RU2700444C1 - Method for production of iodized milk whey proteins for producing a biologically active substance - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to production of iodized whey proteins for preparing a biologically active substance and can be used for preventing iodine deficiency disorders in humans and animals. Process of iodination of initial protein raw material, in form of α-lactalbumin, β-lactoglobulin or a mixture of said proteins, or hydrolysates of said proteins, by mixing with an aqueous solution of inorganic iodine at temperature of 20–40 °C and at ratio of solution of inorganic iodine to total protein selected (2–40):1. Mixture of whey proteins is fermented with an aqueous solution of inorganic iodine by adding a buffer mixture of reagents – a complex of NaCl mineral salts and Na and K phosphates and a mixture of lactoperoxidase-based enzymes containing 16–24 wt%, horseradish peroxidase and from 14 to 21 wt% of catalase. Buffer mixture contains 14–18 wt% sodium phosphate and 22–28 wt% potassium orthophosphate with stability pH=6–8 with a reaction mixture of enzymes immobilized on semi-permeable membranes or on inert carriers. Fermentation process is carried out with continuous monitoring of iodine content in the solution. Aqueous solution of iodinated proteins is purified from macroimpurities and microimpurities, including from inorganic iodine, using macrofiltration, microfiltration with subsequent diafiltration of aqueous solution of iodinated proteins on ultrafiltration unit in tangential flow mode using membrane modules with cut-off limit of 300 to 800 Da at pH 6.0–8.0. Obtained solution of iodated proteins subject to sterilizing microfiltration, then sublimation or spray drying to obtain a finished powder product with content of deterministic covalently bound iodine in amount of 0.5–4 % in form of a mixture of iodised amino acids – monoiodotyrosines in amount of 55–75 wt% in iodated proteins, 24.0–43.5 wt% di-iodotyrosines and 1.0–1.5 wt% of tri-iodotyrosines.

EFFECT: invention enables to obtain a ready powder product with given content of deterministic amount of covalently bound iodine with optimal and specified content of iodised amino acids in it – monoiodotyrosines, di-iodotyrosines and tri-iodotyrosines, production of industrial volumes of iodized milk whey proteins.

1 cl, 6 ex

Description

A method of manufacturing iodized milk whey proteins for a biologically active substance

The invention relates to medicine, in particular to a method for the production of iodinated whey proteins for a biologically active substance, and can be used for the prevention and treatment of iodine-deficient conditions in humans and animals, in particular in the production of food products, dietary supplements, enteral and parenteral nutrition, medicines, veterinary drugs and feed.

A known method of obtaining a biologically active food supplement, including the implementation of the process of iodization of the protein source by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected (2-40): 1, the fermentation process of the initial mixture of whey proteins with water a solution of inorganic iodine by introducing into it a buffer mixture of reagents - a complex of mineral salts NaCl and phosphates Na and K, a reaction mixture of enzymes immobilized on semipermeable membranes or on inert carriers, while the fermentation process is carried out with continuous monitoring of the iodine content in the solution, the aqueous solution of iodinated proteins is purified from macro impurities and trace elements, including

inorganic iodine, using macro filtration, microfiltration and ultrafiltration followed by diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit, the resulting solution of iodized proteins is subjected to sterilizing microfiltration, then freeze-drying to obtain a finished powder product. (see RF patent No. 2212155, IPC A23L 1/30, 09/20/2003).

However, the known method in its use has the following disadvantages:

- does not provide iodinated proteins with the content and ratio of amino acids in them - monoiodotyrosines, diiodotyrosines and triiodotyrosines,

- does not provide a finished powder product with a high content of a determinate amount of covalently bound iodine,

- does not allow to obtain a finished powder product with the necessary characteristics of the distribution and accumulation of organic iodine in humans and animals,

- does not allow to obtain a finished powder product with the necessary characteristics of the distribution and accumulation of organic iodine with the participation of deiodinases in the liver and in tissues and organs,

- does not provide a sufficient amount of industrial volumes of iodized whey milk proteins,

- cannot provide iodized milk whey proteins with hypoallergenic properties.

