RU2318406C1 - Method for production of biologically active supplement (bas) from law molecular milk cation proteins and obtained bas - Google Patents

Abstract

FIELD: biotechnology, medicine industry, pharmaceutical industry, cosmetic.

SUBSTANCE: claimed BAS is obtained by purification of milk raw materials by centrifugation, protein sorption on ion exchanger, chromatography separation thereof, eluation, determination of fraction with optical density more than 0.1 units at 280 nm, dialysis, stabilization, and pre-packing. As raw materials milk-originated native cation protein substances are used. Chromatography separation is carried out CM-cellulose, and dialysis is carried out against distilled water or phosphate buffer. Stabilization and microbial purification are carried out by microfiltration trough semipermeable membrane with selectivity of 0.22-0.45 mum. Obtained BAS is pre-packed, frozen to -20-25°C and freeze dried at temperature of -20±4°C. BAS contains fraction of milk-originated native cation protein fraction enriched with lisosime, angiogenine, pancreatic RNAase, lactoferrine and lactoperoxidase.

EFFECT: BAS with anti-microbial, antioxidant, immunomodulatory, anti-stress properties.

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Description

The invention relates to the dairy, biotechnological, medical, pharmaceutical, cosmetic industries, and in particular to methods for producing biologically active substances that can be used as biologically active additives (BAA).

Known methods of processing milk and obtaining protein polysaccharide concentrate, which consist in the fact that in order to increase the biological value and create a waste-free technology, skim milk in an amount of 5-100% with an acidity of 15-23T is treated at 2-40 ° C with a polysaccharide with a molecular weight of 20,000 -80000 to the separation into the casein and polysaccharide phase [Patent No. 1725803 (Russia). A method of processing milk. / Molochnikov VV, Tolstoguzov VB]. And get a natural casein concentrate (KNK) containing about 97% casein and a caseless fraction containing all the water-soluble part of the milk and the polysaccharide. Subsequently, the caseless phase is thickened, thereby obtaining a food structuring concentrate (PCB) [Patent No. 181876 (Russia). A method of obtaining a protein polysaccharide concentrate. / Molochnikov V.V., Shchanov V.Yu., Anisimov S.V., Khramtsov A.A.]. KSP is recommended for use as an additive to dairy and confectionery products as a structure-forming component, as well as to regulate the content of soluble biologically active components in products that increase the body's resistance to harmful environmental influences [TU 49 1171-85 Structural food concentrate KSP]. The disadvantage of these methods is the content in the finished product (PCB) of biologically active substances in an unconcentrated state, as well as the content along with protein components of minerals, lactose and pectin, and in addition, PCB is subjected to technological processing, as a result of which the native properties of biologically active substances of milk are declining.

Japanese scientists have patented in Russia a method for producing a product containing “active milk protein components” and a product obtained in this way (options) [Patent No. 21-6785 (Russia). A method of obtaining a product containing active milk protein components, and a product obtained by this method (options). / Yoshikazu Yuki (JP), Kumiko Terazawa (JP), Tomoyo Nakagawa (JP), Kazuo Kate (JP)]. The aim of the developed method is to obtain products for preterm infants obtained from breast or cow milk and containing active milk protein components, namely immunoglobulins, lactoferrin and lysozyme. These products can also be used as an additive to breast milk and as pharmaceutical products, for example, in the treatment of resistant diarrhea. The authors of the patent use the term “active protein components” to mean any protein that exhibits physiological and pharmacological properties in minimal amounts. The products are obtained by the method consisting of the steps of adding sugar alcohol or disaccharide or a stabilizer consisting of a disaccharide and amino acid (glycine) to milk plasma or serum, and performing heat treatment of the resulting mixture at pH 6-8, at about 60 ° C for about 10 hours; products containing the active components of milk protein are a precipitate formed by adding ammonium sulfate at a concentration of 30-60% (wt./vol.) to the heat-treated mixture. The heat-treated mixture is cooled, and the precipitated fraction is collected by centrifugation and dissolved in isotonic saline, subjected to adequate dialysis and sterilization using membrane filtration. The disadvantage of this method is the use of prolonged (10 hours) heat treatment, as a result of which not only viruses are destroyed, but also the native substances of milk.

