RU2542123C1 - Method for production of protein additive for food products enrichment with selenium - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method envisages protein isolation from connective tissues, protein hydrolysate production and modification with a selenium preparation. Connective tissue is represented by veins, tendons, fasciae wherefrom protein represented by collagen is isolated by way of peroxide alkaline hydrolysis. Collagen hydrolysate is produced by way of maintenance during 2.5-3.0 hours at a temperature of 36-38°C with "food collagenase" preparation in an amount of 0.02% of the collagen weight; collagen modification with selenium is performed by way of maintenance with 4.4-di[3(5- methylpyrazolil)] at pH value 8.0-8.5 during 2-4 hours.

EFFECT: manufactured product has enhanced moisture-binding and water-retaining capacities.

2 tbl, 2 dwg, 5 ex

Description

The invention relates to the food industry, namely to biotechnology for the production of protein additives - carriers of selenium for the production of food products.

Known methods for producing protein selenium-containing additives, providing for the cultivation of spirulina biomass on a nutrient medium containing an inorganic source of selenium.

A known method of producing selenium-containing spirulina biomass, in which sodium selenite is introduced into the culture fluid as a source of selenium in two portions - in the logarithmic and stationary phases of growth (description of patent RU No. 2146287 IPC C12N 1/12, A61K 35/80, A61K 33 / 04, C12N 1/12, C12R 1:89, 10.03.2000, publ. 10.03.2000).

The disadvantages of the method are the significant (7 days) duration of the spirulina cultivation process, the uneven distribution of selenium by weight of the additive as a result of mixing the resulting concentrate with a bioavailable selenium content of 6.15-6.5 g / kg with dry spirulina biomass to bring the selenium content to the allowed pharmacopoeial norms .

There is a method of obtaining selenium-containing preparation of spirulina biomass, which is obtained by culturing spirulina in an alkaline water-salt medium Zarukha, which is prepared in parts, while selenic acid (H ₂ SeO ₃ ) is additionally added to the first part of the nutrient medium as a source of selenium (description to patent RU 2209077 C2, A61K 35/80, A61K 30/04, publ. 07.27.2003).

A common drawback of these known methods is the specific color of the preparations due to the content of blue-green pigments involved in photosynthesis with the formation of complex organic substances from carbon dioxide and water, which limits its use in food production.

A known method of producing selenium-containing biologically active additives based on dairy raw materials and selenium, providing for the preparation of a nutrient medium, which uses clarified curd whey, the introduction of sodium selenite in an amount of 15-20 μg / ml and an inoculum, which use activated cultures of bifidobacteria or propionic acid bacteria (description of patent RU No. 23333655 C2, A23C 9/12, A23L 1/304, C12N 1/18, publ. 09/20/2008).

The disadvantage of this method is the instability of selenium-containing additives during storage and the need for sterile storage under aseptic conditions, the possibility of residual selenium content in the additive in inorganic form.

An immunocorrecting proteoselenium agent is known in the form of a complex of a low molecular weight component, an ionized organic salt of selenium and organic structures (carbohydrates, phospholipids, phospholipocarbons, caroteno carbohydrates and cyano carbohydrates), while the low molecular weight component is obtained from protein-containing raw materials by extraction with organic solvents and solvents 60 using solvents -90% alcohol, and to obtain the complex, a mixture of a low molecular weight component, ionized organic tion selenium salts and organic structures kept in the electromagnetic field of high frequency sinusoidal 20-30 MHz for 5 min (description of the patent RU 2161034 C1 IPC A61K 33/04, A61P 37/00).

The disadvantages of the method are the complexity of the hardware design and the high cost of the product, which limits the scope of its application by medicine and pharmacology.

The closest in technical essence and the achieved effect is a method of producing a selenium-containing additive for pets, comprising isolating the elastin protein, modifying the elastin hydrolyzate with selenium, followed by sorption of the resulting complex on zeolite (for patent RU, see RU 2321269 C1 A23K 1/16, publ. April 14, 2008).

The disadvantages of this technical solution are the restriction of the use of additives to the area of feed production, the multi-stage process, the limited sources of elastin in the bovine binder, and the high toxicity of sodium selenite used as a source of selenium.

The objective of the invention is the expansion of raw sources of protein supplements, expanding the range of protein supplements enriched with selenium, reducing the toxicity of the source of selenium upon receipt of the additive and its use.

EFFECT: increased yield, enhanced functional properties of the additive by increasing the moisture-binding, water-holding abilities of the protein supplement.

