foods

Review
Protein-Based Fat Replacers: A Focus on Fabrication Methods
and Fat-Mimic Mechanisms
Niloufar Nourmohammadi 1 , Luke Austin 2 and Da Chen 1, *

1 Department of Animals, Veterinary and Food Sciences, University of Idaho, Moscow, ID 83844, USA
2 Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA
* Correspondence: dchen@[Link]

Abstract: The increasing occurrence of obesity and other non-communicable diseases has shifted
the human diet towards reduced calorie intake. This drives the market to develop low-fat/non-
fat food products with limited deterioration of textural properties. Thus, developing high-quality
fat replacers which can replicate the role of fat in the food matrix is essential. Among all the
established types of fat replacers, protein-based ones have shown a higher compatibility with a wide
range of foods with limited contribution to the total calories, including protein isolate/concentrate,
microparticles, and microgels. The approach to fabricating fat replacers varies with their types, such
as thermal–mechanical treatment, anti-solvent precipitation, enzymatic hydrolysis, complexation,
and emulsification. Their detailed process is summarized in the present review with a focus on the
latest findings. The fat-mimic mechanisms of fat replacers have received little attention compared to
the fabricating methods; attempts are also made to explain the underlying principles of fat replacers
from the physicochemical prospect. Finally, a future direction on the development of desirable fat
replacers in a more sustainable way was also pointed out.

Keywords: protein; fat replacer; fat mimic; microparticulation; microgels; thermomechanical treatment

1. Introduction
Citation: Nourmohammadi, N.;
With the prevalence of obesity and obesity-associated chronic diseases, customers
Austin, L.; Chen, D. Protein-Based
become increasingly aware of the calorie intake of their diet. According to the National
Fat Replacers: A Focus on Fabrication
Health and Nutrition Examination Survey (2017–2020), ~42% of U.S. adults aged ≥20
Methods and Fat-Mimic Mechanisms.
have obesity [1]. The Dietary Guideline for Americans, 2020–2025, encourages the public
Foods 2023, 12, 957. [Link]
to consume low- or non-fat foods for healthier diets [2]. Reducing calorie intake by de-
10.3390/foods12050957
creasing the amount of fat in food, especially saturated ones, has been considered one of
Academic Editors: Norbert Raak, the strategies to reduce the occurrence of obesity [3]. Nevertheless, the commitment to
Ruifen Li and Laura Roman consuming low-fat foods or maintaining a low-fat diet remains challenging because of their
Received: 1 January 2023
deteriorated texture and sensorial properties compared to those of full-fat ones. Hence,
Revised: 9 February 2023
the development of fat replacers that imitate not only the functional role of fat but also its
Accepted: 22 February 2023 sensory features is essential to improve the quality attributes of low-fat foods. Depending
Published: 23 February 2023 on the properties and manufacturing approaches, fat replacers fall into two categories:
(1) fat substitute: typically, biomolecules or their degraded products with little or no calo-
ries, functioning similarly to fat. They fall into three main groups based on their sources,
which are carbohydrate-based, which can hold water and impart a creamy texture close
Copyright: © 2023 by the authors. to fat (such as starches and gums), protein-based (such as egg white, milk, and whey),
Licensee MDPI, Basel, Switzerland. and fat-based fat replacers, which are too large to be digested with little contribution to
This article is an open access article calories (such as Caprenin as cocoa butter fat substitute and Olestra) [4]. As a well-known
distributed under the terms and fat substitute example, Olestra is a product composed of sucrose, and hexa-, hepta-, and
conditions of the Creative Commons
octa-esters of saturated and unsaturated fatty acids [5], but its application has been limited
Attribution (CC BY) license (https://
due to the risks of causing gastrointestinal side effects, such as abdominal cramping [6];
[Link]/licenses/by/
(2) fat mimetic (FM): ingredients that partially mimic the organoleptic properties of animal
4.0/).

Foods 2023, 12, 957. [Link] [Link]

Foods 2023, 12, 957 2 of 17

fat, includes mainly food hydrocolloids (gums, cellulose microfibrils, pectins), proteins,
protein aggregates, protein–polysaccharides composites, and emulsion gels.
Protein-based fat replacers have received increasing attention. They boost the protein
nutrition of food products with a low-calorie contribution. According to the dietary refer-
ence, adequate intakes of at least 0.8 g/kg body weight of high-quality protein for sedentary
adults, the optimum amount of 1.2–1.8 g/kg body weight for adults with moderate activity,
and 1.8–2.2 g g/kg body weight for adults with hypertrophy and strength training per day
would be an ideal goal for health enhancement. When it comes to the health-related impact
of a high-protein diet, protein intakes above the recommended amount within a certain
range could limit the appearance of sarcopenia, and reduce the loss of muscle mass [7].
Due to their highly reactive features towards pH, temperature, ions, and enzymes, protein-
based fat replacers can be tuned with distinct physiochemical properties for expanded
food applications, such as in yogurt, cream cheese, salad dressings, and frozen desserts.
Common sources of proteins to develop fat replacers include egg white protein, whey
protein, gelatin, soy, pea, and zein [8]. Animal proteins are considered as higher quality
because of the well-balanced amino acid profiles and high digestibility and bioavailability.
For plant proteins, they commonly lack cysteine and methionine; however, this nutritional
deficiency could be overcome by mixing different types of plant proteins [9]. In addition,
due to the presence of anti-nutritional factors, such as trypsin inhibitors, phenolic com-
pounds, phytates, cyanogenic compounds, lectins, and saponins, the digestibility of plant
proteins can be reduced unless properly processed [10–12]. Furthermore, the incorporation
of protein-based fat replacers levels up the protein content in foods, which enhances protein
nutrition. The protein-based fat replacements have advantages over carbohydrate-based
ones in terms of flavor interactions [13] and the amount of fat that could be replaced [14].
However, protein-based fat replacers may not be suitable to be incorporated into over-
processed food products [15,16] because of protein denaturation and interaction with
other components (e.g., Maillard reaction), resulting in a loss of functionality and fat-like
mouthful feelings [17].
The types of fat replacers, and their characterization and food applications have been
reviewed systematically [12,18,19]. However, the approaches to develop protein-based
fat replacers and the mechanism of their fat-mimic effects, which varies significantly with
the source of proteins, their molecular weight, solubility, and surface chemistry, remain
rarely summarized. This information is essential to design fat replacers with desirable
functionality using the most appropriate approach. We attempt to cover the latest findings
on those within the last five years. Future perspectives on the production of protein-based
fat replacers with improved sustainability are also provided.