In Japan, a method has been patented for isolating angiogenin with physiological activity from milk [Patent No. 2-296000 (Japan)]. Due to the most important function of angiogenin - the ability to induce the growth of blood vessels, it is proposed to use it for the treatment of damaged tissues, ulcers, and organ implantation.

A method for isolating angiogenin is patented, which is a three-step method for chromatographic purification of angiogenin from milk proteins using ion exchange chromatography on CM-cellulose and hydrophobic chromatography on CM Toyopearl 650 S Butyl Toyopearl 650 S [Patent No. 2109784 (RF). The method of isolation of angiogenin / Rogov I.A., Shalygin A.M., Komolova G.S., Mikhailov A.M., Tikhomirova N.A.].

Along with it, a patented method for producing a biologically active concentrate enriched in pancreatic ribonuclease A, angiogenin and lysozyme from milk ultrafiltrate [Patent No. 2204262 (RF). A method of obtaining a biologically active concentrate enriched in pancreatic ribonuclease A, angiogenin and lysozyme from milk ultrafiltrate / Rogov I.A., Shalygin AM, Tikhomirova N.A., Komolova G.S., Fedorova T.V., Mashkov A.E. ., Tsuman VG, Pykhteev DA, Plaksina GV] The method consists in the fact that milk raw materials, which use ultrafiltrate, are first concentrated 10-12 times by reverse osmosis through a membrane with a pore size 1000-5000 D at a pressure of 0.4-0.7 MPa and a temperature of 4-10 ° C, then purify the protein race solution from lactose and minerals by adding potassium phosphate buffer with a pH of 6.7-7.0, by diafiltration, the resulting protein concentrate is sterilized by microfiltration through a membrane with a pore size of 0.10-0.45 μm at a pressure of 0.2 -0.7 MPa and a temperature of 4-10 ° C.

A known method of producing a biologically active additive based on angiogenin, pancreatic RNase, lysozyme and polypeptides obtained by cation exchange chromatography on CM-cellulose [Patent No. 2183935 (RF). The method of obtaining biologically active additives "MILKANG" and dietary supplement "MILKANG" obtained by this method / Rogov I.A., Titov E.I., Shalygina AM, Tikhomirova N.A., Komolova G.S., Rogov S.I.] . The method consists in the fact that first the proteins of milk origin are separated from the fat fraction, then sorption and chromatographic separation, elution, determination of fractions with optical density at a wavelength of 280 nm more than 0.1 units, dialysis against physiological saline are carried out, they are stabilized by microfiltration through a semipermeable membrane with a selectivity of more than 50 kDa, after which the resulting dietary supplement, including angiogenin, pancreatic RNase, lysozyme and polypeptides, is sent to the packaging. This method is a prototype. The disadvantage of this method is the inability to obtain in native form the full composition of biologically active enzymes that are part of the protective complex of milk. In addition, the disadvantages of this method include the receipt of dietary supplements in liquid form, which significantly reduces the shelf life.

Thus, as a result of a patent and literature search, it was found that obtaining biologically active substances from milk is an urgent task, and the use of proteins of angiogenin, lactoferrin, lactoperoxidase, pancreatic ribonuclease, lysozyme in the form of a biologically active drug is widely used not only in medicine, pharmacology, but also in cosmetology.

The problem to which the present invention is directed, is to develop a method for producing dietary supplements from dairy raw materials, including those not used rationally, namely secondary raw materials of the dairy industry, and to obtain by this method dietary supplements containing a concentrated milk protective complex, including cationic protein substances (lactoperoxidase, lactoferrin, lysozyme, angiogenin, pancreatic ribonuclease), which have parapharmaceutical properties.

This is achieved by the fact that to obtain dietary supplements, cationic proteins of milk origin are separated from the fat fraction by centrifugation of milk raw materials, then they are sorbed and chromatographed, eluted, fractions with an optical density at a wavelength of 280 nm greater than 0.1 units are subjected to dialysis. and according to the invention, sorption and chromatographic separation is carried out on CM-cellulose, and the dialysis of the obtained protein fraction is carried out against distilled water, phosphate buffer, while microbi logical purification through microfiltration through a semipermeable membrane, after which the resulting dietary supplements comprising lactoperoxidase, lactoferrin, lysozyme, milk, angiogenin, pancreatic RNase is directed to the packaging of. After packaging in a sterile container to ensure long shelf life, the composition is subjected to freezing to a temperature of minus 20-25 ° C and subsequent freeze-drying at a sublimation temperature of minus 20 ± 4 ° C, a drying temperature of 37 ± 2 ° C.

