violently with water with evolution of heat; can react with
organic materials explosively. Do not mix with hydrochloric acid.
Good general ventilation should be provided to keep vapour and mist
concentrations below the exposure limits.
3. Phenol: Phenol is highly corrosive to the skin and readily absorbed
through it. Work with phenol in a chemical fume hood, especially when
heating it. Never heat or melt phenol in an incubator, microwave,
drying oven, or similar appliance. Change gloves frequently. Wear
chemical goggles to protect the eyes.
Principle The casein is precipitated by addition of acetic acid and sodium acetate
solution at pH 4.6; this is followed by filtration which gives a protein-free
solution of the carbohydrates. The colour is developed in an aliquot portion
of filtrate (by the addition of phenol and concentrated sulphuric acid) which
is proportional to the amount of lactose present. The developed colour is
read at 490 nm is a spectrophotometer.
Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).
2. Conical flasks: 100 mL capacity.
3. Pipettes: 1, 2 and 10 mL capacities.
4. Graduated pipettes: 25 mL capacity.

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 5. Volumetric flasks: 100 mL capacity.
6. Test tubes: 40 mL capacity ground neck and fitted with ground glass
stoppers.
7. Automatic dispenser: Capable of dispensing 5 mL of concentrated
sulphuric acid within 1 sec.
8. Vortex mixer.
9. Micropipettes: 0.2 mL capacity with 0.001 mL divisions.
10. Filter paper: Whatman filter paper Grade 42.
11. Water-baths, maintained at 60 to 70 ℃, and at 20 ℃.
12. Spectrophotometer, suitable for making measurements at a wavelength
490 mm, provided with cuvettes of optical path length of 1 cm.
Materials and Reagents 1. Sodium hydrogen carbonate (NaHCO3): For analysis of acid casein.
2. Pentasodium triphosphate (Na5P3O10): For analysis of rennet casein.
3. Concentrated hydrochloric acid (Approximately 11.6 N).
4. Acetic acid.
5. Sodium acetate.
6. Phenol.
7. Concentrated sulphuric acid (Approximately 36.8 N).
8. Lactose.
Preparation of reagents 1. Dilute hydrochloric acid (0.1 N): Dilute 8.62 mL of concentrated
hydrochloric acid to 1000 mL with distilled water.
2. Dilute sulphuric acid (0.1N): Dilute 2.717 mL of concentrated sulphuric
acid to 1000 mL with distilled water.
3. Dilute acetic acid solution (100 g/L): Mix 100 g of acetic acid with 50
mL of distilled water and makeup the volume to 100 mL with distilled
water.
4. Sodium acetate solution (1 M): Dissolve 82.03 g of sodium acetate in
100 mL of distilled water and makeup the volume to 1000 mL with
distilled water.
5. Lactose (20 g/L) solution: Weigh 2.105 ± 0.001 g of lactose
monohydrate, corresponding to 2.00 g of the anhydrous lactose, into a
100 mL volumetric flask. Dissolve lactose in hot water, make up to
volume and mix well. Store the solution at 0 ℃.

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 6. Pheno 80% by mass: Heat a mixture of 8 g of phenol and 2 g of water
until the mixture is homogeneous,
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis 1. Weigh to the nearest 1 mg about 1 g of the test sample into a 100 mL
conical flask; add the reagents as the case may be:
2. In the case of acid casein, add 0.1 ± 0.001 g of the sodium hydrogen
carbonate.
3. In the case of rennet casein, add 0.1 ± 0.001 g of the pentasodium and
25 mL of water; place the flask in a water-bath controlled at 60 - 70 ℃,
and mix occasionally by shaking.
4. When the test portion is completely dissolved (after 10 to 15 min), cool
and add successively 15 mL of water, 8 mL of dilute hydrochloric acid
or sulphuric acid solution and 1 mL of the dilute acetic acid solution.
5. Stopper and mix the contents by shaking after each addition.
6. Leave for 5 min and then add 1 mL of the sodium acetate solution; mix
by shaking.
7. Allow the casein precipitate to settle, and filter through a dry Whatman
filter paper Grade 42. Discard the first few mL of the filtrate.
8. Simultaneously, carry out a blank by taking 0.1 ± 0.001 g of sodium
hydrogen carbonate or 0.1 ± 0.001 g of pentasodium triphosphate, as
appropriate, (the reagent shall be same as used in case of test sample),
using the same apparatus and reagents.
9. Pipette 2 mL of the filtrate into a test tube, add 0.2 mL of the phenol
solution by means of a micropipette, and mix by shaking.
10. In less than 1 sec, add 5 mL of concentrated sulphuric acid from the
automatic dispenser, directing the stream of acid against the liquid
surface rather than against the side of the test tube in order to obtain
good mixing. Immediately mix, using the mixer, and allow to stand for
15 min. Cool for 5 min in the water bath at 20 ℃.
11. Wipe the tube and precede immediately, measure the absorbance of the
solution at 490 nm using the blank solution as the reference. If the
absorbance is above the upper limit of the calibration graph, repeat using
2 mL of a suitable dilution of the filtrate instead of 2 mL of the filtrate
itself.
Note: If such a dilution is made, the formula given for calculation has
to be modified accordingly.

