Method for producing iodized micellar casein

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] NAME OF INVENTION

[0002] METHOD FOR OBTAINING IODINATED MICELLAR CASEIN

[0003] AREA OF TECHNOLOGY

[0004] The invention relates to the medical and food industries, namely to a method for producing iodinated micellar casein, which can be used to produce agents for the prevention and correction of iodine deficiency conditions in humans and animals.

[0005] PRIOR ART

[0006] A method is known for producing a biologically active food supplement by iodizing the original protein raw material by mixing it with an aqueous solution of molecular iodine at a ratio of the inorganic iodine solution to the total protein content in the range (2-40): 1, wherein the iodination of the whey protein mixture is carried out with a mixture of enzymes immobilized on semipermeable membranes or on inert carriers, and the fermentation process is carried out with continuous monitoring of the iodine content in the solution. The solution of iodized proteins is purified from impurities using macrofiltration, microfiltration and ultrafiltration, followed by diafiltration of the aqueous solution of iodized proteins in an ultrafiltration unit, the purified solution of iodized proteins is subjected to sterilizing microfiltration, then freeze-drying to obtain a finished powder product. (RU 2212155 C1).

[0007] A method is known for producing iodinated whey proteins to obtain a biologically active substance by iodizing the original protein raw material, which is a-lactalbumin, β-lactoglobulin, or a mixture of the listed proteins, or hydrolysates of the listed proteins. Fermentation of the protein mixture with an aqueous solution of inorganic iodine is initiated by introducing a buffer mixture of reagents and a mixture of enzymes based on lactoperoxidase, containing from 16 to 24 wt. %, horseradish peroxidase and from 14 to 21 wt. % catalase. 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 impurities by a combination of macrofiltration, microfiltration and subsequent diafiltration of the aqueous solution of iodinated proteins in an ultrafiltration unit.The resulting solution of iodinated proteins is spray dried to obtain a finished powder product containing 0.5-4% deterministic covalently bound iodine in the form of a mixture of iodinated amino acids contained in iodinated proteins - monoiodotyrosines in the amount of 55-75 wt.%, 24.0-43.5 wt.% diiodotyrosines and with 1.0-1.5 wt.% triiodotyrosines (RU 2700444 C1).

[0008] However, the known methods have disadvantages in the form of a structurally complex process of enzymatic synthesis by immobilized enzymes, which leads to a high cost of the final product when using relatively inexpensive raw materials.

[0009] A harsh method of iodination of casein with iodine monochloride is known (OOO NIK MEDBIOPHARM, TU 9229-001-48363077-99), which results in the production of a drug called "Iodcasein".

[0010] Known methods produce iodinated proteins "Iodcasein" and "Bioiodine", a significant drawback of which is the following: more than 4 / 5 of the total amount of iodine present in the preparation "Iodcasein" is not represented by iodine derivatives of amino acids, but by inorganic iodine compounds and esters of oxygen-containing acids - derivatives of positively charged iodine. Milk protein, heavily oxidized during hard iodination, is broken down by pancreatic proteases of the intestine noticeably worse than the original casein, and only about 1 / 15 of the original amount of iodine in this preparation is released during proteolysis in the form of iodinated di- and tripeptides (with m.v. 0-500 Da), suitable for absorption by enterocytes - intestinal epithelial cells ("Conducting qualitative and quantitative analyses of samples of iodinated proteins "Bioiod" produced by Technovita LLC and "Iodcasein" produced by Med Biofar M LLC". RESEARCH REPORT.NON-GOVERNMENTAL RESEARCH CENTER Center for High Technologies "ChemRar", M., 2011. https: / / refdb.ru / look / 2793682-pall.html).

[0011] A serious drawback of these iodized proteins is their complete insolubility (or extremely low solubility) in water: this feature greatly complicates the technological use of iodized proteins for the purpose of enriching food products with this microelement. A method for obtaining an iodized food product is known, which uses diiodotyrosine in a free or bound state in the protein molecule for the prevention of iodine deficiency in nutrition with food products enriched with it (RU 2134520 C1). The invention considers diiodotyrosine or its bound form in the composition of natural proteins as a chemically stable compound replacing unstable inorganic iodide compounds.

