Abstract (English):
Bacteriocins are antibacterial, mainly complex, substances of protein nature. The promising strains producing bacteriocins used in the food industry are lactic acid microorganisms. This study examines the development of a technology for the production of a recombinant peptide with broad-spectrum antimicrobial properties. An important step is the isolation and purification of the recombinant peptide. It has been proved that the highest antimicrobial activity is manifested by a recombinant peptide isolated by a method based on salting out with ammonium sulfate. During the purification of the recombinant bacteriocin preparation, three kinds of columns were used. In the purification process, the volume of bacteriocin produced decreases 3-fold, while the RU/mL increases 3-fold, and RU/mg increases 6-fold. Purification allows the use of a smaller amount of recombinant bacteriocin in technologies with greater efficacy. Based on the results of determining the molecular weight and purity of the recombinant bacteriocin, it was found that the molecular weight of the recombinant bacteriocin having the amino acid sequence: KYYGNGVTCCKHSCSVDXGKASSCIINNGAMAXATGGH GGNHCCGMSRYIQGIPDFLRGYLHGISSANKHKKGRL, is 13 kDa. A technology for the preparation of a broad-action antimicrobial spectrum peptide has been developed. The process of production of antimicrobial peptide includes such stages as: cultivation of the recombinant strain of Escherichia coli BL21DE3; separation of biomass from the nutrient medium; precipitation of bacteriocins by ammonium sulfate; centrifugation; washing the precipitate; centrifugation at 4200 rpm and separation of the preparation; purification of bacteriocins by HPLC method; packing in bags of polymeric and combined materials; storage at a temperature of 18±2°C for 12 months.

Keywords:
Peptide, production, antimicrobial properties, lactobacilli, biosynthesis, action spectrum

INTRODUCTION Bacteriocins and bacteriocin-like substances are complex substances of a protein nature that possess antibacterial properties. Bacteriocins may differ in many respects: biochemical properties, activity spectrum, genetic control, mode of action. We can say that many bacteriocins have a fairly broad action spectrum, but they also act against closely related bacterial species [10, 18]. According to some sources, bacteriocins are antibiotics, among which there are polypeptide substances. These substances differ from each other in that the bacteriocins are formed on the ribosomes, and antibiotics are formed outside the ribosome under the action of certain enzymes. Recently there has been a widespread allergization of the human body, suppression of immunity factors, rapid formation of drug-resistant forms. This leads to a catastrophic shortage of antibiotics on the planet and the search for new modern pharmaceuticals. A special place in this process is assigned to bacteriocins. It should also be noted that one of the topical issues for countries and regions with a well-developed industrial production of food products is the search for effective and safe drugs that increase the shelf life and sale of these products. Therefore, in order to replace known chemical preservatives, bacterium-producers and bacteriocins actively began to be used. In the food industry, the most promising strains producing bacteriocins are lactic acid microorganisms, the areas of their use are very wide. The first person who began to investigate these microorganisms was Louis Pasteur, so today lactic acid bacteria are well known and well-studied. Gradual development of microbiology every year only expands and improves the possible areas of use of lactic acid bacteria. There are entire industrial productions for their use [3, 4, 7, 12]. Antimicrobial properties of lactic acid microorganisms have been used for many years in various spheres of life. For example, to increase the shelf life of food products through fermentation processes due to the formation of hydroxypropionic acid with a decrease in pH, as well as bacteriocins with antimicrobial properties on certain groups of bacteria, incl. pathogenic microorganisms formed in food products during storage and producing exotoxins that cause gastrointestinal diseases. Please cite this article in press as: Prosekov A.Yu., Babich O. O., Milenteva I.S., and Ivanova S.A. Development of recombinant peptide technology with antimicrobial properties of a broad action spectrum. Science Evolution, 2017, vol. 2, no. 2, pp. 3-14. DOI: 10.21603/2500-1418-2017-2-2-3-14/ Copyright © 2017, Prosekov et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (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. This article is published with open access at http:// science- evolution.ru/. I.I. Mechnikov was the first scientist who discovered this phenomenon and summarized the results of experiments on antagonist microorganisms. The theory of the scientist about the early aging of the population in connection with the steady intoxication of the body with the products of vital activity of intestinal putrefactive microorganisms was recognized by the world and found wide application in practice [2, 6, 15]. Most likely, the ability of bacteria to produce bacteriocins has evolved over time and is conditioned by the desire to adapt to environmental conditions and survive in severe competitive conditions, while taking an appropriate ecological niche. Bacteria synthesizing bacteriocins form a natural microflora of compound feed and various food products, their existence is established in the gastrointestinal tract of both animals and humans in many industrial and natural substrates [2, 14]. However, in this habitat, there are always other saprophyte bacteria that prevail over them, including spore and spontaneous forms, cocci, molds, yeasts, gram-negative bacteria that cause food spoilage during prolonged storage, and which are capable to release inhibitors. The formation of bacteriocins is a feature of organisms that is inherited, consisting in the fact that all strains have the ability to synthesize one or more bacteriocinogenic substances that are