Process for the preparation of iodinated micellar casein

Abstract

To obtain iodinated micellar casein, crystalline potassium iodide is added to an aqueous micellar casein solution in a ratio of 1 :7 - 1 :9, the mixture is held for 5 to 10 minutes, then crystalline potassium persulfate is added to the micellar casein mass in a ratio of 1 :8 - 1 :12, the mixture is held for 4 to 8 hours at 18 - 25 °C and pH 6.5 - 8.9, while continuously monitoring the molecular iodine content, and periodically topping up with crystalline potassium persulfate to continue the iodination, the ratio of crystalline potassium persulfate to mass of micellar casein being 1 :12 - 1 :24; residual inorganic salts in the resulting aqueous solution are removed by microfiltration and diafiltration, and finally sterilising microfiltration, freeze-drying or spray-drying is performed. This method enables the production of iodinated micellar casein products for iodine deficiency prevention, iodine metabolism optimisation, and radiation damage prevention and protection.

Description

Technical Field

[0001] This invention relates to the pharmaceutical and food industries, and more specifically, to a method for producing iodinated micelle casein for use in manufacturing reagents for the prevention and correction of iodine deficiency in humans and animals. Background Technology

[0002] A method for producing a bioactive food additive by iodizing a starting protein material is known, wherein the material is mixed with an aqueous solution of molecular iodine at a ratio of inorganic iodine solution to total protein content in the range of (2-40):1, and an enzyme mixture immobilized on a semi-permeable membrane or inert carrier is used to iodize the whey protein mixture while continuously monitoring the iodine content in the solution during fermentation. Impurities are removed from the iodized protein solution by percolation in a coarse filtration, microfiltration, and ultrafiltration unit, followed by ultrafiltration. The purified iodized protein solution is then subjected to sterile microfiltration and freeze-drying to obtain the final powder product (RU 2212155 C1).

[0003] A method for producing iodized whey protein is known, which involves iodizing α-lactalbumin, β-lactoglobulin, a mixture of the listed proteins, or their hydrolysates as starting protein materials to obtain a bioactive substance. To initiate fermentation, a buffer reagent mixture and a lactoperoxidase-based enzyme mixture are added to a mixture of protein and an aqueous inorganic iodine solution, the enzyme mixture containing 16 to 24 wt% horseradish peroxidase and 14 to 21 wt% catalase. During fermentation, the iodine content of the solution is continuously monitored. Impurities are removed from the iodized protein aqueous solution by percolation of the iodized protein aqueous solution in a combination of coarse filtration and ultrafiltration, followed by percolation in an ultrafiltration unit. The resulting iodized protein solution was spray-dried to produce a final powder product having a definitively covalently bound iodine content of 0.5 to 4% by weight, in the form of a mixture of iodized amino acids contained in the iodized protein, namely 55 to 75% by weight of monoiodotyrosine, 24.0 to 43.5% by weight of diiodotyrosine and 1.0 to 1.5% by weight of triiodotyrosine (RU 2700444 C1).

[0004] However, known methods have drawbacks, including the use of structurally complex enzymatic synthesis processes involving immobilized enzymes, which, despite using relatively inexpensive raw materials, result in high production costs for the final product.

[0005] One known method is to strongly iodinate casein with iodine monochloride (MEDBIOPHARM, TU 9229-001-48363077-99, a research and production company) to produce a drug called 'iodocasein'.

[0006] Known methods for producing iodinated proteins known as 'iodocasein' and 'biological iodine reagents' have a significant drawback: over four-fifths of the total iodine content in 'iodocasein' drugs is not represented by iodinated amino acids, but rather by inorganic iodine compounds and esters of oxyacids, which are positively charged derivatives of iodine. During vigorous iodination, the highly oxidized milk protein is hydrolyzed by pancreatic proteases at a significantly lower rate than the original casein. Therefore, only about 1 / 15 of the initial iodine content in this drug is released during proteolysis as iodinated dipeptides and tripeptides (molecular weights of 0 Da-500 Da) suitable for assimilation by intestinal absorptive cells (intestinal epithelial cells) ("Qualitative and quantitative analysis of iodinated protein samples of 'Bioiod' produced by Technovita LLC and 'Iodocasein' produced by MEDBIOPHARM LLC", Research Report, High Technology Center for Non-Governmental Science "HimRar", Moscow, 2011. https: / / refdb.ru / look / 2793682-pall.html).

[0007] A significant drawback of these iodinated proteins is that they are completely insoluble in water or have extremely low solubility in water. This characteristic greatly hinders the application of iodinated proteins in food production for the enrichment of this trace element.