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 Preparation of the Calibration Graph
12. Pipette 10 mL of the lactose solution (20 g/L solution) into a 100 mL
volumetric flask and dilute to the mark with water (solution A); 1 mL of
solution A corresponds to 2 mg of anhydrous lactose.
13. Prepare three standard solutions by pipetting 1, 2 and 3 mL of solution
A into three 100 mL volumetric flasks and making up the volumes with
water. The anhydrous lactose concentrations of the standard solution
obtained are respectively 20, 40 and 60 µg/mL.
14. Using four test tubes, proceed in accordance, but replace 2 mL of filtrate
respectively, by 2 mL of each of the three standard solutions and 2 mL
of water.
15. Measure the absorbances of the three standard matching solutions using
the solution obtained by treatment of the 2 mL of water as the reference
liquid.
16. Construct a calibration curve by plotting the absorbances of the standard
matching solutions against their anhydrous lactose concentrations in
μg/mL. Draw the best-fitting line through the calibration points.
Calculation with units of The lactose content of the sample, expressed as anhydrous lactose as a
expression percentage by mass, is calculated using the following formula:
C × 10−6 ×50
Lactose (% by mass) = × 100
m

Where,
c =concentration, in μg/mL, of anhydrous lactose in the test solution, read
from the calibration curve.
m = mass in g, of the test portion.
Reference IS 11963: 2005 (Reaffirmed year 2019). Method for determination of
lactose content by photometric method in caseins and caseinates. Bureau of
Indian Standards, New Delhi.

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 Determination of Fixed Ash (ash including P2O5) Content in Edible
Casein Products

Method No. FSSAI 01.118:2022 Revision No. & Date 0.0

Scope This method is used for the determination of fixed ash (ash including P2O5)
content in casein/caseinates.
Caution
Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot crucibles.
2. Hot crucibles will heat air within the desiccator and a vacuum may
form on cooling. Remove desiccator’s cover gradually by sliding to
one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle A test portion of the sample is incinerated at 825 ± 25 ℃ in the presence of
magnesium acetate (added to bind all phosphorus of organic origin). The
resulting ash is weighed and mass of the ash originating due to magnesium
acetate is subtracted.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Pipette: 5 mL capacity.
3. Silica or platinum dishes about 70 mm diameter and 25 to 50 mm deep.
4. Hot air oven (forced air type), capable of being controlled at 102 ± 2 ℃.
5. Electrical furnace with air circulation, capable of being controlled at 825
± 25 ℃.
6. Boiling water-bath .
7. Desiccator, containing an effective desiccant (for example freshly dried
silica gel with a hygroscopic indicator).
Materials and Reagents Magnesium acetate tetra hydrate [Mg (CH3CO2)2.4H2O].
Preparation of reagents Magnesium acetate tetra hydrate (120 g/L solution): Weigh 120 g of
magnesium acetate and dissolved in 1L of water.
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation
Method of analysis 1. Heat two dishes in an electrical furnace controlled at 825 ± 2 ℃ for 30

314 | M o M – D a i r y a n d D a i r y P r o d u c t s
 min.
2. Allow the dishes to cool in the desiccator to the 25 ± 3 Cand weigh to
the nearest 0.1 mg.
3. Weigh to the nearest accuracy (0.1 mg directly in or by difference)
approximately 3 g of the test sample into one of the prepared dishes (A).
4. Using a pipette, add to the dish (A) exactly 5 mL of the magnesium
acetate solution so as to wet the entire portion and allow it to stand for
20 min. To the other prepared dish (B), add 5 mL of magnesium acetate
solution.
5. Evaporate the contents of both dishes (A and B) to dryness on the boiling
water-bath.
6. Place both the dishes in the oven controlled at 102 ± 2 ℃ for 30 min.
7. Heat dish A with its content on a low flame until the test portion is
completely charred, taking care that it does not burst into flame.
8. Transfer both the dishes (A and B) to the electrical furnace controlled at
825 ± 25 ℃, and heat for at least 1 h until all carbon has disappeared
from dish A. Allow both the dishes to cool in the desiccator to the room
temperature (25 ± 3oC) and weigh to the nearest 0.1 mg.
9. Repeat the operations of heating in the electrical furnace, cooling and
weighing, until the mass remains constant to within 1 mg. Record the
minimum mass.
Determination of moisture
Refer method for determination of moisture in edible casein products
(FSSAI 01.113:2022)

Calculation with units of The “fixed ash” of the sample, including phosphorus, as a percentage by
expression mass is calculated by the following formula:

Where,
M0 = mass in g of the test portion;
M1 = mass in g of dish A and residue;
M2 = mass in g of the prepared empty dish A;
M3 = mass in g of the dish B and residue;
M4 = mass in g of the prepared empty dish B.

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 Calculate the “fixed ash” to the nearest 0.01% and report the final result to
the nearest 0.1%.
To calculate the “fixed ash” of the sample on the dry basis, as a percentage
by mass, multiply the result obtained as above by
100
=
100 − M
where,
M is the % moisture content of the sample.
Reference IS 11919: 1987 (Reaffirmed year 2019). Method for determination of fixed
ash in rennet caseins and Caseinate (Reference Method). Bureau of Indian
Standards, New Delhi)

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 Determination of Free Acididity in Edible Casein Products

Method No. FSSAI 01.119:2022 Revision No. & Date 0.0

Scope This method is for the determination of free acidity in casein/caseinates.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
2. Ethanol: Extremely flammable. Use effective fume removal device
when heating or evaporating. Keep away from heat, sparks and open
flame; avoid breathing vapours.
Principle Free acidity in casein is the volume in mL of 0.1 N standard sodium
hydroxide solution required to titrate an aqueous extract of 1 g of casein
sample using phenolphthalein as indicator. This is determined by aqueous
extraction of test portion of the sample at 60 ℃ followed by filtration and
then titration of the filtrate with a standard sodium hydroxide solution to the
end point of phenolphthalein.
Apparatus/Instruments 1. Analytical balance (Readability 0.001 g).
2. Conical flask: 500 mL capacity, with ground neck and fitted with a
ground glass stopper and 200 mL capacity.
3. Burette; 50 mL capacity, graduated in 0.1 mL.
4. Measuring cylinder: 100 mL, 250 mL capacity.
5. Water-bath maintained at a temperature of 60 ± 2 ℃.
6. Appropriate filter.
Materials and Reagents 1. Sodium hydroxide.
2. Phenolphthalein indicator.
3. Potassium hydrogen phthalate.
4. Ethanol.
Preparation of reagents 5. Standard sodium hydroxide solution: 0.1 N standardised against
primary standard potassium hydrogen phthalate.

