RU2544959C1 - Method of production of peptides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of production of peptides is proposed. Yeast autolysis is carried out. The cell membranes are separated by centrifugation. The autolysate is purified on the gel sulphocationite in the hydrogen form, containing 12-16% divinylbenzene. The resulting peptide aqueous solution is passed sequentially through the gel anion-exchange material to obtain the solution at pH 2.0-2.6, and then through the gel cation-exchange material with the divinylbenzene content of 1-2% or macroporous cation-exchange material.

EFFECT: obtaining highly purified peptides that have biological activity.

11 ex

Description

The invention relates to biotechnology and relates to a method for producing peptides from yeast autolysates.

In modern technological processes, yeast is used as a raw material for the production of various products, biologically active substances, amino acids, nucleic components, vitamins, etc. Known methods for processing yeast biomass include autolysis carried out by the yeast's own hydrolytic enzymes. During autolysis, the accumulation in the autolysate of free amino acids, lower (di-, tri-, tetra-) peptides and peptides with a higher molecular weight.

Known methods for producing biologically active peptides-analogues of natural peptide regulators, immunomodulators, morphine-like peptides, antimicrobial peptides and other methods of chemical synthesis or chemical modification of natural peptide regulators or the active part of their structure (RF patents No. 2043769, 2047621, 2060998, 2067000, 2087480, 2107691, 2126014, 2141527, 2145233, 2145234, 2146262, 2316336, 2478105, 2494105, 2182155, 2111215). A disadvantage of the known methods is the need for information on the exact structure of the natural analogue, an expensive and complex peptide synthesis procedure from the initial derivatives of amino acids, which often does not allow obtaining industrial quantities of the product.

Known methods for producing natural peptides from organs and tissues of farm animals (RF patents Nos. 2075944, 2104702, 2161501, 2043364). The disadvantage of this method is the shortage of this type of natural raw material.

A known method of obtaining a product containing antihypertensive peptides and its use as an antihypertensive, edible product (RF patent No. 2276689, IPC С12Р 21.02; A23J 1/20, publication of 2006). The method includes the steps of fermenting a casein-containing starter material with a lactic acid bacterium, nanofiltration of the obtained peptide-containing fermentation product, and isolating the product. The resulting product is used as an antihypertensive agent, as well as an edible product.

The disadvantage of this method is the low degree of purification of peptides.

A known method of producing peptides and amino acids from protein raw materials (RF patent No. 2333663, IPC A23J 3/04, publication 2008), according to which the raw material is casein of milk, and its hydrolysis is carried out in the presence of endogenous enzymes and then subjected to separation processing, including coagulation protein residues.

There is also known a method for producing peptides with a high tryptophan content (RF patent No. 2043364, IPC C07K 1/12; A61K 38/01, publication 1995), comprising suspending the fibrinogen protein in water, subsequent alkaline and enzymatic hydrolysis of the protein, inactivation of the enzyme at 90 -100 ° C for 15-20 minutes, centrifuging the hydrolyzate, precipitating the final product from the supernatant with acetone, and drying the precipitate.

The disadvantage of these methods is the use of scarce raw materials and exogenous enzymes.

Known methods for producing a mixture of amino acids and lower peptides by autolysis of yeast, followed by purification of the target product on ion exchangers (for example, US patent No. 2272982, UK patent No. 952713, ed. USSR certificate No. 957835, 1369300, 1731806).

There is also a method for producing a mixture of amino acids and peptides by filtering an autolysate through weakly basic anion exchange resin IA-1p, followed by sorption of amino acids and lower peptides from the filtrate on sulfonic cationite KU-2 × 8, evaporation of the eluate and drying of the finished product (USSR author's certificate No. 602543).

Significant disadvantages of the known methods is the high content of amino acids in the target product, constituting 70-76% of the total amount of the product, which limits the scope of use of the product as a food and pharmacological purpose, as well as in the form of a peptide component in the creation of microbiological nutrient media and perfume compositions .

