3Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХINTRODUCTIONModern alcohol technologies are based on complexand deep processing of agricultural raw materialsaimed at improving production profitability. Theeffectiveness of biotechnological processing is achievedby developing new biocatalysts of various action andsubstrate specificity. This ensures deep hydrolysis ofhigh molecular weight polymers of grain, especially ryewith its high content of non-starch polysaccharides, gumsubstances, and mucus.As shown by many studies, the use of complexenzyme preparations with broad substrate specificitycan increase the depth of hydrolysis of grain polymersinto ethanol, especially when making concentrated grainwort [1–4]. The complex should contain amylolytic,proteolytic, and hemicellulase enzymes. Amylolyticenzymes play a part in starch conversion: α-amylase instarch dextrinization and liquefaction and glucoamylasein its saccharification). Proteases are beneficial for thegeneration and metabolism of alcoholic yeast, sincetheir catalytic effect on protein enriches the wort witheasily digestible amino acids assimilated by yeast [5].Hemicellulases (β-glucanases and xylanases), catalysingthe hydrolysis of non-starch polysaccharides, decreasethe wort viscosity and lead to the formation of additionalfermented carbohydrates due to the destruction of grainxylans and glucans. The synergic action of amylolytic,proteolytic and hemicellulase enzymes improve thequality of grain wort and its rheological properties,especially when processing difficult-to-ferment rawmaterials, such as rye and barley. Improved biocatalyticconversion of grain polymers intensifies alcoholResearch Article DOI: http://doi.org/10.21603/2308-4057-2019-2-Х-ХOpen Access Available online at http:jfrm.ruEffects of a complex phytase-containing enzyme preparationon the rye wort fermentation processViktor A. Polyakov , Elena M. Serba* , Marina B. Overchenko, Nadezhda I. Ignatova,and Liubov V. RimarevaRussian Scientific Research Institute of Food Biotechnology –a Branch of Federal Research Centre of Nutrition and Biotechnology, Moscow, Russia* e-mail: serbae@mail.ruReceived October 11, 2017; Accepted in revised form March 20, 2018; Published Х Х, 2019Abstract: A complex of amylases, proteases, and hemicellulases is known to enhance deep conversion of polysaccharides andproteins, especially in the processing of difficult-to-ferment raw materials, such as rye, providing grain wort with solublecarbohydrates, amino acids, and peptides. Grain is also a source of phosphorus, whose bioavailability can be increased byhydrolysing the grain with phytase-containing enzyme preparations. However, their catalytic action during the preparation ofgrain wort for alcohol production has hardly been studied. This study aimed to investigate the effect of a new complex phytasecontainingenzyme preparation on yeast metabolism and the efficiency of rye wort fermentation. The work was carried out in theRussian Research Institute of Food Biotechnology. The Glucavamorin complex enzyme preparations derived from recombinantstrains were the object of our research. The preparations differed in the activity level of the main enzyme, glucoamylase, andminor hemicellulase enzymes, as well as in the presence of phytase. The results confirmed their biocatalytic ability to efficientlyhydrolyse polymers of rye grain. An increased content of hemicellulases in Glucavamorin-Xyl improved the rheological propertiesof rye wort. The greatest effect was achieved with the phytase-containing Glucavamorin-Ply. This preparation improved thephosphorus nutrition of yeast, which increased its biomass by 30% and decreased the level of fermentation by-products by 18–20%.Alcohol yield tended to increase and its strength reached 10.5–10.9% vol. When using a phytase-containing enzyme complex, itwas possible to reduce the amount of the main enzyme, glucoamylase, without causing the key fermentation indicators to degrade.Keywords: Rye wort, phytase, enzyme