2. Types of Protein-Based Fat Replacers

2.1. Protein Concentrates and Isolates
Protein concentrates or isolates can be used directly as fat replacers (Figure 1). They
may be slightly denatured during the manufacturing process [20]. The former contains
~30–80% protein, whereas the latter reaches 90–92% [21,22]. Extensive research has been
conducted on developing low-fat dairy products including yogurt [23], ice cream [24],
and cheese [25,26] using protein concentrates/isolates, especially whey protein due to
its high compatibility with other dairy ingredients and a matching flavor profile [27,28].
A higher level (up to 6.8%) addition of whey protein could improve syneresis, yield stress,
storage modulus (G’), viscosity, and creaminess of the low-fat yogurt with reduced serum
separation compared to the control [29,30]. In low-fat cheese, the partial (3–8%) replacement
of fat with whey proteins has been found to improve its hardness because of the formation
of more compact structures [31].
Foods 2023, 12, x FOR PEER REVIEW 3 of 18
Foods 2023, 12, 957 3 of 17

Figure 1. A
Figure 1. schematic showing
A schematic the the
showing types of protein-based
types fat replacers.
of protein-based fat replacers.

2.2.
2.2. Microparticulated
Microparticulated Proteins
Proteins
Protein
Protein microparticulation
microparticulation is a is a process
process of aggregation.
of aggregation. Theof
The size size
theofparticles
the particles
com- com-
monly ranges within 0.1–10 μm, but larger ones >10 μm have also been reported [32]. [32].
monly ranges within 0.1–10 µm, but larger ones >10 µm have also been reported
Protein
Protein particles
particles withwith a size
a size larger
larger thanthan
5 μm5canµmbe can be detected
detected by oralby oral mucosa
mucosa [33];
[33]; thus, a thus,
a smaller size is preferable to provide the smoothening mouthful feelings.
smaller size is preferable to provide the smoothening mouthful feelings. The most widely The most widely
used
used microparticulated
microparticulated proteins
proteins is from
is from whey whey proteins,
proteins, which
which was patented
was patented in and
in 1988 1988 and
later commercialized with the brand name Simplesse ® [34]. Some other proteins, such as
later commercialized with the brand name Simplesse [34]. Some other proteins, such as
®

soy
soy protein,bovine
protein, bovineserum
serumalbumin
albumin[35],[35],egg
egg white
white protein
protein [36],
[36], gelatin
gelatin [37],
[37],zein
zein[38],
[38],wheat
protein [39], pea protein, and potato protein [40], have also been used to
wheat protein [39], pea protein, and potato protein [40], have also been used to produce produce fat replac-
ers, as summarized in Table 1. Due to their spherical shape and size,
fat replacers, as summarized in Table 1. Due to their spherical shape and size, they werethey were claimed
to mimic
claimed fat droplets
to mimic and create
fat droplets a smooth
and create and and
a smooth creamy mouthfeel
creamy through
mouthfeel througha “ball-bearing”
a “ball-
mechanism,
bearing” which which
mechanism, will bewill
discussed later. later.
be discussed

Table
Table A summary
1. summary
1. A of different
of different types types of protein-based
of protein-based fat replacers,
fat replacers, their fabrication
their fabrication methods,
methods, and
and applications.
applications.

Type
Type Protein
ProteinSource
Source Fabrication
Fabrication Method
Method Particle
Particle Size (µm) Application
Application References
References
Size (μm)
Protein Whey concentrates Ultrafiltration at 40–45 ◦ C, - Reduced fat [26]
Protein
concentrate/isolate Whey concentrates Ultrafiltration
membrane at 10
cut-off 40–45
kDa °C, - Reduced fat
cheese [26]
concentrate/isolate membrane cut-off 10 kDa cheese
Soy protein Protein solubilization at pH 10–250 Meat analog [41,42]
Soyfractions
protein 8 and
Protein 9 for 1 h, separate
solubilization at pH 8 and 10–250 Meat analog [41,42]
oil-rich cream by
fractions 9 for 1 h, separate oil-rich cream
centrifuge, protein
by centrifuge,
precipitation in pH protein
4.5 and
precipitation
5 by adding 1 MpH
in 4.5hold
HCl and 5 by
for 1 h
adding 1 M HCl hold for 1 h
Protein Microparticulated Heated 95 ◦ C for 5–15 min 2–20 Drinkable or [36]
Protein
microparticles Microparticulated
soy protein/egg Heated 95 °C for 5–15
with continuous min with
stirring 2–20 Drinkable or
semi-solidprotein- [36]
microparticles soywhite
protein/egg
protein continuous stirring semi-solid
rich foods
white protein
Microparticulated Heating to 10 ◦ C above 20–250
protein-rich foods
- [43]
whey, potato, and denature temperature of
Microparticulated Heating to 10 °C above denature 20–250 - [43]
pea proteins proteins, held for 10 min,
whey, potato, and temperature
and cooling of
in proteins, held for
a concentric
pea proteins 10 min, and cooling −
cylinder with 100–150 s 1 a
in
concentricshear rate with 100–150
cylinder
Microparticulated s−1 shear
Extrusion at 90 ◦rate
C with 2–7 Reduced- [44]
whey proteins a screw speed of fat yogurt
200–1000 rpm
Foods 2023, 12, 957 4 of 17

Table 1. Cont.

