Food Reviews International

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Protein enrichment of biscuits: a review

Amanda de Cássia Nogueira & Caroline Joy Steel

To cite this article: Amanda de Cássia Nogueira & Caroline Joy Steel (2018) Protein
enrichment of biscuits: a review, Food Reviews International, 34:8, 796-809, DOI:
10.1080/87559129.2018.1441299

To link to this article: https://doi.org/10.1080/87559129.2018.1441299

Published online: 01 Mar 2018.

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FOOD REVIEWS INTERNATIONAL
2018, VOL. 34, NO. 8, 796–809
https://doi.org/10.1080/87559129.2018.1441299

Protein enrichment of biscuits: a review

Amanda de Cássia Nogueira and Caroline Joy Steel
Department of Food Technology, School of Food Engineering, University of Campinas, Campinas, São Paulo, Brazil

ABSTRACT KEYWORDS
Protein enrichment of biscuits aims to reach, through a product Pea; protein; protein isolate;
which is widely accepted among consumers, target groups which soybean; whey
have greater needs of this nutrient and also consumers which seek
products which provide more than basic nutrition. Several protein
sources have been studied for this purpose and, although the use of
flours in general is more common, protein concentrates, isolates, and
hydrolysates may be good alternatives. Although this reformulation
seems simple, it is known that small variations in the formulations
can cause important effects on dough rheology, the process, and the
end product (technological and sensorial characteristics).

Introduction
The search for products which offer health benefits is a consolidated trend. Traditional
foods and foods which are widely consumed by the population are effective vehicles for
nutrient incorporation and, thus, are targeted by this growing and increasingly demanding
market. Among these foods, biscuits are potential enrichment vehicles, as they are widely
consumed, present varied forms and flavors, have long shelf life, provide convenience,
have pleasant aroma and taste, and can be consumed as snacks or a complement to other
foods.[1]
Protein enrichment of biscuits fulfills the need for a higher content of this nutrient,
because these products tend to have low protein contents (7–10%). Cereal products are
enriched to improve their nutritional properties, increase protein consumption, or com-
pensate for deficiencies of determined limiting amino acids such as lysine, methionine, or
tryptophan.[2,3]
As the nutritional quality of a protein depends on the content of its essential amino
acids and its digestibility, nutritional quality of biscuits can be improved by mixing wheat
flour with other proteins of high content of limiting amino acids in wheat such as lysine.[4]
Legume and whey proteins can be used as sources for protein enrichment of biscuits
because of their protein quality and nutritional and technological appeal.[5,6]

Biscuits
The name “biscuit” originated in France to describe a type of bread which, baked twice,
became hard for better conservation,[1] and has changed a lot since then. It can be defined

CONTACT Caroline Joy Steel steel@unicamp.br Department of Food Technology, School of Food Engineering,
University of Campinas, Campinas, São Paulo, Brazil.
© 2018 Taylor & Francis
 FOOD REVIEWS INTERNATIONAL 797

as a bakery product made from wheat flour, sugar and fat, and which has moisture content
below 4% (and water activity below 0.3%).
Considered a traditional food with significant presence in many countries, it is con-
sumed by people of almost all socioeconomic levels. Its success can be attributed to some
factors such as: relatively long shelf life which allows large-scale production and wide-
spread distribution, convenience, sensorial properties, and added value, among others.[1,7]
In general, biscuits are consumed for their interesting texture, aroma and taste, and for
the pleasure they bring, as a snack or a complement to other foods. Usually, they are
considered a good source of energy and not an important element of nutrition. Their high
contents of sugar and fat have been a concern in public health, because they are often
linked with risks of some diseases such as cardiovascular diseases, diabetes, and obesity.[1]
Thus, biscuits with health claims have gained more attention in the market, showing an
opportunity to expand production.[8] Biscuits with reduced calories and claiming no fat,
low fat, reduced fat, no added sugar, etc., and enriched with several nutrients and/or
functional ingredients, such as proteins, vitamins and minerals, and fibers, are more and
more common in the market.[1]

