RU2720200C2 - Method for preparing an agent for preventing iodine deficiency disorders and immunodeficiency - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention relates to pharmaceutical industry and represents a method for preparing an agent for preventing iodine deficiency disorders and immunodeficiencies, providing fermentation of protein, introduction of solutions of zinc sulphate and potassium iodide, characterized by that the protein used is soya protein, which is dissolved in hydrochloric acid, fermentation is carried out using pepsin to produce peptides of soya hydrolyzate, which is neutralized, centrifuged, zinc sulphate solution is added to obtained supernatant solution of low-molecular weight peptides in ratio 1:1 with subsequent dialysis, after binding of zinc salts with soya hydrolyzate peptides solution of potassium iodide is added in ratio 1:1 with subsequent dialysis.

EFFECT: invention provides effective regulation of iodine metabolism and immunodeficiencies due to the presence of zinc, possibility of regulating zinc and iodine salt concentrations for different types of consumers, as well as reducing the cost of the method.

3 cl, 2 dwg, 3 tbl, 3 ex

Description

The invention relates to the food industry, and in particular to functional ingredients, to food for the prevention of iodine deficiency conditions and immunodeficiencies. The product contains organically bound iodine, zinc with a soy hydrolyzate. The tool can be used for cooking foods to enrich them with micronutrients - iodine and zinc. In particular, the product can be used in the production of bread, bakery, pasta and confectionery, dairy products, baby food, as well as various drinks: drinking and fruit water, beer, kvass.

It is known to use iodized food salt to compensate for the lack of iodine entering the body. An iodized salt is a mechanical mixture of a common salt (NaCl) and inorganic compounds of potassium iodide (KI) or potassium iodate (KIO ₃ ) (Monitoring Universal Salt Iodization Programs. Published by PAMM / ICCIDD / MI, 1995).

In the Russian Federation, iodized salt with an iodine content of 40 ± 15 μg per 1 g of product is used. Uniform mixing with this ratio of components is a daunting task. In addition, inorganic iodine salts are unstable and gradually volatilize during storage and especially during heat treatment of food. The norms of salt consumption are variable in different groups of the population angle and range from 0 (salt is contraindicated in some diseases) to 10-15 g per day, while 375-825 mcg of iodine can enter the body, which is 2.5-5.5 times exceeds the physiological norm. Exceeding the physiological intake of inorganic iodine can cause thyroid disease. The experience of using iodized salt for the prevention of iodine deficiency in Africa shows an increase in the frequency of iodinated thyrotoxicosis in the elderly (Problems of Endocrinology No. 4, 2005, T 51 p. 36).

Mineral compounds of iodine in vivo act as an auxiliary source of iodine, while the evolutionary mechanism of iodine metabolism is primarily aimed at using the organic form of iodine prevailing in natural food products.

It is known to use dry starch iodide complex for the treatment or prevention of diseases caused by iodine deficiency (RU C1 No. 2110265, IPC 6 A61K 33/18, 1998). Iodine is in this complex mainly in the form of an inclusion compound, so this complex can be called a prolonged form of the inorganic iodine preparation, which is absorbed gradually, as the starch is digested in the gastrointestinal tract.

Thus, the use of starch iodide complex does not allow the regulation of iodine metabolism, which is possible when using the organic form of iodine. In addition, this drug cannot be used in the form of an additive to food subjected to cooking, since it begins to decompose at 40 ° C.

It is known to use a biologically active additive in the form of a complex compound of inorganic iodine and pectin for the prevention of iodine deficiency (RU C1 No. 2265377, IPC 7 A23L 1/30, 2005; RU C1 No. 2265376, IPC 7 A23L 1/30, 2005).

In this case, there is also a complex compound of inorganic iodine, which is gradually absorbed as the pectin of the iodine complex moves along the gastrointestinal tract. Individual adjustment of iodine metabolism in this case is also impossible. The use of this drug as an additive to food is extremely inconvenient, since it is a water-insoluble powder.

Known biologically active food supplement for the prevention of iodine deficiency based on the fruits of chokeberry with the addition of pectin to stabilize the iodine content during storage of the drug (RU C1 No. 2271726, IPC 7 A23L 1/30, 03.20.2006). ^

The main disadvantage of this technical solution is the extremely low specific iodine content in the product: 70 μg per 100 g of product. In addition, since special measures are required to stabilize the iodine content during storage, a significant part of the iodine is in inorganic form.

