RU2416243C2 - Method of extracting low molecular peptides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used in extracting low molecular peptides which are suitable for producing biologically active additives. The method involves preparation of enzymatic hydrolysate from colostric casein - whey mass using pancreatin. Sephadex G-25 is added to the obtained hydrolysate in ratio of 300 g sephadex per 1 l of the enzymatic hydrolysate. The obtained mixture is held for 10 minutes and then centrifuged. The turgid gel is eluted with distilled water and then centrifuged once more. The obtained liquid fractions are sterilised by filtering through fine filters with pore diametre of 0.2 mcm.

EFFECT: invention widens the raw material base for producing low molecular peptides.

2 tbl

Description

The invention relates to biotechnology and medicine, and in particular to methods for isolating low molecular weight peptides, which can serve as the basis for the preparation of biologically active additives (BAA).

It is known that low molecular weight peptides have a molecular weight of 3,000 to 15,000 kDa and play a significant role in maintaining homeostasis of the body, exhibiting a variety of activities - antibacterial, antiviral, antioxidant, regenerating. Low molecular weight peptides include transfer factor proteins with high immunostimulating ability.

There is a method for producing biologically active low molecular weight peptides from milk, colostrum, old-milk milk, cheese and curd whey, ultrafiltrates of skim, whole milk, cheese or curd whey (patent RU No. 2183935). The method includes purification of the fat fraction by centrifugation, sorption of proteins on an ion exchanger, chromatographic separation on CM-cellulose, elution, determination of the fraction with optical density at a wavelength of 280 nm more than 1 unit, dialysis and stabilization by microfiltration through semipermeable membranes with selectivity of more than 50 kDa. The disadvantage of this method is the use of raw materials with high nutritional value.

There is a method for producing dietary supplements from low molecular weight cationic milk proteins and dietary supplements obtained in this way (patent RU 2318406), in which cationic proteins of milk origin are separated from the fat fraction by centrifugation of milk raw materials, their sorption and chromatographic separation on CM-cellulose, dialysis of the obtained protein fraction against distilled water, phosphate buffer, sterilized by microfiltration through a semipermeable membrane. To obtain dietary supplements use secondary raw materials of the dairy industry.

The aim of the present invention is to develop a method for the isolation of low molecular weight peptides from non-utilizable waste products of lactoglobulins.

Lactoglobulins - medical immunobiological preparations (MIBP) for the treatment and prevention of acute intestinal infections (Pe. Certificate No. LS-003127 dated 04/25/2008 for "Lactoglobulin anti-lipoprotein cow" and Peg certificate No. LSR-002636/08 dated 09/09/2008 for "Lactoglobulin against conditionally pathogenic bacteria and bovine salmonella ”), which are produced from colostrum of immunized cows. In the process of producing lactoglobulins, casein-whey mass remains as waste (Industrial Regulation No. PR 01898776-01-06 for the production of Lactoglobulin Anti-Lipid Cow; Industrial Regulation No. PR 01898776-02-06 for the Production of Lactoglobulin Against Conditionally Pathogenic Bacteria and Cow Salmonella "). In the production of 1000 doses of the drug receive (9.3 ± 2) kg of colostrum casein-whey mass containing up to (2.5 ± 0.5) l of lactoserum. The casein-whey colostrum selected for isolation of low molecular weight peptides should have a casein-whey ratio of at least 7: 3, and a pH of at least 4.1 ± 0.2.

The peptide composition of the hydrolysates was studied by chromatographic analysis on a column with Sephadex G-25. Column calibration was performed using substances with known molecular weights. Data on the content in the hydrolysates of peptides with different molecular weights are shown in table 1.

Table 1. The content of peptides of different molecular weights in hydrolysates of colostrum casein-whey mass The quantitative composition of protein fractions of hydrolysates (chromatographic analysis) Number of episodes (% ± m) large peptides 19 10.9 ± 4.4 medium and small peptides 19 47.52 ± 4.9. amino acids 19 41.6 ± 3.1

As can be seen from the data presented in table 1, peptides with medium and low molecular weight prevail in hydrolysates of colostrum casein-whey mass.

The goal - to develop a method for the isolation of low molecular weight peptides from non-utilizable wastes from the production of lactoglobulins - is achieved by the fact that the hydrolyzate obtained by enzymatic hydrolysis of colostrum casein-whey mass using 0.3-0.6 g of pancreatin per 1 liter of hydrolyzable mixture for 3-4 hours and with correction of the medium with 25% aqueous ammonia solution (patent RU 2078811), they are subjected to gel chromatography on Sephadex G-25 for 10 minutes, the mixture of hydrolyzate with Sephadex is centrifuged, the swollen gel is eluted distilled water and re-centrifuged, the resulting liquid fractions are sterilized by filtration through finely porous filters with a pore diameter of 0.2 mmk. The presence of peptides in the obtained fractions is determined by electrophoresis in SDS page.

The method is as follows.

To 1 liter of hydrolyzate obtained by enzymatic hydrolysis of colostrum casein-whey mass using 0.3-0.6 g of pancreatin per 1 liter of hydrolyzable mixture for 3-4 hours and with medium correction with 25% aqueous ammonia, add 300 g Sephadex G-25, left for 10 minutes, the resulting mixture was centrifuged at 3000 rpm for 20 minutes. The supernatant is removed, the swollen gel is eluted with distilled water, centrifuged again at 3000 rpm for 20 minutes. The resulting liquid fractions are sterilized by filtration through finely porous filters with a pore diameter of 0.2 mmk. 118-123 ml of a fraction containing low molecular weight peptides with a molecular weight of 3000 to 15000 kDa are obtained from one liter of hydrolyzate. To obtain a dry form, the fractions are subjected to freeze drying.

The presence of low molecular weight peptides in the fraction is checked by SDS page electrophoresis.

table 2 The content of small molecular weight peptides in the fractions obtained by the proposed method (electrophoresis in SDS page) Number of episodes % ± m Peptides with a molecular weight of 7000 to 14000 kDa 9 9.3 ± 4.7 Peptides with a molecular weight of 3,000 to 7,000 kDa 9 54.3 ± 14.0 Amino acids (molecular weight less than 3000 kDa) 9 36.4 ± 4.2

The data in table 2 show that the use of the proposed method for the isolation of low molecular weight peptides from hydrolysates of colostrum casein-whey mass allows to obtain peptides with a molecular weight of from 3000 to 14000 kDa with a predominance of peptides having a molecular weight of from 3000 to 7000 kDa.

The technical result consists in expanding the raw material base for the production of low molecular weight peptides, in using for this purpose colostrum casein-whey mass - non-utilizable wastes from the production of lactoglobulins.

Claims (1)

A method for isolating low molecular weight peptides, comprising preparing an enzymatic hydrolyzate from colostrum casein-whey mass using pancreatin, adding Sephadex G-25 in a ratio of 300 g Sephadex per 1 liter of enzymatic hydrolyzate, keeping for 10 minutes, centrifuging, eluting with centrifuged, centrifuged, centrifuged distilled gel with distilled gel followed by sterilization of the obtained fractions through finely porous filters with a pore diameter of 0.2 mmk.