The technical result is achieved by the fact that in the method for producing a protein supplement for enriching selenium with food products, comprising isolating protein from connective tissues, obtaining a protein hydrolyzate, modifying it with selenium preparation, veins, tendons, fascia are used as connective tissue, of which protein is isolated collagen by peroxide-alkaline hydrolysis of connective tissues, collagen hydrolyzate is obtained by exposure for 2.5-3.0 hours at 36-38 ° C with food collagenase in an amount of 0.02% to the mass of collagen, and the modification of collagen with selenium is carried out by exposure to 4,4-di [3 (5-methylpyrazolyl)] (DMPS) at a pH of 8.0-8.5 for 2-4 hours at 18-24 ° C.

The use of veins and tendons will increase the mass yield of the protein source (9.7-12.4%) to obtain an additive for beef venation compared with the outcropping of a cattle ligament (cattle) (0.8%). The organization of collagen structures as part of tendons, ligaments, fasciae is characterized by both a parallel arrangement of fibers and bundles, and their interlacing at different angles with a developed intercellular matrix. At the same time, the mass fraction of water-, salt- and alkali-soluble protein fractions in the waste of beef venation is respectively 5.6; 7.4; 20%, including 19.9% of collagen. The specific spatial organization of collagen molecules with the formation of a three-strand left-handed alpha-helix is due to the specific amino acid composition of this protein with a large number of reactive groups. However, the presence of intra- and intermolecular cross-links limit the sorption capacity of collagen in the structure of connective tissues.

To increase the sorption capacity of collagen, it is isolated from connective tissues, for which they perform peroxide-alkaline hydrolysis, and then a collagen hydrolyzate is obtained by holding it with the enzyme preparation “food collagenase” under optimal conditions for the action of this drug (temperature 36-38 ° C, natural pH of the medium for connective tissue of cattle.

For peroxide-alkaline hydrolysis of connective tissues, a peroxide-alkaline composition is used, consisting of a solution of sodium hydroxide with a mass fraction of 10% and a solution of hydrogen peroxide 3% in a ratio of 10: 1. The mechanism of action of sodium hydroxide is the violation and weakening of some hydrogen bridges, the partial rupture of protein-carbohydrate membranes surrounding bundles of collagen fibrils. When processing connective tissues in a solution containing sodium hydroxide and hydrogen peroxide, as a result of the interaction of these two components, an exothermic reaction occurs that intensifies the process of breaking hydrogen bonds, which leads to loosening of the tissue structure and destruction of ballast substances (water and salt-soluble protein and lipid fractions )

To obtain a collagen hydrolyzate, an enzymatic preparation of proteolytic action - “food collagenase” (TU 2639-001-4554109-98, manufacturer of Bioprogress CJSC, Shchelkovo, Moscow Region) is used.

The source of the enzyme preparation “food collagenase” is the king crab hepatopancreas, an organ that combines the functions of the liver and pancreas in their digestive tract and is a complex of collagenolytic proteinases whose molecular weight is in the range of 23-36 kD. The predominant components in the diet of crabs are the skin of fish and the connective tissue of mollusks, worms, containing significant amounts of collagen. This suggests that the digestive enzymes of crab are adapted to the degradation of native collagen, a protein resistant to the action of proteinases from other sources. An electrophoretic study of collagen hydrolysis products showed that trimers and dimers of α-chains disappear. In this case, there is a significant increase in the number of free α-chains, as well as β- and γ-components, and the number of reactive groups increases [G. Rudenko // Bioorganic chemistry. - 2003. - volume 29. - S.117-128].

It was experimentally established that the appropriate duration of the enzymatic treatment is 2.5-3.0 hours (instead of 20 hours compared with the prototype). When the processing time is less than 2.5 hours, insufficient collagen hydrolysis occurs, and therefore, a sufficient sorption capacity and a high level of functional properties (moisture-binding, water-retaining, gel-forming, and fat-holding abilities) are not ensured. An increase in the processing time of more than 3 hours leads to an increase in the loss of the collagen fraction and to a deterioration in the microbiological parameters of the product. The maximum collagenolytic activity of collagenase appears at a pH of 6.5-8.5. Deviations from this range will increase the exposure time of the drug with collagen hydrolyzate.

At temperatures below 36 ° C, the activity of the enzyme preparation decreases, which leads to an increase in the duration of the process. Heating the reaction mixture to a temperature above 38 ° C leads to a deterioration in the functional properties of the additive and the irrational use of energy.

