The work investigated the effect of four enzyme preparations (EP): Bacillolysin, Agroprot, Protozyme and Protozyme C (Russia), on smell, taste, as well as protein and peptide profiles of protein isolate isolated from Focor peas. It has been shown that enzyme treatment improves the odor characteristics of the isolate. Thus, it was possible to significantly reduce the severity of bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to significantly reduce the severity of such disturbing flavors as legume, astringent, bitter and herbal. The results obtained made it possible to select EP (fungal acidic aspartate proteinase) to improve the organoleptic parameters of pea protein isolates intended for the manufacture of analogues of meat and dairy products.

The effect of four enzyme preparations (EP), Bacillolysin, Agroprot, Protozyme, and Protozyme C (Russia), on the protein and peptide profiles of the protein isolate isolated from peas of the Focor variety, as well as on its smell and taste, was investigated in this work. It was shown that enzyme treatment can improve the odor characteristics of the isolate. Thus, it was possible to reduce significantly the severity of the bean and herbal smell. At the same time, enzyme treatment also improved the taste of the isolate: it was possible to reduce significantly the severity of disturbing flavors such as leguminous, astringent, bitter, and herbal. The results obtained allowed us to select EP (fungal acid aspartic protease) to improve the organoleptic parameters of pea protein isolates intended for the production of meat and dairy product analogs.

The effect of four enzyme preparations: bacillolysin, agroprot, protozyme and protozyme C (Russia) on solubility, emulsifying activity, emulsion stability, foaming and foam stability of isolates preparated from two varieties of peas was studied. It is shown that treatment with enzymes can increase the solubility of isolates at pH 5 by more than 7 times, the index of emulsifying activity at pH 5 by 1.5 to 2 times, and at pH 6 by almost 1.5 times; the stability index of the emulsion increased by about 20% at pH 5, and by 1.7 times (in one of the varieties) at pH 6; foaming increased by 2.4 to 3 times at pH 5, and at pH 6 by 1.8 to 3.7 times; foam stability increased by 25 to 33% at pH 5 and by more than 1.5 times (in one of the varieties) at pH 6. The results obtained made it possible to select an enzyme preparation (bacterial alkaline serine protease) to improve the parameters of pea protein isolates intended for the manufacture of analogues of fermented milk products.

Благодаря сбалансированному аминокислотному составу молочные белки являются основным видом сырья для получения белковых гидролизатов, широко используемых в качестве ингредиентов детского, специализированного, лечебного и спортивного питания. Около 80 % белков молока составляет казеин, обладающий высокой биологической ценностью, но также являющийся сильным аллергеном. Для устранения иммуногенных свойств и повышения питательной ценности казеин гидролизуют до низкомолекулярных пептидов и свободных аминокислот с использованием протеолитических ферментных препаратов (ФП). Во ВНИИПБТ был получен ФП Протооризин LAP, который за счет комплексного действия лейцинаминопептидазы с сериновой и аспартатной эндопептидазами обеспечил эффективный гидролиз казеина. Целью исследований являлось изучение влияния дозировок Протооризина LAP и продолжительности гидролиза на эффективность процесса и качество получаемых гидролизатов. Установлено, что применение Протооризина LAP в дозировках 1–4 % при 50 °С, естественном рН 6,0 и концентрации субстрата 10 % дает возможность получать глубокие гидролизаты казеина с высоким содержанием свободных аминокислот. Гидролиз в течение 6 ч обеспечивает переход в растворимое состояние от 83 до 94 % казеина, в зависимости от дозировки препарата в диапазоне 1–4 %. При этом содержание свободных аминокислот в растворимом белке составляет 40–60 %. Кратковременный гидролиз в течение 3 ч при дозировке препарата 2–4 % обеспечивал переход в водорастворимое состояние 73,2–83,7 % казеина при концентрации свободных аминокислот от 30 до 50 %. Электрофоретический анализ показал, что при 3-часовом гидролизе казеина 0,5 % Протооризина LAP в гидролизате наблюдались слабые белковые полосы, соответствующие остаточным количествам αS и β-казеина. При всех остальных вариантах обработки в гидролизатах отсутствовали белковые фракции с молекулярной массой выше 10 кДа. Полученные результаты показали, что применение нового комплексного препарата Протооризина LAP при невысоких дозировках (1–2 % к массе