Method for producing enzymatic lysate of biomass of methane-oxidizing bacteria

Abstract

The invention relates to biotechnology, in particular, to a method for producing an enzymatic lysate of biomass of methane-oxidizing bacteria, the method comprising enzymatic hydrolysis of a bacterial suspension using an enzyme preparation, which is derived from a pancreas, and / or pancreatin obtained from cattle or pigs, in amount of from 5000 to 7000 units of proteolytic activity per 1 kg of absolutely dry substance of the biomass of bacteria, at pH of from 7 to 8.5 and a temperature of 45°C to 55°C for from 4 to 12 hours. The invention also relates to a product obtained by said method. The resulting product has a high content of short-chain peptides with a molecular weight of from 150 to 1000 Da and can be used to produce feed additives and biologically active food supplements. The invention provides for an increase in bioavailability of intracellular bacterial components.

Description

[0001] METHOD FOR PRODUCING ENZYMATIC LYSATE OF BIOMASS OF METHANE-OXIDIZING BACTERIA

[0002] TECHNICAL FIELD

[0003] The present invention relates to biotechnology and can be used in industry to produce feed additives and biologically active food supplements comprising protein, biologically active peptides, amino acids, nucleotides, vitamins and microelements. In particular, the invention relates to a method for producing an enzymatic lysate (hereinafter also called “fermentolysate”) of biomass of methane-oxidizing bacteria, the method comprising enzymatic hydrolysis of a bacterial suspension using an enzyme preparation, as well as to the enzymatic lysate produced by said method.

[0004] BACKGROUND ART

[0005] Biomass of methane-oxidizing bacteria is used as a source of protein for animal feed, and can also be used to produce biologically active food supplements in sports and specialized human nutrition rations.

[0006] Biological value of the biomass of methane-oxidizing bacteria is primarily provided by high content of protein (about 70% of dry weight), essential amino acids, as well as nucleic acids, B vitamins and trace elements. However, effective use of the biomass of methane-oxidizing bacteria is hampered for the following reasons:

[0007] 1) A strong cell wall of methane-oxidizing bacteria comprises difficult-to-digest peptidoglycan (murein), which hampers availability and absorption of intracellular substances.

[0008] 2) The biomass of methane-oxidizing bacteria comprises minimal amounts of free amino acids, short-chain biologically active peptides and oligonucleotides, which perform not only a structural but also a regulatory function in the body.

[0009] To increase the bioavailability of nutrients of the biomass of methane-oxidizing bacteria, a process of enzymatic hydrolysis (also called “enzymolysis” or “fermentolysis”) is currently used, in which enzyme-rich components (enzymatic agents hereinafter also called “enzyme preparations”) are added to the biomass. A method for obtaining enzymatic lysates of Methylococcus capsulatus bacteria is known, wherein a suspension of Methylococcus capsulatus bacteria is placed in a reactor and an enzyme preparation being protosubtilin is added in amount of 4500 to 6000 enzyme units per 1 kg of absolutely dry bacterial biomass, wherein the enzymatic hydrolysis is carried out at a temperature of 55°C to 60°C for 2 hours (patent Rll 2700079 C1 , A23J 3 / 20, published on 12.09.2019). As stated in this document, the method provides disintegration of bacterial cell walls that results in increasing availability of intracellular substances, namely proteins, peptides, amino acids, vitamins, as well as in increasing yield of water-soluble substances up to 80% by weight.

[0010] A method for producing enzymatic hydrolysates of Methylococcus capsulatus bacteria is known, said method comprises enzymatic hydrolysis of a suspension of Methylococcus capsulatus bacteria in the presence of protosubtilin in combination with lipase, wherein protosubtilin is used in amount of 1500 to 2000 enzyme units per 1 kg of absolutely dry bacterial biomass, and lipase in amount of 5000 to 6000 units of activity per 1 kg of absolutely dry bacterial biomass. The enzymatic hydrolysis is carried out at a temperature of 37°C to 45°C and pH of 6.0 to 6.5 for 1 hour (patent Rll 2769286 C1 , A23J 3 / 20, published on 30.03.2022). As indicated in this document, the method provides an increased efficiency of enzymatic hydrolysis of Methylococcus capsulatus bacteria due to liquefying lipid layer of an external membrane shell of bacteria, as well as due to reducing costs for heating and thermostat control of the culture at the hydrolysis step.