Type Protein Source Fabrication Method Particle Size (µm) Application References
Potato protein Extrusion at 80◦Cand 9–110 Fat- [40]
800 rpm screw speed, reduced dessert
pH 6.9
Pea protein Extrusion cooking at 10–75 Milk dessert [45]
100 ◦ C with 600 rpm
screw speed
Egg white protein Heated at 75 ◦ C for 13 min, 9.4 Salad dressing [46]
followed by high-shear
homogenization at
10,000 rpm for 60 s
Microparticulated Heated at 85 ◦ C for 15 min 0.01–2 Reduced-fat [47]
whey protein and sonicated at 20 kHz for cheese emulsion
1 min
Soy protein Alcalase 2.4 L hydrolysate 7.1–9.3 Ice cream [24]
hydrolysate followed by heating at
90 ◦ C for 20 min and
homogenized at 8000 rpm
for 6 min
Pea protein Hydrolysis by Alcalase - - [48]
hydrolysate 2.4 L for 3 min, followed by
heating at 85 ◦ C for 10 min
Zein/ Zein was dissolved in 80% 0.1–0.6 Sausage [49,50]
carboxymethyl ethanol and then added to
dextrin a dextrin solution, followed
by the removal of ethanol
Protein– Oat Oat β-glucan was - Sausage [51]
polysaccharide β-glucan/marine dissolved in buffer solution
complex collagen peptide at pH 3 (glycine–HCl
buffer) at
12% concentration and
mixed with marine
collagen peptide solution at
different ratios. The
samples were stirred at
25 ◦ C for 4 h, then
subjected to high pressure
at 400–500 MPa for 30 min
Pea protein/pectin Pea protein powder was - - [52]
added to solutions
containing different ratios
of pectin to reach 15% (w/v)
of protein concentrate. The
dispersion was stirred at
1500 rpm for 1 h at room
temperature followed by
adjusting the pH to 6.5
using 2 N NaOH
Protein microgels Canola protein Heated at 90 ◦ C under 1–100 Pickering [53]
microgels stirring for 1 h, stored at emulsion
4 ◦ C to form a gel followed stabilization as
by homogenization and a potential
complexation with fat replacer
polysaccharides
Foods 2023, 12, 957 5 of 17

Table 1. Cont.

Type Protein Source Fabrication Method Particle Size (µm) Application References
Whey protein Heated at 90 ◦C
for 20 min, 0.3–300 Low-fat yogurt [54]
emulsion gel followed by the addition of
microparticles oil, homogenization, and
addition of
glucono-delta-lactone
Whey Whey protein was heated - - [55]
protein/alginate at 80 ◦ C for 30 min
microgels followed by
alginate addition
Soy Salt-induced coacervation 1–3 - [56]
protein microgels followed by heating at
80 ◦ C for 30 min
under stirring
Note: The “-“ means “it is not reported”.

2.3. Protein–Polysaccharides Hydrogel

Either proteins or polysaccharides have the capacity to form hydrogels. Protein-based
hydrogels are mainly particle type, whereas those from polysaccharides are “thread or
linear” type. When mixing the two, complexation could occur [57], which tailors protein
functionality in foods by altering its surface chemistry and aggregating behavior [52,58,59]
(Figure 1). Many studies have confirmed that the addition of polysaccharides prevented
the coalescence and interaction between microparticulated proteins, either by shielding
charged groups or by decreasing the collision rate between molecules through an increase
in the viscosity of the system. The presence of polysaccharides can also bind a large amount
of water to provide a creaminess sensation through the oral process [60]. Thus, a protein-
based fat mimetic in combination with polysaccharides is usually formulated to replace
fat to develop low-fat products [27,37,57]. The polysaccharides used in complexation with
protein are gum arabic [61], pectin [52,62], alginate [63], and xanthan [64]. Nowadays,
there is a growing trend to develop plant protein–polysaccharide hydrogel from peas [52],
lentils [65], and soybeans [66] to increase their sustainability.

2.4. Microgel Particles

Protein-based microgel particles are novel types of fat replacers, with a size at nano- to
micro-meter level [32]. They can be categorized into different types: fragments of protein
hydrogels, protein assembles, emulsified gel droplets [67], and protein–polysaccharides
coacervates [68] (Figure 1). The particles have the dual properties of particles and polymers,
which endows ideal lubricating effects. Proteins can be used as the sole component of
the microgel particles, and their elasticity depends on the types of proteins used and the
interactions among protein molecules. In general, plant protein-based microgel particles
have smaller elastic moduli compared to those of animal proteins due to the formation
of less covalent bonds [69]. By changing environmental conditions, such as pH and ionic
strength, the surface charge of protein molecules alters, which further affects their elec-
trostatic interactions, resulting in distinct elasticity. For example, a 15-fold increment of
elasticity of whey protein microgel particles was found at pH 3 and pH 5.5 compared to
those at neutral pH [70]. Besides protein itself, other components could also be incorpo-
rated into microgel particles, such as polysaccharides and/or plant oils. Either of them
contributes to the increased deformability of the particles. When plant oil was used, the
system turns into an emulsion type, with Pickering emulsion being the typical example [53].
The emulsion-type protein-based microgels commonly have a more regular shape and
better lubricating capacity than those of protein-only particles due to the formation of
a lubrication film from the protein nanoparticles and base oils [71]. However, they are
vulnerable to environmental change and long-term stability remains a challenge. Synthetic
 Synthetic polymers or organic solvents might be used to decrease the surface tension or
increase the stability of emulsion-type microgel particles, but they are not suitable for food
Foods 2023, 12, 957 6 of 17
applications.

