Moscow, Moscow, Russian Federation
Moscow, Moscow, Russian Federation
Moscow, Moscow, Russian Federation
Moscow, Moscow, Russian Federation
Moscow, Moscow, Russian Federation
Moscow, Moscow, Russian Federation
Introduction. The study offers a new rational approach to processing cottage cheese whey and using it as a highly nutritional functional ingredient in food production. We proposed a scientifically viable method for hydrolyzing cottage cheese whey with enzyme preparations of acid proteases from Aspergillus oryzae with an activity of 400 units/g and a pH range of 3.0 to 5.0. Study objects and methods. Pre-concentrated whey was enzymatically hydrolyzed at 30°C, 40°C, and 50°C for 60 to 180 min (pH 4.6). Non-hydrolyzed whey protein concentrates were used as a control. The amount of enzyme preparation was determined by calculation. All hydrolysate samples showed an increase in active acidity compared to the control samples. Further, we conducted a full-factor experiment with three levels of variation. The input parameters included temperature, duration of hydrolysis, and a substrate-enzyme ratio; the output parameters were the degree of hydrolysis and antioxidant capacity. Results and discussion. The experiment showed the following optimal parameters for hydrolyzing cottage cheese whey proteins with the enzyme preparation of proteases produced by Aspergillus oryzae: temperature – 46.4°C; duration – 180 min; and the amount of enzyme preparation – 9.5% of the protein content. The antioxidant capacity was 7.51 TE mmol/L and the degree of hydrolysis was 17.96%. Conclusion. Due to its proven antioxidant capacity, the whey protein hydrolysate obtained in the study can be used as a functional food ingredient.
Cottage cheese whey, protein, enzymatic hydrolysis, functional ingredient, Aspergillus oryzae, concentration factor
INTRODUCTION
Processing whey, including cottage cheese whey, is
highly relevant today due to several urgent problems.
Among them are deficiencies in nutrients and raw
materials, as well as low social and environmental
efficiency. According to analytical data, 59% of the
whey produced in Russia is fed to livestock and only
21% is processed for further use. The remaining 20% is
discharged into fields or wastewater, exacerbating the
existing environmental problems [1]. When discharged
into the environment, whey acts as a biochemical
contaminant. It is characterized by high biological
oxygen consumption (50–60 g O2 per one liter annually)
and high chemical oxygen consumption (50.5–54 g O2
per one liter) [2]. Thus, whey entering sewage systems
or, in emergency cases, water bodies can cause serious
environmental problems. Simple calculations show
that the oxidation of organic compounds contained in
25 tons of whey (an output of a medium-sized cheese
factory) needs as much oxygen as the oxidation of
household wastewater in a city with a population of
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Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59
40000 people. Wastewater has a high concentration of
readily oxidizable organic compounds. Therefore, whey
can cause a decrease in dissolved oxygen concentration
in water bodies. Moreover, the presence of suspended
protein particles can lead to the accumulation of bottom
sediments and rotting processes [3].
A positive scenario suggests a growth in production
from using highly efficient technologies for the deep
processing of raw materials, creating “smart” storage
and logistics systems, as well as minimizing losses and
waste. For this, we need to focus on the “intravital”
formation of the composition and properties of
raw materials. It is a prerequisite for modern food
technologies and “smart” agriculture. Only this
approach can lead to potential progress in technologic
development and consistently contribute to positive
trends in the nutrition of the population [4].
A healthy lifestyle requires new ways of increasing
the nutritional value of foods. Intensive food
production often leads to raw materials losing essential
micronutrients at all stages of processing (refining,
pasteurization, etc.) [5].
Another possible cause of nutritional deficiency
is excessive consumption of medicines for chronic
diseases, including gastrointestinal disorders, acute
respiratory viral infections and flue epidemics, etc.
These drugs cause “pharmacological” malabsorption,
contributing to the deficiency of essential nutrients
supplied with food [6, 7]. As a result, they affect
adaptive, compensatory, and regulatory capabilities of
the body, change its physiological functions, and lead
to chronic diseases of not only the digestive system,
but also other organs and systems. These include
atherosclerosis, hypertension, type 2 diabetes, metabolic
immunosuppression, alimentary obesity, autoimmune
pathology, etc. Moreover, the lack of proteins,
polyunsaturated fatty acids, vitamins, and minerals
in the diet leads to impaired immunoreactivity and
resistance to natural and anthropogenic environmental
factors [8].