[0008] A method for producing iodized food products is known, wherein diiodotyrosine in a free or bound state in a protein molecule is used to prevent iodine deficiency in a diet rich in said product (RU 2134520 C1). The present invention contemplates the use of diiodotyrosine or its bound form in natural proteins as a chemically stable compound serving as a substitute for unstable inorganic iodide compounds.

[0009] 'Bioactive additives for iodine deficiency prevention and iodine metabolism optimization, and food products containing the same' (RU 2192150 C1) are known. The advantage of this invention lies in the use of iodine-containing nutrients, which include various classes of chemical compounds containing iodine in a covalently bound form, including compounds iodinated at the 3 or 5 position of a phenolic ring, such as monoiodotyrosine or diiodotyrosine, either in a free state or as part of a protein or peptide molecule. A key characteristic of these compounds is their susceptibility to enzymatic deiodination in releasing iodides to meet the thyroid's requirements for thyroid hormone synthesis. When used as iodine-containing nutrients, these compounds exhibit significant technological advantages over chemically unstable inorganic iodide salts.

[0010] Known methods for 'preventing damage from iodine radionuclides and optimizing iodine metabolism after the prophylactic period' (RU 2323733 C2) and 'methods and drugs for preventing damage to humans or animals from iodine radionuclides' (RU 2796757 C1) propose using plant- or animal-derived proteins containing a 4-hydroxy-3,5-diiodophenyl compound as prophylactic agents, twice daily at 10–14 hour intervals.

[0011] However, the known methods do not disclose specific products, but only provide a list of potential raw materials for their preparation. Another significant drawback is the suggestion to use proteins containing iodine covalently bound at the 5 and 3 positions of the phenol ring. It is well known that a wide range of high molecular weight compounds (especially when contained in 'mixtures' as disclosed in the aforementioned patents) constitute heterogeneous materials, which are very difficult to standardize for the subsequent manufacture of standardized products capable of serving as effective agents against radiation damage.

[0012] Acidic and rennet casein are food-grade or industrial-grade dry concentrates available as granules or finely dispersed powders.

[0013] Their separation mechanism is based on disrupting the colloidal stability of casein micelles, a stability maintained by the 'hair' of surface hydrophilic κ-casein macropeptide residues that prevents micelle aggregation into spheres. During precipitation, monomeric denatured molecules are generated along with protein complexes, including dimers, trimers, and higher-order forms, ultimately resulting in variable particle sizes of the final product. Such traditional protein components (casein, casein salts, coprecipitates) cannot retain their native properties, most notably solubility, which determines their high bioavailability and absorbability in the human gastrointestinal tract.

[0014] Micellar casein production utilizes ultrafiltration membranes with pore sizes that ensure near-complete retention of milk proteins while maximizing the removal of non-protein components. Different membrane sizes can produce micellar casein with specific physicochemical properties, such as solubility, fat and water retention, emulsifying properties, and enable a variety of food system technology functions (Kumar P, Sharma N, Ranjan R, Kumar S, Bhat ZF, Jeong DK. Prospects for membrane technology in the dairy industry: a review. Asia-Oceania Journal of Animal Science. 2013;26(9): 1347–1358. https: / / doi.org / 10.5713 / ajas.2013.13082). Summary of the Invention

[0015] The objective of this invention is to develop a method for preparing iodinated micellar casein with high yield and high quality, the product of which is suitable for the production of bioactive additives and pharmacological preparations. The technical outcome of this invention is the acquisition of iodinated micellar casein and its derivatives for iodine deficiency prevention, iodine metabolism optimization, and radiation damage prevention and protection, exhibiting therapeutic-preventive efficacy and consistent composition throughout the shelf life.

[0016] This problem is addressed by a method for producing iodinated micellar casein suitable for pharmaceuticals and veterinary drugs, bioactive additives, and radiation protection drugs. The method comprises: adding crystalline potassium iodide to an aqueous micellar casein solution at a ratio of 1:7 to 1:9; maintaining the mixture for 5 to 10 minutes; adding crystalline potassium persulfate to the micellar casein material at a ratio of 1:8 to 1:12; maintaining the mixture at 18 to 25°C and pH 6.5 to 8.9 for 4 to 8 hours, while continuously monitoring the molecular iodine content and periodically adding crystalline potassium persulfate fractions to the micellar casein material at a ratio of 1:12 to 1:24 to the micellar casein mass for further iodization; purifying the iodinated micellar casein aqueous solution by microfiltration and percolation; followed by sterile microfiltration; and then freeze-drying or spray-drying.

[0017] Micellar casein used in the methods of this invention exhibits superior physicochemical properties compared to rennet casein and all animal-plant protein mixtures, including ovalbumin, milk or whey protein, hemoglobin, soy protein, and yeast protein and yeast hydrolysates. This is because micellar casein molecules exhibit a uniform size and near 100% solubility.

