RU2400106C1 - Method for production of "l-pfi" dietary supplement from secondary milk stock and "l-pfi" dietary supplement produced by this method - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention relates to diary, biotechnological, medical, pharmaceutical and cosmetological industries, namely to methods of production of biologically active substances which may be used as dietary supplements. The method includes milk stock purification, separation, caseation, two-stage ultrafiltration, proteins chromatographic separation, dialysis, microfiltration with a 0.2-0.3 mcm pore selectivity, separated monofractions mixture in a ratio which is similar to their content in fresh cow's milk, lyophilisation. Separation of the immunoglobulin fraction from the whey (at the first stage of ultrafiltration) is performed through 100 kilodalton membranes. The permeate is subjected to a second ultrafiltration through 30 kilodaltons membranes. The produced protein concentrate is subjected to ion-exchange chromatography illuated with a 0.35 M - 1 M NaCl gradient with lactoferrin and lactoperoxidase fractions production. Mixture of the produced fractions (immunoglobulin, lactoferrin and lactoperoxidase) is performed in the ratio 91:5:4.

EFFECT: invention allows to produce a dietary supplement containing, in native form, immunoglobulins, lactoperoxidase and lactoferrin; the dietary supplement has immunomodulatory, antioxidative, antistress, antibacterial and general tonic actions.

2 cl, 1 tbl, 1 ex

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 antibacterial composition, developed in the form of chewable lozenges, containing as active components lactoferrin and dry colostrum with the addition of citrus pectin [US Patent 6258383 (USA)]. / Dietary supplement consisting of colostrum and lactoferrin / Marcus B. Kokrum (USA), Richard N. (USA)]. The composition is used for the treatment and prevention of infectious diseases, stimulating the immune system and tissue regeneration.

A known method of producing dietary supplements based on immunoglobulins and dietary fiber, which consists in the fact that in the pasteurized milk is introduced the ferment used in the production of cheese, the process of fermentation of milk is carried out, during which changes in pH and fermentation time are carefully monitored to obtain the desired quality of the clot and minimal exposure to the immunoglobulin fraction of milk whey proteins. Separated serum is subjected to separation to remove unbound particles of fat and casein, and then subjected to ultrafiltration, followed by ion exchange chromatography. The obtained concentrate of immunoglobulins is subjected to freeze drying, followed by dry mixing with dietary fiber, such as inulin, pectin, guar gum in the ratio of immunoglobulin - 40%, dietary fiber - 60%. The composition is recommended to be used as a tool for the treatment and prevention of diarrheal infections caused by cryptosporidia, as well as infections of the gastrointestinal tract caused by Candida, Giardia lambda, Entamoeba histolytica, Cholerae, Shigella, Salmonella, Helicobacter pylori, Campylobacter, rotoviruses. Japanese scientists have patented a method for producing lactoperoxidase from milk [Patent JP 2004039704 (Japan). / A method of obtaining lactoperoxidase / Ishikhashi Nobuo (Japan), Yamauchi Skin (Japan), Shin Kouchirow (Japan), Ando Tetsuya (Japan)]. The method includes the following stages: skim milk raw materials are subjected to ion-exchange chromatography, washed with water to wash the non-absorbed components of milk, the fraction containing lactoperoxidase is eluted from the column with a 1.6% sodium chloride solution. The obtained fraction is subjected to ultrafiltration through membranes with a pore selectivity of 20 kDa at a pressure of 0.3 MPa, followed by the introduction of distilled water into the system to desalinate the resulting ultrafiltrate, which is then subjected to cold sterilization and freeze drying.

The disadvantages of the two above analogues can be attributed to the presence of one antibacterial component in the composition of the drugs.

Known in the United States patented method for producing a liquid form of a biologically active composition of a general strengthening effect, as well as aimed at eliminating the existing iron deficiency. The composition contains iron-saturated lactoferrin and sodium bicarbonate [Patent US 5098722 (USA). / Method for the production of iron-fortified drinks / Tanaka (Japan), Maki Shi (Japan), Tojima (Japan), Teichi (Japan), Shun-Ischi (Japan), Tatsumi (Japan), Kiyoshi (Japan)].

