INTRODUCTIONBalanced diet and natural food quality are the mostimportant issues of contemporary food science [1–4].Environmental pollution and such diet-related diseasesas hypertension, diabetes, allergies, etc., require newtypes of diet and functional products [5–8]. Modifiedmilk and whey proteins can serve as basic componentsof functional foods [9–13]. Enzymatic hydrolysis ofdairy proteins is the most popular method of wheymodification, which makes it possible to impartadditional functional and technological properties,e.g. emulsifying, foaming, antioxidant, antihypertensive,immunomodulatory, etc. [14, 15].Whey proteins and their hydrolysates possess highnutritional value, which makes them the most promisingcomponents for diet therapy products. Whey proteinsowe their useful functional properties to bioactivepeptides [16, 17]. Bioactive peptides are amino acidsequences, encoded in the primary structure of nativeproteins. A protein hydrolyzate contains a mix ofbiologically active and inactive peptides, in addition tonon-hydrolyzed proteins. Fractioning can isolate certainbiologically active peptide fractions from hydrolysates.Fractioning relies on such membrane separationprocesses as ultrafiltration and microfiltration [18–22].Membrane separation means that two or morecomponents are separated through a membrane thatacts as a selective semipermeable barrier that partiallyor completely stops one or more substances. Theretained components produce retentate, while those thatpass through the membrane form permeate [23, 24].Membrane processes have several advantages overother separation methods. First of all, they require lessenergy than evaporation or distillation. Second, theydemonstrate high selectivity and are easy to scale.Finally, they are material friendly, which is a veryimportant factor for food industry [24]. Development and design of new membranebioreactors (MBR) is one of the most promisingand dynamic areas of industrial biotechnology.MBR technology combines various membrane andbiochemical separation processes, the latter beinginduced by a catalyst of biological origin, i.e. an enzyme.The main advantage of MBR enzymatic hydrolysisis that it saves expensive enzyme preparations andregulates the molecular composition of hydrolysisproducts by combining membranes with a recommendedmolecular weight cut-off [18].Unfortunately, contemporary food industry usesonly about 50% of the whey produced worldwide,which means that the task of whey recycling is yet to besolved. This issue remains controversial and requirescomprehensive research. The present review describeshow various whey processing MBRs can increase thevalue of whey components [25].STUDY OBJECTS AND METHODSThe present research was based on a direct validatedanalysis and featured the most recent domestic andforeign publications on protein hydrolysis in variousmembrane reactors.RESULTS AND DISCUSSIONFigure 1 illustrates two most common membranereactors (MBR). In the first type, the membranecontrols the mass transfer of the substrate andenzyme preparation to and from the reactor module,thus producing an indirect effect on the hydrolyticdegradation of the substrate (Fig. 1a). In the othertype, the reaction occurs at the membrane level andcomplements the regulation of substrate and enzymemass transfer [26, 27]. Complex as it is, MBRs of thesecond type makes it possible to control proteolysis atthe cellular level (Fig. 2b) [26, 27].Such MBRs are called biocatalytic because themembrane itself acts as a catalyst. They are based oncontinuous stirring: the product either passes throughthe membrane, which retains the enzyme and returns itto the reactor, or remains in the membrane module. Thebiocatalyst is immobilized and separated by a membranein the reaction vessel [26, 28]. As a rule, the membraneimmobilizes the enzymes on membranes becausebiomolecules are covalently attached to the surface ofthe carrier. As a result, the system is more stable, andthe microreactor can be reused while the enzyme is nolonger active. The covalent attachment of enzymes tosolid substrates is very strong and increases the servicelife of the microreactor and immobilized enzymes [29].The numerous advantages of these MBRs make theman alternative to simple bioreactors. The most importantadvantage is that the catalyst (enzyme) can