Foods and Raw Materials Foods and Raw Materials Foods and Raw Materials 2308-4057 2310-9599 48726 10.21603/2308-4057-2022-1-127-136 Research Article Research Article Research Article Enzyme complexes for activating yeast generation and ethanol fermentation Enzyme complexes for activating yeast generation and ethanol fermentation https://orcid.org/0000-0003-3097-0836 Rimareva Liubov V. Rimareva Liubov V. https://orcid.org/0000-0002-1660-2634 Serba Elena M. Serba Elena M. serbae@mail.ru https://orcid.org/0000-0003-0191-5897 Overchenko Marina B. Overchenko Marina B. https://orcid.org/0000-0001-7735-2942 Shelekhova Nataliya V. Shelekhova Nataliya V. https://orcid.org/0000-0002-8416-7478 Ignatova Nadezhda I. Ignatova Nadezhda I. https://orcid.org/0000-0002-8102-0171 Pavlova Anzhelika A. Pavlova Anzhelika A. All-Russian Scientific Research Institute of Food Biotechnology Moscow Россия All-Russian Scientific Research Institute of Food Biotechnology Moscow Russian Federation All-Russian Scientific Research Institute of Food Biotechnology Moscow Россия All-Russian Scientific Research Institute of Food Biotechnology Moscow Russian Federation All-Russian Scientific Research Institute of Food Biotechnology Moscow Россия All-Russian Scientific Research Institute of Food Biotechnology Moscow Russian Federation All-Russian Scientific Research Institute of Food Biotechnology Moscow Россия All-Russian Scientific Research Institute of Food Biotechnology Moscow Russian Federation All-Russian Scientific Research Institute of Food Biotechnology Moscow Россия All-Russian Scientific Research Institute of Food Biotechnology Moscow Russian Federation All-Russian Scientific Research Institute of Food Biotechnology Moscow Россия All-Russian Scientific Research Institute of Food Biotechnology Moscow Russian Federation 30 03 2022 30 03 2022 10 1 127 136 20 04 2021 18 05 2021 https://jfrm.ru/en/issues/7477/7467/

Introduction. Recent studies have shown the benefits of phytolytic enzymes to prepare grain wort in ethanol production. However, there is a lack of data on the effect of phytases and their amount on the conversion of grain polymers, the ionic composition of wort and mash, and the efficiency of yeast generation and ethanol fermentation. Study objects and methods. Wheat and corn wort samples were treated with a complex of hydrolases, including phytases. Capillary electrophoresis determined the ionic composition of wort and mash. Gas chromatography measured the content of volatile metabolites. Results and discussion. The key enzymes were phytases and proteases. They improved the conversion of grain polymers and stimulated the growth and metabolism of yeast cells. Their synergism enriched the wort with assimilable nitrogen, phosphorus, and other valuable minerals. In addition, it intensified the growth of the Saccharomyces cerevisiae yeast, increased the rate of carbohydrate consumption, and reduced the formation of side metabolites 1.7–1.9 times, mainly due to higher and aromatic alcohols. The concentration of phosphates remained practically unchanged during the fermentation of grain wort treated with phytases. However, by the end of fermentation, it was 2.4–5.1 times higher than in the mash samples without phytolytic treatment. Finally, we identified a complex of enzymes and optimal amounts of phytases that have a stimulating effect on ethanol fermentation. Conclusion. Phytases, whether used individually or together with proteases, enriched grain wort with soluble macro- and microelements, improved yeast metabolism, directed ethanol synthesis, and decreased the formation of fermentation by-products.

Introduction. Recent studies have shown the benefits of phytolytic enzymes to prepare grain wort in ethanol production. However, there is a lack of data on the effect of phytases and their amount on the conversion of grain polymers, the ionic composition of wort and mash, and the efficiency of yeast generation and ethanol fermentation. Study objects and methods. Wheat and corn wort samples were treated with a complex of hydrolases, including phytases. Capillary electrophoresis determined the ionic composition of wort and mash. Gas chromatography measured the content of volatile metabolites. Results and discussion. The key enzymes were phytases and proteases. They improved the conversion of grain polymers and stimulated the growth and metabolism of yeast cells. Their synergism enriched the wort with assimilable nitrogen, phosphorus, and other valuable minerals. In addition, it intensified the growth of the Saccharomyces cerevisiae yeast, increased the rate of carbohydrate consumption, and reduced the formation of side metabolites 1.7–1.9 times, mainly due to higher and aromatic alcohols. The concentration of phosphates remained practically unchanged during the fermentation of grain wort treated with phytases. However, by the end of fermentation, it was 2.4–5.1 times higher than in the mash samples without phytolytic treatment. Finally, we identified a complex of enzymes and optimal amounts of phytases that have a stimulating effect on ethanol fermentation. Conclusion. Phytases, whether used individually or together with proteases, enriched grain wort with soluble macro- and microelements, improved yeast metabolism, directed ethanol synthesis, and decreased the formation of fermentation by-products.

 Wort phytase protease mash yeast metabolism ethanol fermentation Wort phytase protease mash yeast metabolism ethanol fermentation The study was subsidized by the Program of Fundamental Scientific Research of the State Academies of Sciences. The study was subsidized by the Program of Fundamental Scientific Research of the State Academies of Sciences.

INTRODUCTIONRecent studies have proven the effectiveness ofmechanical and enzymatic treatment of grains (attemperatures under 100°C) with complex enzymepreparations in ethanol production [1–4]. This “soft”technology for grain wort preparation can significantlyreduce heat and power consumption and increaseprofitability. It is based on controlled biocatalyticconversion of grain polymers (starch, xylans, β-glucans,and proteins) with the formation of easily digestiblecarbohydrates and nitrogenous substances that yeastcells need for normal metabolism [2, 4–7].However, the studies hardly took into accountthe presence of phytic acid and its salts (phytates) ingrains that contain up to 80% of phosphorus in a boundstate [8–10]. The bioavailability of phosphorus can beincreased by using phytolytic enzymes. Grains containenzymes that catalyze the destruction of intracellularpolymers. Normally in a latent state, these enzymesare activated during germination. Studies show that theamount of phytolytic enzymes in grain is insufficient forthe complete release of phosphorus [11, 12]. Therefore,researchers have recently focused on obtainingenzyme preparations – sources of phytases – based onmicroorganisms. Using genetic engineering, they havedeveloped highly productive recombinant strains offungi, yeasts, and bacteria that synthesize phytolyticenzymes [13–15].Copyright © 2022, Rimareva 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, 2022, vol. 10, no. 1E-ISSN 2310-9599ISSN 2308-4057128Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126Phytolytic enzymes of microbial origin have beenwidely studied, namely their use in fodder productionfor the release of phytate phosphorus. The biocatalyticconversion of plant phytates has been shown to improvethe digestibility of nutrients