RU2517734C1 - Method to prepare medicated product from live strains and microorganisms of lactobacilli and bifidobacteria "lb-complex plus" - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method provides for cultivation at 37±1°C of strains of lactobacilli and bifidobacteria in the medium and packing of liquid product with account of daily dose necessary for patients. The medium contains components in the following quantities: caseine hydrolysate dissolved by distilled water, 0.33-0.4 g/l, sodium chloride 5 g/l, fructose 10 g/l, peptone 2 g/l, agar-agar 0.75 g/l for lactobacilli, for bifidobacteria - 1.0 g/l, ascorbic acid 0.25 g/l, distilled water 0.67-0.6 g/l. Strains-producers are Lactobacillus plantarum 8 RA-3, Lactobacillus fermentum 39, Lactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Bifidobacterium bifidum 1. For lactobacilli and bifidobacteria they prepare accordingly media with different content of agar-agar, amine nitrogen in caseine hydrolysate 160-170 and 180-200 mg% and medium pH 7.8-8.0 and 8.5-8.6. Starting from the first generation the bifidobacteria and lactobacilli are cultivated for 24±1 hours, two strains together, one separately. The produced biomass is mixed with the fresh nutrient medium at the ratio of 1:1000, and strains of lactobacilli are cultivated 24±1 hours, strains of bifidobacteria - 48±1 hours. Upon completion of cultivation of biomass of strains they are mixed at the ratio of 2:1:2:1.

EFFECT: production of a product with high content of live microbial cells.

3 tbl

Description

The invention relates to medical and food biotechnology 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.

At present, bacterial preparations and biologically active food additives (BAA) from living strains of lacto- and bifidobacteria have been developed and are widely used both in dry (powders, tablets, etc.) and in liquid forms.

The advantages of dry freeze-dried forms include a long shelf life (up to several years), ease of use and implementation. But during freeze-drying, the structure of surface adhesion proteins is violated, which reduces the colonization ability of bacteria, and also most of the valuable metabolites that positively affect the microflora recovery process are destroyed. In liquid forms of probiotics, the cells are in a physiologically active state and are able to actively colonize the intestine within 2 hours after ingestion, the structure of adhesins is not broken, valuable bacterial metabolites, in particular, organic acids, such as acetic, lactic, vitamins B, C, are preserved , K, which, getting into the intestine, change the properties of the medium in it, which has a beneficial effect on the development of its own microflora and inhibits pathogenic and conditionally pathogenic microorganisms (Bondarenko V.M., Shaposhniko wa LI Clinical effect of liquid probiotic biocomplexes containing physiologically active cells of bifidobacteria and lactobacilli, 2007)

Liquid probiotic preparations are known both on the basis of whole cow's milk and on the basis of hydrolysates, the composition of the strains of lacto and bifidobacteria used as starter cultures. See, for example, a method for preparing a therapeutic and prophylactic drug based on skim milk hydrolyzate (skim milk) from living strains of microorganisms “LB-complex” (RF patent No. 2192269, А61К 35/74, А23С 9/127, С12N 1/20, С12R 1:07, 1: 225, 1:25, published 10.11.2002).

The method includes 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.

Use the HMS the following composition:

Undiluted milk hydrolyzate 1000 ml Sodium chloride 5 g Lactose 10 g Peptone 2 g Agar agar 750 mg Vitamin C 230-250 mg

pH 8.5-8.6

But, firstly, the feedstock used in this method for preparing the hydrolyzate — milk inverse (skim milk) —in the quantitative content of casein protein is an unstable product, and hydrolysis in this way does not provide complete cleavage of casein, in this regard, the resulting intermediate (base medium) has a low level of amino nitrogen (150-160 mg%), which leads to the use of undiluted skim milk hydrolyzate in this way.

Secondly, the use of lactose in the formulation of the medium in an amount of 10 g per liter makes it impossible to use the drug prepared by this method in people with lactase deficiency.

Lactase deficiency is the most common variant of malabsorption syndrome in children. According to Russian researchers, she is responsible for 70% of long-term diarrhea in infants. Lactase deficiency is understood to mean reduced activity of intestinal lactase, an enzyme of parietal digestion that breaks down lactose disaccharide to monosaccharides - glucose and galactose. The enzyme is synthesized by mature enterocytes located on top of intestinal villi. Lactase deficiency in children in Russia occurs in 10-75% of cases depending on the nationality of the population (Samal T.N., Ukraintsev S.E. BSMU. Published in the journal ″ Medical Panorama ’, No. 2 for 2004).