be recoveredand reused in a continuous system, which increasesthe efficiency of the process. The yield rises, while theexpensive enzyme preparation is spared, which lowersthe cost of the final product. In addition, the selectiveremoval from the reaction medium is continuous,and the supply of the reagent to the catalytic reactionmedium is easy to control [26].Ultrafiltration is the most common separationprocess used in this type of MBR. Unfortunately,polarization remains its main disadvantage:eventually, the membrane pores get clogged. Nearlyall membrane filtration processes gradually decrease,as trapped particles accumulate on the surface of theа) membrane bioreactor б) biocatalytic membrane reactorFigure 1 Schematic illustration of membrane reactorsBiocatalyst that passed alongthe membraneBiocatalyst segregated withthe membraneBuilt-in biocatalystGelated biocatalystAdsorption Ionic bondCovalent bond Cross-linkingMolecular recognitionBound biocatalyst273Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280membrane. The rate depends on the operation typeof the membrane, the nature of the flow, the pore size,and charge of the membrane. The flow decreasesbecause of certain physical or chemical interactionsthat occur between the interface of the membraneand the components of the feed stream. The formationrate of the surface layer has to be controlled, as itkeeps accumulating on the side of the membrane thatexperiences excess pressure. No pre-treatment canprevent clogging, and the membrane has to be cleanedregularly [26].In a biocatalytic MBR, the membrane not onlyseparates but also catalyzes. The enzyme enters themembrane matrix and is immobilized there (Fig. 1b),increasing its stability, which is another advantageof this type of MBR [30]. Immobilization increasesthe stability of enzymes during storage, namely, theirresistance to changes in temperature and pH [31].In their study of continuous MBRs, Wang et al.focused on transglutaminase, which was covalentlybound to the surface of the polyethersulfonemembrane. The enzyme cross-linked α-lactalbuminand β-lactoglobulin, thereby retaining them on themembrane [32]. Using transglutaminase for enzymaticmodification of milk protein can prevent protein lossduring whey processing and increase the biologicalvalue of the product [33]. During whey ultrafiltration,α-lactalbumin and β-lactoglobulin can pass through themembrane under transmembrane pressure, in whichcase they block the pores or penetrate into the filteredsolution. As a result, β-lactoglobulin is the main cause ofmembrane clogging during whey filtration [34–36].Wang et al. studied an enzymatic MBR withtransglutaminase, its efficiency, the catalysis of proteincrosslinking, and its separation from whey. The proteinrecovery rate reached 85%, but it decreased over time,as did the relative membrane flow, probably, followingthe decrease in enzymatic activity on the membranesurface after 1365 min of continuous operation. Theoverall specific performance of the enzyme boundmembrane was about 50% less than that of the purepolyethersulfone membrane. Wang et al. concludedthat the efficiency failed because of the repulsion forcesthat appeared between the cross-linked proteins and themembrane [32].Vasileva et al. studied β-galactosidase that wascovalently bound by glutaraldehyde to the surface of themodified polypropylene membrane. They determinedthe optimal hydrolysis conditions for lactose in a batchMBR: enzyme activity 13.6, temperature 40°C, pH 6.8,time 10 h. The scientists compared the resultingdegree of hydrolysis with that obtained by a free nonimmobilizedenzyme. The immobilized enzyme methodproved 1.6 times more effective than the one basedon a free enzyme, as the immobilized enzyme itselfwas twice as stable as the free enzyme. The resultingimmobilized β-galactosidase/polypropylene membranesystem was used to obtain glucose-galactose syrup fromwhey waste. Vasileva et al. carried out hydrolysis ofwhey lactose in a MBR using an immobilized enzymeand a spiral membrane. The optimal membrane surfaceand the whey flow rate were 100 cm2 and 1.0 mL/min,respectively. After 10 h, the lactose hydrolysis reached91%. After cycle 20, the yield was 69.7% [37].Sen et al. focused on skim milk hydrolysis in abatch MBR using β-galactosidase immobilized on apolyethersulfone