A method of obtaining a preparation - mixture based on purified PGA, especially for use in the production of feed and veterinary products

Abstract

A method of obtaining a preparation - mixture based on purified PGA, especially for use in the production of feed and veterinary products, using at least one bacterium and PGA, characterized in that in the first stage, three bacterial cultures are placed in a sealed bioreactor, preferably in equal proportions, i.e.: 1. Bacillus licheniformis, 2. Bacillus subtilis and 3. Bacillus amyloliquefaciens, preferably in liquid form with a CFU from 1*103 to 1*1010, preferably 1 *108; in the second stage a sterile growth medium for bacteria is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at least 7.0 pH, preferably 8.5 pH; and a temperature not higher than 41°C, preferably 38°C - 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for at least 24 hours; The subject of the invention is also preparations-mixtures obtained with this method, and the method of their filtration.

Description

[0001] A method of obtaining a preparation - mixture based on purified PGA, especially for use in the production of feed and veterinary products

[0002] TECHNICAL FIELD

[0003] The subject of the invention is a method of obtaining a preparation-mixture based on purified PGA, especially for use in the production of feed and veterinary products, a system and a mixture, also called the SANSEVERUM mixture.

[0004] BACKGROUND ART

[0005] Polyglutamic acid (PGA) is a highly anionic, natural polymer produced by bacteria of the genus Bacillus spp as a fermentation product secreted into the growth medium. It is a water-soluble, biodegradable, non-toxic substance. PGA stimulates and improves immune activity, making it suitable for the selective delivery of chemotherapeutic agents (Tork et al., 2015; Chatteijee et al., 2018, 2019; Park et al., 2021). Due to its properties, PGA can be used both as a free polymer, as well as a hydrogel and / or polymer nanoparticles. Currently, it is widely used, among others: in sewage treatment, fertilizers, food products, skin care products, surgical adhesives, tissue engineering, drug delivery and other purposes. PGA also has a strong bactericidal effect, especially against gram-negative bacteria (Richard et al., 2001; Chatteijee et al., 2018, 2019; Park et al., 2021).

[0006] Currently, it is widely used, among others: in sewage treatment, fertilizers, food products, skin care products, surgical adhesives, tissue engineering, drug delivery and other purposes. PGA also has a strong bactericidal effect, especially against gram-negative bacteria (Richard et al., 2001; Chatterjee et al., 2018, 2019; Park et al., 2021).

[0007] One of the model bacteria used in broadly understood microbiological and genetic research is E. coli (latin Escherichia coli). It is a gram-negative, relatively anaerobic bacterium belonging to the Enterobacteriaceae family. It is part of the physiological bacterial flora of the large intestine of a human and other warmblooded animals, participating as a symbiont in the breakdown of food, and contributing to the production of B and K vitamins. However, if the body's homeostasis is disturbed, E. coli may be pathogenic to humans and animals. Young organisms are particularly vulnerable to infection with this bacterium, and its various serotypes may also be present in the bodies of sick animals (Kaper et al., 2004; Jang et al., 2017). It is estimated that in the conditions of mass production of poultry and pigs, about half of the deaths of young animals are caused by colibacillosis (Cortes et al., 2010; Ozaki et al., 2017; Maertens et al., 2020; Newman et al., 2021) The resistance of Escherichia coli to environmental factors is relatively low. It dies after 20 minutes of heating at 60°C and is sensitive to all known disinfectants. However, its survival increases at lower temperatures and high humidity (Cho et al., 2010; Ganzle, 2015; Jang et al., 2017).

[0008] A feed comprising gamma-polyglutamic acid is known from the Japanese patent description No. JPH0928309. A feed containing y-polyglutamic acid and / or its degradation product was obtained, effectively increasing the absorption of minerals in the intestines and / or increasing the strength of the egg shell and / or reducing the deposition of fat tissue, especially intra-abdominal fat.