The objective of the invention is to provide a method for the manufacture of iodized milk whey proteins for a biologically active substance.

The technical result is the provision of a finished powder product with a given content of a determinate amount of covalently bound iodine with an optimal and predetermined content of iodinated amino acids — monoiodotyrosines, diiodotyrosines and triiodotyrosines. In addition, the technical result is to obtain industrial volumes of iodized whey milk proteins.

The technical result is achieved by the fact that the proposed method for the production of iodized milk whey proteins for a biologically active substance, including the implementation of the process of iodization of the original protein raw material by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected (2-40): 1, the fermentation process of the initial mixture of whey proteins with an aqueous solution of inorganic iodine by introducing into it a buffer mixture of reagents - a complex of mineral salts NaCl and Na and K phosphates by a reaction mixture of enzymes immobilized on semipermeable membranes or on inert carriers, while the fermentation process is carried out with continuous monitoring of the iodine content in the solution, the aqueous solution of iodized proteins is cleaned of macronutrients and microimpurities, including inorganic iodine using macrofiltration, microfiltration followed by diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit, the resulting solution of iodinated proteins is subjected to microfiltration, then freeze-drying to obtain a finished powder product, while α-lactalbumin, β-lactoglobulin, or a mixture of the listed proteins or hydrolysates of the listed proteins are used as starting protein raw materials during the iodization process, while the process of iodization of the starting whey proteins mixing with an aqueous solution of inorganic iodine is carried out at a temperature of 20 ° C-40 ° C by introducing into it a buffer mixture of reagents - a complex of mineral salts based on NaCl, containing 14-18 wt boiling. % sodium phosphate and 22-28 wt. % potassium orthophosphate at a stability of pH = 6-8 used the initial solution of the reaction mixture of enzymes, which is used as a mixture based on lactoperoxidase containing from 16 to 24 mass. % horseradish peroxidase and from 14 to 21 mass. % catalase, diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit is carried out in a tangential flow mode using membrane modules with a cutoff limit of 300 to 800 Da at pH 6.0-8.0, and after performing diafiltration sterilizing

microfiltration and then freeze-drying or spray drying get the finished powder product with determinate covalently bound iodine in an amount of 0.5-4% in the form of a mixture of iodinated amino acids - monoiodotyrosines in iodinated proteins in the amount of 55-75 mass. %, 24.0-43.5 wt. % diiodotyrosines and from 1.0-1.5 mass. % triiodothyrosine.

The method is as follows. The process of iodination of the initial protein raw material is carried out by mixing it with an aqueous solution of inorganic iodine at a ratio of the solution of inorganic iodine to the total protein selected (2-40): 1. When starting the iodination process, α-lactalbumin, β-lactoglobulin, or a mixture of the listed proteins or hydrolysates of the listed proteins are used as the initial protein raw material.

The process of iodination of the initial whey proteins when they are mixed with an aqueous solution of inorganic iodine is carried out at a temperature of 20 ° C-40 ° C by introducing into it a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 14-18 mass. % sodium phosphate and 22-28 wt. % potassium orthophosphate at stability pH = 6-8 of the used initial solution of the reaction mixture of enzymes, including those immobilized on semipermeable membranes or on inert carriers. Moreover, as the initial solution of the reaction mixture of enzymes using a mixture based on lactoperoxidase containing from 16 to 24 mass. % horseradish peroxidase and from 14 to 21 mass. % catalase, and the fermentation process is carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodized proteins is purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration, followed by diafiltration of the aqueous solution of iodized proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 300 to 800 Yes, at pH 6.0-8.0.

The resulting solution of iodized proteins is subjected to sterilizing microfiltration, then freeze-drying or spray drying to obtain a finished powder product with a deterministic amount of covalently bound iodine in an amount of 0.5-4% in the form of a mixture of iodinated amino acids - monoiodotyrosines in iodinated proteins in the amount of 55-75 mass .,% from 24.0-43.5 mass. % diiodotyrosines and from 1.0-1.5 mass. % triiodothyrosine.

