RU2441907C1 - Method for preparation of therapeutic product out of living microbial strains of lactic bacteria and bifidus bacteria lb-complex l - Google Patents

Abstract

FIELD: biochemistry. ^ SUBSTANCE: method includes separate culturing of strains of lactic bacteria and bifidus bacteria on bouillon containing digest of casein produced by hydrolysis using cattle pancreas, carbohydrate component containing lactose and raffinose in ratio 1:1. Raffinose is both a carbohydrate component and probiotic component. The medium contains peptone, agar-agar, ascorbic acid and distilled water. The mixture of strains is added to sorbing component prepacked in vial in proportion of - from total volume of created complex system. Process of immobilization on sorbent - Litovit M dietary supplement is performed at temperature +6C2C for 18-24 hours. ^ EFFECT: production of immobilized synbiotic product with high content of live microbial cells, several strains of lactic bacteria and bifidus bacteria with increased protective and treatment activity. ^ 3 cl, 2 tbl

Description

The invention relates to biotechnology, namely, medical and food, and can be used for the preparation of therapeutic and prophylactic preparations, biologically active additives and food products using live probiotic strains of microorganisms as starter cultures.

Widely known are drugs aimed at restoring normal intestinal biocenosis, in particular eubiotics - bacterial preparations from living microorganisms - representatives of the obligate intestinal microflora - colibacterin, lactobacterin, bifidumbacterin, bificol, etc. dry preparations (see the book under the editorship of V.A. Knyazhev. Dysbacterioses. Theory and practice. Nizhny Novgorod, 1999, pp. 70-73).

With all the positive qualities of these drugs, when used in dry form, many properties are lost. When the preparation is dried, the number of living strains of microorganisms decreases, and activation of the antagonistic activity of the strains requires a long exposure time in the human intestine.

A known probiotic preparation in dry immobilized form (RF patent No. 2164801, A61K 35/74, A23C 9/12, C12N 1/08, published 04/10/2001), including a carrier, which is a sorbent and eubiotics cells immobilized on the carrier, moreover, the cells of eubiotics are used in conjunction with a nutrient medium, and as a sorbent material is used with antacid properties, a developed meso- and macroporous structure and a macropore volume of at least 0.01 cm ³ / g in the form of a powder with a particle size of at least 0.1 mm or in the form of granules with a size of 0.1-5.0 mm or in the form of tablets, the preparation additionally contains a protective environment, and the cells of eubiotics with a nutrient medium are a dry concentrate of milk-based bifidobacteria with a titer of 10 ⁸ -10 ¹⁰ CFU / g and with an amino acid content (mg / 100 g of dry biomass):

Glutamic acid - 15.0-68.0

Glutamine - 8.0-20.0

Leucine - 10.0-50.0

Arginine - 20.0-30.0

Cysteine - 50.0-80.0

In the following ratio of the components of the drug in dry form, wt.%:

Cells of eubiotics with a nutrient medium and a titer of 10 ⁸ -10 ¹⁰ CFU / g - 0.1-10.0

Protective environment - 0.1-10.0

Sorbent carrier - the rest is up to 100%,

gelatin, sucrose, ascorbic acid and sodium chloride are used as a protective medium in the following components, wt.%:

Gelatin - 8.5-11.5

Ascorbic acid - 0.8-1.2

Sodium Chloride - 1.7-3.0

Sucrose - the rest is up to 100%,

as the sorbent, a porous material based on alumina with hydrophilic-hydrophobic topochemistry is used, for example, carbon modified SUMS enterosorbent;

the preparation as bacteria-eubiotics additionally contains dry sourdough of viscous strains of acidophilic bacteria Lactobacillus acidophilus type AB, the ratio of which with a dry concentrate of bifidobacteria is 1: (8-9), respectively;

as bifidobacteria in a dry concentrate, use Bifidobacterium bfidum N1 or N 791, or LVA-3, or Bifidobacterium longum 379, or Bifidobacterium adolescentis MC-42, or a mixture of these strains, taken in equal weight proportions.

The disadvantages of this drug are as follows.

1. Firstly, in dry preparations, the cells of microorganisms are in deep suspended animation, and the transition from suspended animation to an active state takes 8-10 hours (while the digestion process in children is 4 hours); lyophilization violates the structure of surface adhesin proteins, which reduces the colonization ability of bacteria; during the drying process, most of the metabolites are destroyed, in particular, valuable organic acids: acetic, lactic, vitamins C and K.

