RU2284356C1 - Method for preparing amino acids and lower peptides mixture - Google Patents

Abstract

FIELD: biotechnology, amino acids, peptides.

SUBSTANCE: invention relates to a method for preparing a mixture consisting of amino acids and lower peptides from yeast autolyzates. Method involves autolysis of yeast, separation of cellular debris by centrifugation and purification of autolyzate in ionites resins wherein gel sulfocation-exchange resin in hydrogen form comprising 12-16% of divinylbenzene as a cross-linking agent is used as ionite. Proposed method provides preparing highly purified mixture of amino acids with the low content of peptides.

EFFECT: improved preparing method.

2 tbl, 4 ex

Description

The invention relates to biotechnology and relates to a method for producing a mixture of amino acids and lower 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.

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. 602543, 957835, 1369300, 1731806).

In known methods for the extraction, concentration and purification of amino acid mixtures and lower peptides from yeast autolysates, sulfocationionites and anionites are used.

In the known method according to copyright certificate No. 1731806, the process of isolating from a yeast autolysate a mixture of amino acids and lower peptides, adenine and tyrosine is based on the calculated modes of sorption and desorption of these products on ion exchangers. The method is implemented only under the conditions of a precisely reproducible calculation process using 2 ion exchangers: sulfonic cation exchanger KU-2 × 8 and strongly basic anion exchange resin of the APA type to produce the target product containing at least 20-27% lower peptides in the amino acid mixture. Since, under the conditions realized in the known invention, amino acids and peptides are not adsorbed by ARA anion exchange resin, the presence of a large number of peptides in the target product is due to the sorption properties of KU-2 × 8 sulfocationite.

Closest to the claimed method is a method for producing mixtures of amino acids and lower peptides from a yeast autolysate by filtering the autolysate through weakly basic anion exchange resin IA-1p, followed by sorption of the amino acids and lower peptides from the filtrate on KU-2 × 8 sulfocationite, evaporation of the eluate and drying of the finished product (aut USSR certificate No. 602543 - prototype of the proposed method).

Significant disadvantages of this method are:

- the high content in the target product of biologically active peptides, constituting 24-30% of the total number of amino acids, which limits the scope of use of the product as a food and pharmacological purpose, as well as in the form of an amino acid component in the creation of microbiological nutrient media and perfume compositions;

- low volume load of autolysate V _(aut) on the volume of _{sulfonic cation exchanger} KU-2 × 8 V _{(cation exchanger)} at the maximum volumetric rate of sorption of V _{(aut / hour) of} amino acids and lower peptides by sulfocationion after autolysis, separation of cell membranes, comprising 45-50% from the total volume of the autolysate, and purification of the autolysate on the anion exchange resin IA-1r:

V _(aut) : V _{(cation exchanger)} = 0.8-1.0: 1;

V _{(auto / hour)} : V _{(cation exchanger)} = 0.3: 1;

- a significant consumption of distilled water for washing the ion exchanger KU-2 × 8 after sorption of amino acids and peptides:

V _(water) : V _{(cation exchanger)} = 7.5-8.0: 1,

- sulfonic cation exchanger KU-2 × 8 simultaneously with the sorption of amino acids and peptides almost completely absorbs the soluble ballast substances remaining in the autolysate after filtration through anionite IA-1p, which are responsible for the pigmentation of autolysates, and during desorption of amino acids and peptides from the ion exchanger KU-2 × 8 1 With a 3% solution of ammonia, sorbed ballast and coloring substances enter the eluate, which prevents the obtaining of a high-quality target product.

An object of the present invention is to provide an efficient and economical method for producing a highly purified amino acid mixture with a low peptide content from a yeast autolysate.

The essence of the proposed method for producing a mixture of amino acids and lower peptides is as follows.

The method includes autolysis of yeast, separation of cell membranes by centrifugation, and purification of the autolysate on ion exchangers, using gel sulfocationite in hydrogen form containing 12-16% divinylbenzene as a crosslinking agent.

