RU2020153C1 - Method of preparing of enzyme hydrolysate and nutrient medium for cultivation of eucaryotic cells - Google Patents

Abstract

FIELD: biotechnology, medicine. SUBSTANCE: defatted, dehydrated cattle brain tissue (waste in production of medicinal preparation "Lipocerebrin") is subjected to the mild enzymatic hydrolysis with medicinal pancreatin or homogenate of pig pancreas gland dissolved in water at the ratio 1:25, at 37-38 C, pH 7.6-7.8, for 6-7 days in the presence of chloroform following by heating at 90 C for 5-10 min, precipitation of high-molecular compounds from supernatant with 20-22% calcium chloride solution for 5-10 min, and then at isoelectric point of protein, filtration and drying of the end product. Preparation obtained is a base of nutrient medium "Cerebrat" used for cell cultivation containing, vol.%: hydrolysate of defatted dehydrated cattle brain tissue 0.15-0.20; vitamins 0.0001-0.00012; animal serum blood 5-10, and saline buffer Henx solution - the rest. Obtained preparation is a low-cost valuable product, its amino acid composition is close to the nutrient medium on the basis of enzyme hydrolysate from meat processing waste with regard to qualitative and quantitative respects, it can be used for cultivation of eucaryotic cells. EFFECT: improved method of enzyme hydrolysate preparing. 3 cl, 7 tbl

Description

 The invention relates to biotechnology, the medical and biological industry, and relates to an improved method for producing an enzymatic hydrolyzate, as well as a nutrient medium (PS), intended for growing cells of eukarytes producing biologically active substances (BAS), diagnostic and preventive biological and medical preparations.

A known method of producing an enzymatic hydrolyzate of meat processing waste (FGOMP) [2] by soft hydrolysis by enzyme preparations (medical pancreatin or homogenate of the pancreas of a pig) homogenate of muscle tissue of fetal cows 4.5-5 months of age and / or pigs 2.5-3 -monthly diluted with distilled water 1: 1.5. The process is conducted at a temperature of 37-38 ^{° C} in an alkaline pH 7.6-7.8 zone for 6-7 days. in the presence of chloroform (3 vol.%). The hydrolysis is stopped by heating at a temperature of 90 ^about 5-10 minutes. In this case, non-hydrolyzed proteins coagulate and precipitate. High-molecular compounds remaining in the supernatant are precipitated at the isoelectric point of the protein. The supernatant is filtered and dried in a stream of hot air. Enzymatic hydrolyzate of meat processing wastes is used as a nutrient medium in the cultivation of primary trypsinized animal cells. However, the described method is time-consuming, since it involves the initial separation of the muscle tissue of the fruits of cows or pigs from the bone skeleton with its subsequent homogenization.

 For the cultivation of various heteroploid transplantable animal and human cells, PSs based on FSHMP [1] and PS 199 [3], which are close in growth properties, are known.

PS based on FGOMP has the following composition, vol.%:
Fruit hydrolyzate
4-5 month old cows
age and / or pigs 2.5-3 months of age 0.4-0.5 Vitamin complex 0.0001
Reindeer juvenile blood serum 4.0-6,000 Glutamine 0.025-0.035 Proline 0.025-0.035 Tyrosine 0.025-0.035
Hanks Salt Buffer
This nutrient medium is multi-component. In addition, the amount of available raw materials currently cannot sufficiently ensure the intensive development of biotechnology in the country.

 Nutrient medium 199 contains more than 6% of the components, including 20 amino acids, 17 vitamins, coenzymes, glucose, mineral salts and some other substances; the environment is completely synthetic. PS 199 is used for long-term cultivation of transplantable cell cultures and provides a high degree of standard research. The disadvantage is the complex technology of its manufacture from expensive, mainly imported reagents. In conditions of biotechnological production, where a large amount of nutrient medium is required to obtain cell biomass, PS 199 is unprofitable.

 The aim of the invention is to simplify the process technology, reducing the cost of the final product and the nutrient medium based on it for the cultivation of eukaryotic cells.