[0009] The product preferably comprises 0.01-1% by weight of poly-y-glutamic acid and / or its degradation product, y-polyglutamic acid can be extracted and purified from the mucilage substance NATTO (fermented soybeans) and / or extracted and purified from a substance secreted from a cell of a microorganism of the genus Bacillus, among others Bacillus natto. Preferably, the molecular weight of y-polyglutamic acid is 10,000-1,000,000, the calcium content in the raw material is 0.1-5% by weight, and the iron content in the raw material is 0.005-0.05% by weight %.

[0010] A feed composition containing poly-gamma-glutamic acid is known from the Japanese patent description No. JPH10117701. To obtain a poly-y-glutamic acid-comprising feed composition comprising y-polyglutamic acid (or its decomposition product) to reduce the phosphorus content in the mouths and at the same time increase the suitability of phosphorus in feeds, thereby increasing the yield of slaughtered carcasses. SOLUTION: This feed composition comprising poly-y-glutamic acid, which preferably comprises 0.01-3 % by weight of polyglutamic acid obtained from Bacillus fermentation liquid and / or its dried product, is preferably obtained by adjusting the pH of poly-y- glutamic acid solution comprising Bacillus strains to 1.5-3.5, adding sodium chloride to the solution and separating the salted poly-y- acid glutamic acid from the mycobacterial bodies without any additional treatment and / or with additional drying of the mycobacterial bodies which are comprised in y-polyglutamic acid. A composition comprising poly-y-glutamic acid for cell protection in cell culture is known from the Korean patent description No. KR20110017233.

[0011] The provided composition comprising gamma-polyglutamic acid for addition to the growth medium is intended to induce a reduction in shear force in a serum- and / or protein-free growth medium for animal cells and to improve cell growth. CONSTITUTION: The composition added to the medium to prevent cell damage comprises gamma-polyglutamic acid. The growth medium is a growth medium comprising no serum nor protein. The molecular weight of gammapolyglutamic acid is 50 kDa-500 kDa. The concentration of gamma-polyglutamic acid is 2.5-20% (w / v). Gamma-polyglutamic acid replaces the synthetic product Pluronic F68.

[0012] A detergent - a preparation for animals - is known from the Japanese patent description No. JP2019056037. Developing a pet detergent that can increase the moisture content in animal skin keratin after washing and has excellent moisture retention properties. SOLUTION: The pet detergent of the present invention comprises an ionic complex formed from poly-y-glutamic acid and a cationic surfactant.

[0013] The composition of an immunopatent agent comprising gammapolyglutamic acid is known from the Japanese patent description No. JP2006232799.

[0014] The composition of the immune-boosting agent includes y-polyglutamic acid with a molecular weight of 10-10,000 kDa. The vaccine composition includes a composition of an immune enhancing agent and an antigenic substance. An antigenic substance is any substance selected from the group consisting of a peptide, a polypeptide, a lactobacilli expressing a polypeptide, a protein, a lactobacilli expressing a protein, an oligonucleotide, a polynucleotide, a recombinant bacterium and a recombinant virus.

[0015] Improved viability of probiotic microorganisms using poly-gamma- glutamic acid is known from the American patent description No. US2014248396.

[0016] The invention relates to a probiotic microorganism at least partially coated with a polyglutamic acid, for example poly y-glutamic acid (y-PGA). The invention also relates to a food product comprising a probiotic microorganism at least partially coated with polyglutamic acid and a method of producing a probiotic microorganism at least partially coated with polyglutamic acid. The invention also relates to a method of producing poly-y-glutamic acid (y-PGA).

[0017] From the Chinese patent description No. CN101948785, a bacterium producing gamma-polyglutamic acid and a method of producing gammapolyglutamic acid and its salts using a bacterium producing gamma-polyglutamic acid are known.