strictly defined and specific for it. Synthesis of bacteriocin can be caused by methods of genetic engineering, by many physicochemical effects: peroxides, chemical mutagens, ultraviolet rays, DNA-tropic substances and other methods. The selection of bacteriocins began more than 50 years ago, but it was soon suspended [11, 16, 19]. Formation of bacteriocins was established in the following genera of gram-positive bacteria: Strеptococcus, Bacillus, Micrococcus, Clostridium, Leuconostoc, Mycobacterium, Lactobacillus, Staphylococcus, Sarcina, Corynebacterium, as well as Streptomyces, which synthesize bacteriocin-like compounds. Representatives of lactic acid bacteria are able to synthesize a wide range of bacteriocins: lactococcin, curvacine, plantazin, diacetin, acidodicin, lactocin, plantaricin, etc., from which two groups can be distinguished. The first group includes representatives with a narrow spectrum of antibacterial action - they cause the death of organisms close to the producing organism. These include amylovorin L471, lactocin B and F-27, pedicin N5P, thermophilin A, amylovorin, enterococcin, curvacin A. The second group includes such bacteriocins that inhibit the development of most types of gram-positive microorganisms, including Listeria monocytogenes, Pediococcus acidilactici, Clostridium botulinum, Bacillus spp., Staphylococcus aureus, Enterococcus faecalis, Clostridium sporogenes. These bacteria affect food products, causing their damage. Among them, pathogenic species are distinguished. The bacteriocins of this group include: plantaricin C, pedicin A, mutacin, acidodin B, salivarcine, diacetin B-1, curvacin FS47, sarcacin 674, lacticin 3147, enterococci, nisin. It is proved that most of these bacteriocins do not have toxic and immunogenic properties [1, 6, 17]. Such properties as resistance to high temperatures and inactivation by proteinases characterize their peptide or protein nature. Observation of the synthesis of bacteriocins under certain conditions with lactic acid microorganisms Leuconostoc carnosum, Leuconostoc mesenteroides, Pediococcus acidilocali, Lactococcus lactis, allowed to establish that the conditions under which this process occurs determine the maximum formation of these compounds [13, 17]. Time of the producer incubation, composition of the nutrient medium, temperature, pH, time of incubation of the producer - these cultivation conditions have a direct effect on the biosynthesis of bacteriocins. Some bacteriocins produced by lactic bacteria have been studied very well. Thus, it has been established that Lactococcus lactis is capable of synthesizing bacteriocin diacetin B-1. After isolation and purification, the molecular weight of this bacteriocin was 4300 Da, and 37 amino acid residues were found in the primary structure. Those bacteriocins that are synthesized by strains of lactobacillus L. Lactis, have a broad action spectrum and can impede the growth and development of various species of listeria, including Listeria innocua, and L. Plantarum, P. acidilactici, S. Aureus. Mutacine from Streptococcus mutans (molecular weight 3500 Da) is active against a number of gram- positive bacteria, resistant to high temperatures, sensitive to proteolytic enzymes [7, 9]. Bacteriocin amylovorin 471 under certain conditions is formed by Lactobacillus amylovorus DCE 471. It is mainly used as biopreservative of food and feed products. Bacteriocins are known which are able to exert an inhibitory effect on listeria: enterococcin, curvacin FS47, pedicin, diacetin B-1, sacacine. Some lactic bacteria also possess a similar effect: Leuconostoc gelidum, Lactobacillus hetveticus, L. sake. In the 60s of the last century, a bacteriocin-like substance with a broad spectrum of antimicrobial action and not having a toxic effect on a living organism was isolated from enterococcus. It is shown that enterocins (bacteriocins from enterococci) are able to prevent the development of infection when guinea pigs are infected with Clostridium septicum [18, 19, 21]. The properties and structure of bacteriocin Enterocin A (molecular weight 4289 Da, 47 amino acid residues) isolated from Enterococcus faecium are fully analyzed. In terms of the sequence of amino acids, enterocin A is similar to bacteriocins from lactic acid bacteria, for example, nisin. Enterococci of other species are also capable of synthesizing bacteriocins. E. faecalis S-48 forms bacteriocin with a molecular weight of 80 kDa; the substance is sensitive to proteases, has a narrow spectrum of action - inhibits only E. faecalis. Many bacteriocins produced by lactic acid microorganisms have prospects for use in the food industry [5]. Thus, producers of such bacteriocins act as starter cultures in many food industries. Under the circumstances, bacteriocins favor the development of the necessary microflora and inhibition of foreign bacteria that makes microbiological processes safe. For example, in the process of obtaining dry sausage products, Lactobacillus curvatus, which produces bacteriocin - curvacine, prevents the development of undesirable microflora, while ensuring the safety of the maturation process, acts as a starter culture [8, 15]. At different stages of maturation of sausages of salami varieties, a number of lactic acid bacteria such as Lactobacillus plantarum and L. curvatus, Leuconostoc sp., forming bacteriocin-like compounds that inhibit the growth of extraneous microflora, have been isolated, thus contributing to the safety of the product produced. In the process of obtaining green olives (Spain), the bacteriocin producer Lactobacillus plantarum is used as a starter culture [1, 10, 13]. Producing bacteriocin Streptococcus salivarius is used as a starter culture in the production of yoghurts. The stages of a starter making are presented in a joint use of Lactobacillus delbrueckii. This method ensures the safe storage and transportation of products. Consequently, on the basis of the above research, it has been proved that the use of bacteria producing bacteriocins as a starter ensures the safety of the finished products [9, 14]. Production of yoghurt by the starter culture