Among the essential features characterizing the proposed method for the production of iodinated whey protein proteins for a biologically active substance, the following are distinguishing:

- the use of protein as raw materials in the process of iodination of α-lactalbumin, β-lactoglobulin, or a mixture of these proteins or hydrolysates of these proteins,

- the implementation of the process of iodization of the original whey proteins when they are mixed with an aqueous solution of inorganic iodine at a temperature of 20 ° C-40 ° C by introducing into it a buffer mixture of reagents - a complex of mineral salts based on NaCl containing 14-18 mass. % sodium phosphate and 22-28 wt. % potassium orthophosphate at a stability of pH = 6-8 used the initial solution of the reaction mixture of enzymes, which is used as a mixture based on lactoperoxidase containing from 16 to 24 mass. % horseradish peroxidase and from 14 to 21 mass. % catalase

- the implementation of diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 300 to 800 Da at a pH of 6.0-8.0,

- obtaining, after performing diafiltration, sterilizing microfiltration and then freeze-drying or spray drying of the finished powder product with a content of determined covalently bound iodine in an amount of 0.5-4% in the form of a mixture of iodinated amino acids - monoiodotyrosines contained in iodized proteins in an amount

55-75 mass. % with 24.0-43.5 wt. % diiodotyrosines and from 1.0-1.5 mass. % triiodothyrosine.

Experimental studies of the proposed method for the production of iodinated whey milk proteins for a biologically active substance showed its high efficiency. A method of manufacturing iodinated milk whey proteins for a biologically active substance provided a finished powder product with a given content of a determinate amount of covalently bound iodine with an optimal and predetermined content of iodinated amino acids monoiodotyrosines, diiodotyrosines and triiodothyrosines. In addition, using the proposed method, obtaining specified and sufficient industrial volumes of iodinated milk whey proteins was achieved.

The implementation of the proposed method for the production of iodinated whey protein proteins for a biologically active substance is illustrated by the following practical examples.

Example 1. The process of iodination of the starting protein in the form of α-lactalbumin was carried out by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected 2: 1.

The process of iodination of the initial serumal protein of α-lactalbumin was carried out when it was mixed with an aqueous solution of inorganic iodine at a temperature of 30 ° C by introducing a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 18 wt. % sodium phosphate and 22 wt. % potassium orthophosphate at stability pH = 6 of the used initial solution of the reaction mixture of enzymes immobilized on semipermeable membranes. Moreover, as the initial solution of the reaction mixture of enzymes used a mixture based on lactoperoxidase containing 16 mass. % horseradish peroxidase and 21 wt. % catalase, and the fermentation process was carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodized proteins was purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration, followed by diafiltration of the aqueous solution of iodinated proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 500 Da at pH 8.0.

The resulting solution of iodized proteins was subjected to sterilizing microfiltration, then freeze-drying to obtain a finished powder product with a determinate amount of covalently bound iodine in the amount of 2.5% in the form of a mixture of iodinated amino acids - monoiodotyrosines in iodinated proteins in the amount of 75 masses. % with 24.0 mass. % diiodotyrosines and with 1.0 mass. % triiodothyrosine.

Example 2. The process of iodination of the starting protein in the form of β-lactoglobulin was carried out by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected 10: 1.

The process of iodination of the initial β-lactoglobulin whey protein was carried out by mixing it with an aqueous solution of inorganic iodine at a temperature of 20 ° C by introducing a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 14 wt. % sodium phosphate and 28 wt. % potassium orthophosphate at stability pH = 7 of the used initial solution of the reaction mixture of enzymes immobilized on inert carriers. Moreover, as the initial solution of the reaction mixture of enzymes used a mixture based on lactoperoxidase containing 18 mass. % horseradish peroxidase and 19 wt. % catalase, and the fermentation process was carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodized proteins was purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration, followed by diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit in a tangential

flow mode using membrane modules with a cutoff limit of 300 Da at pH 7.0.