[0012] Known is "Biologically active food supplement for the prevention of iodine deficiency and optimization of iodine metabolism, and a food product containing it" (RU 2192150 C1). The advantage of the invention lies in the proposal to use chemical compounds of different classes containing iodine in a covalently bound form as iodine-containing nutrients, including compounds iodinated at the 3- or 5-positions of the phenolic cycle, such as monoiodotyrosine, diiotyrosine in a free state, or as part of protein, peptide molecules. The main feature of these compounds is their availability for enzymatic deiodination with the release of iodide, intended to meet the needs of the thyroid gland in thyroid hormone synthesis. As iodine-containing nutrients, these compounds have an absolute advantage over chemically unstable inorganic iodide salts.

[0013] The “Method for preventing damage by iodine radionuclides and optimizing iodine metabolism in the post-prophylactic period” (RU 2323733 C2) and “Method and preparation for preventing damage by iodine radionuclides to the human or animal body” (RU 2796757 C1) are known, which propose the use of proteins of plant or animal origin containing 4-hydroxy, 3-, 5- diiodophenyl compound as prophylactic preparations twice a day with a 10-14 hour interval.

[0014] However, the known methods do not assume a specific product and only provide a list of possible raw materials for its creation. Another significant drawback is the proposal to use proteins containing covalently bound iodine in the 5- and 3- or 3- position of the phenolic cycle. It is known that a wide range of high-molecular compounds, especially in the composition of "mixtures" (as indicated in the above-mentioned patents), is a heterogeneous material that is extremely difficult to standardize for the subsequent production of a standardized product capable of acting as an effective means of preventing radiation damage.

[0015] Caseins, acid and rennet, are dry concentrates of food or technical quality, produced in the form of granulated or finely dispersed powders.

[0016] The principle of their isolation is based on the violation of the colloidal stability of casein micelles, caused by the presence of a "hair" layer of hydrophilic macropeptide residues of κ-casein on the surface of the micelles, preventing their spherical convergence. In the process of precipitation, in addition to monomeric denatured molecules, complexes of protein molecules are formed, including dimeric, trimeric and more complex forms, which ultimately causes the presence of particles of different sizes in the final product. Technologies for obtaining such traditional protein ingredients (caseins, caseinates, co-precipitates) do not ensure the preservation of their native properties, the main one of which is solubility, which determines high bioavailability and digestibility in the human gastrointestinal tract.

[0017] In obtaining micellar caseins, ultrafiltration membranes are used, the pore size of which ensures the retention of almost all milk proteins, with the maximum removal of non-protein components. Using membranes of different sizes, it is possible to obtain micellar caseins with specified physicochemical properties, such as solubility, fat and moisture retention, emulsification, and also perform a number of technological functions in food systems (Kumar P, Sharma N, Ranjan R, Kumar S, Bhat ZF, Jeong DK. Perspective of membrane technology in dairy industry: A review. Asian- Australasian Journal of Animal Science. 2013; 26 (9): 1347-1358. https: / / doi.org / 10.5713 / ajas.2013.13082).

[0018] DISCLOSURE OF INVENTION

[0019] The objective of the invention is to develop a method for producing iodinated micellar casein with a high yield and quality of the target product, suitable for the production of biologically active additives and pharmacological preparations.

[0020] The technical result of using the proposed invention consists in obtaining iodinated micellar casein and creating products based on it for the prevention of iodine deficiency and optimization of iodine metabolism and for the prevention and protection against radiation damage, which have a therapeutic and prophylactic effect and an unchanged composition during long-term storage.