preparations, yeast, ethanol, fermentation, metabolitesPlease cite this article in press as: Polyakov V.A., Serba E.M., Overchenko M.B., Ignatova N.I., and Rimareva L.V. Effects ofa complex phytase-containing enzyme preparation on the rye wort fermentation process. Foods and Raw Materials, 2019, vol. 7,no. 2, pp. Х–Х. DOI: http://doi.org/10.21603/2308-4057-2019-2-Х-Х.Copyright © 2019, Polyakov 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 2308-40574Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Хfermentation, increasing the target product (ethanol)yield and decreasing side metabolites formation.A number of studies into the phytase effect onthe processing of sorghum and corn for lager beerproduction showed a potential possibility of improvingnutritional conditions for yeast during the fermentationof raw whole grains [6]. Some researchers noted apositive effect of phytase treatment on the embryoand fibre yield during the dry grinding of yellow dentcorn [7]. A study of the phytolytic effect on the qualityof wheat bread enriched with bran revealed an increasein the bioavailability of iron contained in it [8]. To makethe conversion of phytin-containing raw materials moreefficient, microorganism strains with phytase activitywere selected and identified [9, 10]. Considerableresearch was conducted into the use of phytase toimprove the digestibility of feed nutrients, includingphytate phosphorus [11, 12]. However, there is a lackof studies into the effectiveness of phytolytic enzymesduring the preparation of concentrated grain wort foralcohol production, especially from rye.Currently, extensive studies are underway to obtainenzyme preparations based on recombinant strainsof microscopic fungi using genetic engineering andmutagenesis [13–15]. The preparations contain acomplex of enzymes with an increased biocatalyticcapacity for xylanase, β-glucanase, and cellulase, andthey can be used in the alcohol industry [16]. New highlyactive multienzyme preparations can contribute towardsthe implementation of innovative technologies for deepconversion of grain into ethanol.Of particular interest are phytolytic enzymes.Phytase is an enzyme that breaks down phytic acid.Phytic acid in the form of myo-inositol hexaphosphoricacid or phytate (acid salt) is the main form of mineralphosphorus in plant tissues [17]. Cereal grains have aparticularly high content of phytic acid [18]. Phosphorusis essential for yeast cells to grow and develop. Underanaerobic conditions, yeast assimilates phosphorusmainly in the initial period of fermentation when itsconsumption is 80–90% of the maximum content inyeast. Young, actively breeding yeast cells are richer inphosphorus than non-budding old cells. For example,after 6 hours of fermentation, yeast cells accumulate2.15% of phosphorus per dry matter, while this valueis only 1% at the end of fermentation. Therefore,when making grain wort, it is important to enrich itwith phosphorus to ensure a stable process of yeastgeneration and alcoholic fermentation.Cereals are the main source of phosphorus, whosebioavailability can be enhanced by hydrolysis of grainwith phytase-containing enzyme preparations. Phytatehydrolysis helps reduce the consumption of enzymepreparations, as it inhibits many enzymes and enablesthe release of valuable trace elements, such as calcium,magnesium, zinc, etc. [19, 20]. This way, it providesalcoholic yeast with additional nutrition.Apart from generating the main products offermentation, namely alcohol and carbon dioxide, yeastcells synthesise metabolites called secondary productsor by-products of fermentation. The biosynthesis ofby-products is associated with the cell’s regulatoryfunctions. By-products formation depends on themedium composition, the level of nitrogen, carbon,and phosphorus in the medium, the conditions ofyeast cultivation and the genetic characteristics ofthe strain used [3, 4]. One of the ways to improve theefficiency of alcohol production is to create conditionsto reduce carbohydrate expenditure