The current situation dictates a need for new
directions and technologies for producing healthy
foods, including dairy products. However, one of the
problems here is introducing functional ingredients.
The development of functional foods often involves
enriching foods with functional ingredients and/or
eliminating those substances which cause negative
reactions (food hypersensitivity). For this, we need
adequate scientific data on healthy nutrients used as
ingredients and their effect on the product’s taste and
aroma profile [9, 11].
Controlled biocatalysis with specific enzymes can
undoubtedly help food formulators develop functional
food products [12].
Many researchers suggest using whey protein
hydrolysates as functional ingredients. Due to
bioactive peptides, they enhance the beneficial effect of
traditional foods on public health [13, 15]. According
to many authors, milk proteins modified by enzymatic
hydrolysis have both technological properties (moisture
binding, emulsifying, and foaming abilities) and
functional properties (antioxidant, immunomodulating,
hypotensive, etc.) [16–19].
Whey protein hydrolysates are used in the production
of specialized products, for example, in sports
nutrition [20].
In addition, whey treated with modern methods
can increase the biological value of the end-product
and improve functional and technological properties
of raw materials and meat systems. For example,
introducing whey into the meat system can regulate
certain bio- and physicochemical processes by activating
the biotechnological potential of natural systems in
the ingredients [21]. In one study, hydrated protein
preparations of Belcon Alev I and Lactobel ED were
used to produce high-quality cooked sausages with
a high biological value, digestibility, and prebiotic
properties [22].
Whey protein is successfully used in the production
of sausages as it not only creates a gelatinous mass that
replaces fat, but also retains moisture [23]. This means
that the yield of end-products can be increased without
reducing the content of valuable animal protein or using
additives. In addition, the end-products have better
functional and technological properties, as well as
improved taste characteristics [24].
Another benefit of using concentrated whey proteins
in meat production is improved absorption of the endproduct
by the human body, which, together with a
reduced calorie content, contributes to the physiological
value of the product. Thus, replacing the fat component
of the meat product with a protein fraction of dairy
origin can be a fundamentally new solution to the global
problem of obesity [25].
Cottage cheese whey is currently the main source
of protein hydrolysates. Despite high volumes of whey
produced in Russia and its obvious benefits, there
are insufficient data on its use as a raw material for
functional ingredients [10, 26].
Our study aimed to prove a possibility of using
cottage cheese whey proteins subjected to biocatalysis as
functional food ingredients.
STUDY OBJECTS AND METHODS
The objects of the study included cottage cheese
whey and enzyme preparations – acid proteases from
Aspergillus oryzae with an activity of 400 units/g and a
pH from 3.0 to 5.0.
The initial samples of whey, concentrate, and
hydrolysate were analyzed for active acidity (pH)
potentiometrically according to State Standard 32892-
2014I and for a mass fraction of total protein according
I State Standard 32892-2014. Milk and dairy products. Method of pH
determination. Moscow: Standartinform; 2015. 13 p.
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Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59
to State Standard 23327-98II. The assays were performed
in triplicate.
Whey protein concentrate was obtained on an AL
362 pilot ultrafiltration unit (Altair, Russia) with a
concentration factor of 5.0.
Enzymatic hydrolysis was carried out as follows.
An enzyme preparation was introduced into cottage
cheese whey preheated to the hydrolysis temperature
(namely 30°C, 40°C, and 50°C) for 60–180 min. Active
acidity was 4.6. Whey protein concentrates were used
as a control. The enzyme amount was calculated by the
formula:
(1)
where ME is the amount of the enzyme preparation per
1 g protein;
P is the protein content in 100 g whey;
is the required enzyme activity; and
А is the initial enzyme activity.
The hydrolysis was carried out in a thermostatic
water bath with constant stirring. At the end, the
samples were heated to 85°C and held for 15 min to
inactivate the enzyme preparation.
The samples were then cooled and poured into sterile
dishes.
The degree of hydrolysis was determined
spectrophotometrically according to the method of
Spencer et al. [27]. In particular, we took 2 mL of the
sample into a 15 mL plastic falcon and added 10 mL of a
1% aqueous solution of sodium dodecyl sulfate. For this,
we used an automatic pipette (Eppendof, Germany) with
a measurement range of 500–5000 μL. The resulting
reaction mixture was incubated in a water bath at
75 ± 1°C for 15 min.