[0011] A method for obtaining microbial protein from microbial biomass of a producer of methane-oxidizing bacteria by enzymatic method is also known (patent RU 2781287 C1 , A23J 3 / 20, published on 11.10.2022), said method comprises the following steps. At the first step, a concentrate of denucleated biomass is obtained from native biomass in a form of a suspension of methane-oxidizing bacteria with solids content (SC) of 10-12%, pH of which is adjusted to 3.0-3.5 with sulfuric acid having a concentration of from 45% to 50%. The suspension is heated, pH is adjusted to 8.5-9.5 by adding an alkaline solution, maintained at a temperature of 85°C to 95°C until an alkaline extract is obtained, filtered under pressure and washed with osmotic water to remove nucleic components to obtain a concentrate of denucleated biomass (DNBM). At the second step, hydrolysis is carried out with a complex enzyme preparation comprising amylases, lipases and proteases at the rate of 7000 units of amylase of the complex enzyme preparation per 30-35 g of solids content of the concentrate. At the third step, the obtained suspension is inactivated by raising a temperature, followed by filtration and drying the concentrate and filtrate to obtain a powdered product with a protein content of at least 88%. This complex multi-step process is aimed at obtaining a specific high-protein product with a minimum content of other nutrients necessary for the body, such as biologically active peptides, amino acids, nucleotides, vitamins and microelements.

[0012] A disadvantage of known methods is the use of expensive bacterial preparations. The bacterial protease preparations used in said known methods comprise enzyme mixtures, but conditions for optimal activity of each of components thereof are very different. Neutral protosubtilin protease is the most active at pH of from 5.5 to 6.5, and alkaline serine protease of the same preparation is most active at pH of from 9.2 to 11 .0, so it is difficult to realize the proteolytic potential of each of enzyme components of such a mixture during a technological process. The enzyme preparation protosubtilin comprises only alkaline and neutral proteinases, which do not allow for highly effective hydrolysis of the protein comprised in bacterial cells. Adding lipase to protosubtilin does not change the content of proteases, but allows increasing in a degree of hydrolysis due to partial destruction of lipid membranes of cells and increasing amount of protein available to proteases. The preparations used in the prior art methods are highly selective, but are not capable of providing a high degree of cleavage of a protein substrate, which is necessary to obtain a significant amount of short-chain peptides in a final product. None of prior art methods provide hydrolysis of nucleic acids and, as a consequence, an increase in the accessibility thereof.

[0013] Definitions

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0015] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0016] Terms "includes", "include", "including" as used herein are synonymous with the terms "comprises", "comprise", "comprising" and mean “including but not limited to”, i.e. are not intended to exclude a possibility of presence of further listed elements, such as steps, operations, components, as well as do not exclude or prevent the presence of further, not indicated steps, operations, components, features, elements known in the art or disclosed herein. Unless defined otherwise, percents (%) as used herein are percents by weight, and a term “% by weight” refers to relative weight of a corresponding component based on the total weight of a composition or corresponding element of the composition.

[0017] Indication of numerical ranges as limiting values comprises all integers and fractional numbers within the range, as well as the indicated limiting values, unless otherwise explicitly stated or indicated.

[0018] As used herein, the term “and / or” refers to any possible combinations and encompasses both any possible combinations of one or more of corresponding elements, as well as absence of combinations when interpreted in the alternative form (“or”).

[0019] In the context of the present invention, the terms “enzymatic hydrolysis”, “enzymolysis” and “fermentolysis” are used interchangeably.

[0020] In the context of the present invention, the terms “enzymatic lysate”, “enzyme lysate” “enzymolysate” and “fermentolysate” are used interchangeably and mean a product of enzymatic hydrolysis of a substrate of microbial origin.

