INTRODUCTIONProcessing whey, including cottage cheese whey, ishighly relevant today due to several urgent problems.Among them are deficiencies in nutrients and rawmaterials, as well as low social and environmentalefficiency. According to analytical data, 59% of thewhey produced in Russia is fed to livestock and only21% is processed for further use. The remaining 20% isdischarged into fields or wastewater, exacerbating theexisting environmental problems [1]. When dischargedinto the environment, whey acts as a biochemicalcontaminant. It is characterized by high biologicaloxygen consumption (50–60 g O2 per one liter annually)and high chemical oxygen consumption (50.5–54 g O2per one liter) [2]. Thus, whey entering sewage systemsor, in emergency cases, water bodies can cause seriousenvironmental problems. Simple calculations showthat the oxidation of organic compounds contained in25 tons of whey (an output of a medium-sized cheesefactory) needs as much oxygen as the oxidation ofhousehold wastewater in a city with a population of53Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–5940000 people. Wastewater has a high concentration ofreadily oxidizable organic compounds. Therefore, wheycan cause a decrease in dissolved oxygen concentrationin water bodies. Moreover, the presence of suspendedprotein particles can lead to the accumulation of bottomsediments and rotting processes [3].A positive scenario suggests a growth in productionfrom using highly efficient technologies for the deepprocessing of raw materials, creating “smart” storageand logistics systems, as well as minimizing losses andwaste. For this, we need to focus on the “intravital”formation of the composition and properties ofraw materials. It is a prerequisite for modern foodtechnologies and “smart” agriculture. Only thisapproach can lead to potential progress in technologicdevelopment and consistently contribute to positivetrends in the nutrition of the population [4].A healthy lifestyle requires new ways of increasingthe nutritional value of foods. Intensive foodproduction often leads to raw materials losing essentialmicronutrients at all stages of processing (refining,pasteurization, etc.) [5].Another possible cause of nutritional deficiencyis excessive consumption of medicines for chronicdiseases, including gastrointestinal disorders, acuterespiratory viral infections and flue epidemics, etc.These drugs cause “pharmacological” malabsorption,contributing to the deficiency of essential nutrientssupplied with food [6, 7]. As a result, they affectadaptive, compensatory, and regulatory capabilities ofthe body, change its physiological functions, and leadto chronic diseases of not only the digestive system,but also other organs and systems. These includeatherosclerosis, hypertension, type 2 diabetes, metabolicimmunosuppression, alimentary obesity, autoimmunepathology, etc. Moreover, the lack of proteins,polyunsaturated fatty acids, vitamins, and mineralsin the diet leads to impaired immunoreactivity andresistance to natural and anthropogenic environmentalfactors [8].The current situation dictates a need for newdirections and technologies for producing healthyfoods, including dairy products. However, one of theproblems here is introducing functional ingredients.The development of functional foods often involvesenriching foods with functional ingredients and/oreliminating those substances which cause negativereactions (food hypersensitivity). For this, we needadequate scientific data on healthy nutrients used asingredients and their effect on the product’s taste andaroma profile [9, 11].Controlled biocatalysis with specific enzymes canundoubtedly help food formulators develop functionalfood products [12].Many researchers suggest using whey proteinhydrolysates as functional ingredients. Due tobioactive peptides, they enhance the beneficial effect oftraditional foods on public health [13, 15]. Accordingto many authors, milk proteins modified by enzymatichydrolysis have both technological properties (moisturebinding, emulsifying, and foaming abilities) andfunctional properties (antioxidant, immunomodulating,hypotensive, etc.) [16–19].Whey protein hydrolysates are used in the productionof specialized products, for example, in sportsnutrition [20].In addition, whey treated with modern methodscan increase the biological value of the end-productand improve functional and technological propertiesof raw materials and meat systems. For example,introducing whey into the meat system can regulatecertain bio- and physicochemical processes by activatingthe biotechnological potential of natural systems inthe ingredients [21]. In one study, hydrated proteinpreparations of Belcon Alev I and Lactobel ED wereused to produce high-quality cooked sausages witha high biological value, digestibility, and prebioticproperties [22].Whey protein is successfully