A known method of producing low molecular weight peptides with interferon-stimulating activity (RF patent No. 2409678, IPC С12Р 21/00; C07K 1/34, publication 2011), according to which waste is used as a raw material in the production of lactoglobulin preparation, which is subjected to dialysis on polysulfide membrane filters with a pore diameter of 1 kDa against distilled water until the total protein concentration in the preparation is 3 mg / ml, followed by concentration by gel ion exchange chromatography and elution with a 0.5 M sodium chloride solution . The starting material for the production of low molecular weight peptides is the waste from the production of lactoglobulin against opportunistic bacteria and salmonella (RF patent No. 2298409, publication 2007), according to which the immune colostrum of cows is fermented with pepsin, and the curd is removed. Denatured proteins and fat are removed from the obtained cow colostrum immune serum by ultrafiltration to obtain purified lactoserum. The obtained lactoserum is separated on an ultrafiltration unit into two fractions: a high molecular weight fraction (lactoglobulin) and a filtrate containing low molecular weight (1-5 kDa) peptides as production waste, which is used to obtain the NMP.

A significant disadvantage of this method is the shortage of raw materials (colostrum of cows), the extremely low content of the target product in the waste, requiring preliminary concentration on membrane filters.

A known method for producing a mixture of amino acids and lower peptides, including autolysis of yeast, separation of cell membranes by centrifugation and purification of the autolysate on ion exchangers, characterized in that gel sulfonation in hydrogen form containing 12-16% divinylbenzene as a crosslinking agent is used as an ion exchanger (RF patent No. 2284356, IPC С12Р 13/06; A23J 1/18, 2006 publication - the closest analogue).

The disadvantage of this method is that the waste (breakthrough from cation exchange resin) contains a valuable product - peptides, which can be extracted only with the use of additional isolation and purification operations.

The aim of the present invention is to provide an effective and economical method for producing a highly purified mixture of peptides from a virtually unlimited renewable raw material - yeast.

This goal is achieved by the fact that waste is used as a raw material in the production of a mixture of amino acids and lower peptides from yeast containing peptides.

The essence of the proposed method is as follows.

The method for producing peptides includes yeast autolysis, centrifugal separation of cell membranes, purification of the autolysate on ion exchangers and the preparation of an aqueous solution of peptides (slip), while gel sulfonation in hydrogen form containing 12-16% divinylbenzene as a crosslinking agent is used as an ion exchanger, while the resulting aqueous solution of peptides is passed successively through a gel anion exchange resin to obtain a solution with a pH of 2.0-2.6, and then through a gel cation exchange resin with a content of divinylbenzene 1-2% or macroporous cation exchange resin.

During the production process for the separation of amino acids and lower peptides, the autolysate is processed as follows: cell membranes are separated by centrifugation, the resulting solution is ultrafiltered through membranes with a nominal cut-off molecular weight (HOMM) of 5-15 kDa, the ultrafiltrate is acidified to pH 3.0 and passed through sulfocationion in hydrogen form containing 12-16% divinylbenzene as a crosslinking agent. In this case, amino acids and lower peptides are adsorbed on cation exchange resin, and peptides with a higher molecular weight are not adsorbed and are concentrated in waste (slip). This allows you to almost completely separate individual (free) amino acids from peptides in a one-act process of sorption from an autolysate, while the target product — peptides — remains in solution.

Breakthrough is an aqueous solution of the target product (peptides) with a low pH (1.0-1.2), overloaded with ballast organic (pigmented) and minerals. The essence of the proposed method for producing a mixture of peptides is the selection of peptides from the slip. For this, the breakthrough is filtered through a column with anion exchange resin to obtain a solution with a pH of 2.0-2.6 (preferably 2.4), and then passed through a gel sulfocationionite in hydrogen form with a DVB content of 1-2% or through macroporous sulfocathionite. Desorption from cation exchange resin is carried out with 2.5-3.0% aqueous ammonia solution, the resulting eluate is evaporated to distill off the ammonia and dried on a freeze dryer.

Significant advantages of the invention in comparison with the known is that during the process a high degree of purification of the product from related mineral and organic substances is achieved. The method allows to obtain peptides, not

containing allergens and nucleic components, fats, sugars and salts. The method allows to obtain peptides from almost unlimited renewable raw materials - baker's or brewer's yeast. Obtained by the claimed method, the product has a pronounced biological activity.

Physicochemical analysis of the product showed that the content of peptides in the dry product is 88-92%, amino acids 6-9%. The product consists of a mixture of 22 individual peptides with a molecular weight of from 0.8 to 2.5 kDa. In an experimental study, the product showed pronounced anti-ischemic activity, promising in terms of prevention and treatment of coronary heart disease.