in fodder and stimulatethe growth of farm animals and poultry [16–18]. Muchattention has been paid to the use of phytases in the foodindustry to increase the digestibility and bioavailabilityof food components [19–21]. However, there is a lackof data concerning the catalytic action of microbialphytases on grain polymers in fermentation.Previous studies have shown a positive effect ofphytases on the growth and development of yeast cellscultivated on grain media, including beer productionfrom sorghum [22–24]. Some authors who studied theeffect of phytolytic enzymes reported better rheologicalproperties of rye and triticale wort, as well as improvedfermentation [6, 25, 26].However, there is a lack of research into their effecton the biochemical parameters of grain wort and mash,especially those from wheat and corn [2, 23]. Whatneeds studying is changes in the ionic composition ofgrain wort depending on the substrate specificity ofenzyme systems used for its preparation, as well asthe assimilation of the released phytate phosphorusby the Saccharomyces cerevisiae yeast during ethanolfermentation [1–3, 22–25]. Hardly studied is the effectof phytases on the hydrolytic capacity of enzymes withsubstrate specificity in relation to starch and proteins,on the conversion of these polymers in grain wortpreparation, and on the efficiency of yeast generationand ethanol fermentation.Thus, literature analysis revealed a lack of studiesinto the potential of phytic substances with boundphosphorus for the conversion of grain to ethanol.A number of papers reported that phytates – strongchelating agents – bind not only phosphorus, but alsometal cations (calcium, magnesium, manganese, zinc,iron, etc.) [19, 21, 27–29]. Low bioavailability of macroandmicroelements in grains can have a negative effecton the supply of yeast with minerals and on the catalyticactivity of some metal-dependent enzymes.The S. cerevisiae yeast is a key factor in theefficiency of ethanol fermentation. The yield andquality of ethanol, as well as the process duration,largely depend on the fermentation activity and yeastproductivity. The metabolism of S. cerevisiae issignificantly affected not only by the strains’ geneticcharacteristics, but also by the conditions of theircultivation (nutrient medium with easily assimilatednutrition) [2, 30–32].Metabolism involves all enzymatic reactionsthat occur in the cell to regulate the compositionand synthesis of target and secondary metabolites.Therefore, there is a need for research to select effectiveenzyme systems that contribute to deep destructionof grain polymers. It is especially true of phytolyticenzymes. Recent studies have revealed that phyticacid forms stable complexes with carbohydrates andproteins [33–35]. Apparently, this can reduce thehydrolytic action of carbohydrases and proteases oncarbohydrate and peptide polymers. Phosphate groupsof phytic acid bind to basic amino acids (arginine,histidine, and lysine) and form strong protein-phytatecomplexes.However, the studies of phytate-carbohydratecomplexes have ambiguous results. They show that theinteraction occurs either through direct binding to starchor indirectly, through starch-associated proteins [34–37].Therefore, to ensure a steadily high yield of ethanol, itis important to select optimal parameters for preparinggrain media to produce high-quality wort [1–3].In connection with the above, we can assume thatthe biocatalytic destruction of phytic substances willcontribute to the release of phosphorus and othervaluable microelements. It will also stimulate theconversion of carbohydrate and protein polymers ofgrain through the use of hydrolases with substratespecificity in relation to the main polymers of grain.We aimed to study the effect of hydrolytic enzymeswith proteases and phytases on the efficiency of yeastgeneration and metabolism during the fermentation ofwheat and corn wort.STUDY OBJECTS AND METHODSOur study objects included wheat and corn wortprepared with enzyme preparations that served assources of hydrolases with different substrate specificityand action. They were used for:‒ dextrinization and saccharification of starch α-amylase(EC 3.2.1.1.) and glucoamylase (EC 3.2.1.3.);‒ destruction of xylanase non-starch polysaccharides(EC 3.2.1.8, 3.2.1.32, 3.2.1.37, 3.2.1.72);‒ hydrolysis of protein substances of the proteasecomplex (EC 3.4.11-3.4.15, EC 3.4.21-3.4.24); and‒ hydrolysis of phytase substances (EC 3.1.3.8).Yeast, organic acids, and dietary supplementswere obtained from the Biotechnology Department.Ultraconcentrates of culture liquids were usedas enzyme preparations to produce thermostableα-amylase (Bacillus licheniformis sp., Amilolikheterm),glucoamylase and xylanase (VKM F-4278D,a recombinant strain of Aspergillus awamori,Glucavamorin-Xyl), a protease complex (VKPM F-931,a mutant strain of Aspergillus oryzae, Protoorizin), andphytase (Phytaflow, Novozymes, Denmark) [38–40].Enzyme activity was determined by the existingmethods. A unit of amylolytic activity (AA) was definedas an amount of enzyme that catalyzes the hydrolysisof 1 g of soluble starch to dextrins of various molecularweights under standard conditions (30°C, pH 6.0,10 min). A unit of glucoamylase activity (GA) was anamount of enzyme that is capable of catalyzing starchhydrolysis at 30°C (pH 4.7) and releasing 1 μmol ofglucose per minute. A unit of xylanase activity (XA)was an amount of enzyme that acts on xylan from birchwood and releases 1 μmol of reducing sugars (in glucoseequivalent) per minute under standard conditions129Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126(50°C, pH 5.0). A unit of total proteolytic activity (PA)was an amount of enzyme that brings hemoglobin intoa TCA non-precipitated state equivalent to 1 μmol oftyrosine per minute (30°C, pH 5.3). A unit of phytaseactivity (PhA) was an amount of enzyme that catalyzesthe hydrolysis of sodium phytate to produce 1 μmolof inorganic phosphate per minute under standardconditions (37°С, pH 5.5, 15 min).Grain wort was prepared by the enzymatichydrolyticprocessing of grain (40–90°C, hydromodule1:3). For this, 50 g of grain flour and 150 cm3 of waterwere mixed in 750-cm3 Erlenmeyer flasks and regularlystirred in a PE-4300 water bath (Ekros, Russia).The water-grain mixture was prepared for 2 h(40–90°C) using a thermostable α-amylase (0.6 AAunits/g starch) to liquefy starch. When the mixture wascooled to 60°C (56–60°C, 1 h), EPs with glucoamylase(9.0 GA units/g starch) and xylanase (0.4 XA units/ggrain) activity were used to saccharificate starch andhydrolyze non-starch polysaccharides. They served as acontrol. The experimental samples contained EPs withphytase (1.0–3.0 PhA units/g grain) and proteolyticactivity (0.1 PA units/ g grain), in addition to those withamylolytic, glucoamylase, and xylanase activity.Wort was fermented using a selected race of theSaccharomyces cerevisiae 985-T yeast with thermotolerantand osmophilic properties (34–35°C, 68 h) [41].Methods. The Instructions for the TechnochemicalControl of Alcohol Production were followed todetermine the contents of starch, protein, and nonstarchpolymers of grain, the concentrations of yeastcells and reducing carbohydrates in the grain mash,as well as ethanol concentration and yield [42]. Theconcentration of amine nitrogen in the grain wort wasmeasured by iodometric titration (PharmaceuticalRegulations 1.2.3.0022.15). The ionic composition ofwheat and corn wort, as well as mash, was determinedusing a PrinCE-560 capillary