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 6. Phenolphthalein indicator: Dissolve 1 g of phenolphthalein in 100 mL
of 95% ethanol (v/v).
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis 1. Weigh about 10 g of the test sample to the nearest 10 mg and transfer it
to a conical flask (500 mL capacity).
2. Add 200 mL of water (at 60 ℃ and already boiled). Stopper the flask,
mix it by swirling and place in the water-bath at 60 ℃ for 30 min.
3. Shake the flask at intervals of about 10 min.
4. Filter the contents and cool the filtrate to about 20 ℃. The filtrate must
be clear.
5. Transfer 100 mL of the cool filtrate into the conical flask (200 mL
capacity) using a measuring cylinder.
6. Add 0.5 mL of the ethanolic phenolphthalein solution using a pipette.
7. Titrate with standard Sodium hydroxide solution until a faint pink colour
appears; the colour should persist for at least 30 sec.
8. Record the volume used to 0.01 mL.
Calculation with units of The free acid of the casein is calculated by using the following formula:
expression Free acidity (mL of
20 x V x T
0.1 N sodium hydroxide per g sample)= M
Where,
V = volume, in mL, of the standard sodium hydroxide solution used;
T = normality of the standard sodium hydroxide solution;
M = mass in g, of the test portion.
Calculate the free acid to the nearest 0.01 mL.
Reference IS 11964: 2012 (Reaffirmed year 2018). Method for determination of free
acidity in rennet caseins (Reference Method). Bureau of Indian Standards,
New Delhi).

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 Determination of pH in Edible Casein Products

Method No. FSSAI 01.120:2022 Revision No. & Date 0.0

Scope This method describes the determination of pH in casein/caseinates.
Caution Wear laboratory apron, shoes, safety goggles and mask while working in
laboratory.
Principle In this method, pH of an aqueous extract of casein or an aqueous solution of
caseinate is determined at 20 ℃, using a calibrated pH meter.
Apparatus/Instruments 1. pH meter, minimum sensitivity 0.05 pH unit, with a suitable glass
electrode and a calomel or other reference electrode.
2. Thermometer, with an accuracy of ± 0.5 ℃.
3. Conical flask: 100 mL capacity, fitted with a ground glass stopper.
4. Beaker: 50 mL capacity.
5. Mixer.
6. Beaker (for the mixer), of at least 250 mL capacity.
Materials and Reagents Buffer solutions, for calibration of the pH meter: Two standard buffer
solutions with pH values at 20 ℃ which are known to the second decimal
place and will bracket the pH value of the sample under test, for example
phthalate buffer solution of pH approximately 4 and a borax buffer solution
of pH approximately 9. In addition, a phosphate buffer solution of pH
approximately 7 may be used.
Sample preparation Refer method FSSAI 01.112:2022 for sample preparation.
Method of analysis Calibration of pH meter: Adjust the temperature of the buffer solutions to
20 ℃ and calibrate the pH meter in accordance with the manufacturer’s
instructions.
Notes
If a series of samples is being tested, check the calibration of the pH meter
with one or more of the standard buffer solutions at least every 30 min.
Preparation of test solution
1. Prepare 10% solution of sample by dissolving 10 g of sample in distilled
water and making the final volume to 100 mL.
2. Determine pH with calibrated pH meter.

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 Recording of pH
Caseins: Record (as the pH of the aqueous extract of casein) the value read
to at least one decimal place.
Caseinates: Record (as the pH of the aqueous solution of caseinate) the
value read to at least two decimal places.
Reference IS 11918: 2017. Method for determination of pH in caseins and caseinates
(Reference Method). Bureau of Indian Standards, New Delhi.
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 O. COLOSTRUM

Preparation of Sample of Colostrum

Method No. FSSAI 01.121:2022 Revision No. & Date 0.0

Scope This method describes the preparation of a colostrum sample for analysis
Sample preparation 1. Warm the sample to 37 - 40 ℃ by transferring it to the beaker and
keeping it in a water bath maintained at 40 - 45 ℃.
2. Stir slowly for proper homogenisation, mix sample thoroughly by
pouring back into the bottle, mixing to dislodge any residual fat sticking
to the sides and pour it back in the beaker.
3. During mixing do not shake the bottle vigorously.
4. Allow the sample to come to room temperature (26 - 28 ℃) and withdraw
immediately for analysis.
Reference IS 1479 (Part II):1961 (Reaffirmed year 2018). Methods of test for Dairy
Industry -Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi.

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 Determination of Moisture content in Colostrum

Method No. FSSAI 01.122:2022 Revision No. & Date 0.0

Scope This method describes the determination of moisture content of colostrum.

Caution Follow all safety procedures and wear laboratory apron and safety goggles.