Protein enrichment
Regarding the protein content, commercial biscuits contain around 7–10% protein, which
is considered low.[9,10] Due to such low protein content, protein enrichment of biscuits
and other cereal products is interesting to improve their nutritional properties, increase
protein consumption, or compensate for deficiencies in certain limiting amino acids such
as lysine. In many foods, lysine is the limiting amino acid not only due to the small
amounts which are incorporated during protein biosynthesis, but also as a result of
secondary chemical changes caused by factors such as light, heat, alkali, and reducing
sugars, making lysine nutritionally unavailable. Lysine is nutritionally available when the
ε-amino group is free. If this group is blocked by a chemical bond, the protein segment
next to the affected lysine residue may not be digested.[11,12] Therefore, refined wheat
flour, as the main raw material of bakery products and responsible for their protein
quality, can be improved with the addition of protein-rich ingredients and/or raw
materials.[10]
Another factor to be considered in protein enrichment is protein-calorie malnutrition,
which has been a serious problem in people whose diet consists mainly of starchy cereals
or foods, especially in Afro-Asian countries. In addition, the current trend of population
growth means that this malnutrition will keep growing in the future, unless well-planned
measures are adopted to address this situation.[13]
Another factor is the satiety effect of proteins, in such a way that protein-rich diets are
known to cause positive effects on body composition and metabolism.[14]

Protein sources used to enrich biscuits

Several ingredients and raw materials have been used to improve the protein content of
biscuits. Legumes, such as soybeans, peas, chickpeas, and beans, have been used as protein
sources to fortify biscuits,[5,6,15–26] considering they are rich in starch, protein and fiber,
and contain sufficient amounts of vitamins and minerals, helping to fulfill health-related
798 A. D. C. NOGUEIRA AND C. J. STEEL

demands of conscious consumers.[25,27] In addition, regarding the contents of lysine and

sulfur amino acids, cereal, and legume proteins are nutritionally complementary.[28,29]
Milk and whey proteins have also been used in protein enrichment and fortification of
most bakery products because of their nutritional and functional characteristics.[10,26,30–32]
Some fish species or by-products have also been used for such a purpose,[33–35] as well as
other raw materials such as macambira,[36] Spirulina platensis,[37] brown flax seeds,[38] bee
pollen,[39] sunflower seeds,[40] pumpkin seeds,[41] safflower,[42] and cashew kernel.[43]
In general, there are more studies on composite flours (flours resulting from mixing
two or more types of flour) for protein fortification.[25,44,45] However, protein isolates have
been used as alternative protein enrichment sources, because they present high protein
content (>88%),[46,47] are easily digested by the human body, and some are equivalent to
the protein quality of milk, meats, and eggs.[48] Although protein isolates have been widely
studied separately in terms of functional characteristics, either technological or physiolo-
gical, their functionality in a product depends on the environment in which these proteins
are inserted.[49] Therefore, it is important to study their behavior in the product matrix, in
all stages of the process, and verify the interaction of different protein sources which will
allow the best correlation of their functionality in the final product.[10] Protein isolates
from soybeans and whey have already been widely used in the food industry in general,
mainly because of the high availability of their starting raw materials. Isolates, from
sources such as peas and other legumes, have also been studied.
Considering the aforementioned information, we have decided to analyze in detail
these three protein sources (soy, whey, pea), exposing a literature review focused on the
effects of their products (flour, isolate, and even protein hydrolysate) on the enrichment of
biscuits, in several aspects: dough rheology, process, and technological, sensorial and
nutritional characteristics of the end product.

