[Proteases for obtaining of food protein hydrolysates from proteinaceous by-products].

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.

[Efficiency of an enzyme preparation obtained from a new mutant Bacillus subtilis-96 strain in the hydrolysis of whey and egg white proteins].

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.

[Assessment of the influence of an enzymal preparation - a complex of glucoamylase and xylanase from Aspergillus awamori Xyl T-15 on the intestinal microbiom and immunological indicators of rats].

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15]. Material and methods. In experimental studies carried out for 80 days, Wistar rats (males and females) were used. The study of the effect of EP (a complex of glucoamylase and xylanase from a genetically modified Aspergillus awamori Xyl T-15 strain) in dozes 10, 100 and 1000 mg/kg body mass on the cecum microbiome and the immune status (content of cytokines and chemokines: IL-1a, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF-α, MCP-1, MIP-1a and Regulated on Activation Normal T-cell Expressed and Secreted - RANTES) was carried out. Results. It has been shown that EP - a complex of glucoamylase and xylanase from A. awamori Xyl T-15 at doses of 100 mg/kg or more causes mild disturbances in the composition of gut microbiocenosis. At the same time, these disorders have a significant immunomodulat ory and immunotoxic effect on the body, which manifests itself in a dose-dependent change in the profile of pro-inflammatory cytokines and chemokines in blood and spleen. The adverse effect of EP on the body is probably due to the formation of metabolites that are not formed during usual digestive processes in the gastrointestinal tract. The minimum effective dose (LOAEL) of EP was 100 mg/kg body weight In accordance with established requirements, the activity of the EP should not appear in ready-to-use food. Subject to this requirement, amount of EP entering the body cannot exceed the established LOAEL level. Therefore, a complex of glucoamylase and xylanase can be used in food industry, subject to the establishment of regulations «for technological purposes¼ for A. awamori Xyl T-15 strain. Conclusion. The data obtained on the relationship between the state of the microbiome and the immune status upon the introduction of EP indicate the need to include indicators of the state of gut microbiocenosis in the test protocol of safety.

[Risk assessment of glucoamylase and xylanase complex from Aspergillus awamori Xyl T-15].

The introduction of methods for food production using microbial synthesis, including those obtained with the help of genetically modified (GM) microorganisms, at the present stage, allows to increase production volumes and reduce the cost of food. At the same time, such products in accordance with TR CU 021/2011 "On food safety" are classified as a "novel food"¼ and can be placed on the market only after its risk estimation for health. The emergence of new data and research methods in the last years has made it necessary to improve the risk assessment system for this category of food. The aim of the research is to develope risk assessment approaches of food obtained by microbial synthesis on the example of the GM strain Aspergillus awamori Xyl T-15 and the enzyme preparation (EP) (a complex of glucoamylase and xylanase) produced by it. Material and methods. Outbred ICR mice (CD-1) and Wistar rats (males and females) were used in the experimental studies. Investigations of GM strain A. awamori Xyl T-15 virulence and its ability to disseminate internal organs have been carried out. Acute and subacute (during 80 days) toxicity of EP (a complex of glucoamylase and xylanase) have been studied. Results. The presented experimental data allow us to make a conclusion about the avirulence of the A. awamori Xyl T-15 strain, the lack of ability to disseminate internal organs (invasiveness). At the same time, the strain is characterized by the ability to produce mycotoxins (ochratoxin, fumonisin B2, T-2 and HT-2 toxins). The EP, a complex of glucoamylase and xylanase from A. awamori Xyl T-15, has a low oral acute toxicity for rats (LD50>5000 mg/kg). I ntragastric EP administration at doses of 10, 100 and 1000 mg/kg of body weight during 80 days had not revealed adversely affect on the rate of weight gain in animals, indicators of anxiety and cognitive function, and some studied biochemical indicators. At a dose of 100 mg/kg b.w. or more, there were changes in the relative mass of organs (lungs, kidneys, adrenal glands), small shifts in the parameters of erythropoiesis and leukocyte formula, at a dose of 1000 mg/kg b.w. - an increase in oxidative DNA destruction. T he most pronounced and dose-dependent was the effect of the EP on hepatocyte apoptosis. According to this indicator, the not observed adverse effect level (NOAEL) for EP is not more than 100 mg/kg b.w. in terms of protein. The main target organ for the toxic effect of EP is the liver. Conclusion. The data obtained demonstrate the necessity to conduct an additional analysis of the risks of possible negative effects of EP, namely, to study its impact on the gut microbiocenosis and the immune status of experimental animals, to analyze the presence of determinants of pathogenicity and antibiotic resistance, DNA of selective marker genes of A. awamori Xyl T-15 strain by PCR analysis and DNA sequencing methods.