[0021] The problem was solved by creating a method for obtaining iodinated micellar casein suitable for use in the compositions of pharmacological and veterinary drugs, biologically active additives and radioprotective drugs, including adding crystalline potassium iodide to an aqueous solution of micellar casein at a ratio of 1:7-1:9, holding the mixture for 5-10 minutes, adding crystalline potassium persulfate to the mixture at a ratio of 1:8-1:12 to the weight of micellar casein, then holding the mixture for 4-8 hours at a temperature of 18-25 °C and a pH of 6.5-8.9 with continuous monitoring of the molecular iodine content in the solution and periodic addition of portions of crystalline potassium persulfate at a ratio of 1:12-1 to the weight of micellar casein. :24 to continue the iodination reaction, purification of the resulting aqueous solution of iodinated micellar casein from the remaining inorganic salts by microfiltration and diafiltration,followed by sterilizing microfiltration, freeze-drying or spray drying.

[0022] The micellar casein used in the proposed method is superior in its physicochemical properties to rennet casein and all types of mixtures of animal and vegetable proteins - egg albumin, milk or whey proteins, hemoglobins, soy protein, yeast proteins and yeast hydrolysate. This is due to the fact that micellar casein molecules are uniform in size and have a solubility close to 100%.

[0023] Table 1. Some physicochemical properties of proteins, including the micellar casein fraction (according to our own research).

[0024] This also determines the high efficiency of iodinated micellar casein obtained by the proposed method, since micellar casein has a solubility close to 100% and preserved native properties of the protein, which provides the possibility of carrying out "soft iodination" at pH values ​​of the solution that preserve the native structure of the protein. Preservation of the native structure of the protein is also ensured by the selected ranges of reagent concentrations, fractionality and order of their introduction, which allows avoiding the impact of extreme concentrations of highly active substances on the protein.

[0025] EMBODIMENTS OF THE INVENTION

[0026] The claimed method is carried out as follows.

[0027] The required amount of crystalline potassium iodide is added to the aqueous 5-10% solution of micellar casein, mixed and the mixture is held for 5-10 minutes. Then crystalline potassium persulfate is added in a ratio of 1:8-1:12 to micellar casein and the mixture is held for another 4-8 hours. The content of molecular iodine in the solution is continuously monitored and portions of crystalline potassium persulfate are periodically added to continue the iodination reaction. Iodination is carried out at a temperature of 18-25 °C, in the pH range of 6.5-8.9 with constant pH monitoring and maintaining the required value by titration.

[0028] An aqueous solution of micellar casein with covalently bound iodine is purified from the remaining inorganic salts using microfiltration followed by diafiltration of the aqueous solution. The resulting solution of iodinated micellar casein is subjected to sterilizing microfiltration, then freeze-drying or spray drying to obtain a finished powder product with a covalently bound iodine content of 4-10%.

[0029] As a result of the method implementation, a product is obtained with a content of deterministic covalently bound iodine in an amount of 4-10% in the form of monomers of iodinated amino acids. Iodine is covalently bound in the 5- and 3- or only in the 3- position of the phenolic group of tyrosinyl monomers included in the composition of micellar casein, as well as in the 2-, 2- and 5-, as well as 1-, 2- and 5- positions of histidinyl monomers included in the composition of micellar casein.

[0030] Experimental studies of the proposed method for producing iodinated micellar casein for use in pharmacological and veterinary preparations, as well as biologically active additives, have shown its high efficiency. This was confirmed by experiments conducted on animals. The implementation of the proposed method is illustrated by the following practical examples:

[0031] Example 1.

[0032] The iodination process was performed on a pre-prepared micellar casein solution by adding crystalline potassium iodide to it at a ratio of 1:7 to the total protein, then holding the mixture for 5 minutes. Next, crystalline potassium persulfate was added at a ratio of 1:8 to the protein mass and the mixture was held for 6 hours with continuous monitoring of the molecular iodine content in the solution, adding portions of crystalline potassium persulfate 1.5 hours after the start of iodination in an amount of 1 / 2 of the initial, 3 hours after the start of iodination in an amount of 1 / 2 of the initial, 5 hours after the start of iodination in an amount of 1 / 4 of the initial. The process was carried out at a temperature of 18 °C, in the pH range of 6.5-7.2 with constant pH monitoring and maintaining the required value by titration.