Effects of protein enrichment

On dough rheology
Addition of protein sources to wheat flour tends to alter dough rheology, because it dilutes
gluten proteins, leading to a weakened protein network. The study which analyzed the
addition of 3% to 5% soy protein isolate (SPI) to wheat flour showed increased water
absorption and resistance to extension and reduced development time, stability, and
extensibility.[50] Ammar et al.[31] reported an increase in water absorption and a decrease
in development time and stability by adding 0.7% SPI. Tang and Liu[26] also observed an
increase in water absorption in the Mixolab, adding 5 to 30% soy protein. A higher
amount of water is used to achieve an excellent development of dough containing soy
flour to avoid drying,[51] because this dough tends to be drier and more brittle.[6] Singh
and Mohamed[52] also obtained reduced stability with the addition of gluten and soy
protein isolate. The addition of 10% and 15% whey proteins resulted in decreased water
absorption and reduced dough development time, and it significantly influenced the
viscosity parameters measured in the Rapid Visco Analyser (RVA).[10] Sudha et al.[53]
and Tang and Liu[26] reported a decrease in water absorption with the addition of whey
protein concentrate. Nogueira et al.[54] obtained reduced water absorption and stability by
partially replacing wheat flour with whey protein hydrolysate. Parate et al.[55] observed the
need for increased water addition to achieve an adequate consistency of the biscuit dough
 FOOD REVIEWS INTERNATIONAL 799

with the addition of 20% to 40% whey protein concentrate. The use of pea flour also
resulted in increased water absorption.[3] However, dough prepared with 10%, 20%, and
30% pea flour was very similar to wheat flour dough in terms of consistency and handling
characteristics.[6]
A summary of the studies found and the results obtained can be observed in Table 1. It
can be seen that the addition of the protein sources greatly affects the rheological behavior
of the dough; as there is almost always a change (either increase or decrease) caused by the
addition of the different protein sources. The addition of flours seems to contribute to
increasing WA, also due to the other components (fibers, for example) present in it. On
the other hand, whey products (isolated, concentrated, or hydrolyzed) tend to decrease
this parameter, probably due to a lower water holding capacity or a physical impediment
to the hydration of wheat flour. In general, a weakening of the dough is observed with the
addition of the protein sources. The behavior of the different protein sources in the
pasting properties is very similar, because they influence the parameters mainly by the
dilution of the starch present. It is also important to note here that, in addition to the
influence of the protein sources which can present different abilities to absorb/retain
water, some ingredients of the biscuit formulations may also contribute to a greater or a
lower absorption. This fact also helps to explain the differences found between studies.
Formulations with different levels of sugar and fat, and also of their derivatives, for
example, can strongly influence this parameter.

Table 1. Studies on the effect of soy, whey, and pea as protein sources on rheological parameters of
wheat flour doughs.
Source/
Analysis Concentration (%) Results Reference
[31]
Farinograph SPI/0.7 Increased WA/Decreased DDT and S
Soy flour/0–15 Increased WA, DDT and S [53]
[31]
WPC/0.7 Increased WA/Decreased DDT and S
[10]
WPC/0–15 Decreased WA and S
[53]
WPC/0–15 Decreased WA/Increased DDT
[10]
WPH/0–15 Decreased WA and S
[54]
WPH/0–20 Decreased WA/Increased DDT
Pea flour/0–10 Increased WA [3]
[26]
Mixolab Soy protein/0–30 Increased WA and MST from 10%/Did not change PT
[26]
Measurements Whey protein/0–30 Decreased WA and MST/Increased MDT and PT
Alveograph Soy flour/0–15 Increased P/Decreased L [53]
[53]
WPC/0–15 Decreases P and L
Pasting Soy flour/0–15 Increased PT/Decreased maximum, hold and final viscosities, [53]

properties breakdown and setback

WPC/0–15 Decreased maximum, hold and final viscosities, breakdown and [10]

setback
WPC/0–15 Increased PT/Decreased maximum, hold and final viscosities, [53]

breakdown and setback

WPH/0–15 Decreased maximum, hold and final viscosities, breakdown and [10]

setback
WPH/0–20 Increased PT/Decreased maximum, hold and final viscosities, [54]

breakdown and setback

SPI: soy protein isolate; WPC: whey protein concentrate; WPH: whey protein hydrolyzed; WA: water absorption; DDT: dough
development time; S: stability; PT: pasting temperature; MST: Mixolab stability time; MDT: Mixolab development time; P:
maximum pressure; L: extensibility.
800 A. D. C. NOGUEIRA AND C. J. STEEL