used in the productionof sausages as it not only creates a gelatinous mass thatreplaces fat, but also retains moisture [23]. This meansthat the yield of end-products can be increased withoutreducing the content of valuable animal protein or usingadditives. In addition, the end-products have betterfunctional and technological properties, as well asimproved taste characteristics [24].Another benefit of using concentrated whey proteinsin meat production is improved absorption of the endproductby the human body, which, together with areduced calorie content, contributes to the physiologicalvalue of the product. Thus, replacing the fat componentof the meat product with a protein fraction of dairyorigin can be a fundamentally new solution to the globalproblem of obesity [25].Cottage cheese whey is currently the main sourceof protein hydrolysates. Despite high volumes of wheyproduced in Russia and its obvious benefits, thereare insufficient data on its use as a raw material forfunctional ingredients [10, 26].Our study aimed to prove a possibility of usingcottage cheese whey proteins subjected to biocatalysis asfunctional food ingredients.STUDY OBJECTS AND METHODSThe objects of the study included cottage cheesewhey and enzyme preparations – acid proteases fromAspergillus oryzae with an activity of 400 units/g and apH from 3.0 to 5.0.The initial samples of whey, concentrate, andhydrolysate were analyzed for active acidity (pH)potentiometrically according to State Standard 32892-2014I and for a mass fraction of total protein accordingI State Standard 32892-2014. Milk and dairy products. Method of pHdetermination. Moscow: Standartinform; 2015. 13 p.54Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59to State Standard 23327-98II. The assays were performedin triplicate.Whey protein concentrate was obtained on an AL362 pilot ultrafiltration unit (Altair, Russia) with aconcentration factor of 5.0.Enzymatic hydrolysis was carried out as follows.An enzyme preparation was introduced into cottagecheese whey preheated to the hydrolysis temperature(namely 30°C, 40°C, and 50°C) for 60–180 min. Activeacidity was 4.6. Whey protein concentrates were usedas a control. The enzyme amount was calculated by theformula:(1)where ME is the amount of the enzyme preparation per1 g protein;P is the protein content in 100 g whey;is the required enzyme activity; andА is the initial enzyme activity.The hydrolysis was carried out in a thermostaticwater bath with constant stirring. At the end, thesamples were heated to 85°C and held for 15 min toinactivate the enzyme preparation.The samples were then cooled and poured into steriledishes.The degree of hydrolysis was determinedspectrophotometrically according to the method ofSpencer et al. [27]. In particular, we took 2 mL of thesample into a 15 mL plastic falcon and added 10 mL of a1% aqueous solution of sodium dodecyl sulfate. For this,we used an automatic pipette (Eppendof, Germany) witha measurement range of 500–5000 μL. The resultingreaction mixture was incubated in a water bath at75 ± 1°C for 15 min.A series of dilutions of L-leucine in a 1%SDS aqueous solution with concentrations of0.15–3.0 mmol/dm3 were used as standards fordetermining the degree of hydrolysis according to theSpencer et al. method. In particular, the falcons withdifferent standard concentrations were successivelyfilled with 2 mL of 0.2125 M sodium phosphate buffer(pH 8.20) and 250 μL of a standard solution, as wellas 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 mLof a 0.1% solution of 2,4,6-trinitrobenzenesulfonic acidwas added to all the falcons. The falcons were tightlyclosed and shaken. The samples were then incubatedin a water bath at 50°C for one hour. At the end of theincubation, 4.0 mL of a 0.1 M hydrochloric acid solutionwas added to each falcon to stop the reaction. Thefalcons were tightly closed, shaken, and kept for 30 minat room temperature for cooling. The optical density ofthe solutions was determined on a Synergy 2 microplatephotometer-fluorometer (BioTek, USA) at a wavelengthof 340 nm.To determine the amount of leucine equivalents,we took 0.75 mL of the hydrolysate with the maximumdegree of hydrolysis (100%) and transferred it into a5.0 mL microreaction vessel. Then, we added 0.75 mLof distilled water and 2.4 L of concentrated hydrochloricacid. The vessel was incubated in an oven at 120 ± 2°Cfor 23 h. After incubation, the samples were cooledfor one hour at room temperature and filtered undervacuum in a funnel with a glass filter. The contents ofthe microreaction vessel were quantitatively transferredto the filter and rinsed with distilled water. The pH ofthe wash water entering the Bunsen flask was monitoredusing Lach-Ner universal paper. The contents of theBunsen flask were quantitatively transferred into a100 mL laboratory glass beaker. The active acidity of thefiltrate