of Pediococcus acidilactici, which produces bacteriocin PA-1, is economical and simple enough. Under the influence of temperatures on the culture liquid, the yoghurt starter culture is grown to produce yoghurt, which is subsequently dried and used to create food products [12, 17]. Protection of food and fermented products can occur due to bacteriocins. Bacteriocins and their producing strains (lactic acid bacteria) can be used as natural preservatives for food products. The main goal is to optimize the synthesis of bacteriocins by microorganisms, increase their stability and activity, and directional isolation of bacteriocins with specified properties. But at the same time, the use of bacteriocins as food preservatives is complicated by the fact that bacteria that are resistant to bacteriocins appear and are synthesized. Lactic acid bacteria with antimicrobial properties (Pediococcus, Lactobacillus, Lactococcus, Leuconostoc) can also be used in canned food production. Some bacteriocins, which are lantibiotics, are isolated from lactic acid bacteria. Lantibiotics are bacterial polypeptides that contain rare thioester amino acids like lanthionine and methyllanthionine with a broad spectrum of antimicrobial activity. Lantibiotics, according to the mechanism of biosynthesis, are divided into groups: nisin and subtilis produced by B. subtilis, which are synthesized on ribosomes, and lantibiotics, whose biosynthesis does not occur on ribosomes. The mechanism of the biological effect of lantibiotics, including nisin, is associated with impaired permeability of bacterial cytoplasmic membranes. The disturbance of the membrane potential is initiated by the formation of pores through which the molecules of lantibiotics pass [21]. The lantibiotics formed by lactic acid bacteria include: variacin, lacticin 481, streptococcin AFF22, salivarimycin A, lacticin 3147, bacteriocin from Streptococcus salivarius SBT1277, nisin. One structural group of lantibiotics includes salivarimycin A, lacticin 481, varienzin, streptococcin AFF22. It was found that lacticin 481 consists of 27 amino acid residues, including methyllanthionine, dehydrobutyrin and lanthionine. The complete structure of the compound was also studied. This group of bacteriocins can be used in the canned- foods industry [11, 18]. The synthesized L. lactis 3147, lactitin 3147 has a broad spectrum of antimicrobial activity and resistance to elevated temperature. The producing strain is extracted from kefir grains and is used to create cheddar cheese. Methods of genetic engineering allow to optimize the production process in some way. Thus, a plasmid carrying the genetic determinants of lacticin 3147 was introduced into the production starter strain L. lactis subsp. cremoris 4268; the resulting transconjugant was used in the starter for the production of cheddar cheese. For half a year, while the cheese was at the stage of maturation, the bacteriocin content did not change and was in proportion to the foreign lactic acid microorganisms. Thus, these starter cultures can serve as a way to control the development of microflora in maturing fermented products [8, 13, 16]. Nisin is the most studied and safe biological preservative (E234). Since 1997, it is the only antibiotic recognized by the European Parliament as safe. It is a membranotropic polypeptide that inhibits the development of bacteria due to disruption of the formation of the membrane potential. In the course of observation of the action of bacteriocin, it was established that it acts on the cytoplasmic membrane. Nisins A, B, C, D, E, Z, R, Q and similar lactitins are well-known. These bacteriocins differ in the composition of amino acids, physicochemical properties, spectra of antimicrobial activity. Their producers are different strains of the same species Lactococcus lactis subsp. lactis [19, 20]. A peptide antibiotic (nisin Z) was also isolated from the L. lactis 10-1. It suppresses the growth of various gram-positive bacteria, but not the strain-producer of nisin A. Its molecular weight is 3337.9 Da, its structure differs from that of nisin A by one amino acid substitution (histidine-27 to asparagine-27). The substance is a lantibiotic [6, 7]. From the foregoing it follows that at the current stage of the research only a small part of the bacteriocins of lactobacilli have been isolated, purified and characterized. The use of bacteriocin-producing starter cultures in fermented food products is investigated not only in vitro, but also with the help of pilot lines. The obtained results of experiments emphasize one of the main roles of lactic acid microorganisms synthesizing bacteriocins in preserving and improving the quality of finished products and their safety during production and storage. The search for natural preservatives that meet the requirements of consumers and producers is an urgent issue for the Russian Federation. Unfortunately, the study of bacteriocins in Russia is insufficient. Based on the foregoing, the aim of the work was to develop a technology for producing a peptide of a broad antibacterial action spectrum. MATERIALS AND METHODS The object of research was a recombinant strain- superproducent antimicrobial peptide obtained by transformed cells Escherichia coli BL21DE3. The optimal method for isolation of bacteriocins was selected by three methods. The first method consisted in concentrating the culture liquid on hollow fibers, followed by the addition of dry NaCl and shaking with further centrifugation of the suspension. The pH of the supernatant was adjusted to 3.0, the resulting suspension was centrifuged, water was added to the precipitate, then the precipitate was suspended, alcohol was added, the mixture was incubated at 0 ± 2 C for 30 minutes and centrifuged. The alcohol was removed from the solution by evaporation, water and activated charcoal were added, the mixture was centrifuged with removal of the adsorbed impurities on the charcoal, after which the aqueous solution was passed through the membrane. The second method of isolating bacteriocins was to separate the cells from the cultural liquid by centrifugation at 