According to the proposed method, after separation of the cell membranes, the yeast autolysate is acidified to pH = 2.0-3.0, passed through hydrogen sulfation in the form of hydrogen containing 12-16% divinylbenzene (hereinafter DVB) as a crosslinking agent, and the sulfated ion is washed with acidified water with pH = 2-3 and the sorbed amino acid mixture is eluted with a 2-3% ammonia solution, and the eluate is filtered through anion exchange resin in a known manner, evaporated and dried to obtain the desired product.

The basis of the proposed method for the separation of amino acids and lower peptides contained in the yeast autolysate, based on modern ideas about the kinetic-diffusion features of the sorption of organic ions on gel ion exchangers of various "density" (or permeability).

In the present invention, the use of weakly swelling (K _(nab) = 1.1-1.3) "dense" sulfocationionites with 12-16% DVB allowed us to almost completely separate individual (free) amino acids from lower peptides in a one-act sorption process from autolysate. Under the conditions of a dynamic column process on such sulfocationionites, predominant sorption of individual amino acids occurs until the full exchange capacity of the ion exchanger is exhausted. Moreover, the absorption of lower peptides is significantly hampered due to kinetic-diffusion limitations due to the reduced permeability of the “dense” sorbent matrix for larger organic ions — in our case, peptides. At the same time, since in the multicomponent composition of the autolysate, the physicochemical properties of lower peptides, in particular some tri- and dipeptides, differ little from the properties of individual amino acids, there is a slight sorption of peptides on ion exchangers with 12-16% DVB.

In contrast to the proposed method in the prototype, as in the known method according to ed. certificate 1731806, when using KU-2 × 8 gel sulfocationite with 8% DVB, which has a significantly less “dense” matrix and K _(nab) = 3.0, diffusion to the sorption centers of cation exchanger of free amino acids and lower peptides consisting of 2 , 3, 4 or 5 amino acids, passes without apparent difficulties, and upon desorption, these peptides together with individual amino acids enter the target product, accounting for 24-30% of the total amount of amino acid mixture.

This is the fundamental difference between the proposed method from the known.

Significant advantages of the proposed method in comparison with the known are:

- the final product contains no more than 5-9% of the lower peptides against 24-30% in the known method;

- the content of free amino acids in the final product is at least 90% compared with 65-75% in the known method;

- expanding the scope of use of the target product due to the low content of biologically active peptides in the product;

- the resulting mixture of individual amino acids and lower peptides is a highly purified preparation, since the use of strongly "crosslinked" sulfocationionites with 12-16% DVB significantly inhibits the sorption of ballast substances due to steric and kinetic diffusion restrictions, while sulfonic cationite KU-2x8 simultaneously with sorption amino acids almost completely absorbs soluble ballast substances from the autolysate, which during desorption fall into the finished product;

- simplification of the process, since the volume load of the autolysate V _(aut) on the volume of sulfocationite V _(cationite) with 12-16% DVB and the volumetric rate of sorption V _{(auth / hour) of a} mixture of amino acids and lower peptides by sulfocationite directly from the autolysate after separation of the cell shells compared with sulfonic cation exchanger KU-2x8 increase 2.3-2.6 and 3 times, respectively;

V _(aut) : V _{(cation exchange resin)} = 2.3-2.6: 1;

V _{(auto / hour)} : V _{(cation exchange resin)} = 1.0-1.2: 1;

- a decrease of 12-15 times the flow rate of distilled water for washing sulfocationite with 12-16% DVB compared with washing the ion exchanger KU-2 × 8 after sorption of amino acids and peptides in the known method:

V _(water) : V _{(cation exchanger)} = 0.5-0.7: 1.

Example.

400 ml of distilled water are poured into a sterilized reactor and 2.0 kg of fresh yeast with a moisture content of 75% is charged. The mixture is heated to 48 ° C and with a working stirrer stand for 30 hours. The resulting autolysate suspension is centrifuged and, after separation of insoluble cell membranes, the supernatant is acidified with a 5N sulfuric acid solution to pH = 2.9 and fed to a column (5 × 25 cm) with sulfocathionite in hydrogen form with 12-16% DVB for sorption of amino acids and peptides. The optical density (OD) of the “slip” after the column at 420 nm is 79-92% of the optical density of the original autolysate. After sorption is completed, the ion exchanger is washed with 250-350 ml of a 0.002 N solution of sulfuric acid, and a mixture of amino acids and lower peptides is stripped with a 2.5% ammonia solution. Further, in a known manner, the eluate is filtered through an ion exchanger, evaporated and dried.