1. Use thermal protection gloves, tongs and protective eyewear while

handling hot moisture dishes.
2. Hot moisture dishes will heat air within the desiccator and a vacuum
may form on cooling. Remove desiccator’s cover gradually by sliding
to one side to prevent a sudden inrush of air at the end of cooling period.
3. Open and close desiccator slowly in order to avoid the danger of glass
breakage.
Principle In this method, first total solids are determined in the sample. For estimating
moisture content total solids are subtracted from 100.
Apparatus/Instruments 1. Analytical balance (Readability 0.0001 g).
2. Flat-bottomed dishes: Made of aluminum alloy, nickel, stainless steel,
porcelain, 50 to 75 mm diameter, 20 to 25 mm depth, and provided with
easily removable but closely fitting lids.
3. Boiling water-bath (100 ± 2 oC).
4. Hot air oven (forced air type), capable of being controlled at 102 ± 2 ℃.
5. Desiccator with efficient desiccant (for example freshly dried silica gel
with a hygroscopic indicator).
6. Short glass stirring rods: Flattened at one end and fitting in to the dish.
Sample preparation Refer method FSSAI 01.121:2022 for sample preparation.
Method of analysis 1. Take a dish with the lid and stirring rod and weigh to the nearest 0.1 mg.
2. Place 2.0 g of the prepared test sample in the dish, spread evenly with
the help of stirring rod and weigh the dish to the nearest 0.1 mg.
3. Heat the dish on a boiling water-bath, for approximately 30 min while
stirring
4. Dry the bottom of the dish and heat the dish with its lid alongside, in a
hot air over maintained at 102 ± 2 ℃ for 4 h.

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 5. Place the lid on the dish, allow the dish to cool in the desiccator and
weigh to the nearest 0.1 mg. Repeat the operations described above
(heating the dish for 1 h) until the difference in mass between two
successive weighing does not exceed 0.5 mg.
6. Record the lowest mass.
Calculation with units of M1 - M2
Moisture % by mass = × 100
expression M1 -M
Where,
M is mass in g, of the empty dish with lid along with glass rod;
M1 is initial mass in g of the dish with lid, glass rod, and test portion;
M2 is the mass in g of the dish with lid, glass rod, and dried test portion
Express the results to the nearest 0.01% (m/m).
Reference IS 1479 (Part II):1961 (Reaffirmed year 2018). Methods of test for Dairy Industry
-Chemical Analysis of Milk. Bureau of Indian Standards, New Delhi.

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 Determination of Fat Content in Colostrum

Method No. FSSAI 01.123:2022 Revision No. & Date 0.0

Scope This method describes the determination of fat content in colostrum.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer
to MSDS (Material Safety Data Sheets) for specific information.
1. Ammonia solution: Do not breathe vapor or mist. Use only with
adequate ventilation. Although ammonia is not flammable, it may ignite
and burn with explosive force. It reacts violently with halogens (such as
fluorine, bromine, and chlorine), acids (such as hydrochloric acid,
hydrogen fluoride, hydrogen bromide).
2. Ethyl alcohol: Extremely flammable. Use effective fume removal
device when heating or evaporating. Keep away from heat, sparks and
open flame; avoid breathing vapours.
3. Diethyl ether: Store protected from light. It is extremely flammable and
can react explosively when in contact with Cl2, O3, LiAlH4 or strong
oxidizing agents.
4. Petroleum ether: Extremely flammable. Avoid breathing fumes.
Perform all operations in a fume hood.
Principle Rose-Gottlieb Method is a gravimetric method in which fat globule
membrane of milk fat globules is ruptured by addition of ammonia to milk
sample to liberate the fat. The liberated fat is extracted using combination
of solvents viz., diethyl ether and petroleum ether. The solvents are
evaporated and obtained fat is dried and weighed. This method is considered
suitable for reference purposes. Strict adherence to details is essential in
order to obtain reliable results.
Apparatus/Instruments 1. Mojonnier fat extraction flask or any other suitable extraction tube (as
per IS specification).
2. Cork or stopper of synthetic rubber unaffected by usual fat solvents.
3. 100 mL flat bottom flask with G/G joint or stainless steel or aluminium
dishes of 5.5 cm height and 9 cm diameter or glass bowl.
Materials and reagents 1. Ammonia solution (Specific gravity - 0.91).
2. Ethyl alcohol (95%).
3. Diethyl ether, peroxide-free.
4. Petroleum ether, boiling range 40-60 ℃.
Sample preparation Refer method FSSAI 01.120:2022 for sample preparation.
Method of analysis 1. Weigh accurately about 10 g of sample in a beaker.

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 2. Add 1.25 mL of ammonia sp. gr. 0.91 (or an equivalent volume of a
more concentrated ammonia solution may be used), mix and shake
thoroughly.
3. Transfer the content to extraction tubes.
4. Proceed for the extraction of fat by Rose-Gottlieb method as prescribed
for milk from the step indicating addition of 10 mL ethyl alcohol
(Method No FSSAI 01.23:2022).
Calculation with units of
expression (𝑀1 − 𝑀2 ) − (𝑀3 − 𝑀4 )
𝐹𝑎𝑡 % (𝑚⁄𝑚) = × 100
𝑤

Where,

M1 is the mass, in g, of the fat-collecting flask and extracted matter after

drying
M2 is the mass, in g, of the empty fat-collecting flask
M3 is the mass, in g, of the fat-collecting flask used in the blank test after
drying
M4 is the mass, in g, of the empty fat-collecting flask used in the blank test
w is the weight of the sample in g
Reference 1. F.A.O. Manual of Food Quality Control, 14/8, page 8.

2. IS 1479 (Part II): 1961 (Reaffirmed Year: 2018). Methods of test for
Dairy Industry – Chemical Analysis of Milk. Bureau of Indian
Standards, New Delhi.

3. AOAC 905.02. 21st Edn. (2019). Official method –Fat in milk. AOAC
International, USA.

4. ISO 1211: 2010. Milk — Determination of fat content — Gravimetric

method (Reference method). International Organization for
Standardization, Geneva.

