RU2409291C1 - Method for production of water-soluble polypeptide complex of salmon fishes liver - Google Patents

Abstract

FIELD: food industry. ^ SUBSTANCE: invention relates to food industry, namely, to production of biologically active additives based on water-soluble proteins contained in salmon fishes liver. The method involves initial raw material selection, extraction, solid fraction separation, target product decontamination and drying. The initial raw material is represented by salmon fishes liver (fresh, frozen or dried). Extraction is performed with distilled water, the raw material to extractive agent ratio being 1:5 -1:30 which is followed by the suspension alkalisation till pH is 8-9 and infusion at room temperature during 2-4 h accompanied by stirring. Then one proceeds with the target product decontamination by way of the solid fraction treatment with a 0.3% solution of chitosan in a 3% solution of an organic food acid, pH being 2.5-3.0 in a 1:20 ratio during 1 h accompanied by stirring with subsequent incubation of the mixture during 2-3 h. One performs double separation into the solid and the liquid fractions with subsequent depletion of the liquid fractions. ^ EFFECT: invention allows to produce a water-soluble polypeptide complex of salmon fishes liver which is characterised by a balanced amino acid composition, an enhanced taurine content and a high antioxidant activity. ^ 2 cl, 3 dwg, 1 tbl, 4 ex

Description

The invention relates to the food industry, and in particular to methods for producing biologically active additives (BAA) from animal by-products, in particular to producing BAA based on water-soluble proteins of the liver of salmon fish with a balanced amino acid composition, high taurine content and high antioxidant activity.

In world practice, there are many examples of the detection and isolation of various biologically active substances from the liver of aquatic organisms. Thus, in the liver and other organs of the Japanese flounder Paralichtys olivaceus, a cysteine-rich peptide was found that exhibits high antimicrobial activity and is called hepsidin (Hirono I. et al., 2005). Cells producing estrogen and xenoestrogen were found and localized in the liver of Atlantic salmon Salmo salar (Arukwe A. et al., 1999), a protein possessing antimicrobial activity was isolated from the same object (Richards R.C., 2001). 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 enzymes that can be used both for the manufacture of biologically active food additives with a complex effect and highly purified preparations that are useful as reagents for scientific research and have a very high cost.

One of the main objects of marine fishing in the Far East are salmon. Salmon liver makes up from 1.2 to 3% of the waste mass (Basin norms of waste, losses, yield of finished products and raw material costs in the production of fish products from the Far East. - M., 2007).

As food raw materials, the salmon liver passed state standardization, however, its use in the food industry for the preparation of products similar to those obtained from cod and pollock liver is hampered by the difficulty in separating the gall bladder, which gives the salmon liver a bitter aftertaste.

A known method of producing an inhibitor of proteinases "Ingiprol" (Russia, 2197252 C1, CL 7 A61K 35/12, 2003), including grinding and homogenization of cattle, extraction in an acidic medium, separation of cake and subsequent purification of the target product by salting out of the extract with using sodium chloride, precipitation of impurity proteins with trichloroacetic acid, cation exchange chromatography, precipitation with acetone and sterilizing filtration of the solution, and after separation of the meal, chitosan is added to the supernatant to adsorb negatively on it charged proteins and carbohydrates, the clarified extract is concentrated by ultrafiltration on hollow fibers with a transmission limit of 5 kDa, the eluate obtained after chromatographic purification on carboxymethyl cellulose treated with 1% solution of phenol, precipitate was removed. And from the supernatant, the target product is precipitated twice with acetone, followed by dissolution of the precipitate in a 0.9 M sodium chloride solution to a concentration of 0.9-1.1 mg / ml.

The disadvantage of this method is the large number of working stages, the use of chromatography and ultrafiltration, which does not allow the use of this method to obtain the final product on an industrial scale, as well as the use of a fire hazardous organic solvent at the final stage.

A known method of obtaining a water-soluble complex of polypeptides from the liver of fish, namely from shark liver, used as an immunostimulant (USA, 5840342 A, class A61K 35/407, 1998), 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.

However, equipment for high-speed centrifugation for several hours is so expensive that it is hardly possible to use it for industrial production.

