RU2563816C1 - Method for producing immune stimulant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for producing an immune stimulant from cephalopods nerve tissue, marine mammal and fish brain, involving defrosting and grinding the raw material; thereafter, the ground raw material is processed in protein-degrading enzymes; the produced supernatant is separated, and the end product is dried in the specific environment.

EFFECT: method enables increasing the end product yield.

2 cl, 5 tbl, 4 ex

Description

The invention relates to the field of food and medical industry, in particular to a method for producing an immunostimulant from marine animals and aquatic organisms, and can be used as a biologically active food supplement (oral) and as a therapeutic injection for humans and animals.

A known method of producing an immunostimulant from hydrobionts, for example, mussel Dreissena (RF Patent No. 2374891 from 07/16/2008, IPC A23J 1/04). In this case, an immunostimulant is obtained by fermentolysis. The raw materials are crushed together with the valves and subjected to hydrolysis with an enzyme preparation taken in an amount of 0.1-0.2% by weight of the forcemeat mixture, the process is carried out for 360-480 minutes, at a pH of 3-11 and a temperature of 54-56 ° C, then the enzyme is inactivated by heating the mass to 70-75 ° C for 10 min, evaporated and filtered to separate the hydrolyzate from the precipitate, enzymes of plant, animal or microbial origin are used as the enzyme preparation.

A known method of obtaining an immunostimulant from marine aquatic organisms (RF patent No. 2468593 from 09/29/2011, IPC A23L 1/33). At the same time, mussels are used as raw materials, which are crushed, dried and mixed with the enzyme preparation. As an enzyme preparation, the drug "Protozyme" with an activity of at least 490 PE / g of proteolytic action is used. Hydrolysis is carried out. Water is added to the mixture, while the raw materials, enzyme preparation and water are taken in a ratio of 20: 1: 400. The resulting homogenate is heated for 30-40 minutes to a temperature of 50 ° C and maintained at this temperature with constant stirring for 10 hours. The liquid fraction is separated and concentrated to a moisture content of not more than 20%. In this case, a product is obtained having a high content of low molecular weight peptides.

A known method of producing an immunostimulant from cucumaria (RF Patent No. 2147239, MKI 7 A61K 35/56 “General strengthening non-specific immunomodulating agent”). The crushed muscle tissue of cucumaria is mixed with water, proteolytic enzyme, sodium chloride and 0.1% sodium benzoate are added, the mixture is heated and thermostatic, then the enzyme is inactivated by heating, filtered and canned with 96% ethanol or an alcohol extract from the entrails of cucumaria.

The disadvantage of the above analogues is that muscle tissue or whole hydrobions were used as raw materials, in which, as is known, the peptide content does not exceed 1%, and the biological activity is due to the presence of triterpene glycosides or other components in the tissue. The disadvantages also include the duration and multi-stage process of obtaining, a low level of immunostimulating activity.

An immunostimulant obtained from the nervous tissue of marine aquatic organisms, fish and marine mammals contains a complex of low molecular weight peptides and amino acids with immunostimulating activity, stimulation of cerebral circulation, and increased resistance to infectious diseases.

A known method of obtaining an immunostimulant from the nervous tissue of marine aquatic organisms with immunostimulating action (RF Patent No. 2091072 of 03/10/1993, IPC A61K 35/50). In this method, squid or cuttlefish ganglia are homogenized in water acidified with acetic acid to a pH of 3.5-4.5 with 0.1% zinc chloride. The mixture is incubated for 28-48 hours. After separation of the supernatant, it is subjected to sterilizing filtration. The sterile filtrate is concentrated 5.5 times by ultrafiltration through a porous material with a separation limit of 1000-5000 D and subjected to purification in triplicate volume of water, alkalized to pH 9.0-10.0. The purified concentrate is again concentrated 2 times on the same porous material and subjected to sterilizing filtration. Then sterile filling of the preparation is carried out and freeze-dried.

A known method of obtaining a biologically active substance with an immunostimulating effect (RF Patent No. 2105504 from 05.06.1996, IPC A61K 35/50). The immunostimulant is derived from squid or cuttlefish nerve tissue. Ganglia (nerve tissue) are washed with water and acetone, then placed in acetone and kept in acetone at a temperature of 4-10 ° C, homogenized in water, acidified to pH 3.5-4.5 with 0.1% zinc chloride solution, stand for 24-48 hours, the supernatant is separated, which is treated with acetone at a ratio of the supernatant and acetone 1: 6 by volume, at a temperature of 3-5 ° C, the precipitate is separated, dried, dissolved in water with a pH of 6.0-7, 0 and filter.