5. Pearson’s Composition and analysis of foods, 9th edn., 1991 page 538.
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 Determination of Milk Protein in Colostrum

Method No. FSSAI 01.124:2022 Revision No. & Date 0.0

Scope This method describes the determination of milk protein in colostrum.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer to
MSDS (Material Safety Data Sheets) for specific information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalies as solids or
concentrated solutions. While making solution add pellets to water and not
vice versa.
2. Concentrated sulphuric acid: Concentrated sulphuric acid is extremely
corrosive and can cause serious burns when not handled properly. It reacts
violently with water with evolution of heat; can react with organic
materials explosively. Do not mix with hydrochloric acid. Good general
ventilation should be provided to keep vapour and mist concentrations
below the exposure limits.
3. Ethanol: Extremely flammable. Use effective fume removal device when
heating or evaporating. Keep away from heat, sparks and open flame;
avoid breathing vapours.
4. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical splash
goggles at all times when handling Hydrochloric acid to protect your eyes
and skin. Concentrated hydrochloric acid is toxic if inhaled, so avoid
breathing it in and always handle it while under a fume hood. It reacts with
strong oxidizing agents to form toxic chlorine gas.
5. Ammonium sulphate: Thermal decomposition products can lead to release
of irritating gases and vapours. Minimize dust generation and
accumulation.
Principle Refer the method for determination of protein by Kjeldahl method in milk and
Apparatus/Instruments milk products (FSSAI 01.026/027:2022)
Materials and reagents
Preparation of Reagents
Sample preparation Refer method FSSAI 01.121:2022 for sample preparation.

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Method of analysis 1. Weigh quickly about 3-4 g of the prepared colostrum sample and transfer
to a 500 or 800 mL Kjeldahl flask/digestion tube taking care to see that no
portion of the sample clings to the neck of the flask/tube.
2. Refer the method for determination of protein in milk and milk products
(FSSAI 01.026/027:2022).
Calculation with units of Refer the method for determination of protein in milk and milk products
expression (FSSAI 01.026/027:2022)
Reference 1. ISO 8968-1/IDF 020-1:2001. Milk - Determination of nitrogen content
(Part 1): Kjeldahl method. International Organization for Standardization,
Geneva.

2. ISO 8968-2/IDF 020-2:2001. Milk - Determination of nitrogen content

(Part 2): Block-digestion method (Macro method). International
Organization for Standardization, Geneva.
3. IS 7219: 1973 (Reaffirmed year 2015). Method for determination of
protein in foods and feeds. Bureau of Indian Standards, New Delhi.

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 Determination of Melamine in Milk, Milk Products and Infant formula

Method No. FSSAI 01.125:2022 Revision No. & Date 0.0

Scope This method describes the determination of melamine in milk and milk
products and is applicable for infant formula.
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Perform work in fume hood while working with solvents. Refer to
MSDS (Material Safety Data Sheets) for specific information.

1. Melamine: Keep away from heat. Avoid contact with skin and eyes.
Keep away from incompatibles such as oxidizing agents, acids.

2. Acetonitrile: Do not inhale substance/mixture. Avoid generation of

vapours/aerosols. Keep away from open flames, hot surfaces and sources
of ignition. Keep container tightly closed in a dry and well-ventilated
place (+5 °C to +30 °C).

3. Formic acid: Do not inhale substance/mixture. Keep away from open

flames, hot surfaces and sources of ignition. May decompose forming
gaseous products, especially when stored over long periods, so place in
close containers in such a way to enable internal pressure to escape (e.g.
excess pressure valve). Protected from light. Keep container tightly
closed in a dry and well-ventilated place.

4. Ammonium formate: Ensure adequate ventilation. Avoid dust formation.

Keep containers tightly closed in a dry, cool and well-ventilated place.
Incompatible with strong oxidizing agents, strong acids and strong bases.

Principle A liquid chromatography triple quadrupole tandem mass spectrometry (LC-

MS/MS) method for residues of melamine consists of an initial extraction
with 2.5% aqueous formic acid, followed by a series of filtration,
centrifugation, and dilution steps. The method detects both melamine and
cyanuric acid using a Hydrophilic Interaction Liquid Chromatography
(HILIC) column and mass spectroscopy. Melamine is detected in positive ion
mode and cyanuric acid in negative ion mode. Analyte concentrations are
calculated using an external standard calibration curve prepared using pre-
fortified control matrix, which has been carried through the extraction
procedure.
Apparatus/Instruments 1. Liquid chromatograph: Binary LC pump is recommended for accurate
mixing at low flow rate and rapid response to mobile phase gradient.
2. Column: ZIC-HILIC, 2.1 × 150 mm, 5 μm, 200 Å or equivalent.

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 3. Mass Spectrometer: Triple quadrupole capable of meeting system
suitability.
4. Analytical balance (Readability 0.0001 g).
5. Centrifuge: Capable of 4000 rpm (approximately 3750 g) with 50 mL
tubes.
6. Microcentrifuge: Capable of 13,000 rpm (16000 g) with 1.5 /2 mL tubes.
7. Mixers and shakers: Single and multi-tube vortex mixers platform shaker
8. Ultrasonic bath: Including timer and heater.
9. Centrifuge tubes: 50 mL disposable polypropylene with caps, with
graduations from 5 to 50 mL and 1.5 mL microcentrifuge tubes.
10. Syringe Filters: Polyvinylidene fluoride (PVDF), 13 mm, 0.22 µm.
11. Syringes: Three mL polypropylene.
Materials and Reagents 1. Melamine (CAS #: 108-78-1).
2. Acetonitrile (LC grade).
3. Formic acid (Reagent grade > 95%).
4. Water (LC grade, or purified by Millipore Milli-Q system to > 18 Mohm
resistivity, or equivalent).
5. Ammonium formate (Purity > 97%).
Preparation of reagents 1. 0.1% Formic acid in water: One mL formic acid is transferred to one L
graduated flask and diluted to volume with LC water.
2. Mobile phase A: 0.1% Formic acid in acetonitrile (5:95 v/v). Mix 50 mL
of 0.1% formic acid in water with 950 mL acetonitrile in a one L solvent
bottle.
3. Mobile phase B: 20 mM Ammonium formate in acetonitrile (50:50 v/v).
Mix 500 mL of 20 mM ammonium formate and 500 mL of acetonitrile
in a one L solvent bottle.
4. 2.5% Formic acid in water: 25 mL formic acid is transferred to one L
volumetric flask and diluted to volume with LC grade water.
5. 20 mM Ammonium formate: 0.63 gm of ammonium formate is weighed
and dissolved in 500 mL LC grade water.
Sample preparation Refer appropriate method for sample preparation for the particular product.
Method of analysis 1. Sample of milk/milk products (2.0 ± 0.1g) is weighed in a 50 mL
polypropylene centrifuge tube.
2. Pre-fortify control and matrix calibration standards
3. 14 mL of 2.5% Formic acid in water is added to samples. Tube is tightly
sealed. Dissolve sample by shaking for 15-30 seconds (vortex as needed),