3. Production of Protein-Based Fat Replacers

polymers or organic solvents might be used to decrease the surface tension or increase the
The way to fabricate fat replacers associates closely with their physicochemical prop-
stability of emulsion-type microgel particles, but they are not suitable for food applications.
erties and functionalities, such as particle size and shape, viscosity, gelling, emulsifying,
foaming,
3. andof
Production water-holding
Protein-Basedcapacity. This further affects the color, texture, and other sen-
Fat Replacers
soryThecharacteristics of the
way to fabricate fat final products.
replacers For closely
associates proteins with
with high
their water solubility,
physicochemical prop-such as
wheyand
erties proteins, thermalsuch
functionalities, treatment to trigger
as particle size andprotein aggregation
shape, viscosity, under
gelling, the controlled
emulsifying,
shear is commonly
foaming, used. Forcapacity.
and water-holding those with
Thislimited
furtherwater solubility,
affects the color,microparticulation
texture, and other could
sensory characteristics
be achieved of the
by breaking final large
down products. For proteins
aggregates into with highones
smaller water
orsolubility,
improving such
their sol-
as whey proteins, thermal treatment to trigger protein aggregation under the controlled
ubility/dispersibility first followed by precipitation. In the following sections, the common
shear is commonly
approaches used to used. For those
produce with limited
fat replacers arewater solubility,
discussed withmicroparticulation
a focus on proteincould
micropar-
be achieved by breaking down large aggregates into smaller ones or improving their
ticles and microgels.
solubility/dispersibility first followed by precipitation. In the following sections, the
common approaches used to produce fat replacers are discussed with a focus on protein
3.1. Protein Concentrate or Isolate
microparticles and microgels.
The production of a protein concentrate or isolate has been well summarized [72–75]
3.1.
andProtein Concentrate
will not or Isolate
be detailed here. Animal proteins, especially dairy proteins, are concentrated
The production[26]
by ultrafiltration of aorprotein concentrate
diafiltration or isolate
followed byhas been well
optional summarized
evaporation of [72–75]
the retentate
and will not be detailed here. Animal proteins, especially dairy proteins, are
prior to spray drying (Figure 2). The temperature of spray drying associates closely with concentrated
by
theultrafiltration
denaturation [26]oforthe
diafiltration
proteins,followed
and theirby optional evaporation
aggregation of the retentate
and particle prior The
morphology.
to spray drying (Figure 2). The temperature of spray drying associates closely with the
mean diameter of whey protein powders has been reported to increase from ~15 μm to
denaturation of the proteins, and their aggregation and particle morphology. The mean
~20 μm when the outlet temperature was increased from 60 °C to 100 °C [76]. The mor-
diameter of whey protein powders has been reported to increase from ~15 µm to ~20 µm
phology
when the of whey
outlet protein concentrate
temperature was increasedparticles
from was
60 ◦ Cchanged from
to 100 ◦ C [76].spherical at a lower inlet
The morphology
of whey protein concentrate particles was changed from spherical at a lower inlet air and
air temperature to a deflated shape at high temperatures [77]. How the morphology
size of particles
temperature in a milk
to a deflated protein
shape isolate
at high or concentrate
temperatures affect
[77]. How thetheir fat-mimic
morphology andcapacity
size re-
mains
of poorly
particles in a studied. It has
milk protein to beornoted
isolate that the
concentrate sizetheir
affect andfat-mimic
morphology of the
capacity particles in
remains
powders
poorly may It
studied. change
has to after
be notedtheythat
arethe
incorporated into the food
size and morphology of thematrix due
particles in to the presence
powders
may change
of water, after
salt, or they
otherare incorporated
food [Link] the food matrix due to the presence of water,
salt, or other food components.

[Link]
Figure schematic showing
showing thethe production
production of protein
of protein isolates
isolates and concentrates.
and concentrates.
Foods 2023, 12, 957 7 of 17

For plant proteins, wet and dry fractionation has been widely used (Figure 2). Wet
methods include mainly alkaline-isoelectric point precipitation, or water, salt, or acid
extraction. By using acid or alkaline, the protein recovery efficiency is high due to the
Foods 2023, 12, x FOR PEER REVIEWincreased solubility of proteins at the pH values far away from its isoelectric point [41,42]. 8 of 18
Alkaline-isoelectric precipitation is more commonly used than other methods in food
industries to extract proteins followed by spray drying to produce a protein isolate [78], but
it uses a large amount of water and generates excessive salt in wastewater, which potentially
addition, the protein content in the fed materials can be adjusted to higher values (≥20%),
threatens the freshwater ecosystem. In addition, high pH used during extraction may cause
which significantly
denaturation, increasesand
racemization, thelysinoalanine
yield of protein microparticles
formation of plant[85]. Wheyresulting
proteins, protein parti-in
deteriorated quality [78,79]. Dry fractionation is more environmentally friendly andof
cles produced through extrusion have been found to enhance the creaminess low-fat,
specific
plain,
to stirred
produce yogurt concentrate
a protein while maintaining
(40–50%itsprotein
consumer acceptance
content). It takeswith a less than
advantage of the0.5%
sizead-
dition
and [44,85].
charge Similarly,
of proteins thediffered
that incorporation of pea
from those of protein
starch andmicroparticles
dietary fiberderived from the
and achieves
same
the method into
separation fat-reduced
by sieving milk desserts
or electrostatic resultedforces
interactions in approximately
[72]. Since nothe same
high pHcreamy
and
perception as the full-fat milk dessert [45]. The conventional extrusion
extensive heat are involved during isolation, the proteins tend to have better functionality method consumes
a large
than amount
those from theof energy
alkalinetomethod.
unfold proteins for subsequent aggregation. A hybrid tech-
nology, which takes advantage of the expanding properties of supercritical carbon diox-
3.2. Microparticulated
ide (SC-CO Proteins
2) to induce aggregation at lower temperatures (<100 °C), is more energy effi-
3.2.1. Thermal–Mechanical
cient [86]. In supercritical fluid Treatments
extrusion, proteins tend to expose the hydrophobic regions
Thermomechanical treatment
and aggregates driven by the surrounding is the mostnon-polar
common approach
environment, to develop micropartic-
resulting in distinct
ulated
propertiesproteins as fat replacers.
of microparticles, In general,
such a heating sourceinteractions,
as the protein–protein is needed tosurface
unfold hydropho-
proteins
by heating
bicity, and above their denaturation
rheological properties [87]. temperature (Figure 3). Unfolded proteins are then
aggregated via covalent (S-S bonds)
High-pressure homogenization combined and non-covalent interactions
with heat treatment (mainly hydrophobic
is another technique
interactions). Animal proteins are commonly heated at 75–95 ◦ C for 20–40 min for mi-
capable of applying a strong shear force and a sudden pressure modulation to tune pro-
croparticulation ◦
tein aggregationto(Table occur1).[80–82].
Liu et More extensive
al. used such a heating
method(80–95
(10,000 C, rpm~30 min)
for 60 s,is75
required
°C for 13
for
min) to produce microparticulated egg white proteins as a fat replacer in salad heating
plant proteins due to their higher thermal stability [43,54]. The duration of dressings,
shortens with the increase of temperature. For instance, pea protein particles were formed
which are comparable to the commercial salad dressings’ features [46]. Ultrasound-as-
within a minute at 135 ◦ C [45]. The morphology and size of the protein particle changes
sisted heating could also induce protein microparticulation. The ultrasonication could be
with the heating conditions. At lower temperatures, particles commonly have a smaller size
conducted simultaneously with the heating or after. A recent study found large whey pro-
with higher compactness [45,82–84]. Besides temperature, the shear force also applies to
tein aggregates (60–600 μm) were formed by heating, whose size was reduced to 0.01–2
the system during heating unless at a low protein concentration (≤5%), otherwise, gelation
μm upon
could sonication
occur. The size[47]. Theparticles
of the treatment also resulted
negatively in higher
correlates withsurface hydrophobicity
the shear force. High of
the particles, which is possibly due to the exposure of more interior
shear force results in more rigorous breaking of the aggregates and produces particles regions of protein
with
aggregates.
smaller sizes [43], as demonstrated in whey proteins [82].

