RU2472517C1 - Method for preparing peptide complex of cod liver - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely a method for preparing a biologically active peptide complex of Gadidae fam. liver. The method involves grinding a raw material, extracting in mixed oil and water in proportions of raw material: extractant 1:5-1:30 at room temperature for 2-4 hours with agitation. Further, the end product is purified by treating in an organic solvent in relation of 1:0.5-2 followed by separation and drying of an aqueous residue. The raw material is presented by cod liver either fresh, frozen or dry, while the organic solvent is specified in a number of diethyl ester, ethyl acetate, butanol, chloroform.

EFFECT: invention provides preparing the peptide complex of cod liver possessing the balanced amino acid composition, the high content of peptides and high antioxidant activity.

3 cl, 4 ex

Description

The invention relates to the field of medicine and veterinary medicine, and in particular to methods for producing biologically active substances from offal of animal origin, in particular to producing biologically active complexes based on water-soluble proteins of the liver of fish of the Gadidae family, having a balanced amino acid composition, high peptide content and high antioxidant activity.

One of the main components of the system of internal organs, rich in high-quality proteins, is the liver. The protein content in the liver, depending on the species of fish, can reach 20% (Bechtel et al. Chemical characterization of liver lipid and protein from cold-water fish species. J. Food Sci. 2006. 71 (6): 480-485) . The most studied peptides from internal organs are peptides isolated from intestinal and liver hydrolysates of fish bonito Gymnosarda unicolor (Ahn et al. Enzymatic production of bioactive protein hydrolysates from tuna liver: effects of enzymes and molecular weight on bioactivity. Int. J. Food Sci. Technol. 2010. 45: 562-568; Karaki et al. Oral administration of peptides derived from bonito bowels decreases blood pressure in spontaneously hypertensive rats by inhibiting angiotensin converting enzyme. Comp. Biochem. Physiol. C. 1993. 104 (2): 351-353). In the liver and other organs of the Japanese flounder Paralichtys olivaceus, a cysteine-rich peptide hepsidin was found to exhibit high antimicrobial activity (Hirono et al. Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus. FEBS J. 2005.272 (20): 5257-5264 ) Cells producing estrogen and xenoestrogen (Arukwe et al. Fish model for assessing the in vivo estrogenic potency of the mycotoxin zearalenone and its metabolites. Sci. Total Environ. 1999. 236 (1-3) were found and localized in the liver of Atlantic salmon Salmo salar. : 153-161), an antimicrobial protein was isolated from the same object (Richards et al. Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar). Biochem. Biophys. Res. Commun. 2001.284 (3) : 549-555). Thus, the liver of various fish can serve as a source of a large number of both low molecular weight substances, and a whole set of peptides that can be used for the preparation of medicines.

A method for producing a water-soluble complex of polypeptides from the liver of fish, namely from shark liver, used as an immunostimulant, comprising cooling the shark liver to 0 ° C; spraying chilled shark liver to produce finely ground material; mixing thoroughly ground material with an equal amount of water, provided that the mixture is kept at 0 ° C for 20 minutes; centrifuging the mixture at 0-4 ° C for 4-6 hours to separate the mixture into three different layers, which are the cell layer, the aqueous layer and the oil layer; separation of the water layer from other layers; filtering the aqueous layer to obtain a filtered solution; and spraying the filtered solution in a vacuum chamber at 0-4 ° C in order to obtain a dry powder substance (Pat. US 5840342 (A), publ. 11.24.1998).

The use of equipment for spraying a chilled liver, as well as low-temperature high-speed centrifugation for several hours is expensive. Its use for the production of products on an industrial scale leads to high material costs and cost of production.

A known method of obtaining the drug "Squaacan" - a complex of polypeptides from the liver of fish, namely from shark liver, which consists in the fact that the catran shark liver is homogenized, degreased by cold sedimentation at 1 - + 5 ° C for 20-24 hours, then fat-free homogenate is sent for thermal and chemical coagulation of proteins at pH 1.0-6.0, thawed at room temperature for 5-6 hours to obtain a stabilized extract, which after centrifugation and membrane filtration is amplified and autoclaved (Pat. RU 2211700 (C2), publ. 09/10/2003).