When the dosage of the drug “collagenase food” is less than 0.02%, the destruction of native collagen is not achieved, accompanied by an increase in the number of free α-chains, which provide a high sorption capacity.

The dosage of the food collagenase drug over 0.02% does not lead to a further increase in the sorption capacity of collagen hydrolyzate and is not economically feasible.

As a source of selenium, a DMDPS solution was used with a content of 1000 μg of selenium in 1 ml This is an antioxidant drug containing the organic form of selenium [Sanotsky I.V. // Essential Selenium. Prevention and treatment of diseases. - M., 2001. - S.18-21].

DMDPA is a preparation of toxicity class 4 (low hazard), less toxic than sodium selenite, which belongs to toxicity class 1 (extremely dangerous) according to GOST 12.1.007-76. The drug refers to compounds where the valency of selenium is 2. The drug, in addition to antioxidant properties, through the pyrazolyl ring (in its structure) has a pronounced effect on the enzyme systems of humans and animals, which are responsible for the formation of immunity.

Figure 1 shows a diagram of the effect of the exposure time of the collagen hydrolyzate with DMDPS on the concentration of bound selenium; figure 2 - the effect of the pH of the collagen hydrolyzate on the concentration of bound selenium upon exposure to DMDPS with collagen hydrolyzate for 3 hours. From the experimental data on the sorption of DMDPS on collagen hydrolyzate it follows that it is advisable to modify collagen with selenium by aging collagen hydrolyzate from 4.4 -di [3 (5-methylpyrazolyl)] (DMDPS) at the rate of 1.2 μg of selenium per 1 g of protein at a pH of 8.0-8.5 for 2-4 hours

A method of obtaining a protein supplement for fortifying selenium foods is as follows.

The feedstock - veins, tendons, fascia, allocated at the stage of beef trimming in sausage or canning production, is washed with running water, placed in a peroxide-alkaline solution containing 10% sodium hydroxide and 3% hydrogen peroxide. The processing time is 6-10 hours with a water module of 1: 2-2.5, which ensures uniform interaction of the peroxide-alkaline solution with the feedstock. Next, the liquid fraction is separated by decantation or centrifugation, and the solid collagen residue is washed with water and neutralized to a pH of 8.0-8.5, which falls within the pH range favorable for the manifestation of collagenolytic activity of the food collagenase preparation.

To obtain a collagen hydrolyzate, the solid residue is heated to 36-38 ° C, a food collagenase solution is added in an amount of 0.02% by weight of collagen, the hydromodule is 1: 2-2.5, and it is kept under periodic stirring for 2.5 -3.0 hours. After this, the liquid fraction of the hydrolyzate is separated by decantation or centrifugation, washing with water, remove the remains of the enzyme preparation and carry out the collagen modification with selenium, for which the resulting mass is kept with 4,4-di [3 (5-methylpyrazolyl)] (DMPS) based on 1.2 μg of selenium per 1 g of collagen at a pH of 8.0-8.5 in t chenie 2-4 hours. The resultant mass was neutralized to pH 7,0, using edible organic acid such as acetic or ascorbic. When used to neutralize ascorbic acid, the resulting product is additionally enriched with sodium ascorbate.

Example 1

10 kg of beef veins, tendons, fascias are washed with running water for 10 minutes. Then, peroxide-alkaline treatment is carried out, for which 20 liters of sodium hydroxide solution are prepared with a mass fraction of 10% and 2 liters of a 3% hydrogen peroxide solution, the solutions are mixed, introduced into a container with ground veins, tendons, fasciae and kept for 6 hours, at this mass is stirred every hour for 5 minutes The liquid fraction is separated by centrifugation at 45 s ^-1 for 5 minutes, 15 L of water are added to the solid residue, mixed and centrifuged again to separate the liquid fraction. The solid collagen residue is neutralized with acetic acid to pH = 8.0, poured with 20 l of water and heated to 37 ° C. The drug "collagenase food" weighing 2 g is dissolved in 200 cm ^{3 of} water with a temperature of 37 ° C. The resulting solution was introduced into a container with collagen and kept at 37 ° C for 3 hours, stirring every hour for 5 minutes. The liquid fraction is separated by centrifugation at 45 s ^-1 for 5 minutes, 12 L of water is added to the solid residue, mixed and centrifuged again to separate the liquid fraction. Then, 33 cm ³ of DMDPS solution with a concentration of 1000 μg / cm ³ , which corresponds to a concentration of selenium of 1.1 μg per 1 g of collagen, is introduced into the container, kept for 3 hours at 18 ° C and neutralized with a 5% ascorbic acid solution to pH = 7.