As a prototype, a method for the preparation of a therapeutic and prophylactic preparation based on live strains of the microorganisms lactobacilli and bifidobacteria "LB-complex L" (RF patent No. 2441907, C12N 1/20, А61К 35/74, А23С 9/127, publ. 10.02. 2012).

The method includes cultivating a mixture of microorganism strains on a nutrient medium containing a nutrient base — casein hydrolyzed by the pancreas of cattle and distilled water to dilute the fermented hydrolyzate to a predetermined amine nitrogen content as a carbohydrate component containing lactose and raffinose in a 1: 1 ratio. and raffinose is both a carbohydrate component and a prebiotic component. The mixture was added to strains of sorbing component prepacked rate of _^1/2 - _^1/4 part of the total set up an integrated system. The process of immobilization on the sorbent carrier of dietary supplements for food “Litovit M” occurs at a temperature of + 6 ° ± 2 ° C for 18-24 hours. L.plantarum 8 RA-3, L.fermentum 39, L are used as producer strains .fermentum 90 TC-4, B.bifidum 1, B.bifidum 791, B.longum 379. The method allows to obtain an immobilized preparation - a synbiotic with a high content of live microbial cells of several strains of lactobacilli and bifidobacteria, which has an increased protective therapeutic effect.

The disadvantage of this method is the use of lactose as a carbohydrate component in an amount of 5 g per liter and a lengthy three-stage process for scaling the biomass of producer strains with a high concentration of living microbial cells 10 ¹⁰ -10 ¹² CFU / ml, taking a total of 84 hours.

This disadvantage is eliminated by the proposed solution.

The problem to be solved is the improvement of the method for preparing a hypoallergenic lactose-free multicomponent probiotic. EFFECT: obtaining a synbiotic preparation for children with fermentopathies (lactase deficiency as a variant of malabsorption syndrome) by replacing the carbohydrate component of lactose with natural fruit sugar fructose (synonyms: levulosis, b-D-fructofuranose), while reducing the time of the technological process.

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 at a temperature of 37 ± 1 ° C on a nutrient medium containing a nutrient base - casein hydrolyzate diluted with distilled water, sodium chloride, carbohydrate component , agar-agar, acidic component, L. producerarum 8 RA-3, L.fermentum 39, L.fermentum 90 TC-4, B. bifidum 791, B.longum 379, B. bifidum 1, are used as producer strains liquid product packaging, taking into account the necessary for patients with a daily dose, for the cultivation of lactobacilli and bifidobacteria, two different media are used: one medium for lactobacilli with an amine nitrogen content of 160-170 mg%, the other for bifidobacteria - 180-200 mg%, different agar-agar contents - 750 ± 10 mg / l and 1000 ± 10 mg / l, different pH of the finished medium 7.8 - 8.0 and 8.5 - 8.6, respectively, as the carbohydrate component in both environments use fructose diluted in the medium for lactobacilli strains of L. plantarum 8 RA-3 and L. fermentum 39 are combined in a 1: 1 ratio and 24 ± 1 h are cultivated together, the obtained biomass of the first the 1st generation is mixed with fresh nutrient medium in a ratio of 1: 1000 and cultivation is continued for 24 ± 1 h, and the strain L. fermentum 90 TC-4 is cultivated separately using the same method, strains B. bifidum 791 and B. longum 379, diluted in a medium for bifidobacteria, they are combined in a ratio of 1: 1 and cultivated for 24 ± 1 h, the obtained biomass of the first generation is mixed with fresh nutrient medium in a ratio of 1: 1000 and cultivation is continued for 48 ± 1 h, and strain B. bifidum 1 is cultivated according to the same method separately, at the end of the cultivation of biomass strain s grown on various media are mixed in a ratio of 2: 1: 2: 1

Thus, an optimal complex has been proposed, which allows reducing the process time to 72 hours (i.e., 1.2 times) to obtain a hypoallergenic, lactose-free, multi-component probiotic in the form of a bacterial concentrate with a high content of living microbial cells of all 6 starter cultures per unit volume with a long shelf life.

The method is as follows:

1. Prepare a single intermediate product - the basis of the nutrient medium according to the following scheme: casein is diluted in warm drinking water, ground cattle pancreas is added, the temperature is adjusted to 48 ± 1 ° C, pH is adjusted to 10-20% with NaOH solution, mixed, 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.