субстрата) обеспечивает расщепление иммуногенных белков казеина и позволяет получать гидролизаты с высоким содержанием низкомолекулярных пептидов и свободных аминокислот. Варьируя дозировку ФП и продолжительность обработки, можно получать гидролизаты с разным количеством свободных аминокислот, в зависимости от требований к продукту. Due to their balanced amino acid composition, milk proteins represent the main raw material for the production of protein hydrolysates, widely used as ingredients for baby food, specialized, medical and sports nutrition. Casein makes up about 80 % of milk proteins, has high biological value and possesses strong allergenicity. To eliminate immunogenic properties and increase nutritional value, casein is hydrolyzed to low molecular weight peptides and free amino acids using proteolytic enzyme preparations (EP). Earlier we obtained Protoorizin LAP proteolytic preparation, which, due to the synergistic action of leucine aminopeptidase with serine and aspartate endopeptidases, ensured effective hydrolysis of casein. The purpose of this research was to study the effect of dosages of Protoorizin LAP and the duration of hydrolysis on the efficiency of the process and the quality of the resulting hydrolysates. It was found out that Protoorizin LAP in dosages of 1–4 % at 50 °C, natural pH 6.0 and substrate concentration of 10% allows to obtain extensive casein hydrolysates with high content of free amino acids. Hydrolysis for 6 hours ensures the transition into a soluble state from 83 to 94 % of casein, depending on the dosage of the EP in the range of 1–4 %. The content of free amino acids in soluble protein accounts to 40–60 %. Short-term hydrolysis for 3 hours at Protoorizin LAP dosage of 2–4 % ensured the transition of 73.2–83.7 % of casein into a water-soluble state and a concentration of free amino acids from 30 to 50 %. Electrophoretic analysis showed that after 3-hour hydrolysis of casein with 0.5 % Protoorizin LAP, weak protein bands were observed in the hydrolyzate, corresponding to residual amounts of αS and β-casein. All other processing options provided hydrolysates without protein fractions with a molecular weight above 10 kDa. The results obtained showed that the use of the new complex EP Protoorizin LAP at low dosages (1–2 % by weight of the substrate) ensures the breakdown of immunogenic casein proteins and allows one to obtain hydrolysates with a high content of low molecular weight peptides and free amino acids. By varying the EP dosage and the duration of treatment, it is possible to obtain hydrolysates with different amounts of free amino acids, depending on the product requirements.

Белки молочной сыворотки являются перспективным сырьем для получения ферментативных гидролизатов, широко применяемых в детском, спортивном питании, при создании продуктов профилактического и терапевтического назначения. Учитывая, что основные белки молочной сыворотки – β-лактоглобулин и α-лактальбумин – обладают сильным иммуногенным потенциалом, для использования их в качестве компонентов функционального и специализированного питания требуется проведение глубокого гидролиза, обеспечивающего расщепление основных белковых фракций до низкомолекулярных пептидов и свободных аминокислот. Цель исследований заключалась в определении эффективности отечественных протеолитических ферментных препаратов (ФП): промышленного препарата Протосубтилина и опытных образцов ФП Протолихетерма и Протооризина LAP в процессе гидролиза сывороточных белков. В качестве основного критерия рассматривали полноту гидролиза белков молочной сыворотки, определяемую по исчезновению белковых полос на ПААГ-электрофореграммах. Также определяли содержание в гидролизатах свободных аминокислот. Установлено, что наиболее эффективным при гидролизе белков молочной сыворотки оказался комплексный препарат Протооризин LAP, который в дозировках 0,5 и 1,0 % ФП к массе субстрата обеспечил устранение α-лактоглобулина. Для полного расщепления всех основных аллергенов молочной сыворотки требовалась предварительная термообработка субстрата. Протосубтилин в дозировке 11,6 ед/г субстрата (3,2 %) полностью прогидролизовал β-лактоглобулин и α-лактальбумин в предобработанном концентрате сывороточных белков. Протолихетерм был менее эффективен при гидролизе сывороточных белков по сравнению с Протооризином LAP и Протосубтилином. Максимальный выход аминокислот обеспечило применение Протооризина LAP, ключевым компонентом которого является лейцинаминопептидаза, и составило 15,5 % от массы субстрата или 19,3 % от содержания белка в концентрате сывороточного белка. Варьируя дозировку