electrophoresis system(Netherlands) equipped with a conductometric detector.The composition and content of volatile metabolitessynthesized by yeast were measured using an HPAgilent 6850 gas chromatograph (USA).Data obtained in triplicate were statisticallyprocessed in Microsoft Excel using the Student’scoefficient with a 0.95 confidence interval.RESULTS AND DISCUSSIONGround wheat and corn were used as a substrateto prepare wort. Table 1 compares the compositions ofthe main polymers contained in the grains. Accordingto their caryopsis composition, wheat and corn, like allgrains, are classified as multicomponent starchy plantraw materials, in which starch is the main polymer thatdetermines ethanol yield [10, 25, 42].Corn had a higher content of starch (65.8%), whilewheat was richer in protein (13.9%). In addition, thegrains under study contained non-starch polysaccharides(hemicelluloses, cellulose), protein substances, andphytates (Table 1). This multicomponent compositionof grain polymers determined the selection of enzymepreparations with a given substrate specificity to preparegrain wort (Table 2). Amilolicheterm (thermostableα-amylase, 330 AA units/g) was used to liquefy anddextrinize starch. Glucavamorin-Xyl (glucoamylase,7700 GA units/g; xylanase, 350 XA units/g) wasused to saccharify starch and hydrolyze non-starchpolysaccharides. Protoorizin (protease, 580 PAunits/g) was used for protein proteolysis and Phytaflow(30 000 PhA units/g) was selected to convert phyticsubstances.Protoorizin contained at least five proteolyticenzymes that differ in action [39]. In previous studies,the authors used neutral protease or Glucavamorin-Xyl,which contained proteinases to catalyze the hydrolysisof proteins to peptides with different molecularweights [24, 25]. In contrast to them, Protoorizincontains a complex of proteinases and peptidases thathydrolyze proteins to low-molecular-weight peptides andfree amino acids [40, 43].Table 1 Biochemical composition of wheat and cornComponents Content, %Wheat CornProteins 13.9 ± 0.5 10.2 ± 0.4Mono- and disaccharides 3.0 ± 0.1 4.1 ± 0.2Starch 57.4 ± 2.6 65.8 ± 3.2Hemicellulose 4.4 ± 0.2 3.0 ± 0.1Cellulose 2.9 ± 0.1 3.3 ± 0.1Phytates 1.30 ± 0.05 2.30 ± 0.09Values are expressed as means ± SDTable 2 Enzyme preparations by activity of major and minor enzymesEnzyme preparation Enzyme activity, units/gАA GA XA PA PhAAmilolicheterm 330 ± 8 0 25 ± 5 7.0 ± 0.1 0Glucavamorin-Xyl 110 ± 4 7700 ± 240 350 ± 13 6.0 ± 0.1 0Protoorizin 0 0 0 580 ± 26 0Phytaflow 0 0 0 0 30000 ± 1200AA – Amylolytic activity; GA – Glucoamylase activity; XA – Xylanase activity; PA – Proteolytic activity; PhA – Phytase activity130Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126Enzyme preparations were used at the stage ofgrain wort preparation in the amounts specified above(see “Study objects and methods”). The control wortcontained Amilolicheterm and Glucavamorin-Xylpreparations, the sources of carbohydrases that catalyzethe hydrolysis of polysaccharides. Their proteolyticenzymes had practically no effect on the degree ofprotein conversion due to the low level of their activity.The concentration of proteases in the control was under0.02 PA units/g grain, which was 5 times lower thanin the experimental samples. Therefore, Protoorizin(0.1 PA unit/g grain) and Phytaflow (1.0–3.0 PhA units/ggrain) were additionally added to the experimentalsamples to ensure an efficient conversion of grainpolymers and activate yeast generation and ethanolfermentation.We found that the ionic composition of grainwort changed depending on the type of grain and thesubstrate specificity of the enzymes used to hydrolyzegrain polymers. In the wheat wort treated with acomplex of amylolytic and xylanase enzymes (AA +GA + XA), the phosphate content was 2.7 times higherthan in the corn wort (Fig. 1). The concentrations ofother cations, such as potassium, calcium, sodium, andmagnesium, were also different. The wheat wort wasricher in potassium, while the corn wort had a highercontent of calcium and magnesium (Table 3).Phytolytic enzymes had a more significant effecton the release of phytate phosphorus. Increasing theiramount to 1.5–2.0 PhA units/g grain led to higherconcentrations of released phosphates due to thecatalytic destruction of phytic substances. We found thatthe content of phosphorus ions increased 1.6 times inthe wheat wort and 3.8 times in the corn wort. A furtherincrease in the phytase concentration to 3.0 PhA units/ggrain had practically no effect on the content of solublephosphates in the wort (Fig. 1).The catalytic synergism of phytases and proteasesstimulated the release of phosphorus ions. Their contentincreased 1.8 times in the wheat wort and 4.9 times inthe corn wort (Table 3).In addition to the release of phosphorus ions,phytases increased the concentration of cations in thewheat and corn wort samples: potassium by 12 and 13%,AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PhA – phytase activityFigure 1 Effects of phytolytic enzymes on phosphate release in wheat (1) and corn (2) wortTable 3 Effects of hydrolytic enzymes on amine nitrogen and basic ions in wheat and corn wortEnzyme composition Type ofwortAmine nitrogen,mg%Ion content, mg/dm3Phosphorus Potassium Calcium Sodium MagnesiumControl (АA+GA+XA) Wheat 53.0 ± 2.5 787.1 ± 28.2 811.4 ± 33.4 12.1 ± 0.2 16.5 ± 0.4 131.1 ± 3.7Corn 40.3 ± 1.2 290.0 ± 13.2 780.3 ± 24.7 16.1 ± 0.4 16.1 ± 0.3 179.6 ± 5.4Control+0.1 PA units Wheat 85.7 ± 3.2 960.5 ± 29.4 825.1 ± 34.5 12.9 ± 0.3 16.9 ± 0.5 141.9 ± 4.2Corn 70.4 ± 2.6 330.4 ± 13.3 834.6 ± 28.8 18.0 ± 0.4 19.0 ± 0.7 199.3 ± 8.2Control+1.5 PhA units Wheat 53.5 ± 1.8 1242.1 ± 44.2 907.0 ± 34.9 13.9 ± 0.5 23.0 ± 0.8 151.8 ± 5.8Corn 41.3 ± 1.6 1109.0 ± 38.4 878.0 ± 26.3 21.8 ± 0.6 20.9 ± 0.7 218.5 ± 8.9Control+2.0 PhA units Wheat 53.9 ± 2.1 1232.3 ± 51.2 909.3 ± 31.4 14.1 ± 0.3 23.9 ± 0.8 152.1 ± 6.7Corn 41.1 ± 1.6 1112.3 ± 36.7 881.0 ± 28.4 22.9 ± 0.7 21.7 ± 0.7 222.3 ± 6.4Control +1.5 PhAunits+0.1 PA unitsWheat 93.9 ± 3.8 1379.6 ± 51.4 918.7 ± 34.6 14.0 ± 0.4 24.2 ± 0.8 152.9 ± 4.8Corn 76.5 ± 2.6 1423.6 ± 56.7 888.9 ± 42.5 23.4 ± 0.8 23.9 ± 0.7 225.6 ± 7.4AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity120200400600800100012001400АС+ГлС+КС(К)(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФСmg/dm32340246810121416180 8 16 g/100 cm3 12 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm3123 40100200300400500600mg/dm3120200400600800100012001400АС+ГлС+КС(К)(mg/dm312 3 40300600900120015001800АС+ГлС+КС (К) К+mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 mg/dm31600700mg/dm3AA+GA+XAControlControl+1.0PhA unitsControl+1.5PhA unitsControl+2.0PhA unitsControl+2.5PhA unitsControl+3.0PhA units131Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126calcium by 15 and 35%, sodium by 39 and 30%, andmagnesium by 16 and 22%, respectively (Table 3).We found that the content of cations depended onthe substrate specificity of the enzymes involved in thebioconversion of grain polymers. The combined actionof proteolytic and phytolytic enzymes contributed notonly to the accumulation of phosphates and minerals,but also to a significant increase in amine nitrogen. Itsconcentration was 1.8 times as high in the wheat wortand 1.9 times as high in the corn wort, compared to thecontrol.Grain wort enriched with phosphates and mineralswas used as a nutrient medium to cultivate the ethanolyeast Saccharomyces cerevisiae (race 985-T). Westudied the process of yeast generation on grain wortprepared with