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

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.

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The invention relates to medical and food biotechnology and can be used for the preparation of therapeutic and preventive drugs, dietary supplements and food products using as starter cultures of live probiotic strains of microorganisms.

Currently developed and widely used bacterial drugs and biologically active food additives (BAA) of live strains of lacto - and bifidobacteria in dry (powders, tablets etc), and liquid forms.

The advantages of dry lyophilized forms can be attributed to long term storage (up to several years), ease of use and implementation. But when the freeze-drying process affects the structure of surface proteins adhesins that reduces colonization ability of the bacteria, and also destroyed a large part of valuable metabolites, positively influencing the process of restoration of microflora. In the liquid forms of probiotics cells are physiologically active and is able to colonize the intestine in 2 hours after the ingestion, the structure of adhesins not disturbed, nutrient-based saves valuable bacterial metabolites, in particular organic acids: acetic, lactic, vitamins b, C, K, which, once in the intestine, change the properties of the environment, is it beneficial to the development of native flora and inhibits pathogenic and conditionally pathogenic microorganisms (Bondarenko V.M., Shaposhnikova LI Clinical effect of liquid probiotic bio-complexes containing physiologically active cells of bifidobacteria and lactobacilli, 2007)

Known liquid preparations, probiotics on the basis of whole cow's milk, and based hydrolysates, as starter cultures used in the composition of strains of lacto - and bifidobacteria. See, for example, the method of preparation of preventive and curative medicine on the basis of hydrolyzed skim milk (skim milk) from living microorganisms "LB-complex (patent RF №2192269, AK 35/74, AS 9/127, 12N 1/20, 12R 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 the ratio 1:1 at the end of cultivation. The cultivation is performed on hydrolysate-dairy environment (HMS). As the basis HMS use hydrolyzed skim milk and impose additional ascorbic acid in the amount of 230 to 250 mg per 1000 ml of the hydrolysate. The finished drug is Packed in bottles taking into account required daily dose. The method allows to get ready to use the drug, affecting bifido - and lacecommunity microflora of man, with a long shelf life.

Use the GMR of the following composition:

pH 8.5 and 8.6

But, first used in this way the raw materials for the preparation of hydrolysate - skimmed milk milk (skim milk), quantitative protein casein unstable product, and conducting hydrolysis at the specified method does not provide a full breakdown of casein, in this regard, the resulting intermediate (base medium) has a low level of amino nitrogen (150-160 mg%), which causes the application of undiluted skim milk hydrolysate used in this method.

Secondly, the use in the formulation of environment lactose in an amount of 10 g per liter makes impossible the use of the drug prepared in this way, people with lactase deficiency.

Lactase deficiency is the most common syndrome of malabsorption in children. According to Russian researchers,it is responsible for 70% of long-term diarrhea in infants. Under lactase deficiency see reduced activity of intestinal lactase - enzyme parietal digestion that breaks down the disaccharide lactose to the monosaccharides glucose and galactose. The enzyme synthesize Mature enterocytes located on the top of the intestinal villi. Deficiency of lactase in children in Russia occurs in 10-75% of cases, depending on the nationality of the population (Samal T.N., Ukrainians S.E. bgmu. Published in the journal "Medical view", No. 2, 2004).

As the prototype accepted method of preparation of therapeutic and preventive medicine on the basis of live strains of microorganisms lacto - and bifidobacteria "LB-complex L" (patent RF №2441907, C12N 1/20, AK 35/74, AS 9/127, publ. 10.02. 2012).