Протооризина LAP, можно получать гидролизаты с различным содержанием свободных аминокислот, в зависимости от требований к конечному продукту. Проведенные исследования показали возможность использования отечественного промышленного ФП Протосубтилина и опытного образца ФП Протооризина LAP при получении белковых гидролизатов на основе концентрата белков молочной сыворотки. Whey proteins are promising raw materials for the production of enzymatic hydrolysates, widely used in baby food and sports nutrition, in the obtaining of products for prophylactic and therapeutic purposes. The major whey proteins – β-lactoglobulin and α-lactalbumin – have a strong immunogenic potential. So for using them as components of functional and specialized nutrition, extensive hydrolysis is required to ensure the breakdown of the main protein fractions into low molecular weight peptides and free amino acids. The purpose of the research was to determine the effectiveness of the following domestic proteolytic enzyme preparations (EP): the industrial preparation Protosubtilin; experimental samples of EP Protolichetherm and Protooryzin LAP, in the process of hydrolysis of whey proteins. The main indicator was the completeness of whey proteins hydrolysis, evaluated by the disappearance of protein bands on SDS-PAGE electropherograms. The content of free amino acids in the hydrolysates was also determined. It was found that the most effective in the whey proteins hydrolysis was the complex preparation Protooryzin LAP, which in dosages of 0.5 and 1.0 % to the weight of the substrate ensured the elimination of α-lactoglobulin. To completely break down all the main whey allergens, preliminary heat treatment of the substrate was required. Protosubtilin at a dosage of 11.6 units of proteolytic activity/g substrate (3.2 % of EP to the weight of the substrate) completely hydrolyzed β-lactoglobulin and α-lactalbumin in pretreated whey protein concentrate. Protolichetherm was less effective in hydrolyzing whey proteins compared to Protooryzin LAP and Protosubtilin. Protooryzin LAP, containing leucine aminopeptidase as the key component of proteolytic complex, ensured the maximum amino acids yield amounted to 15.5 % of the substrate weight or 19.3 % of the protein content in whey protein concentrate. By varying the dosage of Protooryzin LAP, it is possible to obtain hydrolysates with different free amino acids contents, depending on the requirements for the final product. The conducted studies showed the feasibility of using the domestic industrial EP Protosubtilin and experimental sample of EP Protooryzin LAP in the production of protein hydrolysates from whey protein concentrate.

The development of technologies for producing bacterial concentrates and enzyme preparations using domestic microbial strains is an urgent task. The use of whey protein hydrolysates as components of nutrient media for probiotic bacteria consortia for the cultivation of lactic acid and bifidobacteria makes it possible to improve and develop innovative processes for obtaining bacterial concentrates with the required functional properties for the production of dietary supplements. A consortium of probiotic microorganisms (lactic acid and bifidobacteria) was created in the All-Russian Scientific Research Institute of Food Biotechnology as a starter culture for specialized dairy products. Using strain Aspergillus oryzae 21-154 LAP a new complex enzyme preparation with a laboratory name Protoorizin LAP has been obtained providing the extensive hydrolysis of protein substrates. The purpose of the research was to evaluate the possibility of using the new domestic proteolytic enzyme preparation Protoorizin LAP in preparing whey-based nutrient media for culturing a consortium of probiotic microorganisms to obtain bacterial concentrates. Material and methods. The object of the research was a symbiotic consortium, including lactic acid bacteria strains (Lactobacillus delbrueckii ssp. bulgaricus Д-16, Lactobacillus plantarum 578/25, Lactobacillus helveticus 842(D)-2, Lactococcus lactis subsp. lactis М-12, Streptococcus thermophilus В-92) and bifidobacteria (Bifidobacterium longum Б-2). Unclarified curd whey and whey protein concentrate were taken as the nutrient medium basis. The media were treated with β-galactosidase to reduce the lactose content. In order to hydrolyze proteins, the control culture medium was treated with commercial preparations: serine protease - Alcalase® 2.4 L and leucine aminopeptidase - Flavourzyme® 1000 L. In the experimental medium, two imported preparations were replaced with a laboratory sample