RU2712747C1 - Method for producing enzymatic hydrolyzate from sea hydrobiont processing wastes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and the food industry. Raw material is prepared from sea hydrobionts processing wastes, which is represented by fish processing wastes, Kamchatka crabs and sea urchin. Preparation is carried out in stages using ultrasonic treatment. At the first stage, fish processing wastes are prepared. If necessary, defrosted, milled, loaded into a reactor with a mixing device in amount of 60–80 % of the total weight of the ingredients. Fish insides are separately homogenized and added to the reaction mixture in amount of 10–15 %. Water of room temperature is added at ratio of 1:1 to 1:2 to the weight of the material, mixed, placed in a reactor an ultrasonic device and subjected to ultrasound for 10–15 minutes with an intensity of exposure of 100–200 W/dm³ at initial temperature of 20–25 °C and pH 6-7. At the second stage prepared and homogenized crab insides are added to the reactor, including hepatopancreas in amount of 10–15 % of the initial weight of fish processing wastes. Ultrasonic treatment is carried out for 10–15 minutes with intensity of exposure of 100–200 W/dm. At the third stage, prepared and homogenous sea urchin insides are added to the reactor in amount of 1–5 % of the initial weight of fish processing wastes. After fermentation is completed, ultrasound is performed for 10–15 minutes with intensity of exposure of 100–200 W/dm³. At the fourth stage ultrasound treatment is performed for 15 minutes at intensity of exposure of 500–600 W/dm³. Obtained mixture is settled, insoluble precipitate is filtered, fat is removed by separation. Hydrolyzate solution undergoes ultrafiltration or tangential filtration. Filtered solution is evaporated till dry residue content is not less than 30 %. It is dried lyophilic or with the help of spray drier.

EFFECT: invention provides developing an accelerated method of producing, having a balanced amino acid and protein composition.

1 cl, 5 tbl, 3 ex

Description

The invention relates to food, feed and microbiological industries, and in particular to methods for producing enzymatic hydrolysates from protein-containing waste from the processing of marine hydrobionts.

A known method of producing protein enzymatic hydrolysates (RF patent for the invention No. 2312514), including grinding fish stock, washing it with water, hydrolysis with a NaCl solution in the chamber of a diaphragm electrolyzer with an electrode connected to the negative pole of a direct current source, separation of insoluble precipitate, solution neutralization, drying packing. Hydrolysis is carried out with the introduction of a proteolytic enzyme in an amount of 1.0-2.0% by weight of the raw material at optimal pH and temperature values corresponding to the maximum activity of the enzyme for 5-6 hours. In this case, the optimal hydrolysis conditions are obtained by conducting electrolysis in an electrolyzer, to which an alternating current source is additionally connected, and the electrolyzer is connected to an alternating current source to achieve and maintain the required temperature of the solution, and to a direct current source to achieve and maintain the required pH value of the solution. After hydrolysis, insoluble precipitate is precipitated in the same chamber as hydrolysis by lowering the pH of the solution to 4.0-5.0, for this, disconnect the electrode from the negative pole of the direct current source and connect it to the positive pole of the direct current source. After reaching the precipitation pH, the solution is heated to 98-100 ° C, disconnecting the electrolyzer from a direct current source and connecting it to an alternating current source, and leave the solution for 5-6 hours, while the enzyme is thermally inactivated simultaneously with the precipitation. Then the insoluble precipitate is separated, the solution is neutralized by increasing the pH to 7.5-8.0.

The disadvantages of this method is the use not of waste products from aquatic organisms, but low-value fish species (for example, blue whiting), the duration of the process is 5-6 hours, the use of expensive enzyme preparations, the use of a specialized electrochemical chamber, and the process is carried out in a mixed enzymatic-acid way, since with the passage of direct electric current hydrochloric acid is formed, which is the reason for the change in the pH of the medium and makes the hydrolysis process non-ecological (requires stage of neutralization).

A known method of producing a biologically active additive from marine aquatic organisms used in the patent of the Russian Federation for invention No. 2623738, including the homogenization of raw materials, enzymatic hydrolysis using enzyme preparations Protamex and / or CelloLux-F in an amount of 0.5-2% by weight of the raw material with a hydraulic module equal to 1 : 0.5-1: 1.5, subsequent inactivation of the enzymes, purification of the obtained hydrolyzate and drying to a water content of 8-10%. Secondary cheese and / or wastes are used as raw materials during the primary processing of cephalopods, or sharks, or stingrays, or sturgeons, or salmon fish, or gobies.