4200 rpm for 30 minutes. Bacteriocins were precipitated by ammonium sulfate to 90% of the saturating concentration. Precipitate and cultural liquid were separated by centrifugation at 4200 rpm for 40 min. The precipitate was dissolved in 20 mmol acetate buffer рН 5.0. Undissolved precipitate was separated by centrifugation at 4200 rpm for 30 minutes. The precipitate was then washed again in 20 mmol acetate buffer (pH 5.0) and repeatedly separated by centrifugation. The precipitate was discarded, and the product obtained as a result of two washes was used to determine the antimicrobial activity. The third method of isolation of bacteriocins was carried out as follows: the supernatant was added with chloroform and agitated vigorously on a magnetic stirrer for 20 minutes. The mixture was then centrifuged at 10.400 g for 20 minutes at a temperature of 1 ± 2°C. The upper aqueous layer was carefully poured off, keeping the floating precipitate in a pipette, and the organic layer was drained off. For resuspension, a buffer of 5-10 ml Tris 0.1 mole, pH 7.0 was used. The contents of the vials (precipitates, surface precipitate, chloroform and cultural medium residues) and mixtures were stirred and centrifuged again at 12.100 g for 15 min. The precipitate was separated. Antimicrobial activity was determined using the diffusion method. The bacilli were grown on a medium prepared on a Hottinger broth in the ratio of 1 to 2 with the following composition (g/l): glucose - 10.0; NaCl - 2.0; agar - 20.0; E. coli was grown on MPA medium. The fungi were grown on a Saburo medium (g/l): glucose - 40.0; peptone - 10.0; agar - 20.0. Bacilli, staphylococci and micrococci were cultivated at 37°C, E. coli at 42°C, B. coagulans at 55°C, microscopic fungi, including yeast, at 28°C. For seeding Petri dishes with test cultures, daily cultures of bacteria and five-day cultures of fungi in the form of a suspension of cells and spores (respectively) in phosphate buffer (pH 5.5) in the amount of 1·109 cells/ml by bacterial turbidity standard (optical turbidity standard from L.A. Tarasevich State Research Institute for Standardization and Control of Medical Biological Preparations) were used. For the experiment, a solid nutrient medium was poured into Petri dishes and the same wells were made at a distance of 20 mm from the edge of the dish into which the recombinant peptide suspension was placed. The zone of growth retardation made it possible to judge the antibiotic activity of the strain under investigation against different groups of microorganisms. Solutions of 10, 20, 30, 40, 50 IU/ml of Nisaplin commercial drug (Aplin & Barrett, Ltd, UK) with activity of 1000 IU/mg, for fungi - nystatin (Sigma) with an activity of 4670 IU/mg were used as a standard. Purification of the recombinant bacteriocin was carried out by HPLC method. The amount of bacteriocin forming the lysis zone with a diameter of 1 cm was taken as a relative unit of activity. Specific activity was calculated as the ratio of activity to protein concentration. Purification was carried out using the AKTAfplc system (Amersham Biosciense, Sweden). In the first stage of purification, a XK16 column with a Phenyl Sepharose 6 Fast Flow carrier was used. The Phenylsepharose column was balanced by initial buffer - 20 mmol acetate buffer, pH 5.0 + 1 mole (NH4)2SO4. (NH4)2SO4 concentration in the applied preparation was made up to 1 mol. The preparation was applied onto the column at 3 ml/min. The column was washed with two volumes of the initial buffer and two volumes of 20 mmol acetate buffer, pH 5.0. The elution of the recombinant bacteriocin from the hydrophobic carrier was carried out with an ethanol gradient of 0-50% for 0.5 column volume (20 ml), and an elution rate of 3 ml. The second stage of purification was performed on an Octyl HR 16/60 column (GE Healthcare, USA). The column was balanced by initial buffer: 20 mmol acetate buffer рН 5.0 + 1 mol (NH4)2SO4. (NH4)2SO4 concentration in the applied preparation was made up to 1 mol. The preparation was applied onto the column at 1 ml/min. The column was then washed with two volumes of the initial buffer to remove components that had not contacted the carrier, and were washed with two volumes of 20 mmol acetate buffer, pH 5.0. Elution of bacteriocins was carried out with a gradient of 0-50% ethanol. The length of the gradient was 0.3 column volume (8 ml). The final stage of purification was performed at ENRichS column (BioRad, USA). The column was balanced by initial buffer - 20 mmol acetate buffer, pH 5.0. The application rate was 1 ml/min. Further, the column was washed with three volumes of the initial buffer and three volumes of buffer - 20 mmol acetate buffer, pH 5.0 + 1 mol NaCl. Elution of recombinant bacteriocin from the column was carried out with a linear gradient of ethanol, the length of the gradient was 8 ml. Purification of bacteriocins was carried out by a second method, using ammonium sulfate precipitation. To determine the molecular weight of the preparation of bacteriocins and the degree of purity, electrophoresis was performed in the Tricin-SDN system in 16% PAAG using a Mini-PROTEAN II device (BioRad, USA). The preparation of the buffer consists in weighing of glycine - 28.80 g; tris - 6.05 g, then adding a solution of 20% sodium disulphate - 20.00 ml and bringing distilled water to 2000.00 ml. Before use, the solution is diluted two-fold. To obtain a 2% sample buffer, add the following reagents to the eppendorf tube: mercaptoethanol - 100 μl; 10% solution of sodium dodecyl sulfate (SDS) - 40.00 μl; 50% glycerol solution - 40.00 μl; bromophenol blue - 50.00 μl; 1 mol Tris-HCl pH 6.8 - 50.00 μl. A solution for fixing and staining the gel is prepared using: 125 ml of isopropanol; 50 ml of acetic acid; 325 ml of water; 1.25 g Coomassie R250, washing solution: 50 ml of acetic acid; 50 ml of isopropanol; 400 ml of water. To prepare the samples, 20 μl of protein, 10 μl of sample buffer, 10 μl of distilled water were added to the eppendorf tube. The samples were then vortexed and heated to 95C for 5 minutes, then again mixed and added to the sample wells. After