[0033] An aqueous solution of iodinated micellar casein was purified from macro and micro impurities, including inorganic iodine, using macrofiltration followed by diafiltration of the aqueous solution in an ultrafiltration unit on microfiltration membranes with a pore size of 0.1 μm. The resulting solution of iodinated micellar casein was subjected to freeze drying to obtain a finished powder product containing a determined amount of covalently bound iodine in the amount of 8% in the form of a mixture of iodinated amino acids contained in iodinated proteins. Iodine is covalently bound in the 5- and 3- or only in the 3-position of the phenolic group of tyrosinyl monomers, which is part of micellar casein, as well as in the 2-, 2- and 5-, as well as 1-, 2- and 5-positions of the histidinyl monomers, which is part of micellar casein.

[0034] Example 2.

[0035] The iodination process was performed on a pre-prepared micellar casein solution by adding crystalline potassium iodide to it at a ratio of 1:9 to the total protein, then the mixture was held for 5 minutes. Next, crystalline potassium persulfate was added at a ratio of 1:12 to the protein mass and the mixture was held for 8 hours with continuous monitoring of the molecular iodine content in the solution, adding portions of crystalline potassium persulfate 1.5 hours after the start of iodination in an amount of 1 / 2 of the initial, 3 hours after the start of iodination in an amount of 1 / 2 of the initial, 4.5 hours after the start of iodination in an amount of 1 / 4 of the initial and 6.5 hours after the start of iodination in an amount of 1 / 4. The process was carried out at a temperature of 25 °C, in the pH range of 7.2-7.9 with constant pH monitoring and maintaining the required value by titration.

[0036] An aqueous solution of iodinated micellar casein was purified from macro and micro impurities, including inorganic iodine, using macrofiltration followed by diafiltration of the aqueous solution in an ultrafiltration unit on microfiltration membranes with a pore size of 0.1 μm. The resulting solution of iodinated micellar casein was subjected to freeze drying to obtain a finished powder product containing a determined amount of covalently bound iodine in the amount of 7% in the form of a mixture of iodinated amino acids contained in iodinated proteins. Iodine is covalently bound in the 5- and 3- or only in the 3-position of the phenolic group of tyrosinyl monomers, which is part of micellar casein, as well as in the 2-, 2- and 5-, as well as 1-, 2- and 5-positions of the histidinyl monomers, which is part of micellar casein.

[0037] Example 3.

[0038] The iodination process was performed on a pre-prepared micellar casein solution by adding crystalline potassium iodide to it at a ratio of 1:6 to the total protein, then the mixture was held for 5 minutes. Crystalline potassium persulfate was then added at a ratio of 1:7 to the protein mass, and the mixture was held for 4 hours with continuous monitoring of the molecular iodine content in the solution, adding portions of crystalline potassium persulfate 1 hour after the start of iodination in an amount of 1 / 2 of the initial, 2 hours after the start of iodination in an amount of 1 / 2 of the initial, 3 hours after the start of iodination in an amount of 1 / 2. The process was carried out at a temperature of 25 °C, in the pH range of 8.0-8.9 with constant pH monitoring and maintaining the required value by titration.

[0039] An aqueous solution of iodinated micellar casein was purified from macro and micro impurities, including inorganic iodine, using macrofiltration followed by diafiltration of the aqueous solution in an ultrafiltration unit on microfiltration membranes with a pore size of 0.1 μm. The resulting solution of iodinated micellar casein was subjected to freeze drying to obtain a finished powder product containing a determined amount of covalently bound iodine in the amount of 8% in the form of a mixture of iodinated amino acids contained in iodinated proteins. Iodine is covalently bound in the 5- and 3- or only in the 3-position of the phenolic group of tyrosinyl monomers, which is part of micellar casein, as well as in the 2-, 2- and 5-, as well as 1-, 2- and 5-positions of the histidinyl monomers, which is part of micellar casein.