[0021] In the context of the present invention, the terms "methane-oxidizing bacteria", "methanotrophic bacteria", "methane-utilizing bacteria", "methane-fixing bacteria" are interchangeable and mean a group of bacteria capable of using methane as a sole source of both carbon and energy.

[0022] In the context of the present invention, the terms "pancreas", "pancreatic enzyme", "pancreatic enzyme preparation", "pancreatin" and "pancreatin preparation" are used interchangeably and refer to any type of enzymes present in pancreatic secretion, or a mixture thereof, or any extract derived from the pancreas and comprising pancreatic enzymes, as described in details below.

[0023] SUMMARY OF INVENTION

[0024] An objective of the present invention is to develop a method that provides effective hydrolysis of proteins, nucleic acids and lipids comprised in cells of methane-oxidizing bacteria with obtaining the maximum amount of short-chain peptides with a molecular weight of up to 1000 Da in the final product.

[0025] An advantageous effect provided by the present invention consists in increased efficiency of enzymatic hydrolysis of biomass of methane-oxidizing bacteria due to the use of an enzyme preparation derived from a pancreas and / or pancreatin, with obtaining an enzymatic lysate comprising at least 48% of free amino acids and short-chain peptides with a molecular weight of up to 1000 Da based on the total amount of amino acids and peptides with a molecular weight of up to 5000 Da. The inventive method for obtaining the enzymatic lysate of biomass of methane-oxidizing bacteria provides for a solution to problems that hamper effective digestion, uptake and provision of physiological action of valuable nutrients thereof.

[0026] The method according to the invention comprises enzymatic hydrolysis of biomass of methane-oxidizing bacteria with an enzyme preparation, which is an enzyme preparation derived from a pancreas, and / or pancreatin obtained from cattle or pigs.

[0027] The pancreas and pancreatin obtained therefrom are a complex enzyme preparation comprising proteolytic enzymes (trypsin, chymotrypsin, carboxypeptidases, elastase, aminopeptidase, collagenase, dipeptidase), nucleolytic enzymes (nucleases such as DNAase and RNAase), amylolytic enzymes (amylase, maltase, lactase, invertase) and lipolytic enzymes (lipase, phospholipase, cholinesterase, carboxylesterase, monoglyceride lipase, alkaline phosphatase).

[0028] Pancreatin is an enzyme system that comprises several proteolytic enzymes of different selectivity that are capable of functioning simultaneously, under the same conditions and parameters of medium. Pancreatin is less selective than individual bacterial enzymes or complexes thereof, but it is capable of much effective hydrolyzing a protein substrate. Pancreatin comprises several endoproteases with a rather extensive list of amino acid pairs hydrolysable by them. One of the pancreatin proteases, trypsin, hydrolyzes bonds between lysine and arginine residues. Another protease, chymotrypsin, brakes peptide bonds of many amino acids: tyrosine, phenylalanine, tryptophan, leucine, methionine, aspartic acid and glutamic acid. Thus, the two main pancreatin endoproteases, at pH of 7 to 8, are capable of destroying more than half of all peptide bonds in a protein. Pancreatin also comprises other proteolytic enzymes: elastases, which cleave bonds formed by aliphatic amino acids, and carboxypeptidases, which cleave fragments of several monomeric amino acids from the C-terminus of a peptide sequence. Therefore, pancreatin preparations, unlike bacterial protease preparations, allow obtaining enzymatic lysates with a high degree of cleavage, particularly, with the maximum content of di-, tri- and tetrapeptides in the final product. Non-proteolytic enzymes comprised in the pancreatin provide hydrolysis of nucleic acids to mono- and oligonucleotides, and hydrolysis of phospholipids, glycolipids and lipids to fatty acids, glycerol and carbohydrate residue, thereby generally allow increasing efficiency of absorption of cellular components of methane-oxidizing bacteria by the body and reducing the load on a digestive system (A.V. Aripovsky, V.N. Titov. "Biologically active peptides in the regulation of metabolism: oligopeptides, peptones, insulin and arterial pressure". Clinical laboratory diagnostics. 2019, Vol. 64, p. 14.).