The closest to the achieved result is a method of obtaining a biologically active substance with an immunostimulating effect (RF Patent No. 2222337 from 03.07. 2002, IPC A61K 35/50). In this case, an immunostimulant is obtained from the nervous tissue of cephalopods: squid, cuttlefish, octopuses; the brain of marine mammals: beluga whales, sperm whales, whales, walruses, larghas, seal, lahtak, akiba; fish brain: salmon, sturgeon, herring, cod, flounder, perch, etc. The raw materials are thawed, crushed, extracted with a 3% solution of acetic acid with the addition of zinc chloride, centrifuged, the supernatant is separated and the target product is isolated by ultrafiltration and diafiltration, which are carried out in two stages on two membranes with different molecular weight separation limits. The immunostimulant obtained by the proposed method has clearly limited molecular weight limits of 1000 -10000-15000 Yes. A concentrated solution with the target substance with a molecular weight of 1000-10000 Yes is sterile filtered on membranes with a pore size of 0.22 μm, poured under sterile conditions into bottles, followed by freeze drying.

The disadvantages of these methods are:

- the use of acetic acid;

- the use of zinc chloride;

- the use of acetone;

- sediment separation and ultrafiltration process;

- the duration of the process;

- low yield of the final product (less than 1%).

The problem solved by the invention is to simplify the method of obtaining an immunostimulant from the nervous tissue of marine aquatic organisms and mammals and increase the yield of the final product through the use of exogenous proteolytic enzymes.

 The problem is solved as follows.

A method of obtaining an immunostimulant, including thawing the raw material, grinding it, obtaining a supernatant, separating it and drying the final product, while the crushed raw material is fermented with a hydraulic module of 1: 1-5, pH 5.5-7.5, temperature 22-45 ° С, during 1.5-3.5 hours under the action of proteolytic enzymes in the amount of 15-45 units. activity per 1 g of raw material, and the supernatant is separated by filtration.

As raw materials, the nervous tissue of cephalopods is used: squid, cuttlefish, octopus; the brain of marine mammals: beluga whales, sperm whales, whales, walruses, larghas, seal, lahtak, akiba; fish brain: salmon, sturgeon, cod, herring, flounder, perch, etc.).

As proteolytic enzymes, proteolytic enzymes (for example, protamex, collagenase, megateria, trypsin, chymotrypsin, pylorine) are used, and the final product is either sublimated or dried on a drum-type vacuum dryer. The yield of the final product is 5-10% by weight of the feedstock.

New in the claimed technical solution is that an immunostimulant is obtained by fermenting raw materials (nervous tissue of marine aquatic organisms and mammals) with proteolytic enzymes, while the biological activity of the final product is preserved, and the yield from the mass of the starting material is 5-10%, depending on the type of raw material, while in the prototype it is less than 1%.

The use of enzymes with a proteolytic effect promotes the formation of an additional amount of low molecular weight peptides that have a stimulating effect.

The increase in the yield of the final product through the use of proteolytic enzymes is determined experimentally. The achieved result is illustrated by the data.

Tables 1
The yield of the final product, in% by weight of the feedstock Raw materials Immunostimulator prototype The inventive immunostimulant (ZI) cephalopods 0.97 10.0 fish 0.88 7.0 mammals 0.54 5,0

The biological activity of the claimed immunostimulant was determined by studying the phagocytic activity of peripheral blood neutrophils

Heparinized venous blood was placed in a thermostat for 20-30 minutes to sediment red blood cells. Using the pipette, the upper (light) fraction containing leukomass was selected, 0.85% NaCl was washed three times (10 min at 1000 rpm). The supernatant was drained, the cells were resuspended in Hanks solution, adjusted to a concentration of 2 × 106 / ml by neutrophils and their phagocytic activity was examined.

The phagocytic activity of neutrophils was determined in relation to latex. Initially, blood neutrophils from healthy donors were incubated with the studied biologically active substances for 60 minutes at 37 ° C at a final concentration of 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml. Then a suspension of cells in a volume of 100 μl was combined in centrifuge tubes with 100 μl of latex (particle size of latex 1.5-2 μm) in a ratio of 1:20 and incubated for 30 minutes at 37 ° C. Then the tubes were centrifuged at 1000 rpm for 5 minutes and the supernatant was drained. Smears were prepared from the precipitate, fixed with methanol, stained with azur-II-eosin and microscopic, determining the phagocytic index (FP%) - the percentage of cells involved in phagocytosis, and phagocytic number (PS) - the average number of latex particles absorbed by one phagocyte.

table 2
The percentage of cells involved in phagocytosis (AF%) FT Average value Standard deviation p The control 49.0 5.97 Tinrostim 1 mcg / ml 47.0 3.06 0.89 Tinrostim 0.1 μg / ml 51.0 4.98 0.11 Tinrostim 0.01 mcg / ml 53.5 0.88 0.01 ZI 1 m kg / ml 47.5 5.41 0.50 ZI 0.1 μg / ml 50,0 14.68 0.26 ZI 0.01 μg / ml 53.50 3.63 0.05

For AF - Statistically significant differences with the control - Tinrostim 0.01 μg / ml and the inventive immunostimulant (ZI) 0.01 μg / ml; mcg - micrograms; 1 mg - 1000 mcg