membrane with a pore diameter of30 kDa. The study featured aqueous solutions ofskim milk in the concentration range of 30–80 kg/m3.The solutions underwent deproteinization throughtwo membrane ultrafiltration modules with pore sizes30 kDa and 5 kDa. As a result, 95–97% of lactosebecame permeate. The permeates obtained weresubjected to hydrolysis in a batch MBR equipped withan enzyme-immobilized membrane. The enzyme wasimmobilized by cross-linking on an ultrafiltrationmembrane using 3 and 4% glutaraldehyde. The 4%glutaraldehyde solution provided a greater enzymeactivity retention (94.2%) and enzyme loading (98%).The final conversion of lactose was 45.2 and 21.4%when β-galactosidase was immobilized with 4 and 3%glutaraldehyde, respectively. The control experimentwith an immobilized enzyme showed a significantdecrease in the flow of pure water: 27.5 for 3%glutaraldehyde and 67.5 for 4% glutaraldehyde [38]When the biocatalyst is confined to the membranemodule, not the reservoir with the reagents, it is notrecirculated into the outlet flow; with that, low molecularweight products and inhibitors leave the system directlythrough the membrane. This type of MBR findsapplication in bio-artificial pancreas or extracorporealdetoxification devices [26].Biocatalytic MBRs are undoubtedly more efficient,since both the reaction and the separation occurin the same membrane module. However, currentknowledge about the nanoscale processes withinthe microenvironment of the membrane remainsinsufficient. Equally lacking is the knowledge aboutthe control of continuous hydrolysis at the macroscopiclevel. As a result, biocatalytic MBRs cannot be used forcommercial production [39–41].Biocatalytic MBRs, or bioreactors, are integratedwith such membrane processes as microfiltration,ultrafiltration, reverse osmosis, membrane extraction,etc. They are especially effective for food and beverageproduction, e.g. wine, fruit juices, milk, etc. [42, 43].In the dairy industry, MBRs were first used to producelow lactose milk [43]. Such MBRs are still widely usedto produce functional products for patients with lactasedeficiency. However, lactose is not the only substancethat causes milk intolerance: some people cannot absorbhigh molecular proteins (≥ 5 kDa) due to inadequateimmune response. MBRs are also used to produce lowallergenicmilk [44].MBRs are getting more popular in food industryas a result of industrial demand for functional foods,e.g. hypoallergenic, nutraceutical, or alternative foods,ingredients that are part of dietary and preventive274Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280menus, etc. MBRs are actively used in whey processing.The physicochemical composition of whey makes ita very promising raw material for functional foodproduction. Whey contains 0.4–0.8% of protein and4.4–5.5% of lactose. Whey proteins possess a good latentpotential of biofunctional properties [43].Batch MBRs are simple enough to gain extensive usein the production of protein hydrolysates. However, theyneed a lot of enzyme, energy, and labor, which makes itexpensive [19]. American scientists from the Departmentof Food Science (Pennsylvania, USA) attempted toprocess food substrates using batch-type enzymereactors with an immobilized enzyme. They identifieda number of additional disadvantages, e.g. high lossesin the activity of the biocatalyst, the expensive enzymeimmobilization, etc. [44].Continuous stirred tank membrane reactors(CSTMR) are an alternative to batch MBRs. They arebased on the difference in molecular weight betweenthe enzyme and the hydrolysis products. CSTMRscan separate products from the reaction medium toincrease the yield. The soluble enzyme is confined tothe retentate side of the membrane, where it comes incontact with the substrate. CSTMRs make it possibleto reuse the enzyme and select a suitable membranepore size, which facilitates the control of the molecularweight of the final product [44].Ewert et al. used a two-stage enzymatic membranebioreactor (EMBR) to obtain sodium caseinatehydrolyzate with improved antioxidant capacity andreduced bitterness (Fig. 2) [44]. At the first stage, sodiumcaseinate was hydrolyzed at 65°C and pH 6.7 usingendopeptidase Sternzym BP 25201. The stage took 12 hand involved hydrolysis and filtration through a ceramicultrafiltration