of the enzyme preparation Protoorizin LAP. In the prepared nutrient media, the content of amine nitrogen, free amino acids and soluble protein was determined, and electrophoretic analysis of proteins and peptides was carried out. The consortium growth was monitored by the content of dry substances and reducing sugars, by active and titratable acidity, as well as by microscopy. The number of viable cells of lactic acid bacteria and bifidobacteria at the end of fermentation and in the resulting bacterial concentrates were determined by sieving on the appropriate selective agar media using an automatic colony counter. Results. The effectiveness of Protoorizin LAP in the hydrolysis of whey proteins significantly exceeded the result of the combined action of Alcalase® 2.4 L and Flavourzyme® 1000 L both in terms of reducing the undigested protein content, including immunogenic fractions, and in terms of the yield of soluble protein, amine nitrogen and amino acids. The nutrient media obtained using proteases ensured good growth and development of the probiotic consortium. Due to the high content of free amino acids, the dynamics of carbohydrate consumption, titratable acidity, and the number of viable cells were higher in the medium obtained using Protoorizin LAP than when using commercial preparations. At the same time, a high titer of probiotic strains and good cultural and morphological characteristics were obtained on all media. The experimental preparation Protoorizin LAP provided the increase in viability of bacterial cells after lyophilization. Conclusion. The technological method that include application of the new proteolytic preparation Protoorizin LAP in preparing nutrient media based on whey proteins was developed. The method can be used in the technology of producing bacterial concentrates at the stage of culturing of the created lactic acid and bifidobacteria consortium. The bacterial concentrate can be recommended as a recipe ingredient in the manufacture of dietary supplements or foods for special dietary uses containing probiotics.

Микроскопические грибы широко используются для промышленного производства технических ферментов; важным условием для этого является создание высокоактивных штаммов – продуцентов ферментов. Показана возможность использования для получения таких штаммов двух подходов: индуцированного мутагенеза и методов генетической инженерии. Разработаны схемы индуцированного мутагенеза, обеспечивающие получение высокоактивных продуцентов технических ферментов. В результате гамма-мутагенеза получены мутантные штаммы грибов рода Trichoderma – продуценты целлюлаз, ксиланаз и пектиназы, с различным компонентным составом карбогидразного комплекса, позволяющие получать в культуральной жидкости (КЖ) до 35–40 г/л внеклеточного белка. С помощью комбинированной обработки УФ и гамма-облучением получен стабильный мутантный штамм Aspergillus awamori с увеличенной продукцией глюкоамилазы. Создана система экспрессии Penicillium verruculosum В1-537 (ΔniaD), позволяющая трансформировать реципиентный штамм экспрессионными конструкциями, содержащими целевые гетерологичные или гомологичные гены, функционально связанные с промотором и терминатором сильного индуцибельного промотора гена мажорного секреторного белка целлобиогидролазы I (cbh1) P. verruculosum. Технология создания и отбора активных рекомбинантных штаммов проста и надежна, состав питательной среды и условий культивирования рекомбинантных штаммов – продуцентов различных целевых ферментов стандартизирован. Затраты времени на получение продуцента целевого фермента составляют от 3 до 6 мес. Реципиентный штамм P. verruculosum характеризуется высокой секреторной способностью (до 60 г/л внеклеточного белка в КЖ), а рекомбинантные штаммы-продуценты – высокой продуктивностью целевых ферментов; полученные с их помощью ферментные препараты (ФП) содержат 30‒70 % целевых рекомбинантных ферментов (в некоторых случаях до 80 %) от общего пула белка. Созданы рекомбинантные штаммы – продуценты целлюлаз, β-глюканаз, ксиланаз, фитазы, кислой протеазы, пектин-лиазы, β-глюкозидазы, экзо- и эндоинулиназ. Мутантные и рекомбинантные штаммы – продуценты различных ферментов – используются для промышленного производства на заводе ООО «Агрофермент» различных технических ФП для пищевой промышленности и кормопроизводства. Microscopic fungi are widely used for the industrial production of technical enzymes; an important condition for this is the creation of highly active strains – producers of enzymes. The possibility of using such approaches to obtain highly active strains as induced mutagenesis and genetic