Example 2

Beef veins, tendons, fascia weighing 20 kg are washed with running water for 15 minutes. Then, peroxide-alkaline treatment is carried out, for which 40 liters of sodium hydroxide solution are prepared with a mass fraction of 10% and 4 liters of a 3% hydrogen peroxide solution, the solutions are mixed, introduced into a container with crushed veins, tendons, fascias and incubated for 10 hours, at this mass is stirred every hour for 5 minutes The liquid fraction is separated by centrifugation at 60 s ^-1 for 5 minutes, 30 L of water are added to the solid residue, mixed and centrifuged again to separate the liquid fraction. The solid collagen residue is neutralized with acetic acid to pH = 8.5, poured with 30 l of water and heated to 38 ° C. The drug "collagenase food" weighing 4 g is dissolved in 400 cm ^{3 of} water with a temperature of 38 ° C. The resulting solution was added to a collagen container and kept at 38 ° C for 2.5 hours, stirring every hour for 5 minutes. The liquid fraction is separated by centrifugation at 60 s ^-1 for 5 minutes, 30 L of water are added to the solid residue, mixed and centrifuged again to separate the liquid fraction. Next, 74 cm ³ of DMDPS solution with a selenium concentration of 1000 μg / cm ³ , which corresponds to a concentration of selenium of 1.2 μg per 1 g of collagen, is introduced into the container, incubated for 3 hours at 18 ° C and neutralized with a 5% ascorbic acid solution to pH = 7 .

Example 3

The conditions for producing the additive correspond to Example 1, characterized in that after centrifugation and separation of the liquid fraction, 40 cm ³ of DMDPS solution with a concentration of 1000 μg / cm ^{3 is} introduced, which corresponds to a concentration of selenium of 1.3 μg per 1 g of collagen.

Example 4

The conditions for the preparation of the additive correspond to Example 1, characterized in that after centrifugation and separation of the liquid fraction, 30.6 cm ³ of DMDPS solution with a concentration of 1000 μg / cm ^{3 is} introduced, which corresponds to a concentration of selenium of 1 μg per 1 g of collagen.

Example 5

The conditions for obtaining the additive correspond to example 1, characterized in that at the final stages the additive is dehydrated by freeze-drying.

The additive obtained in examples No. 1-5, is characterized by the indicators presented in table 1, and can be used in the formulations of products based on meat, fish, milk based as a builder and stabilizer in the amount of 3-20% by weight of the main raw material.

The advantages of the proposed technical solutions in comparison with the prototype are presented in table 2.

Table 1 The name of indicators Characteristic and value indicators for additive samples Example Number one 2 3 four 5 Consistency Gel-like Powdered (d meas .≤100 μm) Color Yellowish Creamy white Mass fraction,% moisture 82.70 87,20 82.8 82.9 4.42 squirrel 16.56 12.05 16.55 16.57 90.28 ash 0.71 0.72 0.71 0.71 4.92 fat 0.3 0.3 0.3 0.3 0.38 The concentration of selenium in the additive, μg / g 1,1 1,2 1,2 1,0 7.9 BCC,% 7.69 7.91 7.69 7.71 26,42 VUS,% 39.37 39.55 39.38 39.37 88.7

table 2 Indicators The claimed method Prototype Scarcity of raw materials No Yes Toxicity class of selenium source DMDP, 4 low hazard Sodium Selenite, 1 extremely dangerous Destination area Food industry Feed production Functional corrective properties in food systems Yes No The ability to control the concentration of selenium in products Yes No

Claims (1)

A method of producing a protein supplement for enriching selenium with food products, comprising isolating protein from connective tissues, obtaining a protein hydrolyzate and modifying it with a selenium preparation, characterized in that veins, tendons, fascia are used as connective tissue, from which collagen is isolated by peroxide as protein alkaline hydrolysis, collagen hydrolyzate obtained by exposure for 2.5-3.0 hours at 36-38 ° C with the drug "collagenase food" in the amount of 0.02% by weight of collagen, while the modification of the coll ene selenium is carried out by exposure to 4,4-di [3- (5-methylpyrazolyl)] (DMDPS) at pH 8,0-8,5 for 2-4 hours.

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