membrane made of hollow fiber with amolecular weight cut-off of 10 kDa. The antioxidantactivity of the resulting permeate increased by 33%,compared to sodium caseinate. The volume of permeatethat left EMBR-1 was automatically compensated for byadding a new substrate to the reactor vessel.At the second stage, the main objective was toremove bitterness. The hydrolysis was carried outin EMBR-2 using Flavorzyme at 50°C and pH 6.7.After 12 h of hydrolysis, it was filtered through a UVpolyethersulfone membrane with a molecular weight cutoffof 10 kDa. EMBR-2 also increased the antioxidantcapacity of the permeate to its half-maximal inhibitionconcentration (IC50) of 13.8 μg/mL, which was 39%more than that of sodium caseinate. The experimentmade it possible to avoid the mutual effect of peptidasesby separating endo- and exopeptidases at the two stagesof hydrolysis. The selected conditions proved optimaland ensured a stable production for three days. Theresearch featured the degree of hydrolysis of biocatalysisproducts. The hydrolyzate obtained in EMBR-1 had thefollowing parameters: degree of hydrolysis – 8.0 ± 0.2%,permeate – 8.7 ± 0.4%, sediment fraction – 2.9 ± 0.3%.The permeate hydrolyzed in EMBR-2 had a degreeof hydrolysis of 21.8 ± 0.8%. The loss of enzymaticactivity in both reactor vessels was compensated by thedaily addition of the corresponding enzyme. The wholeprocess took 110 h [45].Due to the applied temperature, the relative activityof peptidase in EMBR-1 decreased to 82 ± 6.9% of itsinitial value during the preliminary hydrolysis. As forEMBR-2, its initial activity remained the same duringthe preliminary hydrolysis (26–38 h) and decreased to82% after 24 h of filtration (38–62 h). The two reactorsmaintained stable conditions because the activitieswere adjusted every 24 h. The experiment proved thatCSTMRs can be used for commercial production offunctional antioxidant ingredients based on sodiumcaseinate [45].Guadix et al. studied hydrolyzate production ofhypoallergenic whey [44]. The research objective wasFigure 2 Block diagram of a two-stage installation of a two-stage enzymatic membrane bioreactor with continuous hydrolysisEnzymatic membrane bioreactor – 1Retentate 1Substrate EnzymatichydrolysisUV-filtration HeatingSupernatantSedimentation SedimentPermeate 1Enzymatic membrane bioreactor – 2Retentate 2Permeate 2Enzymatichydrolysis UV-filtration275Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280to create a stable long-term process for the productionof whey protein hydrolysates with low antigenicity. Thestudy was based on other scientific schools of continuoushydrolysis. For instance, specialists from the Universityof Illinois (USA) studied continuous hydrolysis ofsoy protein from Promin-D in a CSTMR with hollowmembrane fibers. At the initial stage, the conversion ratewas 90%, which dropped to 60% after 10 h because ofthe leakage of the enzyme through the membrane andthermal deactivation. The Illinois team also studiedmilk protein hydrolysis. They hydrolyzed casein withalkalase, also in a CSTMR with hollow fibers. Theirexperiments determined the efficiency of the reactor at50 and 37°C. After a 15-h fermentative treatment, thedegree of conversion dropped from 96 to 62% at 50°Cand from 75 to 51% at 37°C. Like in the first case, theefficiency fell down because of enzyme leakage, thermaldeactivation, and enzyme-membrane interactions.French scientists studied the effect of operatingvariables on the performance of hollow fiber CSTMRsfor hydrolysis of blood plasma proteins using alcalase.After 35 h of operation, the permeate flow dropped dueto membrane clogging, which occurred as a result ofthe polarizing layer that accumulated on the membranesurface. Spanish and Colombian biochemists hydrolyzedwhey proteins with alcalase using the same CSTMRswith hollow fibers. They managed to maintain anuninterrupted process only for 7 h because of therapid clogging and the inactivation of enzymes. Boththe proteolysis regimes and the design features of themembranes obviously needed correction.A team from Taiwan managed to maintainuninterrupted operation for 16 h. In addition to alcaslase,they also included Flavuerzyme into the enzymepreparation. The Laboratory of New Dairy Technologies(France) used CSTMRs to obtain specific bioactivepeptides by hydrolysis of