engineering methods is shown. Schemes of induced mutagenesis have been developed that ensure the production of highly active producers of technical enzymes. As a result of gamma-mutagenesis, mutant fungal strains of Trichoderma genus were obtained – producers of cellulases, xylanases and pectinase, with different component composition of the carbohydrase complex. The methods used made it possible to obtain up to 35–40 g/l of extracellular protein in the cultural liquid (CL). Using UV and gamma rays combined treatment, a stable mutant Aspergillus awamori strain with increased glucoamylase production was obtained. An expression system for Penicillium verruculosum B1-537 (ΔniaD) has been developed, which makes it possible to transform a recipient strain with expression constructs containing target heterologous or homologous genes functionally linked to the promoter and terminator of the strong inducible promoter of the P. verruculosum major secretory protein cellobiohydrolase I (cbh1) gene. The technology for creating and selection of active recombinant strains is simple and reliable, the composition of the nutrient medium and cultivating conditions for recombinant strains producing various target enzymes are standardized. The time spent on obtaining the target enzyme producer is from 3 to 6 months. The recipient P. verruculosum strain is characterized by a high secretory capacity (up to 60 g/l of extracellular protein in CL), and the recombinant strains are characterized by a high productivity of target enzymes. Enzyme preparations (EP) derived from the recombinant strains contain 30–70 % of the target recombinant enzymes in the total protein pool (in some cases, up to 80 %). Recombinant strains producing cellulases, β-glucanases, xylanases, phytase, acid protease, pectin-lyase, β-glucosidase, exo- and endo-inulinases have been developed. Mutant and recombinant strains – producers of various enzymes for the food industry and feed production are successfully used for industrial production of various technical EPs at the plant LLC «Agroferment».

Пшеничный глютен является источником незаменимых аминокислот и биоактивных пептидов, а его ферментативные гидролизаты широко используются в качестве пищевых добавок и усилителей вкуса. В то же время высокое содержание пролина препятствует гидролизу глютена пищеварительными протеазами, что приводит к возникновению аллергических реакций и нарушениям пищеварительной системы. Глубокий ферментативный гидролиз способствует снижению аллергенности глютена, повышает его пищевую ценность и расширяет область применения в пищевой промышленности. Некоторые комплексные ферментные препараты (ФП), содержащие эндо- и экзопептидазы, за счет их синергетического действия обеспечивают эффективный гидролиз глютена. На основе штамма Aspergillus oryzae 21-154 LAP – продуцента комплекса эндо- и экзопептидаз с увеличенной активностью лейцинаминопептидазы (LAP) – получен комплексный ФП Протооризин LAP, по активности LAP сравнимый с широко используемым в пищевой промышленности ФП Flavourzyme® 1000L, при высоком уровне общей протеолитической активности, обусловленном действием эндопептидаз. По увеличению содержания растворимого белка и свободных аминокислот при гидролизе пшеничного глютена Протооризин LAP превосходил импортные ФП Flavourzyme® 1000L и Alcalase 2.4L как при индивидуальном, так и при совместном применении. Электрофоретический анализ полученных гидролизатов показал, что Протооризин LAP интенсивно расщеплял все основные белки глютена. Через 24 ч гидролиза оставалась только слабая полоса белка с молекулярной массой ~37 кДа. ФП Alcalase 2.4L расщеплял пшеничный белок с меньшей эффективностью, чем Протооризин LAP, оставляя через 24 ч, помимо полосы на уровне ~37 кДа, некоторое количество пептидов с молекулярной массой менее 14,4 кДа. Наименьшей способностью к гидролизу белков пшеницы обладал Flavourzyme® 1000L, однако при совместном использовании с Alcalase 2.4L препарат эффективно гидролизовал низкомолекулярные белковые фракции. Протооризин LAP успешно заменял совместное действие ФП Flavourzyme 1000L и Alcalase 2.4L, что показывает перспективность его использования при получении гидролизатов пшеничного глютена. Wheat gluten is a good source of essential amino acids and bioactive peptides. Its hydrolysates are widely used as food additives and flavor enhancers. At the same time, the high proline content interferes with the gluten hydrolysis by digestive proteases, which leads to allergic reactions and digestive system disorders. Deep enzymatic hydrolysis contributes to the reduction of gluten allergenicity, increases its