2. Secondly, the biomass of starter cultures is increased on a milk nutrient medium to the number of live microbial cells in the biomass of 10 ⁹ -10 ¹¹ CFU / ml, then the liquid biomass is mixed with a dry SUMS sorbent, the immobilization process takes place within 1.0-1, 5 hours at a temperature of + 4 ° C until the cells are completely immobilized on a carrier. Next, the resulting product is washed in isotonic NaCl solution according to MUK 4.2.577-96 p. 7.1. Clause 7.10, check the number of living microorganisms per unit volume, and sorption is considered complete if no more than 50% of the initial number of microorganisms remains in the selected sample of the washing solution, which will certainly reduce the number of living to 2 · 10 ⁸ -2 · 10 ¹⁰ CFU / ml A protective medium is then added and dried under conditions of low temperature freeze drying. It is known that when using any vacuum-drying equipment with existing technologies for drying the preparation, the number of living microbial cells is reduced by two orders of magnitude, that is, as a result, in the proposed preparation, the number of live CFU / ml in the control of the finished preparation when laying biomass 2 · 10 ⁸ -2 · 10 ¹⁰ CFU / ml in the output will be at best 10 ⁶ -10 ⁸ CFU / ml. That is, the difference between the immobilized forms and the dry ones in terms of yield of the number of living microbial cells cannot be expected.

3. Thirdly, the cultivation of the original strains in a classic dairy environment does not give a large yield of biomass.

4. Fourth, SUMS-1 is specially prepared artificially from aluminum oxide coated with a black carbon film

As a prototype, a method of preparing a therapeutic and prophylactic preparation based on live strains of microorganisms "LB-complex" (RF patent No. 2192269, A61K 35/74, A23C 9/127, C12N 1/20, C12R 1:07, 1: 225, 1:25, published 10.11.02), including separate cultivation of strains of bifidobacteria and lactobacilli and mixing them in a ratio of 1: 1 at the end of cultivation. Cultivation is carried out on a hydrolyzate-dairy medium (HMS). As a basis for HMS, a skim milk hydrolyzate is used and ascorbic acid is additionally added in an amount of 230-250 mg per 1000 ml of hydrolyzate. The finished drug is packaged in bottles, taking into account the required daily dose. The method allows to obtain a ready-to-use drug that affects the bifidobacteria and lactic components of human microflora, with a long shelf life.

The disadvantages of this method are as follows.

1. Firstly, the feedstock for the preparation of the hydrolyzate is milk return, an unstable product, its quality depends on many factors. Pancreatin, used as an enzyme for hydrolysis, does not provide complete hydrolysis of the protein due to the absence of minor enzymatic components. In this regard, the prepared hydrolyzate has a low level of amine nitrogen (150-250 mg%, table 1), which leads to the use of undiluted milk hydrolyzate by this method.

From table 1 it is seen that pancreatin does not provide complete protein breakdown. As a marker of the completeness of hydrolysis, it is customary to use the determination of the level of tryptophan in the finished intermediate, i.e. the higher the tryptophan content, the more complete the hydrolysis.

2. Secondly, this method involves the preparation of a classic multi-strain probiotic preparation without a prebiotic component, which reduces the likelihood of engraftment of microorganisms in the large intestine and does not provide an additional substrate for their reproduction.

3. Thirdly, the absence of a carrier sorbent does not ensure the immobilization of strains of microorganisms, which reduces the likelihood of the preservation of biomass when passing through an aggressive-acidic environment of the stomach.

These shortcomings are eliminated by the proposed solution.

The problem to be solved is the improvement of the method for preparing an immobilized probiotic enriched in fructooligosaccharide (FOS) in the form of a bacterial concentrate based on live strains of microorganisms.

EFFECT: obtaining a synbiotic preparation with a high content of live microbial cells of several strains of lacto- and bifidobacteria, which has an increased protective therapeutic effect due to the introduction of fructooligosaccharide as a prebiotic component and the immobilization of probiotic strains on a sorbent.

This result is achieved by the fact that in the method of preparing a therapeutic and prophylactic drug from live strains of the microorganisms lactobacilli and bifidobacteria by culturing them on a nutrient medium containing a nutrient base - casein hydrolyzate diluted with distilled water, sodium chloride, carbohydrate component, agar-agar, acidic component, packaging of the liquid preparation, taking into account the daily dose necessary for patients, casein hydrolyzate is used as a hydrolyzate of the nutrient base, split by the mammary gland of cattle (Table 1), lactose and raffinose in a ratio of 1: 1 (Table 2) are used as the carbohydrate component, and raffinose is both a carbohydrate component and a prebiotic component, at the end of separate cultivation, the mixture of strains is added to the sorbent component, packaged in vials, so that its share is 1 / 2-1 / 4 of the total volume of the created complex system, which is then kept for immobilization on the sorbent at a temperature of + 6 ± 2 ° C for 18-24 hours; as a sorbent component, dietary supplement “Litovit M” is used, and L. plantarum, L.fermentum, B. bifidum, B.longum are used as producer strains.

