METHOD FOR OBTAINING CASEIN FROM IODIZED MICELLE

Abstract

Iodized micelle casein is obtained by adding crystalline potassium iodide to an aqueous solution of micelle casein in a ratio of 1:7-1:9; allowing the mixture to stand for 5-10 minutes; adding crystalline potassium persulfate to the mixture at a ratio of 1:8-1:12 to the mass of micelle casein; maintaining the mixture at 18-25 °C and pH 6.5-8.9 for 4-8 hours, during which time the molecular iodine content in the solution is continuously monitored and the iodization reaction is maintained by periodically adding portions of crystalline potassium persulfate at a ratio of 1:12-1:24 to the mass of micelle casein; purifying the resulting aqueous solution from remaining inorganic salts by microfiltration and diafiltration, followed by sterilizing microfiltration and lyophilization or spray drying.The method enables the production of products aimed at preventing iodine deficiency and optimizing iodine metabolism, as well as obtaining iodized micelle casein suitable for use in preventing and protecting against radiation damage.

Description

METHOD FOR OBTAINING CASEIN FROM IODIZED MICELLE TECHNICAL FIELD The invention has implications for the medical and food industries, particularly for conditions related to iodine deficiency in humans and animals. Iodized micelles that can be used in the production of products for prevention and correction of disease. This relates to the method of obtaining casein. STATE OF THE ART Ratio of starting protein feedstock to total protein content of inorganic iodine solution (2-40):1 biological iodization by mixing with an aqueous solution of molecular iodine to maintain a range within the specified range. A method for obtaining an active food supplement is known. In this method, cheese Iodization of water protein mixtures, onto semipermeable membranes or inert carriers. This is carried out with a mixture of enzymes immobilized on it, and the fermentation process, The process involves continuously monitoring the iodine content in the solution. Iodized protein solution is processed using macrofiltration, microfiltration, and ultrafiltration. It is purified from impurities by an ultrafiltration of an aqueous solution of iodized proteins. monitoring diafiltration in the setup; sterilizing the purified iodized protein solution. It undergoes microfiltration, followed by lyophilization to obtain the final powder product (RU). 2212155 C1). Iodization of the starting protein raw material to obtain a biologically active substance. a method for preparing iodized milk whey proteins through this It is known that α-lactalbumin, β-lactoglobulin, and a mixture of these are used as starting protein raw materials. or hydrolyzates of the proteins in question are used. The protein mixture is mixed with aqueous inorganic iodine. fermentation with the solution, a buffered reagent mixture based on lactoperoxidase, 16% by mass. by adding an enzyme mixture containing 24 horseradish peroxidases and 14-21% by mass of catalase The fermentation process is initiated, and the iodine content in the solution is continuously monitored. Aqueous solution of iodized proteins; by combination of macrofiltration and microfiltration and this followed by diafiltration of an aqueous solution of iodized proteins in an ultrafiltration setup. It is purified from impurities. The resulting iodized protein solution is dried by spray drying, thus separating the iodized proteins. It contains 0.5-4% covalently bonded iodine in the form of an iodized amino acid mixture. A final powder product is obtained. The mixture in question contains 55-75% monoiodotyrosine by mass, and... It contains 24.0–43.5% diiodotyrosine and 1.0–1.5% triiodotyrosine by mass (RU 2700444 C1). 1 However, the disadvantage of known methods is that enzymatic treatments are performed with immobilized enzymes. The synthesis process is structurally complex, and this results in a relatively low initial cost. Despite using the raw materials, it increases the cost of the final product. One method is known for the iodization of casein with iodine monochloride under harsh conditions (OOO «NIK MEDBIOFARM», TU 9229-001-48363077-99); as a result of this method, the preparation called «Iodkazein» It is produced. Iodized proteins, known as "Iodcasein" and "Bioiod," are produced using conventional methods. An important aspect of these is... The disadvantage is that more than 4 / 5 of the total iodine in the "Iodkazein" preparation is amino acids. not in the form of iodine derivatives of acids, but inorganic iodine compounds and esters of oxygen-containing acids - Positively charged iodine derivatives exist in the form of -. Milk protein, which is intensely oxidized during hard iodization, is then processed by pancreatic proteases in the intestine. It is significantly less well-degraded compared to the starting casein, and in this preparation... Only about 1 / 15 of the initial iodine amount is absorbed by enterocytes during proteolysis in the intestinal epithelium. Iodized di- and tripeptides (molecular weight 0-500 Da) suitable for absorption by cells It is released in the form of ("OOO Tekhnovita produced Bioiod and OOO