RU2262859C2 - Method for obtaining fermentative hydrolyzate based upon fish proteins - Google Patents

Abstract

FIELD: food industry, medicinal and veterinary biotechnology.

SUBSTANCE: the present innovation deals with obtaining curative-prophylactic means, food and fodder additives, nutritive media for cultivating microorganisms and eukariotic cells. In the method suggested one should obtain protein-containing mass out of fish viscera and fish bodies, as for its hydrolysis it should be performed directly or due to mixing it with water, moreover, as an alkaline agent one should apply calcium hydroxide, and as an acidic agent - orthophosphoric acid. Final purification of the product (removal of pigments, specific odor and taste of fish) should be carried out due to chromatography upon carriers of Amberlight XAD type (France). The innovation provides to obtain final product at low ash (salts) content, shortens the duration of technological modes to conduct the process and improves palatability of the product obtained due to removing specific fish odor.

EFFECT: higher efficiency.

7 cl, 5 ex, 1 tbl

Description

The invention relates to food industry, medical and veterinary biotechnology, in particular to a method for the manufacture of enzymatic hydrolyzate of fish proteins, and can be used to obtain therapeutic and prophylactic agents, food and feed additives, culture media for the cultivation of microorganisms and eukaryotic cells.

Currently, it is known that as a source of protein for the production of enzymatic hydrolysates are mainly animal proteins: non-merchandise meat of animals, cuts of cattle skins, milk proteins. Extracts of the pancreas of cows and pigs are most often used as a source of proteolytic activity, less often, due to their availability and / or cost, proteases of microbial origin (L. Telishevskaya, “Protein Hydrolysates”, ed. M., 2000, p. .65).

In addition, the restrictions imposed on food and pharmaceutical products obtained by processing animal raw materials lead to the need to develop technologies based on alternative raw materials.

The closest in technical essence to the present invention is a method for producing an enzymatic hydrolyzate based on fish proteins, including preparing a protein-containing mass, mixing it with water 1: 1, keeping at a temperature of 40-45 ° C at a pH of 7.6-8.0 in the presence of chloroform with stirring for 18 hours, followed by heating the biomass at 90 ° C for 5-10 minutes, precipitating high molecular weight compounds in the acidic pH zone, filtering and drying the final product (see RF Patent No. 2103360, class A 23 J 3 / 04, 1998).

In the known method as the protein-containing mass use the intestines and carcasses of commercial fish.

The disadvantages of the known invention are:

- the use of a system of hydrochloric acid - sodium hydroxide to regulate the acidity of the reaction mixture, which leads to a high content of ash (salts) in the final product - more than 10%, due to the formation of sodium chloride;

- the duration (18 hours) of the technological modes of the process and, as a result, the use of chloroform for protection against microbial contamination;

- the product has a specific smell, which affects the taste of the resulting product.

The tasks to be solved by the proposed method are the elimination of the above disadvantages.

The technical result in the present invention is achieved by creating a method for producing an enzymatic hydrolyzate based on fish proteins, including preparing a protein-containing mass, hydrolyzing the mass by alkalization and heating with stirring, acidification of the resulting biomass by subsequent decantation of fat and obtaining a product in which according to the invention the protein-containing mass is obtained from the insides fish or by mixing the entrails and carcasses of fish, and the hydrolysis of the mass is carried out directly or by mixing it with water, while calcium hydroxide is used as an alkaline agent, and phosphoric acid is used as an acid one.

The use of alkaline and acidic agents, such as calcium hydroxide, orthophosphoric acid, to regulate the acidity of biomass allows the final product to be obtained with a low ash content. This is due to the fact that at neutral pH values, the added reagents precipitate in the form of a water-insoluble precipitate of CaHPO ₄ , which allows additional purification of the final product from high molecular weight compounds and heavy metals.

Hydrolysis of the protein-containing mass without adding water allows the process to be compacted as much as possible, which, combined with preservation of the obtained product by adding phosphoric acid, makes it possible to use the proposed method in the conditions of fishing vessels and seasonal fishing and to carry out long-term storage of the product without energy costs.

The invention is characterized in that the protein-containing mass is alkalinized to pH 8.5-8.8 with calcium hydroxide, heated to 50-60 ° C with stirring for 3-6 hours, the biomass is acidified to pH 3.2-3.5.

As a result of the experiments, it was found that the proposed method with the given characteristics is obtained when conducting it with the modes within the specified limits.

If the value of any mode goes beyond the specified limits, then the necessary characteristics will not be achieved, i.e. the technical result in the invention will not be obtained.

This allows us to conclude that these parameters of the modes of the method relate to the essential features of this invention.

Separation of insoluble sediment after decantation of fat from the resulting mixture is necessary for the primary clarification of the solution and separation of ballast, because as an initial product, crude fish entrails and carcasses were used.