329 | M o M – D a i r y a n d D a i r y P r o d u c t s
 then sonicate in ultrasonic bath and mix on multi vortex mixer for 30 min
each.
4. Centrifuge at 4000 rpm (3750 g) for 10 min at 25 ± 3 C.
5. Approximately 1.4 mL of the supernatant is transferred into a 1.5 mL
micro centrifuge tube.
6. Centrifuge at 13,200 rpm (16100 g) for 30 min.
7. Load aqueous extract into a plastic 3 mL syringe and force through a 13
mm, 0.22 µm PVDF filter into a micro centrifuge tube. (Note: some
formulations may require some force, or two filtration steps to obtain a
clear solution before the next step.). Possible stopping point: aqueous
extracts can be stored at 5 - 10 °C for future dilutions.
8. Vortex mix for 30 seconds and centrifuge at 13200 rpm (16100 g) for 30 min.
9. Supernatant is transferred to a 2 mL autosampler vial, avoiding the
precipitate.

Instrumental Analysis
The column is equilibrated in mobile phase A at 0.4 mL/min for 30 - 60 min.
It is necessary to evaluate system suitability, solvent blank (1x) and mixed
standard are injected at 7.0 ng/mL (3-4x).

Data should meet the signal-to-noise and ion ratio criteria before continuing.
It is recommended to inject the standards and sample in following sequence:

(i) solvent blank (mobile phase A), (ii) extracted matrix standards from 0.25
to 5 μg/g, (iii) solvent blank, (iv) control extracts, (v) post-fortified extracts
and solvent standards for calculation of recoveries and matrix effects, (vi)
solvent blank, (vii) unknown samples, and (viii) continuous calibration
standards (an extracted matrix standard as well as solvent standard at 7
ng/mL), to verify that instrument response was maintained during the run.

Calculation with units of Use external standard calibration. The calibration curve should not include
expression the origin, but does include a matrix blank with a concentration of 0. Export
the processed data into Microsoft Excel or equivalent spreadsheet program
for further calculations:
Recovery (%) = calculated from extracted calibration curve
Matrix effect (%) = 100 x Post-fortified sample / solvent standard (same
concentration)
The limit of quantification (LOQ) for each analyte is defined as the
concentration of the lowest calibration standard used, or the lowest calibration
standard which shows > 10-fold higher response than background signals in
negative control sample.

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 Calculations for Confirmatory Analysis
Calculate ion ratios as percent relative abundances. The melamine ion ratio is
m/z 68/85.
Reference Turnipseed, S., Casey, C., Nochetto, C., & Heller, D. N. (2008).
Determination of melamine and cyanuric acid residues in infant formula
using LC-MS/MS. US FDA laboratory information bulletin, 24, 4421.
Approved by Scientific Panel on Methods of Sampling and Analysis

331 | M o M – D a i r y a n d D a i r y P r o d u c t s
 ANNEXURES

Preparation of Standard Solution of Sodium Hydroxide

Method No. FSSAI 01.126:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of standard solution of sodium hydroxide
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Refer to MSDS (Material Safety Data Sheets) for specific
information.
1. Sodium hydroxide: It is extremely caustic and can cause severe burns.
Protect skin and eyes when working with these alkalis as solids or
concentrated solutions. While making solution add pellets to water and
not vice versa.
Principle Since sodium hydroxide is not a standard substance, so to prepare a standard
solution of sodium hydroxide, it is required to be standardized against the
primary standard viz., potassium acid phthalate to accurately determine its
normality. When an acid reacts with an alkali, salt and water are formed and
the reaction is known as neutralization. An indicator indicates the end point
by its characteristic colour change, such as colourless to pink when
phenolphthalein is used as indicator and change in color occurs at pH 8.3.

Apparatus/ Instruments 1. Beakers of various capacity.

2. Volumetric flasks of various capacity.
3. Conical flasks of various capacity.
4. Burette (50 mL).
5. Analytical balance (Readability 0.0001 g).
Materials and reagents 1. Distilled water: Prior to use, boil distilled water for 20 min and cool to
room temperature (25 ± 3 °C).
2. Sodium hydroxide.
3. Potassium acid phthalate.
4. Phenolphthalein indicator.
Preparation of reagents A) Preparation of stock solution of sodium hydroxide
Mix 1 part of sodium hydroxide pellets and 1 part of water in an
appropriate flask. Swirl until solubilization of pellets. Close the flask

332 | M o M – D a i r y a n d D a i r y P r o d u c t s
 with rubber stopper and set aside (approximately 10 days) until sodium
carbonate has settled, leaving perfectly clear liquid.
B) Preparation of sodium hydroxide solution of approximate normality
In a laboratory, standard sodium hydroxide solutions of various strengths
may be required. For preparing a particular strength of sodium hydroxide
solution, dilute appropriate volume of stock solution of sodium
hydroxide (Table 1) to 10 L with distilled water.
Table 1: Volume of stock solution of sodium hydroxide required to
prepare solution of different normality.
mL of stock solution of sodium
Approximate Normality
hydroxide to be diluted to 10 L
0.01 5.4
0.02 10.8
0.10 54.0
0.50 270.0
1.0 540.0