[0018] Table 1. Physicochemical properties of some proteins, including the micellar casein moiety (based on the inventors' research). ovalbumin 380±50 80-800 milk protein 1,000±180 5-90 yeast protein 550±120 5-150 Soy protein 150±80 10-300 micellar casein 20,000±1,000 80-90 This is also why the iodinated micellar casein obtained by the method of this invention is highly efficient, because micellar casein exhibits near 100% solubility and retains the characteristics of the native protein, thus enabling "mild iodization" under solution pH conditions that maintain the native protein structure. The selected reagent concentration range, the method of addition in stages, and the order of addition also ensure the preservation of the native protein structure, avoiding the influence of extreme, highly reactive substance concentrations on the protein. Detailed Implementation

[0019] The method is implemented as follows.

[0020] Add the required amount of crystalline potassium iodide to a 5-10% aqueous solution of micellar casein and stir the mixture for 5-10 minutes. Next, add crystalline potassium persulfate to the micellar casein at a mass ratio of 1:8 to 1:12, and maintain the mixture for another 4-8 hours. Continuously monitor the molecular iodine content of the solution, periodically adding crystalline potassium persulfate to maintain iodization. During iodization, maintain a temperature of 18-25°C while continuously monitoring and adjusting the pH by titration (6.5-8.9).

[0021] Residual inorganic salts were removed from the resulting aqueous solution of iodinated micelle casein by microfiltration followed by percolation. The resulting iodinated micelle casein solution was then sterilized by microfiltration, followed by freeze-drying or spray-drying. The resulting powder product contained 4 to 10% covalently bound iodine.

[0022] This method produces a product containing 4 to 10% definitively covalently bound iodine in the form of iodinated amino acid monomers. The iodine is covalently bound at positions 5 and 3 of the phenolic ring of the micelle-casein tyrosine monomer, or only at position 3, and at positions 2, 2, and 5, or positions 1, 2, and 5, of the micelle-casein histidine monomer.

[0023] Experimental studies on the method for producing iodinated micelle casein of this invention, used in pharmacological preparations, veterinary preparations, and bioactive additives, have demonstrated its high efficacy. Animal experiments have confirmed this.

[0024] The following examples illustrate the implementation of the method of the present invention: Example 1

[0025] The pre-prepared micellar casein solution was subjected to iodination: crystalline potassium iodide was added to the total protein at a ratio of 1:7, and the mixture was held for 5 minutes. Next, crystalline potassium persulfate was added to the protein mixture at a ratio of 1:8, and the mixture was held for 6 hours under continuous monitoring of the molecular iodine content in the solution. Additional crystalline potassium persulfate was added at 1.5 hours, 3 hours, and 5 hours after the start of iodination, at amounts of 1 / 2, 1 / 2, and 1 / 4 of the initial amount, respectively. Throughout this process, the temperature was maintained at 18°C, and the pH was continuously monitored and titrated to adjust to 6.5–7.2.

[0026] Macromolecules and trace impurities, including inorganic iodine, are removed from the resulting solution by microfiltration, followed by percolation in an ultrafiltration unit equipped with a 0.1 µm microfiltration membrane. The resulting solution is freeze-dried to produce a finished powder product containing 8% definitively covalently bound iodine, which is a mixture of protein-bound iodinated amino acids. Iodine is covalently bound at positions 5 and 3, or only at position 3, of the phenolic ring of the micelle-casein tyrosine monomer, and at positions 2, 2, and 5, or positions 1, 2, and 5, of the micelle-casein histidine monomer. Example 2

[0027] The pre-prepared micellar casein solution was iodized by adding crystalline potassium iodide to the total protein at a ratio of 1:9, and the mixture was held for 5 minutes. Next, crystalline potassium persulfate was added to the protein material at a ratio of 1:12, and the mixture was held for 8 hours under continuous monitoring of the molecular iodine content in the solution. Additional crystalline potassium persulfate fractions were added at 1.5, 3, 4.5, and 6.5 hours after the start of iodization, at amounts of 1 / 2, 1 / 2, 1 / 4, and 1 / 4 of the initial amount, respectively. Throughout this process, the temperature was maintained at 25°C, and the pH was continuously monitored and titrated to adjust to 7.2–7.9.