hydrolytic enzymes differing in substratespecificity and found that the presence of phytase in theenzyme complex had a positive effect on the growth andreproduction of yeast cells (Fig. 2).As we know, mineral and nitrogenous substancesare essential for the biochemical reactions of yeastcells [24, 25, 43]. Magnesium and calcium ionsactivate the catalytic ability of almost all intracellularmetalloenzymes, including phosphofructokinase,which is involved in glucose metabolism. Potassium,sodium, and calcium ions have a regulatory effect onthe metabolism of yeast cells. Potassium also plays anessential role in oxidative phosphorylation and glycolysisprocesses [43]. It activates yeast aldolase and, togetherwith magnesium ions, catalyzes pyruvate carboxylase.Like nitrogen, potassium can also affect yeast lipidmetabolism.We analyzed the processes of yeast generation andcarbohydrate consumption and found that the presenceof phosphorus and other minerals in the mediumintensified the growth of S. cerevisiae yeast. In the lagphase (first 18–24 h of growth), the concentration ofyeast cells increased 1.2–1.3 times, alongside a risingrate of carbohydrate consumption. A 1.3-fold increasein phytolytic enzymes (up to 2.0 PhA units/g grain)during grain wort preparation had no significant effecton the yeast growth and the assimilation of reducingcarbohydrates (Fig. 2).The catalytic synergism of phytase and proteasesignificantly enriched the wheat wort with mineral andnitrogenous nutrition and resulted in the most activegrowth of yeast cells (2-fold) and a more intensiveconsumption of carbohydrates (Fig. 2, curves 4 and 8).A similar pattern was observed with the corn wort.Thus, our results confirmed that the nutrientmedium has a significant effect on yeast generationand physiological activity, particularly the presence ofsoluble macro- and microelements in addition to easilydigestible carbohydrates and nitrogenous substances.At the next stage, we analyzed changes in theconcentration of phosphates in the mash against theamount of phytolytic enzymes used in the preparationof wheat and corn wort (Figs. 3a and 3b). We found thatthe content of phosphorus ions significantly decreasedduring the fermentation of the control wort, which wasnot treated with phytases. In the logarithmic phaseof yeast cell growth (on day 1), it declined 1.9 timesin the wheat wort and 2.3 times in the corn wort. Thewort samples treated with protease in addition tocarbohydrases showed the same trend – a sharp decreasein phosphates after 24 h of fermentation, followed by aslight rise.In the experimental samples treated with phytolyticenzymes, the concentration of phosphates hardlychanged during the fermentation of grain wort. Thismight be due to the ongoing biocatalytic hydrolysis ofphytic substances and an extra release of phosphorus.By the end of fermentation, the content of phosphatesslightly increased, which might be associated withautolytic processes in the cell (Fig. 3).We found an increase of 2.4–2.6 times and 4.3–5.1 times in the residual content of phosphates in thewheat mash and the corn mash, respectively. Thisindicated that phytolytic enzymes not only enrichedthe grain wort with assimilable phosphorus and othervaluable minerals, but also improved the value of grainstillage, a waste product of ethanol production that isused in the diet of farm animals.Apart from the main fermentation products(ethanol and carbon dioxide), yeast cells synthesizeaccompanying metabolites: secondary (organicacids, aldehydes, and esters) and by-products (higheralcohols) [43].We studied the metabolism of S. cerevisiae 985-Tyeast during its cultivation on wheat and corn worttreated with phytolytic enzymes and found a decreaseof 18–21 and 20–23%, respectively, in total metaboliteformation that accompanies ethanol synthesis (Fig. 4).1 and 5 – treated with amylases and xylanase (Control);2 and 6 – treated with phytases (Control + 1.5 PhA units/g grain);3 and 7 – treated with phytases (Control + 2.0 PhA units/g grain);4 and 8 – treated with phytases and proteases(Control + 1.5 PhA units + 0.1 PA units)PA – proteolytic activity; PhA – phytase activityFigure 2 Changes in carbohydrate consumption (1–8)and Saccharomyces cerevisiae 985-T yeast growth (5–8)during wheat wort fermentation for 68 h2.5ФС(К)+3.0ед.ФС123456780204060801001201400246810121416180 8 16 24 32 40 72g/100 cm3 mln./cm3hед.ФС+0,1ПС12 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПСmg/dm3(К)+1,5ед.ФС+0,1ед.ПС(К)+1,5ФС+0,1ед.ПС)+1,5 ед.ФС+0,1ед.ПС120200400600800100012001400АС+ГлС+КС(К)(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФСmg/dm312340246810121416180 8 16 24 32 g/100 cm3 12 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm31600mg/dm3120200400600800100012001400АС+ГлС+КС(К)(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФСmg/dm3123456780204060801001201400246810121416180 8 16 24 32 40 72g/100 cm3 mln./cm3h12 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПСmg/dm31 26008001000mg/dm3123456780204060801001201408 16 24 32 40 72cm3 mln./cm3h3 4ГлС+КСК)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПС(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФС123456780204060801001201400246810121416180 8 16 24 32 40 72g/100 cm3 mln./cm3hК+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПС12 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПСmg/dm3К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПС0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСК)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС68132Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126These results indirectly confirmed the improvement ofphosphorus metabolism in the cell.The use of proteases reduced the synthesis of sideand secondary metabolites by more than 30%. Thegreatest effect was caused by the combined catalyticaction of phytases and proteases during yeast generationon the media enriched with assimilable phosphatesand amine nitrogen. In particular, the total contentof accompanying volatiles in the wheat and corn mashdecreased by 44 and 42%, respectively, compared to thecontrol.Higher alcohols (mostly isoamylol, isobutanol,and 1-propanol) dominated among side and secondarymetabolites synthesized by S. cerevisiae 985-T duringthe wort fermentation. In addition, the mash containedaromatic alcohols (β-phenylethyl, p-hydroxyphenylethyl)and a small amount of secondary by-products, primarilyaldehydes and esters (Fig. 5).Our study showed that treating the nutrient mediawith phytolytic and proteolytic enzymes decreasedthe synthesis of higher and aromatic alcohols (1.9 and1.4 times, respectively) (Fig. 5). Exposure to phytasesreduced the amount of higher alcohols and proteases(1.3–1.4 and 1.5–1.6 times, respectively). In addition, weobserved a slight decrease in aldehydes and esters.Thus, our study showed that providing a yeast cellwith a balanced nitrogenous and mineral nutritioncreated conditions for synthesizing ethanol with areduced amount of fermentation by-products. Byregulating yeast metabolism we can improve the qualityand sensory properties of the target product.According to our results, the biochemical changesin the grain wort affected the yield of ethanol, themain fermentation product. The destruction of phyticAA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activityFigure 4 Total side metabolites during the cultivation of Saccharomyces cerevisiae 985-T yeast on wheat (1) and corn (2) wort120200400600800100012001400АС+ГлС+КС(К)(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФСmg/dm312340246810121416180 