[0018] The invention discloses a strain of bacteria producing gamma-polyglutamic acid. The gamma-polyglutamic acid-producing bacterium is classified and named Bacillussubtilis PGS-1, and is collected in the China General Microbial Culture Collection Center (CGMCC), where the bacterial strain collection number is CGMCC No. 4034; and the date of receipt is July 23, 2010. The invention also discloses a process of producing gamma-polyglutamic acid and its salts using a gamma-polyglutamic acid-producing bacterium. Compared to the prior art, the gamma-polyglutamic acid synthesized by the gamma-polyglutamic acid-producing bacterium of the invention has the advantages of lower molecular weight (300 to 400 KDa), narrower molecular weight distribution, suitability for applications with low molecular weight requirements, and extremely wide application prospects.

[0019] A method of producing medical- and commercial-grade high-molecular- weight polygamma-glutamic acid and a system for microfiltration of PGA is known from US patent description US7364875.

[0020] Methods for producing high molecular weight polyglutamic acid (PGA). PGA is produced by fermentation and purified by tangential flow filtration followed, if necessary, by diafiltration to yield a product of the desired purity. The product obtained may be of very high purity using all the prescribed purification steps. Product of this purity is suitable for in vivo medical applications. Other applications, such as food or agricultural, may utilize lower purity levels, and hence do not require all the purification steps specified.

[0021] DISCLOSURE OF THE INVENTION

[0022] The aim of the invention is to develop a new method of obtaining, a system and a new mixture of bacterial metabolites with cytoprotective properties for selected strains and antibacterial properties for strains undesirable for the livestock microbiome; whereby the new approach is particularly novel when three different bacterial strains start competing for shared resources and are stimulated to produce higher concentrations of antimicrobiotic peptides (bacteriocins) (than if they were stimulated separately in separate enclosed e.g. bioreactors).

[0023] A method in accordance with the invention consists in that in the first stage, three bacterial cultures are placed in a sealed bioreactor, preferably in equal proportions, three bacterial cultures i.e.: 1. Bacillus licheniformis, 2. Bacillus subtilis and 3. Bacillus amyloliquefaciens, preferably in liquid form with a CFU from l* 103to l*10ilJ, preferably 1 * 108; in the second stage, a sterile growth medium for bacteria is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at least 7.0 pH, preferably 8.5 pH; and a temperature not higher than 41 °C, preferably 38°C - 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for at least 24 hours; and then the obtained cultivated mixture - the preparation is hygienically bottled.

[0024] Preferably after the second stage, preferably before bottling, the obtained mixture is additionally subjected to at least three-stage filtration.

[0025] Preferably in the first filtration stage the mixture is sterilized based on microfiltration (A), and then the remaining filtrate is purifi ed of low-molecular substances using filters (B), allowing to obtain a fraction of pure gamma-polyglutamic acid with a particle size of lMDa-100 kDa, in the second stage, the remaining filtration fraction is transferred to the nanofiltration system, where a low-molecular-weight fraction of gammapolyglutamic acid enriched with antimicrobial peptides (bacteriocins) is obtained, and at the same time, waste in the form of simple sugars, dextrins and lactic acid is removed, then, in the third stage, both sediment fractions after ultra and nanofiltration are mixed together to create a mixture of the final product with cytoprotective (IMDa- lOOkDa fraction) and antimicrobial (80kDa-300Da fraction) properties.

[0026] Preferably the filtration is carried out for 2 hours.

[0027] Preferably the once-filtered mixture is re-filtered in the same manner as the first filtration was carried out until up to 99.95% of cell bodies and bacteria are removed, and the mixture-filtrate is monitored for presence of bacteria in the filtrate.