The disadvantage of this method is the long duration of the process and the use of centrifuges and membrane equipment to clarify the final extract, which does not allow to process large volumes of the extract. The process is carried out at low pH, which requires the treatment of wastewater from acids.

A known method of producing a water-soluble polypeptide complex from the liver of fish of salmon species, including grinding the feedstock, extraction with distilled water at a ratio of raw materials: extractant 1: 5-1: 30, followed by alkalization of the suspension to pH 8-9 and insisting it at room temperature for 2 -4 hours with stirring, purification of the target product by processing the liquid fraction with a 0.3% solution of chitosan in a 3% solution of edible organic acid at a pH of 2.5-3.0 in a ratio of 1:20 with stirring for 1 hour and then nkubirovaniem mixture for 2-3 hours and double separation by separation into solid and liquid fractions, followed by a compound liquid fractions (Pat. RU 2409291 (C1), publ. 20.01.2011).

The disadvantage of this method is the large number of working stages, the use of acids and alkalis, which leads to a large number of effluents requiring treatment, the use of expensive chitosan, which has its own biological activity.

The closest in technical essence and the achieved effect is the method of complex processing of the liver of the cod fish family and obtain a lipid complex, hydrophilic complex and feed product (Pat. RU 2420213 (C1), published on 10.06.2011), selected by the authors for the prototype. The liver of the codfish family is crushed, the mass is transferred to a reactor with a stirrer, and vegetable oil is added in small portions with stirring at a ratio of crushed liver and oil of 1: 0.2 ÷ 5.0. After that, with stirring in small portions, water is introduced into the reactor with a ratio of crushed liver and water 1: 0.2 ÷ 5.0. The resulting mixture was stirred at room temperature for 1-15 minutes. The process is conducted until the appearance of a phase boundary, determined visually. The resulting suspension is centrifuged to separate fractions that differ in density: hydrophilic, lipophilic and dense residue. The hydrophilic fraction, which is an aqueous solution of biologically active compounds, including peptides, and the lipophilic fraction are sent for separation. Dense residue is used as a feed product. The hydrophilic fraction after separation is directed to coagulation. The resulting lipophilic fraction is purified from impurities of non-fat nature. After that, the lipophilic fraction is cleaned of sterols and waxes by keeping it at a temperature of from -15 to + 5 ° С (6) for 1-24 hours until the ballast substances precipitate and then filter the lipophilic fraction. The content of peptides in the hydrophilic fraction is about 1-1.15%.

The disadvantage of this technology is the difficult separation of fractions and the need for centrifugation, the low content of peptides in the hydrophilic fraction, the presence of a significant amount of lipids. This circumstance limits the solubility of the target product, which can lead to a decrease in activity, faster spoilage. In addition, this complex is insoluble in water, which limits its bioavailability.

The objective of the invention is the industrial processing of the liver of fish of cod species with obtaining a water-soluble complex of peptides with a balanced amino acid composition, a high content of peptides and high antioxidant activity.

The objective of the invention is solved in that in the known method, including grinding the liver, mixing with oil at a ratio of liver and oil 1: 0.2 ÷ 5.0 and water at a ratio of liver and water 1: 0.2 ÷ 5.0, delamination at room temperature for 1-15 minutes, centrifugation, separation, maintaining the lipid complex at a temperature of from -15 to + 5 ° C for 1-24 hours and filtering, according to the invention, the extraction is carried out with a mixture of oil and water at a ratio of raw material: extractant 1: 5-1: 30 at room temperature for 2-4 hours with stirring, target th product was purified by treatment with an organic solvent in the ratio 1: 0,5 ÷ 2 followed by separation and drying of an aqueous residue.

Moreover, as a raw material, the liver of cod fish is used fresh, ice-cream or dry.

The organic solvent is selected from the series diethyl ether, ethyl acetate, butanol, chloroform.

The essence of the method is as follows. Fresh or frozen liver of cod fish (family Gadidae) (cod, haddock, pollock, blue whiting, etc.) is ground in a meat chopper or grinder. As shown by preliminary experiments, more thorough grinding with cell destruction is not required. It is not necessary to grind the dried liver, since it is crushed before drying. Stuffing fresh or frozen liver or crushed pieces of dried liver is poured with a mixture of oil and water at a ratio of raw materials: extractant 1: 5-1: 20 for fresh or frozen liver and 1:30 in the case of sublimated liver.