The disadvantages of this method is the non-use of waste processing of crab and sea urchins, the use of expensive enzyme preparations, the need to additionally inactivate enzymes, which increases the duration of the process.

The closest analogue is a method for producing enzymatic protein hydrolysates from marine hydrobionts for microbiological and / or feed purposes (RF patent for the invention No. 2215425). The method involves the processing of protein-containing industrial waste and the processing of aquatic organisms and includes the simultaneous grinding of raw materials and hepatopancreas-raw king crab, diluting with water a homogenized mixture with a ratio of the mass of raw materials and water 1: 0-1: 2, heating to 45-55 ° C, mainly 50 ° C at natural pH for 0.5-8 hours with a mass ratio of raw materials and proteolytic agent from 1: 0.001 to 1: 0.100. The conditions of enzymatic hydrolysis are set depending on the desired degree of hydrolysis, and with a degree of hydrolysis of at least 30%, the solution is separated by centrifugation and purified using a polymer coagulant with a concentration of from 0.1 to 2% in a solution of hydrochloric acid from 0.5 to 2 mol / L . After adding the coagulant, the solution is kept for 0.5 to 2 hours, the suspension is centrifuged and filtered. The transparent hydrolyzate is dried and the target product is obtained — the protein base for microbiological media, and the remaining precipitate is dried and used as the protein base of the feed mixtures.

The disadvantages of this method are the duration of the process (up to 10 hours in total), the incomplete use of crab processing waste (only hepatopancreas is used), a high temperature of 500 ° C, at which proteolytic enzymes can be inactivated, and the use of hydrochloric acid.

The inventive method for producing enzymatic hydrolysates from marine hydrobiont processing waste, as well as known, includes processes for preparing raw materials, grinding it, adding water, introducing marine hydrobionts as proteolytic agents, carrying out enzymatic hydrolysis, separating sediment, evaporating and drying.

The technical problem of the invention is the development of technology for the integrated rational use of waste products from aquatic organisms.

The technical result of the invention is the development of an accelerated, energy-efficient method for producing protein hydrolyzate with a balanced amino acid and protein composition.

The technical result is achieved by the fact that the raw materials used are fish processing wastes, the inside of the Kamchatka crab and the inside of the sea urchin, it is carried out in stages using ultrasonic treatment. At the same time, at the first stage, fish processing waste is prepared, if necessary, it is thawed, crushed, loaded into a reactor with a mixing device in the amount of 60-80% of the total mass of ingredients. Separately, the insides of the fish are homogenized, and added to the reaction mixture in an amount of 10-15%, water is added at room temperature in a ratio of 1: 1 to 1: 2 to the mass of raw materials, mixed, an ultrasonic apparatus is placed in the reactor, and ultrasonic treatment is performed for 10 -15 min with an exposure intensity of 100-200 W / dm3 at an initial temperature of 20-25 ° C and a pH of 6-7. At the second stage, prepared and homogenized crab entrails, including hepatopancreas, are added to the reactor in the amount of 10-15% of the initial weight of fish processing waste, and sonication is performed for 10-15 minutes with an exposure intensity of 100-200 W / dm3. At the third stage, prepared and homogenized insides of the sea urchin are added to the reactor in the amount of 1-5% of the initial weight of fish processing waste, and ultrasonic treatment is performed for 10-15 minutes with an exposure intensity of 100-200 W / dm3. At the next stage, at the end of fermentation, in order to inactivate the enzymatic complex, sonication is carried out for 15 minutes at an exposure intensity of 500-600 W / dm3. The resulting mixture was defended, the insoluble precipitate was filtered, the fat was removed by separation. The hydrolyzate solution is subjected to ultrafiltration or tangential filtration through a membrane with a pore size of 5-10 kDa. The filtered solution is evaporated to a solids content of not less than 30% and freeze-dried or using a spray dryer.

Fawn sea urchin with entrails and gonads can also be used as raw material.

The proposed technology provides an integrated approach to solving the problem of the rational use of waste products from aquatic organisms (fish and invertebrates) and helps to reduce environmental pollution, due to the fact that waste from processing commercial species of fish, for example, cod and / or waste from processing salmon species, is used as raw material. artificially grown, for example, Arctic char, as well as waste from marine aquatic organisms, namely Kamchatka crab, green sea urchin, as well as non-fishing This type of hedgehog is a fawn hedgehog, often found in by-catch. In the processing of Kamchatka crab, the waste consists of the insides of the crab, including hepatopancreas; in the processing of the sea urchin, the waste includes the insides after the removal of caviar (gonads). An important property of the internal digestive organs of marine aquatic organisms is their proteolytic activity.