A known composition containing trivalent chromium isolated from milk of cows and other mammals is chloride, acetate, sulfate, picolinate, nicotinate, GTF Cg (glucose tolerance factor), chromium from yeast, mineral and chromium salts and combinations thereof [US Patent US 20070010426 ) / Composition for the prevention and treatment of cardiovascular diseases / Mao Frank Chiahung (Taiwan), Chen Wen-Ying (Taiwan), Chiang Yi Chung (Taiwan), Chiang Liu Cheng (Taiwan)]. The composition is applicable as an agent in the treatment of hypertension, hyperlipidemia, obesity, as well as cardiovascular diseases, for example, myocardial infarction. The composition has a synergistic effect in the ratio of lactoferrin and compounds Cr {3+} 1: 200-1: 10. Vitamin and mineral salts are also administered in oral formulations, in the form of solid nutritional supplements and in beverages.

A known method of obtaining a biologically active product from colostrum [Patent CN 1112394 (China) / Method for preparing a biologically active product from colostrum of cows / Zhaokoung Deng (CN); Yiyun Liu (CN); Liu Shengwu (CN)]. The method includes the following sequential operations: removing fat and casein by centrifugation, adding pepsin or hydrochloric acid, removing the clot by centrifugation, precipitating immunoglobulin with saturated ammonium sulfate or hyperfiltration, collecting and separating immunoglobulin and lysozyme. The resulting product has a significant effect for the prevention and treatment of diarrhea, as well as chronic bronchitis in newborns. The disadvantage of this method is that when pepsin or hydrochloric acid is added, part of the biologically significant components of colostrum lose their activity.

There is a method for 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). A method of obtaining a biologically active additive "MILKANG" and dietary supplement "MIZHANG" obtained by this method / Rogov IA, Titov EI, Shalygina AM, Tikhomirova NA, Komolova GS, Rogov SI] . 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. The disadvantages of this method include obtaining dietary supplements in liquid form, which significantly reduces the safety level of the components that make up the dietary supplement during storage.

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 a pH of 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 disadvantages of this method are the content of biologically active components in an unstandardized ratio, as well as the use of long-term (10 hours) heat treatment, which results in not only the destruction of viruses, but also a partial or complete loss of nativeness of biologically active milk proteins. A known method of producing dietary supplements from low molecular weight milk proteins, containing in concentrated form a protective complex of milk, including cationic protein substances (lactoperoxidase, lactoferrin, lysozyme, angiogenin, pancreatic ribonuclease), with para-pharmaceutical properties [Patent No. RU 2318406 C1 (Russia). A method of producing dietary supplements from low molecular weight cationic proteins of milk and dietary supplements obtained by this method. / Rogov I.A. (RU), Titov E.I. (RU), Semenov G.V. (RU), Tikhomirova N.A. (RU), Komolova G.C. (RU), Ionova G.G. (RU), Lepikhina O.E. (RU)]. The method includes isolating the protein fraction of milk by centrifugation of milk raw materials, then sorption and chromatographic separation, elution, determination of fractions with optical density at a wavelength of 280 nm more than 0.1 units are carried out, dialysis, sorption and chromatographic separation are carried out on CM-cellulose and dialysis of the obtained protein fraction is carried out against distilled water, phosphate buffer, while microbiological purification is carried out by microfiltration through a semipermeable membrane well, after which the obtained dietary supplement, including lactoperoxidase, lactoferrin, milk lysozyme, angiogenin, pancreatic RNase, is sent for packaging. 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.

This method is a prototype.

The disadvantages of this method is the selection of biologically active cationic milk proteins in the form of a single fraction and the absence of technological steps aimed at obtaining purified forms of biologically active proteins. What is extremely important, given that the component composition of raw milk depends on many factors and is unstable during the year, and accordingly, the dietary supplement obtained in this way cannot be characterized by a constant component composition and a given level of biological properties.

It is also worth noting that the proposed method does not provide for the production of immunoglobulins, which are also found in milk raw materials and play an important role in the regulation of immune processes in the body.

Thus, as a result of patent and literary searches, it has been established that obtaining biologically active substances from milk is an urgent task, and the use of immunoglobulin, lactoferrin, lactoperoxidase proteins in the form of a biologically active drug is widely used in medicine and pharmacology.