[0029] According to the present invention, a method for producing enzymatic lysate of biomass of methane-oxidizing bacteria comprises the following steps:

[0030] - adding an enzyme preparation to a suspension of methane-oxidizing bacteria;

[0031] - enzymatic hydrolysis of the suspension of biomass of the methane-oxidizing bacteria with the enzyme preparation; and

[0032] - spray drying of a resulting product, wherein the enzyme preparation is an enzyme preparation derived from a pancreas and / or pancreatin obtained from cattle or pigs, in amount of from 5000 to 7000 units of proteolytic activity (UPA) per 1 kg of absolutely dry substance of the biomass of bacteria, and the enzymatic hydrolysis is carried out at pH of from 7 to 8.5 and a temperature of from 45°C to 55°C with continuous stirring for from 4 to 12 hours.

[0033] In order to ensure a longer shelf life, the method preferably further comprises a step of thermal inactivation of enzymes carried out before the spray drying to stop the enzymatic hydrolysis. The enzymatic hydrolysis is preferably stopped by inactivating the enzymes in the biomass by heating to a temperature of from 85°C to 95°C for from 15 to 20 minutes. If inactivation is not carried out, then a residual enzymatic activity is retained in the dried product after drying, that can contribute to a deeper cleavage of the biomass components and even greater increase in digestibility therof. However, in this case, when used in feed, the risk of higher bacterial contamination of the feed increases.

[0034] The enzyme preparation is preferably added in a form of crumbled pancreas or pancreatin (which is usually supplied in powder form), suspended in water.

[0035] The enzyme preparation can be added either once at the beginning of the hydrolysis, or in equal portions at equal intervals during the enzymatic hydrolysis process.

[0036] Due to the wide range of enzymes comprised in the pancreas and / or pancreatin, the method according to the invention allows for the enzymatic hydrolysis of various methane-oxidizing bacteria, in particular bacteria of genus Methylocystis (such as Methylocystis echinoides, Methylocystis rosea, Methylocystis parvus), genus Methylosinus (such as Methylosinus trichosporium, Methylosinus sporium), genus Methylomonas (such as Methylomonas methanica), genus Methylobacter, genus Methylocaldum, genus Methylotuvimicrobium (such as Methylomicrobium buryatense 5G, Methylotuvimicrobium alkaliphilum 20Z), and genus Methylococcus (such as Methylococcus capsulatus), to obtain a product comprising components having high bioavailability, which allows using thereof to produce effective feed additives and biologically active food supplements.

[0037] The method is simple and does not require using sophisticated equipment, expensive or toxic reagents.

[0038] Another subject of the invention is a product obtained by the method described above, namely, an enzymatic lysate of biomass of methane-oxidizing bacteria. Due to advantages of the method according to the invention described above, the resulting product has a high content of oligopeptides (short-chain peptides) with a molecular weight of from 150 to 1000 Da. Said content can be 48% or more based on the total amount of amino acids and peptides with a molecular weight of up to 5000 Da. Such a high content of oligopeptides could not be obtained by the methods of the prior art due to aforesaid disadvantages and specificities of said methods.

[0039] The product according to the invention can be used to obtain feed additives and biologically active food supplements. Said additives can be manufactured by conventional methods known to persons skilled in the art.

[0040] The invention is further described with reference to illustrative examples demonstrating implementation of the invention, but not intended to limit the claimed scope of the invention defined in the appended claims.

[0041] EXAMPLES OF CARRYING OUT THE INVENTION

[0042] Example 1

[0043] The method according to the invention is carried out as follows.

[0044] A suspension of methane-oxidizing bacteria and crumbled pancreas or pancreatin of cattle or pigs in a ratio of 5000 to 7000 units of proteolytic activity of the pancreas or pancreatin per 1 kg of absolutely dry substance (ADS) of a biomass of methane-oxidizing bacteria, suspended in water, is loaded while stirring into a thermostatically controlled reactor with a possibility to control pH. pH of the suspension is adjusted to 7-8.5 and the suspension is incubated with continuous stirring at a temperature of from 45°C to 55°C for from 4 to 12 hours.