The use of ultrasonic treatment with an intensity of exposure of 100-200 W / dm ³ at all stages of the fermentation stage provides an intensification of the fermentation process by increasing the activity of proteolytic enzymes that are part of the waste used (entrails of aquatic organisms), a significant reduction in fermentation time (up to 10-15 min per each stage), allows to achieve a high degree of hydrolysis up to obtaining free amino acids. Carrying out ultrasonic treatment with an intensity of exposure of 500-600 W / dm ³ after the end of fermentation ensures the inactivation of the enzyme complex.

The proposed technology does not provide for the addition of additional energy resources. At the first stage, fish processing wastes are prepared, namely, the head, skin, fins, musculoskeletal residue, as well as the viscera (gastrointestinal tract). If necessary, the raw materials are thawed, crushed, loaded into the reactor with a mixing device in the amount of 60-80% of the total mass of ingredients. The insides of the fish are homogenized separately and added to the reaction mixture in an amount of 10-15%, water is added at room temperature in a ratio of 1: 1 to 1: 2 to the entire mass of raw materials, mixed, placed in an reactor with an ultrasonic apparatus, and sonicated for 10 -15 min with an exposure intensity of 100-200 W / dm ³ at an initial temperature of 20-25 ° C and a pH of 6-7.

At the second stage, prepared and homogenized crab entrails, including hepatopancreas, are added to the reactor in the amount of 10-15% of the initial weight of fish processing wastes, and sonication is performed for 10-15 minutes with an exposure intensity of 100-200 W / dm ³ . When cutting crabs and producing raw-frozen or boiled-frozen limbs of the crab, 30 to 50% of the waste is generated (carapax crab with abdomen and entrails), which averages from 12 to 20 thousand tons per year. They contain such high-value components as protein (12.5%), chitin (2.6%), lipids (0.24%) and mineral elements (4.96%) (calculated on raw material). It is reasonable to use these wastes for the production of protein hydrolysates using biotechnological methods.

Sea urchin processing waste is of great value. In the intracavitary fluid and waste from the internal organs of the sea urchin, up to 42% of proteins, 20% of fats, 5% of glycogen were found. At the third stage, prepared and homogenized insides of the sea urchin are added to the reactor in the amount of 1-5% of the initial mass of fish processing waste, and ultrasonic treatment is performed for 10-15 minutes with an exposure intensity of 100-200 W / dm ³ .

Then, at the end of the fermentation, an ultrasonic treatment is carried out for 15 minutes at an exposure intensity of 500-600 W / dm ³ . The resulting mixture was defended, the insoluble precipitate was filtered, the fat was removed by separation. The hydrolyzate solution is subjected to ultrafiltration or tangential filtration through a membrane with a pore size of 5-10 kDa. The filtered solution is evaporated to a solids content of not less than 30% and freeze-dried or using a spray dryer.

Instead of a green sea urchin, a fawn urchin can be used, which is often by-catch of a green sea urchin (by-catch can be from 30 to 90%). In this case, the gonads do not separate. Because they are not a valuable food product.

Confirmation of the possibility of obtaining the claimed technical result by this method is the development of an accelerated, energy-efficient method for producing protein hydrolysates with a balanced amino acid and protein composition, which can be included in microbiological media as a source of amino acids (purified hydrolyzate), or used as a component of fish feed, increasing its effectiveness, is given in the following specific examples.

Example 1. Obtaining an enzymatic protein hydrolyzate for microbiological protein culture media from waste cutting salmon (char) using the insides of king crab and the insides of a sea urchin (green).

0.65 kg of musculoskeletal remainder of arctic char, including heads, bones, fins and skin, were thawed in air at room temperature. Grind the raw materials in an electric meat grinder. In the blender, the insides of the fish were crushed in an amount of 0.15 kg to a homogeneous mass. The crushed raw materials were loaded into a 5 L flask with a mixing device. Poured 1.0 kg of water.

The mixture was mixed, the ultrasonic apparatus of the Volna series UZTA-0.4 / 22-OM was placed in a flask, the apparatus was turned on at a power of 96 W, and fermented for 15 min. Subsequently, thawed and homogenized crab entrails, including hepatopancreas, were added in an amount of 0.15 kg. Re-enabled ultrasound under the same conditions and processing time. At the next stage, 0.05 kg homogenized insides of the sea urchin were added. We performed sonication under the same conditions. At the end of the fermentation, the ultrasound apparatus was turned on at full power and the reaction mixture was processed for 15 minutes to inactivate the enzymes. The resulting mixture was left to stand for 1 hour at a temperature of 4-70 ° С, the precipitated precipitate (non-hydrolyzable insoluble) was filtered through a suction filter (under vacuum), and dried on a freeze dryer.