A series of dilutions of L-leucine in a 1%
SDS aqueous solution with concentrations of
0.15–3.0 mmol/dm3 were used as standards for
determining the degree of hydrolysis according to the
Spencer et al. method. In particular, the falcons with
different standard concentrations were successively
filled with 2 mL of 0.2125 M sodium phosphate buffer
(pH 8.20) and 250 μL of a standard solution, as well
as 50 μL of a hydrolysate sample in the first case,
250 μL of a UV concentrate sample in the second case,
and a blank sample in the third case. In addition, 2 mL
of a 0.1% solution of 2,4,6-trinitrobenzenesulfonic acid
was added to all the falcons. The falcons were tightly
closed and shaken. The samples were then incubated
in a water bath at 50°C for one hour. At the end of the
incubation, 4.0 mL of a 0.1 M hydrochloric acid solution
was added to each falcon to stop the reaction. The
falcons were tightly closed, shaken, and kept for 30 min
at room temperature for cooling. The optical density of
the solutions was determined on a Synergy 2 microplate
photometer-fluorometer (BioTek, USA) at a wavelength
of 340 nm.
To determine the amount of leucine equivalents,
we took 0.75 mL of the hydrolysate with the maximum
degree of hydrolysis (100%) and transferred it into a
5.0 mL microreaction vessel. Then, we added 0.75 mL
of distilled water and 2.4 L of concentrated hydrochloric
acid. The vessel was incubated in an oven at 120 ± 2°C
for 23 h. After incubation, the samples were cooled
for one hour at room temperature and filtered under
vacuum in a funnel with a glass filter. The contents of
the microreaction vessel were quantitatively transferred
to the filter and rinsed with distilled water. The pH of
the wash water entering the Bunsen flask was monitored
using Lach-Ner universal paper. The contents of the
Bunsen flask were quantitatively transferred into a
100 mL laboratory glass beaker. The active acidity of the
filtrate was adjusted to 7.00 ± 0.02 pH by adding a 40%
aqueous solution of sodium hydroxide. The neutralized
filtrate was quantitatively transferred into a 100 mL
volumetric flask and the volume was adjusted to the
mark with a 1% SDS aqueous solution. The contents of
the flask were thoroughly mixed.
After foam collapse, a 0.25 mL sample was taken
from the volumetric flask and analyzed. The degree of
hydrolysis of the hydrolysate protein was calculated
according to the equation:
(2)
where
is the optical density in the hydrolysate
sample at 340 nm;
is the optical density in the blank sample
at 340 nm;
is the optical density in the
UV - concentrate sample at 340 nm;
30 is the hydrolysate dilution factor;
6 is a dilution factor for raw materials to obtain a
hydrolysate;
K is the slope of the calibration graph showing the
dependence of the optical density of the solution at
340 nm on the concentration of the standard in the
sample (0.1733 L/mol);
II State Standard 23327-98. Milk and milk products. Determination of
mass fraction of total nitrogen by Kjeldahl method and determination
of mass fraction of protein. Moscow: Standartinform; 2009. 11 p.
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Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59
is the optical density in the acid hydrolysate
sample (100% hydrolysis) at 340 nm; and
133.33 is the acid hydrolysate dilution factor.
The in vitro antioxidant capacity (TEAC) was
measured using the ABTS radical cation.
The ABTS radical cation was obtained
according to the Re et al. method by incubating a
solution of 7 mM ABTS and 2.45 mM potassium
peroxodisulfate in the dark at room temperature
for 12–18 h [28]. The concentrated solution of
ABTS radical cation was diluted with а 50 mM
phosphate-buffered saline (with 100 mM sodium
chloride), pH 7.4, to OD734 = 0.70 ± 0.02. This value
corresponded to the final concentration of ABTS radical
cation = 47 μM (ε734 = 1.5×104 mol–1·L·cm–1).
To determine the antioxidant capacity (AОC),
20 μL of the test samples or a Trolox solution and 180 μL
of the ABTS radical cation solution were added to the
wells of 96-well non-absorbing polystyrene microplates
with a flat bottom. The control was 180 μL of the ABTS
radical cation solution and 20 μL of a 50 mM phosphatebuffered
saline (with 100 mM sodium chloride), pH 7.4.