Thus, an optimal complex has been proposed that allows one to obtain an immobilized preparation with a high content of live microbial cells of all six starter cultures per unit volume with a long shelf life and with a prebiotic component.

The method is as follows: prepare the basis of a liquid immobilized synbiotic hydrolyzate-casein medium (GCS) according to the following scheme: dilute casein in warm drinking water, add ground pancreas, bring the temperature to 48 ± 1 ° C, set the pH to 10-20% NaOH solution , stirred, chloroform is added and placed in a thermostat for 72 hours, the first hour is constantly stirred, then filtered through a paper filter. In the finished hydrolyzate, amine nitrogen is controlled, the hydrolyzate is diluted with distilled water to amine nitrogen 160-170 mg%, agar-agar, sodium chloride, peptone, lactose, raffinose, ascorbic acid are added and sterilized.

The cultivation of producer strains of lactobacilli and bifidobacteria is carried out according to a single method - each strain separately in three stages. At the end of cultivation, the biomass of individual strains is mixed, preferably in a 1: 1 ratio, and packaged in vials with a pre-dosed and sterilized sorbent, taking into account the daily dose of 5-10 ml, and kept in a refrigerator at t = 6 ± 2 ° C. Then the bottles are marked, packaged in boxes of 25 pieces (minimum course of treatment). A natural zeolite belonging to aluminosilicates (clinoptilolite of the heilandite group) of the Kholinsky deposit in the form of dietary supplements for Litovit M food is used as a sorbent. This drug has been used as an sorbent in medical practice since the 80s of the last century, has unique selective ion exchange properties, supplies the body with the missing macro, micro and ult microelements, nano and picoelements, if they are missing and removes them from the body if they are in excess [V. Bgatov. Approaches to ecogeology ”, Novosibirsk, NSU, 1993]. The catalytic property of zeolite is also important, which contributes to the normalization of all biochemical processes in the body. It is proved that the use of natural zeolite (dietary supplement “Litovit”) provides a number of positive effects, such as antitoxic effect, excretion of heavy metals from the body, immunomodulating effect, antianemic effect, normalization of lipid, protein and carbohydrate metabolism, hepatoprotective effect, optimizes the work enzymes, etc. [Agadzhanyan N.A. et al. “Natural minerals in the service of man, Novosibirsk, 2000].

An example implementation of the method.

At the first stage, a semi-product is prepared for future use - casein hydrolyzate as follows: drinking water (GOST 2874-82) is heated to 48 ° C, casein is poured (food acid casein according to OST 4960-74) in an amount of 60-70 g per liter, stirred, adjusted pH to 7.8-8.2 IU with a 20% NaOH solution, put in a thermostat at 48 ° C for two hours to swell, then add the chopped pancreas (GOST 11285-93) in an amount of 60-70 g / l and chloroform in amount of 10 ml / l, adjust the pH to 7.9-8.0 UNITS of 10-20%) with NaOH solution and put in a thermostat at a temperature of 48 ° C. During the first hour, the contents are mixed several times (the plug after shaking is pierced to remove chloroform vapor) and left to carry out the hydrolysis process for 72 hours, after exposure to thermostat, the supernatant is poured through a paper filter. The finished hydrolyzate should contain 450-500 mg% of amine nitrogen. The hydrolyzate is stored for future use under chloroform 1% by volume at a temperature of 4 ± 1 ° C.

The nutrient base of the probiotic (GCS) is prepared from the obtained intermediate product according to the following recipe:

Probiotic base formulation Casein Hydrolyzate 0.333 L 346.32 g 33.58% Distilled water 0.667 L 667.0 g 64.7% Sodium chloride 5.0 g 5.0 g 0.48% Lactose 5.0 g 5.0 g 0.48% Raffinose 5.0 g 5.0 g 0.48% Peptone 2.0 g 2.0 g 0.19% Agar agar 0.75 0.75 g 0.07% Vitamin C 0.25 0.25 g 0.02% Total: 1031.32 g one hundred%

To prepare GCS, the whole hydrolyzate is diluted with distilled water to an amine nitrogen index of 160-170 mg%. A decrease in amino nitrogen inhibits the process of reproduction of microorganisms, that is, the intensity of biomass growth slows down.