The suspension is incubated at room temperature with stirring for 2-4 hours, then the precipitate is separated, which is advisable to carry out on an industrial separator or continuous flow centrifuge. The liquid fraction is separated, the aqueous residue is dried in one way or another, with the exception of high-temperature evaporation. Next, the target product is purified by treatment with butanol in a ratio of 1: 1, followed by separation and drying.

The final product is a brown substance, not hygroscopic, with a brackish taste and without an unpleasant odor, does not contain toxic elements in amounts exceeding the MPC, and pathogenic microflora, i.e. is safe.

A product of 30 percent or more consists of peptides. When conducting polyacrylamide gel electrophoresis, it was found that peptide fractions with a molecular weight of 20-15 kDa, 15-3.5 kDa and low molecular weight remain in the final product from a wide range of proteins and polypeptides present in the aqueous extract of the liver of fish of the Gadidae family fraction less than 3.5 kDa, which includes oligopeptides, free amino acids and glycerophosphate. In the peptide substance there are no high molecular weight protein compounds (according to the qualitative reaction to the protein by the precipitation method with trichloroacetic acid). The content of free amino acids was 20-55%, among which the main ones are: glutamic acid, aspartic acid, proline, series, threonine, lysine, glutamine, valine, tyrosine, leucine and methionine.

Example 1

2 kg of fresh cod liver (Gadus morrhua L.) is ground using an electric meat grinder, after which the minced meat is placed in an apparatus with a stirrer and 20 l of a mixture of vegetable oil and water are poured in a ratio of 1: 1, stirred at room temperature for 2 hours. The resulting suspension shared on a separator. The solid fraction is sent to the manufacture of feed flour, the liquid fraction is sent to further purification from fatty impurities and high molecular weight protein impurities. The resulting aqueous fraction was freeze-dried and the resulting residue was dispersed in water and purified from water-insoluble impurities by extraction with diethyl ether in a ratio of 1: 1. The aqueous fraction is decanted and dried. The result is 18 g of dry peptide complex.

The final product is a light brown powder, non-hygroscopic, with a brackish taste and no unpleasant odor, does not contain toxic elements in quantities exceeding the MPC, and pathogenic microflora, i.e. complies with safety indicators.

Example 2

0.5 kg of sublimated haddock liver (Melanogrammus aeglefinus L.) is loaded into a reactor with a stirrer and pour 25 l of a mixture of vegetable oil and water in a ratio of 1: 1, stirred at room temperature for 4 hours. Then proceed analogously to example 1, but clean from water-insoluble impurities by extraction with butanol in a ratio of 1: 0.5.

The yield of the peptide complex is 5.3 g.

Example 3

5 kg of frozen blue whiting liver (Micromesistius poutassou) is crushed, placed in an apparatus with a stirrer and pour 3.5 l of a mixture of vegetable oil and water in a ratio of 1: 1, stirred at room temperature for 3 hours. Then proceed analogously to example 1, but clean from water-insoluble impurities by extraction with ethyl acetate in a ratio of 1: 2.

The yield of dry polypeptide product 43 g.

Example 4

Antioxidant activity against the radical DPPH.

The antioxidant activity of the composition of example 1 was investigated in relation to the radical DPPH spectrophotometric method. Alflutop, a preparation obtained from small sea fish, was used as a comparison drug.

It was found that the IC50 for water-soluble polypeptides isolated from the liver of codfish fish is about 4.3 mg / ml, which is comparable to the comparison drug Alflutop, for which there was an IC50 (4.6 mg / ml).

Claims (3)

1. A method of obtaining a peptide complex from the liver of codfish fish, including grinding the feedstock, extraction with a mixture of oil and water at a ratio of raw materials: extractant 1: 5-1: 30 at room temperature for 2-4 hours with stirring, purification by treatment with an organic solvent in a ratio of 1: 0.5-2, followed by separation and drying of the aqueous residue. 2. The method according to claim 1, characterized in that the liver of the fish cod species is used as raw material in fresh, ice cream or dry form. 3. The method according to claim 1, characterized in that the organic solvent is selected from the range of diethyl ether, ethyl acetate, butanol, chloroform.