2. Next, to prepare the hydrolyzate-casein medium GKS-L, the intermediate prepared according to claim 1 is diluted with distilled water to amine nitrogen 160-170 mg%, agar-agar 750 ± 10 mg / l, sodium chloride, peptone, fructose are added , ascorbic acid, set the pH to 10-20% NaOH solution and sterilized, the pH of the finished SCS-L should be 7.8-8.0.

3. To prepare the hydrolyzate-casein medium GKS-B, the intermediate prepared according to claim 1 is diluted with distilled water to amine nitrogen 180-200 mg%, agar-agar 1000 ± 10 mg / l, sodium chloride, peptone, fructose are added, ascorbic acid, set the pH to 10-20% NaOH solution and sterilized, the pH of the finished medium GKS-B should be 8.5-8.6.

Cultivation of the producer strains of lactobacilli and bifidobacteria is carried out on different media according to a single method - two strains together, one separately in two stages so that the time spent on building up the biomass of lactobacilli is 48 ± 1 h, and bifidobacteria - 72 ± 1 h, thus, the whole process takes 72 hours. At the end of the cultivation, the biomass of the strains is mixed in a ratio of 2: 1: 2: 1 and packaged in vials. Then the bottles are marked, packaged in boxes of 25 pieces (minimum course of treatment).

An example 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 ± 1 ° C, casein is poured (food acid casein according to OST 4960-74) in an amount of 60-70 g per liter, stirred , adjust the pH to 7.8-8.2 IU with a 20% NaOH solution, put in a thermostat at 48 ± 1 ° С for two hours to swell, then add the baked cattle pancreas (GOST 11285-93) in an amount of 60- 70 g / l and chloroform in an amount of 10 ± 0.5 ml / l, adjust the pH to 7.9-8.0 with a 10-20% NaOH solution and put in a thermostat at a temperature of 48 ± 1 ° 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 /% amine nitrogen. The hydrolyzate is stored for future use under chloroform 1% by volume at a temperature of 4 ± 10 ° C.

Two nutrient media GKS-L and GKS-B are prepared from the obtained intermediate product according to the following formulations (Table 1.).

To prepare GCS-L, the whole hydrolyzate is diluted with distilled water to an amine nitrogen index of 160-170 mg /%. 5 g of sodium chloride, 2 g of peptone, 10 g of fructose, ascorbic acid 0.24 ± 0.1 g and 0.75 ± 0.1 g of previously standardly prepared agar agar are added to the diluted hydrolyzate. NaOH 20% solution is added to establish a pH of 7.8-8.0.

To prepare GCS-B, the whole hydrolyzate is diluted with distilled water to an amine nitrogen index of 180-200 mg /%. 5 g of sodium chloride, 2 g of peptone, 10 g of fructose, ascorbic acid 0.24 ± 0.1 g and 1.0 ± 0.1 g of previously standardly prepared agar agar are added to the diluted hydrolyzate. Add NaOH 20% solution to establish a pH of 8.5-8.6.

Ready-made media GKS-L and GKS-B are sterilized according to a single method at 0.5 atm. 30 minutes.

The use of a standard product, acid casein, for the preparation of the hydrolyzate provides a high level of amine nitrogen of the resulting intermediate - 450-500 mg%, which allows the intermediate to be diluted to the required level of amine nitrogen in nutrient media, which provides a high biomass yield of the drug.

Ascorbic acid is used as a growth promoter 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.

Fructose - a carbohydrate - an energy substrate and a carbon source.

Sodium chloride maintains the optimal osmotic pressure of cells.

As producer strains, L.plantarum 8RA-3, 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 the following method:

Generation I: lactobacilli - add 1 ml of GCS-L to ampoules with dry strains of lactobacilli L.plantarum 8RA-3 and L.fermentum 39, then the contents of both ampoules are mixed in a bottle with 18 ml of GCS-L; dry strain L.fermentum 90TC-4 is diluted with 1 ml of GCS-L, then the contents of the ampoule are transferred to a bottle with 9 ml of GCS-L.

Strains of bifidobacteria are prepared according to the same method on GKS-B medium, strains B.bifidum 791 and B.longum 379 are mixed in a bottle, B.bifidum 1 is diluted separately. Then the producer strains are cultured for 24 + 1 h at a temperature of 37 ± 1 ° C (table. 2).