Further clarification of the solution after separation of the insoluble precipitate by alkalizing it to pH 7.2-7.5 with calcium hydroxide, heating to a temperature of 80-90 ° C for 15-30 minutes, followed by separation of the precipitate and concentration of the solution allows to obtain a product that meets the standards presented to food products, and depending on the intended purpose, can be directly used in the production of food products, culture media, premixes, etc.

Precipitation obtained after the first and second clarification is combined, dried and crushed for use as a feed additive and high-quality fertilizer.

Drying after concentration of the clarified solution allows to obtain a product with a shelf life of at least two years in the absence of moisture access.

Adding inert (dietary) fibers to the concentrated solution before drying, and then grinding the dried mass, allows you to create a product with a long shelf life that does not have increased hygroscopicity.

The invention is also characterized in that after concentration of the solution or after drying, the product is further purified by chromatography at pH 4.1-4.5 with filtration of the product through a chromatographic carrier at room temperature, which allows to obtain a product with improved organoleptic properties.

The proposed method is the most unified and does not require the use of equipment other than that used in the conditions of food and / or biotechnological production.

Patent studies showed that technical solutions with the indicated set of essential features are not known in similar methods for producing an enzymatic hydrolyzate based on fish proteins, i.e. the proposed solution meets the criterion of "novelty."

In the analysis of known analogues and prototype, no proposal was found with a combination of essential features set forth in the claims, which implies that for specialists involved in methods for producing enzymatic hydrolyzate based on fish proteins, it does not explicitly follow from the prior art and, therefore, corresponds to the criteria of the invention is "inventive step".

We believe that the information set forth in the application materials is sufficient for the practical implementation of the invention, which is confirmed by tests in a production environment using reactors with a volume of 1 m ³ and 20 m ³ .

The proposed method for producing an enzymatic hydrolyzate based on fish proteins is as follows:

First, a protein-containing mass is prepared.

In the proposed method, the interiors of commercial fish (sea or river) are used as raw materials: salmon, mackerel, cod, pikeperch, pike, cyprinids, etc. An additional source of protein in the mass are carcasses of any non-commercial fish.

Fresh or freshly frozen fish internals washed with main water are crushed and homogenized. Similarly, carcasses of fish are treated.

The resulting paste is pumped into the reactor, where, if necessary, mixed with the product obtained from carcasses in a ratio of 1: 2. Then carry out the hydrolysis of the protein-containing mass.

At this stage, depending on production conditions, two further ways of carrying out the hydrolysis process are possible.

In the first embodiment, the hydrolysis of the resulting mass is directly carried out without adding water.

The protein-containing mass, while stirring, is made alkaline with an aqueous suspension of calcium hydroxide to a pH of 8.5-8.8 and heated to 50-55 ° C. Depending on the nature of the feedstock, the process is carried out with stirring for 4-6 hours until the reaction depth reaches at least 80% of the maximum possible. Upon completion of the hydrolysis process, 80-90% is added to the resulting biomass orthophosphoric acid, bringing the pH of the medium to 3.2-3.5, and then the fat is decanted.

The resulting product can be directly used in the subsequent stages of the proposed method or pumped into inert containers for storage and / or transportation at ambient temperature.

The application of the proposed method in this form eliminates the long-term use of refrigeration units for storing the feedstock, provides economical preservation and transportation of the concentrated intermediate product, and allows efficient use of this technology both in the conditions of sea commercial fishing and seasonal coastal fishing.

In addition, the product obtained by using the proposed method has independent value, since after neutralization it can be used as an effective feed additive and plant growth stimulator.

According to the second variant of hydrolysis, an equivalent volume of purified water is added to the protein-containing mass in the reactor. The mixture is made alkaline with vigorous stirring with calcium hydroxide to pH 8.5 and heated to 50-55 ° C.

Depending on the nature of the feedstock, hydrolysis takes 3-5 hours. Monitoring the progress of the hydrolysis process in both the first and second options is carried out hourly, taking samples and determining the content of amine nitrogen in them.

Upon completion of the process, 80-90% of the reaction mixture is acidified with phosphoric acid to a pH of 3.2-3.5 and the fat is decanted.

Then, the product obtained directly without adding water according to the first hydrolysis variant or after adding an equal volume of purified water, the product obtained in the second variant is separated from the insoluble precipitate and fat residues, for example, using a flow centrifuge and the solution is pumped to the next reactor.

In the reactor, the process of clarification of the solution by alkalization to pH 7.2-7.5 using calcium hydroxide and heating at 90-95 ° C for 15-30 minutes is carried out.

The precipitate formed is separated, for example, using a flow centrifuge or filtration.

Then the clarified solution containing about 10% of a mixture of amino acids and short peptides (no more than 2000 D) is concentrated two to three times using a vacuum evaporator.