On the process
Effects of protein sources on different biscuit production processes are still not very well
understood. Scarce information is available in the literature on how these protein sources
impact process parameters, and it is not clear if there is any process with better results for
such enrichment. In addition, submitting these protein sources to processing, that is, to
factors which interfere with the interactions of protein molecules and their conformational
structure, leads to changes in their technological and physical-chemical properties,[56]
which can be positive or negative for the process. For instance, an increase in water
absorption can be considered a negative point in biscuit production, as it will lead to
longer baking times. Shorter process times are desired to achieve greater productivity.
Soy flour improves biscuit shaping. In the lamination stage, it helps to prevent
hardening, producing more homogeneous dough, because it reduces dough elasticity
and increases dough extensibility, as a result of the emulsifying power of lecithin found
in soy flour.[1,51] During the dough modeling stage for biscuit production, formulations
with higher content of defatted soy flour presented harder and more brittle dough and,
therefore, of difficult modeling and handling,[6,19] which may also reduce the biscuit
diameter.[19] As previously mentioned, the fact that the pea flour dough behaves very
similarly to wheat flour dough means that no alteration would be required in the
formulation for easier handling of biscuit dough containing this flour.[6]
Most of the studies found in the literature do not address the biscuit manufacturing
process, because, in them, the biscuits are produced more manually. This fact often makes
it impossible to acquire information which translates into a level of production on a larger
scale. There are also different types of biscuits, which are derived from different processes
(molded, laminated, and wire-cut biscuits, for example). Thus, the behavior of these
sources can be quite distinct in each of them, which makes the study of processes an
important gap to be filled.

On the technological characteristics of biscuits

Besides increasing protein content, the sources used for such purpose can have different
effects on the technological characteristics of biscuits. One example is biscuit dimensions.
Gani et al.[10] did not observe any alteration in the spread factor (width/thickness) of
biscuits enriched with up to 15% whey protein concentrate, unlike Wani et al.[57] and
Tang and Liu[26] who mentioned an increase in this parameter with whey protein.
However, Parate et al.[55] observed reduced diameter and increased thickness of biscuits
with 20% to 40% of the same protein. These variations can be due to the formulations as
well as the processes used. Singh and Mohamed[52] also observed a negative correlation
between biscuit diameter and protein content. McWatters,[6] when using pea flour, did not
observe any alteration to biscuit dimensions or weight; however, with soy flour, the author
found reduced diameter and spread factor (width/height). With soy protein, Tang and
Liu[26] found a decrease of the expansion factor with additions of 5% and 10% and an
increase with 30%. At intermediate concentrations (15%, 20%, and 25%), this parameter
was similar to the control test (without soy protein). The incorporation of other proteins
interferes in the development of the gluten network and negative effects, such as the
decrease in the spread ratio, are expected, especially when the addition of other proteins is
greater than 5%.[6] However, when the addition is much greater than the gluten content,
there can be a different behavior.[26] These authors, at higher concentrations of soy
 FOOD REVIEWS INTERNATIONAL 801