A known method for producing the drug "Squaacan" - a complex of polypeptides from the liver of fish, namely from the shark liver (Russia, 2211700 C2, class 7 А61К 35/60, 2003), which consists in the fact that the catran shark liver is homogenized, degreased by cold sludge at 1- + 5 ° C for 20-24 hours, then fat-free homogenate is sent to 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 of the ampoule comfort and autoclaved.

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.

A known method of producing a complex of polypeptides from fish, used as an immunostimulant (Russia, 2137390 C1, class 7 A23J 1/04, A23L 1/326), while the raw materials used are washings from fish processing plants that are poor in amino acid composition and therefore to obtain an immunomodulatory effect, chitosan is added to the final product.

The closest in technical essence and the achieved effect is a method for producing protein concentrate from marine objects of animal origin (USSR, No. 772518, Cl. А23J 1/04, 1980), where any waste from the fishing industry is used as raw material, including the entire entrails, and the process includes aqueous extraction in a strongly alkaline medium, neutralization with mineral acid and precipitation of a lipid-protein complex, and further processing of this complex with a mixture of ethyl alcohol and an organic halide solvent.

As a result of this process, degraded fragments of native proteins in a wide range of molecular weights are obtained as the final product; in addition, the use of organic solvents poorly meets modern labor protection requirements and creates an increased fire hazard in the workplace.

The objective of the invention is the industrial processing of the liver of fish of salmon species to obtain a water-soluble polypeptide complex with a balanced amino acid composition with a high content of taurine and high antioxidant activity.

The objective of the invention is solved in that in the proposed method for producing a water-soluble polypeptide complex from the liver of salmon fish, including grinding the raw material, extraction, separation of the solid fraction, purification of the target product and drying, according to the invention, the salmon fish liver is used as the raw material, extraction carried out with distilled water at a ratio of raw materials: extractant 1: 5-1: 30, followed by alkalization of the suspension to a pH of 8-9 and insisting it at room temperature for 2-4 hours s with stirring, the target product is purified by treating 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 incubating the mixture for 2 -3 hours and double separation by separation into solid and liquid fractions, followed by combining the liquid fractions.

Moreover, the liver of fish of salmon breeds is used as raw material in fresh, frozen or dry form.

A product is obtained, 40% or more consisting of a protein-peptide material. When conducting polyacrylamide gel electrophoresis, it was found that from a wide range of proteins and polypeptides present in the salmon liver water extract, two polypeptide fractions with a molecular weight of 30 and 60 kDa and a low molecular weight fraction, which include small peptides and free peptides, remain in the final product after treatment with chitosan amino acids. Among the latter, taurine (about 30 mg / g of product) is very valuable for humans and other warm-blooded vertebral sulfoamino acids. In the obtained samples, the antioxidant activity was determined according to the Glevind method using the stable free radical of α-diphenyl-α-picrylhydrazine as a substrate. The dipeptide carnosine (medium strength antioxidant) was used as a reference antioxidant. It was shown that the antioxidant activity of samples obtained from whole frozen and sublimated pink salmon liver is 6.5 and 4.9 times higher than the activity of carnosine, respectively. Moreover, the activity of the aqueous extract obtained from the frozen liver of pink salmon without treatment with chitosan is only 90% of the activity of carnosine.

A search by patent and scientific and technical sources did not reveal a known method that matches the set of essential features of the proposed method, in connection with which we can conclude that the proposed technical solution meets the criteria of the invention of "novelty" and "inventive step".

Salient features of the proposed method in comparison with the prototype are:

1. The following raw materials are used: fresh, frozen or dry liver of salmon fish, as a rule, for food purposes.

2. The extraction of raw materials is carried out with distilled water at a ratio of raw materials: extractant 1: 5 -1: 30, followed by alkalization of the suspension to a pH of 8-9 and insisting it at room temperature for 2-4 hours with stirring. It was experimentally established that at such a pH ratio of raw materials: extractant and the time of exposure to raw materials, the protein-peptide material is most fully extracted.

3. purification of the target product is carried out by treating 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, followed by stirring the mixture for 1 hour and incubation for 2-3 hours. This operation allows you to completely rid the extract of lipid material and high molecular weight proteins, while mineral reagents are not used. In addition, treatment with chitosan completely eliminates the characteristic odor inherent in the fish internals.