The resulting product can be used as phosphorus-calcium fertilizer. The mixture was again left to stand for 0.5-1 hours to separate the fat and water phases, the fat was removed on a separator. The hydrolyzate solution was subjected to tangential filtration through a membrane with a pore size of 5-10 kDa. In this case, the filtered solution was evaporated to a solids content of not less than 30% and freeze-dried. Got a purified fraction containing amino acids and oligopeptides, which can be used in microbiological environments as a source of free amino acids. A concentrated solution containing soluble protein and polypeptide macromolecules formed after ultrafiltration was sent for freeze drying. Received crude protein hydrolyzate for feed purposes. The chemical composition of the obtained products is shown in table 1. The amino acid composition is presented in table 2.

Example 2. Obtaining an enzymatic protein hydrolyzate for food purposes as a premix for fish feed from cod cutting waste using the entrails of king crab and the entrails and caviar of a sea urchin (fawn).

0.80 kg of musculoskeletal residue of cod fish, including heads, bones, fins and skin, were thawed in air at room temperature. Grind the raw materials in an electric meat grinder. In a blender, the guts of fish were crushed in an amount of 0.10 kg to a homogeneous mass. The crushed raw materials were loaded into a 5 L flask with a mixing device. Poured 2.0 kg of water.

The mixture was mixed, the ultrasonic apparatus of the Volna series UZTA-0.4 / 22-OM was placed in a flask, the apparatus was turned on at a power of 96 W, and fermented for 15 min. Subsequently, thawed and homogenized crab entrails, including hepatopancreas, were added in an amount of 0.05 kg. Re-enabled ultrasound under the same conditions and processing time. At the next stage, homogenized insides and caviar of a yellow sea urchin in the amount of 0.04 and 0.01 kg, respectively, were added. We performed sonication under the same conditions. At the end of the fermentation, the ultrasound apparatus was turned on at full power and the reaction mixture was processed for 15 minutes to inactivate the enzymes. The resulting mixture was filtered through a suction filter (under vacuum), the solution was separated on a separator to remove fat, concentrated on a rotary evaporator and dried on a freeze dryer.

Received crude protein hydrolyzate for feed purposes. The chemical composition of the obtained products is shown in table 1. The amino acid composition is presented in table 2.

The concept of the ideal protein for feeding fish. An ideal protein can be defined as a component that provides the most accurate balance between the content of amino acids necessary for optimal activity and maximum growth rates (achieving size, carcass weight and body composition). Developing a feed based on such an ideal protein is an effective way to use less of this component to meet the needs for a full range of amino acids.

In their diet, fish make demands not on proteins, as such, but rather on the specificity of the amino acids that make them up. When proteins are digested, single amino acids are released and absorbed, either as part of dipeptides, tripeptides and other chain molecules. There are about 20 known amino acids used as original building blocks for proteins in all living organisms. Ten of them are indispensable, they are necessarily included in the feed, as the fish either can not synthesize them, or can, but at a pace insufficient for its needs. The quality of feed additives for growing fish is largely determined by the amount of true protein according to Barnstein (table 1), the difference between the level of protein and non-protein nitrogen, the content of soluble protein, ash, as well as the level of amino acids, especially irreplaceable. Table 2 presents the data of the analysis of amino acids of hydrolysates obtained by the methods described in examples 1 and 2. When comparing the results with reference data [Ponomarev, S.V. Feed and feeding fish in aquaculture. Textbook / S.V. Ponomarev, Yu.N. Grosescu, A.A. Bahareva - M .: Publishing House. “Morkniga”, 2013 - 417 pp.] In terms of the number and ratio of interchangeable and irreplaceable amino acids, it is obvious that the developed technology of enzymatic hydrolysis of waste products of hydrobiont processing ensures a balanced composition of the products obtained.

Example 3. Experimental confirmation of the effectiveness of the obtained hydrolysates for feeding sturgeon.

In the experiment, we compared the use of the widely used Coppens Supreme-10 compound feed (Holland), the composition of which is shown in Table 3, and the same feed, but enriched with the hydrolyzate obtained by the proposed method (Example 2). The duration of the experiment was 26 days. Object of study - sterlet producers (21 pcs. Per pool). The Coppens Supreme-10 compound feed was first crushed, then the hydrolyzate was added in the amount of 3 and 5%, then granules were formed, dried and the fortified feed was used as well as the industrial Coppens Supreme-10, but not enriched with the additive. The experimental results are presented in tables 4 and 5.