Figure 3. AAschematic
Figure schematicillustrating
illustratingthe
theproduction
productionofofmicroparticulated
microparticulatedproteins
proteinsusing
usingthermal–me-
thermal–
chanical treatments.
mechanical treatments.

3.2.2. Enzymatic Hydrolysis

Protein aggregation and microparticulation could also be triggered by enzymatic
treatment with or without additional heating. Transglutaminase catalyzes acyl transfer
between ε-amino groups of lysine residues and the γ-carboxyamide groups to form cova-
Foods 2023, 12, 957 8 of 17

Multiple strategies have been adopted to provide heat and shear simultaneously
(Figure 3). The simplest setup is to stir the sample on a temperature-controlled water bath.
The strength of the shear can be fulfilled through adjusting the stirring speed, but the
heating and cooling rate may be beyond control unless a sophisticated heating/cooling
system is used. The other shortcoming of the method is it fails to provide ideal mixing of
the concentrated protein suspension because of the high viscosity. These can be overcome
by using a concentric cylinder accessory (bob-cup) equipped on a rheometer. The shear
rate of the bob can be adjusted to the required value while the temperature is controlled by
the cup. Such a method has been used to design microparticulated structures from whey,
potato, and pea proteins [43] (Table 1). Within a certain range, the increase of the shear
rate reduces the size of the formed particles as it disrupts the accumulated aggregation of
proteins. The concentric cylinder is also able to monitor the microparticulation progress
and explore the effects of shear force and temperature on the viscoelasticity, but the large-
scale production of microparticles using a concentric cylinder remains a challenge as
the volume of the cup is limited (e.g., 25 mL or less). The extrusion process, another
thermomechanical treatment, is widely applied in food industries with the features of
large-scale and continuous production. Proteins are unfolded and aggregated under
the control of heating and screw rotation. Compared to the water bath and concentric
cylinder, the temperature inside the extruder can achieve above 100 ◦ C due to the high-
sealed environment [45]. This facilitates protein microparticulation within a short duration.
In addition, the protein content in the fed materials can be adjusted to higher values
(≥20%), which significantly increases the yield of protein microparticles [85]. Whey protein
particles produced through extrusion have been found to enhance the creaminess of low-
fat, plain, stirred yogurt while maintaining its consumer acceptance with a less than
0.5% addition [44,85]. Similarly, the incorporation of pea protein microparticles derived
from the same method into fat-reduced milk desserts resulted in approximately the same
creamy perception as the full-fat milk dessert [45]. The conventional extrusion method
consumes a large amount of energy to unfold proteins for subsequent aggregation. A hybrid
technology, which takes advantage of the expanding properties of supercritical carbon
dioxide (SC-CO2 ) to induce aggregation at lower temperatures (<100 ◦ C), is more energy
efficient [86]. In supercritical fluid extrusion, proteins tend to expose the hydrophobic
regions and aggregates driven by the surrounding non-polar environment, resulting in
distinct properties of microparticles, such as the protein–protein interactions, surface
hydrophobicity, and rheological properties [87].
High-pressure homogenization combined with heat treatment is another technique
capable of applying a strong shear force and a sudden pressure modulation to tune protein
aggregation (Table 1). Liu et al. used such a method (10,000 rpm for 60 s, 75 ◦ C for 13 min) to
produce microparticulated egg white proteins as a fat replacer in salad dressings, which are
comparable to the commercial salad dressings’ features [46]. Ultrasound-assisted heating
could also induce protein microparticulation. The ultrasonication could be conducted si-
multaneously with the heating or after. A recent study found large whey protein aggregates
(60–600 µm) were formed by heating, whose size was reduced to 0.01–2 µm upon soni-
cation [47]. The treatment also resulted in higher surface hydrophobicity of the particles,
which is possibly due to the exposure of more interior regions of protein aggregates.