The technical result obtained according to this production method is that the dietary supplement contains in its native form lactoperoxidase, lactoferrin, lysozyme, angiogenin, pancreatic RNase, which have angiogenic, antimicrobial, immunomodulatory, antioxidant, anti-stress and general strengthening effect.

A fairly simple modern method for producing dietary supplements based on cationic milk proteins with biological activity has been developed. For the first time, a fraction of cationic proteins of milk origin was obtained, enriched, along with lysozyme, angiogenin, pancreatic RNase, with unique proteins lactoperoxidase, lactoferrin, which have antimicrobial, antioxidant, immunomodulatory, anti-stress properties. Due to the receipt of the fraction of proteins in the native state, this composition is advisable to use as the basis of dietary supplements for food products or in the technologies for the production of medical, pharmaceutical and cosmetic preparations. To obtain a fraction of cationic protein substances of milk, ion-exchange chromatography on CM-cellulose was used. Microfiltration through semipermeable membranes was used for sterilization and microbiological purification of dietary supplements. To ensure long shelf life of the obtained dietary supplement, freeze-drying is used. Raw milk, as well as secondary dairy raw materials of the dairy industry (skim milk), were used as a source of cationic protein substances of milk with antimicrobial, antioxidant, wound healing, immunomodulatory, general strengthening properties. Milk, in particular secondary dairy resources, is a promising source of biologically active substances [N.A. Tikhomirova, G.S. Komolova, I.I. Ionova. Biologically active milk proteins: Textbook. manual - M .: MGUPB, 2004, - 80 pp.], which can be used as an active base for dietary supplements for special-purpose products and drugs with a therapeutic and prophylactic orientation of a wide spectrum of action. In particular, of interest are cationic proteins that are practically not used in traditional technologies and have multifunctional properties: lactoperoxidase (78 kDa, has antimicrobial activity), lactoferrin (78-80 kDa, multifunctional protein: bactericidal and bacteriostatic activity, stimulates cell growth, acute phase protein , inhibits immune and inflammatory reactions, participates in the processes of internal digestion, an immunomodulator), lysozyme (about 14 kDa, has a bactericidal effect), angiogenin (14.5 to and a multifunctional protein: vascular growth factor, osteogenesis active factor, immunoregulator, bactericidal action, acute phase protein, is involved in the transfer of passive immunity), pancreatic RNAse (12 kDa antiviral effect). The developed method allows to obtain a dietary supplement containing in concentrated, but in this native form, the above cationic milk proteins. Due to the preservation of their native properties, dietary supplements have antimicrobial, antioxidant, immunomodulatory, angiogenic properties, which allows you to use it either directly or as an additive to dairy and other food products, which can be recommended as a dietary, functional, therapeutic and preventive nutrition.

The protein composition of dietary supplements is checked electrophoretically in PAAG, and the protein's nativeness by enzymatic activity (lactoperoxidase, lysozyme, pRNAse, lactoferrin) and by interaction with a placental inhibitor (angiogenin).

The method is as follows. To obtain dietary supplements, milk raw materials with native cationic protein substances are taken, degreased by centrifugation, then a fat-free protein fraction is applied to a column with a sorbent CM-cellulose and ion-exchange chromatography is carried out, eluted by collecting fractions with A ₂₆₀ > 0.1. Fractions are combined and dialyzed against distilled water, phosphate buffer. The resulting dialysate is subjected to microfiltration through semipermeable membranes with a selectivity of 0.22-0.45 μm. The result is a dietary supplement in liquid form, which is then sent to the packaging. In order to ensure longer shelf life, the obtained dietary supplement after packaging is subjected to freezing to temperatures of minus 20-25 ° C and subsequent freeze-drying at sublimation temperature of minus 20 ± 4 ° C and a maximum drying temperature of plus 37 ± 2 ° C, thereby obtaining a dry form Dietary supplement. The enzymatic composition of the obtained dietary supplement is presented in table 1.