was adjusted to 7.00 ± 0.02 pH by adding a 40%aqueous solution of sodium hydroxide. The neutralizedfiltrate was quantitatively transferred into a 100 mLvolumetric flask and the volume was adjusted to themark with a 1% SDS aqueous solution. The contents ofthe flask were thoroughly mixed.After foam collapse, a 0.25 mL sample was takenfrom the volumetric flask and analyzed. The degree ofhydrolysis of the hydrolysate protein was calculatedaccording to the equation:(2)whereis the optical density in the hydrolysatesample at 340 nm;is the optical density in the blank sampleat 340 nm;is the optical density in theUV - concentrate sample at 340 nm;30 is the hydrolysate dilution factor;6 is a dilution factor for raw materials to obtain ahydrolysate;K is the slope of the calibration graph showing thedependence of the optical density of the solution at340 nm on the concentration of the standard in thesample (0.1733 L/mol);II State Standard 23327-98. Milk and milk products. Determination ofmass fraction of total nitrogen by Kjeldahl method and determinationof mass fraction of protein. Moscow: Standartinform; 2009. 11 p.55Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59is the optical density in the acid hydrolysatesample (100% hydrolysis) at 340 nm; and133.33 is the acid hydrolysate dilution factor.The in vitro antioxidant capacity (TEAC) wasmeasured using the ABTS radical cation.The ABTS radical cation was obtainedaccording to the Re et al. method by incubating asolution of 7 mM ABTS and 2.45 mM potassiumperoxodisulfate in the dark at room temperaturefor 12–18 h [28]. The concentrated solution ofABTS radical cation was diluted with а 50 mMphosphate-buffered saline (with 100 mM sodiumchloride), pH 7.4, to OD734 = 0.70 ± 0.02. This valuecorresponded to the final concentration of ABTS radicalcation = 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 μLof the ABTS radical cation solution were added to thewells of 96-well non-absorbing polystyrene microplateswith a flat bottom. The control was 180 μL of the ABTSradical cation solution and 20 μL of a 50 mM phosphatebufferedsaline (with 100 mM sodium chloride), pH 7.4.The reaction was recorded as OD734 decreased during40.5 min with a measurement interval of 60 s at 25°C ona Synergy 2 photometer-fluorimeter (BioTek, USA). Theassays were performed in quadruplicate.The calibration curve of decreased optical densityversus Trolox concentrations varying within 1–10 μMcan be seen in Fig. 1. Equivalent concentrations ofantioxidants in the samples were determined in relationFigure 1 Decrease in optical density of ABTS radical cationsolutions vs. Trolox concentrations in the samplesto the decrease in optical density of the reaction mediumin the presence of the studied compounds. The AOCof the samples was expressed in μM TE. When testingthe antioxidant activity of hydrolysate samples withrespect to the ABTS radical cation, the working range ofdilution factors for a 50 mM phosphate-buffered saline(pH 7.4) was 150.Sensory analysis described by Spellman was used todetermine bitterness in enzymatic hydrolysates [29].To optimize the conditions for enzymatic hydrolysisof cottage cheese whey, we conducted a full-factorexperiment with three variables: temperature (X1),Trolox μmol/LTable 1 Variation levels of independent parametersin multifactorial experiments to optimize the hydrolysisof cottage cheese wheyFactor Variable Level of variation–1 0 +1Temperature, °С Х1 30 40 50Hydrolysis duration, min Х2 60 120 180E/S, % Х3 0.5 4.5 9.5Table 2 Enzyme amounts per 1 g proteinEnzyme-substrate ratio,%Enzyme amount per 100 g whey,mg0.5 4.2254.5 38.0259.5 80.275Table 3 Results of full-factor experiments to optimizeenzymatic hydrolysis of cottage cheese whey proteinsSample Bitter taste Degree of hydrolysis(DH), %TEAC, TEmmol/Lcontrol samplesControl 1 – 0 5.30Control 2 – 0 4.17Control 3 – 0 2.65fermented samples4 – 7.82 4.175 – 10.02 7.586 + 12.68 7.207 – 8.41 4.558 – 11.29 5.689 + 13.07 6.7410 – 8.59 5.1911 + 11.92 6.3612 ++ 13.06 8.7113 – 7.31 7.5814 – 11.43 6.0615 – 13.13 8.7116 – 8.21 5.3017 – 11.88 9.0918 – 15.35 9.8119 – 8.24 1.1420 – 12.08 8.7121 + 15.75 4.5522 – 8.43 3.4123 – 11.28 5.3024 – 15.08 4.5525 + 9.01 3.0326 – 13.07 4.9227 – 18.01 4.5528 – 9.59 3.7929 – 14.96 5.6830 – 19.66 8.71D734k – D734oEquation y = a + b&#39;xAdj. R-Square 0.9959Value Standard ErrorB Intercept 0.00615 0.0022B Slope 0.00132 3.78856E-556Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59duration of hydrolysis (X2), and enzyme-substrate ratioE/S (X3). Each of the parameters varied at three levels(Table 1). The output parameters were the degree ofhydrolysis (DH) and antioxidant capacity (TEAC).The variation levels of independent parameters in themultifactorial experiments conducted to optimize thehydrolysis of cottage cheese whey are shown in Table 1.The results of the multifactorial