The resulting solution of iodized proteins was subjected to sterilizing microfiltration, then spray drying to obtain a finished powder product with a determinate amount of covalently bound iodine in the amount of 1.7% in the form of a mixture of iodinated amino acids - monoiodotyrosines in iodinated proteins in the amount of 55 mass. % with 43.5 wt. % diiodotyrosines and with 1.5 wt. % triiodothyrosine.

Example 3. The process of iodination of the starting protein in the form of a mixture of α-lactalbumin and β-lactoglobulin was carried out by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected 30: 1.

We performed the process of iodination of the initial whey protein in the form of a mixture of α-lactalbumin, β-lactalbumin, when it was mixed with an aqueous solution of inorganic iodine at a temperature of 35 ° C by introducing a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 18 wt. % sodium phosphate and 24 wt. % potassium orthophosphate at stability pH = 6 of the used initial solution of the reaction mixture of enzymes immobilized on inert carriers. Moreover, as the initial solution of the reaction mixture of enzymes used a mixture based on lactoperoxidase containing 19 mass. % horseradish peroxidase and 16 wt. % catalase, and the fermentation process was carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodinated proteins was purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration, followed by diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 700 Da at pH 6.0.

The resulting solution of iodinated proteins was subjected to sterilizing microfiltration, then freeze-drying to obtain a finished powder

a product with a determinate amount of covalently bound iodine in an amount of 2.0% in the form of a mixture of iodinated amino acids - monoiodotyrosines contained in iodinated proteins in an amount of 68 mass. % with 31.0 mass. % diiodotyrosines and with 1.0 mass. % triiodothyrosine.

Example 4. The process of iodination of the starting protein was performed in the form of an α-lactalbumin hydrolyzate by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected 25: 1.

We performed the process of iodization of the initial whey protein in the form of an α-lactalbumin hydrolyzate when it was mixed with an aqueous solution of inorganic iodine at a temperature of 20 ° C by introducing a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 15 wt. % sodium phosphate and 27 wt. % potassium orthophosphate at stability pH = 7 of the used initial solution of the reaction mixture of enzymes immobilized on semipermeable membranes. Moreover, as the initial solution of the reaction mixture of enzymes used a mixture based on lactoperoxidase containing 17 mass. % horseradish peroxidase and 20 wt. % catalase, and the fermentation process was carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodized proteins was purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration, followed by diafiltration of the aqueous solution of iodized proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 300 Da at pH 7.0.

The resulting solution of iodized proteins was subjected to sterilizing microfiltration, then spray drying to obtain a finished powder product with a determinate amount of covalently bound iodine in an amount of 4.0% in the form of a mixture of iodinated amino acids based on monoiodotyrosines in iodinated proteins in an amount

70 mass. % with 29.0 wt. % diiodotyrosines and with 1.0 mass. % triiodothyrosine.

Example 5. The iodination process of the starting protein was performed in the form of a β-lactoglobulin hydrolyzate by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected 40: 1.

We performed the process of iodization of the initial whey protein in the form of a β-lactoglobulin hydrolyzate when it was mixed with an aqueous solution of inorganic iodine at a temperature of 40 ° C by introducing a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 14 wt. % sodium phosphate and 25 wt. % potassium orthophosphate with stability pH = 8 of the used initial solution of the reaction mixture of enzymes immobilized on inert carriers. Moreover, as the initial solution of the reaction mixture of enzymes used a mixture based on lactoperoxidase containing 20 mass. % horseradish peroxidase and 21 wt. % catalase, and the fermentation process was carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodinated proteins was purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration, followed by diafiltration of the aqueous solution of iodinated proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 800 Da at pH 8.0.

The resulting solution of iodized proteins was subjected to sterilizing microfiltration, then freeze-drying to obtain a finished powder product with a determinate amount of covalently bound iodine in the amount of 3.1% in the form of a mixture of iodinated amino acids - monoiodotyrosines in iodinated proteins in the amount of 65 wt. % with 33.5 wt. % diiodotyrosines and with 1.5 wt. % triiodothyrosine.

