Estimation of neutralizers, adulterants and
preservatives in milk and milk products
DTC 311 (Chemical quality Assurance)
Binod Kumar Bharti
Assistant Professor cum Jr. Scientist
Department of Dairy Chemistry
S.G.I.D.T., (BASU) Patna
Detection of Neutralizer
Rosalic Acid Test
Principle
Rosalic acid develops a rose red colour with milk containing carbonate and bicarbonate.
Apparatus
Test Tube, Pipette
Reagents
1. Rosalic Acid –one percent (w/v) in ethyl alcohol
2. 2. Ethyl alcohol-95percent by volume
Procedure
3. Take 5 ml milk in a test tube of capacity 20 ml.
4. Add 5 ml ethanol (95%,v/v) and
5. 4 drops of Rosalic acid solution (0.05%; w/v in ethanol). Mix the contents. Observe
the colour.
Interpretation
If carbonate or bicarbonate is present a rose red color will appear. If NaOH is present a
pink colour will appear.
Alkalinity of ash test
Apparatus-
Muffle Furnace, Silica crucible
Chemicals-
N/10 HCl, Phenolphthalein
Principle-
1. Take 20 ml milk in a silica crucible.
2. First evaporate the water to dryness, and then burn the content to ash
in a muffle furnace at 550°C.
3. Disperse the ash in 10 ml distilled water and titrate the ash content
against N/10 HCl using phenolphthalein as indicator.
Interpretation-
If the volume of N/10 HCl exceeds 1.20 ml, then the milk contains the
added neutralizers.
Determination of Sugar
Materials required
Boiling water bath, Test tube
Chemicals required
Resorcinol solution, Hydrochloric acid
Methods
[Link] 1ml of milk sample
2. Add 1 ml of 0.5% resorcinol solution (0.5% resorcinol solution in 3N
Hydrochloric acid).
3. Mix the contents and heat in boiling water bath for 5 min and observe
the colour
Observation
If sugar is present in the sample, the red color will be produced. Blank
shows pale yellow or brownish color.
Determination of Starch
Materials required
Test tube
Chemicals required
1% iodine solution
Methods
1. Take 3.0 ml of suspected milk sample in a test tube.
2. Add one drop of 1% iodine solution (1gm of iodine crystals and 5gm
Potassium iodide in 100 ml distilled water, mixed by warming to
dissolve the iodine crystals).
3. Mixing the contents and observe the colour
Interpretation:
Development of blue color indicates the presence of starch.
Determination of Glucose
Reagents
a) Barfoed‘s reagent ( Prepared by dissolving 24 gm of cupric acetate in
450 ml boiling distilled water and immediately adding 25 ml of 8.5%
lactic acid to the hot solution and cooling and diluting the contents to
500 ml).
b) Phosphomolybdic acid reagent. (Dissolve 35gm of Ammonium
molybdate and 5 gm of sodium tungstate in 400ml of 5% (W/V)
Sodium hydroxide solution. Boil the contents vigorously for 20 – 40
min so that the reactants are dissolved properly. While boiling, the
ammonia is released. No change in the color of litmus paper
indicates that reagent is free from ammonia. As a result of boiling,
water gets evaporated, now cool the contents and dilute to about 350
ml and add 125ml of concentrated (85%) phosphoric acid. Finally
make the volume to 500ml with distilled water).
Apparatus
Test tube, boiling water bath
Methods
1. Take 1ml of adulterated milk sample in a test tube
2. Add 1 ml of Barfoed‘s reagent.
3. Heat the mixture for 3 min in a boiling water bath and cool for 3min
under tap water.
4. Add 1ml of phosphomolybdic acid reagent and
5. Mix the contents and observe the colour.
Interpretation:
Formation of blue color indicates the presence of glucose.
Determination of Dextrin/ Maltodextrin
Reagents
Iodine solution, % Potassium iodide solution
Apparatus
Test tube,
Methods
1. Take 1ml of suspected milk sample in a test tube.
2. Add 1 ml of iodine solution (0.005N Iodine solution prepared using
3% Potassium iodide solution in water).
3. Mix the contents and observe the colour.
Interpretation:
Development of chocolate red brown color indicates the presence of
dextrin/ maltodextrin.
Determination of Formaldehyde
Hehner Test:
Reagents
Sulfuric acid, Ferric chloride
Apparatus
Test tube,
Methods
1. Take 10 ml of the suspected milk sample in a test tube.
2. Add 0.5 ml of ferric chloride solution.
3. Add gently 5 ml of sulfuric acid along the side of the tube so that it forms
a layers at the bottom without mixing the milk. Observe the colour.