for the formationof fermentation by-products through the use of mediawith a balanced amino acid composition. The amountof ethanol impurities can also be reduced by regulatingtechnological processes in such a way that conditions areprovided to promote ethanol synthesis with decreasedformation of fermentation by-products [4]. Therefore,complex enzyme preparations contribute to a morerational use of high-molecular components of grain rawmaterials.Our aim was to study the effect of a new complexphytase-containing preparation on yeast metabolism andthe efficiency of rye wort fermentation.STUDY OBJECTS AND METHODSThis research was conducted at the Department forBiotechnology of Enzyme Preparations, Yeast, OrganicAcids and Biologically Active Substances of the RussianResearch Institute of Food Biotechnology, a branch ofthe Federal Research Centre of Nutrition, Biotechnologyand Food Safety.The study objects included rye grain, theSaccharomyces cerevisiae 985T alcohol yeast, andcomplex enzyme preparations (EP), GlucavamorinXyl and Glucavamorin Ply. The preparations wereobtained in the Laboratory of New Enzyme Producersof the Russian Research Institute of Food Biotechnologyfrom the transformants of the commercial Aspergillusawamori strain [21]. They differed in the activity levelof the major enzyme (glucoamylase) and minor enzymes(hemicellulases).We used the following methods to determine thecatalytic activity of the enzyme preparations.Amylolytic and glucoamylase activity was determinedaccording to State Standard 54330-2011*. The methodfor determining amylolytic activity is based on thequantification of starch hydrolysed by amylolytic enzymesto dextrins of various molecular weight under standardconditions (temperature 30°С; pH 6.0 for bacterial and4.7 for fungal α-amylase; hydrolysis duration 10 min). Themethod for determining glucoamylase activity is basedon the quantification of glucose formed during starchhydrolysis by glucoamylase under standard conditions(temperature 30°С; pH 4.7; hydrolysis duration 10 min).β-glucanase activity (β-GcS) was determinedaccording to State Standard 53973-2010**. The method* State Standard 54330-2011. Enzyme preparations for foodindustry. Methods for determination of amylase activity. Moscow:Standartinform, 2012.** State Standard 53973-2010. Enzyme preparations for food industry.Methods for determination of β-glucanase activity. Moscow:Standartinform, 2011.5Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Хis based on the quantitative determination of reducingsugars resulting from β-glucanase action on β-1,4 bondsof β-glucan under standard conditions (temperature50°С; pH 4.7; hydrolysis duration 10 min).Cellulase activity was determined according toState Standard 55293-2012 (Enzyme preparations forfood industry. Method for determination of cellulaseactivity. Moscow: Standartinform, 2014). The methodis based on the quantitative determination of reducingsugars resulting from cellulase action on the substrate ofsodium carboxymethylcellulose (CMC) at 50°C.Xylanase activity was determined according to StateStandard 55302-2012***. The method is based on thequantitative determination of reducing sugars resultingfrom xylanase (exoxylanase) action on β-1,4 bonds of xylan.Phytase activity was determined according to StateStandard 31487-2012****. One unit of phytase activityis the amount of enzyme that catalyses the hydrolysis ofsodium phytate to form 1 μmol of inorganic phosphateper minute under standard conditions (temperature37°C; pH 5.5; hydrolysis duration 15 min).Rye, used as a raw material to make grain wort, wasprepared by “soft” enzymatic cooking at a water ratioof 1 : 3. At the mash stage, thermostable α-amylase wasused for starch dextrinization at the rate of 0.5 unit/gstarch. At the saccharification stage, the test samplesincluded complex enzyme preparations, namelyGlucavamorin Xyl and Glucavamorin Ply in an amountof 6–10 units/g starch each. The grain wort used as acontrol sample was made with commercial enzymepreparations (EP) without phytase: Glucomil L-706 as asource of glucoamylase and BrewZyme BGX as a sourceof xylanase, β-glucanase, and cellulase. The amountof EPs for the biocatalysis of rye wort polymers in thecontrol sample was 10; 0.4; 0.05; and 0.2 unit/g starchfor glucoamylase, xylanase, β-glucanase, and cellulase,respectively.The wort was fermented with the Saccharomycescerevisiae 985T alcohol yeast, which has thermotolerantand osmophilic properties, by the fermentation samples*** State Standard 55302-2012. Enzyme preparations for foodindustry. Method for determination of xylanase activity. Moscow:Standartinform, 2013.**** State Standard 31487-2012. Enzyme preparations. Methods ofphytase enzyme activity determination. Moscow: Standartinform, 2012.method. The fermentation was carried out at 35°Cfor 72 hours. The Guidelines for the Technical andChemical Control of Alcohol Production were followedto determine the biochemical indicators of rye grain, wortconcentration, the number of yeast cells, the percentage ofbudding cells, the content of total and residual reducingcarbohydrates (RS, reducing substances), and ethanolconcentration and yield [3, 5]. The composition andlevel of side metabolites formed during fermentationwas analysed on an Agilent 6850 gas chromatographaccording to State Standard 55792-2013*****.RESULTS AND DISCUSSIONGround rye grain, whose biochemical compositionis given in Table 1, was used as a substrate forfermentation. Rye is known to be a multicomponentsubstrate characterised by a high content ofhemicelluloses and gum substances. The studied ryegrain contained 56.4% of starch, 8.4% of hemicellulose,and 2.2% of cellulose. The presence of non-starchpolysaccharides complicates the process of preparingconcentrated wort that has good rheological propertiesand contains soluble carbohydrates in a form that isaccessible to yeast cells.Therefore, new enzyme preparations ofglucoamylase and hemicellulase action were tried toprepare media ensuring stable yeast generation andalcohol fermentation.To prepare the grain for fermentation, we used theGlucavamorin complex enzyme preparations basedon A. Аwamori recombinant strains and containingxylanase, β-glucanase, and cellulase, along withamylolytic enzymes.The Glucavamorin enzyme preparations hadglucoamylase (GlS), xylanase (XS), β-glucanase(β-GcS), and cellulase (ClS) activities. Of particularinterest was the Glucavamorin-Ply preparation, whichadditionally exhibited a high level of phytase (PhS)activity (4,300 units/g). Glucavamorin-Xyl had ahigher level of xylanase (810 units/g) and glucoamylase(9,640 units/g) activity. The results are in Table 2.The complex enzyme preparations were used assources of glucoamylase and concomitant enzymes.***** State Standard 55792-2013. Brew from food raw material.Gas-chromatographic method for determination of volatile organicadmixtures. Moscow: Standartinform, 2014.Table 1. Biochemical indicators of rye grainIndicator Content, %Proteins 15.7Mono- and disaccharides 1.5Starch 56.4Cellulose 2.2Hemicellulose 8.4Gum substances 2.5β-glucan 0.18Arabinoxylan 0.65Table 2. Comparative activity of enzyme preparations derivedfrom Aspergillus awamori recombinant strainsand commercial preparationsEnzyme preparation Enzyme activity, units/gGlS XS ClS β-GcS PhSGlucavamorin-Xyl 9,640 810 120 285 0Glucavamorin-Ply 7,580 260 130 310 4,300BrewZyme BGX 0 3,600 2,000 500 0Glucomil L-706 8,000 0 0 0 06Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХTherefore, their amount was based on the glucoamylasequantity of 6–10 units/g starch. The level of concomitantenzymes contained in the complex EPs was alsomonitored (Table 3).In the control sample (No. 1) we used Glucomil, forstarch saccharification, and BrewZyme, for the catalytichydrolysis of hemicelluloses. Glucavamorin-Xyl wasused in test samples No. 2, 3, and 4 in an amount of 6.0,8.0, and 10.0 units/g starch, respectively, at the stage ofsaccharification at 58–60°C. Glucavamorin-Ply wasused in test samples No. 5, 6, and 7 in