[0045] Pancreas or pancreatin of cattle or pigs is added to the biomass of methaneoxidizing bacteria once at the beginning of the hydrolysis or in portions throughout the process.

[0046] Enzymatic hydrolysis of bacterial biomass is accompanied by an increase in viscosity at the first step, which viscosity then decreased. pH decreased throughout the hydrolysis process.

[0047] Hydrolysis is stopped by inactivating enzymes by heating the mixture to a temperature in the range of from 85°C to 95°C for from 15 to 20 minutes and then spray dried.

[0048] Several specific examples of obtaining enzymatic lysate are given below.

[0049] Example 2

[0050] 20 liters of a suspension comprising a fraction of absolutely dry substance (ADS) of biomass of methane-oxidizing bacteria in amount of 25% by weight in a weakly alkaline medium at pH of 7.5-8.5 is placed in a thermostatically controlled reactor, pancreatin is added in amount of 5000 units of proteolytic activity per 1 kg of ADS of the biomass of methane-oxidizing bacteria. A process of enzymatic hydrolysis is carried out for 8 hours at a temperature of 55°C. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.3 kg of the enzymatic lysate with the following characteristics:

[0051] - a - protein cleavage degree: 0.21 ,

[0052] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 31 % of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0053] Example 3

[0054] Enzymatic hydrolysis is carried out according to the procedure described for Example 2 for 8 hours at a temperature of 55°C and pH of 7.5-8.5. Pancreatin in amount of 6000 units of proteolytic activity per 1 kg of ADS of the biomass of methane-oxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.25 kg of the enzymatic lysate with the following characteristics:

[0055] - a - protein cleavage degree: 0.36,

[0056] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 37.1% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0057] Example 4

[0058] Enzymatic hydrolysis is carried out according to the procedure described for Example 2 for 8 hours at a temperature of 55°C and pH of 7.5-8.5. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methane-oxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.18 kg of the enzymatic lysate with the following characteristics:

[0059] - a - protein cleavage degree: 0.39,

[0060] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 47.4% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0061] A product of the invention is obtained in a similar way using various parameters of the method according to the present invention.

[0062] Example 5

[0063] Enzymatic hydrolysis is carried out according to the procedure described for Example 2 for 8 hours, but at a temperature of 50°C and pH of 7.0-7.5. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methane- oxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.35 kg of the enzymatic lysate with the following characteristics:

[0064] - a - protein cleavage degree: 0.45,

[0065] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 42.6% of total amino acids and peptides with a molecular weight of up to 5000 Da. Example 6

[0066] Enzymatic hydrolysis is carried out according to the procedure described for Example 2 for 8 hours, but at a temperature of 50°C and pH of 7.5-8.0. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methaneoxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.28 kg of the enzymatic lysate with the following characteristics:

[0067] - a - protein cleavage degree: 0.55,

[0068] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 46.2% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0069] Example 7

[0070] Enzymatic hydrolysis is carried out according to the procedure described for Example 2 for 8 hours 2, but at a temperature of 50°C and pH of 8.0-8.5. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methaneoxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.55 kg of the enzymatic lysate with the following characteristics:

[0071] - a - protein cleavage degree: 0.05,

[0072] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 43.7% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0073] Example 8

[0074] Enzymatic hydrolysis is carried out according to the procedure described for Example 2, but for 12 hours, at a temperature of 45°C and pH of 7.0-7.5. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methaneoxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.8 kg of the enzymatic lysate with the following characteristics: a - protein cleavage degree: 0.9, - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 29.0% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0075] Example 9

[0076] Enzymatic hydrolysis is carried out according to the procedure described for Example 2, but for 12 hours, at a temperature of 45°C and pH of 7.5-8.0. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methaneoxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.6 kg of the enzymatic lysate with the following characteristics:

[0077] - a - protein cleavage degree: 0.14,

[0078] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 31 .5% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0079] Example 10