8 16 24 g/100 cm3 12 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm312 3 4100200300400500600700mg/dm312 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПС1 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm3123 40100200300400500600АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПСmg/dm3AA+GA+XAControlControl+0.1 PAunitsControl+1.5 PhAunitsControl+1.5PhA units+0.1PA unitsControl+2.0 PhAunitsAA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activityFigure 3 Changes in the concentration of phosphates (mg/dm3) after 0 (1), 24 (2), 48 (3), and 68 h (4) during the fermentationof wheat (a) and corn (b) wort depending on the amount of enzymes0200АС+ГлС+КС(К)(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФС1340240 8 16 24 12 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm3123 40100200300400500600АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПСmg/dm3(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФС123456780204060801001201400246810121416180 8 16 24 32 40 72g/100 cm3 mln./cm3hПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПС12 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПСmg/dm3(К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПС(К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПС(К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПС12020040060080010001200АС+ГлС+КС(К)(К)+1.0ед.ФСmg/dm312 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm312200300400500600mg/dm3120200400600800100012001400АС+ГлС+КС(К)(К)+1.0ед.ФС(К)+1.5ед.ФС(К)+2.0ед.ФС(К)+2.5ед.ФС(К)+3.0ед.ФСmg/dm3123456780204060801001201400246810121416180 8 16 24 32 40 72g/100 cm3 mln./cm3h12 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 4030060090012001500АС+ГлС+КС(К)К+0,1 ед.ПС К+1,5 ед.ФС К+1,5ед.ФС+0,1ед.ПСmg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm31400500600mg/dm3(а)(b)AA+GA+XAControlControl+0.1 PAunitsControl+1.5 PhAunitsControl+1.5 PhAunits+0.1 PA unitsAA+GA+XAControlControl+0.1 PAunitsControl+1.5 PhAunitsControl+1.5PhA units+0.1PA units133Rimareva L.V. et al. Foods and Raw Materials, 2022, vol. 10, no. 1, pp. 117–126substances enriched the wort with mineral nutritionand activated the physiological activity of yeast cells.It also led to a slight increase in ethanol yield duringthe fermentation of the wheat and corn wort (by 2.3–2.4 and 1.5–1.7%, respectively) (Table 4). The ethanolconcentration in the mash varied due to a higher starchcontent in corn (Tables 1 and 4).The metabolic processes in yeast cells improvedon the media treated with amylolytic, xylanase, andproteolytic enzymes, as well as a complex of enzymeswith phytases. This improvement contributed to acomplete fermentation of carbohydrates and an increasein ethanol yield, with a simultaneous decrease inassociated metabolites (Table 4, Figs. 4 and 5).The highest concentration of ethanol generated inthe wort treated with a full complex of enzymes was11.15% in the wheat mash and 12.74% in the corn mash.The ethanol yield from the fermentation of wheat andcorn wort increased by 3.2 and 2.6%, respectively, whentreated with proteases and by 4.3 and 3.2%, respectively,when treated with proteases and phytase (Table 4).The rise in ethanol synthesis in the experimentalsamples was probably associated with an improvedconversion of polymers of grain wort and its enrichmentwith assimilable amine nitrogen. Another reason wasa release of macro- and microelements that was vitalfor yeast cells. These findings were consistent with anumber of previous studies [2, 7, 25–27].AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activityFigure 5 Concentrations of higher alcohols (1), aromatic alcohols (2), esters (3), and aldehydes (4) during yeast cultivationon wheat (a) and corn (b) wort treated with various enzymatic complexesTable 4 Ethanol yield during fermentation of wheat and corn wort treated with various enzymatic complexesEnzyme composition Ethanol concentration in mash, % vol. Ethanol yield, cm3/100 g starchWheat Corn Wheat CornАA+GA+XA (Control) 9.80 ± 0.04 11.81 ± 0.05 65.6 ± 0.3 64.8 ± 0.4Control + 0.1 PA units 10.87 ± 0.06 12.57 ± 0.07 67.7 ± 0.2 66.5 ± 0.3Control + 1.5 PhA units 10.65 ± 0.05 12.20 ± 0.06 67.1 ± 0.1 65.8 ± 0.3Control + 2.0 PhA units 10.72 ± 0.05 12.21 ± 0.07 67.2 ± 0.3 65.9 ± 0.3Control + 1.5 PhA units + 0.1 PA 11.15 ± 0.06 12.74 ± 0.07 68.4 ± 0.2 66.9 ± 0.2AA – amylolytic activity; GA – glucoamylase activity; XA – xylanase activity; PA – proteolytic activity; PhA – phytase activity0200АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПС12 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm3123 40100200300400500600АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПСmg/dm3 (а)(b)AA+GA+XAControlControl+0.1 PAunitsControl+1.5 PhAunitsControl+1.5PhA units+0.1PA unitsControl+2.0 PhAunits0200АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПС12 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (К)+1,5ед.ФС+0,1ед.ПСmg/dm3123 40100200300400500600АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 ед.ФС+0,1ед.ПСmg/dm312 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.mg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm3123 40100200300400500600АС+ГлС+КС (К) (К)+0,1 ед.ПС mg/dm312 3 40300600900120015001800АС+ГлС+КС (К) К+0,1 ед.ПС К+1,5 ед.ФС К+1,5 ед.ФС+ед.ПСmg/dm31 202004006008001000АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС ед.ФС+mg/dm312 3 40100200300400500600700АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.ФС (ед.ФС+mg/dm3123 40100200300400500600АС+ГлС+КС (К) (К)+0,1 ед.ПС (К)+1,5 ед.ФС (К)+2,0 ед.Ф(СК )+1,5 mg/dm3AA+GA+XAControlControl+0.1 PAunitsControl+1.5 PhAunitsControl+1.5PhA units+0.1PA unitsControl+2.0 PhAunitsCONCLUSIONWe found almost no changes in the concentration ofphosphates during the fermentation of phytases-treatedwort, with a slight increase by the end of the process.It was probably caused by the continuing biocatalytichydrolysis of phytic substances and the release ofphosphorus, as well as autolytic processes in the cell.The control samples (without phytolytic enzymes) hada significantly lower residual content of phosphates inthe wheat and corn mash (2.4–2.6 and 4.3–5.1 times,respectively).Our results confirmed that nitrogen and phosphorusnutrition played a regulatory role in the generation andmetabolism of ethanol yeast. The catalytic action ofphytases and proteases ensured the accumulation ofeasily assimilable phosphates, minerals, and aminoacids in the wort. Also, it intensified the growth ofyeast cells and increased the rate of carbohydrateconsumption. Finally, it decreased the formation of sidemetabolites 1.7–1.9 times, mainly due to higher andaromatic alcohols. At the same time, the Saccharomycescerevisiae 985-T yeast synthesized ethanol, whose yieldincreased by 1.5–4.3%, depending on the type of grainand enzyme complex. The greatest effect was achievedby a full complex of enzymes (carbohydrase, protease,and phytase).CONTRIBUTIONThe authors were equally involved in writing themanuscript and are equally responsible for plagiarism.CONFLICT OF INTERESTThe authors declare that there is no conflict ofinterest.