The method includes culturing a mixture of strains of microorganisms in a nutrient medium containing a nutrient basis - casein, hydrolyzed pancreas of cattle, and distilled water for cultivation fermented hydrolysate to a specified content of amino nitrogen, as the carbohydrate component containing lactose and raffinose in the ratio of 1:1, and raffinose is as a carbohydrate component and prebiotic component. A mixture of strains add to sorbing component, pre-packaged based¹/₂-¹/₄the portion of the total the volume of created a complex system. The process of immobilization on the sorbent carrier BAA "Litovit M" occurs at a temperature of +6°±2°C for 18-24 hours as of strains-producers use L.plantarum 8 RA-3, L.fermentum 39, L.fermentum 90 TC-4, B.bifidum 1, B.bifidum 791, B.longum 379. The method allows to obtain the immobilized drug - synbiotic with a high content of live microbial cells of several strains of lactobacilli and bifidobacteria, which has a higher 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 long three-step process to scale biomass producer strains with a high concentration of live microbial cells 10¹⁰-10¹²CFU/ml, occupying a total of 84 hours.

This disadvantage is eliminated by the proposed solution.

Task - improving method of cooking Allergy-free lactose-free multicomponent probiotic. Technical result - receiving synbiotic preparation for children with fermentopathia (lactase deficiency as a variant of malabsorption syndrome) due to the replacement of the carbohydrate component of lactose in natural fruit sugar fructose (synonyms: levulose, b-D-fructofuranose), reduction of the time of the process.

This result dostigaet the same time, a method for preparing therapeutic and prophylactic drug of live strains of lactobacilli and bifidobacteria by culturing at a temperature of 37±1°C in a nutrient medium containing a nutrient base - hydrolyzed casein, diluted with distilled water, sodium chloride, carbohydrate component, agar-agar, an acidic component, as producer strains used L.plantarum 8 RA-3, L.fermentum 39, L.fermentum 90 TC-4, B.bifidum 791, B.longum 379, B.bifidum 1, the filling of the liquid preparation with the necessary facilities for patients, daily dose, for the cultivation of lactobacilli and bifidobacteria use two different environments: one medium for lactobacilli with the content of amino nitrogen 160-170 mg%, the other for bifidobacteria - 180-200 mg%, different content of agar-agar - 750±10 mg/l and 1000±10 mg/l, different pH ready environment of 7.8 to 8.0 and 8.5 and 8.6, respectively, as the carbohydrate component in both environments use fructose dissolved in the medium for lactobacilli strains L. plantarum 8 RA-3 and L. fermentum 39 are combined in a ratio of 1:1 and jointly cultivate 24±1 h, the resulting biomass the first generation mixed with fresh nutrient medium in a ratio of 1:1000 and continue culturing for 24±1 h, and the strain L. fermentum 90 TC-4 is cultivated in the same way separately, strains Century bifidum 791 a and B. longum 379, diluted in medium for bifidobacteria, the connection is Aut in the ratio of 1:1 and jointly cultivate 24±1 h, the resulting first generation biomass is mixed with fresh nutrient medium in a ratio of 1:1000 and continue culturing for 48±1 h, and the strain Century bifidum 1 cultivated in the same way separately, at the end of cultivation, the biomass of the strains grown on different media, mixed in the ratio 2:1: 2:1

Thus, the proposed optimal set, which allows reduction of the time process up to 72 hours (i.e. by a factor of 1.2) to obtain hypoallergenic, lactose-free multicomponent probiotic in the form of bacterial concentrate with a high content of live microbial cells all 6 starter cultures per unit volume with a long shelf life.

The method is as follows:

1. Prepare a single intermediate - based nutrient medium according to the following scheme: bred casein in warm water, add ground pancreas of cattle, bring the temperature up to 48±1°C, set pH 10-20% solution of NaOH, mix, add chloroform and placed in a thermostat at 72 hours, the first hour is constantly stirred, and then filtered through filter paper. Ready hydrolysate control amine nitrogen.

2. Next to cooking hydrolysate-casein environment GCS-L-bred intermediate prepared according to claim 1., distilled water to minnesoata 160 - 170 mg%, add the agar-agar 750±10 mg/l, sodium chloride, peptone, fructose, ascorbic acid, establish pH 10-20% solution of NaOH and sterilized, pH ready GCS-L must be of 7.8 to 8.0.