Medbiofarm produced Iodkazein"). "Qualitative and quantitative analysis of iodized protein samples." SCIENTIFIC RESEARCH REPORT ON THE STUDY. Non-State Scientific Center, High Technologies Center «KhimRar», Moscow, 2011. (https: / / refdb.ru / look / 2793682-pall.html). A serious disadvantage of these iodized proteins is that they are not completely soluble in water (or only partially soluble). (their solubility). This property ensures that food products contain these microelements. The technological use of iodized proteins for enrichment purposes has significantly increased. It makes things more difficult. In order to prevent iodine deficiency in the diet through the use of iodine-fortified food products, An iodized food that uses diiodotyrosine either in a free state or bound within a protein molecule. The method of obtaining the product is known (RU 2134520 C1). In this invention, diiodotyrosine or natural The bound form in proteins is chemically stable, replacing unstable inorganic iodide compounds. It is considered as a compound. "Biologically active food for preventing iodine deficiency and optimizing iodine metabolism." It is also known as "supplement and food product containing it" (RU 2192150 C1). The advantage of the invention is that it contains iodine. As nutritional components, different classes of chemical compounds contain iodine in a covalently bonded form. These include monoiodotyrosine iodinated at the 3- or 5-positions of the phenolic ring and free monoiodotyrosine. compounds such as diiodotyrosine found in this form or within protein or peptide molecules Its use is recommended. The main characteristic of these compounds is that they meet the thyroid gland's need for thyroid hormone synthesis. They are amenable to enzymatic deiodination, which occurs with the release of 35 iodide. (Iodine-containing) 2 As food components, these compounds are chemically unstable compared to inorganic iodide salts. It has an undeniable advantage. "Prevention of damage caused by iodine radionuclides in the post-prophylaxis period and iodine..." Method of optimizing metabolism of iodine radionuclides in humans or Methods and preparations for preventing damage caused by the disease in animal organisms» (RU 2796757) C1) is known. In these methods, plant or animal compounds containing 4-hydroxy-3,5-diiodophenyl are used. Proteins of the origin are used as a prophylactic preparation twice daily at intervals of 10-14 hours. It is recommended. However, the known methods do not foresee a specific product, only its creation. A list of possible raw materials is presented. Also included are those with the phenolic ring in the 5- and 3- or 3- positions. Another significant disadvantage is the recommendation to use covalently bonded iodine-containing proteins. Especially in "mixtures" (as specified in the patents mentioned above), a wide range of high molecular weight compounds. The composite spectrum is known to be a heterogeneous material. This material is then subjected to radiation. to obtain a standardized product that can be an effective tool in preventing damage It is extremely difficult to standardize. Acid and rennet caseins are dry concentrates in the food or technical grade category, available in granular or fine form. They are produced in the form of dispersed powders. Their separation principle is based on their action on the surface of casein micelles. hydrophilic κ-casein macropeptides that are located and prevent them from approaching each other globally. Colloidal casein micelles resulting from the presence of a "hairy" layer formed from remnants It is based on the disruption of its stability. During precipitation, monomeric denatured molecules are also present. In order, protein molecule complexes are formed, including dimeric, trimeric, and more complex forms, and these This leads to the presence of particles of different sizes in the final product. Obtaining these types of traditional protein components (caseins, caseinates, co-precipitated proteins) Technologies aimed at this, primarily those with high bioavailability and in the human gastrointestinal system It does not ensure the preservation of its native properties, including solubility that allows absorption. In the production of caseins with micelles, pore size allows for the maximum removal of non-protein components. ultrafiltration membranes that retain almost all proteins in milk during the process It is used. By using membranes of different sizes, solubility, oil and moisture retention, Micelles with specific physicochemical properties, such as emulsification, can be obtained from caseins and used in food. A number of technological functions can be performed in their 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). ANNOUNCEMENT OF THE INVENTION The aim of the invention is to create a target-grade material suitable for the production of biologically active supplements and pharmacological preparations. 35. The goal is to develop a method for obtaining iodinated mycelial casein with high product yield and quality. 