The amino acid composition of the target product and the operating parameters of the invention are presented in tables 1 and 2 in examples No. 1, 2, 3. For comparison, example No. 4 shows the corresponding data when using KU-2 × 8 ion exchanger for sorption of a mixture of amino acids and peptides from autolysate.

Table 1 No. Example No. 1 Example No. 2 Example No. 3 Example No. 4 ion exchange with 12% TWO ion exchange with 13% DVB ion exchange with 16% DVB IONITE KU-2 × 8 Amino Acid (AK) target product target product target product target product hydrolysis. * no hydrolysis. ** hydrolysis. * no hydrolysis. ** hydrolysis. * no hydrolysis. ** hydrolysis. * no hydrolysis. ** mg / g mg / g mg / g mg / g one Arginine 39.0 34.6 37,4 33.3 31,4 30.1 12,2 6.4 2 Lysine 98.1 89.2 100,2 91.6 75.8 74.3 52.6 30.2 3 Tyrosine 38.6 34.7 50,5 50,0 15,2 14.8 27.5 19.6 four Phenylalanine 33,2 31.8 25.8 24.6 35,4 34.3 39.2 29.6 5 Histidine 21.6 20.3 19,4 16.3 20.6 20,2 14.0 5.2 6 Leucine 81.8 71,4 93.2 83,2 64,2 61.2 69.3 58.9 7 Isoleucine 52.3 50.9 58.5 58.2 50,0 45.5 45.9 33.5 8 Methionine 17.1 16.3 11.2 11.0 17,2 16.9 15,5 13.6 9 Valine 53.3 51.7 52,2 51.9 51.5 51.9 58.8 42.3 10 Proline 29.2 27,2 30,0 29.2 25.7 25.3 42.8 21,4 eleven Threonine 108.6 98.5 133.0 132.6 77.1 76.1 35.5 27.4 12 Serine 33.7 30.9 35.3 34.1 39.7 32,0 33.6 35.9 13 Alanine 88.2 86.4 107.4 102.6 78,4 77.5 73,4 63.5 fourteen Glycine 39.3 35.7 41.7 41.1 36,4 37.1 36.8 14.1 fifteen Cysteine 5,6 4.2 5.3 5.3 3.9 2.9 27.9 14.9 16 Glutamic acid 67.2 61.6 65.6 60.5 60.9 52,5 76.0 56.1 17 Aspartic acid 50.6 37.6 49.4 32,2 45.8 42.8 63.6 61.2 eighteen Tryptophan 5.3 2.9 0.1 0.1 5,4 4.9 0 0 Total in product 862.7 785.9 916.2 857.8 734.2 700,1 724.6 533.8 The content in the mixture of AK and lower peptides: - free AK 91.1 93.6 95.3 73.7 lower peptides 8.9 6.4 4.7 26.3 * - total amino acid content in the hydrolyzed product
** - the content of individual (free) amino acids in the product without hydrolysis.

table 2 Options Example No. 1 Example No. 2 Example No. 3 Example No. 4 Ionite with 12% DVB Ionite with 13% DVB Ionite with 16% DVB Ionite KU-2 × 8 Volumetric load, l / l 2.6: 1 2.5: 1 2.3: 1 0.9: 1 V _(autol) : V _{(cation exchanger)} Volumetric sorption rate, l / h: l 1.2: 1 1.1: 1 1.0: 1.0 0.3: 1 V _(autol) : V _{(cation exchanger)} OP * "slip" after sorption with respect to the OD of the autolysate, 420 nm,% 79 84 92 19 Water consumption for flushing. ion exchange resin after sorption, l / l 0.7: 1 0.6: 1 0.5: 1 7.5: 1 V _(water) : V _{(cation exchanger)}

Claims (1)

A method of producing a mixture of amino acids and lower peptides, including yeast autolysis, centrifugal separation of cell membranes, and ion-exchange autolysate purification, characterized in that gel sulfonation in hydrogen form containing 12-16% divinylbenzene as a crosslinking agent is used.