Example No. 1

35 L of the slip obtained after autolysis and extraction of the amino acid product from 50 kg of baking yeast is passed through a column with strongly crosslinked (12% DVB) Dowex 550A strongly basic anion exchange resin, the volume of the anion exchange resin is 3.5 L, the flow rate is 0.7 L / h, then the anion exchange resin washed with 1.0 l of distilled water. Get 36 l of a solution with a pH of 2.2. The resulting solution is filtered through a column with Dowex MSC-1C macroporous sulfocationionite in H + form, cation exchanger volume 10 l, volume flow 10 l / h, then cation exchanger is washed with 4 l of acidified water (pH 1-2) and desorption is carried out with 3% ammonia. Get 3 l of eluate (pH 10.2), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. 0.43 kg of product is obtained containing 90% peptides and 7% amino acids.

Example No. 2

35 L of slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with highly basic high-capacity polyacrylic gel anion exchange resin Purolite A830 in OH ^- form, the volume of the anion exchanger is 2.0 l, feed 0.7 l / h, then the anion exchange resin is washed with 0.5 l of distilled water. Obtain 35.5 l of a solution with a pH of 2.4. The resulting solution was filtered through a column with Dowex MSC-1C macroporous sulfocationionite in H + form, cation exchanger volume 10 l, volume flow 10 l / h, then cation exchanger was washed with 4 l of acidified water (pH 1-2) and desorption was carried out with 3% ammonia. Get 3 l of eluate (pH 10.2), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. 0.44 kg of product is obtained containing 89% peptides and 7% amino acids.

Example No. 3

35 L of slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with Purolite A830 anion exchange resin in the OH form, the volume of the anion exchange resin ^is 2.0 L, the flow rate is 0.7 L / h, then the anion exchange resin is washed with 0.5 l of distilled water. Obtain 35.5 l of a solution with a pH of 2.4. The resulting solution is filtered through a column with macroporous sulfonic cation exchanger KU-23 in H + form, the volume of cation exchanger is 10 l, the volumetric flow is 10 l / h, then the cation exchanger is washed with 4 l of acidified water (pH 1-2) and desorption is carried out with 3% ammonia. Obtain 3.2 l of eluate (pH 10.4), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. 0.3 kg of product is obtained containing 76% peptides and 8% amino acids.

Example No. 4

35 L of slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with Purolite A830 anion exchange resin in the OH form, the volume of the anion exchange resin ^is 2.0 L, the flow rate is 0.7 L / h, then the anion exchange resin is washed with 0.5 l of distilled water. Obtain 35.5 l of a solution with a pH of 2.4. The resulting solution was filtered through a column of gel weakly crosslinked (1% DVB) Dowex W50 × l cation exchanger in H + form, cation exchanger volume 13 l, volume flow 10 l / h, then cation exchanger was washed with 4 l acidified (pH 1-2) water and carry out desorption of 3% ammonia. Obtain 3.2 l of eluate (pH 10.3), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. 0.42 kg of product is obtained containing 91% peptides and 5% amino acids.

Example No. 5

35 l of the slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with anion exchange resin, the volume of the anion exchange resin is 2 l, the flow rate is 0.7 l / h, then the anion exchange resin is washed with 0, 5 liters of distilled water. Obtain 35.5 l of a solution with a pH of 2.4. The resulting solution is filtered through a column of Dowex W50 × 2 gel sulfocationionite containing 2% crosslinking (DVB) in the H + form, the volume of cation exchanger is 10 l, the volumetric flow is 10 l / h, then the cation exchanger is washed with 4 l of acidified water (pH 1-2) and carry out desorption of 3% ammonia. Obtain 2.7 l of eluate (pH 10.1), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. Obtain 0.4 kg of a product containing 88% peptides and 9% amino acids.

Example No. 6

35 l of the slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with anion exchange resin, the volume of the anion exchange resin is 2 l, the flow rate is 0.7 l / h, then the anion exchange resin is washed with 0, 5 liters of distilled water. Obtain 35.5 l of a solution with a pH of 2.4. The resulting solution was filtered through a column of Dowex W50 × 4 gel sulfation cationite (4% DVB) in H + form, the volume of cation exchanger was 10 L, the volumetric flow was 10 l / h, then the cation exchanger was washed with 4 l of acidified water (pH 1-2) and desorption 3% ammonia. Obtain 2.7 l of eluate (pH 10.1), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. Obtain 0.22 kg of a product containing 75% peptides and 8% amino acids.