protein, observed a possible aggregation which induced the formation of a network, which
helped increase the elasticity and extensibility of wheat flour, leading to an increase in the
spread ratio. The soy protein network formed may also have conferred a protective effect
on the partial structure of gluten, contributing to the gain of quality.[26] Kissel and
Yamazaki[5] stated that the phenomenon of biscuit horizontal expansion is primarily
physical and controlled by the ability of the components to absorb water.
Biscuit color may also change. The addition of whey protein resulted in darker
biscuits,[10,26,55,57] as did the addition of SPI.[26,51] The replacement of wheat flour by
soy flour and whey protein concentrate also had a significant effect on color
development.[58] However, these authors stated that soy flour had a much less sig-
nificant effect than whey protein concentrate. Whey protein concentrate probably
favored the development of cookie color due to its high lactose content. The darkening
of biscuits can be attributed to a higher degree of the Maillard reaction, due to a
higher protein content and consequent interaction with sugars.[10] It is known that
flavors developed through the Maillard reaction during the last stage of baking con-
tribute to biscuit acceptance. However, it should be noted that, besides biscuit darken-
ing, sometimes excessive, the condensation reaction between reducing sugars and the
amino group of the lysine side chain during the Maillard reaction leads to a great loss
of available lysine.[58]
Increased protein concentrations in biscuits can also lead to increased hardness.[10,19]
McWatters et al.[59] attributed a harder texture of biscuits to increased protein content and
protein interaction with sugars during dough preparation and baking. Tang and Liu[26]
observed greater hardness in their biscuits with whey protein up to 15% addition. With
soy protein, only the concentrations of 20%, 25%, and 30% differed from the control,
presenting lower hardness, probably due to a greater spread factor (and lower thickness).
Also regarding physical characteristics, biscuits containing pea flour were very similar
to those produced with wheat flour, with no significant difference in diameter, height,
spread factor, external appearance, texture, and moisture content.[6]
Table 2 shows the main results obtained with the addition of these protein sources on
technological characteristics of biscuits. In general, the moisture content of enriched
biscuits increased. The variations observed in spread ratio and texture results may be
due, not only to the addition levels, but also the formulations and processes used.
However, the protein sources seem to greatly influence these important quality parameters
for biscuits. And with regard to color, the darkening caused by the addition of these
protein sources is an already expected effect.

On the sensorial characteristics of biscuits

Regarding sensorial characteristics, in most cases, the overall low acceptance of biscuits
containing higher protein contents is attributed to the taste and darkening. According to
Gani et al.,[10] consumers perceived a bitter taste in biscuits with concentrations above 5%
whey protein concentrate, which can be attributed to the presence of bitter peptides,
especially when using protein hydrolysates. Tang and Liu[26] also observed a decrease in all
sensory parameter scores with the addition of whey protein, reporting that the highest
concentration accepted was 10%. Parate et al.[55] also mentioned the development of
roughness on the surface of biscuits with increased whey protein concentrate level, leading
to reduced acceptance of biscuit appearance. Also, according to these authors, an addition
802 A. D. C. NOGUEIRA AND C. J. STEEL

Table 2. Studies on the effect of soy, whey, and pea as protein sources on composition and
technological characteristics of cookies.
Analysis Source/Concentration (%) Results Reference
[17]
Moisture SPI/0–20 Increased
Soy flour/0–10 Increased [25]

Soy flour/0–20 Decreased [24]

Soy flour/0–30 Without change [6]

[55]
WPC/0–40 Increased
[57]
WPC/0–6 Increased
Pea flour/0–30 Without change [6]

Pea flour/0–10 Increased [25]

[17]
Protein SPI/0–20 Increased/At the highest level of addition: increase of 39.5%
Soy flour/0–10 Increased/At the highest level of addition: increase of 27.8% [25]

Soy flour/0–20 Increased/At the highest level of addition: increase of 38% [24]

Soy flour/0–30 Increased/At the highest level of addition: increase of 49.3% [6]
[55]
WPC/0–40 Increased/At the highest level of addition: increase of 65%
[57]
WPC/0–6 Increased/At the highest level of addition: increase of 31.3%
Pea flour/0–30 Increased/At the highest level of addition: increase of 25.7% [6]