3.2.2. Enzymatic Hydrolysis

Protein aggregation and microparticulation could also be triggered by enzymatic treat-
ment with or without additional heating. Transglutaminase catalyzes acyl transfer between
ε-amino groups of lysine residues and the γ-carboxyamide groups to form covalent cross-
links, but the reaction is slow and requires hours to complete, which hinders large-scale
production [88]. The breaking down of proteins to expose hydrophobic regions or reactive
amino acid residues also induces aggregation [48], which can be fulfilled through limited
enzymatic hydrolysis. Compared to heat-induced aggregation, proteolysis is a greener
approach, which consumes less energy. Depending on the types of proteases and the
Foods 2023, 12, 957 9 of 17

proteins, the conditions to promote aggregation vary. For instance, small aggregates were
formed in Bacillus licheniforms (BLP) hydrolyzed whey proteins when 70% of the proteins
were intact [89]. Using the same enzyme, whey proteins were found to form soft and turbid
aggregate gels at 50 ◦ C for 1 h [90]. For pea proteins, when the degree of hydrolysis was
controlled to around 6–7% by a short time (2–3 min) hydrolysis with alcalase at 50 ◦ C,
the obtained hydrolysates were aggregated rapidly in response to heat. Analysis of the
aggregates found that non-covalent interactions were the dominant forces that drive aggre-
gation [48]. This suggests the aggregates might be deformed easily to provide lubricating
effects. Zang et al. studied the influence of limited enzymatic hydrolysis of rice bran pro-
teins by trypsin, at the ratio of 1:100 (Enzyme:Substrate)(v/w), on the emulsifying properties.
They found a significant enhancement of the emulsifiying properties of hydrolysates with
a 3% degree of hydrolysis. This was due to the release and exposure of soluble peptides
from insoluble aggregates resulting in the exposure of more ionizable amino groups [91].

3.2.3. Anti-Solvent Precipitation

Anti-solvent precipitation has been used to produce fine particles at the micro- and
nano-scale level. The size and morphology of the particles could be well controlled by
adjusting the protein concentration and polarity of the solvent [92]. The method is mainly
applied to fabricate prolamin-based composites, such as zein and wheat gluten. Zein
is the dominating protein found in maize, which can be extracted from dry-milled corn
(DMC) or distillers-dried grains by using aqueous ethanol, acetic acid, or alkaline [93,94].
The hydrophobic nature of zein and its incomplete amino acid profile limit the food
applications. Nevertheless, the inherent hydrophobicity and the heat-softening capacity of
zein make it an ideal candidate for fat analog [95]. To achieve this, zein or its aggregates are
suggested to micronize to a level similar to those of oil droplets by anti-solvent precipitation.
Firstly, hydrophobic proteins are solubilized in an organic solvent, such as aqueous ethanol
(70 to 90% v/v) or acetic acid/ethanol solution (e.g., at 55/45 ratio, v/v, pH 3.0) [96] under
agitation [97,98]. Then, the concentration of the organic solvents is lowered down by adding
water, resulting in increased polarity of the environment [99]. This triggers the aggregation
of zein or wheat gluten mediated by hydrophobic interactions and precipitates out from
the system when gravity overcomes electrostatic repulsions. Cui et al. have used zein
nanoparticles prepared from anti-solvent precipitation as a stabilizer in low-fat emulsions.
They solubilized zein in an 85% (v/v) aqueous ethanol at room temperature followed by
the addition of phytic acid (PA) solutions to improve its stability. By stirring the mixture
continuously for 30 min at 600 rpm, zein protein nanoparticles with a mean size of ~160 nm
were formed [100]. The concentration of the organic solvent and the drying temperature
directly links to the size and stiffness of the formed nanoparticles. Bisharat et al. (2018)
found that the size of zein particles was increased from an average of 200–500 nm to 1 µm
as the ethanol concentration was decreased from 90% to 70%. When the drying temperature
was decreased from 55 ◦ C to 40 ◦ C and then to room temperature, the Young’s modulus
of the particles was dropped continuously, corresponding to a higher flexibility and less
resistance to stretching [101,102]. Zein could also form particles containing polysaccharides
using anti-solvent precipitation. The particles have been shown to stabilize oil droplets as
a potential fat replacer in sausages [49,50].

3.2.4. Protein–Polysaccharides Hydrogel

When proteins and polysaccharides co-exist, depending on the environmental condi-
tions and the concentration of each component, they behave distinctly. Thermodynamic
incompatibility occurs mainly at high protein and polysaccharide concentrations due to
their differed chemical nature and affinity towards solvent. Nevertheless, phase separation
may not occur because of the high viscosity of the system. When gelation occurs fast, for
instance, heating under a high temperature, the extent of phase separation or inhomogene-
ity of the gel would become less [60]. This results in higher consistency of the gel texture.
Even though heating is not the sole condition to form a protein–polysaccharides hydrogel,
Foods 2023, 12, 957 10 of 17

it is the most common approach. Depending on the types of proteins and polysaccharides,
they can both gel under heat, but not always. Most of the proteins are heat-sensitive and
tend to unfold and aggregate to form a gel under heat, whereas polysaccharides could
remained unchanged [103]. The proportion of the polysaccharides affects the rheological
and fat-mimic capacity of the mixed gel and can be tuned accordingly. For instance, when
mixing soy protein isolate and cellulose nanofibrils (CF), with the increasing of CF, the
creaminess was increased [104]. If strong electrostatic interactions occur during the initial
heating of proteins and polysaccharides, they can be gelled without extra heating. Since the
complexation occurs rapidly, the protein or polysaccharides solution needs to be prepared
separately before mixing. A high-pressure homogenization might be required to facilitate
a homogenous distribution of polysaccharides and proteins [51]. Using this approach, Fan
et al. (2020) developed oat β-glucan/marine collagen peptides mixed gels to replace the
fat in sausage products [51]. Adjusting the environmental pH or ionic strength enables
us to modify the surface charge of proteins, which further tunes the interactions between
proteins and polysaccharides [103], and the textural properties of the gel. As non-covalent
interaction are weak forces, the formed gels commonly possess high deformability against
force and heat as desirable fat mimics.