nutritional value and expands its scope in the food industry. Some complex enzyme preparations (EP) containing endo- and exopeptidases, due to their synergistic action, provide effective hydrolysis of gluten. Using Aspergillus oryzae 21-154 LAP strain, a producer of endo- and exopeptidases complex with increased leucine aminopeptidase (LAP) activity, the EP Protoorizin LAP was obtained, in terms of LAP activity comparable to Flavourzyme® 1000L widely used in the food industry, and possessing a high level of total proteolytic activity due to endopeptidases presence. During wheat gluten hydrolysis Protoorizin LAP was superior to Flavourzyme® 1000L and Alcalase 2.4L, both when used individually and when combined, by the increase in the soluble protein and free amino acids content. Electrophoretic analysis of the obtained hydrolysates showed that Protoorizin LAP intensively cleaves all major gluten proteins. After 24 h of hydrolysis, the ~14 kDa faint band remained at the level of ~37 kDa. Alcalase 2.4L cleaved wheat protein with less efficiency than Protoorizin LAP, leaving after 24 h, in addition to the ~37 kDa band, some peptides with a molecular weight of less than 14.4 kDa. Flavourzyme® 1000L had the least ability to hydrolyze gluten, however, when used together with Alcalase 2.4L, the EP contributed to a more complete hydrolysis of low molecular weight protein fractions. Protoorizin LAP successfully replaced the combined action of Flavourzyme 1000L and Alcalase 2.4L, which shows the promise of its use in the wheat gluten hydrolysates production.

Whey and hen egg white proteins are characterized by high nutritional value, but possess antigenic properties, which limit their use in the production of dietary products. Enzymatic hydrolysis decreases significantly the allergenicity of proteins. The efficiency of hydrolysis depends on the specificity of the proteases used. The aim of this work was to determine the effectiveness of EP-96 enzyme preparation obtained from Bacillus subtilis-96 culture liquid in the hydrolysis of whey and egg white proteins in comparison with commercial bacterial proteases preparations - Alcalase, Neutrase, and Protosubtilin. Material and methods. Whey and egg white protein concentrates were used as substrates. Commercial enzyme preparations Alcalase, Neutrase, and Protosubtilin, and an experimental sample of EP-96 preparation obtained from Bacillus subtilis-96 culture liquid were used for hydrolysis. Hydrolysis was carried out at a substrate concentration of 100 g/L for 3 h at 55 °C or for 24 h at 50 °C. After hydrolysis, the reaction mixture was incubated at 90 °C for 15 min to inactivate the enzymes. The content of peptides with a molecular weight of less than 10 kDa was determined in the obtained hydrolysates. The hydrolysis of the main allergenic proteins was assessed by the disappearance of the corresponding protein bands on the hydrolysate supernatants electrophoregrams. Results and discussion. All the studied preparations showed high efficiency in the hydrolysis of whey proteins and provided the yield of low molecular weight peptides at the level of 18.8-22.8% after 3 h of hydrolysis and 39.4-41.6% after 24 h. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a residual amount of protein with a molecular weight of about 14 kDa, corresponding to α-lactoalbumin, after 3 h of hydrolysis when using Neutrase. The preparations containing serine protease, including EP-96, provided more intensive hydrolysis of whey proteins. In the hydrolysis of egg white protein, Neutrase showed the greatest efficiency. The efficiency of EP-96 was comparable to Neutrase both in the yield of low molecular weight peptides and in the intensity of cleavage of the main allergenic proteins. The effectiveness of preparations with predominant content of serine proteases - Alcalase and Protosubtilin was significantly lower. Conclusion. The optimal ratio of neutral and serine proteases in the EP-96, obtained on the basis of the B. subtilis-96 strain, provided the high efficiency and its versatility in the hydrolysis of the main allergenic proteins of whey and egg white. The parameters of the hydrolysis technology using EP-96 are recommended, which provide intensive conversion of the main immunogenic proteins of whey and egg white to soluble and low molecular weight fractions (duration 3 h at a temperature of 55 °C and the proteolytic activity of the preparation is not less than 2 units per g of substrate) and an increase of subsequent ultrafiltration efficiency in the production of protein hydrolysates for foods for special dietary uses.