Table 3
The values of the phagocytic number (PS) - the average number of latex particles absorbed by one phagocyte Average Standard value deviation R The control 1.75 0.13 Tinrostim 1 mcg / ml 2.00 0.37 0.563 Tinrostim 0.1 μg / ml 2.05 0.18 0.056 Tinrostim 0.01 mcg / ml 2,30 0.25 0,020 ZI 0.1 μg / ml 2.15 0.20 0,036 ZI 0.01 μg / ml 2.05 0.48 0,023 ZI 0.001 mcg / ml 2,50 0.47 0.003

For PS - Statistically significant differences with control - Tinrostim 0.01 mg / kg and 0.01 μg / ml; The inventive immunostimulant (ZI) is 0.01 μg / ml and 0.01 μg / ml.

The results presented in tables 2 and 3 indicate that there are no significant differences in the immunostimulating activity of tinrostim preparations and the claimed immunostimulant. Thus, the activity of the drug obtained by enzymatic hydrolysis, coincides with the activity of the drug obtained by the method of the prototype.

Example 1

10 kg of frozen squid ganglia are thawed to a temperature of minus 4 ° C in the center of the briquette and ground in an electric meat grinder. The crushed raw materials are placed in a reactor and water is added in a ratio of 1: 2, pH 7.5. The mixture is stirred and add a solution of the enzyme protamex at the rate of 25 units. per 1 g of raw materials. The temperature is brought to 25 ° C and fermented for 1.5 hours. After the end of the fermentolysis process, the mixture is filtered on a suction filter through calico. The liquid fraction is sent to freeze-drying. The yield of the final product is 1 kg, which is 10% of the weight of the feedstock.

The resulting final product is a light gray powder, readily soluble in water and insoluble in organic solvents and contains 45% free amino acids and 35% peptides with a molecular weight of less than 15 kDa.

Table 4
Composition of free amino acids from nerve tissue Amino acid ZI, mol% prototype,% Taurine 9.67 1,56 Aspartic acid 22.86 3.69 Threonine 1,67 0.27 Series 2.66 0.43 Glutamic acid 18.59 3.0 Glycine 2.66 0.43 Alanya 2.85 0.46 Valine 2.17 0.35 Methionine 1.30 0.21 Isoleucine 1.24 0.20 Leucine 3.66 0.59 Histidine 2.11 0.34 Lysine 28.56 4.61

Table 5
The output fractions of Tinrostim and ZI during ultrafiltration Sample, fraction No. Tinrostim ZI The yield of fractions, g The yield of fractions, g Centrifugate / filtrate 85.0 ± 2.5 1000 ± 5.0 more than 100 kD 26.6 ± 0.2 220 ± 4.0 from 10 to 100 kD 21.8 ± 0.1 310 ± 4.0 from 1 to 10 kD 36.6 ± 0.2 375 ± 3,5 less than 1 kD - 95 ± 1.7.0

The output of tinrostim from 10 kg of raw materials are shown in table 1.

Example 2

10 kg of frozen nerve tissue of the brain of marine mammals are thawed to a temperature of minus 4 ° C in the center of the briquette and ground in an electric meat grinder. The crushed raw materials are placed in a reactor and water is added in a ratio of 1: 5. The mixture is stirred and a collagenase enzyme solution is added at a rate of 15 units. per 1 g of raw materials. The temperature was adjusted to 45 ° C and fermented for 3.5 hours at pH 5.5. After the process of fermentolysis, the mixture is cooled to room temperature and filtered on a suction filter through calico. The drying of the liquid fraction is carried out on a drum-type vacuum dryer. The yield of the final product is 0.5 kg, which is 5% of the weight of the feedstock.

Example 3

10 kg of frozen nerve tissue of the fish brain is thawed to a temperature of minus 4 ° C in the center of the briquette and crushed in an electric meat grinder. The crushed raw materials are placed in a reactor and water is added in a ratio of 1: 3. The mixture is stirred and trypsin enzyme solution is added at the rate of 45 units. per 1 g of raw materials. The mixture was fermented for 2.5 hours at a temperature of 22 ° C and a pH of 7.5. After the end of the fermentolysis process, the mixture is filtered on a suction filter through calico. The liquid fraction is lyophilized. The yield of the final product is 0.7 kg, which equals 7% of the weight of the feedstock.

Claims (2)

1. A method of obtaining an immunostimulant from the nervous tissue of cephalopods, the brain of marine mammals and fish, including thawing raw materials, grinding them, obtaining a supernatant, separating them and drying the final product, characterized in that the crushed raw materials are treated with proteolytic enzymes in an amount of 15-45 units activity per 1 g of raw material with a water module of 1: 1-5, pH 5.5-7.5, temperature 22-45 ° C, for 1.5-3.5 hours 2. The method according to p. 1, characterized in that as the proteolytic type enzymes use protomex, or trypsin, or chymotrypsin.