Known food products, including iodine-containing food additives: bread, bakery products, milk, butter, meat products (Collection of recipes and technological instructions for the preparation of dietetic and prophylactic varieties of bakery products, State Research Institute of Bakery Industry, Moscow, Pishcheproduct, 1997, Iodine-fortifying food supplement "Amiton", TU, TI, RC 9110-273-05747158-98). Inorganic compounds of iodine, kelp (seaweed), and yeast grown in iodized aqueous medium are introduced into these products as food additives.

A disadvantage of the known products is that in yeast when grown on iodized medium, the metabolism can change, and the products have a pronounced unpleasant aftertaste and smell. This is due to the decomposition of inorganic iodine compounds in the light with the release of free iodine. Sea kale also contains a large number of inorganic iodine compounds and the ratio of organic to inorganic forms of iodine varies greatly depending on the location and growing conditions, processing and transportation methods.

A known method of obtaining a biologically active food supplement for optimizing iodine metabolism, which is the germination of grain in water containing various trace elements: iodine, selenium, cobalt, molybdenum, zinc, copper, manganese in a certain concentration, drying the sprouted grain, grinding and compounding it in various combinations (RU C1 No. 2271725, IPC 7 A23L 1/30, 03.20.2006).

The advantage of this method is that, along with iodine, concomitant micronutrients are introduced that improve the metabolism of iodine in the body. The disadvantage of this method is that 20-50% of the average daily need for iodine is in 40-70 g of flour. Receiving such an amount daily is clearly inconvenient, and the low-tech production method does not allow replacing a significant part of the flour produced with its biologically active analogue.

A known method of obtaining a biologically active food supplement by enzymatic hydrolysis of an animal protein - elastin and iodization of the resulting hydrolyzate, followed by drying of the product (RU C1 No. 2266021, IPC 7 A23L 1/30, 2005). The daily rate of iodine contains 1 to 4 g of the resulting powder.

The disadvantage of this method is the need to dissolve the powder before adding to food products, and the use of additives leads to a change in the taste of the products. In addition, in this case, only iodine deficiency is compensated for without the introduction of other micronutrients involved in its metabolism.

In a number of technical solutions, for the prevention of iodine deficiency, it is proposed to use a synthetic organic compound with covalently bound iodine, selected from various groups of natural organic compounds, including proteins of plant, animal or microbiological origin and various mixtures thereof (RU C1 No. 2134520, IPC 6 A23J 1/20 , 1999; RU C1 No. 2141205, IPC 6 A21D 2/02, 1999; RU C1 No. 2163127, IPC 7 A61K 33/18, 2001; RU C1 No. 2192150, IPC 7 A23L 1/304, 2002).

The advantage of this proposal is the lack of hormonal activity of the obtained synthetic organic compounds and the ease of their addition to food products. The disadvantages include the use of xenobiotics for the synthesis, such as chloramine T, iodine chloride or iodine trichloride. Special measures are needed to control their absence in the final product. Known methods also do not introduce trace elements accompanying iodine.

A known method for producing iodized protein (RU C1 No. 2188648, IPC 7 A61K 33/18, 2002) and a means for regulating iodine metabolism or prevention of iodine deficiency conditions (RU C1 No. 2151611, IPC 7 A61K 38/16, 2000), where the protein is iodinated by adding to a solution of a protein of an iodinating agent - iodine chloride, dissolved in hydrochloric or acetic acid. As a result, iodine joins to the aromatic rings of amino acids - tyrosine, phenylalanine and tryptophan. During the reaction, the pH of the solution decreases and a precipitate of iodinated protein forms, which is separated by centrifugation or filtration and then dried. The resulting product contains iodinated protein or its low molecular weight component, the structure of which includes at least one of the following amino acids - phenylalanine, tryptophan. In the particular case, it is proposed to use milk protein - casein for iodination.

When using this tool, an overdose is impossible, since in the gastrointestinal tract the iodized protein is decomposed to iodinated amino acids, which enter the liver. In the liver, iodine is cleaved from them under the influence of the deiodinase enzyme, the activity of which depends on the degree of iodine deficiency and the functional state of the thyroid gland. Excessive amount of iodinated amino acids, turning into glucuronides, leaves the body.