Example No. 7

35 L of slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with Purolite A830 anion exchange resin in the OH ^- form, the volume of the anion exchange resin ^is 2.2 L, the flow rate is 0.7 L / h, then the anion exchange resin is washed with 0.5 l of distilled water and get 35.5 l of a solution with a pH of 2.7. The resulting solution is filtered through a column with Dowex MSC-1C macroporous sulfonated cation exchanger in the H + form, a cation exchanger volume of 10 l, a flow rate of 10 l / h, then cation exchanger is washed with 4 l of acidified water (pH 1-2) and desorption is carried out with 3% ammonia. Get 3 l of eluate (pH 10.2), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. 0.40 kg of product is obtained containing 87% peptides and 9% amino acids.

Example No. 8

35 L of slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with Purolite A830 anion exchanger in OH ^- form, anion exchanger volume 1.8 L, 0.7 L supply / h, then the anion exchange resin is washed with 0.5 l of distilled water and get 35.5 l of a solution with a pH of 1.9. The resulting solution is filtered through a column with Dowex MSC-1C macroporous sulfonated cation exchanger in the H + form, a cation exchanger volume of 10 l, a flow rate of 10 l / h, then cation exchanger is washed with 4 l of acidified water (pH 1-2) and desorption is carried out with 3% ammonia. Get 3 l of eluate (pH 10.2), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, the resulting solution is dried on a freeze dryer. 0.39 kg of product is obtained containing 85% peptides and 9% amino acids.

Example No. 9

35 L of a slip with a pH of 1.2 obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with Dowex MSC-1C macroporous sulfocathionite in H + form, the volume of cation exchanger is 10 L, the volumetric flow is 10 l / h , and act, as in example No. 1. 0.15 kg of product is obtained containing 91% peptides and 8% amino acids.

            Example No. 10

28 L of slip (pH 1.2) obtained after autolysis and extraction of the Polypharmine amino acid product from 50 kg of baking yeast is passed through a column with Purolite A830 anion exchange resin in the OH form, the volume of the anion exchange resin ^is 1.6 L, the flow rate is 0.7 L / h, then the anion exchange resin is washed with 0.5 l of distilled water. Get 28.5 l of a solution with a pH of 2.3. The resulting solution is filtered through a column with Dowex MSC-1C macroporous sulfocationionite in H + form, cation exchanger volume 10 l, volumetric flow 10 l / h, then cation exchanger is washed with 4 l of acidified water (pH 1-2) and desorption is carried out with 3%> ammonia. 2.9 L of eluate is obtained (pH 10.1), ammonia is distilled off in a vacuum evaporator to obtain a pH of 8.5, and the resulting solution is dried on a freeze dryer. 0.33 g of product is obtained containing 90% peptides and 7% amino acids.

Example No. 11

The product "Polycardin", obtained according to example No. 2, was used to study biological activity during experimental myocardial infarction. Myocardial infarction was simulated by ligation of the rat coronary artery. The reference drug was the standard antihypoxic drug Renitec. The drugs were administered intragastrally 1 time per day in the course of 5 days before the experimental myocardial infarction and in the course of 7 days after, in the form of aqueous solutions, Renitec at a dose of 1 mg / kg. Polycardine at a dose of 100 mg / kg. It was found that dietary supplement "Policardin" reliably has anti-ischemic action comparable to that of the drug "Renec", prevents the formation of a focus of necrosis and significantly increases the survival of experimental animals in conditions of acute hypoxia.

Claims (1)

A method for producing peptides, including autolysis of yeast, centrifugal separation of cell membranes, purification of autolysate on ion exchangers, and the preparation of an aqueous solution of peptides, using gel sulfocationite in hydrogen form containing 12-16% divinylbenzene as a crosslinking agent, characterized in that the resulting aqueous solution of peptides is passed successively through a gel anion exchange resin to obtain a solution with a pH of 2.0-2.6, and then through a gel cation exchange resin with a content of divinylbenzene 1-2% or macroporous nit.