electrophoresis, staining and washing of the gel were performed. The following markers were used, kDa: 1 - myoglobin - 16.95; 2 - alpha-lactalbumin- 14.437; 3 - aprotinin - 6.512; 4 - oxidized insulin b- chain - 3.496. To determine the amino acid sequence, 10 μl of 0.1% TFA was added to 10 μl of the sample, washing was carried out in 5 volumes (10 μl) of 0.1% TFA and elution with 10 μl of 60% acetonitrile in 0.1% TFA. Further, the samples were dried and subjected to proteolysis with 0.1 M HCl. Hydrolysis was carried out for 5 hours at 37°C, then the proteolysis was stopped by the addition of 20 μl of 0.5% NaOH in a 10% solution of aqueous acetonitrile. This solution was used to prepare MALDI-mass spectra. On a mass spectrometric target, 1 μl of sample solution and 0.5 μl of a solution of 2,5-dihydroxybenzoic acid (Aldrich, 20 mg/ml in 20% aqueous acetonitrile, 0.5% TFA) were mixed, and the resulting mixture was air dried. Mass spectra were obtained on a MALDI-time-of-flight mass spectrometer made by Ultraflextreme BRUKER (Germany) equipped with a UV laser (Nd) in a positive ion mode using a reflectron; the accuracy of the measured monoisotopic masses after the calibration by trypsin autolysis peaks was 0.005% (50 ppm). Spectra were obtained in the range of 600-5000 m/z, choosing the laser power, which is optimal for achieving the best resolution. To obtain the fragmentation spectra, the tandem mode of the instrument was used, the accuracy of measuring the fragment ions was at least 1 Da. Thermostability of the recombinant antimicrobial peptide was researched by the diffusion method. Strains of Bacillus subtilis ATCC 6633 and Escherichia coli B-6954 were used as test cultures. Processing of experimental data obtained during the research was carried out by standard methods of regression and correlation analysis using the Microsoft Office Excel 2010 and Statistica 6.0 application software package. To compare the reproducibility characterizing the proximity to each other of the results obtained in performing the research under different conditions, the F- ratio test was used. RESULTS AND DISCUSSION The main stage of the process of microbial synthesis is the stage of cultivation of the producer strain. In the process of cultivation, the biosynthesis of the target metabolites occurs. For this purpose, the cultivation conditions (nutrient medium composition, duration, temperature, aeration, mixing parameters) of the recombinant super-producer strain of the antimicrobial peptide optimized to ensure the maximum yield of the desired product were optimized. The most accessible sources of carbon and nitrogen were selected. The selection of the nutrient medium was carried out based on the yield of the recombinant protein in the medium for 4- 20 hours, measuring the composition every 2 hours. Extraction and purification of the recombinant peptide with antimicrobial properties consisted in the selection of the optimal of the three proposed methods. The cultures were incubated for 24 hours at 37°C. At the end of the incubation, growth inhibition in the medium was detected due to the bacteriostatic action of the recombinant peptide. With a diameter of the growth inhibition zone up to 10 mm, the culture was classified as insensitive, 11-14 mm - as a medium sensitive and more than 15 mm - as highly sensitive. Findings are presented in Table 1. Table 1. Diameters of the zones of growth inhibition of test cultures due to the bacteriostatic action of the recombinant peptide obtained as a result of different methods of isolation Test culture Strain characteristic Growth inhibition zone diameter, mm Method 1 Method 2 Method 3 Bacillus subtilis ATCC 6633 Gram+ 19.8 26.0 21.7 Bacillus coagulans B3042 Gram+ 11.1 17.3 13.0 Bacillus fastidiosus B-5651 Gram+ 19.1 25.2 20.9 Bacillus cereus B-2893 Gram+ 18.2 24.4 20.3 Leuconostoc mesenteroides B-8404 Gram+ 17.3 23.5 19.2 Micrococcus luteus B7846 Gram+ 15.3 21.8 17.5 Staphylococcus aureus ATCC 25923 Gram+ 18.9 25.1 20.8 Comamonas terrigena ATCC 8461 Gram- 10.0 16.3 12.4 Escherichia coli B-6954 Gram- 12.5 18.7 14.2 Pseudomonas fluorescens B-3502 Gram- 11.5 17.7 13.5 Pseudomonas aeruginosa ATCC 9027 Gram- 8.4 15.0 10.3 Fusarium verticilloides F446 Microscopic fungi 1.8 5.0 3.7 Candida albicans ATCC 885-653 Microscopic fungi 0.8 4.0 2.2 Candida tropicales Y1633 Microscopic fungi 1.0 5.0 2.5 Aspergillus niger F586 Microscopic fungi 0 4.6 2.3 Penicillium chrysogenum F605 Microscopic fungi 1.3 5.5 3.5 Note. * diameter of wells: 7 mm. In case of the first method of isolation, the findings shown in Table 1 indicate that the recombinant peptide has the maximum inhibitory effect on gram-positive strains, the inhibition zone of which varies from 11.1 to 19.8 mm (with a well diameter of 7 mm). The recombinant peptide acts less efficiently against gram- negative strains whose inhibition zone varies from 8.4 to 12.5 mm and the recombinant peptide is the least efficient in the inhibition of the growth of microscopic fungi which inhibition zone varies from 0 to 1.8 mm. In case of the second method of isolation, the recombinant peptide exhibits a higher antibacterial activity than all other methods against all test cultures. The diameter of the zones of gram-negative bacteria growth inhibition is: Comamonas terrigena ATCC 8461 - 16.5 mm, Escherichia coli B-6954 - 18.5 mm, Pseudomonas fluorescens B-3502 - 17.5 mm, Pseudomonas aeruginosa ATCC 9027 - 15.0 mm. Antibacterial peptide has a less pronounced effect on the suspensions of microscopic fungi: Fusarium verticilloides F446 - 5.0 mm, Candida albicans ATCC 885-653 - 4.0 mm, Candida tropicales Y1633 - 5.0 mm, Aspergillus niger F586 - 4.6 mm, Penicillium chrysogenum F605 - 5.5 mm. The most sensitive to the action of the recombinant peptide were gram-positive microorganisms which diameter of inhibition zones was: Bacillus subtilis ATCC 6633 - 26.0 mm, Bacillus coagulans B3042 - 17.5 mm, Bacillus fastidiosus B- 5651 - 25.5 mm, Bacillus cereus B-2893 - 24.5 mm, Leuconostoc mesenteroides B-8404 - 23.5 mm, Micrococcus luteus B7846 - 21.5 mm, Staphylococcus aureus ATCC 25923 - 25.5 mm. Using the third