The product obtained after clarification and its concentration meets the standards for food products and, depending on the purpose, can be directly used in further production (drinks, preserves, nutrient media, etc.).

The process according to the proposed method can be continued further, namely the clarified and concentrated solution is dried, for example, by spraying.

A drying option is possible, when inert (dietary) fibers are added to the solution before drying to a thick paste-like state, and then they are dried and crushed using the appropriate technological equipment.

Further purification (removal of pigments, specific smell and taste of fish) is carried out by chromatography on media such as Amberlight XAD (France). Cleaning can be carried out both after concentration of the solution, and after its subsequent drying by spraying.

The concentrated solution or dry product after diluting it 3-5 times with water is acidified to pH 4.1-4.5 and filtered at room temperature through a column containing the appropriate carrier in the ratio of the volume of the swollen carrier / mass of dry solution contents 1/1.

The yield of the target product is at least 80% with the practical preservation of the ratio of amino acids (see table).

The resulting solution is concentrated using a vacuum evaporator and dried, for example, by spraying at a spray temperature of not higher than 140 ° C.

Examples of a method for producing an enzymatic hydrolyzate based on fish proteins.

Example 1. The salmon entrails are washed with water, homogenized, mixed with purified water in a ratio of 1: 1 and the pH of the mixture is adjusted to 8.8 with a suspension of calcium hydroxide.

The process is carried out with stirring for 3-4 hours at 55 ° C, monitoring the progress of the reaction by increasing the content of amine nitrogen (N _am ). Upon completion of the process at 84%, the pH of the reaction mixture was adjusted to 3.5 with phosphoric acid.

The fat is decanted, the precipitate is separated using a flow centrifuge, and the solution is pumped to the next reactor.

By adding calcium hydroxide, with stirring, the pH of the medium is adjusted to 7.0-7.5 and heated at 90 ° C for 30 minutes.

The precipitate and residual fat are removed using a flow centrifuge, the solution is concentrated 2–3 times using a vacuum evaporator and dried by spray drying.

The result is a product that is characterized by the following parameters, wt.%:

N _am 6.4 N _total 10.5 Amine ratio, N _am / N _total 0.61 Ash 2,3 Fat 1.8 Amino acids (acid hydrolysis) 53 Free amino acids 86

Example 2. The viscera and carcasses of pollock are washed with water, mixed in a ratio of 1: 2, homogenized, mixed with purified water in a ratio of 1: 1 and adjusted to pH 8.8 with a suspension of calcium hydroxide. The process is carried out with stirring for 4-5 hours at 55 ° C, monitoring the progress of the reaction by increasing the content of amine nitrogen (N _am ). Upon completion of the process, 80-85% of the reaction mixture is treated as described above.

The result is a product that is characterized by the following parameters, wt.%:

N _am 7.4 N _total 10,4 Amine ratio, N _am / N _total 0.71 Ash 1.9 Fat 1,5 Amino acids (acid hydrolysis) 89 Free amino acids 58

Example 3. The entrails of carp and carcasses of freshwater partial fish are washed with water, mixed in a ratio of 1: 2, homogenized, mixed with purified water in a ratio of 1: 1 and adjusted to pH 8.8 with a suspension of calcium hydroxide. The process is carried out with stirring for 4-5 hours at 55 ° C, monitoring the progress of the reaction by increasing the content of amine nitrogen (N _am ). Upon completion of the process, 80-85% of the reaction mixture is treated as described above.

The result is a product that is characterized by the following parameters, wt.%:

N _am 6.6 N _total 12.1 Amine ratio, N _am / N _total 0.55 Ash 2,8 Fat 0.25 Amino acids (acid hydrolysis) 85 Free amino acids 54

Example 4. The entrails of carp and carcasses of freshwater partial fish are washed with water, mixed in a ratio of 1: 2, homogenized and adjusted to pH 8.8 with a suspension of calcium hydroxide. The process is carried out with stirring for 5-6 hours at 55 ° C, monitoring the progress of the reaction by increasing the content of amine nitrogen (N _am ). Upon completion of the process, 80-85% of the biomass is acidified with phosphoric acid to pH 3.5 and the fat is decanted. An equal volume of purified water is added to the resulting product with stirring, the precipitate and fat residues are separated using a flow centrifuge and pumped to the next reactor.

Next, the resulting solution is treated with calcium hydroxide, concentrated and dried, as described above.

The result is a product that is characterized by the following parameters, wt.%:

N _am 6.7 N _total 13,2 Amine ratio, N _am / N _total 0.51 Ash 3.9 Fat 0.3 Amino acids (acid hydrolysis) 93 Free amino acids 61

Example 5. The insides of salmon and carcasses of salmon are washed with water, mixed in a ratio of 1: 2, homogenized, mixed with purified water in a ratio of 1: 1 and adjusted to pH 8.8 with a suspension of calcium hydroxide. The process is carried out with stirring for 4-5 hours at 55 ° C, monitoring the progress of the reaction by increasing the content of amine nitrogen (N _am ). At the end of the process, 80-85% of the biomass is treated as described above, concentrated and dried.