Pea flour/0–10 Increased/At the highest level of addition: increase of 15.8% [25]
[26]
Spread ratio Soy protein/0–30 Decreased up 10%/Without change in the other concentrations
Soy flour/0–30 Without change with 10%/Decreased with 20 and 30% [6]
[10]
WPC/0–15 Without change
[55]
WPC/0–40 Decreased
[57]
WPC/0–6 Without change with 2%/Increased with 4 e 6%
[10]
WPH/0–15 Without change
[26]
Whey protein/0–30 Without change up 5%/Decreased in the other concentrations
Pea flour/0–30 Increased [6]
[26]
Texture Soy protein/0–30 Less hard from 20%
[10]
WPC/0–15 Harder
[10]
WPH/0–15 Harder
[26]
Whey protein/0–30 Harder from 15%
[26]
Color Soy protein/0–30 Darkening
Soy flour/0–15 Darkening [58]
[10]
WPC/0–15 Darkening
[57]
WPC/0–6 Darkening
[58]
WPC/0–15 Darkening
[10]
WPH/0–15 Darkening
[26]
Whey protein/0–30 Darkening
SPI: soy protein isolate; WPC: whey protein concentrate; WPH: whey protein hydrolyzed.

of up to 25% whey protein concentrate allows the production of good quality biscuits. As
taste has a great effect on sensory acceptance, it can be said that formulations richer in
ingredients tend to have greater acceptance. This explains the higher level of protein
substitution achieved by Parate et al.,[55] compared to Gani et al.[10] and Tang and Liu,[26]
for example. The formulation of Parate et al.[55] included diverse ingredients which
collaborate with the taste (sugar, condensed milk, invert syrup, liquid glucose, and flavor),
whereas the others counted only with sugar and/or dextrose.
As for soy protein, Tang and Liu[26] achieved good sensory acceptance of cookies
with concentrations of 10%, 20%, and 30%. Using SPI, Mohsen et al.[17] did not find
significant differences in color acceptance among all cookie samples with up to 15%
SPI, although supplementation of cookies with up to 10% SPI increased acceptance of
all other sensory attributes (aroma, taste, crispness, and overall acceptability). The low
overall acceptability of cookies containing 20% SPI was attributed to less crispiness and
more darkening. And even after three months, the sample supplemented with 10% SPI
was the most preferred. With soy flour, Farzana and Mohajan,[24] incorporating 10% to
 FOOD REVIEWS INTERNATIONAL 803

20% soybean flour, achieved good overall acceptability. However, McWatters[6]

obtained, for cookies containing 20% and 30% soy flour, low scores for appearance,
texture, and flavor, because of the thickness, hardness, raw bean flavor, and low
expansion properties. These properties also prevented the development of a typical
surface of biscuits, that is, without roughness.
The addition of protein sources may also alter the aroma of cookies. Mohsen et al.,[17]
for example, observed variations in the number and content of most of the volatiles
identified by adding SPI in this product. According to these same authors, among the
investigated samples, the cookies containing 10% SPI had a high content of volatile
compounds, such as pyrazines and pyranones, which are considered aroma markers of
these products. The concentration of pyrazines is related to the development of roasted
flavor in thermally treated cereal products,[60] whereas that of pyranones, such as maltol
(3-hydroxy-2-methyl-4(H)-pyran-4-one) or DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-
4(H)-pyran-4-one), is associated with a pleasant sweet aroma.[61] Regarding the volatiles
from lipid degradation, the fortification with 5% SPI revealed a considerable increase of
these volatiles, followed by a gradual decrease with the increase of SPI content. This
apparent decrease in the volatiles derived from lipids can be attributed to an increase in
the formation of products derived from the Maillard reaction.[17] According to Mohsen
et al.,[17] the differences in aroma and taste among the cookie samples could be correlated
to the changes in generated volatiles during the baking process.
McWatters,[6] when adding 10%, 20%, and 30% pea flour to the biscuit dough, obtained
high-quality sensorial scores for appearance, with no significant difference in relation to
the control (100% wheat flour). In color and aroma scores, pea flour practically had no
effect, and in texture scores, a slight reduction was observed with higher contents of this
flour. These results showed the possibility to add up to 30% pea flour and still maintain
biscuit quality. According to Kamaljit et al.,[3] the addition of up to 5% pea flour did not
affect the sensorial characteristics of biscuits and improved their nutritional quality. Amin
et al.[25] also obtained high sensory acceptance scores for cookies with 5% and 10% pea
flour.
A summary of the results obtained regarding the sensorial properties can be observed
in Table 3. Sensory acceptance of soybean sources presented different results, where some