Zueva NV, Agafonov GV, Korchagina MV, Dolgov AN. Effect of enzyme preparations on the main parameters of products in the development of processing technology of concentrated wort on ethanol. Proceedings of the Voronezh State University of Engineering Technologies. 2017;79(2):191-197. (In Russ.). https://doi.org/10.20914/2310-1202-2017-2-191-197. Zueva NV, Agafonov GV, Korchagina MV, Dolgov AN. Effect of enzyme preparations on the main parameters of products in the development of processing technology of concentrated wort on ethanol. Proceedings of the Voronezh State University of Engineering Technologies. 2017;79(2):191-197. (In Russ.). https://doi.org/10.20914/2310-1202-2017-2-191-197. Serba EM, Abramova IM, Rimareva LV, Overchenko MB, Ignatova NI, Grunin EA. Influence of enzymatic preparations on technological parameters of grain wort and quality of alcohol. Beer and beverages. 2018;(1):50-54. (In Russ.). https://doi.org/10.24411/2072-9650-2018-00002. Serba EM, Abramova IM, Rimareva LV, Overchenko MB, Ignatova NI, Grunin EA. Influence of enzymatic preparations on technological parameters of grain wort and quality of alcohol. Beer and beverages. 2018;(1):50-54. (In Russ.). https://doi.org/10.24411/2072-9650-2018-00002. Zueva NV, Agafonov GV, Korchagina MV, Dolgov AN, Chusova AE. Selection of enzyme preparations and temperature-time regimes of water-heat and enzymatic treatment in the development of complex technology of processing of grain raw materials. Proceedings of the Voronezh State University of Engineering Technologies. 2019;81(1):112-119. (In Russ.). https://doi.org/10.20914/2310-1202-2019-1-112-119. Zueva NV, Agafonov GV, Korchagina MV, Dolgov AN, Chusova AE. Selection of enzyme preparations and temperature-time regimes of water-heat and enzymatic treatment in the development of complex technology of processing of grain raw materials. Proceedings of the Voronezh State University of Engineering Technologies. 2019;81(1):112-119. (In Russ.). https://doi.org/10.20914/2310-1202-2019-1-112-119. Caspeta L, Coronel J, Montes de Oca A, Abarca E, González L, Martínez A. Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae. Biotechnology and Bioengineering. 2019;116(10):2587-2597. https://doi.org/10.1002/bit.27103. Caspeta L, Coronel J, Montes de Oca A, Abarca E, González L, Martínez A. Engineering high-gravity fermentations for ethanol production at elevated temperature with Saccharomyces cerevisiae. Biotechnology and Bioengineering. 2019;116(10):2587-2597. https://doi.org/10.1002/bit.27103. Fang L, Wang T, Lamsal B. Use of surfactant and enzymes in dry-grind corn ethanol fermentation improves yield of ethanol and distillers corn oil. Industrial Crops and Products. 2018;111:329-335. https://doi.org/10.1016/j.indcrop.2017.10.031. Fang L, Wang T, Lamsal B. Use of surfactant and enzymes in dry-grind corn ethanol fermentation improves yield of ethanol and distillers corn oil. Industrial Crops and Products. 2018;111:329-335. https://doi.org/10.1016/j.indcrop.2017.10.031. Franco JH, Minteer SD, de Andrade AR. Product analysis of operating an ethanol/O2 biofuel cell shows the synergy between enzymes within an enzymatic cascade. Journal of the Electrochemical Society. 2018;165(9):H575-H579. https://doi.org/10.1149/2.0931809jes. Franco JH, Minteer SD, de Andrade AR. Product analysis of operating an ethanol/O2 biofuel cell shows the synergy between enzymes within an enzymatic cascade. Journal of the Electrochemical Society. 2018;165(9):H575-H579. https://doi.org/10.1149/2.0931809jes. Guillaume A, Thorigné A, Carré Y, Vinh J, Levavasseur L. Contribution of proteases and cellulases produced by solid-state fermentation to the improvement of corn ethanol production. Bioresources and Bioprocessing. 2019;6(1). https://doi.org/10.1186/s40643-019-0241-0. Guillaume A, Thorigné A, Carré Y, Vinh J, Levavasseur L. Contribution of proteases and cellulases produced by solid-state fermentation to the improvement of corn ethanol production. Bioresources and Bioprocessing. 2019;6(1). https://doi.org/10.1186/s40643-019-0241-0. Benesova K, Belakova S, Mikulikova R, Svoboda Z. Survey of the analytical methods for the phytic acid determination. Kvasny Prumysl. 2013;59(5):127-133. https://doi.org/10.18832/kp2013013. Benesova K, Belakova S, Mikulikova R, Svoboda Z. Survey of the analytical methods for the phytic acid determination. Kvasny Prumysl. 2013;59(5):127-133. https://doi.org/10.18832/kp2013013. Mikulski D, Klosowski G. Phytic acid concentration in selected raw materials and analysis of its hydrolysis rate with the use of microbial phytases during the mashing process. Journal of the Institute of Brewing. 2015;121(2):213-218. https://doi.org/10.1002/jib.221. Mikulski D, Klosowski G. Phytic acid concentration in selected raw materials and analysis of its hydrolysis rate with the use of microbial phytases during the mashing process. Journal of the Institute of Brewing. 2015;121(2):213-218. https://doi.org/10.1002/jib.221. Serba EM, Overchenko MB, Ignatova NI, Belokopytova EN, Rimareva LV. Composition of concentrated grain wort, prepared from various types of grain raw materials. Aktualʹnye voprosy industrii napitkov [Current issues of the beverage industry]. 2018;(2):166-170. (In Russ.). Serba EM, Overchenko MB, Ignatova NI, Belokopytova EN, Rimareva LV. Composition of concentrated grain wort, prepared from various types of grain raw materials. Aktualʹnye voprosy industrii napitkov [Current issues of the beverage industry]. 2018;(2):166-170. (In Russ.). Zhulʹkov AYu, Vitol IS, Karpilenko GP. Rolʹ zernovoy fitazy pri proizvodstve i sbrazhivanii rzhanogo susla. Chastʹ i. Issledovanie fitaznogo kompleksa rzhi [The role of grain phytase in the production and fermentation of rye wort. Part I. A study of the phytase complex of rye]. Storage and Processing of Farm Products. 2009;(5):50-55. (In Russ.). Zhulʹkov AYu, Vitol IS, Karpilenko GP. Rolʹ zernovoy fitazy pri proizvodstve i sbrazhivanii rzhanogo susla. Chastʹ i. Issledovanie fitaznogo kompleksa rzhi [The role of grain phytase in the production and fermentation of rye wort. Part I. A study of the phytase complex of rye]. Storage and Processing of Farm Products. 2009;(5):50-55. (In Russ.). Sapna, Singh B. Phytase production by Aspergillus oryzae in solid-state fermentation and its applicability in dephytinization of wheat ban. Applied Biochemistry and Biotechnology. 2014;173(7):1885-1895. https://doi.org/10.1007/s12010-014-0974-3. Sapna, Singh B. Phytase production by Aspergillus oryzae in solid-state fermentation and its applicability in dephytinization of wheat ban. Applied Biochemistry and Biotechnology. 2014;173(7):1885-1895. https://doi.org/10.1007/s12010-014-0974-3. Hesampour A, Ranaei O, Malboob MA, Harati J, Mohandes N. Comparison of biochemical properties of recombinant phytase expression in the favorable methylotrophic platforms of Pichia pastoris and Hansenula polymorpha. Progress in Biological Sciences. 2014;4(1):97-111. https://doi.org/10.22059/pbs.2014.50309. Hesampour A, Ranaei O, Malboob MA, Harati J, Mohandes N. Comparison of biochemical properties of recombinant phytase expression in the favorable methylotrophic platforms of Pichia pastoris and Hansenula polymorpha. Progress in Biological Sciences. 2014;4(1):97-111. https://doi.org/10.22059/pbs.2014.50309. Raman S, Abdullah N, Boo LJ, Azizi S, Mohamad R. Improvement of phytase biosynthesis by new bacterial isolate, Pediococcuspentosaceus C4/1A via continuous cultivation. Journal of Microbiology, Biotechnology and Food Sciences. 2019;8(5):1118-1124. https://doi.org/10.15414/jmbfs.2019.8.5.1118-1124. Raman S, Abdullah N, Boo LJ, Azizi S, Mohamad R. Improvement of phytase biosynthesis by new bacterial isolate, Pediococcuspentosaceus C4/1A via continuous cultivation. Journal of Microbiology, Biotechnology and Food Sciences. 