The reaction was recorded as OD734 decreased during
40.5 min with a measurement interval of 60 s at 25°C on
a Synergy 2 photometer-fluorimeter (BioTek, USA). The
assays were performed in quadruplicate.
The calibration curve of decreased optical density
versus Trolox concentrations varying within 1–10 μM
can be seen in Fig. 1. Equivalent concentrations of
antioxidants in the samples were determined in relation
Figure 1 Decrease in optical density of ABTS radical cation
solutions vs. Trolox concentrations in the samples
to the decrease in optical density of the reaction medium
in the presence of the studied compounds. The AOC
of the samples was expressed in μM TE. When testing
the antioxidant activity of hydrolysate samples with
respect to the ABTS radical cation, the working range of
dilution factors for a 50 mM phosphate-buffered saline
(pH 7.4) was 150.
Sensory analysis described by Spellman was used to
determine bitterness in enzymatic hydrolysates [29].
To optimize the conditions for enzymatic hydrolysis
of cottage cheese whey, we conducted a full-factor
experiment with three variables: temperature (X1),
Trolox μmol/L
Table 1 Variation levels of independent parameters
in multifactorial experiments to optimize the hydrolysis
of cottage cheese whey
Factor Variable Level of variation
–1 0 +1
Temperature, °С Х1 30 40 50
Hydrolysis duration, min Х2 60 120 180
E/S, % Х3 0.5 4.5 9.5
Table 2 Enzyme amounts per 1 g protein
Enzyme-substrate ratio,
%
Enzyme amount per 100 g whey,
mg
0.5 4.225
4.5 38.025
9.5 80.275
Table 3 Results of full-factor experiments to optimize
enzymatic hydrolysis of cottage cheese whey proteins
Sample Bitter taste Degree of hydrolysis
(DH), %
TEAC, TE
mmol/L
control samples
Control 1 – 0 5.30
Control 2 – 0 4.17
Control 3 – 0 2.65
fermented samples
4 – 7.82 4.17
5 – 10.02 7.58
6 + 12.68 7.20
7 – 8.41 4.55
8 – 11.29 5.68
9 + 13.07 6.74
10 – 8.59 5.19
11 + 11.92 6.36
12 ++ 13.06 8.71
13 – 7.31 7.58
14 – 11.43 6.06
15 – 13.13 8.71
16 – 8.21 5.30
17 – 11.88 9.09
18 – 15.35 9.81
19 – 8.24 1.14
20 – 12.08 8.71
21 + 15.75 4.55
22 – 8.43 3.41
23 – 11.28 5.30
24 – 15.08 4.55
25 + 9.01 3.03
26 – 13.07 4.92
27 – 18.01 4.55
28 – 9.59 3.79
29 – 14.96 5.68
30 – 19.66 8.71
D734k – D734o
Equation y = a + b'x
Adj. R-Square 0.9959
Value Standard Error
B Intercept 0.00615 0.0022
B Slope 0.00132 3.78856E-5
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Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59
duration of hydrolysis (X2), and enzyme-substrate ratio
E/S (X3). Each of the parameters varied at three levels
(Table 1). The output parameters were the degree of
hydrolysis (DH) and antioxidant capacity (TEAC).
The variation levels of independent parameters in the
multifactorial experiments conducted to optimize the
hydrolysis of cottage cheese whey are shown in Table 1.
The results of the multifactorial experiments were
statistically processed using the DOE block of Statistica
10.0 (StatSoft Inc., USA).
RESULTS AND DISCUSSION
The protein content was 0.56% in the initial whey
and 1.35% in the concentrate. The enzyme amounts per
1 g of protein are shown in Table 2.
The results of the full-factor experiments conducted
to optimize enzymatic hydrolysis of cottage cheese
whey proteins are demonstrated in Table 3.