[0028] Preferably the bacteria sterile growth medium comprises per 300 liters of bioreactor - monosodium glutamate in an amount from 12 to 18 kg, preferably 15 kg, glucose in an amount from 18 to 24 kg, preferably 21 kg, sodium citrate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, ammonium sulfate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, sodium nitrate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, yeast extract in amount from 4.3 kg to 5.7 kg, preferably 5 kg, peptone in an amount from 4.1 kg to 5.9 kg, preferably 5 kg, zinc sulfate in an amount from 0.18 kg to 0.24 kg, preferably 0.21 kg, dipotassium phosphate in an amount from 0.17 kg to 0.25 kg, preferably 0.21 kg.

[0029] The object of the invention is also a method of filtration of the preparation- mixture described above, which is characterized in that the filtration is carried out in a system of three filters with different pore sizes, where in the first filtration stage the mixture is sterilized based on microfiltration

[0030] (A), and then the remaining filtrate is purified of low-molecul r substances using filters

[0031] (B), allowing to obtain a fraction of pure gamma-polyglutamic acid with a particle size of lMDa-100 kDa, in the second stage, the remaining filtration fraction is transferred to the nanofiltration system, where a low-molecular-weight fraction of gammapolyglutamic acid enriched with antimicrobial peptides (bacteriocins) is obtained, and at the same time, waste in the form of simple sugars, dextrins and lactic acid is removed, then, in the third stage, both sediment fractions after ultra and nanofiltration are mixed together to create a mixture of the final product with cytoprotective (IMDa- lOOkDa fraction) and antimicrobial (80kDa-300Da fraction) properties.

[0032] Preferably at the microfiltration stage the pressure is 3 Ba, and at the ultrafiltration and nanofiltration stage the pressure is over 4 Ba.

[0033] A mixture based on purified PGA, especially for use in the production of feed and veterinary products according to the invention is characterized in that it comprises by volume: volumetric mixture of bacterial cell bodies (Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens) - up to 20% by volume, high molecular weight glutamic acid homopolypeptides (>I MDa) - from 2 to 10% by volume, medium molecular weight glutamic acid homopolypeptides - from 4 to 25, preferably 12% by volume, low molecular weight glutamic acid homopolypeptides - from 1 to 10, preferably 5% by volume, bacteriocins, preferably licenifensin - from 0.001% to 1.5% to preferably 1% by volume, and low molecular weight metabolites such as glucose, fructose, dextrins, lactic acid - up to 5% by volume and the rest is water.

[0034] A mixture based on purified PGA, especially for use in the production of feed and veterinary' products after filtration according to the invention is characterized in that it comprises by volume: a mixture of medium molecular weight glutamic acid homopolypeptides from 5 to 35%, preferably 20% by volume, low molecular weight glutamic acid homopolypeptides from 5 to 40, preferably 20% by volume, bacteriocins, preferably licenifensin, from 0.001% to 2% by volume, the rest is water. The essence of the solution according to the invention is to develop a mixture - metabolites having a high concentration (up to 30% solution) of gamma-polyglutamic acid enriched with antibacterial peptides produced as a result of competition (for resources - growth medium in a sealed bioreactor) of the three strains used (up to 1% by weight). When we have three different strains, the bacteria begin to compete for limited resources and are stimulated to produce higher concentrations of antimicrobial peptides (bacteriocins) (than if they were stimulated separately in separate closed e.g. bioreactors), and in consequence the liquid - mixture has significantly enhanced antibacterial properties, inhibiting the development of pathogenic bacteria, including MRSA or E. coli.

[0035] Before filtration, the "dirty" preparation comprises approx. 400 different (active) chemical compounds, being among others metabolites of the transformations of the above-mentioned three bacteria (approximate results from the mass spectrometer).

[0036] Properties of the preparation - mixture according to the invention (which are also its additional advantages):

[0037] 1. bactericidal,

[0038] 2. immunomodulatory,

[0039] 3. cytoprotective (protection of appropriate bacterial strains against unfavorable environmental factors).