The experimental results confirm the effectiveness of the use of the new hydrolyzate (example 2) for the enrichment of industrial feed from the Dutch company Coppens Supreme-10: for all mass indicators, dose-dependent indicators increased, including an increase in average daily weight.

Blood is a sensitive and informative indicator of the state of the body, quickly responding to changes in exogenous and endogenous factors and reflecting the effect both on a single individual and on the population as a whole [Stroganov N.S. Ecological physiology of fish / N.S. Stroganov. M .: Moscow Publishing House. University, 1962. T. 1. 444 p.]. The dynamics of biochemical parameters can serve as a marker of the state of the fish organism in artificial conditions, characterize the quality and quantity of food, population density, adaptive abilities of fish, and growth rate. There is a method of selecting physiologically complete females of stellate sturgeon during factory reproduction by determining physiological and biochemical tests (Metallov G.F., Geraskin P.P., Aksenov V.P. Physiological and biochemical aspects of assessing the fish-breeding quality of females stellate stellate Asipenser stellatus Pall. In the book. : Information Package. Fisheries. Aquaculture Series. Aquaculture: Problems and Achievements. M, 1997, issue 7, pp. 4-14). Apply tests characterizing red blood (hemoglobin concentration, erythrocyte sedimentation rate - ESR), protein metabolism (concentration of whey protein and beta-lipoproteins). Significant differences between the groups were found in the study of ESR, which is an informative indicator of damage to the body of traumatic, inflammatory and hypoxic genesis. In the experimental group, the value of this indicator was in most cases within 2.7-3.55 mm / hour at the end of the experiment, and in the control group - 4.05 mm / hour. The average ESR value in the experimental group was 0.5-1.35 mm / h lower than in the control. A decrease in ESR in fish of the experimental group also indicates a positive effect of enriched feed. The value of ESR is an informative, although relatively insensitive criterion for inflammatory, necrotic and other pathological processes in the body. Some of the metabolic effects detected (changes in serum beta-lipoproteins) reflect, apparently, an increase in protein metabolism in experimental groups of fish.

The proposed method is simple, fast and energy efficient, since it completely eliminates the use of any reagents, except for waste processing of marine hydrobionts and water, does not require the addition of additional energy resources (steam, refrigerants) or the use of complex expensive devices, does not pollute the environment.

Protein hydrolysates obtained by this method have a balanced amino acid and protein composition, can be included in microbiological media as a source of amino acids, or used as a component of fish feed, increasing its effectiveness.

Claims (2)

1. A method of producing an enzymatic hydrolyzate from waste products from marine aquatic organisms with the preparation of raw materials, their grinding, adding water, introducing the interior of marine aquatic organisms as proteolytic agents, enzymatic hydrolysis, sediment separation, evaporation and drying, characterized in that waste is used as raw material processing of fish, the interior of Kamchatka crab and sea urchin, is carried out in stages using ultrasonic treatment, while at the first stage they prepare of fish processing, if necessary, thawed, crushed, loaded into the reactor with a mixing device in the amount of 60-80% of the total weight of the ingredients, separately insulated the inside of the fish, and added to the reaction mixture in the amount of 10-15%, add room temperature water in the ratio from 1: 1 to 1: 2 to the mass of raw materials, mix, place an ultrasonic apparatus in the reactor, and perform ultrasonic treatment for 10-15 minutes with an exposure intensity of 100-200 W / dm ³ at an initial temperature of 20-25 ° C and pH 6-7; at the second stage, prepared and homogenized crab entrails, including hepatopancreas, are added to the reactor in the amount of 10-15% of the initial weight of fish processing waste, and sonication is performed for 10-15 minutes with an exposure intensity of 100-200 W / dm ³ ; at the third stage, the prepared and homogenized insides of the sea urchin are added to the reactor in the amount of 1-5% of the initial mass of fish processing waste, and sonication is performed for 10-15 minutes with an exposure intensity of 100-200 W / dm ³ ; at the fourth stage, sonication is carried out for 15 minutes at an intensity of exposure of 500-600 W / dm ³ ; the resulting mixture is defended, the insoluble precipitate is filtered, the fat is removed by separation, the hydrolyzate solution is subjected to ultrafiltration or tangential filtration, the filtered solution is evaporated to a solids content of not less than 30% and freeze-dried or using a spray dryer. 2. The method according to claim 1, characterized in that the fawn sea urchin with entrails and gonads is used as a raw material of a sea urchin.