casein-macropeptide.Cow’s milk whey is not the only type of whey insuch studies. Cambridge specialists studied hydrolysatesof goat whey from the point of view of the formation ofbiologically active peptide compounds. Goat whey washydrolyzed with pepsin in an enzymatic reactor. Theultrafiltration polymer membrane was combined with amineral membrane with a cut-off of 30 kDa. Peptides inthe permeate were separated by reversed-phase HPLC,which is the most common method for separatingmilk peptides [46, 47]. As β-lactoglobulin is resistantto pepsin, most opioid and antihypertensive peptideswere derived from α-lactalbumin. Pepsin exhibited aconsiderable substrate specificity; the molecular weightsof the obtained peptides ranged from dipeptides to verylarge peptides with disulfide bridges (150–6900 Da). Asa result of the α-lactalbumin hydrolysis, the amount ofpeptides with a molecular weight of ≤ 600 Da was 36%,600–2000 Da – 24%, and ≥ 2000 Da – 40%.Guadix et al. hydrolyzed diluted milk wheyconcentrate (50 g protein/L) in a CSTMR at 50°C andpH 8.5 using Protex 6L bacterial protease obtained fromBacillus licheniformis. The design of the membranereactor included a 3-L vessel, an automatic controllerof pH and temperature, a recirculation pump, anda frame membrane ultrafiltration module with apolyethersulfone plate with an effective area of 0.07 m2and a molecular weight cut-off of 3 kDa. The reactionmix was continuously recirculated at a rate of 1.5 L/minwith a pump at a rate of 0–15 L/min. The pump wasinstalled between the reaction vessel and the inlet of themembrane module.As a result of membrane clogging, the permeateflow dropped from 10 mL/min to 6.3 mL/min after16 h. After 10 h of operation, the degree of hydrolysisstabilized at about 80%, while the permeate flowstabilized after 13 h. As the permeate flow decreasedduring the first 13 h, the enzymes demonstrated signsof thermal inactivation. The resulting hydrolyzatecontained peptides that consisted of four amino acids.The content of antigenic whey protein decreased by99.97% in the final product, which means that it canbe used in hypoallergenic diets, baby food, and enteralfeeding. However, the authors had to compensate for theloss of enzymatic activity by feeding small amounts offresh enzyme [44].O’Halloran et al. developed an EMBR in whichthe whey protein isolate was subjected to enzymatichydrolysis to obtain antidiabetic peptides that inhibitdipeptidyl peptidase-IV (DPP-IV). The efficiency grewFigure 3 Method of gradient dilution feeding substrate in an enzymatic membrane reactorDairy proteinGradientdilution feedingsubstrateOptimal feeding modeStable hydrolysisPeptidesHydrolysisin a continuousstirred tankmembranereactorEnzymatic efficiency276Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280by 7.2 and 8.7% when using Protamex and Korolase2TS, respectively, compared to the standard method ofbatch processing. Previously, neither of the enzymeswas considered effective for obtaining peptides withantidiabetic activity. Protamex and Korolaza 2TS provedcapable of producing peptides that inhibit DPP-IV. Thepermeate hydrolyzate obtained with Protamex showed a33.7% higher DPP-IV inhibition value compared to thehydrolyzate obtained using Korolase 2TS. J. O’Halloranand colleagues proved that Protamex can be used toproduce protein substrates with antidiabetic activity [48].Huang et al. used a CSTMR to improve the yield ofpeptides that inhibit angiotensin-converting enzymefrom milk protein. The research employed a newmethod of gradient dilution feeding substrate (GDFS)(Fig. 3) [49]. The scientists compared the stabilityof the hydrolysis process, enzymatic efficiency, andkinetics of the method with the traditional modes offeeding, when adding water after feeding the substrate,or feeding the substrate with a constant concentration.The GDFS method showed the highest membraneflow rate and the lowest fluctuations in the proteinconcentration in the reactor. GDFS also had a higherrate of protein hydrolysis, which increased by 67.58%.The yield of peptides reached 138.51 g/g neutrase, andthe angiotensin-converting enzyme inhibitory activityof hydrolysates was 0.74 mg/mL. The optimal operatingtime was 720 min. The GDFS method can serve asan alternative method for obtaining highly efficientbioactive peptides [49].German