Table 3. Studies on the effect of soy, whey, and pea as protein sources on sensorial characteristics of
cookies.
Source/Concentration
Analysis (%) Results Reference
[17]
Sensory SPI/0–20 Desirable scores/Maximum amount accepted: 20%
[26]
properties Soy protein/0–30 Desirable scores/Maximum amount accepted: 30%
Soy flour/0–10 Decreased of scores/Maximum amount accepted: 5% [25]

Soy flour/0–20 Desirable scores/Maximum amount accepted: 15% [24]

Soy flour/0–30 Decreased of scores from 20%/Maximum amount accepted: [6]

10%
[10]
WPC/0–15 Decreased of scores/Maximum amount accepted: 5%
[55]
WPC/0–40 Decreased of scores/Maximum amount accepted: 25%
[26]
Whey protein/0–30 Decreased of scores/Maximum amount accepted: 10%
[10]
WPH/0–15 Decreased of scores/Maximum amount accepted: 5%
Pea flour/0–30 Decreased of scores/Maximum amount accepted: 10% [6]

Pea flour/0–10 Decreased of scores/Maximum amount accepted: 5% [25]

SPI: soy protein isolate; WPC: whey protein concentrate; WPH: whey protein hydrolyzed.
804 A. D. C. NOGUEIRA AND C. J. STEEL

studies presented good scores for the parameters analyzed, whereas others did not (even
with similar levels of addition). Despite this, the soy sources had greater sensory accep-
tance than those of whey and pea. The studies with whey proteins and pea flour showed a
decrease of the scores for the sensorial parameters and a great variation in the maximum
quantity accepted. This variation can be linked to differences in the formulation. It is
known that differences in sugar and fat contents, as well as their derivatives, in the
formulations, can influence the taste and, consequently, the sensorial acceptance of the
final product. A great challenge in the sensorial aspect is to mask the residual flavor of the
protein-enriched products and, thus, to allow greater incorporations.

On the nutritional characteristics of biscuits

As expected, the addition of protein sources caused a great increase of this nutrient in the
biscuits (Table 2). However, protein quality is related to its content of essential amino
acids, which should be higher than the reference levels defined by the Food and
Agriculture Organization/World Health Organization/United Nations University (FAO/
WHO/UNU),[62] and protein digestibility. In this sense, the nutritional quality of a protein
with essential amino acid deficiency can be improved by mixing the protein with another
protein which is rich in that specific amino acid, such as when cereal proteins (with lysine
deficiency) are mixed with legume proteins (with methionine deficiency). However, it
should be noted that an excessive consumption of any specific amino acid can lead to
“amino acid antagonism” or toxicity, which results in a greater need for other essential
amino acids, in addition to possible growth inhibition, leading to pathological
conditions.[4]
Although the content of amino acids is the primary indicator of protein quality, the
true quality also depends on the level of utilization of these amino acids in the body. The
protein availability of milk, peas, wheat flour, and SPI, for example, is 95%, 88%, 86%, and
95%, respectively.[62] Several factors, such as protein conformation, anti-nutritional fac-
tors, and processing, affect this availability.[4] Partial thermal denaturation of proteins
usually improves the digestibility and bioavailability of essential amino acids.[4]
Most plant-based protein isolates and concentrates present anti-nutritional factors,
such as trypsin and chymotrypsin inhibitors, which may impair the complete hydrolysis
of legume proteins by pancreatic proteases,[4] affect the digestibility, absorption, or
utilization of nutrients, and cause harmful effects to health if ingested at high
concentrations.[63] Peas have much lower activity levels of trypsin inhibitors and hemag-
glutinins or lectins when compared to soybeans.[64] Other anti-nutritional factors are also
present in plant-based proteins, such as tannins and phytates. Tannins are considered as
anti-nutrients because of the adverse effect on protein digestibility. Phytates interact with
basic residues of proteins, participating in the inhibition of digestive enzymes such as
pepsin, pancreatin, and α-amylase.[4,65] However, when these proteins are subjected to a
thermal treatment, these compounds are partially or totally eliminated.[4,66]
The presence of allergens in various protein raw materials can cause allergic reactions
in children and adults.[4] Cow’s milk is the most common allergen for infants, with α-
lactalbumin considered the main allergen.[67] Among legumes, some of the soybean
fractions present allergenic activity, and the 2S fraction was reported as the most allergenic
among the components of soy proteins.[68] However, these allergens, such as those from
 FOOD REVIEWS INTERNATIONAL 805