3 The technical result obtained from the use of the proposed invention is the production of iodinated micelle casein and Based on this, preventing iodine deficiency and optimizing iodine metabolism with radiation. To prevent and protect against damage, long-term therapeutic and prophylactic drugs are used. The goal is to create products whose composition does not change during storage. The stated aim is to analyze pharmacological and veterinary preparations, biologically active supplements, and Obtaining iodinated micelle casein suitable for use in the composition of radioprotective preparations. This was achieved by developing the method. The method involves adding micelles to an aqueous solution of casein in a 1:7-1:9 ratio. Adding crystalline potassium iodide in a specific ratio, letting the mixture stand for 5-10 minutes, and then adding to the mixture... Adding crystalline potassium persulfate to the micelles in a ratio of 1:8-1:12 to the mass of casein, then... The mixture should be kept at a temperature of 18-25 °C and pH 6.5-8.9 for 4-8 hours; the molecular iodine content in the solution... To maintain the iodination reaction while being continuously monitored, the micelles should have a casein mass ratio of 1:12-1:24. periodic addition of portions of crystalline potassium persulfate; the resulting iodized Micelles are separated from the remaining inorganic salts of the aqueous solution of casein by microfiltration and diafiltration. purification and sterilization by microfiltration and lyophilization or spray drying It includes monitoring. The micelles used in the proposed method are casein, which differs from rennet casein in terms of its physicochemical properties. and all protein mixtures of animal and plant origin - egg albumin, milk or whey. Water proteins, hemoglobins, soy protein, yeast proteins, and yeast hydrolysate are superior. The reason is that micelles are homogeneous in size from casein molecules and are approximately 100% It has high resolution. Table 1. Physicochemical properties of some proteins, including casein fraction in micelles (from our own research) (according to the data). The solubility of dry granules in water, Proteins Particle size in dry matter, µm mg per 1000 ml Egg proteins 380±50 80-800 Milk proteins 1000±180 5-90 Yeast proteins 550±120 5-150 Soy proteins 150±80 10-300 Micellar casein 20000±1000 80-90 The high efficacy of iodized micelle casein obtained by the proposed method stems from this. This is because micelles contain approximately 100% casein solubility and conserved native protein. It possesses these characteristics. This situation results in a "soft" solution pH value that preserves the protein's native structure. It allows for "iodization" to be carried out. Preservation of the protein's native structure; selected reagent concentration ranges, reagent portions This is also achieved by adding them in a specific order and in a particular way; thus, highly active substances The effects of excessive concentrations on the protein are avoided. 4 METHODS OF IMPLEMENTING THE INVENTION The requested method is implemented as follows. To a 5-10% aqueous solution of micelle casein, the required amount of crystalline potassium iodide is added. The mixture is stirred and left to stand for 5-10 minutes. Then, the micelles are mixed with casein to achieve a ratio of 1:8-1:12. Crystalline potassium persulfate is added, and the mixture is left to stand for another 4-8 hours. The molecular iodine content in the solution is then measured. It is constantly monitored and crystalline potassium persulfate is used to maintain the iodination reaction. Portions are added periodically. Iodization is performed at a temperature of 18-25 °C, within the pH range of 6.5-8.9, with pH continuously monitored and the required value maintained. This is achieved by preserving it through titration. Micelles containing covalently bonded iodine are used to prepare an aqueous solution of casein, followed by microfiltration of the aqueous solution. It is purified from remaining inorganic salts by diafiltration. The resulting iodized micelles are casein. The solution is subjected to sterilizing microfiltration, followed by lyophilization or spray drying. By processing, a final powder product containing 4-10% covalently bonded iodine is obtained. As a result of applying the method, 4-10% is obtained in the form of iodized amino acid monomers. A product containing specified covalently bonded iodine is obtained. The iodine binds to the tyrosinyl group in the casein structure of the micelles. monomers at the 5- and 3- positions of the phenolic group or only at the 3- position, and also histidinyl The monomers are covalently bonded at the 2-, 2-, and 5- positions, as well as at the 1-, 2-, and 5- positions. Used in the composition of pharmacological and veterinary preparations and biologically active supplements. Experimental studies on the preparation of casein using iodinated micelles by the proposed method, The method has demonstrated high effectiveness. This has been proven through experiments on animals. It has been confirmed. The application of the proposed method is explained with the following practical examples. Example 1 The pre-prepared micelles are added to the casein solution in a crystalline to total protein ratio of 1:7. Iodization was carried out by adding potassium iodide, then the mixture was left for 5 minutes. It has been delayed. Then, crystalline potassium persulfate was added to the mixture at a ratio of 1:8 to the protein mass, and the mixture was... The molecular iodine content in the solution was continuously monitored for 6 hours. 