5 g of sodium chloride, 2 g of peptone, 5 g of lactose, 5 g of raffinose, ascorbic acid 0.24 ± 0.1 g and 0.75 ± 0.1 g of previously standardly prepared agar agar are added to the hydrolyzate based on 1 l . Add NaOH 20% solution to establish a pH of 8.4-8.5 UNITS.

The finished medium is sterilized at 0.5 atm. 30 minutes.

Ascorbic acid is used as a growth stimulant for bifidobacteria and lactobacilli.

Peptone is a mixture of poly- and oligopeptides, amino acids, salts and trace elements, respectively, is a source of nutrients.

Agar increases the viscosity of the medium, which leads to a uniform growth of microorganisms throughout the thickness of the medium and a uniform consumption of growth factors and nutrients.

Lactose is an energy substrate and carbon source.

Raffinose (fructooligosaccharide), in addition to satisfying the cell’s need for carbohydrate, promotes selective growth stimulation and metabolism of beneficial representatives of intestinal microflora (lactobacilli and bifidobacteria)

Sodium chloride maintains the optimal osmotic pressure of cells.

As producer strains, L.plantarum 8RA3 and L.fermentum 90-TC-4 and L.fermentum 39, B.bifidum 791, B.bifidum 1, B.longum 379 are used.

The cultivation of producer strains of lactobacilli and bifidobacteria is carried out according to a single method.

Generation I — the dry strain is diluted in 10 ml of GCS and cultured at 37 ± 1 ° C for 18 hours.

II generation - 10 ml of the 1st generation is poured into 100 ml of GCS and cultured for 18 hours at 37 ± 1 ° C.

Generation III - 3 ml of generation II are added to 300 ml of GCS medium (i.e. 9.9 liters of generation III are obtained from 100 ml of generation II) and cultivated for 48 hours at a temperature of 37 ± 1 ° C.

At the end of the cultivation, the biomass of the individual strains is mixed at a ratio of 1: 1.

In parallel with the preparation of starter culture generations in penicillin vials, 2 ± 0.5 g of the sorption component of dietary supplement for food “Litovit M” manufactured by ZAO NPF “Nov” is packaged (TU 9197-001-16925875-05, registration certificate No. 77.99.23.3.U .9488.11.07, release date 11/29/2007), which amounts to the daily dose, according to the manufacturer's recommendations, and the proportion of the sorbent is 1 / 2-1 / 4 of the total volume of the complex system, sterilized in an “air sterilizer” at a temperature of 180 ± 1 ° C 1 hour At the end of the sterilization, the vials cool, pour a mixture of biomass starter cultures of lactobacilli and bifidobacteria into 5-10 ml, taking into account the daily dose of probiotic.

Seal tightly with a rubber stopper and an aluminum cap, shake. Further, the immobilization process takes place at a temperature of + 6 ± 2 ° C for 18-24 hours ± 2 hours. Then the bottles are marked, packaged in boxes of 25 pieces (minimum course of treatment). The drug is stored at a temperature of + 6 ± 2 ° C for up to 60 days with the safety of live microbial cells in 1 ml of a liquid preparation 10 ¹⁰ -10 ¹² CFU / ml.

The drug can be obtained by a reactor method.

The drug can be used orally in 1 or 2 doses, 1 bottle per day before meals with water, compote, fruit drink, juice, etc. with a temperature not exceeding 30 ° C. Before use, the vial with the drug is thoroughly shaken.

For children of the first months of life, the “LB-complex L” is introduced into any adapted artificial mixture in the amount of 2.5-5 ml per day (fractional for 3 feedings).

Indications for use:

- diseases complicated by intestinal dysbiosis: long-term treatment with antibiotics, chemo-and hormonal drugs; allergic diseases (allergic dermatoses, eczema, etc.); chronic diseases of the gastrointestinal tract, acute intestinal infections of bacterial and viral etiology (desinteria, coli enteritis, salmonellosis, acute intestinal infections of unknown etiology, rota- and enterovirus infection, etc.), foodborne toxicoinfection;

- intoxication of various origins, including alcohol;

- in acute and chronic liver diseases;

- as a probiotic and sorption component of diet therapy in postoperative patients;

- chronic diseases accompanied by immunodeficiency;

- radiation and chemotherapy for cancer.

Clinical testing of a probiotic prepared according to the method presented in the application was carried out in the following medical institutions of the city of N. Novgorod: MLPU Children's Clinic No. 49 of the Prioksky Rayzdravotdel, MLPU Children's City Clinical Hospital No. 27 Aibolit, GUZ Nizhny Novgorod Regional Clinical TB Dispensary. ”

The indicated results were confirmed. Obtained by the proposed method, the drug is conditionally called "LB-complex L".