an amount of6.0–10.0 units/g starch.The studies showed that the concentration of solublesolids (SS) in the rye wort prepared with various amountsof the Glucavamorin complex enzymes was 25.8–26.7%,and the content of reducing carbohydrates was 14.9–15.9%.The highest rates of reducing substances were achievedwith the use of Glucavamorin-Ply. Apparently, this wasdue to a higher β-glucanase activity of the preparation.The catalytic action of β-glucanase contributed tothe hydrolysis of grain glucans and the formation ofadditional reducing carbohydrates (Table 3).Further studies showed that the quality of the grainwort made with the complex enzyme preparationsaffected the processes of yeast generation and alcoholfermentation.The comparative studies into the fermentation ofrye wort treated with phytase-free enzyme preparationsrevealed a higher efficiency of Glucavamorin-Xyl,especially in sample No. 4, where it was used at themaximum amount (10 units/g starch). As seen inTable 4, the yield of ethanol reached 65.7 cm3/100 gstarch, exceeding the rate in the control sample(65.3 cm3/100 g starch).Thus, the above confirmed that the synergic actionof the enzymes was determined by their catalytic effecton the grain structural polymers interrelated witheach other. We found that to improve the technologicalparameters of concentrated grain wort, it was necessaryto use a complex of hemicellulase enzymes (xylanase,β-glucanase, and cellulase), along with the traditionallyused amylases. The effective destruction of non-starchpolymers improved the rheological properties of the ryewort, which had a positive effect on the fermentationTable 3. Effects of enzyme complexes derived from recombinant strains on the concentration of rye wort and content of reducingcarbohydratesRye wortsamplesEnzyme complex and amount, unit/g Rye wort indicatorsGlucomil +BrewZyme(control)Glucavamorin-XylGlucavamorin-PlySolublesolids (SS), %Reducingcarbohydrates (RC), %1 GlS – 10.00XS – 0.40ClS – 0.22β-GcS – 0.05– – 26.2 15.52 – GlS – 6.00XS – 0.50ClS – 0.08β-GcS – 0.18– 25.8 14.93 – GlS – 8.00XS – 0.70ClS – 0.10β-GcS – 0.23– 26.1 15.34 – GlS – 10.00XS – 0.81ClS – 0.12β-GcS – 0.31– 26.4 15.75 – – GlS – 6.00XS – 0.21ClS – 0.12β-GcS – 0.24PhS – 3.4126.2 15.06 – – GlS – 8.00XS – 0.31ClS – 0.14β-GcS – 0.32PhS – 4.5226.4 15.57 – – GlS – 10.00XS – 0.37ClS – 0.18β-GcS – 0.40PhS – 5.6326.7 15.97Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–Хprocess. The use of Glucavamorin-Xyl with an increasedconcentration of hemicellulases contributed to a slightrise in ethanol yield compared to the control sample withthe same amount of glucoamylase (10 units/g starch;Table 4).The greatest effect was achieved with Glucavamorin-Ply, which included phytase, apart from a hemicellulasecomplex. For example, the use of Glucavamorin-Plyin samples No. 5, 6, and 7 in amounts of 6.0; 8.0; and10.0 units/g starch, respectively, intensified the processesof yeast generation and alcohol fermentation, whichincreased the ethanol yield to 66.0 cm3/100 g anddecreased the concentration level of residual carbohydratesin the mash to 0.64 g/100 cm3 (Fig. 1, Table 4).Such enzyme complexes contribute to a morerational use of grain components, reduce the wortviscosity, enrich the wort with nutrients, increase thephysiological and fermentation activity of yeast and, asa result, accelerate the processes of yeast generation andalcohol fermentation.The phytolytic action of the preparation had apositive effect on yeast generation and led to a higherconcentration of yeast cells compared to the control (I)and those samples where a phytase-free glucoamylaseEP was used (Table 2, samples No. 2–4). There wasa tendency towards an increase in ethanolyield to65.7–66.0 cm3/100 g starch, with alcohol concentrationof 10.5–10.9% vol., even though the amount ofglucoamylase was reduced by 20–40% (from 10.0 to6.0–8.0 units/g starch) in samples No. 2 and 4.By catalysing the hydrolysis of phytic acid in theraw material, phytase contained in Glucavamorin-Plyappeared