This is achieved by the fact that as a protein-containing raw material, fat-free, dehydrated cattle brain tissue diluted with distilled water in a ratio of 1:25 is used, which is subjected to treatment with medical pancreatin or native pig pancreas in an alkaline zone of pH 7.6-7.8 in the presence of chloroform at a temperature of 37-38 ^about C for 6-7 days. followed by heating at ⁹⁰ ° C for 5-10 min 20-22% hydrolyzate by treatment with a solution of calcium chloride for 5-10 min, by precipitation of high-molecular compounds in the isoelectric point of the protein, the desired product by filtration and drying.

Fat-free and dehydrated brain tissue of cattle is a waste product of the drug "Lipocerebrin" and has the following standard parameters:
mass fraction of protein is at least 60%;
mass fraction of fat not more than 15%;
does not contain bacteria of E. coli and Proteus, as well as pathogenic microorganisms, including salmonella.

 The release of the medicinal product "Lipocerebrin" ensures the stability of obtaining large quantities (tons) of protein raw materials for the enzymatic hydrolyzate. The latter makes it possible to make the production of the Lipocerebrin medical product waste-free, thereby improving the environmental conditions for its production, and, in addition, to use protein raw materials in the future as an enzymatic hydrolyzate - the basis of the Cerebrate nutrient medium.

The nutrient medium based on the enzymatic hydrolyzate of fat-free, dehydrated brain tissue of cattle contains the following components, vol.%:
Enzymatic hydro
fat free lysate,
dehydrated brain tissue of cattle 0.15-0.20 Vitamin complex 0.0001-0.00012 blood serum of cattle 5.0-10.0 Hanks solution The rest
In contrast to the known nutrient medium 199, the described nutrient medium contains an amino acid base obtained by enzymatic hydrolysis of available complete proteins, eliminates the need for expensive imported ingredients.

PRI me R 1. Fat-free, dehydrated cattle brain tissue, diluted with distilled water in a ratio of 1:25, is subjected to gentle hydrolysis by enzyme preparations (medical pancreatin or native pig pancreas). The process is conducted at a temperature of 38 ^{° C} in an alkaline pH 7,6 zone for 6 days in the presence of chloroform (1.5 vol.%). The hydrolysis is stopped by heating at a temperature of 90 ^about 5 minutes In this case, non-hydrolyzed proteins coagulate and precipitate. High-molecular compounds remaining in the supernatant are precipitated first in the presence of a 20-22% solution of calcium chloride, and then at the isoelectric point of the protein. The supernatant is filtered and spray dried in a stream of hot air. Drying mode (inlet 150-160 ^{° C} and outlet 95-100 ° ^C) provides a dry product with a residual humidity not more than 3-4%.

0,55-0,60
Коэффициент протео- лиза

0,9-1,0
Качественный состав и количественное содержание аминокислот в 0,2%-ном растворе питательной среды с гидролизатом обезжиренной, обезвоженной мозговой ткани КРС ("Церебрат") в сравнении с составом питательной среды 199 и 0,5%-ным раствором гидролизата отходов мясопереработки - ФГОМП представлены в табл.1.The obtained enzymatic hydrolyzate of fat-free, dehydrated brain tissue of cattle has the following physicochemical characteristics:
Indicators:
The concentration of hydrogen ions in a 1% solution of 6.2-7.0
Solubility,% not less than 5.0
Mass fraction of moisture,% no more than 3.0-4.0
Mass fraction of total nitrogen,% in the range of 10.0-11.0
Mass fraction of amine nitrogen,% in the range 5.5-6.0 Amine coefficient

0.55-0.60
Proteolysis coefficient

0.9-1.0
The qualitative composition and quantitative content of amino acids in a 0.2% solution of a nutrient medium with a hydrolyzate of fat-free, dehydrated cattle brain tissue (Cerebrate) in comparison with a composition of a nutrient medium 199 and a 0.5% solution of a meat processing waste hydrolyzate - FGOMP are presented in table 1.

 Strict adherence to hydrolysis conditions and regular monitoring of the physicochemical parameters of the hydrolyzate in the process make it possible to obtain a sufficiently standard product with a quantitative amino acid spread of no more than 5-6%.