researchers developed a stable process forobtaining specific hydrolysates with selected biologicalproperties. They developed and tested a continuousreactor system with a ceramic capillary modulewith various combinations of enzymes and proteinsubstrates (Fig. 4) [49]. Alcalase was immobilized on thesurface of capillaries modified with aminosilane with apore size of 1.5 μm. The loading capacity was 0.3 μg ofenzyme per 1 mg of capillary with a residual enzymeactivity of 43%. They tested controlled hydrolysisof casein, sunflower, and lupine isolates. Caseinhydrolysates proved to possess the largest amount ofpeptides with enhanced biological properties [50].A continuous reactor consists of a ceramic capillarywith one enzymatic filler. The filler is made of yttriumstabilizedzirconium oxide. It is fixed in a specialstainless steel casing (Fig. 4). In a way, this systemis a plug flow reactor system. The protein solution ispumped through the capillary module with a peristalticpump. The capillary module is part of the column oven,which makes it possible to keep the temperature at 37°C.The end of the capillary is sealed with cyanoacrylatecement to inject the flow from the intracapillaryspace into the extracapillary space. The enzyme isimmobilized on the activated surface of the ceramiccapillary with an APTES linker. The protein movesthrough ceramic capillaries by forced convective flow.The immobilization makes it possible to use the entireavailable capillary surface. As a result, enzymes canbe immobilized on the inner and outer surfaces, as wellas on the pore walls. One capillary is 10 cm long andhas an outer diameter of 1.8 mm, an inner diameter of1 mm, and an average pore size of 1.5 μm. The ceramiccapillary was replaced with a new immobilized enzymeto prevent protein contamination. The residence time ofthe substrate appeared to be inversely proportional to theflow rate: the longer the residence time of the substratein the capillary filled with the enzyme, the higher thecontinuous yield. These continuous reactors producedspecific peptides with the desired biologically activeproperties [50].New combined hypoallergenic functional productsneed new methods of gluten reduction. For example,MBRs can be used for wheat processing to create dairyproducts fortified with vegetable protein, but withhypoallergenic proteins and a low content of lactoseand gluten.Merz et al. developed a 96-h continuous hydrolysisof wheat gluten with flavurzim in an EMBR [51].Figure 4 Capillary module that immobilizes enzymes on a ceramic substrate APTESProtein substrateCeramic capillariesAPTES linkerAlcalaseProtein PeptidesCyanoacrylate cementHydrolyzate277Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–280Temperature, pump load, and enzyme flow throughthe membrane were the main criteria for hydrolysisstability and direction. The scientists optimized thehydrolysis to maximize the space-time yield. Formicrobial stability, they included 8% ethanol with asubstrate concentration of 100 g/L at 37°C and pH 7.5for 96 h (Fig. 5) [51].A diaphragm pump (P 1) circulated one liter ofsubstrate. The flow rate was 3.3 L/min. Hollow fiberceramic membranes were 45 mm in length, 6 mmin diameter, and 0.0085 m2 in surface area. Theyperformed cross-flow ultrafiltration of hydrolysates (F 1)on a membrane with a pore size of 1, 5, or 10 kDa.The hydrolyzate inside the reactor was stirred usinga magnetic stirrer (R 2). A constant transmembranepressure of 2 Bar was adjusted with a ball valve (V 1)and measured with barometers (PI 1, PI 2). The substratewas fed continuously using a tubular pump (P 2). Thefeed container was kept in an ice bath during the entiretest [51]. This EMBR hydrolysis scheme can be costeffectivein the industrial production of hydrolysatesfrom grain proteins.Russian specialists also developed a CSTMRthat produced a hydrolyzate of whey proteins withlow residual antigenicity. The installation was basedon enzyme preparation alcalase 2.4 L (Fig. 6) [52].Hydrolysis products were accumulated in an enzymaticmedium, which was followed by membrane separationinto a purified hydrolyzate (permeate) and an insolubleresidue (retentate). The experiment aimed at completeseparation of the enzyme to keep it active inside thereactor core.The scientists reproduced the process describedin foreign