2019;8(5):1118-1124. https://doi.org/10.15414/jmbfs.2019.8.5.1118-1124. Tarutina MG, Kashirskaya MD, Lazareva MN, Lapteva AR, Dobrynin VYu, Gordeeva TL, et al. Comparative characteristics of phytases from Citrobacter freundii and Yersinia intermedia expressed in Ogataea polymorpha and Pichia pastoris methylotrophic yeasts. Biotechnology in Russia. 2019;35(6):51-56. (In Russ.). https://doi.org/10.21519/0234-2758-2019-35-6-51-56. Tarutina MG, Kashirskaya MD, Lazareva MN, Lapteva AR, Dobrynin VYu, Gordeeva TL, et al. Comparative characteristics of phytases from Citrobacter freundii and Yersinia intermedia expressed in Ogataea polymorpha and Pichia pastoris methylotrophic yeasts. Biotechnology in Russia. 2019;35(6):51-56. (In Russ.). https://doi.org/10.21519/0234-2758-2019-35-6-51-56. Kulova FM. Effect of the enzymatic preparation fitasa in diets with different mineral content on zootechnical calves’ indexes. Proceedings of Gorsky State Agrarian University. 2016;53(1):71-76. (In Russ.). Kulova FM. Effect of the enzymatic preparation fitasa in diets with different mineral content on zootechnical calves’ indexes. Proceedings of Gorsky State Agrarian University. 2016;53(1):71-76. (In Russ.). Lenkova TN, Egorova TA, Menʹshenin IA, Sysoeva IG. Otechestvennaya fitaza v kombikormakh dlya kur-nesushek [Domestic phytase in compound feed for laying hens]. Poultry and Poultry Processing. 2016;(1):37-40. (In Russ.). Lenkova TN, Egorova TA, Menʹshenin IA, Sysoeva IG. Otechestvennaya fitaza v kombikormakh dlya kur-nesushek [Domestic phytase in compound feed for laying hens]. Poultry and Poultry Processing. 2016;(1):37-40. (In Russ.). Kryukov VS, Glebova IV, Zinoviev SV. Reevaluation of phytase action mechanism in animal nutrition. Biochemistry (Moscow). 2021;86:S152-S165. https://doi.org/10.1134/S000629792114011X. Kryukov VS, Glebova IV, Zinoviev SV. Reevaluation of phytase action mechanism in animal nutrition. Biochemistry (Moscow). 2021;86:S152-S165. https://doi.org/10.1134/S000629792114011X. Zyba SJ, Wegmüller R, Woodhouse LR, Ceesay K, Prentice AM, Brown KH, et al. Effect of exogenous phytase added to small-quantity lipid-based nutrient supplements (SQ-LNS) on the fractional and total absorption of zinc from a millet-based porridge consumed with SQ-LNS in young Gambian children: a randomized controlled trial. American Journal of Clinical Nutrition. 2019;110(6):1465-1475. https://doi.org/10.1093/ajcn/nqz205. Zyba SJ, Wegmüller R, Woodhouse LR, Ceesay K, Prentice AM, Brown KH, et al. Effect of exogenous phytase added to small-quantity lipid-based nutrient supplements (SQ-LNS) on the fractional and total absorption of zinc from a millet-based porridge consumed with SQ-LNS in young Gambian children: a randomized controlled trial. American Journal of Clinical Nutrition. 2019;110(6):1465-1475. https://doi.org/10.1093/ajcn/nqz205. Castro-Alba V, Lazarte CE, Bergenståhl B, Granfeldt Y. Phytate, iron, zinc, and calcium content of common Bolivian foods and their estimated mineral bioavailability. Food Science and Nutrition. 2019;7(9):2854-2865. https://doi.org/10.1002/fsn3.1127. Castro-Alba V, Lazarte CE, Bergenståhl B, Granfeldt Y. Phytate, iron, zinc, and calcium content of common Bolivian foods and their estimated mineral bioavailability. Food Science and Nutrition. 2019;7(9):2854-2865. https://doi.org/10.1002/fsn3.1127. Bangar P, Glahn RP, Liu Y, Arganosa GC, Whiting S, Warkentin TD. Iron bioavailability in field pea seeds: Correlations with iron, phytate, and carotenoids. Crop Science. 2017;57(2):891-902. https://doi.org/10.2135/cropsci2016.08.0682. Bangar P, Glahn RP, Liu Y, Arganosa GC, Whiting S, Warkentin TD. Iron bioavailability in field pea seeds: Correlations with iron, phytate, and carotenoids. Crop Science. 2017;57(2):891-902. https://doi.org/10.2135/cropsci2016.08.0682. Kruger J, Oelofse A, Taylor J, Taylor JRN. Potential for improvement in yeast nutrition in raw whole grain sorghum and maize lager brewing and bioethanol production through grain genetic modification and phytase treatment. Journal of the Institute of Brewing. 2012;118(1):70-75. https://doi.org/10.1002/jib.86. Kruger J, Oelofse A, Taylor J, Taylor JRN. Potential for improvement in yeast nutrition in raw whole grain sorghum and maize lager brewing and bioethanol production through grain genetic modification and phytase treatment. Journal of the Institute of Brewing. 2012;118(1):70-75. https://doi.org/10.1002/jib.86. Rimareva LV, Overchenko MB, Ignatova NI, Krivova AYu, Serba EM. Generation of alcoholic yeast on media prepared from cereals with different phytate content. Food Industry. 2019;(4):83-85. https://doi.org/10.24411/0235-2486-2019-10042. Rimareva LV, Overchenko MB, Ignatova NI, Krivova AYu, Serba EM. Generation of alcoholic yeast on media prepared from cereals with different phytate content. Food Industry. 2019;(4):83-85. https://doi.org/10.24411/0235-2486-2019-10042. Kovaleva TS, Agafonov GV, Yakovlev AN, Yakovleva SF. Effect of protease and phytase on the physiological state of alcoholic yeast in cultivation. Proceedings of the Voronezh State University of Engineering Technologies. 2019;81(4):98-102. (In Russ.). https://doi.org/10.20914/2310-1202-2019-4-98-102. Kovaleva TS, Agafonov GV, Yakovlev AN, Yakovleva SF. Effect of protease and phytase on the physiological state of alcoholic yeast in cultivation. Proceedings of the Voronezh State University of Engineering Technologies. 2019;81(4):98-102. (In Russ.). https://doi.org/10.20914/2310-1202-2019-4-98-102. Polyakov VA, Serba EM, Overchenko MB, Ignatova NI, Rimareva LV. The effect of a complex phytase-containing enzyme preparation on the process of rye wort fermentation. Foods and Raw Materials. 2019;7(2):221-228. http://doi.org/10.21603/2308-4057-2019-2-221-228. Polyakov VA, Serba EM, Overchenko MB, Ignatova NI, Rimareva LV. The effect of a complex phytase-containing enzyme preparation on the process of rye wort fermentation. Foods and Raw Materials. 2019;7(2):221-228. http://doi.org/10.21603/2308-4057-2019-2-221-228. Rimareva LV, Krivova AYu, Shelekhova NV, Overchenko MB, Serba EM. Catalytic features of proteas and fitaz when processing politicals of the trititical-without in manufacture of alcohol. Aktualʹnye voprosy industrii napitkov [Current issues of the beverage industry]. 2019;(3):184-190. (In Russ.). https://doi.org/10.21323/978-5-6043128-4-1-2019-3-184-190. Rimareva LV, Krivova AYu, Shelekhova NV, Overchenko MB, Serba EM. Catalytic features of proteas and fitaz when processing politicals of the trititical-without in manufacture of alcohol. Aktualʹnye voprosy industrii napitkov [Current issues of the beverage industry]. 2019;(3):184-190. (In Russ.). https://doi.org/10.21323/978-5-6043128-4-1-2019-3-184-190. Lonnerdal B. Phytic acid-trace element (Zn, Cu, Mn) interactions. International Journal of Food Science and Technology. 2002;37(7):749-758. https://doi.org/10.1046/j.1365-2621.2002.00640.x. Lonnerdal B. Phytic acid-trace element (Zn, Cu, Mn) interactions. International Journal of Food Science and Technology. 2002;37(7):749-758. https://doi.org/10.1046/j.1365-2621.2002.00640.x. De Carli L, Schnitzler E, Ionashiro M, Szpoganicz B, Rosso ND. Equilibrium, thermoanalytical and spectroscopic studies to characterize phytic acid complexes with Mn(II) and Co(II). Journal of the Brazilian Chemical Society. 2009;20(8):1515-1522. https://doi.org/10.1590/S0103-50532009000800019. De Carli L, Schnitzler E, Ionashiro M, Szpoganicz B, Rosso ND. Equilibrium, thermoanalytical and spectroscopic studies to characterize phytic acid complexes with Mn(II) and Co(II). Journal of the Brazilian Chemical Society. 