[0028] Macromolecules and trace impurities, including inorganic iodine, are removed from the resulting solution by microfiltration, followed by percolation in an ultrafiltration unit equipped with a 0.1 µm microfiltration membrane. The resulting solution is freeze-dried to produce a finished powder product containing 7% definitively covalently bound iodine, which is a mixture of protein-bound iodinated amino acids. Iodine is covalently bound at positions 5 and 3, or only at position 3, of the phenolic ring of the micelle-casein tyrosine monomer, and at positions 2, 2, and 5, or positions 1, 2, and 5, of the micelle-casein histidine monomer. Example 3

[0029] The pre-prepared micellar casein solution was iodized by adding crystalline potassium iodide to the total protein at a ratio of 1:6, and the mixture was held for 5 minutes. Next, crystalline potassium persulfate was added to the protein material at a ratio of 1:7, and the mixture was held for 4 hours under continuous monitoring of the molecular iodine content in the solution. Additional crystalline potassium persulfate fractions were added at 1, 2, and 3 hours after the start of iodization, at half the initial amount. Throughout this process, the temperature was maintained at 25°C, and the pH was continuously monitored and titrated to adjust to 8.0–8.9.

[0030] Macromolecules and trace impurities, including inorganic iodine, are removed from the resulting solution by microfiltration, followed by percolation in an ultrafiltration unit equipped with a 0.1 µm microfiltration membrane. The resulting solution is freeze-dried to produce a finished powder product containing 8% definitively covalently bound iodine, which is a mixture of protein-bound iodinated amino acids. Iodine is covalently bound at positions 5 and 3, or only at position 3, of the phenolic ring of the micelle-casein tyrosine monomer, and at positions 2, 2, and 5, or positions 1, 2, and 5, of the micelle-casein histidine monomer.

[0031] Purified iodinated micellar casein is used to manufacture pharmaceutical and veterinary drug compositions designed to prevent iodine deficiency and to prevent and protect against radiation damage.

[0032] To manufacture these compositions, particularly bioactive additives for iodine deficiency prevention and radiation protection, iodinated micellar casein monoforms and combinations thereof with other physiologically active substances are provided to enable personalized iodine metabolism disease prevention formulated as components of capsules, tablets, powders, solutions, or vitamin-mineral complexes.

[0033] The resulting drug based on iodinated micelle casein exhibits high water solubility and stable iodine content covalently bound to its shelf life.

[0034] Table 2. Indicators of iodine content in iodinated micellar casein (IMC) during storage

[0035] To demonstrate the efficacy of the drug, biological experiments were conducted in accordance with the guideline "MUK 2.3.2.721 - Determination of safety and efficacy of bioactive food additives". Male Wistar rats (200 g weight, n = 8-10 rats / group) were used in the experiment.

[0036] To experimentally simulate its main symptoms, a hypothyroidism model was established based on the pathogenesis of hypothyroidism. For this purpose, a specific dose of methimazole (25 mg / kg) was administered to accelerate thyroid iodine elimination, inhibit peroxidase, and block tyrosine iodination during thyroid hormone synthesis.

[0037] Thyroid hormone concentrations in the serum of experimental animals were measured under induced hypothyroidism conditions and then corrected using the iodinated micelle casein drug and potassium iodide reference drug of this invention. The iodine dose per animal body weight was calculated, corresponding to 200 µg of the WHO recommended human dose. Enzyme-linked immunosorbent assay (ELISA) was performed to determine hormone levels.

[0038] Administering methimazole to rats for two consecutive weeks halved the concentrations of thyroxine and triiodothyronine, while doubling the level of thyroid-stimulating hormone (TSH) (see Table 3).

[0039] Table 3. Indicators of decreased thyroxine and triiodothyronine concentrations

[0040] Note: p<0.01 (mean measurement error).

[0041] When exposed to iodinated micelle casein, the concentrations of thyroxine, triiodothyronine, and thyroid-stimulating hormone reached 99.8%, 95.5%, and 95.2% of the levels in the intact animal control group, respectively.

[0042] Compared with the hypothyroidism group, administration of KI-200 for experimental hypothyroidism correction reduced thyroid-stimulating hormone concentration by 35%, while increasing thyroxine and triiodothyronine concentrations by 55% and 45%, respectively.

[0043] Therefore, experimental data show that iodinated micelle casein completely eliminated the effects of hypothyroidism and exhibited significantly higher efficacy than KI.

Claims

1. A method for producing iodinated micellar casein suitable for use in pharmacological and veterinary drug formulations, bioactive additives, and radiation protection agents, comprising: Add crystalline potassium iodide to the micelle casein aqueous solution at a ratio of 1:7 to 1:9 and keep the mixture for 5 to 10 minutes. Crystalline potassium persulfate was added to the micellar casein material at a ratio of 1:8 to 1:

12. The mixture was maintained at 18 to 25°C and pH 6.5 to 8.9 for 4 to 8 hours, while the molecular iodine content was continuously monitored. Crystalline potassium persulfate was periodically added, with the ratio of crystalline potassium persulfate to micellar casein mass being 1:12–1:24, to continue the iodination reaction. The residual inorganic salts in the resulting iodinated micellar casein aqueous solution were purified and removed by microfiltration and percolation, followed by sterile microfiltration, and then freeze-drying or spray drying.

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