[A New Enzyme Preparation with High Penicillopepsin Activity Based on the Producer Strain Penicillium canescens].

The producer of fungal penicillopepsin, an aspartate protease, has been created by genetic engineering. The biochemical and physicochemical properties of the penicillopepsin enzyme preparation obtained from the culture liquid of the producer were studied. Properties of the new enzyme preparation and the commercially available aspergillopepsin were compared. Their proteolytic activities were found to be 670-680 U/g of the preparation. The soluble protein yield upon the wheat flour hydrolysis with penicillopepsin was 2.7 times higher than with aspergillopepsin. It is probably caused by the presence of the xylanase activity in the penicillopepsin preparation.

[Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain].

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.

[Increase in glucoamylase productivity of Aspergillus awamori strain by combination of radiating mutagenesis and plasmid transformation methods].

Increase in the level of amylolytic genes activator protein encoded by amyR gene was shown to result in enhancement of glucoamylase productivity of A. awamori strain by 30%. However, the same effect equal to 30% increase can be achieved by introduction of extra copies of gla gene encoding glucoamylase. These two effects were not additive, which gave the possibility to suggest an additional limitation in the egulation mechanism of glucoamylase gene expression in Aspergillus family strains while introducing an additional copies of amyR and gla genes.

[Enzymatic hydrolysis of keratin-containing stock for obtaining protein hydrolysates].

Optimization of the process of enzymatic hydrolysis of keratin-containing stock aimed at obtaining hydrolysates of high biological value has been performed. The increasing of the stock/water weight ratio, the amount of the alkaline protease preparation from Acremonium chrysogenium added and the temperature of the reaction mixture resulted in an increase in the yield and antioxidant capacity of hydrolysis products. The molecular masses of soluble products obtained under optimal hydrolysis conditions ranged from 3.55 to 3.60 kDa. High antioxidant capacity, 100% bioavailability and a well-balanced amino acid composition was characteristic of the hydrolysis products.

[Hydrolysis of wheat flour with amylase preparations and individual enzymes].

Rheological properties of wheat flour were studied in the course of its processing (cooking and saccharification). The effects of commercial alpha-amylase preparations were compared during flour preparation. Test preparations were equally potent in decreasing the viscosity of an all-grain batch. Homogenous glucoamylases isolated from Aspergillus differed in the presence or absence of the starch-binding domain. The starch-binding domain provided for the high activity of glucoamylase on insoluble starch, but gave no advantages in saccharification of pretreated wheat flour.

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

[Effect of the proteolytic enzymes of Bacillus licheniformis and the lysoamidase of Lysobacter sp. XL1 on Proteus vulgaris and Proteus mirabilis].

Preparations of culture liquid of three Bacullus licheniformis strains (S, 103, and 60.4) and the enzymatic preparation lysoamidase from culture liquid of Lysobacter sp. strain XL1 actively lysed preliminarily autoclaved cells of gram-negative bacteria Proteus vulgaris and P. mirabilis. Living Proteus cells treated with these enzymatic preparations were lysed during their subsequent autoclaving. Inoculation of enzyme-treated Proteus cells, taken either separately or in combination with one another and polymyxin B, into a rich medium led to cell repair and restoration of viability of culture.

[Preparation of an active strain of Bacillus licheniformis--producer of thermostable alpha-amylase]. / Poluchenie aktivnogo shtamma Bacillus licheniformis--produtsenta termostabil'noi alpha-amilazy.

A highly potent strain of Bacillus licheniformis 103 that synthesized thermostable alpha-amylase with temperature and pH optima of 90-95 degrees C and 6.0-8.5, respectively, was obtained by mutagenesis and selection. The composition of fermentation media and conditions for submerged cultivation of the producer were optimized. alpha-Amylase whose activity reached 260 U/ml was obtained in laboratory fermenters.