[0080] Enzymatic hydrolysis is carried out according to the procedure described for Example 2, but for 12 hours, at a temperature of 45°C and pH of 8.0-8.5. Pancreatin in amount of 7000 units of proteolytic activity per 1 kg of ADS of the biomass of methaneoxidizing bacteria is used as an enzyme preparation. Enzymatic hydrolysis is stopped, the biomass is inactivated by heating (85-95°C for 15-20 minutes). Enzymatic lysate is dried in a spray dryer to obtain 4.75 kg of the enzymatic lysate with the following characteristics:

[0081] - a - protein cleavage degree: 0.12,

[0082] - proportion of short-chain peptides with a molecular weight from 150 Da to 1000 Da: 30.0% of total amino acids and peptides with a molecular weight of up to 5000 Da.

[0083] Parameters of the process of enzymatic hydrolysis of the biomass of methaneoxidizing bacteria under various conditions and characteristics of the obtained products are given in Table 1 below, wherein a is the protein cleavage degree, which is the ratio of the number of cleaved peptide bonds to the total number of all peptide bonds of the substrate.

[0084] As noted above, the advantageous effect of the present invention consists in increased efficiency of enzymatic hydrolysis of the biomass of methane-oxidizing bacteria due to the use of an enzyme preparation derived from a pancreas and / or pancreatin, with obtaining an enzymatic lysate comprising at least 48% of free amino acids and short-chain peptides with a molecular weight of up to 1000 Da based on the total amount of amino acids and peptides with a molecular weight of up to 5000 Da. The obtained results show that the method according to the invention ensures obtaining a product with a high content of short-chain peptides with a molecular weight of up to 1000 Da, which have high bioavailability, that allows increasing the efficiency of absorption of cellular components of methane-oxidizing bacteria by the body and reducing the load on a digestive system. Experimental data presented in Table 1 clearly corroborate that aforesaid advantageous effect can be obtained within the entire range of amounts of the enzyme preparation from 5000 to 7000 units of proteolytic activity (UPA) per 1 kg of absolutely dry substance of the bacterial biomass, pH from 7 to 8.5, temperatures from 45°C to 55°C and hydrolysis time from 4 to 12 hours specified in the appended set of claims.

[0085]

Claims

CLAIMS1. A method for producing an enzymatic lysate of biomass of methaneoxidizing bacteria, comprising the following steps: adding an enzyme preparation to a suspension of methane-oxidizing bacteria; enzymatic hydrolysis of the suspension of biomass of the methane-oxidizing bacteria with the enzyme preparation; and spray drying of a resulting product, wherein the enzyme preparation is an enzyme preparation derived from a pancreas, and / or pancreatin obtained from cattle or pigs, in amount of from 5000 to 7000 units of proteolytic activity per 1 kg of absolutely dry substance of the biomass of bacteria, and the enzymatic hydrolysis is carried out at pH of from 7 to 8.5 and a temperature of from 45°C to 55°C with continuous stirring for from 4 to 12 hours.

2. The method according to claim 1 , wherein the method further comprises a thermal inactivation of enzymes carried out before the spray drying in order to stop the enzymatic hydrolysis.

3. The method according to claim 2, wherein the enzymatic hydrolysis is stopped by thermal inactivation of enzymes by heating to a temperature of from 85°C to 95°C for from 15 to 20 minutes.

4. The method according to any of claims 1 , 2 or 3, wherein the enzyme preparation is added in a form of crumbled pancreas or pancreatin suspended in water.

5. The method according to any of claims 1 , 2, 3 or 4, wherein the enzyme preparation is added once at the beginning of the hydrolysis.

6. The method according to any of claims 1 , 2, 3 or 4, wherein the enzyme preparation is added in equal portions at equal intervals during the enzymatic hydrolysis process.

7. An enzymatic lysate of biomass of methane-oxidizing bacteria for producing feed additives and biologically active food supplements, obtained by the method according to any of claims 1 to 6, wherein said enzymatic lysate comprises at least 48% of free amino acids and short-chain peptides with a molecular weight of up to 1000 Da based on the total amount of amino acids and peptides with a molecular weight of up to 5000 Da.

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