[0040] Purified iodinated micellar casein is used to produce pharmaceutical and veterinary compositions used in cases of iodine deficiency prevention and prevention and protection against radiation damage.

[0041] For the production of compositions, in particular biologically active additives (BAA), for the prevention of iodine deficiency and the prevention of radioactive contamination, both monoforms of iodinated micellar casein and its combinations with other physiologically active substances are offered, providing an individual approach to the prevention of diseases associated with iodine metabolism disorders, made in the form of capsules, tablets, powders, solutions, or included in vitamin-mineral complexes.

[0042] Preparations obtained on the basis of iodinated micellar casein have good water solubility and an unchanged content of covalently bound iodine during storage.

[0043] Table 2. Iodine content in iodinated micellar casein (IMC) during storage.

[0044] To prove the effectiveness of preparations with iodinated micellar casein, biological experiments were conducted in accordance with the recommendations of MUK 2.3,2.721 "Determination of the safety and effectiveness of biologically active food supplements". The experiment was conducted on male Wistar rats weighing 200 g, n = 8-10 in each group.

[0045] Taking into account the pathogenetic mechanisms of hypothyroidism development, a hypothyroidism model was created for experimental modeling of its main symptoms. For this purpose, the drug mercazolil was used, which accelerates the removal of iodides from the thyroid gland, inhibits peroxidase and suppresses the processes of tyrosine iodination with the formation of thyroid hormones, at a dosage of 25 mg / kg of animal weight.

[0046] In the experiment, the concentration of thyroid hormones in the blood serum of experimental animals was determined against the background of hypothyroidism and with subsequent correction with a preparation of iodinated micellar casein and a comparison preparation - potassium iodide. The concentration of iodine was calculated for the animal's weight, and it corresponded to the dosage recommended by WHO for humans - 200 mcg of iodine. The level of hormones was determined by the enzyme immunoassay method.

[0047] Chronic administration of mercazolil to rats for 2 weeks caused a 2-fold decrease in the concentration of thyroxine and triiodothyronine, while the level of thyroid-stimulating hormone increased 2-fold (Table 3).

[0048] Table 3. Indicators of decrease in thyroxine and triiodothyronine concentrations.

[0049] Note: p < 0.01 (mean error).

[0050] When exposed to the iodinated micellar casein preparation, the hormone level was 99.8% of thyroxine, 95.5% of triiodothyronine, and 95.2% of thyroid-stimulating hormone, compared to the level of hormone concentration in the intact group of animals.

[0051] The use of KI-200 for the correction of experimental hypothyroidism reduced the concentration of thyroid-stimulating hormone by 35% and increased the concentrations of thyroxine by 55% and triiodothyronine by 45% compared to the experimental hypothyroidism group.

[0052] Thus, experimental data show that iodinated micellar casein completely eliminates the effects of hypothyroidism and is much more effective than KI.

Claims

Invention formula A method for producing iodinated micellar casein suitable for use in the formulations of pharmacological and veterinary drugs, biologically active additives and radioprotective drugs, comprising adding crystalline potassium iodide to an aqueous solution of micellar casein at a ratio of 1:7-1:9, holding the mixture for 5-10 minutes, adding crystalline potassium persulfate to the mixture at a ratio to the weight of micellar casein of 1:8-1:12, then holding the mixture for 4-8 hours at a temperature of 18-25°C and a pH of 6.5-8.9 with continuous monitoring of the content of molecular iodine in the solution and periodically adding portions of crystalline potassium persulfate at a ratio to the weight of micellar casein of 1:12-1:24 to continue the reaction iodination, purification of the resulting aqueous solution of iodinated micellar casein from the remaining inorganic salts by microfiltration and diafiltration followed by sterilizing microfiltration,sublimation or spray drying,