The disadvantage of this method is the use of chlorine iodide to obtain iodinated protein (an admixture of this xenobiotic in the final product is possible). The finished form in the form of a powder is not always convenient for addition to food products - an additional stage of dissolution is necessary. This tool introduces only iodine into the body, and this is not always enough to regulate iodine metabolism, especially at the initial stage of correction of iodine deficiency states.

The closest in technical essence to the claimed invention is a method of producing a biologically active food supplement for the prevention of iodine deficiency conditions and optimization of iodine metabolism, made in the form of casein containing both iodine and zinc in the composition of organic molecules (RU C2 No.2328878, IPC A23L 1 / 30, 2012). The method includes adding to the casein solution a solution of tetrasodium salt of ethylenediaminetetraacetic acid. A saturated solution of fine crystalline iodine in a solution of potassium iodide is used for iodination of aromatic amino acids, and a solution of a water-soluble zinc salt is used to saturate the phosphorus groups of casein with zinc ions.

The disadvantage of this method is that there is a simultaneous mixing of iodine and zinc without the possibility of regulating the concentration of trace elements.

The technical result of the invention is to obtain a means for the prevention of iodine deficiency conditions, which provides effective regulation of iodine metabolism and immunodeficiencies, the ability to control the concentration of zinc and iodine salts for different types of consumers, as well as the cheaper method.

The specified technical result is achieved by the fact that in the method of obtaining funds for the prevention of iodine-deficiency conditions and immunodeficiencies, which involves protein fermentation, the introduction of solutions of zinc sulfate and potassium iodide. According to the invention, soy protein is used as a protein, which is dissolved in hydrochloric acid, fermentation is carried out using pepsin to obtain peptides of a soy hydrolyzate, which are neutralized, centrifuged, zinc sulfate solution is added to the resulting supernatant solution of low molecular weight peptides, followed by dialysis, after binding of zinc salts to In a ratio of 1: 1 with peptides of a soy hydrolyzate, a solution of potassium iodide in a ratio of 1: 1 was added, followed by dialysis.

Distinctive features of the proposed method are the use of soy protein, its pepsin fermentation with the formation of low molecular weight peptides, as well as the sequential introduction of zinc and iodine salts in a solution of low molecular weight peptides.

At a soy protein concentration of 0.5%, the concentration of soluble peptides in the supernatant was low (see table 1). At protein concentrations of 2% and higher, the volume of the supernatant was small compared with concentrations of less than 2%, which would be irrational in view of the increased cost of raw materials. Therefore, in further studies, it was decided to use low molecular weight peptides of soy hydrolyzate, formed when pepsin acts on soy protein at a concentration of 1-1.5%.

At a concentration of ZnSO _{4 of} 0.05 M and higher, aggregation and precipitation of peptides of soy hydrolyzate occurred. The optimal concentration of ZnSO ₄ was a concentration of 0.01 M, the content of bound zinc at this concentration was 34.7 ± 3.12 μg / ml. With a decrease in concentration of less than 0.01. The amount of bound zinc decreased significantly: at a concentration of ZnSO _{4 of} 0.005 M, the amount of bound zinc was 11.56 ± 1.1 μg / ml.

It was experimentally revealed that at a concentration of 0.002 M the content of bound iodine is most optimal. At a concentration of less than 0.002 M, the content of bound iodine is too low, and at a concentration above, no significant increase in bound iodine was detected.

With the simultaneous introduction of zinc and iodine salts, the salts interacted with each other with the appearance of a precipitate. With the sequential administration of iodine and then zinc, the concentration of bound zinc was 2.85 μg / ml, and with the sequential administration of zinc salts and then iodine, the concentration of zinc was 34.7 μg / ml. This is apparently due to the fact that the formation of chemical bonds with zinc requires more energy. Thus, it was found that, first of all, it is necessary to conduct the interaction of peptides of soy hydrolyzate with zinc salts, and then with iodine salts. The sequential binding of zinc and iodine salts allows you to control the number of associated trace elements, which allows you to apply the obtained additive depending on the factors of consumers: starting from various constituent entities of the Russian Federation with different needs for micronutrients, ending with consumers having restrictions or contraindications in the dose of micronutrients.