soybeans and milk, also become innocuous by denaturation when submitted to thermal
treatment.[4,67]
Proteins can undergo several chemical changes during processing.[4] The interaction of
proteins with polysaccharides and fibers can reduce the rate and degree of protein
hydrolysis.[69] Lysine, for example, which is already limiting in cereal proteins, can have
its availability further reduced. The condensation reaction between reducing sugars and
the amino side-chain of lysine during the Maillard reaction leads to a severe loss of lysine
availability.[58] Pérez et al.[58] observed that the loss of lysine availability increased linearly
with the incorporation of whey protein concentrate and decreased with increasing water
in the formulation. According to these same authors, the positive correlation between loss
of lysine availability and whey protein concentrate was expected because the whey protein
concentrate used contained high levels of lactose. Another interaction which affects
protein structure is the protein-phenolic interaction, which may decrease protein
bioavailability.[70,71] Phenolic compounds can bind to proteins and thus their bioavail-
ability may be significantly reduced.[72]
Based on the aforementioned considerations, the protein digestibility of biscuits will
depend on the protein content of the sources used, the other ingredients used in product
formulation, and the processing to which the products will be submitted. In addition,
when stored, these proteins may also undergo some alterations, requiring further study of
the factors previously mentioned. Thus, the study of the nutritional characteristics of
protein-enriched biscuits is very important and should not be underestimated, as it
ensures that the primary objective has been achieved: biscuits with higher content and
better quality protein which offer consumers the benefits of this nutrient.

Conclusion
The addition of proteins to enrich biscuits, whether in the form of composite flours (more
common) or protein concentrates, isolates, or hydrolysates, greatly influences dough
behavior and the technological characteristics of the final product. In general, it leads to
a weaker dough, changing its viscosity (pasting properties), because the gluten proteins
and the starch are diluted. During the process, among the scarce information found, the
emulsifying effect of soy flour is observed during lamination. Regarding technological
characteristics, protein enrichment causes changes in biscuit dimensions, color and tex-
ture, and it often generates a bitter aftertaste, in a sensorial perspective. Nutritional
strategies chosen involve the challenge of obtaining biscuits with higher protein content
and quality which also allow the availability of this nutrient to the human body.
Studies that used soybean and pea flours, SPI, as well as whey hydrolysate and concentrate,
were found in the literature. However, even with the evidence from the studies which used
these protein sources, their effects on biscuit production processes are still not very well
understood, because few studies have analyzed these effects or compared the various
processes, especially regarding the molding steps. In addition, there is need for studies
which analyze the technological effects and the protein availability in the final product.
806 A. D. C. NOGUEIRA AND C. J. STEEL

Acknowledgments
We would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for
the Doctorate Program scholarship granted to author Amanda de Cássia Nogueira and the Research
Productivity scholarship granted to author Caroline Joy Steel, and the Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) for funding. The authors also thank Espaço da Escrita –
Coordenadoria Geral da Universidade – UNICAMP – for the language services provided.

ORCID
Amanda de Cássia Nogueira http://orcid.org/0000-0002-1450-5574
Caroline Joy Steel http://orcid.org/0000-0002-0911-5889

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