3. To prepare hydrolysate-casein environment GKS-bred B intermediate prepared according to claim 1., distilled water to amine nitrogen 180-200 mg%, add the agar-agar 1000±10 mg/l, sodium chloride, peptone, fructose, ascorbic acid, establish pH 10-20% solution of NaOH and sterilized, pH ready environment GKS-B should reach 8.5 and 8.6.

Cultivation of strains producing lactobacilli and bifidobacteria are conducted in different environments on a single method - two strains together, separately in two stages so that the time spent on increased biomass of lactobacilli is 48±1 h and bifidobacteria - 72±1 h, thus, the whole process takes 72 hours. After cultivation, the biomass of the strains are mixed in the ratio 2:1:2:1 and filled into vials. Then the bottles are labeled, Packed in boxes of 25 pieces (minimum treatment).

An example of the method

In the first stage, preparing for the future intermediate - hydrolyzed casein as follows: drinking water (GOST 2874-82) is heated to 48±1°C, poured casein (casein, edible acid according to OST 4960-74) in the amount of 60-70 grams per liter, stirred, adjusted pH to 7.8 to 8.2 IU 20% solution of the m NaOH, put in a thermostat at 48±1°C for two hours to swell, then add promolotogo pancreas of cattle (GOST 11285-93) in the amount of 60-70 g/l and chloroform in an amount of 10±0.5 ml/l, adjusted pH to 7.9-8.0 a 10-20% solution of NaOH and placed in a thermostat at a temperature of 48±1°C. during the first hour the contents several times mix (tube after shaking pierce to remove vapors of chloroform) and leave for carrying out the process of hydrolysis for 72 hours after exposure in thermostat decant the supernatant through a paper filter. Ready hydrolyzate must contain 450-500 mg/% amino nitrogen. Keep hydrolysate for the future under chloroform 1% by volume at a temperature of 4±10°C.

From the obtained intermediate is prepared two nutrient medium GCS-L and SCS-B according to the following formulations (table 1.).

For the preparation of CSG-L bred solid hydrolyzed with distilled water to amine nitrogen 160-170 mg/%. In the diluted hydrolysate added per 1 l of 5 g of sodium chloride, 2 g peptone, 10 g fructose, ascorbic acid 0.24±0.1 g and 0.75±0.1 g pre-standard prepared agar-agar. Add NaOH 20% solution to establish a pH of 7.8 to 8.0.

For the preparation of SSC-bred B solid hydrolyzed with distilled water to amine nitrogen 180-200 mg/%. In the diluted hydrolysate type of R is to 1 liter of 5 g of sodium chloride, 2 g of peptone, 10 g fructose, ascorbic acid 0.24±0.1 g and 1.0±0.1 g pre-standard prepared agar-agar. Add NaOH 20% solution to establish a pH of 8.5 and 8.6.

Ready environment GCS-L and SCS-B are sterilized in a unified way at 0.5 ATM. 30 minutes.

Use for the preparation of the hydrolysate standard product acid casein, provides a high level of amino nitrogen of the resulting intermediate - 450-500 mg%, which allows to dissolve the intermediate to the desired level of amine nitrogen in the nutrient environments, providing a high biomass yield of the drug.

Ascorbic acid is used as a stimulant for the growth of 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, resulting in uniform growth of microorganisms through the thickness of the medium and uniform consumption growth factors and nutrients.

Fructose is a carbohydrate energy substrate and the carbon source.

Sodium chloride maintains the optimal osmotic pressure of the cell.

As producer strains used L.plantarum 8RA-3, and L.fermentum 90-TS-4 and L.fermentum 39, B.bifidum 791, B.bifidum 1, B.longum 379.