publications, i.e. protein hydrolysis,combined with the separation of hydrolysis products onultrafiltration membranes. The resulting hydrolyzatehad a low solids content (1.5%). The technology provedcommercially unprofitable and expensive. The low solidscontent resulted from the low cut-off of membranes(5 and 10 kDa). In this case, a portion of hydrolysisproducts was retained by the elective membranes andremained in the concentrate. Another disadvantageof membranes with a low molecular weight cut-off(≤ 10 kDa) was the low filtration rate and hightransmembrane pressure. The latter triggered theformation of a polarization layer and, eventually,membrane clogging [52].The molecular weight of the enzyme used forprotein biocatalysis is the most important parameterfor determining the cut-off threshold of membranes.Alcalase, which we used for hydrolysis of whey proteinsin our research, has a molecular weight of 24–27 kDa.Membranes with a cut-off threshold of 20 kDa couldeasily separate an enzyme with such a molecularweight [22]. Such membranes could significantlyreduce the transmembrane pressure, thus minimizingthe formation of a polarization layer and subsequentmembrane clogging.Separate hydrolysis and filtration made itpossible to provide optimal conditions for each of theprocesses (Fig. 6).The hydrolysis was carried out under the previouslyestablished conditions: substrate concentration – 4.5%;enzyme concentration – 0.5%, hydrolysis temperature –* – the gray line indicates a membrane restart, which is activated if the pressure exceeds 6 barFigure 5 Enzymatic membrane reactor with two stirred reactors (B 1, B 2), a water bath (W 1) with a thermostat (TIC),a membrane pump (P 1), a feed pump (P 2), a transverse filtration unit flow (F 1), two barometers (PI 1, PI 2), level indicator (LIC),and valves (V 1, V 2)Product / PermeateRetentateReactorIce bathFeed278Ryazantseva K.A. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 271–28065°C; hydrolysis time – 1 h. The proteolysis did notinclude pH-statisation. The initial active acidity ofthe reaction mix was 10. As for the molecular weightdistribution of hydrolysis products, the residues ofunhydrolyzed protein were retained during fractionation,which decreased the hydrolyzate yield. However, adouble filtration made it possible to increase the yield ofthe finished product by an average of 6%.The whey protein hydrolyzate had the followingparameters: degree of hydrolysis – 18–25%; massfraction of ash – 6.5–6.9%; osmolality of a 10% solution– 280–300 mmol/L of water; residual antigenicity –≤ 2×10–5 of the protein mass. The resulting hydrolyzatein the form of a 10% aqueous solution had a clear,moderately bitter taste, without off-flavors. Its antigenicproperties make it possible to use it in therapeutic andprophylactic functional foods based on enzymaticprotein hydrolysates [30].CONCLUSIONIn addition to batch enzymatic reactors, bioactivepeptides are obtained by a semi-continuous reaction ora continuous reaction in an enzymatic membrane reactor(EMBR) [31, 39, 40, 42–45].Considering the enzymatic efficiency and cost ofenzymatic hydrolysis, continuous reaction has obviousadvantages. Hydrolysates can promptly be separatedfrom the substrate, the yield of biological peptides canbe significantly increased, and enzymes can be usedmore than once. In addition, the production process isquite simple, which reduces labor costs [47, 48]. As aresult, this method is popular in food industry.Membrane reactors can process a variety of proteinfood media of plant and animal origin. They havegood prospects for whey processing in functionalfood production. Bioreactors can also be used for theproteolysis of whey proteins with maximal antigenic,antihypertensive, and antidiabetic properties.Protein hydrolysis in continuous EMBRs isattracting scientific attention because it can simplifythe technological process and reduce the cost of thefinal product while increasing the yield, despite highoperating costs. Therefore, the need to improve anddevelop these technologies is obvious.CONTRIBUTIONK.A. Ryazantseva supervised the project.E.Yu. Agarkova and O.B. Fedotova conducted thetheoretical research, processed the data, and preparedthe manuscript.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterests regarding the publication of this article.