[0040] Uses

[0041] Thanks to the preparation-mixture according to the invention, it will be possible to offer on the market a sub stance / semi -finished product based on purified gam ma-poly glutamic acid (PGA), as a qualitative and economical alternative to the overuse of antibiotics in the feed and veterinary industry; where in particular a mixture of bacterial metabolites (Bacillus subtilis, bacillus licheniformis, Bacillus atnyloliquefaciens) will be used in the feeding of farm animals.

[0042] In the feeding of farm animals, it is optimal to administer 5 liters of the preparation-mixture according to the invention - per 1 ton of feed.

[0043] The subject of the invention is explained in the examples and drawings in which:

[0044] Fig. 1 show's a block diagram of micro-ultra and nanofiltration and hygienic pouring of the finished preparation (under the name "Sansverum"),

[0045] Fig. 2 show's the filtration diagram of the obtained product (under the name "Sansverum"),

[0046] Fig. 3 shows the results of the experiment showing the level of inhibition of E. colli bacteria depending on the level of filtration, w'ith filtration C (nanofiltration, fig.2) inhibiting pathogenic bacteria developing in chicken embryos to the greatest extent.

[0047] EXAMPLES

[0048] Example 1 (method of preparation and ’’dirty” mixture - before filtration)

[0049] Method of obtaining

[0050] In the first stage, three bacterial cultures are placed in a sealed bioreactor in equal proportions, i.e.: 1. Bacillus licheniformis, 2. Bacillus subtilis and 3. Bacillus amyl oliquefaci ens, in liquid form w'ith a CFU around 1 *108.

[0051] In the second stage, bacteria sterile growth medium is placed in the tank.

[0052] Wherein using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at 8.5 pH; and a temperature not higher than 41 °C, preferably 38°C, while ensuring oxygenation of the mixture with additional oxygen to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for 24 hours. Then the obtained cultivated mixture - the preparation is hygienically bottled.

[0053] The sterile growth medium for bacteria was used having composition per 300 liters of bioreactor: sodium glutamate (15 kg), glucose (21 kg), sodium citrate (2.1 kg), ammonium sulfate (2.1 kg), sodium nitrate (2.1 kg), yeast extract (5 kg), peptone (5 kg), zinc sulfate (0.21 kg), dipotassium phosphate (0.21 kg).

[0054] Obtained mixture ( “dirty”):

[0055] The mixture comprises by volume: volumetric mixture of bacterial cell bodies (Bacillus licheniformis, Bacillus subtilis and Bacillus amyl oliquefaci ens) - 20% by volume, high molecular weight glutamic acid homopolypeptides (>lMDa) - 2% by volume, medium molecular weight glutamic acid homopolypeptides - 12% by volume, low molecular weight glutamic acid homopolypeptides - 5% by volume, bacteriocins, preferably lichenifensin - 1% by volume; and low molecular weight metabolites such as glucose, fructose, dextrins, lactic acid - 5% by volume and the rest is water.

[0056] Example 2 (method of obtaining and ’’pure” mixture after first filtration and filtration method)

[0057] Method of obtaining In the first stage, three bacterial cultures are placed in a sealed bioreactor in equal proportions, i.e.: 1. Bacillus licheniformis, 2. Bacillus subtilis and 3. Bacillus amyloliquefaciens, in liquid form with a CFU around 1 *108.

[0058] In the second stage, bacteria sterile growth medium is placed in the tank.

[0059] Wherein using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at 8.5 pH; and a temperature not higher than 41°C, preferably 38°C, while ensuring oxygenation of the mixture with additional oxygen to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for 24 hours. Then the obtained cultivated mixture - the preparation is hygienically bottled.

[0060] The sterile growth medium for bacteria was used having composition per 300 liters of bioreactor: sodium glutamate (15 kg), glucose (21 kg), sodium citrate (2.1 kg), ammonium sulfate (2.1 kg), sodium nitrate (2.1 kg), yeast extract (5 kg), peptone (5 kg), zinc sulfate (0.21 kg), dipotassium phosphate (0.21 kg).