 PRI me R 2. The concentration of enzymatic hydrolyzate of fat-free, dehydrated brain tissue of cattle in the nutrient medium "Cerebrate", which provides optimal living conditions of the transplanted human kidney cell line RH-PA. The results are presented in table.2.

 From the data of Table 2 it follows that the optimal conditions for cell growth and development are provided by PS containing 0.15-0.20 vol.% Enzymatic hydrolyate of fat-free, dehydrated brain tissue of cattle (which corresponds to the content of amine nitrogen 14-15 mg.%, according to the content of amine nitrogen in PS 199).

 PRI me R 3. We tested the growth-promoting properties of PS "Cerebrate", supplemented with SCRS in various concentrations, during the cultivation of heteroploid transplantable human kidney cell lines RH-PA and SPEV in comparison with PS 199 manufactured by IPM and VE of the USSR Academy of Medical Sciences. The results are presented in table. 3. As can be seen from table 3, the optimal concentration of SCRS is 5-10 vol.%.

 PRI me R 4. The biological growth properties of the described PS was tested during cultivation of heteroploid transplantable human kidney cell lines RH-PA and SPEV compared with PS 199 produced by IPM and VE AMS USSR and supplemented with 5% vol. Cattle serum (table 4 ) From the data presented in table 4 it follows that the substitute "Cerebrate" (0.15-0.20 vol.% Enzymatic hydrolyzate in Hanks solution) with 5 vol. % SCRS has growth-stimulating properties identical to those of PS 199 manufactured by IPM and VE of the USSR Academy of Medical Sciences.

 In all subsequent examples, the composition of the Cerebrate substation is given with the following content of components, vol%: FG 0.15-0.2; SCRS 5; a complex of vitamins 0.0001-0.00012; Hanks solution rest.

 The possibility of using the Cerebrate PS in the cultivation of the transplantable diploid line of fibroblasts of a light embryo of a cow (LEC) with its introduction into the composition of PS MEM in the amount of 50 vol.% Was studied (Table 5). The data presented in the table illustrate the possibility of using the Cerebrat nutrient medium as part of the PS MEM produced by IPM and VE AMS of the USSR when cultivating transplantable diploid lines of human cells (M-19) and animals (LEC).

 PRI me R 5 (see table 6).

 The data obtained indicate that with prolonged passage of the transplanted human kidney cell culture RH-PA, the growth-promoting activity of Cerebrate PS is similar to that in PS 199.

 PRI me R 6.

 The data presented in table. 7 indicate that the cells grown in the Cerebrate substation maintain good viability and completely restore their properties by passage 5 after freezing.

 The Cerebrate nutrient medium based on the obtained enzymatic hydrolyzate of fat-free, dehydrated cattle brain tissue has high growth-promoting abilities for some primary as well as transplantable heteroploid animal and human cell lines, which allows it to be used to obtain large amounts of cellular biomass instead of a multicomponent synthetic nutrient medium 199 .

Claims (2)

1. A method of obtaining an enzymatic hydrolyzate, including the processing of protein-containing raw materials diluted with water with medical pancreatin or native pig pancreas, in the alkaline zone, pH 7.6 - 7.8 in the presence of chloroform at 37 - 38 ^o C for 6 - 7 days, heating at 90 ^o C for 5 to 10 minutes, subsequent precipitation of high molecular weight compounds at the isoelectric point of the protein, filtering the target product and drying it, characterized in that, in order to simplify the process technology and reduce the cost of the final product, fat-free, dehydrated brain tissue of cattle is used as a protein-containing raw material, the enzyme is dissolved in water in a ratio of 1: 25, and before precipitation of high molecular weight compounds, the hydrolyzate is treated with a 20-22% calcium chloride solution for 5-10 minutes. 2. Nutrient medium for culturing eukaryotic cells, including an amino acid base, a complex of vitamins, Hanks solution and cattle blood serum, characterized in that, in order to reduce the cost, an enzymatic hydrolyzate of fat-free, dehydrated cattle brain tissue is used as the amino acid base in the following ratio of components, vol.%:
Enzymatic hydrolyzate of fat-free, dehydrated brain tissue of cattle 0.15 - 0.20
Vitamin Complex 0.0001 - 0.00012
Cattle blood serum 5 - 10
Hanks Solution Else

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