II generation - the obtained biomass of the 1st generation is each separately mixed with fresh nutrient medium (GCS-L for lactobacilli and GCS-B for bifidobacteria) in a ratio of 1: 1000 (i.e. 10 ml of the 1st generation are added to 9.99 l of medium and receive 10 l of the second generation) and continue to cultivate lactobacilli for 24 ± 1 h at a temperature of 37 ± 1 ° C, bifidobacteria for 48 ± 1 h at 37 ± 1 ° C (Table 2).

At the end of cultivation, the obtained biomass is mixed in a ratio of 2: 1: 2: 1.

A mixture of biomass of starter cultures of lactobacilli and bifidobacteria is poured into sterile vials of 2.0, 2.5, and 5.0 ml, taking into account the daily dose of the probiotic.

Hermetically sealed with a rubber stopper and an aluminum cap. 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 (Table 3).

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, “LB-COMPLEX plus” is introduced into any adapted artificial mixture in the amount of 2-2.5 ml per day (fractionally for 3 feedings).

Indications for use:

- as a probiotic component of diet therapy for any diseases complicated by intestinal dysbiosis, as a means of normalizing microflora: with prolonged treatment with antibiotics, chemo and hormonal drugs; with allergic diseases (allergic dermatoses, eczema, etc.); in case of chronic diseases of the gastrointestinal tract, acute intestinal infections of bacterial and viral etiology (desinteria, coli enteritis, salmonellosis, acute intestinal infections of unknown etiology, company and enterovirus infection, etc.), foodborne infections; and etc.

It can be used for lactase deficiency and diabetes mellitus, as well as against the background of antibiotic therapy, taking into account the pharmacokinetics and pharmacodynamics of the antibiotic / chemotherapy.

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 City Clinical Hospital No. 27 "Aibolit", MLPU "Children's Clinic No. 49" Prioksky Rayzdravotdela, State Government Healthcare Institution Nizhny Novgorod Region "Nizhny Novgorod Specialized Orphanage".

The indicated results were confirmed. Obtained by the proposed method, the drug is conditionally named "LB-COMPLEX plus."

The tables show that the replacement of the carbohydrate component did not affect the number of living microbial cells per unit volume of the finished probiotic and the safety of the drug.

Claims (1)

A method of preparing a therapeutic and prophylactic drug from live strains of the microorganisms lactobacilli and bifidobacteria by culturing them at a temperature of 37 ± 1 ° C on a nutrient medium containing a nutrient base: casein hydrolyzate diluted with distilled water, sodium chloride, carbohydrate component, peptone, agar-agar, the acidic component is ascorbic acid, distilled water, Lactobacillus plantarum 8 RA-3, Lactobacillus fermentum 39, Lactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Bif are used as producer strains bifidum 1, the packaging of the liquid preparation, taking into account the daily dose necessary for patients, while fructose is used as the carbohydrate component of the nutrient media in the following ratio of components:
casein hydrolyzate 0.33-0.4 g / l distilled water 0.67-0.6 g / l agar agar 0.75-1.0 g / l sodium chloride 5 g / l fructose 10 g / l peptone 2 g / l vitamin C 0.25 g / l
for the cultivation of lactobacilli and bifidobacteria, respectively, a medium is prepared with an agar-agar content of 0.75 and 1.0 g / l, amine nitrogen in casein 160-170 and 180-200 mg% diluted with distilled water and the pH of the prepared medium is 7.8-8.0 and 8.5 -8.6, Lactobacillus plantarum 8 RA-3 and Lactobacillus fermentum 39 strains diluted in the medium for lactobacilli are combined in a 1: 1 ratio and cultivated for 24 ± 1 h, the first biomass obtained is mixed with fresh nutrient medium in a ratio of 1: 1000 and cultivation continues within 24 ± 1 h, and the strain of Lactobacillus fermentum 90 TC-4 culture strain according to the same method, strains of Bifidobacterium bifidum 791 and Bifidobacterium longum 379, diluted in a nutrient medium for bifidobacteria, are combined in a ratio of 1: 1 and cultivated for 24 ± 1 h, the obtained biomass of the first generation is mixed with fresh nutrient medium in a ratio of 1: 1000 and continue cultivation for 48 ± 1 h, and the Bifidobacterium bifidum 1 strain was cultivated separately in the same way, after the cultivation of the biomass of the strains grown on various media, mixed in a ratio of 2: 1: 2: 1.