[0061] After the second stage, preferably before bottling, the obtained mixture is subjected to three-stage filtration and the sequence shown in Fig. I, where up to 99.9% of cell bodies and bacteria are removed within 2 hours..

[0062] A method of three stage filtration

[0063] The filtration is carried out in a system of three filters with different pore sizes, where

[0064] In the first filtration stage the mixture is sterilized through microfiltration (A), and then the remaining filtrate is purified of low-molecular substances using filters (B), allowing to obtain a fraction of pure gamma-polyglutamic acid with a particle size of lMDa-100 kDa,

[0065] In the second stage, the remaining filtration fraction is transferred to the nanofiltration system, where a low-molecular-weight fraction of gamma- polyglutamic acid enriched with antimicrobial peptides (bacteriocins) is obtained, and at the same time, waste in the form of simple sugars, dextrins and lactic acid is removed.

[0066] In the third stage, both sediment fractions after ultra and nanofiltration are mixed together to create a mixture of the final product with cytoprotective (IMDa-l OOkDa fraction) and antimicrobial (80kDa-300Da fraction) properties; the filtration properties are shown in Fig. 2.

[0067] The filtration system allows for filtering the fermentation product of three bacterial cultures (Bacillus licheniformis, Bacillus subtilis and Bacillus amyl oliquefaci ens) in the sequence shown in Fig. 1.

[0068] The mixture obtained (after three-stage filtration "pure"):

[0069] The mixture comprises by volume: a mixture of medium molecular weight glutamic acid homopolypeptides - 20% by volume, low molecular weight glutamic acid homopolypeptides - 20% by volume, bacteriocins, preferably licenifensin - 2% by volume, the rest is water.

[0070] In the feeding of farm animals, it is optimal to administer 5 liters of preparationmixture per 1 ton of feed. Example 3 (mixture after two time three-stage filtration)

[0071] The mixture was prepared and filtered as in Example 2.

[0072] The once filtered mixture was subjected to re-filtration according to the method described in Example 2 and the sequence given in Fig, 2, until 99.95% of cell bodies and bacteria wore removed. The mixture-filtrate was subjected to monitoring for the presence of bacteria in the filtrate, optimally 1 ml of the mixture per microbiological plate with general medium.

Claims

Patent claims1. A method of obtaining a preparation - mixture based on purified PGA, especially for use in the production of feed and veterinary products, using at least one bacterium and PGA, characterized in that in the first, stage, three bacterial cultures are placed in a sealed bioreactor, preferably in equal proportions, i.e.:

1. Bacillus licheniformis, 2. Bacillus subtilis and 3. Bacillus amyloliquefaciens, preferably in liquid form with a CFU from l*103to 1*1O10, preferably 1 *108; in the second stage a sterile growth medium for bacteria is placed in the tank, while using sodium base (NaOH), the alkaline pH of the mixture in the tank is maintained at least 7.0 pH, preferably 8.5 pH; and a temperature not higher than 41 °C, preferably 38°C - 40°C, while ensuring oxygenation of the mixture with additional oxygen, preferably to a saturation of 70%, while maintaining the above-mentioned conditions in the tank for at least 24 hours; and then the obtained cultivated mixture - the preparation is hygienically bottled.

2. The method of obtaining the preparation-mixture according to claim. 1, characterized in that after the second stage, preferably before bottling, the obtained mixture is additionally subjected to at least three-stage filtration.

3. The method according to claim 2, characterized in that in the first filtration stage the mixture is sterilized through microfiltration (A), and then the remaining filtrate is purified of low-molecular substances using filters (B), allowing to obtain a fraction of pure gamma-polyglutamic acid with a particle size of lMDa-100 kDa, in the second stage, the remaining filtration fraction is transferred to the nanofiltration system, where a low-molecular-weight fraction of gammapolyglutamic acid enriched with antimicrobial peptides (bacteriocins) is obtained, and at the same time, waste in the form of simple sugars, dextrins and lactic acid is removed, then, in the third stage, both sediment fractions after ultra and nanofiltrationare mixed together to create a mixture of the final product with cytoprotective (IMDa-lOOkDa fraction) and antimicrobial (80kDa-300Da fraction) properties.