3.3. Microgel Particles

For protein-only microgel particles, a hydrogel needs to be prepared followed by
size reduction via homogenization or microfluidization to form micro- or nano-particles.
Gelation could be conducted by using either the hot or cold method. Heat gelation oc-
curs normally at 80–95 ◦ C at a relatively high protein concentration (≥10%) for 20–30 min
to induce protein aggregation and cross-linking (Figure 4). Cold gelation also requires
heat to denature the protein first, followed by aggregating using calcium [105], salt [56],
polysaccharides [55], or acid [106] at room temperature (Table 1). By changing the con-
centration of the reactants, the microgel particles display distinct physicochemical prop-
erties. For instance, when the concentration of CaCl2 was increased from 0.02 to 0.1 M,
the size of whey protein microgel particles was decreased with the increment of their
viscoelasticity [105]. When using salt, the concentration should be high enough to screen
the surface charge of proteins to promote coacervation [56], but not cause the “salt in”
effects. Similar to salt, the charged polysaccharides could also mediate the aggregation
of proteins by reducing their surface charge. The hydrogen bonding between proteins
Foods 2023, 12, x FOR PEER REVIEW 11 of 18
and polysaccharides may also contribute and possibly play more significant roles than the
electrostatic interaction on large-scale aggregation [107].

Figure 4. A
Figure4. A schematic
schematic showing
showing the
the formation
formation of
of protein-based
protein-based microgels.
microgels.

Besides protein-only or protein-polysaccharides microgel particles, there is increas-

ing interest to fabricate emulsion-based ones (Figure 4). Proteins could be gelled [53] or
denatured via heating (e.g., 90 °C for 20 min) [54] followed by emulsification with plant
oil using homogenization. Heating enables the exposure of the buried hydrophobic re-
gions of proteins for increased non-polarity favoring the stabilization of oil droplets.
Foods 2023, 12, 957 11 of 17

Foods 2023, 12, x FOR PEER REVIEW 12 of 18

Besides protein-only or protein-polysaccharides microgel particles, there is increasing
interest to fabricate emulsion-based ones (Figure 4). Proteins could be gelled [53] or
denatured via heating (e.g., 90 ◦ C for 20 min) [54] followed by emulsification with plant oil
usingFor the particles with
homogenization. irregular
Heating shapes
enables theand larger sizes,
exposure of thesuch as aggregates
buried hydrophobic from plant
regions
protein
of or protein
proteins hydrolysates,
for increased non-polaritytheir favoring
deformation or even disintegration
the stabilization would
of oil droplets. contrib-
When the
ute to a soft and/or
concentration smooth proteins
of denatured texture (Figure
is too low5). to
Mechanical
form gels,deformation of foodsteps
additional gelling throughare
chewing and a biting motion in the oral cavity helps facilitate
required, such as acidification [54] and complexation with polysaccharides [108]. In some organoleptic perception,
such as
other flavor
cases, releaseare
proteins or mixed
the textural attributes
with oil in theirofnative
food. states
Theseto movements,
form emulsions alongside
withthe or
forces that
without thethe tongue makes
assistance by bouncing
of an emulsifier, then and excreting
heated against to
or acidified thetrigger
palate,the
could deform
gelation of
or even [109,110].
proteins break the Thermal
protein aggregates
gelation hasthrough
been showndisrupting
to delivernon-covalent
aggregatesinteractions
or heterogeneous[116].
microgels,
Klost et [Link]
(2020) havegelation by acidification
changed the proportion resulted in spherical
of soluble and more
and insoluble peahomogenous
protein ag-
particles
gregates with
in thea fermented
better fat-mimic
gels tocapacity
mimic the [110]. To facilitate
texture of yogurt. industrial
They found applications,
the gels with the
microgel particles can be harvested by centrifugation or membrane
higher insoluble aggregates showed higher flexibility and were easier to deform, showing filtration followed by
spray
smallerdrying
elastictomoduli.
form powders.
After reducing the size of the aggregates by applying a larger in-
tercycle strain, the system was converted from predominantly elastic to plastic behavior
4. TheInFat-Mimicking
[58]. a recent study,Mechanisms
Chen and Campanella (2022) observed a substantial reduction of
An ideal
viscosity of peafatprotein
replacer should possess
hydrolysate lubrication,
gels under shear. flow
Suchproperties,
shear-thinningand behavior
heat melting was
capacity [111] analog to fat. Unfortunately, protein-based fat replacers
partially due to the breaking down of the formed aggregates in addition to the realign- can hardly mimic
all of them.
ment Providing lubrication
of the aggregates toward the shear for theflow
reduced friction
direction [48].of food products is the main
function of a fat replacer. One of the theories
Another assumption for the fat-mimic mechanism is the to explain theemulsification
changes in textures is the
effects where
“ball-bearing”
the fat replacers effect.
mayIt form
refersan to emulsifier
the protein layer
microparticles
or oil layer or microgels employing
on the respective a rolling
surfaces for
mechanism
lubrication, similar
depending to “ball
on thebearings”
stability (Figure
of emulsion5). When force is applied,
gel microparticles the 5).
(Figure balls
Forrotate
those
and/or
with highslide to decrease
stability [117],frictions of the surrounding
the oil droplets are entrapped matrix [112]. The surrounding
by interacting with the inner matrix
sur-
could be the neighboring protein networks or the boundary regime
face of protein particles during mastication. The outer surface of protein particles contacts between proteins and
non-protein components [113–115].
with other macromolecules The shape
(e.g., starch, and in
proteins) size
theoffood
the protein
[Link] allow
Since the oil droplet
them
inside the emulsion gel microparticles has a low glass transition and high flexibility,and
to entrain in the narrow space between the tongue and palate in the mouth the
provide creaminess
outer layer that made and
uptheof perception of smoothness.
protein particles can act as aThe spherical
filler. shape and size
When experiencing of
force
microparticulated
such as chewing, fat thereplacers
particlesranging
slide tofrom
each0.2 to 9 and
other µm are the key
deform to factors
reduce in providing
the friction andthe
ball-bearing effect. This mechanism has been claimed to be dominant
increase the creaminess. For emulsion-type particles with limited stability, during masti- for microparticulated
whey proteins in liquid and semi-liquid model foods [113]. Besides size and shape, the
cation, they can be broken down into small fragments at a small deformation with the
interior compactness of the protein particles is also highly desirable for the ball-bearing
release of entrapped oil. The released oil lubricates the foods and provides a smooth
effect to occur. Sarkar et al. (2017) found negligible changes in the microstructure and
mouth perception [118]. For a certain type of fat replacers, a set of fat-mimic mechanisms
size of whey protein microgel particles after the tribology test, implying their resistance
may occur which requires a comprehensive assessment [32,119].
towards friction force [115].