Cultivation of strains producing lactobacilli and bifida is clear is performed in the following way:

First generation: lactobacilli in capsules with dry lactobacilli strains L.plantarum 8RA-3 and L.fermentum 39 add 1 ml of CSG-L, then the contents of both vials are mixed in a bottle with 18 ml of CSG-L; dry strain L.fermentum TS-4 bred 1 ml GCS-L, then the contents of the ampoule is transferred into the vial containing 9 ml of SSC-HP

Strains of bifidobacteria are prepared in the same way on the environment GKS-B strains B.bifidum 791 and B.longum 379 mix in a bottle, B.bifidum 1 bred separately. Then the strains-producers cultured for 24+1 hours at a temperature of 37±1°C (table. 2).

II generation - derived biomass I generate each separately mixed with fresh nutrient medium (GCS-L for lactobacilli and SCS-B for bifidobacteria) in the ratio 1:1000 (i.e. 10 ml of I generation make 9,99 l environment and receive a 10 l II generation) and continue to cultivate lactobacilli for 24±1 hours at a temperature of 37±1°C, bifidobacteria within 48±1 hours at 37±1°C (table 2).

After culturing the obtained biomass are mixed in the ratio 2:1:2:1.

Pour the mixture of biomass starter cultures of lactobacilli and bifidobacteria in sterile vials 2,0, 2,5, 5,0 ml given a daily dose of the probiotic.

Sealed with a rubber stopper and aluminium cap. Then the bottles are labeled, Packed in boxes of 25 pieces (minimum treatment). Store the drug at the temperature of +6±2°C up to 60 days from the safety of the living microbial cells in 1 ml of liquid preparation 10 ¹⁰-10¹²CFU/ml (table. 3).

The drug can be obtained reactor method.

The drug can be used orally in 1 or 2 doses 1 bottle a day before meals with water, juice, juice, juice, etc. with temperature not exceeding 30°C. Before use, the bottle of medication was thoroughly shaken.

For children in their first months of life "LB-COMPLEX plus" is entered in any adapted artificial mixture in the amount of 2-2,5 ml per day (fractional to 3 feeding).

Indications for use:

as the probiotic component of the diet in all diseases, complicated by intestinal dysbiosis, as a means of normalizing microflora: prolonged treatment with antibiotics, chemotherapy and hormonal therapy; allergic diseases (allergic, eczema and the like), chronic gastrointestinal diseases, acute intestinal infections of bacterial and viral etiology (dezinteria, coli-enteritis, salmonellosis, OKA unknown etiology, Rota-and enterovirus infection and others), food poisoning; and others

Can be used with lactase deficiency and diabetes, as well as on a background of antibacterial therapy with consideration of the pharmacokinetics and pharmacodynamics of antibiotic/chemical.

Clinical testing of probiotic prepared according to the method presented in the VCE, was conducted in the following therapeutic-prophylactic institutions of the city N. Novgorod: at Children's City clinical hospital №27 "Aibolit", at "Children's polyclinic №49" Priokskaya of district medical Department, Public health institution of the Nizhny Novgorod region Nizhny Novgorod specialized children's home".

These results were confirmed. Received the proposed method, the drug is conventionally called "LB-COMPLEX plus".

The tables indicate that replacing carbohydrate component did not affect the number of live microbial cells in a unit volume of the finished probiotic and on the safety of the drug.

The method of preparation of the treatment-and-prophylactic preparation of live strains of lactobacilli and bifidobacteria by culturing at a temperature of 37±1°C in a nutrient medium containing a nutrient basis: casein hydrolysate, diluted with distilled water, sodium chloride, carbohydrate component, peptone, agar-agar, an acidic component is ascorbic acid, distilled water, as strains producing use of Lactobacillus plantarum 8 RA-3, Lactobacillus fermentum 39, Lactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Bifidobacterium bifidum 1, the filling of the liquid preparation with regard to heart and soul is for my patients daily dose, at the same time as the carbohydrate component of the nutrient media use fructose in the following ratio of components: