INTRODUCTIONThese days, biochemists and food industry workersare facing an important task: they have to providepopulation with high-quality protein. Introducing dairyproducts into bakery formulae can solve the problem,since milk proteins are biologically valuable accordingto the content and ratio of essential amino acids. Theamino acid composition of whey proteins is closest tothat of human muscle tissue. Whey proteins are superiorto all other animal or plant proteins in terms of essentialamino and branched-chain acids, i.e. valine, leucine, andisoleucine [1–3].However, there is the problem of people with lactoseintolerance. According to the Institute of Immunology(Ministry of Health of the Russian Federation), 65% ofallergic patients demonstrate intolerance to some kindof food, e.g. dairy products. This problem is especiallycommon among children [4–7]. Therefore, dairyproducts as additives require a thorough research [8].Although people of any age can digest unalteredmilk proteins, cow’s milk remains one of the strongestand most common allergen [6–8]. It contains about20 proteins with different degrees of antigenicity,including those with the highest clinical relevance,such as β-lactoglobulin, α-lactalbumin, bovine serumalbumin (BSA), γ-globulin, and α- and β-caseins [9–11].β-lactoglobulin is the predominant whey protein incow’s milk: 50% of whey protein and about 10% of totalprotein. It is considered one of the main milk allergens,while α-lactalbumin and BSA have a lower immunereactivity [12]. Sensitization to β-lactoglobulin is causedby numerous continuous epitopes located along theentire length of its molecule [2, 12, 13].A β-lactoglobulin molecule consists of 162 amino acidresidues and has a molecular weight of about 18300 Da.Copyright © 2019, Savkina et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix,transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.Foods and Raw Materials, 2019, vol. 7, no. 2E-ISSN 2310-9599ISSN 2308-4057Research Article DOI: http://doi.org/10.21603/2308-4057-2019-2-X-XOpen Access Available online at http:jfrm.ruDegradation of β-Lactoglobulin during sourdough bread productionOlesya A. Savkina1,* , Olga I. Parakhina1, Marina N. Lokachuk1, Elena N. Pavlovskaya1,and Vadim K. Khlestkin1,21 St. Petersburg Branch of the State Research Institute of Baking Industry, St. Petersburg, Russia2 Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia* e-mail: o.savkina@gosniihp.ruReceived December 11, 2018; Accepted in revised form December 28, 2018; Published X X, 2019Abstract: The research featured various types and strains of lactic acid bacteria (LAB) and yeast. The research objective was tostudy their ability to utilize β-lactoglobulin during sourdough fermentation. The present paper also described the effect of sourdoughfermentation and baking on β-lactoglobulin degradation. A set of experiments with various types and strains of LAB showed thatβ-lactoglobulin decreased in gluten-free sourdough with 30%, 60%, and 90% of skimmed milk powder (SMP). L.plantarum E36demonstrated the highest biodegradation of β-lactoglobulin (by 53%) with SMP = 30%. L.helveticus ATCC8018T showed the lowestcontent of β-lactoglobulin with SMP = 60% and 90%: the content fell by 48% and 40%, respectively. The largest decrease in thecontent of β-lactoglobulin was observed in the sourdough with Saccharomyces cerevisiae 17 (by 28–42%) and Candida milleriPushkinsky (by 25–41%). The content of total protein increased, which was not associated with yeast biomass growth. The contentwas determined after fermentation in sourdoughs with SMP = 60% and 90% using a bicinchoninic acid reagent kit. The content ofβ-lactoglobulin in the control and experimental samples did not exceed 1 μg/g in the finished bakery products. This fact indicated asignificant effect of thermal treatment on β-lactoglobulin degradation in baking. Thus, temperature processing (baking) had a greaterimpact on the destruction of β-lactoglobulin than enzymatic processing (fermentation).Keywords: β-lactoglobulin, enzyme-linked immunosorbent assay, lactic acid bacteria, milk, sourdough, breadPlease cite this article in press as: Savkina OA, Parakhina OI, Lokachuk MN, Pavlovskaya EN, Khlestkin VK. Degradation ofβ-Lactoglobulin during sourdough bread production. Foods and Raw Materials. 2019;7(2):X–X. DOI: http://doi.org/10.21603/2308-4057-2019-2-X-X.66Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХAt pH 6.8–7, β-lactoglobulin can be found in milk as adimer [14].β-Lactoglobulin is relatively resistant to acidhydrolysis and intestinal proteases. As a result, whenconsumed with food, part of the protein remains intactin the gastrointestinal tract and can penetrate throughthe intestinal wall. Heat treatment reduces the IgEbindingability in proportion to the degree of heating.However, new antigenic sites may form in denaturedproteins. These sites were unavailable for binding in thenative molecule or appeared during a chemical reactionwith other food molecules. IgE obtained from patientswith an allergy to β-lactoglobulin was found specific toboth native and denatured proteins [2, 10].Like any proteins, milk proteins are exposed totemperature, pressure, and enzymes. The followingscheme is generally accepted for the thermaldenaturation of β-lactoglobulin: deployment of proteinmolecules – dissociation of dimer – aggregation ofdenatured protein. Dimeric β-lactoglobulin reversiblydissociates into monomers at 30–55°C. At 80°C, themolecule is almost completely unfolded [11, 13]. Thereversibility of β-lactoglobulin denaturation depends onthe heating degree and time. After a low temperatureheating, a small part of the denatured (unfolded)β-lactoglobulin molecules can restore their nativestructure. However, an hour at 95–97 °C leads to anactive aggregation of β-lactoglobulin molecules. As aresult, protein denatures irreversibly. After denaturationat ≥ 70°C, the β-lactoglobulin structure can partiallystabilize as the chains re-clot and disulfide bridges areformed. At 130–140°C, the disulfide bonds break, andthe protein polypeptide chains deploy completely andirreversibly [2, 15–17]. Denaturation and hydrolysis ofβ-lactoglobulin is possible when exposed to microwaveradiation [18, 19]. Denatured or hydrolyzed milkproteins used in dairy mixes are known to be lessallergenic [20, 21]During baking, the temperature of the crust canreach 180–230°C, while the core crumb warms up to nomore than 95°C for several minutes [22]. In this regard,the effect of the baking process on the β-lactoglobulincontent in bread with dairy products remainsunderstudied.In fermented milk products, most milk proteinsare destroyed by various microorganisms, includingLAB. Prebiotic cultures of LAB are known to reducethe allergenicity of cow’s milk due to the partialdenaturation of allergenic proteins [24, 25].Microorganisms play an important part in baking.For instance, fermentation process takes place insourdough and dough. Various types of LAB are widelyused in sourdough [22, 23]. Hence, it is necessaryto study the effect of LAB sourdough and doughfermentation on the destruction of cow milk allergenprotein. The research can result in a method of reducingthe allergenicity of dairy products and creating new,safer bakery products.Thus, the research objective was to study the effectof LAB and yeast on the destruction of β-lactoglobulinduring baking.STUDY OBJECTS AND METHODSEffect of LAB on the β-lactoglobulin contentand acidity of the sourdough. The research featuredsourdough of 8 LAB strains: Lactobacillus plantarumE36, Lactobacillus plantarum E4, Lactobacillusplantarum E1, Lactobacillus parabuchneri E7,Lactobacillus paracasei/casei E31, Lactobacillusparacasei E3, Lactobacillus acidophilus 22n2, andLactobacillus helveticus ATCC 8018T. As for theyeast strains, 8 types were employed: Saccharomycescerevisiae – strains L-1, 90, 512, 17, XII, andKrasnodarsky; Candida milleri Chernorechensky; andKluyveromyces marxianus Pushkinsky. The sampleswere obtained from the Collection of the St. PetersburgBranch of the State Research Institute of BakingIndustry (St. Petersburg, Russia) [26].Preparing the sourdough: The nutritional mixtureconsisted of rice flour and SMP (30%, 60%, and 90%per 100 kg of mixture). The moisture content w as 75%.The LAB culture fluid had a cell content of 108 CFU/mlcultivated in SMP for 48 h. During the first phase, itwas added to a mixture of raw materials and water,stirred, and placed in a thermostat for 24 h at 30°C. Thefermented sourdough was then added to the nutrientmixture in the ratio of 1:3 and allowed to ferment for 24 hat 30°C. Table 1 shows the formulae for sourdough of thepropagating and production cycles. A nutritional mixturedevoid of any LAB served as a control sample.The quality of the sourdoughs was assessedaccording to their acidity. The acidity was determinedby the common method used in baking industry.The sourdough suspension was titrated in water atH = 0.1 with NaOH solution and phenolphthalein [27].Table 1 Formulae for sourdough with SMP and pure LABcultures in the propagating and production cyclesMaterial Raw materials in the sourdough with thecontent of SMP, % to dry solids30 60 90 30 60 90Phase I of thepropagating cycleProduction cycleLAB culturefluid, ml10.0 10.0 10.0 – – –Sourdough(PhaseI of thepropagatingcycle), g– – – 50.0 50.0 50.0Rice flour, g 35.0 20.0 5.0 29.0 16.6 4.1SMP, g 15.0 30.0 45.0 12.4 24.9 37.3Water, g 121.0 121.0 121.0 108.6 108.6 108.6Total: 181.0 181.0 181.0 200.0 200.0 200.067Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХEffect of the yeast on the content ofβ-lactoglobulin in the sourdough. Preparing thesourdough: Yeast strains grown on malt wort slant agar(8% DS) were used to screen the allergen reducingactivity of the yeast. 10 ml of yeast culture wereintroduced into an aqueous suspension with 10 CFU/mlcell content in the nutritional mixture (Table 2). Themixture consisted of rice flour, SMP (30%, 60%,and 90% per 100 kg of the mixture), and water. Themoisture content of the mixture was 75%. To preventthe development of extraneous microflora, L.helveticusATCC 8018T was added to the nutrient mixtures. Thestrain had been selected during the first stage of theexperiment. It demonstrated the highest allergenreducingactivity.The sourdoughs were fermented for 24 h at 30°C andthen examined for acidity, temperature, and moisturecontent.The effect of sourdough and dough fermentationand baking on the content of β-lactoglobulin.Laboratory baking was used to study of the effect of thetechnological process (fermentation and baking) on thecontent of β-lactoglobulin in dough and gluten-free bread.Preparing the sourdough: The nutritional mixtureconsisted of rice flour and SMP (0%, 30%, 60%,90%, and 100%). The moisture content was 75%.LAB of L.helveticus ATCC 8018T strain and yeast ofS.cerevisiae 17 and C.milleri Pushkinsky were added tothe mixture in the quantities indicated in Table 3.Preparing the dough: The dough for the controlsample was kneaded from corn starch, extrusion starch,soy protein isolate, rice flour, and SMP in the amount of3%, 6%, 9%, and 10% to the weight of the mixture. Themixture contained sugar, salt, pressed baking yeast, andvegetable oil. The moisture content was 53.5%.The dough for the samples was prepared from thesourdough obtained at phase II of the propagating cycle(10% of the mixture in the intermediate product), cornstarch, extrusion starch, rice flour, sugar, salt, pressedbaking yeast, vegetable oil, and water. Table 4 shows theformulae of the dough.The dough was poured into 250-gram moulds andallowed to rise at 35–40°C at an average humidity ofTable 2 Sourdough formulae with SMP and pure culturesof yeast and LABMaterial Raw materials in the sourdough withthe content of SMP, % to dry solids30 60 90Yeast suspension, ml 10.0 10.0 10.0Culture fluid ofL.helveticusATCC 8018T, ml10.0 10.0 10.0Rice flour, g 35.0 20.0 5.0SMP, g 15.0 30.0 45.0Water, g 111.0 111.0 111.0Total: 181.0 181.0 181.0Table 3 Sourdough formulae with SMP in propagatingand production cyclesMaterial Raw materials in the sourdough with thecontent of SMP, % to dry solids30 60 90 30 60 90Phase I of thepropagating cycleProduction cycleCulture fluidof L.helveticusATCC 8018T, ml10.0 10.0 10.0 – – –Yeast suspension,ml: S.cerevisiae 175.0 5.0 5.0C.milleriPushknsky5.0 5.0 5.0Sourdough,g – – – 50.0 50.0 50.0Rice flour, g 35.0 20.0 5.0 29.0 16.6 4.1SMP, g 15.0 30.0 45.0 12.4 24.9 37.3Water, g 111.0 111.0 111.0 98.6 98.6 98.6Total: 181.0 181.0 181.0 200.0 200.0 200.0Table 4 Dough formulaeMaterial Consumption of raw materials per 100 kg of the mixture with the SMP content, % to the weight of themixture in the doughControl sample Experimental sample3 6 9 10 3 6 9 10Corn starch, g 64.2 61.2 58.2 57.2 64.2 61.2 58.2 57.2Extrusion starch, g 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0Rice flour, g 20.0 20.0 20.0 20.0 13.0 16.0 19.0 20.0SMP, g 3.0 6.0 9.0 10.0 – – – –Sourdough, g – 36.0Pressed baking yeast, g 2.5Vegetable oil, g 3.8Salt, g 0.8Sugar, g 2.0Water, g 110.6 84.7Total 217.068Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Х80 ± 2%. After that, the samples were baked in an ovenat 210°C for 18 min with a 5-second steam supply.Preparing samples for the immunoassay and gelelectrophoresis. Preceding the analysis, the samplesunderwent the following procedures. 9 ml of phosphatesalinebuffer (PBS, pH = 7.4) was added to 1g of thetest sample (sourdough, dough, or bread). The buffercontained sodium azide to protect the samples frommicroorganisms. After that, a 12-hour extractionwas performed using a shaker at 20 ± 1°C. After theextraction, the samples were centrifuged at 40°C and14000 rpm in an Eppendorf Centrifuge 5417R to removemicroorganisms and undissolved components. After thecentrifugation, the samples were diluted 10 thousandtimes in a phosphate-buffered saline (20 mM phosphate,150 mM NaCl, pH 7.2). The dilution was adapted to theconcentration range defined by the test system.The method of enzyme-linked immunosorbent assay(ELISA method) was used to measure the content ofβ-lactoglobulin in the sourdoughs at the onset and at theend of fermentation. The process involved antibody No.362-beta-lactoglobulin – a set of reagents provided byOOO Hema (St. Petersburg, Russia).Electrophoresis in a sodium dodecyl sulphatepolyacrylamide denaturing gel was employed to confirmthe presence of β-lactoglobulin in the sourdoughs at theonset and at the end of fermentation, as well as in the bread.A bicinchoninic acid reagent kit (BCA, Pierce) wasused to define the total protein in the sourdoughs at theonset and at the end of fermentation and in the producedbread. The disc electrophoresis was conducted in nonreducingconditions according to Laemmli method. Thesamples were diluted to a protein concentration of 1 mg/ml before they were applied to a 13% separating gel.Statistics. The statistical analysis was performedusing Excel software. The method of two-way ANOVAwas used to compare the effects of the SMP amountand the type of strain on the content of β-lactoglobulinin the sourdoughs, dough, and bread. The research alsoassessed the correlation and covariance between theβ-lactoglobulin content and the sourdough acidity.The data show the confidence intervals, which provethe accuracy of the methods for determining proteincontent and acidity.RESULTS AND DISCUSSIONThe experiment measured the acidity in thesourdoughs based on various strains with differentcontent of SMP. Acidity reflects the development ofmicroorganisms in the environment. A high level ofacidity improves the absorption of nutrients from theenvironment. High acidity values accelerate proteolysis,which is important for the destruction of protein andits constituents, including the allergenic ones. Duringphases I and II of fermentation, L.acidophilus 22n2 andL.helveticus ATCC 8018T showed the highest titratedacidity indicators at the end of phase II (Table 5). Thesestrains demonstrated the maximum titratable aciditywith SMP = 60%.All the LAB strains had different effects onβ-lactoglobulin (Fig. 1). The degree of β-lactoglobulindegradation decreased with the increase in the SMPconcentration in the nutritional mixture, while differentstrains reacted differently to the increase in the SMPconcentration. At SMP = 30%, the sourdough samplewith L.plantarum E36 showed the biggest drop inβ-lactoglobulin content in the fermentation process –by 53%. However, at SMP = 60% and 90%, it was theL.helveticus ATCC8018T sample that showed the biggestdrop in the content of the allergen – by 48 and 40%,respectively. In the sourdoughs, the SMP amount mighthave a different effect on the vital activity of lactic acidbacteria, since they normally live in silage and flour,except L.acidophilus 22n2 and L.helveticus ATCC8018T.The two-way ANOVA method gave the followingresults. The SMP amount had a significant effect onthe β-lactoglobulin content in the sourdough afterfermentation: alpha = 0.05, P < 0 .001, F = 2 7.78,Fcritical = 3.63. However, the type of LAB strainfactor produced no effect: alpha = 0.05, P = 0 .25,F = 1.46, Fcritical = 2.59. A strong positive correlationand covariance was revealed between the finalβ-lactoglobulin content and the final acidity level ofthe sourdough for L.plantarum E4 and L.acidophilus22n2. The correlation coefficients were 0.99 and 0.91,Table 5 Effect of various LAB strains on the sourdoughacidityLAB strain in thesourdough at differentSMP amountsTitrated acidity of the sourdough, degreePhase I Phase IIonset final onset finalSMP = 30%L.paracasei E3L.paracasei E31L.plantarum E36L.plantarum E4L.parabuchneri E7L.acidophilus 22n2L.helveticus ATCC 8018TL.plantarum E13.0 ± 0.33.0 ± 0.32.5 ± 0.33.0 ± 0.33.0 ± 0.33.0 ± 0.33.0 ± 0.32.7 ± 0.312.9 ± 1.312.3 ± 1.27.7 ± 0.87.5 ± 0.86.8 ± 0.715.8 ± 1.612.8 ± 1.39.5 ± 1.04.5 ± 0.54.2 ± 0.42.9 ± 0.32.9 ± 0.32.8 ± 0.33.5 ± 0.43.3 ± 0.35.0 ± 0.513.5 ± 1.415.3 ± 1.59.8 ± 1.08.6 ± 0.911.0 ± 1.118.5 ± 1.916.5 ± 1.712.2 ± 1.2SMP = 60%L.paracasei E3L.paracasei E31L.plantarum E36L.plantarum E4L.parabuchneri E7L.acidophilus 22n2L.helveticus ATCC 8018TL.plantarum E13.5 ± 0.43.5 ± 0.33.0 ± 0.33.4 ± 0.33.8 ± 0.44.5 ± 0.54.5 ± 0.54.1 ± 0.413.7 ± 1.414.2 ± 1.49.5 ± 1.08.2 ± 0.88.5 ± 0.921.0 ± 2.117.2 ± 1.710.5 ± 1.15.3 ± 0.56.5 ± 0.73.6 ± 0.45.0 ± 0.54.0 ± 0.46.0 ± 0.65.5 ± 0.64.5 ± 0.519.8 ± 2.020.4 ± 2.011.9 ± 1.211.2 ± 1.111.8 ± 1.328.0 ± 2.822.5 ± 2.310.2 ± 1.0SMP = 90%L.paracasei E3L.paracasei E31L.plantarum E36L.plantarum E4L.parabuchneri E7L.acidophilus 22n2L.helveticus ATCC 8018TL.plantarum E14.7 ± 0.54.2 ± 0.44.5 ± 0.54.5 ± 0.55.2 ± 0.55.7 ± 0.65.5 ± 0.65.0 ± 0.513.2 ± 1.312.5 ± 1.39.5 ± 1.011.0 ± 1.19.1 ± 0.923.9 ± 2.420.0 ± 2.010.5 ± 1.16.0 ± 0.65.6 ± 0.65.5 ± 0.65.3 ± 0.55.7 ± 0.67.0 ± 0.76.0 ± 0.65.8 ± 0.616.2 ± 1.619.6 ± 2.017.4 ± 1.716.3 ± 1.617.0 ± 1.727.5 ± 2.822.5 ± 2.315.3 ± 1.569Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Хrespectively. The covariance coefficients were 3270and 2449, respectively. L.paracasei E31 demonstrated aweak inverse correlation (coefficient = 0.25).The screening of the allergen-reducing activity ofvarious yeast strains (Fig. 2) showed that the strainsproduced a different effect. As for Saccharomycescerevisiae, strain 17 demonstrated the highest allergenreducingactivity: the β-lactoglobulin content fell by28–42%. As for the Candida milleri, it was Pushkinskystrain: the β-lactoglobulin content fell by 25–41%.The two-way ANOVA method gave the followingresults. The SMP amount had a significant effect onthe β-lactoglobulin content in the sourdough afterfermentation: alpha = 0.05, P < 0 .001, F = 9 3.60,Fcritical = 3.56. However, the type of yeast strain factorproduced no effect: alpha = 0.05, P = 0 .37, F = 1 .17,Fcritical = 2.46.Lactic acid bacteria strain L.helveticus ATCC 8081Tand two yeast strains, S.cerevisiae 17 and C. milleriPushkinsky, were selected for further research, whichfeatured the effect of fermentation and baking on theβ-lactoglobulin content in sourdough, dough, and bread.The enzyme immunoassay showed a decreasein β-lactoglobulin at the end of phases I and II by1.4–1.8 times, if compared with its content in thenutrient mixture immediately after mixing (Fig. 3).Thus, the allergen was destroyed by the LAB enzymes.Figure 1 Content of β-lactoglobulin in the sourdoughs with various LAB strains after fermentationFigure 2 Content of β-lactoglobulin in the sourdoughs various yeast strains after fermentation08001600240032004000control L.paracasei E3 L.paracaseiE31L.plantarumE36L.plantarumE4L.parabuchneriE7L.acidophilus22n2L.helveticusATCC 8018TL.plantarumE1Content of β-lactoglobulin, μg/g30% SMP 60% SMP 90% SMP01500300045006000without yeast,S.cerevisiae L-1S.cerevisiae L-1 S.cerevisiae 17 S.cerevisiae 512 S.cerevisiae 90 S.cerevisiaeKrasnodarskyS.cerevisiae XII C.milleriPushkinskC.milleriChernorechenskyContent of β-lactoglobulin, μg/g30% SMP 60% SMP 90% SMP08001600240032004000control L.paracasei E3 L.paracaseiE31L.plantarumE36L.plantarumE4L.parabuchneriE7L.acidophilus22n2L.helveticusATCC 8018TL.plantarumE1Content of β-lactoglobulin, μg/g30% SMP 60% SMP 90% SMP01500300045006000without yeast,S.cerevisiae L-1S.cerevisiae L-1 S.cerevisiae 17 S.cerevisiae 512 S.cerevisiae 90 S.cerevisiaeKrasnodarskyS.cerevisiae XII C.milleriPushkinskC.milleriChernorechenskyKluyveromycesmarxianusContent of β-lactoglobulin, μg/g30% SMP 60% SMP 90% SMPFigure 3 Content of β-lactoglobulin in the sourdough beforeand after fermentation at the end of phases I and II02500500075001000030% SMP 60% SMP 90% SMPContent of β-lactoglobulin,μg/gSourdough before fermentation End of phase I End of phase II01530456030% Total protein content, mg/gSourdough before 03006009001200150030% SMP 60% SMP 90% SMPContent of β-lactoglobulin, μg/gbefore fermentation after fermentationFigure 4 Total protein in the sourdough before fermentationand after phases I and II02500500075001000030% SMP 60% SMP 90% SMPContent of β-lactoglobulin,μg/gSourdough before fermentation End of phase I End of phase II01530456030% SMP 60% SMP 90% SMPTotal protein content, mg/gSourdough before fermentation End of phase I End of phase II030060090012001500Content of β-lactoglobulin, μg/g70Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХDespite the destruction of β-lactoglobulin, the totalprotein content in samples with SMP = 60% and 90%increased in the fermentation process, if compared withthe initial amount (Fig. 4). The total protein contentwas determined using a bicinchoninic acid reagentkit. Presumably, there are two ways additional proteincould appear during the experiment. First, it couldincrease during fermentation due to the accumulationof yeast biomass. Second, it could be released fromany supramolecular or covalent complexes with othermacromolecules – proteins or polysaccharides. Tounderstand how the increase in the microbial biomassaffected the increase in total protein, an experiment wasconducted with sourdough based on rice flour, withoutSMP. In this case, the amount of total protein in thesourdough without SMP remained virtually unchangedduring the fermentation. It was 3.8 mg/g beforefermentation and 4.0 mg/g at the end of phase I. Theincrease in the total protein in the sourdoughs with SMPmight have been caused by the release of the previouslybound protein. It happened under the influence of yeastand LAB enzymes, not because their biomass increased.The experiment revealed a decrease inβ-lactoglobulin in the dough after fermentation,compared with its content immediately after kneading(Fig. 5). Due to the fact that the kneading involvedpressed yeast, the decrease in β-lactoglobulin couldbe explained by the combined effect of fermentingmicroflora enzymes and industrial yeast.As for the finished products, the content ofβ-lactoglobulin in the control and experimental breadsamples did not exceed 1 μg/g. Hence, the temperaturedegradation of β-lactoglobulin proved highly efficientfor bakery products.The electrophoresis was conducted accordingto Laemmli’s method in sodium dodecyl sulphatepolyacrylamide gel with non-reducing conditions. It alsoconfirmed a decrease in the content of β-lactoglobulin(Fig. 6 and 7 ). N either b lotting o f p olyacrylamide g elproteins to nitrocellulose, nor detection of β-lactoglobulinby antibodies from the ELISA test system gave anyresults. Neither of the antibodies was able to identify theantigen after electrophoresis in such conditions. Thatproved that the content of β-lactoglobulin in the finishedproducts was extremely low.Thus, the research proved that thermal treatment hasa greater impact on the destruction of β-lactoglobulinthan enzymatic treatment.CONCLUSIONThe research investigated the effect of various LABand yeast strains on the β-lactoglobulin content ingluten-free sourdough with SMP. Increasing the amountof SMP had an inhibitory effect on the utilization ofβ-lactoglobulin by L.plantarum E36, L.plantarumE1,and L.helveticus ATCC8018T. The last demonstrated thehighest allergen-reducing activity when SMP equalled60% and 90% of the solid weight: β-lactoglobulindecreased by 48% and 40%, respectively. The yeaststrains Saccharomyces cerevisiae 17 and Candida milleriPushkinsky showed the biggest decrease in the content ofβ-lactoglobulin: by 28–42% and 25–41%, respectively.Figure 5 Content of β-lactoglobulin in the dough before andafter fermentationFigure 6 Electrophoregramme samples: sourdough beforefermentation: SMP = 30% (1), SMP = 60% (2), SMP = 90% (3);sourdough after fermentation: SMP = 30% (4),SMP = 60% (5), SMP = 90% (6), and the marker (M)Figure 7 Electrophoregramme samples: control bread samplewith SMP = 30% (1), SMP = 60% (2), SMP = 90% (3);experiment bread samples with SMP = 30% (4), SMP = 60%(5), SMP = 90% (6), and the marker (M)02500500030% SMP 60% SMP 90% SMPContent of Sourdough before fermentation End of phase I End of phase II01530% SMP 60% SMP 90% SMPTotal protein Sourdough before fermentation End of phase I End of phase II03006009001200150030% SMP 60% SMP 90% SMPContent of β-lactoglobulin, μg/gbefore fermentation after fermentation1 2 3 4 5 6 M97 kDa66 kDa45 kDa30 kDa20 kDa14 kDa1 2 3 4 5 6 M 97 kDa66 kDa45 kDa30 kDa20 kDa14 kDa71Savkina O.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХS.cerevisiae L-1, S.cerevisiae 512, S.cerevisiae 90,and S.cerevisiae XII demonstrated an increase in thecontent of β-lactoglobulin at SMP concentration of90%. This might have been connected with a release ofβ-lactoglobulin, previously bound to other proteins.The content of β-lactoglobulin in the controland experimental samples of bread did not exceed1μg/g, which proved a high efficiency of temperaturedegradation of β-lactoglobulin in the baking process.Therefore, temperature processing (baking) had agreater impact on the destruction of β-lactoglobulin thanenzymatic processing (fermentation).CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest related to this article.ACKNOWLEDGMENTSThe authors would like to express their deepestgratitude to Yevgeny Alexandrovich Kozhevnikov(OOO Hema) for his consultations on the biochemistryof cow’s milk proteins, Pavel Pavlovich Kornev(St. Petersburg Branch of the State Research Institute ofBaking Industry) for the baking, and Vasily MikhailovichMatveyev (St. Petersburg Branch of the State ResearchInstitute of Baking Industry) for IT support.FUNDINGThe research was conducted on the premises ofthe St. Petersburg branch State Research Institute of aBaking Industry within the framework of the followingresearch topic: 0593-2014-0017 ‘Biotechnologies forsourdoughs based on the microbial composition oflactic acid bacteria and yeast with an allergen-reducingabilities to develop technology and assortment of bakedgoods with reduced allergenicity’, a basic program offundamental scientific researches of the state academies.The research employed microorganisms from theCollection of the St. Petersburg branch State ResearchInstitute of a Baking Industry (St. Petersburg, Russia).The Collection is on the list of collections that depositnon-pathogenic microorganisms for government use,as approved by the Decree of the Government of theRussian Federation (June 24, 1996 No. 725-47) and theOrder of the Ministry of Agriculture and Food of Russia(August 15, 1996 No. 14c).