In FIG. 1 shows that the immobilization of zinc in a ratio of 1: 1 with the formation of stable bonds with low molecular weight peptides of the hydrolyzate occurs within 2 hours, while the content of bound zinc was 34.7 μg / ml.

The dependence of the binding of iodine ions with soy hydrolyzate in a ratio of 1: 1 depending on the incubation time was studied (Fig. 2). Based on the results of the studies, the exposure time was selected with an aqueous solution of potassium iodide - 24 hours at room temperature. At these treatment parameters, 1 mg of soy protein hydrolyzate bound 1.6 μg / ml of iodine.

It was experimentally revealed that the maximum binding of soy hydrolyzate and salts of trace elements occurs at a ratio of 1: 1.

Thus, the distinguishing features of the proposed method, namely the use of soy protein, its pepsin fermentation, the sequential introduction of zinc and iodine salts into a solution of low molecular weight peptides, provide effective regulation of iodine metabolism and immunodeficiencies, the ability to control salt and iodine concentrations, as well as the cheaper method. The maximum concentration of zinc sulfate is a value of 0.01 M. The maximum concentration of potassium iodide is 0.002 M.

A method of obtaining an agent for the prophylaxis of iodine deficiency conditions and immunodeficiencies involves adding soy protein to a solution of 0.1 M hydrochloric acid, mixing them, and subsequent fermentation at a temperature of 37-38 ° C for 24 hours using the pepsin enzyme. The resulting solution is neutralized with 10% sodium carbonate to a pH of 6.5-7.5. The solution is then centrifuged at 2000 rpm for 20 minutes for the subsequent separation of the supernatant. To the obtained supernatant solution of low molecular weight peptides of soy hydrolyzate with a concentration of 19-20 mg / ml, 0.05 M zinc sulfate was added in the ratio of soybean gyrolyzate: zinc sulfate solution 1: 1, followed by dialysis for 2 hours. After the zinc salts are bound to the peptides of the soy hydrolyzate, 0.002 M potassium iodide is added in the ratio of the hydrolyzate and zinc: potassium iodide solution 1: 1, followed by dialysis for 24 hours.

The possibility of implementing the proposed method is illustrated by the following examples.

Example 1

5 g of soy protein is mixed with 500 ml of hydrochloric acid (0.1 M), 250 mg of pepsin are added and fermented at 37 ° C for 24 hours. The resulting solution with a concentration of 1% protein is neutralized with 10% sodium carbonate to pH 7.0. The solution is then centrifuged at 2000 rpm for 20 minutes for the subsequent separation of the supernatant. In the resulting solution of peptides of soy hydrolyzate with a concentration of 19.47 mg / ml, 0.01 M zinc sulfate is introduced in a ratio of 1: 1, followed by dialysis for 2 hours. After the zinc salts are bound to soy protein peptides, 0.002 M potassium iodide is introduced in a 1: 1 ratio, followed by dialysis for 24 hours. The result is 800 ml of a solution containing in its composition: 19.47 mg / ml protein, 34.7 μg / ml zinc, 1.6 μg / ml iodine.

Example 2

7.5 g of soy protein is mixed with 750 ml of hydrochloric acid (0.1 M), 375 mg of pepsin is added and fermented at 37 ° C for 24 hours. The resulting solution with a concentration of 1.5% protein is neutralized with 10% carbonate sodium to pH 7.0. The solution is then centrifuged at 2000 rpm for 20 minutes for the subsequent separation of the supernatant. In the resulting solution of peptides of soy hydrolyzate with a concentration of 20.61 mg / ml, 0.05 M zinc sulfate was added in a ratio of 1: 1, followed by dialysis for 2 hours. As a result of increasing zinc concentration, soy hydrolyzate precipitates, which affects the quality of the solution and shelf life. After the zinc salts are bound to soy protein peptides, 0.005 M potassium iodide is added, followed by dialysis for 24 hours. The result is 520 ml of a solution containing in its composition: 24.37 mg / ml protein, 41.5 μg / ml zinc, 1.8 μg / ml iodine.