C) Phenolphthalein indicator (1%): Dissolve 1 g phenolphthalein in 100

mL 95% ethanol.
Method of analysis Standardization of prepared sodium hydroxide solution of approximate
normality
1. Fill the burette with prepared standard sodium hydroxide solution up to
a known mark.
2. Transfer accurately weighed appropriate quantity of potassium acid
phthalate (Molecular weight: 204.229) in 300 ml flaks and add 50 ml of
distilled water. Swirl the flask gently until potassium acid phthalate
dissolves.
3. To it add 2-3 drops of phenolphthalein indicator.
4. Note down the initial reading on the scale of burette and then titrate with
sodium hydroxide solution until the colour changes from colourless to
pink.
5. Note down the final reading on the burette.

333 | M o M – D a i r y a n d D a i r y P r o d u c t s
 6. Determine the actual normality of the prepared sodium hydroxide
solution using following equation:
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 potassium acid phthalate (g) 𝑥 1000
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑑𝑖𝑢𝑚 ℎ𝑦𝑑𝑟𝑜𝑥𝑖𝑑𝑒 𝑢𝑠𝑒𝑑 (𝑚𝐿) 𝑥 (204.229)

Note:
If the Normality of the sodium hydroxide calculated is not same as
intended, then dilute it accordingly to have required normality using the
following equation
NaVa = NbVb

Where,
Na = required normality of the solution
Va = required volume of solution
Nb = Normality of prepared solution
Vb = Volume of prepared solution to be dilute

To check the normality of the final sodium hydroxide solution, again repeat
the standardization as indicated above.
Reference 1. AOAC International. 1995. Official Methods of Analysis of AOAC
International, 16th ed. Method 936.16, App. A, p. 7. The Association,
Arlington, VA.
Approved by Scientific Panel on Methods of Sampling and Analysis

334 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Preparation of Standard Solution of Hydrochloric Acid

Method No. FSSAI 01.127:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of standard solution of hydrochloric acid
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Refer to MSDS (Material Safety Data Sheets) for specific
information.
1. Concentrated hydrochloric acid: Take precautions like wearing a
chemical-resistant apron, chemical-resistant gloves and chemical
splash goggles at all times when handling Hydrochloric acid to protect
your eyes and skin. Concentrated hydrochloric acid is toxic if inhaled,
so avoid breathing it in and always handle it while under a fume hood.
It reacts with strong oxidizing agents to form toxic chlorine gas.
2. Sodium Carbonate: If improperly handled, or in the event of accidental
exposure, sodium carbonate can pose several hazards to people’s
health and safety. Inhalation of this chemical can lead to adverse
effects such as respiratory tract irritation, coughing, shortness of
breath, and pulmonary edema.
3. Safety glasses are to be worn whenever working with acids, bases, or
any flammable materials.
4. Let the instructor know immediately about any glassware breakage or
chemical spills that may occur so that proper cleanup procedures can
be instituted.
5. Use pipet fillers whenever pipetting any fluid. Whenever working with
acids or bases, have the chemicals available at your work station, do
not carry pipets with fluid across the room.
6. When diluting acids, pour the acid into the water, NOT water into acid
as this may cause spattering of the acid.
7. When acid is mixed with another reagent, an exothermic reaction may
occur which heats up the container, this is natural, so do not panic and
spill contents.
Principle The solution of hydrochloric acid is required to be standardized against the
primary standard viz., sodium carbonate to accurately determine its
concentration. When an acid reacts with an alkali, salt, water is formed and
carbon dioxide is released. When a solution of a weak alkali is titrated
against a strong acid, methyl orange indicator is used.

335 | M o M – D a i r y a n d D a i r y P r o d u c t s
Apparatus/ Instruments 1. Beakers of various capacity.
2. Volumetric flasks of various capacity.
3. Conical flasks of various capacity.
4. Burette (50 mL).
5. Analytical balance (Readability 0.0001 g).
Materials and reagents 1. Concentrated hydrochloric acid.
2. Sodium carbonate.
3. Methyl orange indicator.
Preparation of reagents A. Preparation of hydrochloric acid solution of approximate normality
In a laboratory, standard hydrochloric acid solutions of various strengths
may be required. For preparing a particular strength of hydrochloric acid
solution, dilute appropriate volume of concentrated solution of
hydrochloric acid (Table 1) to 10 L with distilled water.
Table 1: Volume of concentrated hydrochloric acid (36.5-38%)
required to prepare solution of different normality.
mL concentrated hydrochloric
Approximate Normality
acid to be diluted to 10 L
0.01 8.6
0.02 17.2
0.10 86.0
0.50 430.1
1.0 860.1

B. Methyl orange indicator (0.1 g/100 mL): Dissolve 0.1 g of methyl

orange in 50 mL of distilled water and make up the volume to 100 mL
with distilled water.
C. Reference Solution: 80 mL distilled water containing 3 or 4 drops
methyl orange.
Method of analysis Standardization of prepared hydrochloric acid solution of approximate
normality
1. Fill the burette with prepared standard hydrochloric acid solution up
to a known mark.