Table 1 The enzymatic composition of dietary supplements from low molecular weight cationic milk proteins No. p / p Enzyme Yield of 1 liter of milk mg mg / mg protein * one Lactoferrin 10.3 ± 1.4 0.269 2 Lactoperoxidase 17.9 ± 1.2 0.471 3 Pancreatic RNase 2.7 ± 0.1 0,070 four Angiogenin 0.9 ± 0.1 0,023 5 Lysozyme 0.09 ± 0.01 0.018 6 Total protein 38.2 ± 4.9 * - based on 1 mg of the total protein of the cationic protein fraction.

Example 1

Whole milk in an amount of 5 L defatted by centrifugation, then degreased fraction was applied to a column of CM-cellulose sorbent and conduct ion-exchange chromatography, eluted, collecting fractions with A _280> 0.1. Fractions are combined and dialyzed against distilled water. The resulting dialysate is subjected to microfiltration through semipermeable membranes. The result is 50 ml of dietary supplement, the composition of which in comparison with the prototype is presented in table 2. The finished dietary supplement in liquid form is Packed in a sterile container. In dry form, dietary supplements are obtained using freeze-drying with preliminary freezing of the finished dietary supplement.

Example 2 is carried out according to example 1, while skim milk is used as raw material and a dietary supplement is obtained, the composition of which is presented in table 2.

Example 3 is carried out according to example 1, while the dialysis of the obtained fraction of cationic proteins of milk is carried out against physiological saline and get a dietary supplement, the composition of which is presented in table 2.

Example 4 is carried out as in example 1, while the dialysis of the obtained fraction of cationic proteins of milk is carried out against phosphate buffer and get a dietary supplement, the composition of which is presented in table 2.

Thus, in comparison with the prototype, dietary supplements include a wider range of biologically active substances, which, in addition to wound healing and general strengthening effects, also has antimicrobial, antioxidant and immunomodulatory properties due to the additional content of lactoperoxidase and lactoferrin, which makes it possible to use the obtained dietary supplement as the basis for parapharmaceutical preparations .

table 2 The composition of dietary supplements in comparison with the prototype Sample Prototype Example 1 Example 2 Example 3 Example 4 Angiogenin, mg / ml 0.1-0.3 1.0 ± 0.1 0.9 ± 0.09 1.5 ± 0.5 0.97 ± 0.09 rRnase, mg / ml 0.7-2.4 2.7 ± 0.3 2.4 ± 0.15 2.9 ± 0.1 2.5 ± 0.1 Lysozyme, mg / ml 0.001-0.004 0.09 ± 0.01 0.083 ± 0.01 0.095 ± 0.01 0.086 ± 0.01 Lactoperoxidase mg / ml - 17.9 ± 1.2 17.1 ± 1.3 18.01 ± 1.3 17.6 ± 1.1 Lactoferrin, mg / ml - 10.5 ± 1.4 9.9 ± 1.4 10.2 ± 1.3 10.3 ± 1.0

Claims (2)

1. Dietary supplement containing angiogenin, lysozyme and pancreatic RNase, lactoperoxidase and lactoferrin in the following ratio, mg / ml: Lactoperoxidase 17.9 ± 1.2 Lactoferrin 10.3 ± 1.4 Angiogenin 0.9 ± 0.1 Pancreatic RNase 2.7 ± 0.1 Lysozyme 0.09 ± 0.01 2. A method for producing a biologically active additive (BAA) according to claim 1, including the purification of milk raw materials by centrifugation, sorption of proteins on an ion exchanger, their chromatographic separation, elution, determination of fractions with optical density at a wavelength of 280 nm more than 0.1 units, dialysis while dialysis of the obtained protein fraction is carried out against phosphate buffer or distilled water, stabilization and microbiological purification are carried out by microfiltration through semipermeable membranes with a selectivity of 0.22-0.45 μm obtained by dietary supplement asovyvayut in sterile packaging, freeze-drying at a sublimation temperature minus 20 ± 4 ° C and the maximum final drying temperature plus 37 ± 2 ° C.