1.5 hours after the start of iodization... half of the initial amount after 1 hour, half of the initial amount after 3 hours, and the initial amount after 5 hours. A portion of crystalline potassium persulfate equal to 1 / 4 of the amount was added. The process is carried out at 18 °C, with a pH range of 6.5-7.2, pH is continuously monitored, and the required value is determined by titration. It was carried out under protection. Iodized micelles are aqueous solutions of casein free from macro and micro impurities, including inorganic iodine; Macrofiltration is applied, followed by microfiltration on 0.1 µm pore size membranes. The aqueous solution was purified by diafiltration in an ultrafiltration system. The resulting iodized micelle casein solution was subjected to lyophilization, revealing the presence of iodized proteins. The final powder contains 8% covalently bonded iodine in the form of an iodized amino acid mixture. The product has been obtained. Iodine is present in micelles at the 5- and 3- positions of the phenolic group of tyrosinyl monomers in the casein structure. only at the 3- position and also at the 2-, 2- and 5- positions of histidinyl monomers and also at the 1-, 2- and They are covalently bonded at positions 5 and 5. Example 2 The pre-prepared micelles are added to the casein solution in a crystalline to total protein ratio of 1:9. Iodization was carried out by adding potassium iodide, then the mixture was left for 5 minutes. It has been delayed. Then, crystalline potassium persulfate was added to the mixture at a ratio of 1:12 to the protein mass, and the mixture was... The molecular iodine content in the solution was continuously monitored for 8 hours. 1.5 hours after the start of iodization... half of the initial amount after 1 / 4 hours, half of the initial amount after 3 hours, initial amount after 4.5 hours 1 / 4 of the initial amount and 1 / 4 of the initial amount of crystalline potassium persulfate after 6.5 hours. Portions have been added. The process is carried out at 25 °C, with a pH range of 7.2-7.9, pH is continuously monitored, and the required value is determined by titration. It was carried out under protection. Iodized micelles are aqueous solutions of casein free from macro and micro impurities, including inorganic iodine; Macrofiltration is applied, followed by microfiltration on 0.1 µm pore size membranes. The aqueous solution was purified by diafiltration in an ultrafiltration system. The resulting iodized micelle casein solution was subjected to lyophilization, revealing the presence of iodized proteins. The final powder contains 7% covalently bonded iodine in the form of an iodized amino acid mixture. The product has been obtained. Iodine is present in micelles at the 5- and 3- positions of the phenolic group of tyrosinyl monomers in the casein structure. only at the 3- position and also at the 2-, 2- and 5- positions of histidinyl monomers and also at the 1-, 2- and They are covalently bonded at positions 5 and 5. Example 3 The pre-prepared micelles are added to the casein solution in a crystalline solution with a total protein ratio of 1:6. Iodization was carried out by adding potassium iodide, then the mixture was left for 5 minutes. It has been delayed. 6 Then, crystalline potassium persulfate was added to the mixture at a ratio of 1:7 to the protein mass, and the mixture was... The molecular iodine content in the solution was continuously monitored for 4 hours. 1 hour after the start of iodization. Then half of the initial amount, half of the initial amount again after 2 hours, and the initial amount again after 3 hours. Half the amount of crystalline potassium persulfate was added. The process is carried out at 25 °C, with a pH range of 8.0-8.9, pH is continuously monitored, and the required value is determined by titration. It was carried out under protection. Iodized micelles are aqueous solutions of casein free from macro and micro impurities, including inorganic iodine; Macrofiltration is applied, followed by microfiltration on 0.1 µm pore size membranes. The aqueous solution was purified by diafiltration in an ultrafiltration system. The resulting iodized micelle casein solution was subjected to lyophilization, revealing the presence of iodized proteins. The final powder contains 8% covalently bonded iodine in the form of an iodized amino acid mixture. The product has been obtained. Iodine is present in micelles at the 5- and 3- positions of the phenolic group of tyrosinyl monomers in the casein structure. only at the 3- position and also at the 2-, 2- and 5- positions of histidinyl monomers and also at the 1-, 2- and They are covalently bonded at positions 5 and 5. Purified iodized micelle casein is used to prevent iodine deficiency and radiation damage. It is used in the production of pharmaceutical and veterinary compounds used