method of isolation, the recombinant peptide showed larger growth inhibition zones compared to the first type of isolation, which was from 17.5 to 21.7 mm for gram-positive bacteria, from 10.3 to 14.2 mm for gram-negative bacteria and from 2.2 up to 3.7 mm for microscopic fungi. However, these results are less effective than in the case of the second method for extracting the recombinant peptide. The results given in Table 1.1 show that the highest antimicrobial activity is manifested by a recombinant peptide extracted by a second method based on salting out by ammonium sulfate. The minimum antimicrobial activity is observed in the isolation of bacteriocins by the first method, based on the concentration of the cultural liquid on hollow fibers. Based on the results obtained, a method for salting out by ammonium sulfate was chosen to further isolate the bacteriocins. As a result of the study of the antibacterial action spectrum, it was revealed that the recombinant peptide exerts the strongest action against gram-positive bacteria, less pronounced action against gram-negative bacteria and weak action against microscopic fungi. The recombinant peptide exerts the most powerful action against strains of Bacillus subtilis ATCC 6633, Bacillus fastidiosus B-5651, Bacillus cereus B-2893, Leuconostoc mesenteroides B-8404 and Staphylococcus aureus ATCC 25923. The peptide showed the weakest antibacterial activity against Candida albicans ATCC 885-653, Candida tropicales Y1633 and Aspergillus niger F586. To further work with the preparation of recombinant bacteriocin, its activity was determined. The results of determining the activity of the bacteriocin preparation, as well as data on the concentration and final yield of bacteriocin are given in Table 2. The data given in Table 2 indicate that three kinds of columns were used to purify the recombinant bacteriocin preparation. In the first stage of purification on a PhenylSepharose column with a hydrophobic carrier, the recombinant peptide was eluted by ethanol and was not eluted by an aqueous buffer solution, indicating their hydrophobic nature. During this purification stage, a recombinant bacteriocin preparation was prepared in a volume of 18 ml, the activity of the preparation was 523 RU/ ml, the total activity was 9414 RU, the protein concentration was 1.9 mg/ml, the specific activity was 275.3 RU/mg. According to the results of the second stage of purification of bacteriocins on a column with Octyl HR 16/60 carrier, it was found that bacteriocins, just as in the first stage of purification, were eluted only by ethanol that confirms their hydrophobic nature. As a result of the second stage of purification, recombinant bacteriocin preparation was prepared in a volume of 12 ml, the activity of the preparation was 1021 RU/ml, the total activity was 12252 RU, the concentration of the protein was 1.6 mg/ml, the specific activity was 638.1 RU/mg. At the final stage of purification using the ENRich S anion exchange column, it was found that the recombinant bacteriocin was not eluted by a sodium chloride solution, but was eluted by ethanol. This fact additionally indicates the hydrophobic nature of the bacteriocins of this strain. After this stage, the protein concentration was 0.9 mg/ml, activity - 1546 RU/ml. The total activity of bacteriocins after the final stage of purification was 9276 RU, the specific activity was 1717.8 RU/mg. In the purification process, the volume of bacteriocin produced decreases 3-fold, while the RU/mL increases 3-fold, and RU/mg increases 6-fold. Purification allows the use of a smaller amount of recombinant bacteriocin in technologies with greater efficacy. The results of determining the molecular weight and purity of recombinant bacteriocin are shown in Fig. 1. When analyzing the results obtained, it was concluded that the molecular weight of the recombinant bacteriocin is 13 kDa. No trace amounts of other proteins were found, which indicates a high degree of purity of the preparation obtained. Further, the physico-chemical properties of the purified recombinant peptide were evaluated. As a result of the determination, the following sequence of recombinant bacteriocin was obtained: KYYGNGVTCCKHSCSVDXGKASSCIINNGAMAXA TGGHGGNHCCGMSRYIQGIPDFLRGYLHGISSANK HKKGRL. The amino acid composition of the recombinant peptide is shown in Table 3. Table 2. Results of purification of the recombinant bacteriocin preparation Column used Volume, ml Activity, RU/ml Total activity, RU Protein concentration, mg/ml Specific activity, RU/mg Phenyl Sepharose 18 523 9414 1.9 275.3 Octyl HR 16/60 12 1021 12252 1.6 638.1 ENRich S 6 1546 9276 0.9 1717.8 1 2 16.950 14.437 6.512 3.496 Fig. 1. Results of electrophoresis of recombinant bacteriocin: (1) markers, kDa; (2) purified recombinant bacteriocin preparation. Table 3. Amino acid composition of the recombinant peptide Amino acid Designation Number in structure % of the total number of amino acids Glycine G 13 17.3 Serine S 7 9.3 Cysteine C 6 8.0 Lysine К 6 8.0 Alanine A 5 6.7 Histidine H 5 6.7 Isoleucine I 5 6.7 Asparagine N 5 6.7 Thirosine Y 4 5.3 Arginine R 3 4.0 Leucine L 3 4.0 Methionine M 2 2.7 Valine V 2 2.7 Threonine T 2 2.7 Asparagine acid D 2 2.7 Not specified X 2 2.7 Glutamine Q 1 1.3 Proline P 1 1.3 Phenylalanine F 1 1.3 According to the results presented in Table 3, the highest amount in the composition of recombinant peptide has the amino acid glycine - 17.3%. Serine is present in the amount of 9.3%, cysteine and lysine - 8.0%, alanine, histidine, isoleucine, asparagine - in the amount of 6.7%. The nature of one amino acid could not be established, most likely, it belongs to a non-trivial series of proteinogenic amino acids. According to radical classification, the amino acid refers to the polar uncharged types of amino acids, which also include serine, threonine, cysteine, methionine, asparagine, glutamine. Thermostability of the recombinant antimicrobial peptide was researched by the diffusion method. The recombinant peptide suspension was heated prior to addition to a well at a temperature of 40, 50, 60 and 80C for a period of 2 to 20 