experiments werestatistically processed using the DOE block of Statistica10.0 (StatSoft Inc., USA).RESULTS AND DISCUSSIONThe protein content was 0.56% in the initial wheyand 1.35% in the concentrate. The enzyme amounts per1 g of protein are shown in Table 2.The results of the full-factor experiments conductedto optimize enzymatic hydrolysis of cottage cheesewhey proteins are demonstrated in Table 3.The experiments showed an increase in the degreeof hydrolysis and antioxidant activity of cottagecheese whey proteins with larger amounts of enzymepreparations and longer fermentation at 30°C and50°C. The maximum degree of hydrolysis (19.66%)was recorded at 50°C, 180 min fermentation, andTable 4 Effects of variable factors on the hydrolysis degree of cottage cheese whey proteinsFactor Effect Std. dev. Student t-test P –95, % +95,%11.95086 0.098745 121.0273 0.000000 11.74253 12.15920Temperature, °C (L) 2.54302 0.241874 10.5138 0.000000 2.03271 3.05333Temperature, °C (Q) –0.51056 0.209255 –2.4399 0.025942 –0.95204 –0.06907Duration, min (L) 1.88078 0.241874 7.7759 0.000001 1.37047 2.39109Duration, min (Q) 0.30944 0.209255 1.4788 0.157487 –0.13204 0.75093E/S (L) 6.68667 0.241627 27.6735 0.000000 6.17688 7.19645E/S (Q) 0.61926 0.209685 2.9533 0.008897 0.17686 1.061661L by 2L 1.06167 0.295931 3.5875 0.002269 0.43731 1.686031L by 3L 1.97152 0.295324 6.6758 0.000004 1.34844 2.594602L by 3L 0.77102 0.295324 2.6108 0.018268 0.14795 1.394109.5% enzyme. However, higher temperatures led toa noticeable, almost two-fold decrease in antioxidantactivity in the control samples, which were nothydrolyzed. Thus, temperature had a significant effecton this indicator (Table 4).According to sensory evaluation, the most bittertaste was registered in the sample that was hydrolyzedat 30°C with the maximum duration and enzymeamount (13.06% degree of hydrolysis). However, sampleNo. 30, which was obtained at the maximumtemperature, duration of hydrolysis, and enzymeamount, did not taste bitter. It means that theseconditions make the process more directional, producinghydrolysates that do not contain peptides with bitteramino 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 thatvariable factors have on the degree of hydrolysis ofcottage cheese whey proteins. As we can see, all thevariable factors, except for the quadratic duration factor,have a significant (P < 0.05) effect on the degree ofhydrolysis.The relation between the degree of whey proteinhydrolysis 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 averagethird factor: (a) Degree of hydrolysis versus duration and temperature; (b) Degree of hydrolysis versus duration and enzymesubstrateratio; (c) Degree of hydrolysis versus temperature and enzyme-substrate ratiodegree of hydrolysis, %duration, mintemperature, °Cdegree of hydrolysis, %duration, mindegree of hydrolysis, %temperature, °CE/SE/S57Agarkova E.Yu. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 52–59illustrated in Fig. 2. The graphs show no local maximaor minima, suggesting that the degree of hydrolysis riseswith an increase in each of the parameters.Table 5 shows the statistical analysis of effectsthat variable factors have on the antioxidant activityof cottage cheese whey proteins. As we can see, onlythe linear factor of enzyme amount has a significant(P > 0.05) effect on the antioxidant capacity.The surfaces of equal response of hydrolysatesTEAC versus variable parameters of whey concentratehydrolysis are presented in Fig. 3. We can clearly see thepresence of a local maximum of antioxidant activity inFigs. 3a and 3b.Finally, we correlated the key factors in themultifactorial experiments in order to select optimalconditions for the enzymatic hydrolysis of theUV-concentrate of cottage cheese whey using theenzyme preparation from Aspergillus oryzae.CONCLUSIONBased on the statistical analysis, we selected thefollowing optimal conditions for the hydrolysis ofcottage cheese whey proteins: temperature – 46.4°C;duration – 180 min; and the enzyme amount – 9.5%of the protein content. These conditions provided theantioxidant capacity of 7.5 TE mmol/L with a 17.96%degree of hydrolysis.The given data open up new prospects forprocessing acid cottage cheese whey and using wheyproteins as potential functional components withincreased antioxidant activity. We showed that targetedbiocatalytic conversion can make whey proteins morefunctional. The obtained hydrolysate of cottage cheesewhey proteins can be used to develop new functionalfoods, including meat and dairy products.CONTRIBUTIONE.Yu. Agarkova led the research. A.G. Kruchininstatistically processed the data. N.A. Zolotarevdeveloped and analyzed the test samples.N.S. Pryanichnikova checked the data reliability.Z.Yu. Belyakova systematized the data. T.V. Fedorovasummarized the data.CONFLICT OF INTERESTThe authors state that there is no conflict of interest.