2009;20(8):1515-1522. https://doi.org/10.1590/S0103-50532009000800019. Lee K-M, Kang H-S, Yun C-H, Kwak H-S. Potential in vitro protective effect of quercetin, catechin, caffeic acid and phytic acid against ethanol-induced oxidative stress in SK-Hep-1 cells. Biomolecules and Therapeutics. 2012;20(5):492-498. https://doi.org/10.4062/biomolther.2012.20.5.492. Lee K-M, Kang H-S, Yun C-H, Kwak H-S. Potential in vitro protective effect of quercetin, catechin, caffeic acid and phytic acid against ethanol-induced oxidative stress in SK-Hep-1 cells. Biomolecules and Therapeutics. 2012;20(5):492-498. https://doi.org/10.4062/biomolther.2012.20.5.492. Kumar K, Venkatraman V, Bruheim P. Adaptation of central metabolite pools to variations in growth rate and cultivation conditions in Saccharomyces cerevisiae. Microbial Cell Factories. 2021;20(1). https://doi.org/10.1186/s12934-021-01557-8. Kumar K, Venkatraman V, Bruheim P. Adaptation of central metabolite pools to variations in growth rate and cultivation conditions in Saccharomyces cerevisiae. Microbial Cell Factories. 2021;20(1). https://doi.org/10.1186/s12934-021-01557-8. Meng L, Liu H-L, Lin X, Hu X-P, Teng K-R, Liu S-X. Enhanced multi-stress tolerance and glucose utilization of Saccharomyces cerevisiae by overexpression of the SNF1 gene and varied beta isoform of Snf1 dominates in stresses. Microbial Cell Factories. 2020;19(1). https://doi.org/10.1186/s12934-020-01391-4. Meng L, Liu H-L, Lin X, Hu X-P, Teng K-R, Liu S-X. Enhanced multi-stress tolerance and glucose utilization of Saccharomyces cerevisiae by overexpression of the SNF1 gene and varied beta isoform of Snf1 dominates in stresses. Microbial Cell Factories. 2020;19(1). https://doi.org/10.1186/s12934-020-01391-4. Zhang Y, Lin Y-H. Metabolic flux analysis of Saccharomyces cerevisiae during redox potential-controlled very high-gravity ethanol fermentation. Biotechnology and Applied Biochemistry. 2020;67(1):140-147. https://doi.org/10.1002/bab.1861. Zhang Y, Lin Y-H. Metabolic flux analysis of Saccharomyces cerevisiae during redox potential-controlled very high-gravity ethanol fermentation. Biotechnology and Applied Biochemistry. 2020;67(1):140-147. https://doi.org/10.1002/bab.1861. De Stefano С, Giuffre O, Milea D, Rigano C, Sammartano S. Speciation of phytate ion in aqueous solution. Non covalent interactions with biogenic polyamines. Chemical Speciation and Bioavailability. 2003;15(2):29-36. https://doi.org/10.3184/095422903782775235. De Stefano S, Giuffre O, Milea D, Rigano C, Sammartano S. Speciation of phytate ion in aqueous solution. Non covalent interactions with biogenic polyamines. Chemical Speciation and Bioavailability. 2003;15(2):29-36. https://doi.org/10.3184/095422903782775235. Kumar V, Sinha AK, Makkar HPS, Becker K. Dietary roles of phytate and phytase in human nutrition: A review. Food Chemistry. 2010;120(4):945-959. https://doi.org/10.1016/j.foodchem.2009.11.052. Kumar V, Sinha AK, Makkar HPS, Becker K. Dietary roles of phytate and phytase in human nutrition: A review. Food Chemistry. 2010;120(4):945-959. https://doi.org/10.1016/j.foodchem.2009.11.052. Zhang YY, Stockmann R, Ng K, Ajlouni S. Revisiting phytate-element interactions: implications for iron, zinc and calcium bioavailability, with emphasis on legumes. Critical Reviews in Food Science and Nutrition. 2020. https://doi.org/10.1080/10408398.2020.1846014. Zhang YY, Stockmann R, Ng K, Ajlouni S. Revisiting phytate-element interactions: implications for iron, zinc and calcium bioavailability, with emphasis on legumes. Critical Reviews in Food Science and Nutrition. 2020. https://doi.org/10.1080/10408398.2020.1846014. Nielsen AVF, Tetens I, Meyer AS. Potential of phytase-mediated iron release from cereal-based foods: A quantitative view. Nutrients. 2013;5(8):3074-3098. https://doi.org/10.3390/nu5083074. Nielsen AVF, Tetens I, Meyer AS. Potential of phytase-mediated iron release from cereal-based foods: A quantitative view. Nutrients. 2013;5(8):3074-3098. https://doi.org/10.3390/nu5083074. Yu S, Cowieson A, Gilbert C, Plumstead P, Dalsgaard S. Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin. Journal of Animal Science. 2012;90(6):1824-1832. https://doi.org/10.2527/jas.2011-3866. Yu S, Cowieson A, Gilbert C, Plumstead P, Dalsgaard S. Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin. Journal of Animal Science. 2012;90(6):1824-1832. https://doi.org/10.2527/jas.2011-3866. Tsurikova NV, Nefedova LI, Kostyleva EV, Zvenigorodskii VI, Yasinovskii VG, Voeikova TA, et al. Selection of a potent Bacillus licheniformis strain producing thermostable α-amylase. Applied Biochemistry and Microbiology. 2002;38(5):427-432. https://doi.org/10.1023/A:1019960216770. Tsurikova NV, Nefedova LI, Kostyleva EV, Zvenigorodskii VI, Yasinovskii VG, Voeikova TA, et al. Selection of a potent Bacillus licheniformis strain producing thermostable α-amylase. Applied Biochemistry and Microbiology. 2002;38(5):427-432. https://doi.org/10.1023/A:1019960216770. Rimareva LV, Tsurikova NV, Kostyleva EV, Sereda AS. Recombinant mycelial strain aspergillus awamori producer of enzymatic complex of glucoamylase and xylanase. Patent RU 2457246C1. 2012. Rimareva LV, Tsurikova NV, Kostyleva EV, Sereda AS. Recombinant mycelial strain aspergillus awamori producer of enzymatic complex of glucoamylase and xylanase. Patent RU 2457246C1. 2012. Serba EM, Sokolova EN, Borshcheva YuA, Rimareva LV, Overchenko MB. The effect of chemical mutagenesis on physiological characteristics and productivity of Aspergillus oryzae. Mycology and Phytopathology. 2018;52(1):49-54. (In Russ.). Serba EM, Sokolova EN, Borshcheva YuA, Rimareva LV, Overchenko MB. The effect of chemical mutagenesis on physiological characteristics and productivity of Aspergillus oryzae. Mycology and Phytopathology. 2018;52(1):49-54. (In Russ.). Rimareva LV, Overchenko MB, Ignatova NI, Serba EM, Martynenko NN. Features of selected races of alcohol yeast with osmophilic and thermotolerant properties. Storage and Processing of Farm Products. 2016;(10):29-34. (In Russ.). Rimareva LV, Overchenko MB, Ignatova NI, Serba EM, Martynenko NN. Features of selected races of alcohol yeast with osmophilic and thermotolerant properties. Storage and Processing of Farm Products. 2016;(10):29-34. (In Russ.). Instruktsiya po tekhnokhimicheskomu i mikrobiologicheskomu kontrolyu spirtovogo proizvodstva [Instructions for the technochemical and microbiological control of alcohol production]. Moscow: DeLi print; 2007. 479 p. (In Russ.). Instruktsiya po tekhnokhimicheskomu i mikrobiologicheskomu kontrolyu spirtovogo proizvodstva [Instructions for the technochemical and microbiological control of alcohol production]. Moscow: DeLi print; 2007. 479 p. (In Russ.). Serba EM, Overchenko MB, Rimareva LV. Biotekhnologicheskie osnovy mikrobnoy konversii kontsentrirovannogo zernovogo susla v ethanol [Biotechnological principles of microbial conversion of concentrated grain wort into ethanol]. Moscow: Biblio-Globus; 2017. 120 p. (In Russ.). https://doi.org/10.18334/9785950050169. Serba EM, Overchenko MB, Rimareva LV. Biotekhnologicheskie osnovy mikrobnoy konversii kontsentrirovannogo zernovogo susla v ethanol [Biotechnological principles of microbial conversion of concentrated grain wort into ethanol]. Moscow: Biblio-Globus; 2017. 120 p. (In Russ.). https://doi.org/10.18334/9785950050169.