Due to the increasing shortage of food protein in the world, the most effective and complete use of proteinaceous substrates is an urgent task. The most promising way to utilize secondary protein-containing raw materials is to increase its nutritional value through enzymatic hydrolysis. The use of protein hydrolysates obtained from protein-containing by-products has great potential in various areas of the food industry, as well as in the production of foods for medical and special dietary uses. The aim of the research was to propose optimal methods for processing protein substrates to obtain hydrolysates with desired properties, taking into account the characteristics of the main types of proteinaceous by-products and the specificity of proteases used. Material and methods. We used the data contained in PubMed, WoS, Scopus, and eLIBRARY.RU databases, which meet the requirements of scientific reliability and completeness. Results. Collagen-containing wastes from the meat, poultry and fish processing industries, whey, soy protein and gluten are the main types of protein-containing by-products successfully used to produce food and functional hydrolysates. The molecular structure, basic biological and physico-chemical properties of collagen, whey proteins, various protein fractions of wheat gluten and soy protein are described. The expediency of enzymatic treatment of protein-containing by-products using proteases is shown to reduce antigenicity and eliminate anti-nutritional properties, improve nutritional, functional, organoleptic and bioactive properties for subsequent use in food production, including those for medical and special dietary uses. Information is presented on the classification of proteolytic enzymes, their main properties, and the effectiveness of their use in the processing of various types of proteinaceous by-products. Conclusion. Based on the literature data analysis, the most promising methods for obtaining food protein hydrolysates from secondary protein-containing raw materials are proposed, including pretreatment of substrates and the selection of proteolytic enzymes with a certain specificity.

One of the ways to solve the problem of whey utilization is to obtain enzymatic hydrolysates with bioactive properties and reduced antigenicity. A wide range of enzyme preparations are used for whey hydrolysis. The aim of the study was to select the conditions for enzymatic processing of whey protein concentrate to provide the most complete hydrolysis of all basic whey proteins using the most popular proteolytic enzyme preparations for the food industry – Alcalase and Flavourzyme. It was found that the completeness of whey proteins hydrolysis, determined by the yield of soluble and low molecular weight protein, as well as by the disappearance of protein bands on electrophoregrams of the obtained hydrolysates, largely depends on the enzyme preparations dosages and preliminary heat treatment of the substrate. Combined hydrolysis of the pretreated whey protein concentrate with 1% Alcalase and 2% Flavourzyme for 20.5 h at 50°C with stirring allows one to convert about 70% of whey proteins to a stable soluble state, not denatured by heating at 90°C, and 50% of the hydrolyzed protein has a molecular weight of less than 10 kDa. This treatment provides the elimination of the main antigenic whey proteins and obtaining the hydrolysates with a degree of hydrolysis of 36%, determined using the formol titration method.