to release additional mineral phosphorus,assimilated by alcohol yeast. This improved the growth,activity and productivity of yeast cells.We studied Saccharomyces cerevisiae 985Tyeast cultured under anaerobic conditions on ryemedia with enzyme preparations that differed in thecontent of phytolytic enzymes. The study showed thatGlucavamorin-Ply contributed to increased physiologicalactivity of yeast cells (Fig. 1).As seen in Fig. 1, the presence of phosphorus in themedium led to intensified yeast development, especiallyin the lag phase (first 18–24 hours of growth), acceleratedTable 4. Effects of enzyme complexes derived from recombinant strains on the rye wort fermentationRye wortsamplesEnzyme complex and amount, unit/g Fermentation indicators per 72 hGlucomil +BrewZyme(control)Glucavamorin-XylGlucavamorin-PlyYeast, mln/cm3 Soluble solids,g/100 cm3Alcohol concentration,% vol.Ethanol yield,18 h 44 h cm3/100 g starch1 GlS – 10.00XS – 0.40ClS – 0.22β-GcS – 0.05– – 85 94 0.80 10.2 65.32 – GlS – 6.00XS – 0.50ClS – 0.08β-GcS – 0.18– 68 83 0.74 10.2 65.33 – GlS – 8.00XS – 0.70ClS – 0.10β-GcS – 0.23– 82 100 0.72 10.3 65.54 – GlS – 10.00XS – 0.81ClS – 0.12β-GcS – 0.31– 100 107 0.68 10.5 65.75 – – GlS – 6.00XS – 0.21ClS – 0.12β-GcS – 0.24PhS – 3.41108 135 0.68 10.5 65.76 – – GlS – 8.00XS – 0.31ClS – 0.14β-GcS – 0.32PhS – 4.52115 138 0.66 10.8 65.97 – – GlS – 10.00XS – 0.37ClS – 0.18β-GcS – 0.40PhS – 5.63120 140 0.64 10.9 66.08Polyakov V.A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. Х–ХHigher alcohols Aldehydes Esters Organic acidsSample 1 Sample 3 Sample 6carbohydrate consumption, and increased concentrationof yeast cells (1.4–1.5 times). The fermentationprocess was more complete, with the minimalamount of residual carbohydrates (0.64 g/100 cm3)and the maximum ethanol yield (Table 4).The analysis of the phytolytic effect of theenzyme preparations on the metabolism of yeast cellsshowed that Glucavamorin-Ply contributed to an18–20% decrease in the formation of side metabolitesaccompanying ethanol synthesis, thereby improving thequality of the target product (Fig. 2).We compared the metabolites synthesised during thefermentation of rye wort made with the glucoamylaseenzyme preparations. We found that the phytasecontainingGlucavamorin-Ply (sample No. 4) lowered thecontent of volatile substances by the end of fermentation,compared to the control (sample No. 1) and the samplewith the phytase-free Glucavamorin-Xyl. It did so byreducing the synthesis of major impurities: higheralcohols, aldehydes, and esters (Fig. 3). This improvedthe organoleptic and analytical indicators of the finalproduct, i.e. ethanol.CONCLUSIONThe study showed that the use of the Glucavamorincomplex enzyme preparations, derived from Aspergillusawamori recombinant strains, at the stage of preparingrye wort for fermentation enhanced the efficiencyof yeast generation and alcohol fermentation. Theincreased content of minor hemicellulase enzymes inGlucavamorin-Xyl improved the rheological propertiesof the rye wort and had a positive effect on thefermentation process.The catalytic effect of the phytase-containingGlucavamorin-Ply enzyme preparation improvedthe phosphorus nutrition of yeast. This intensifiedyeast generation, increased the concentration of yeastcells in the rye wort by 30%, reduced the level of sidemetabolites by 18–20%, and enhanced ethanol yield.The study revealed that using a phytase-containingenzyme complex made it possible to reduce the amountof the main enzyme, glucoamylase, from 10.0 to6.0–8.0 units/g starch without causing the keyfermentation indicators to degrade.Thus, the study confirmed that the synergic effectof enzymes with different substrate specificity on thepolymers of grain raw materials enhanced the efficiencyof their conversion when fermenting rye wort.CONFLICT OF INTERESTThe authors declare no conflict of interest.ACKNOWLEDGEMENTSThe study was financed with a federal subsidy withinthe framework of the Programme for Basic ScientificResearch of the State