Figure 5.
Figure 5. A
A schematic
schematic showing
showing the
the mechanisms
mechanisms of
offat-mimic
fat-mimiceffects.
effects.

For the particles

5. Conclusions with irregular
and Future Trends shapes and larger sizes, such as aggregates from plant
protein or protein hydrolysates, their deformation
In summary, designing protein-based or evenisdisintegration
fat replacers would the
essential to improve contribute
quality
attributes of low-fat and fat-free food products; although, they are more costly compared
to those of carbohydrate-based ones. Microparticulated proteins, as the dominant fat re-
placers, are produced mainly by thermal–mechanical treatment. Other fabrication
Foods 2023, 12, 957 12 of 17

to a soft and/or smooth texture (Figure 5). Mechanical deformation of food through
chewing and a biting motion in the oral cavity helps facilitate organoleptic perception,
such as flavor release or the textural attributes of food. These movements, alongside the
forces that the tongue makes by bouncing and excreting against the palate, could deform
or even break the protein aggregates through disrupting non-covalent interactions [116].
Klost et al. (2020) have changed the proportion of soluble and insoluble pea protein aggre-
gates in the fermented gels to mimic the texture of yogurt. They found the gels with higher
insoluble aggregates showed higher flexibility and were easier to deform, showing smaller
elastic moduli. After reducing the size of the aggregates by applying a larger intercycle
strain, the system was converted from predominantly elastic to plastic behavior [58]. In
a recent study, Chen and Campanella (2022) observed a substantial reduction of viscosity
of pea protein hydrolysate gels under shear. Such shear-thinning behavior was partially
due to the breaking down of the formed aggregates in addition to the realignment of the
aggregates toward the shear flow direction [48].
Another assumption for the fat-mimic mechanism is the emulsification effects where
the fat replacers may form an emulsifier layer or oil layer on the respective surfaces for
lubrication, depending on the stability of emulsion gel microparticles (Figure 5). For those
with high stability [117], the oil droplets are entrapped by interacting with the inner surface
of protein particles during mastication. The outer surface of protein particles contacts with
other macromolecules (e.g., starch, proteins) in the food matrix. Since the oil droplet inside
the emulsion gel microparticles has a low glass transition and high flexibility, the outer
layer that made up of protein particles can act as a filler. When experiencing force such as
chewing, the particles slide to each other and deform to reduce the friction and increase the
creaminess. For emulsion-type particles with limited stability, during mastication, they can
be broken down into small fragments at a small deformation with the release of entrapped
oil. The released oil lubricates the foods and provides a smooth mouth perception [118].
For a certain type of fat replacers, a set of fat-mimic mechanisms may occur which requires
a comprehensive assessment [32,119].

5. Conclusions and Future Trends

In summary, designing protein-based fat replacers is essential to improve the quality
attributes of low-fat and fat-free food products; although, they are more costly compared to
those of carbohydrate-based ones. Microparticulated proteins, as the dominant fat replacers,
are produced mainly by thermal–mechanical treatment. Other fabrication methods, such
as anti-solvent precipitation and enzymatic hydrolysis, have also been used with less heat
requirement. Microgel particles as a novel type of fat replacement receives increasing
attention. With the incorporation of plant oil, it could provide a better lubrication effect
and creaminess. The mechanisms of protein-based fat replacers remain unambiguous. The
ball-bearing effect, protein aggregates/particle deformation and disassembly, and their
emulsification contribute significantly to the lubrication and flow properties of fat-replacer
incorporated food products for desirable texture and mouthful feeling.
Although much progress has been made in the production, modification, and/or ap-
plication of fat replacers, the development of novel protein-based fat replacers in a greener
way is still in its infancy. The thermal–mechanical treatment requires a large amount of
energy input to trigger protein unfolding and aggregation, which, in turn, levels up the cost
of the final products and is not environmentally friendly. The modification of the protein
structure using processing or bioprocessing to promote their aggregating capacity under
heat requires further investigation. Most of the protein-based fat replacers do not have the
heat melting property except the prolamin-based ones. The incorporation of zein particles
to those of others with high compatibility and ideal fat-mimic effects is worthwhile to
explore. There is a growing trend of using plant proteins as fat replacers and each of them
has its own structural, physiochemical, and mechanical properties. Yet, the maximum
amount of fat that can be replaced with plant-based protein is ambiguous. The evaluation
of different protein sources, as well as combining plant-based with other animal-based
Foods 2023, 12, 957 13 of 17

proteins, such as whey, or other non-protein ingredients, such as carbohydrates [38], to

test their fat-mimicking potential, needs further attention. In addition, tribology combined
with rheological textural analysis is dominant to assess the lubrication of fat replacer-
incorporated low-fat products [119]. The difference between instrumental analysis and
human mouth sensing implies a combination of the two is more accurate to reflect the true
fat-mimic capacity of protein-based fat replacers and needs focused attention.

Author Contributions: N.N. and D.C. designed and wrote the manuscript. L.A. edited the manuscript.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data availability Statement: Not applicable.
Acknowledgments: The authors would like to acknowledge the support from the Department of
Animal, Veterinary and Food Sciences in the University of Idaho.
Conflicts of Interest: The authors declare no conflict of interest.

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