Гидролизаты коллагена широко используются в пищевой, фармацевтической и косметической промышленности. Для обработки коллагена используют различные протеолитические ферменты растительного, животного и микробного происхождения, из которых самыми перспективными являются бактериальные протеазы. Наиболее известные и часто используемые в пищевой отрасли коммерческие препараты бактериальных протеаз различаются по компонентному составу и могут содержать сериновые, металлопротеазы либо их смесь. С целью определения эффективности препаратов бактериальных протеаз с различным компонентным составом при получении гидролизатов коллагена мы провели ферментативную обработку говяжьего коллагена с использованием коммерческих препаратов Alcalase, Neutrase, Protamex и двух образцов ферментных препаратов, полученных в лабораторных условиях на основе новых отечественных штаммов Bacillus. Эффективность гидролиза оценивали по накоплению аминного азота, содержанию низкомолекулярного белка и по интенсивности белковых полос на электрофореграммах полученных продуктов. Все исследуемые препараты обеспечивали эффективный гидролиз коллагена. Наиболее интенсивно коллаген расщепляли препараты, содержащие только сериновые протеазы: Alcalase и ФП-145, полученный из культуральной жидкости штамма B. licheniformis-145. Высокую эффективность показали коммерческий препарат Protamex и ФП-96, полученный на основе мутантного варианта штамма B. subtilis-359 - продуцента субтилизина BPN’ и бациллолизина. Наименьший выход аминного азота и низкомолекулярных белков наблюдался в варианте с препаратом Neutrase, протеолитическая активность которого полностью представлена действием нейтральной протеазы бациллолизина. Лабораторные образцы ферментных препаратов, полученные на основе новых отечественных продуцентов, не уступали зарубежным коммерческим аналогам, а ФП-96 обеспечил более высокий выход аминного азота и низкомолекулярных белков в сравнении с широко используемым в пищевой промышленности препаратом Protamex. Collagen hydrolysates are widely used in the food, pharmaceutical and cosmetic industries. For collagen hydrolysis, various proteolytic enzymes of plant, animal and microbial origin are used, of which the most promising are bacterial proteases. The most well-known and commonly used in the food industry commercial preparations of bacterial proteases differ in their component composition and may contain serine protease, metalloproteases, or their mixture. In order to determine the effectiveness of bacterial protease preparations with different component composition in obtaining collagen hydrolysates, we carried out hydrolysis of beef collagen using commercial preparations Alcalase, Neutrase, Protamex and two laboratory samples of enzyme preparations obtained from new domestic Bacillus strains. The efficiency of hydrolysis was assessed by amine nitrogen accumulation, low-molecular-weight protein content, and by the intensity of the protein bands on the electrophoregrams of the hydrolysates. All investigated preparations provided effective collagen hydrolysis. Collagen was hydrolyzed most intensively by preparations containing only serine proteases: Alcalase and FP-145, obtained from B. licheniformis-145 culture liquid. The commercial preparation Protamex and FP-96, obtained from a mutant variant of the B. subtilis-359 strain, a producer of subtilisin BPN' and bacillolysin, showed high efficiency. The lowest yield of amine nitrogen and low molecular weight proteins was observed when using Neutrase, which contains only the neutral protease bacillolysin. Laboratory samples of enzyme preparations obtained from the new domestic producers were not inferior to foreign commercial counterparts, and FP-96 provided a higher yield of amine nitrogen and low molecular weight proteins in comparison with Protamex, which is widely used in the food industry.

Studies were conducted to obtain a complex enzyme preparation that allows efficient hydrolysis of the main plant nonstarch polysaccharides (cellulose, xylans, and pectin). The goal of the studies was to optimize the composition of a fermentation medium for the submerged cultivation of the new mutant strain T. reesei Co-44, a highly active producer of endo-carbohydrases. A concentrate of low molecular soy components was chosen as the inducer of the key carbohydrase biosynthesis by the strain. This concentrate provided the maximum (more than eightfold) increase in polygalacturonase activity and an increased level of endoglucanase and xylanase biosynthesis. After the cultivation of T. reesei Co-44 under optimized conditions, a complex enzyme preparation, Xylorizin K4, was obtained, and its physicochemical properties were studied. The presence of endopolygalacturonase (GH28) T. reesei in Xylorizin K4 was confirmed via electrophoresis and MALDI⎯TOF mass spectrometry. The studies show the potential of Xylorizin K4 application for the production of soy-protein concentrates to eliminate the main soybean nonstarch polysaccharides.