The objective of the present invention is directed to the development of a method of producing dietary supplements from dairy raw materials, including those not rationally used, namely secondary raw materials of the dairy industry and the production of dietary supplements by this method, which contains a concentrated milk protective complex, including biologically active milk proteins (immunoglobulins, lactoferrin, lactoperoxidase), with a wide spectrum of parapharmaceutical action.

The problem is solved in the developed method for producing a biologically active dietary supplement, including purification of dairy raw materials from mechanical impurities, separation, acid precipitation of casein, dialysis, cold sterilization, freeze drying, according to the invention, the obtained serum after acid precipitation of casein is made alkaline to pH 6.8 and subjected ultrafiltration, passing it through a membrane of 100 kDa, after which the selected immunoglobulin fraction is subjected to microfiltration using ceramic membranes from villages pore activity of 0.2 μm and a pressure of 0.05-0.2 MPa and sent for intermediate storage, and the permeate is subjected to repeated ultrafiltration through 30 kDa membranes, the resulting protein concentrate is applied to a column with a sorbent CM-agarose and ion exchange chromatography is carried out, elute with a NaCl gradient from 0.35 M to 1 M, separating the resulting concentrate into two protein fractions (one of which contains lactoperoxidase, the other lactoferrin), both fractions obtained with A ₂₈₀ > 0.1 are collected and dialyzed against distilled water, and then hold mic ltratsiyu fractions obtained through membranes with pore selectivity 0.2-0.3 microns, after which the three resulting monofraction (immunoglobulin, laktoferrinovuyu and lactoperoxidase) were mixed in a ratio similar to the content of their svezhevydoennom in cow's milk.

The technical result obtained according to this production method is that the dietary supplement “L-PFI” has a constant component composition and in its native form contains such multifunctional milk components as immunoglobulins, lactoferrin and lactoperoxidase, which have immunomodulatory, antioxidant, anti-stress, antimicrobial and tonic effect.

A fairly simple and modern method for producing dietary supplements based on whey proteins of milk with biological activity has been developed. For the first time, a fraction of cationic proteins of milk origin enriched with lactoperoxidase, lactoferrin and immunoglobulins was obtained. 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 production technologies of medical, pharmaceutical and cosmetic preparations.

To obtain a fraction of the protein substances of milk, stepwise ultrafiltration and ion exchange chromatography on CM agarose were used. Microfiltration through semipermeable membranes was used for sterilization, microbiological cleaning, and dietary supplement. 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 protein substances of milk with antimicrobial, antioxidant, immunomodulatory 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. allowance. - 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 (immunomodulator), immunoglobulin (antimicrobial and immunomodulatory activity).

The developed method allows to obtain a dietary supplement containing in a concentrated, but at the same time native form, the above biologically active milk proteins. Due to the safety 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 PAGE, and the protein's nativeness by enzymatic activity (lactoperoxidase, lactoferrin), immunoglobulins by ELISA (enzyme-linked immunosorbent assay).

The method is as follows.

To obtain dietary supplements, milk raw materials with native biologically active protein substances purified from mechanical impurities and contaminants are taken, separated, then the defatted protein fraction is subjected to acid coagulation to produce casein and serum. After acid precipitation of casein, the obtained serum is alkalinized to pH 6.8 and ultrafiltered by passing it through 100 kDa membranes to obtain an immunoglobulin fraction, which is microfiltered using ceramic membranes with pore selectivity of 0.2 μm and a pressure of 0.05-0.2 MPa and sent for intermediate storage, and the permeate is subjected to repeated ultrafiltration through 30 kDa membranes, the protein concentrate obtained is applied to a column with a CM-agarose sorbent and ion exchange chromatography is performed, eluting with g radiant NaCl from 0.35 to 1 M, two fractions (lactoferrin and lactoperoxidase) with A ₂₈₀ > 0.1 are collected and dialyzed against distilled water. To ensure microbiological purity, microfiltration of the obtained fractions through membranes with a pore selectivity of 0.2-0.3 microns is carried out.