for protection against this. Compounds, especially biologically active supplements (BAAs), can help with iodine deficiency and radioactivity. For the production of iodized micelles to prevent contamination, single-component casein is used. both forms and an individualized approach in the prevention of diseases related to iodine metabolism disorders. Combinations with other physiologically active substances are recommended. These are in capsule and tablet form. It can be prepared in powder or solution form, or included in the composition of vitamin-mineral complexes. Iodized micelles, preparations obtained based on casein, have good water solubility and storage capacity. It has the characteristic of remaining unchanged in its covalently bonded iodine content during this process. Table 2. Indicators of iodine content in iodized micelle casein (IMC) during storage. Iodine content, It is given as a percentage of the initial value. Preparation after 2 months, after 3 months, after 4 months, after 5 months, after 6 months Initial iodine content of 8% 100 100 100 100 100 IMK Initial iodine content of 6% 100 100 100 100 100 IMK Initial iodine content of 5% 100 100 100 100 100 IMK 7 To prove the efficacy of the preparation containing iodized micelles casein, MUK 2.3.2.721 "Recommendations for determining the safety and efficacy of biologically active food supplements" Biological experiments were carried out accordingly. The experiment involved administering 200 g to each group, with n=8-10 participants. The study was conducted on male Wistar rats weighing [weight not specified]. Experimental studies of key symptoms, taking into account the pathogenetic mechanisms of hypothyroidism development. A hypothyroidism model was created for the purpose of modeling. For this, iodides were removed from the thyroid gland. accelerates its removal, inhibits peroxidase, and results in the formation of thyroid hormones. Mercazolil, which suppresses tyrosine iodination processes, is administered at a dose of 25 mg / kg per kilogram of animal body weight. It has been used. In the experiment, the concentration of thyroid hormones in the blood serum of the experimental animals was found to be hypothyroid. under these conditions and subsequently with iodized micelle casein preparation and comparison preparation. The iodine concentration was determined after correction with potassium iodide. Iodine concentration was calculated as a percentage of the animal's body weight. This was calculated based on and corresponds to the WHO's recommended dose of 200 µg of iodine for humans. Hormone The level was determined by immunoenzymatic analysis. Chronic administration of mercazolil to rats for 2 weeks resulted in increased efficacy against thyroxine and triiodothyronine. This leads to a twofold decrease in concentration, while thyrotropic hormone levels increase by twofold. has opened (Table 3). Table 3. Indicators of decreased thyroxine and triiodothyronine concentrations. Group T-4, nmol / L T-3, nmol / L TSH, µIU / mL Intact 110.2±10.1 1.5±0.05 0.69±0.02 Mercazolid, 25 mg / kg, 14 54.77±3.8 0.75±0.02 1.41±0.03 day Iodized micelles contain casein, hypothyroidism model 109.98±10.1 1.43±0.4 0.66±0.02 then for 14 days oral 4 µg / kg KI-200, hypothyroidism model. subsequently, for 14 days, 79.4±5.8 1.16±0.03 0.92±0.03 oral 4 µg / kg Note: p <0.01 (error in determining the mean). Hormone levels under the influence of iodized micelle casein preparation; 99.8% for thyroxine, In the intact animal group, the results were 95.5% for triiodothyronine and 95.2% for thyrotropic hormone. The hormone concentration level has been reached. The use of KI-200 preparation for the correction of experimental hypothyroidism, experimental hypothyroidism Compared to the group, it reduced thyrotropic hormone concentration by 35%; thyroxine concentration by 55% and It increased triiodothyronine concentration by 45%. 8 Thus, experimental data show that iodized micelle casein completely eliminates the consequences of hypothyroidism. It shows that it removes and is much more effective when compared to KI. 9

Claims

1. Pharmacological and veterinary preparations, biologically active supplements and radioprotective agents. Method for obtaining iodinated micelle casein suitable for use in the composition of preparations This involves adding crystalline potassium iodide to an aqueous solution of micelle casein in a ratio of 1:7-1:

9. The mixture should be left to stand for 5-10 minutes, with a micelle to casein mass ratio of 1:8-1:

12. Adding crystalline potassium persulfate as described, then maintaining the mixture at a temperature of 18-25 °C and pH 6.5- Allow to stand for 4-8 hours at 8.9°C; iodization while continuously monitoring the molecular iodine content in the solution. To maintain the reaction, the micelles contain crystalline potassium with a mass ratio of 1:12-1:24 to casein. periodic addition of persulfate portions; the resulting iodized micelles are casein. purification of the aqueous solution from remaining inorganic salts by microfiltration and diafiltration, and This is followed by sterilizing microfiltration, lyophilization, or spray drying. method involving.