minutes with step of 3. The result is shown in Fig. 2. The temperature of 40С is the optimum for the recombinant peptide action, so that the duration of pretreatment at this temperature does not affect the antibacterial activity of the peptide. Partial inactivation of the enzyme occurs at a temperature of 50С. 30 Inhibition zone diameter, mm 25 20 15 10 5 0 2 5 8 11 14 17 20 Duration, min 40С 50С 60С 70С 80С (a) 20 Inhibition zone diameter, mm 18 16 14 12 10 8 6 4 2 0 2 5 8 11 14 17 20 Duration, min 40С 50С 60С 70С 80С (b) Fig. 2. Effect of temperature and duration of treatment on the recombinant peptide activity: (a) for Bacillus subtilis ATCC 6633; (b) for Escherichia coli B-6954. During the 5-minute treatment, no changes in the antibacterial activity of the peptide are observed for both strains. When the enzyme is preheated for more than 5 minutes, the activity decreases and the pathogen inhibition zone reaches 20.2 mm for Bacillus subtilis ATCC 6633, which is 77.7% of the initial value, and 12.9 mm for Escherichia coli B-6954, which is 67% of the value of the inhibition zone at the optimum temperature of the action of the recombinant enzyme. When the enzyme is pretreated for 2 min at a temperature of 60С, the enzyme activity decreases by a factor of 2 and is 16 mm for Bacillus subtilis ATCC 6633 and 10.7 mm for Escherichia coli B-6954. Throughout the time interval, the size of the pathogen inhibition zones is uniformly reduced and the size of wells at 20 minutes of preliminary inactivation of the enzyme is 6.9 mm (26.5%) for Bacillus subtilis ATCC 6633 and 1.6 mm (8.5%) for Escherichia coli B-6954. At a temperature of 70С, the activity of the enzyme decreases from 42.3% (11 mm) at 2 min treatment to 0% at 17 min treatment for Bacillus subtilis ATCC 6633, and from 32% (6 mm) to 0% at 14 min pretreatment for Escherichia coli B-6954. Upon heating to 80С, complete inactivation of the enzyme occurs within 11 minutes (for Escherichia coli B-6954, no inhibition was observed at 8 min). The obtained data testify that the recombinant peptide possesses an increased thermostability, which considerably expands the scope of its use. Within the framework of this work, the effect of proteolytic enzymes on the activity of recombinant peptide was studied. The gastrointestinal tract is an aggressive medium for substances of a protein nature; therefore, to study the possibilities of using a recombinant peptide for medical purposes, the influence of the main types of human proteolytic enzymes on the activity of the recombinant enzyme was analyzed. The following types of recombinant enzymes were studied: Pepsin is a proteolytic enzyme of the hydrolase class (CF 3.4.23.1), produced by the main cells of the gastric mucosa, splits food proteins to peptides. It is present in the gastric juice of humans, mammals, birds, reptiles and most fish. Pepsin is an endopeptidase, that is, an enzyme that splits the central peptide bonds in protein and peptide molecules (except for keratin and other scleroproteins) to form simpler peptides and free amino acids. Pepsin hydrolyses the peptide bonds formed by aromatic amino acids - tyrosine and phenylalanine - with the highest speed, however, unlike other proteolytic enzymes, trypsin and chymotrypsin, it does not have strict specificity. Rennin (chymosin) is an enzyme from the hydrolase class that is produced in the stomach glands of mammals, including humans. In ruminant animals, the abomasum is produced by the rennet stomach glands (the 4th section of the stomach), hence one of its trivial names is the rennet enzyme. The original substrate of rennin is milk protein casein, which under the action of the enzyme is hydrolyzed and splits to an insoluble protein of casein. As a result, the main milk protein remains in the stomach for a long time and is slowly split by pepsin. Trypsin is an enzyme of the hydrolase class, splitting peptides and proteins; it also possesses esterase activity (hydrolysis of esters). It catalyzes the hydrolysis of proteins and peptides, waxes-esters. Trypsins belong to the group of serine proteases and contain the residues of serine and histidine in the active center. Trypsins easily undergo self-digestion (autolysis), which leads to contamination of trypsin preparations with inactive products (the industrial preparation contains up to 50% of inactive impurities). Preparations of high purity trypsin are obtained by chromatographic methods. Carboxypeptidase A (CF 3.4.17.1) is a proteolytic enzyme, exopeptidase. Carboxypeptidase A is synthesized in the pancreas as a pro-enzyme pro- carboxypeptidase A and in this form, in the pancreatic juice enters the duodenum, where, under the action of trypsin, pro- carboxypeptidase A is converted to carboxypeptidase A. Carboxypeptidase A consists of 307 amino acid residues, has a molecular weight of about 34 4000 and splits from peptides all C-amino acid residues, with the exception of arginine, lysine, proline and hydroxyproline, catalyzing mainly aromatic and aliphatic amino acids. Carboxypeptidases belong to Zn-metalloenzymes. In order to analyze the effect of the recombinant peptide, the components were dissolved in a phosphate buffer (pH 5.5). The pH of the buffer was adjusted to 1.7 by addition of HCl in the case of pepsin (Sigma-Aldrich, P7125-500G) to 4.5 in the case of chymosin (Sigma- Aldrich, R4877) by adding CaCl2 to 0.01 M concentration. In the case of trypsin (Sigma-Aldrich, T1426), the pH was adjusted to 7.9 by addition of NaOH, in the case of trypsin supplemented with CaCl2 to 0.01 M, to 7.5 for carboxypeptidase A (Sigma-Aldrich, C9268). All preparations were diluted in the ratio of 1 to 2,000 and incubated with the recombinant peptide for 3 hours at 37С. After that, the action of pepsin and chymosin was inhibited by increasing the pH to a neutral value with NaOH, and the action of trypsin and carboxypeptidase A was neutralized by lowering the pH using HCl to 4.0. The activity of the recombinant peptide after incubation was verified by the diffuse method using strains of Bacillus subtilis ATCC 6633 and Escherichia coli B-6954. Findings are presented in Table 