The proposed technical solution meets the criterion of "industrial use", since the use of fresh and ice cream or dry raw materials allows you to organize the production of a water-soluble polypeptide complex in any fish processing industry, including those located at a distance from the fishing site.

The essence of the method is as follows. Fresh or frozen liver of salmon fish (pink salmon, chum salmon, chinook salmon, etc.) is ground in a meat chopper or meat 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. Stuffed fresh or frozen liver or crushed pieces of dried liver are poured with water in a ratio of 1: 5 for fresh or frozen liver and 1:30 in the case of freeze-dried liver. Then, the resulting suspension is made alkaline with sodium hydroxide to a slightly alkaline pH (8-9).

It was established experimentally that at this pH (8-9), the protein-peptide material from fresh or frozen liver is extracted most fully (Figs. 1-3).

When using a dried liver, it was found that when alkalizing the extractant, an increase in the yield of protein-peptide material is not achieved. In this case, it is advisable to add sodium hydroxide solution to achieve an alkaline pH immediately before the liquid fraction is treated with a chitosan solution, since the formation of a chitosan precipitate occurs only in an alkaline medium.

The suspension is incubated with stirring for about 4 hours, then the precipitate is separated, which is advisable to carry out on an industrial separator or continuous flow centrifuge - this allows you to handle very large volumes of the suspension. The use of centrifuges with rotary loading does not give such an opportunity, filtering under vacuum also requires a lot of time, since any filter material is quickly clogged with small and greasy particles of the suspension. The resulting solid fraction can be used to make fat, and 1/20 of the volume of 0.3% solution of chitosan in 3% acetic acid, pH 2.5, is added to the liquid fraction. For one hour, the mixture was stirred and left for 2-3 hours to form a precipitate. The supernatant was decanted, and the precipitated suspension was again separated in a separator or flow centrifuge. Chitosan sediment can be sent for further processing to extract proteins and lipids of independent value. The resulting filtrate was combined with a previously selected supernatant and dried in one way or another, with the exception of high-temperature evaporation. The experiments showed that cyclone-type spray drying is best suited for this. Freeze-drying allows you to get the drug in even milder conditions, but currently all known samples of such equipment have loading restrictions.

Thus, in the presence of containers of the corresponding volume, from 100 to 200 kg of fresh or frozen liver of salmon fish, or up to 20 kg of dried liver, can be processed per shift. In this case, the finished product yield is approximately 0.5% of the initial mass of fresh or frozen liver or 5% of the initial mass of dried liver.

The final product is a brown powder, non-hygroscopic, with a brackish flavor and no unpleasant odor. It does not contain heavy or toxic elements in amounts exceeding the MPC and pathogenic microflora, i.e. complies with safety indicators. A product of 40 percent or more consists of a protein-peptide material. When conducting polyacrylamide gel electrophoresis, it was found that from a wide range of proteins and polypeptides present in the salmon liver water extract, two polypeptide fractions with a molecular weight of 30 and 60 kDa and a low molecular weight fraction, which include small peptides and free peptides, remain in the final product after treatment with chitosan amino acids. Among the latter, there is especially a lot of taurine, very valuable for humans and other warm-blooded vertebrate sulfoamino acids (table).

In the obtained samples, the antioxidant activity was determined according to the Glevind method using the stable free radical of α-diphenyl-α-picrylhydrazine as a substrate. The dipeptide carnosine (medium strength antioxidant) was used as a reference antioxidant. It was shown that the antioxidant activity of samples obtained from whole frozen and sublimated pink salmon liver is 6.5 and 4.9 times higher than the activity of carnosine, respectively. Moreover, the activity of the aqueous extract obtained from the frozen liver of pink salmon without treatment with chitosan is only 90% of the activity of carnosine.

EXAMPLE 1

10 kg of fresh pink salmon liver were taken, minced using an electric meat grinder, after which the minced meat was placed in a container and poured with 50 L of distilled water, stirred and the pH of the resulting suspension was adjusted with a 1 M sodium hydroxide solution to pH 8-9, then left at room temperature for 4 hours with stirring. The resulting suspension was separated on a separator (56 plates). The solid fraction was sent to the production of feed flour, the liquid fraction was sent for further cleaning, for which it was again placed in a vessel with a stirrer and 3 L of a 0.3% solution of chitosan in 3% acetic acid, pH 2.5, was added. The mixture was stirred for one hour, then stirring was stopped and left in the vessel for 3 hours to form a precipitate. The supernatant was decanted. The resulting wet cake was again separated on a separator into solid and liquid fractions. The latter was combined with previously selected supernatant and dried on a cyclone-type spray dryer. The result is 45 g of dry product.