336 | M o M – D a i r y a n d D a i r y P r o d u c t s
 2. Transfer accurately weighed appropriate quantity of sodium carbonate
(Molecular weight: 105.988) in 300 ml flaks and add 50 ml of distilled
water. Swirl the flask gently until sodium carbonate dissolves.
3. To it add 2-3 drops of methyl orange indicator.
4. Note down the initial reading on the scale of burette and then titrate
with sodium hydroxide solution until the colour begins to deviate from
reference solution.
5. Note down the final reading on the burette.
6. Determine the actual normality of the prepared hydrochloric acid
solution using following equation:
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑑𝑖𝑢𝑚 𝑐𝑎𝑟𝑏𝑜𝑛𝑎𝑡𝑒 𝑥 1000
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑐ℎ𝑙𝑜𝑟𝑖𝑐 𝑎𝑐𝑖𝑑 𝑢𝑠𝑒𝑑 (𝑚𝐿) 𝑥 (105.988/2)

Note:
1. If the Normality of the hydrochloric acid calculated is not same as
intended, then dilute it accordingly to have required normality (0.1 N
hydrochloric acid solution) using the following equation
NaVa = NbVb

Where,
Na = required normality of the solution
Va = required volume of solution
Nb = Normality of prepared solution
Vb = Volume of prepared solution to be dilute

To check the normality of the final hydrochloric acid solution, again repeat
the standardization as indicated above.
Reference 1. AOAC International. 1995. Official Methods of Analysis of AOAC
International, 16th ed. Method 936.15; Appendix A, p. 3. The
Association, Arlington, VA.
Approved by Scientific Panel on Methods of Sampling and Analysis

337 | M o M – D a i r y a n d D a i r y P r o d u c t s
338 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Preparation of Standard Solution of Silver Nitrate

Method No. FSSAI 01.128:2022 Revision No. & Date 0.0

Scope This scope is for the preparation of standard solution of silver nitrate
Caution Follow all safety procedures while handling and disposing solutions. Wear
laboratory apron, shoes, safety goggles and mask while working with
chemicals. Refer to MSDS (Material Safety Data Sheets) for specific
information.
1. Silver nitrate: It is corrosive. It is not combustible, but it is a strong
oxidizer that enhances the combustion of other substances.
2. Potassium chromate: It is a strong oxidizing agent. It is a carcinogen
and mutagen and should be handled with extreme caution.
Principle The solution of silver nitrate is required to be standardized against the
primary standard viz., potassium chloride and indicator potassium chromate
to accurately determine its concentration. Silver nitrate reacts with sodium
chloride and forms a white precipitate of silver chloride. When all the
chloride ions are precipitated as silver chloride, excess silver nitrate reacts
with potassium chromate indicator and forms a reddish-brown precipitate
of silver chromate.
2Ag+ + CrO42- Ag2CrO4(s)

Apparatus/ Instruments 1. Beakers of various capacity.

2. Volumetric flasks of various capacity.
3. Conical flasks of various capacity.
4. Burette (50 mL).
5. Analytical balance (Readability 0.0001 g).
Materials and reagents 1. Potassium chloride.
2. Silver nitrate.
3. Potassium chromate indicator.
Preparation of reagents A. Preparation of silver nitrate solution of approximate normality
In a laboratory, standard silver nitrate solutions of various strengths may
be required. For preparing a particular strength of silver nitrate solution,
dissolve slightly more than theoretical weight of silver nitrate

339 | M o M – D a i r y a n d D a i r y P r o d u c t s
 (Molecular weight: 169.87) in distilled water and dilute to appropriate
volume.
D. 5% Potassium Chromate Solution: Weigh 5 g of potassium chromate
in a beaker and dissolve it in distilled water. Transfer the contents of
beaker to a 100 mL volumetric flask and make up the volume up to the
mark.
Method of analysis Standardization of prepared silver nitrate solution of approximate
normality
1. Fill the burette with prepared standard silver nitrate solution up to a
known mark.
2. Accurately weigh enough potassium chloride (e.g., 0.3 g for 0.1N
solution; Molecular weight = 74.555) and transfer to 250 mL flask and
add 40 ml of distilled water. Swirl the flask gently until potassium
chloride dissolves.
3. To it add 1 mL potassium chromate solution.
4. Note down the initial reading on the scale of burette and then titrate with
silver nitrate solution until first precipitable pale red-brown appears.
5. Repeat the titration for blank solution containing 75 mL distilled water
and 1 mL potassium chromate.
6. Note down the volume of silver nitrate required until first precipitable
pale red-brown appears.
7. Subtract the titration volume of silver nitrate used for blank from
volume of silver nitrate used for standard potassium chloride.
8. Determine the actual normality of the prepared silver nitrate solution
using following equation:
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑜𝑡𝑎𝑠𝑠𝑖𝑢𝑚 𝑐ℎ𝑙𝑜𝑟𝑖𝑑𝑒 (𝑔) 𝑥 1000
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑖𝑙𝑣𝑒𝑟 𝑛𝑖𝑡𝑟𝑎𝑡𝑒 𝑢𝑠𝑒𝑑(𝑚𝐿) 𝑥 74.555
Note:
1. If the Normality of the silver nitrate calculated is not same as intended,
then dilute it accordingly to have required normality using the
following equation
NaVa = NbVb

340 | M o M – D a i r y a n d D a i r y P r o d u c t s
 Where,
Na = required normality of the solution
Va = required volume of solution
Nb = Normality of prepared solution
Vb = Volume of prepared solution to be dilute
To check the normality of the final silver nitrate solution, again repat the
standardization as indicated above.
Reference AOAC International. 1995. Official Methods of Analysis of AOAC
International, 16th ed. Method 941.18; Appendix A, p. The Association,
Arlington, VA.
Approved by Scientific Panel on Methods of Sampling and Analysis

Food Safety and Standards Authority of India

(Ministry of Health and Family Welfare)
FDA Bhawan, Kotla Road,
New Delh i-110002
[Link]
For any query contact: sp-sampling@[Link]

341 | M o M – D a i r y a n d D a i r y P r o d u c t s
342 | M o M – D a i r y a n d D a i r y P r o d u c t s