4. The method of obtaining the preparation-mixture according to claim 2 or 3, characterized in that the filtration is carried out for 2 hours.

5. The method of obtaining the preparation-mixture according to claim 2 or 3 or 4, characterized in that the once-filtered mixture is re-filtered in the same manner as the first filtration was carried out until up to 99.95% of cell bodies and bacteria are removed, and the mixture-filtrate is monitored for presence of bacteria in the filtrate.

6. The method of obtaining the preparation-mixture according to any of the claims 1-5, characterized in that the bacteria sterile growth medium comprises per 300 liters of bioreactor - monosodium glutamate in an amount from 12 to 18 kg, preferably 15 kg, glucose in an amount from 18 to 24 kg, preferably 21 kg, sodium citrate in an amount from 1, 7 kg to 2.5 kg, preferably 2.1 kg, ammonium sulfate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, sodium nitrate in an amount from 1.7 kg to 2.5 kg, preferably 2.1 kg, yeast extract in amount from 4.3 kg to 5.7 kg, preferably 5 kg, peptone in an amount from 4.1 kg to 5.9 kg, preferably 5 kg, zinc sulfate in an amount from 0.18 kg to 0.24 kg, preferably 0.21 kg, dipotassium phosphate in an amount from 0.17 kg to 0.25 kg, preferably 0.21 kg.

7. A method of filtration of the preparation-mixture described in claims 1-6, characterized in that the filtration is carried out in a system of three filters with different pore sizes, where in the first filtration stage the mixture is sterilized based on microfiltration (A), and then the remaining filtrate is cleaned of low-molecular substances using filters (B), allowing to obtain a fraction of pure gamma-polyglutamic acid with a particle size of lMDa-100 kDa, in the second stage, the remaining filtration fraction is transferred to the nanofiltration system, where a low-molecular-weight fraction of gamma-polyglutamic acid enriched with antimicrobial peptides (bacteriocins) is obtained, and at the same time, waste in the form of simple sugars, dextrins and lactic acid is removed, then, in the third stage, both sediment fractions after ultra and nanofiltration are mixed together to create a mixture of the final product with cytoprotective (IMDa-lOOkDa fraction) and antimicrobial (80kDa-300Da fraction) properties.

8. The method according to claim 7, characterized in that at the microfiltration stage the pressure is 3Ba, and at the ultrafiltration and nanofiltration stage the pressure is over 4Ba.

9. A mixture based on purified PGA, especially for use in the production of feed and veterinary products, characterized in that it comprises by volume: volumetric mixture of bacterial cell bodies (Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens) - up to 20% by volume, high molecular weight glutamic acid homopolypeptides (>lMDa) - from 2 to 10% by volume, medium molecular weight glutamic acid homopolypeptides - from 4 to 25, preferably 12% by volume, low molecular weight glutamic acid homopolypeptides - from 1 to 10, preferably 5% by volume, bacteriocins, preferably lichenifensin - from 0.001% to 1.5% to preferably 1 % by volume; and low molecular weight metabolites such as glucose, fructose, dextrins, lactic acid - up to 5% by volume and the rest is water.

10. A mixture based on purified PGA, especially for use in the production of feed and veterinary products after filtration, characteri ed in that it comprises by volume: a mixture of medium molecular weight glutamic acid homopolypeptides from 5 to 35%, preferably 20% by volume, low molecular weight glutamic acid homopolypeptides from 5 to 40,preferably 20% by volume, bacteriocms, preferably lichenifensin, from 0.001% to 2% by volume, the rest is water.

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