After that, the three monofractions obtained (immunoglobulin, lactoperoxidase and lactoferrin) are mixed in a predetermined ratio equal to the ratio of these proteins in freshly milked cow's milk 91: 5: 4, respectively.

The sterile composition is packaged in penicillin vials and subjected to freezing to a temperature of minus 20-25 ° C, followed by freeze drying at a temperature of minus 20 ± 4 ° C and a subsequent drying temperature plus 37 ± 2 ° C.

The enzyme composition of the obtained dietary supplement "L-PFI" are presented in table 1.

Table 1
Enzyme composition of dietary supplement “L-PFI” from biologically active milk proteins No. Enzyme Exit from Composition p / p 1 liter of milk mg mg mg / mg protein * one Immunoglobulin 414.3 ± 38.4 531.3 ± 38.3 0.91 one Lactoferrin 14.4 ± 1.4 21.4 ± 1.8 0.35 2 Lactoperoxidase 13.4 ± 1.2 29.6 ± 3.2 0.49 6 Total protein 444.1 ± 41.0 585.9 ± 43.3 one * - per 1 mg of the total protein of the obtained protein composition.

Example.

Whole milk in an amount of 5 l is purified from mechanical impurities, separated, then a weak solution of hydrochloric acid is added to the fat-free fraction and casein is precipitated. The resulting serum was alkalinized to pH 6.8 and ultrafiltered, passing it successively through 100 kDa membranes, after which the isolated immunoglobulin fraction was microfiltered using ceramic membranes with a pore selectivity of 0.2 μm and a pressure of 0.05-0.2 MPa and sent for intermediate storage, and the permeate is subjected to repeated ultrafiltration through 30 kDa membranes, the resulting protein concentrate is applied to a column with a CM-agarose sorbent and ion exchange chromatography is performed, eluting with a NaCl gradient 0.35 m to 1 M, two fractions were obtained (laktoferrinovuyu and lactoperoxidase) with A _280> 0.1 were collected and dialyzed against distilled water. To ensure microbiological purity, microfiltration of the obtained fractions through membranes with a pore selectivity of 0.2-0.3 microns is carried out. After that, the obtained monofractions (immunoglobulin, lactoperoxidase and lactoferrin) are mixed in a ratio similar to their content in freshly milked cow's milk (91: 5: 4, respectively), and L-PFI dietary supplement is obtained. The finished dietary supplement "L-PFI" in liquid form is Packed in penicillin vials and subjected to freezing to a temperature of minus 20-25 ° C followed by freeze drying at a temperature of minus 20 ± 4 ° C and a subsequent drying temperature plus 37 ± 2 ° C, as a result get a dry form of dietary supplement "L-PFI".

Thus, in comparison with the prototype, dietary supplements include biologically active proteins in their native state, which, in addition to wound healing and general strengthening effects, also have antimicrobial, antioxidant and immunomodulatory properties due to the additional content of immunoglobulins, which makes it possible to use the obtained dietary supplement as the basis for parapharmaceutical preparations.

Claims (2)

1. A method of producing a biologically active dietary supplement additive, including purification of milk raw materials from mechanical impurities, separation, casein acid precipitation, dialysis, cold sterilization, freeze drying, characterized in that the obtained serum after acid casein precipitation is alkalinized to pH 6.8 and ultrafiltered passing it through 100 kDa membranes, after which the isolated immunoglobulin fraction is subjected to microfiltration using ceramic membranes with pore selectivity of 0.2 μm and a pressure of 0.05-0.2 MPa and from They are separated into intermediate storage, and the permeate is subjected to repeated ultrafiltration through 30 kDa membranes, the protein concentrate obtained is applied to a column with a CM-agarose sorbent and ion-exchange chromatography is performed, eluting with a NaCl gradient from 0.35 M to 1 M, two fractions (lactoferrin and lactoperoxidase) with a _280> 0.1 were collected and dialyzed against distilled water and then subjected to microfiltration the fractions obtained through a membrane with a selectivity of pores of 0.2-0.3 microns, is mixed in a ratio similar to their content in svezhevydoe Mr cow's milk 91: 5: 4, respectively. 2. The dietary supplement obtained according to claim 1, characterized in that it contains biologically active milk proteins in a ratio corresponding to their content in freshly milked cow's milk.