From table 1 it can be seen that the maximum level of amino nitrogen and the most complete breakdown of cow's milk protein is achieved by hydrolysis of casein by the pancreas of cattle (an indicator of the completeness of hydrolysis is the level of tryptophan).

table 2 Comparative characteristics of the growth properties of nutrient bases with various combinations of carbohydrate components and fructooligosaccharide (lactose and raffinose) Name of basis The content of lactose, g / l The content of raffinose, g / l Producer strain The number of CFU / ml of producer strains after 48 hours of incubation Hydrolyzate Casein Medium Method 1 10 - L.plantarum 8RA3 10 ⁹ 10 ¹⁰ 10 ⁹ L.fermentum 39 10 ¹⁰ 10 ⁹ 10 ⁹ L.fermentum 90-TC-4 10 ¹⁰ 10 ¹⁰ 10 ⁹ B.bifidum 1 10 ⁹ 10 ⁸ 10 ⁹ B.bifidum 791 10 ¹⁰ 10 ¹⁰ 10 ¹¹ B.longum 379 10 ⁹ 10 ¹⁰ 10 ¹⁰

Continuation of table 2 Hydrolyzate Casein Medium Method 2 7.5 2.5 L.plantarum 8RA3 10 ¹⁰ 10 ¹⁰ 10 ¹¹ L.fermentum 39 10 ¹⁰ 10 ¹¹ 10 ¹⁰ L.fermentum 90-TC-4 10 ⁹ 10 ¹⁰ 10 ¹⁰ B.bifidum 1 10 ¹⁰ 10 ⁹ 10 ¹⁰ B.bifidum 791 10 ¹⁰ 10 ¹¹ 10 ¹⁰ B.longum 379 10 ¹¹ 10 ¹⁰ 10 ¹¹ Hydrolyzate Casein Medium Method 3 5,0 5,0 L.plantarum 8RA3 10 ¹² 10 ¹⁰ 10 ¹¹ L.fermentum 39 10 ¹¹ 10 ¹² 10 ¹² L.fermentum 90-TC-4 10 ¹² 10 ¹¹ 10 ¹¹ B.bifidum 1 10 ¹¹ 10 ¹¹ 10 ¹² B.bifidum 791 10 ¹¹ 10 ¹² 10 ¹² B.longum 379 10 ¹² 10 ¹⁰ 10 ¹¹ Hydrolyzate Casein Medium Method 4 - 10 L.plantarum 8RA3 10 ¹⁰ 10 ⁹ 10 ¹⁰ L.fermentum 39 10 ⁹ 10 ⁹ 10 ¹⁰ L.fermentum 90-TC-4 10 ⁹ 10 ⁹ 10 ¹⁰ B.bifidum 1 10 ¹⁰ 10 ⁹ 10 ¹⁰ B.bifidum 791 10 ¹⁰ 10 ¹⁰ 10 ¹⁰ B.longum 379 10 ¹⁰ 10 ¹⁰ 10 ⁹

From table 2 it can be seen that the maximum biomass yield of all starter cultures was recorded with method No. 3 for preparing a nutrient base (10 ¹⁰ -10 ¹² CFU / ml) containing 5 g of lactose and 5 g of raffinose.

Claims (3)

1. A method of preparing a therapeutic and prophylactic drug from live strains of the microorganisms of lactobacilli and bifidobacteria by culturing them on a nutrient medium containing a nutrient base — a hydrolyzate diluted with distilled water, sodium chloride, a carbohydrate component, agar-agar, an acidic component, and packaging the liquid preparation with taking into account the daily dose necessary for patients, characterized in that casein hydrolyzate, split by the large horned pancreas, is used as the basis of the nutrient medium cattle, and distilled water to dilute the fermented hydrolyzate to a given amine nitrogen content, lactose and raffinose in a ratio of 1: 1 are used as the carbohydrate component, and raffinose is both a carbohydrate component and a prebiotic component, after the completion of separate cultivation, the strain mixture is added the sorbent component, packaged in bottles, so that its share is 1 / 2-1 / 4 of the total volume of the created complex system, which is then maintained for immobilization II on a sorbent at a temperature of + 6 ± 2 ° C for 18-24 hours 2. The method according to claim 1, characterized in that as a sorbent component use dietary supplement for food "Litovit M". 3. The method according to claim 1, characterized in that L.plantarum 8RA-3 and L.fermentum 39, L.fermentum 90 TC-4, B. bifidum 1, B. bifidum 791, B. are used as producer strains. longum 379.