Interpretation
Formation of a violet to purple colored ring at the junction of the two liquids
indicates the presence of formalin in the milk sample.
Determination of Formaldehyde
Leech test:
Reagents
concentrated HCl, ferric chloride solution
Apparatus
Test tube, Bunsen burners
Methods:-
1. Take about 5.0 ml of milk in a test tube.
2. Add to it equal volumes of concentrated HCl containing 1.0 ml of 10%
ferric chloride solution to each 500 ml of the acid.
3. Heat over a flame for about five minutes. Rotate the tube to breakup the
curd, and observe the color.
Interpretation
Development of violet color indicates the formaldehyde added as a
preservative.
Detergents in Milk
Chemicals:
Methylene blue solution, chloroform
Methods
i) Take 2.0 ml of milk in a test tube and
ii) add 1.0 ml of 0.025% methylene blue solution in water.
iii) Mix the contents and add 5.0 ml of chloroform and shake the
contents vigorously.
iv) Keep the tubes undisturbed for 15 min or centrifuge the contents for
10 min. and observe the color in lower layer.
Observation
Intense blue color in lower layer indicates the presence of detergent.
Detection of Vanaspati in Ghee
Isolate the fat from milk by heat clarification method.
Reagents
concentrated HCl, furfural solution,
Methods:
i. Take about 5 g of the melted fat in a test tube.
ii. Add 5 ml of concentrated HCl (AR grade).
iii. Add 0.4 ml furfural solution (2% in alcohol) and shake the tube
thoroughly for 2 min.
iv. Allow the mixture to separate.
v. The development of pink or red color in the acid layer indicates
presence of vanaspati.
vi. Confirm by adding 5 ml distilled water and shaking again. If the
color in acid layer persists, vanaspati is present. If the color
disappears, it is absent SP:18 (1987).
Detection of animal body fats and vegetable oils/fats by the Opacity Test
in ghee
i. Melt the sample of fat (5 gm) isolated by heat clarification method at
50 +1°C in a test tube and maintain for 3 min to equilibrate.
ii. Then transfer the test tube at 23 + 0.2°C water bath and record the
opacity time (Time taken by fat sample to acquire either O.D. at 570
nm between 0.14-0.16 or Klett reading using red filter between 58-
62 after adjusting the instrument to 100% transmittance).
iii. The opacity time of pure buffalo ghee is 14-15 min, cow ghee is 18-
19 min and that of ghee from cotton tract area is 11-12 min.
iv. The opacity time of buffalo ghee adulterated at 10% level with
vanaspati is 10-11 min, with pig body fat is 8-9 min, with buffalo
body fat is 2-3 min, with cow body fat is 3-4 min and with refined
oils is 20-25 min (Singhal, 1980).
Test for Skimmed Milk Powder in Natural milk (Cow, Buffalo, Goat,
Sheep)
i. Take 50 ml of milk in a 60 ml centrifuge tube.
ii. Place the tube in the centrifuge and balance it properly.
iii. Centrifuge at 3000 rpm for 15 minutes. Decant the supernatant
creamy layer carefully.
iv. Add 0.5 ml of 4 % acetic acid for coagulation and then add 2 ml of 1
% phosphomolybdic acid in water.
v. Mix the contents thoroughly and heat in a water bath at boiling
temperature for 15 minutes and then cool.
vi. The curd obtained from pure milk shall be greenish in color whereas
the curd of sample containing skimmed milk powder shall be bluish
in color.
vii. The intensity of bluish color depends on the amount of the skim
milk powder present in the sample.
Determination of Hydrogen peroxide
Reagents
2% paraphenylene diamine solution
Apparatus
test tube,
Methods
i. Take 5 ml milk in a test tube and
ii. Add 5 drops of 2% paraphenylene diamine solution and shake it
well.
iii. Change of the color of milk to blue confirms that the milk is added
with hydrogen peroxide.
Determination of Common salt
Principle
Detection of common salt is based on the principle of argentometric
titrations. In this case excess of silver nitrate is made to react with
potassium chromate and resulted in the formation of reddish brown
precipitates of silver chromate.
Reagents:
Silver nitrate solution, potassium chromate indicator
Methods:
i. Take 5ml of milk in a test tube.
ii. Add 1 ml of silver nitrate solution (4.3 gm/100ml double distilled
water).
iii. Mix it and add few drops of 5% potassium chromate indicator in
water and mix the contents. Observe the color change.
Observation: Development of yellow color indicates the presence of
excess salt and red color indicates the absence of excess sodium chloride.
Thank You