Example 6. The iodination process of the starting protein in the form of a mixture of α-lactalbumin and β-lactoglobulin was carried out by mixing it with an aqueous solution

inorganic iodine at a ratio of inorganic iodine solution to the total protein selected 40: 1.

We performed the process of iodination of the initial whey protein in the form of a mixture of α-lactalbumin, β-lactalbumin, when it was mixed with an aqueous solution of inorganic iodine at a temperature of 40 ° C by introducing a buffer mixture of reagents - a complex of NaCl-based mineral salts containing 16 masses. % sodium phosphate and 27 wt. % potassium orthophosphate at stability pH = 7 of the used initial solution of the reaction mixture of enzymes immobilized on semipermeable membranes. Moreover, as the initial solution of the reaction mixture of enzymes used a mixture based on lactoperoxidase containing 24 mass. % horseradish peroxidase and 14 wt. % catalase, and the fermentation process was carried out with continuous monitoring of the iodine content in the solution.

The aqueous solution of iodized proteins was purified from macro impurities and microimpurities, including inorganic iodine, using macro filtration, microfiltration and ultrafiltration, followed by diafiltration of the aqueous solution of iodized proteins in an ultrafiltration unit in a tangential flow mode using membrane modules with a cutoff limit of 400 Yes at pH 8.0.

The resulting solution of iodized proteins was subjected to sterilizing microfiltration, then spray drying to obtain a finished powder product with a determinate amount of covalently bound iodine in an amount of 0.5% in the form of a mixture of iodinated amino acids based on monoiodotyrosines in iodinated proteins in an amount of 70 masses. % with 28.5 wt. % diiodotyrosines and with 1.5 wt. % triiodothyrosine.

Claims (1)

A method of manufacturing iodized milk whey proteins for a biologically active substance, including the process of iodizing the original protein raw material by mixing it with an aqueous solution of inorganic iodine at a ratio of inorganic iodine solution to the total protein selected (2-40): 1, performing the fermentation process of the initial whey protein mixture with an aqueous solution of inorganic iodine by introducing into it a buffer mixture of reagents - a complex of mineral salts NaCl and Na and K phosphates by a reaction mixture of enzymes izilizirovannykh on semipermeable membranes or on inert carriers, while the fermentation process is carried out with continuous monitoring of the iodine content in the solution, the aqueous solution of iodinated proteins is cleaned of macronutrients and microimpurities, including inorganic iodine, using macrofiltration, microfiltration followed by diafiltration of the aqueous solution iodized proteins in the ultrafiltration unit, the resulting solution of iodinated proteins is subjected to sterilizing microfiltration, then sublimation su ke to obtain a finished powder product, characterized in that as the source of protein raw materials when performing the iodination process using α-lactalbumin, β-lactoglobulin or a mixture of these proteins, or hydrolysates of these proteins, while the process of iodization of the original whey proteins when mixed with water a solution of inorganic iodine is carried out at a temperature of 20 ° C-40 ° C by introducing into it a buffer mixture of reagents - a complex of mineral salts based on NaCl, containing 14-18 mass. % sodium phosphate and 22-28 wt. % potassium orthophosphate at a stability of pH = 6-8 used the initial solution of the reaction mixture of enzymes, which is used as a mixture based on lactoperoxidase containing from 16 to 24 mass. % horseradish peroxidase and from 14 to 21 mass. % catalase, diafiltration of an aqueous solution of iodinated proteins in an ultrafiltration unit is carried out in a tangential flow mode using membrane modules with a cutoff limit of 300 to 800 Da at pH 6.0-8.0, and after performing diafiltration sterilizing microfiltration and then sublimation or spray drying, a finished powder product is obtained with a content of determinate covalently bound iodine in an amount of 0.5-4% in the form of a mixture of iodinated amino acids contained in iodized proteins - monoiod irozinov in an amount of 55-75 wt. %, 24.0-43.5 wt. % diiodotyrosines and from 1.0-1.5 mass. % triiodothyrosine.