The experiments showed an increase in the degree
of hydrolysis and antioxidant activity of cottage
cheese whey proteins with larger amounts of enzyme
preparations and longer fermentation at 30°C and
50°C. The maximum degree of hydrolysis (19.66%)
was recorded at 50°C, 180 min fermentation, and
Table 4 Effects of variable factors on the hydrolysis degree of cottage cheese whey proteins
Factor Effect Std. dev. Student t-test P –95, % +95,%
11.95086 0.098745 121.0273 0.000000 11.74253 12.15920
Temperature, °C (L) 2.54302 0.241874 10.5138 0.000000 2.03271 3.05333
Temperature, °C (Q) –0.51056 0.209255 –2.4399 0.025942 –0.95204 –0.06907
Duration, min (L) 1.88078 0.241874 7.7759 0.000001 1.37047 2.39109
Duration, min (Q) 0.30944 0.209255 1.4788 0.157487 –0.13204 0.75093
E/S (L) 6.68667 0.241627 27.6735 0.000000 6.17688 7.19645
E/S (Q) 0.61926 0.209685 2.9533 0.008897 0.17686 1.06166
1L by 2L 1.06167 0.295931 3.5875 0.002269 0.43731 1.68603
1L by 3L 1.97152 0.295324 6.6758 0.000004 1.34844 2.59460
2L by 3L 0.77102 0.295324 2.6108 0.018268 0.14795 1.39410
9.5% enzyme. However, higher temperatures led to
a noticeable, almost two-fold decrease in antioxidant
activity in the control samples, which were not
hydrolyzed. Thus, temperature had a significant effect
on this indicator (Table 4).
According to sensory evaluation, the most bitter
taste was registered in the sample that was hydrolyzed
at 30°C with the maximum duration and enzyme
amount (13.06% degree of hydrolysis). However, sample
No. 30, which was obtained at the maximum
temperature, duration of hydrolysis, and enzyme
amount, did not taste bitter. It means that these
conditions make the process more directional, producing
hydrolysates that do not contain peptides with bitter
amino acids at the end of the chain. At the same time,
this sample had the highest degree of hydrolysis.
Table 4 shows the statistical analysis of effects that
variable factors have on the degree of hydrolysis of
cottage cheese whey proteins. As we can see, all the
variable factors, except for the quadratic duration factor,
have a significant (P < 0.05) effect on the degree of
hydrolysis.
The relation between the degree of whey protein
hydrolysis and variable parameters is graphically
(a) (b) (c)
Figure 2 Degree of hydrolysis in UV concentrate hydrolysates of cottage cheese whey versus variable parameters with an average
third factor: (a) Degree of hydrolysis versus duration and temperature; (b) Degree of hydrolysis versus duration and enzymesubstrate
ratio; (c) Degree of hydrolysis versus temperature and enzyme-substrate ratio
degree of hydrolysis, %
duration, min
temperature, °C
degree of hydrolysis, %
duration, min
degree of hydrolysis, %
temperature, °C
E/S
E/S
57
Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59
illustrated in Fig. 2. The graphs show no local maxima
or minima, suggesting that the degree of hydrolysis rises
with an increase in each of the parameters.
Table 5 shows the statistical analysis of effects
that variable factors have on the antioxidant activity
of cottage cheese whey proteins. As we can see, only
the linear factor of enzyme amount has a significant
(P > 0.05) effect on the antioxidant capacity.
The surfaces of equal response of hydrolysates
TEAC versus variable parameters of whey concentrate
hydrolysis are presented in Fig. 3. We can clearly see the
presence of a local maximum of antioxidant activity in
Figs. 3a and 3b.
Finally, we correlated the key factors in the
multifactorial experiments in order to select optimal
conditions for the enzymatic hydrolysis of the
UV-concentrate of cottage cheese whey using the
enzyme preparation from Aspergillus oryzae.
CONCLUSION
Based on the statistical analysis, we selected the
following optimal conditions for the hydrolysis of
cottage cheese whey proteins: temperature – 46.4°C;
duration – 180 min; and the enzyme amount – 9.5%
of the protein content. These conditions provided the
antioxidant capacity of 7.5 TE mmol/L with a 17.96%
degree of hydrolysis.
The given data open up new prospects for
processing acid cottage cheese whey and using whey
proteins as potential functional components with
increased antioxidant activity. We showed that targeted
biocatalytic conversion can make whey proteins more
functional. The obtained hydrolysate of cottage cheese
whey proteins can be used to develop new functional
foods, including meat and dairy products.
CONTRIBUTION
E.Yu. Agarkova led the research. A.G. Kruchinin
statistically processed the data. N.A. Zolotarev
developed and analyzed the test samples.
N.S. Pryanichnikova checked the data reliability.
Z.Yu. Belyakova systematized the data. T.V. Fedorova
summarized the data.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
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