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

EXAMPLE 2

12 kg of frozen chum salmon liver were taken, thawed and crushed using a top cutter, after which the minced meat was placed in a container with a stirrer and filled with 60 l of distilled water, stirred and the pH of the resulting suspension was adjusted with a 1 M sodium hydroxide solution to pH 8-9, then left at room temperature for 4 hours with stirring. Then the suspension was separated in a centrifuge at 2500-3000 rpm and centrifuged for 25-30 minutes at a temperature of 10 ° C for one load, the precipitate was sent to feed preparation, the supernatant (liquid fraction) was sent for further cleaning, for which it was again placed 3.5 l of a 0.3% solution of chitosan in 3% acetic acid, pH 2.5, was added to a vessel with a stirrer. The mixture was stirred for one hour, then stirring was stopped and left for 2 hours to form a precipitate. The supernatant was decanted; the resulting wet cake was again centrifuged into solid and liquid fractions under the same conditions. The resulting supernatant was combined with a previously selected supernatant and dried on freeze-drying. The result is 50 g of dry preparation.

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

EXAMPLE 3

1.5 kg of sublimated pink salmon liver was placed in a container with a stirrer, after breaking up large lumps, 45 l of distilled water was poured, stirring was started and left for 4 hours at a temperature of 15-18 ° C. Then the suspension was separated on a separator (56 plates), the precipitate was discarded or sent to the production of feed flour. The liquid fraction was again placed in a container with a stirrer, made basic with 1 M sodium hydroxide to pH 8-9 and 2.5 L of a 0.3% solution of chitosan in 3% acetic acid, pH 2.5, was added to it. Then stirring was started, which was carried out for one hour, stirring was stopped and left for 3 hours to form a precipitate. The supernatant was decanted, and the wet cake separated from it was again separated on a separator into solid and liquid fractions. The latter was combined with previously selected supernatant and dried on a cyclone-type spray dryer. The result is 70 g of dry preparation.

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

EXAMPLE 4

2 kg of freeze-dried liver of chum salmon was placed in a container with a stirrer, after breaking up large lumps, 60 l of distilled water was poured and stirring was started for 4 hours at room temperature. The resulting suspension was separated in a centrifuge at 2500-3000 rpm and the duration of centrifugation for one load of 25-30 minutes at a temperature of 10 ° C, the solid fraction was discarded or sent to the production of feed flour. The liquid fraction was again placed in a container with a stirrer, made basic with 1 M sodium hydroxide to pH 8-9 and 2.5 L of a 0.3% solution of chitosan in 3% acetic acid, pH 2.5, was added to it. Then stirring was started, which was carried out for one hour, stirring was stopped and left for 2 hours to form a precipitate. The supernatant was decanted, and the wet cake separated from it was again separated in a centrifuge into solid and liquid fractions. The latter was combined with previously selected supernatant and dried on freeze-drying. The result is 85 g of dry preparation.

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

Using the proposed method will allow to establish industrial processing of the liver of fish of salmon species to obtain a water-soluble complex of polypeptides with a balanced amino acid composition with a high content of taurine and high antioxidant activity.

Claims (2)

1. The method of obtaining a water-soluble polypeptide complex from the liver of salmon fish, including the selection of feedstock, extraction, separation of the solid fraction, purification of the target product and drying, characterized in that the salmon fish liver is used as feedstock, the extraction is carried out with distilled water at the ratio of raw materials: extractant 1: 5-1: 30, followed by alkalization of the suspension to a pH of 8-9 and insisting it at room temperature for 2-4 hours with stirring, the target product is purified the treatment of 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 h and then incubating the mixture for 2- 3 hours, double separation into solid and liquid fractions, followed by combining the liquid fractions. 2. The method according to claim 1, characterized in that the liver of fish of salmon species is used as raw material in fresh, ice cream or dry form.

Images