The concentrate (1) is acidified to a pH of 4.1-4.5 or the dried product (2) is diluted with water 4-5 times and the pH of the solution is also adjusted to a pH of 4.1-4.5. The resulting solution was filtered at room temperature through a column containing an appropriate Amberlight XAD chromatographic support (France) in a ratio of the volume of the swollen support / dry weight of the solution 1/1. The yield of the target product is at least 80%. The resulting solution is concentrated using a vacuum evaporator and dried by spray drying at a spray temperature of no higher than 140 ° C (3).

As a result, products are obtained that are characterized by the following parameters, wt.%:

Table 1 Product (1, 2) Product (3) N _am 6.1 8.9 N _total 9,4 11.5 Amine ratio, N _am / N _total 0.65 0.76 Ash 3.2 1,2 Fat 1.95 0.8 Amino acids (acid hydrolysis) 85 90 Amino Acid Free 72 78

Enzymatic protein hydrolysates of various fish obtained by the proposed method, in their characteristics, are not inferior to the drugs obtained by the known method, differing from them in two to three times lower content of inorganic substances (see TU 9160-001-08627543-98).

Thus, the proposed method is universal and allows for a large-scale accelerated and economical process for producing an enzymatic hydrolyzate from fish processing waste, which is a valuable raw material for food, feed and biotechnological industries.

Dry protein hydrolyzate in the form of a food supplement showed its high physiological activity, for example, in the treatment of patients with gastrointestinal bleeding.

table 2
Amino acid composition of the enzymatic hydrolyzate of salmon proteins before (I) and after chromatographic purification (II), g / 100 g of dry preparation. Acid hydrolysis Free amino acids I II I II Taurine 3.19 3.05 1,67 2.12 Aspartic acid 5.53 5.67 2.96 3.12 Threonine 3.42 3.79 3.04 3.18 Serine 3,54 3.75 3.24 3.34 Glutamic acid 10.8 12.45 4.02 3.41 Proline 5.01 6.41 2.23 1.17 Glycine 7.72 8.23 3.03 2.92 Alanine 6.74 8.02 6.55 6.36 Valine 5.06 5.76 5.52 5.93 Methionine 2.12 2.73 1.93 1.95 Isoleucine 4.07 4.32 4.74 4.29 Leucine 6.88 7.28 7.76 7.93 Tyrosine 4.87 2.75 4.08 2.82 Phenylalanine 3.77 3.11 3.71 2.72 Hydroxylysine 1.05 0.67 0.52 0.47 Ornithine 1,67 2.25 2.04 2.15 Lysine 4.89 6.08 5.2 5.67 Histidine 0.74 0.92 0.92 0.8 Tryptophan 0.76 0.36 10.15 7.29 Arginine 3.52 2.72 4.66 4.36 Total: 85.35 90.32 77.97 72

Claims (9)

1. A method of producing an enzymatic hydrolyzate based on fish proteins, including the preparation of a protein-containing mass, hydrolysis by alkalization and heating with stirring, acidification of the resulting biomass, characterized in that the protein-containing mass is prepared from the insides of fish or by mixing the insides of fish and carcasses, and hydrolysis the mass is carried out directly or mixing it with water at a temperature of 50-55 ° C for 3-6 hours, while alkalizing the protein-containing mass with calcium hydroxide to a pH of 8.5-8.8, and acidifying the resulting biomass to a pH of 3.2-3.5 with phosphoric acid and the fat is decanted. 2. A method of obtaining an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that the interiors of the fish and carcasses of fish are mixed in a ratio of 1: 2. 3. The method for producing an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that after decanting the fat from the mixture, an insoluble precipitate is separated, while if the hydrolysis of the mass is carried out directly, the mass is pre-mixed with an equal volume of purified water before separation. 4. A method of producing an enzymatic hydrolyzate based on fish proteins according to claim 1 or 3, characterized in that after separation of the insoluble precipitate, the solution is clarified by alkalizing it to pH 7.2-7.5 with calcium hydroxide, heating to a temperature of 80-90 ° C for 15-30 minutes 5. A method of obtaining an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that the clarified solution is concentrated. 6. A method for producing an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that the clarified solution is dried after concentration. 7. A method of producing an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that dietary fiber is added to the solution before drying, and then the dried mass is ground. 8. A method for producing an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that after concentration of the solution or after drying, the product is further purified by chromatography. 9. A method of producing an enzymatic hydrolyzate based on fish proteins according to claim 1, characterized in that the chromatography is carried out at a pH of 4.1-4.5, filtering the product through a chromatographic medium at room temperature.