Example 3

2.5 g of soy protein is mixed with 250 ml of hydrochloric acid (0.1 M), 125 mg of pepsin are added and fermented at 37 ° C for 24 hours. The resulting solution, with a protein concentration of 0.5%, is neutralized with 10% sodium carbonate to a pH of 7.0. The solution is then centrifuged at 2000 rpm for 20 minutes for the subsequent separation of the supernatant. In the resulting solution of peptides of soy hydrolyzate with a concentration of 10-11 mg / ml, 0.005 M zinc sulfate is introduced in a ratio of 1: 1, followed by dialysis for 2 hours. After zinc is bound to soy protein peptides, 0.001 M potassium iodide is added in a 1: 1 ratio, followed by dialysis for 24 hours. The result is 1200 ml of a solution containing in its composition: 10.21 mg / ml protein, 11.56 μg / ml zinc, 0.8 μg / ml iodine. The protein content is extremely low with the large volumes of supernatant obtained.

The advantages of the funds obtained by the claimed method were confirmed in experimental models of laboratory animals.

Experimental hypothyroidism was simulated using thyrosol tyrosol control group for 14 days. Then, for 21 days, the test additive was orally administered to the experimental group of animals.

The biological effectiveness of the supplement was evaluated by the level of hormones (thyroxine T4, triiodothyronine T3 and thyrotropic hormone TSH) in the blood serum of animals by enzyme-linked immunosorbent assay. The results of the study are presented in table 2.

* - significantly relative to the intact group

** - significantly relative to the control group

As can be seen from the results obtained, the administration of tyrosol relative to the intact group caused an increase in the TSH hormone by 1.47 times, the level of T3 hormones decreased by 1.19 times, and the level of T4 hormones decreased by 1.46 times, which indicated the occurrence of hypothyroidism in experimental animals .

With the introduction of funds obtained by the present method, there was a restoration of the level of triiodothyronine and thyroid-stimulating hormone in the blood serum of animals against the background of tyrosol hypothyroidism to indicators of the intact group. However, as can be seen from table 2, the level of the hormone T4 was not significantly different from the control, although the increase in the hormone T3 reached the level of intact animals. It is possible that the level of T4 decreased as a result of the synthesis of T3 hormone and could not recover to that in the intact group of animals at a given administered dosage of bound iodine. Based on the data obtained, it can be concluded that the dose of organic forms of trace elements does not cause restoration of the hormonal status of animals by T4 for 21 days of rehabilitation therapy.

Experimental immunodeficiency was simulated using the azathioprine immunosuppressor control group for 7 days. Then, for 14 days, the test additive was orally administered to the experimental group of animals.

The biological effectiveness of the additive was evaluated by the concentration of immunogldobulin in the blood serum of animals by enzyme-linked immunosorbent assay. The results are presented in table 3.

The results of studies of the concentration of serum immunoglobulins in experimental animals showed that the administration of azathioprine in concentration causes a decrease in the production of immunoglobulin A by 86.2%, immunoglobulin M by 98.2%, immunoglobulin G by 82.9%. The administration of the test agent against the background of immunodeficiency caused by the administration of azathioprine caused a sharp increase in the production of antibodies of immunoglobulin A, immunoglobulin G, immunoglobulin M. The level of antibodies exceeded the corresponding indices of intact living animals by 1.7-4 times (immunoglobulin A - 4 times, immunoglobulin M - 2 times, immunoglobulin G - 1.7 times), which indicates the stimulating nature of the action of dietary supplements obtained by the present method.

Claims (3)

1. The method of obtaining funds for the prevention of iodine deficiency and immunodeficiency, involving protein fermentation, the introduction of solutions of zinc sulfate and potassium iodide, characterized in that the protein used is soy protein, which is dissolved in hydrochloric acid, fermentation is carried out using pepsin to obtain soy peptides the hydrolyzate, which is neutralized, centrifuged, in the resulting supernatant solution of low molecular weight peptides add a solution of zinc sulfate in a ratio of 1: 1, followed by dialysis m, after the binding of zinc salts with peptides of soy hydrolyzate add a solution of potassium iodide in a ratio of 1: 1, followed by dialysis. 2. The method according to p. 1, characterized in that they use a solution of zinc sulfate with a concentration of 0.01 M. 3. The method according to p. 1, characterized in that they use a solution of potassium iodide with a concentration of 0.002 M.

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