4. According to the data obtained in Table 2, rennin has practically no effect on the antibacterial activity of the recombinant peptide. The effect of pepsin and carboxypeptidase A is approximately equivalent and causes the enzyme activity to drop by 3.2-3.5 and 4.2-4.8%, respectively. The strongest effect was shown by trypsin, the inactivation percentage of which was 6.9-7.4%, respectively. The technological process for producing a broad antimicrobial spectrum peptide is carried out in accordance with the flowchart shown in Fig. 3. The process of production of antimicrobial peptide includes such stages as: cultivation of the recombinant strain Escherichia coli BL21DE3; separation of biomass from the nutrient medium by centrifugation at 4200 rpm for 30 minutes; precipitation of bacteriocins with ammonium sulfate to 90% of the saturating concentration; centrifugation at 4200 rpm for 40 minutes, dissolving the precipitate in 20 mmol acetate buffer, pH 5.0; washing the precipitate in 20 mmol acetate buffer, pH 5.0; centrifugation at 4200 rpm for 40 minutes and separation of the preparation; purification of bacteriocins by HPLC method; packing in bags from polymeric and combined materials; storage at a temperature of 18 ± 2°C for 12 months. The recipe for the preparation of the recombinant peptide includes the reagents necessary to produce 1000 dm3 of cultural medium for the cultivation of microorganisms, and the reagents necessary for purification of the cultural liquid. The reagents needed to produce a broad antimicrobial spectrum peptide are shown in Table 5. Table 4. Effect of proteolytic enzymes on the activity of the recombinant peptide Factor Bacillus subtilis ATCC 6633 Escherichia coli B-6954 Inhibition zone diameter % inactivation from control Inhibition zone diameter % inactivation from control Pepsin 25.2 3.5 18.2 3.2 Rennin 26.0 0.4 18.7 0.5 Trypsin 24.3 6.9 17.4 7.4 Carboxypeptidase A 25.0 4.2 17.9 4.8 Control 26.1 0.0 18.8 0.0 Cultivation of the recombinant strain Escherichia coli BL21DE3: 40 ± 2°С, 18 h, 200 rpm и 1 l/l·min Separation of biomass from the nutrient medium by centrifugation (4200 rpm, 30 min) Precipitation of bacteriocins with ammonium sulfate to 90% of the saturating concentration Centrifugation (4200 rpm, 40 min) Dissolution of the precipitate in 20 mmol acetate buffer, pH 5.0 Separation of the insoluble precipitate by centrifugation (4200 rpm, 30 min) Washing of the sediment in 20 mmol acetate buffer, pH 5.0 Centrifugation (4200 rpm, 40 min) and separation of the formed preparation Purification of recombinant bacteriocin by HPLC method, column XK16 with Phenyl Sepharose 6 Fast Flow, Octyl HR 16/60, ENrich S carrier was balanced by initial buffer: acetate buffer рН 5.0 + 1М (NH4)2SO4, application rate 1-3 ml/min, washed with acetate and initial buffer, elution was carried out with an ethanol gradient of 0-50%, a gradient length is 3-8 ml Packing in bags of polymeric and combined materials Storage at a temperature of 18 ± 2 C for 12 months Fig. 4. Process flowsheet for the production of a broad antimicrobial spectrum peptide. Table 5. Reagents required to prepare a purified recombinant peptide preparation Components Weight, kg (dm3) Composition of the cultural medium for strain culturing trypton 12.0 saccharose 12.0 acetic sodium 0.5 magnesium sulfate 0.2 ammonium sulfate 0.5 sodium chloride 6.0 calcium chloride 2.0 Reagents for recombinant peptide purification Sodium acetate 5.6 Acetic acid 2.5 Ammonium sulfate 8.0 Ethanol 8.0 Water 1000.0 CONCLUSIONS AND RECOMMENDATIONS Based on the research, the following results were obtained. A method for the isolation and purification of a recombinant peptide with antimicrobial properties has been developed. The highest antimicrobial activity is manifested by a recombinant peptide extracted by a second method based on salting out by ammonium sulfate. As a result of the study of the antibacterial action spectrum, it was revealed that the recombinant peptide exerts the strongest action against gram-positive bacteria, less pronounced action against gram-negative bacteria and weak action against microscopic fungi. The recombinant peptide exerts the most powerful action against strains of Bacillus subtilis ATCC 6633, Bacillus fastidiosus B-5651, Bacillus cereus B-2893, Leuconostoc mesenteroides B-8404 and Staphylococcus aureus ATCC 25923. The peptide showed the weakest antibacterial activity against Candida albicans ATCC 885-653, Candida tropicales Y1633 and Aspergillus niger F586. During the purification of the recombinant bacteriocin preparation, three kinds of columns were used. In the purification process, the volume of bacteriocin produced decreases 3-fold, while the RU/mL increases 3-fold, and RU/mg increases 6-fold. Purification allows the use of a smaller amount of recombinant bacteriocin in technologies with greater efficacy. The results of determining the molecular weight and purity of recombinant bacteriocin suggest that the molecular weight of the recombinant bacteriocin is 13 kDa. No trace amounts of other proteins were found, which indicates a high degree of purity of the preparation obtained. Based on the studies, the following sequence of recombinant bacteriocin was obtained: KYYGNGVTCCKHSCSVDXGKASSCIINNGAMAXA TGGHGGNHCCGMSRYIQGIPDFLRGYLHGISSANK HKKGRL. A technology for the preparation of a broad-action antimicrobial spectrum peptide has been developed. The process of production of antimicrobial peptide includes such stages as: cultivation of the recombinant strain Escherichia coli BL21DE3; separation of biomass from the nutrient medium; precipitation of bacteriocins with ammonium sulfate; centrifugation; washing the precipitate; centrifugation at 4200 rpm and separation of the preparation; purification of bacteriocins by HPLC method; packing in bags from polymeric and combined materials; storage at a temperature of 18±2°C for 12 months. ACKNOWLEDGEMENT The research was carried out with partial financial support of the Russian Foundation for Basic Research within the framework of the scientific project No. 15-08-02003A.

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