RU2163131C2 - Method of preparing composition "enterogs" for therapeutic destination and production of therapeutic and dietetic nutrition preparation "bifilakt" - Google Patents

Abstract

FIELD: food industry and medicine. SUBSTANCE: invention provides composition called "Enterogs" that can be used as therapeutic and preventive agent in cases of gastrointestinal and nervous diseases, in oncology, in cases of immunodepressive states, and also in dietetic nutrition of adults and children. Invention consists in single preparation of "Bifilakt" and using it to precipitate proteins of defatted milk. Resulting suspension is filtered and filtrate is supplemented by glucose to concentration 0.2-0.4% to give nutrient medium. The latter is used for separately growing lactic and bifid bacteria, which are added to skimmed milk to concentrations 1.0-1.2 and 0.5-0.6%, respectively. After culturing to morphologic ripeness mixing procedures are used to prepare ferment composition "Enterogs". The latter is used for preparing preparation "Bifilakt TU", which further replaces previously prepared "Bifilakt". Composition is high-quality product for production of dietetic and therapeutic product useful in treatment of dysbacteriosis and s series of associated gastrointestinal diseases, for correction of metabolic disorders, immunodepressive conditions, for treatment of nervous system and blood. EFFECT: Extended assortment of gastrointestinal drugs. 3 tbl

Description

 The invention relates to food industry and medicine and can be used as a therapeutic and prophylactic agent in the complex treatment of gastrointestinal diseases, diseases of the nervous system, oncological diseases, immunosuppressive conditions, food allergies, as well as for the production of therapeutic diet food Bifilact TU 9222- 001-34960875-97, which is also used in the complex therapy of the above conditions and in the diet of adults and children. The widespread use of drug treatment, stress conditions, disturbances in dietary patterns, environmentally unfavorable factors entails a serious problem of cleaning the body, eliminating the side effects of medications, restoring normal microflora, and immunocorrection of the body.

 A known method of producing bifidumbacterin (Pharmacopoeia article 42-132 BC-94, M3 and the medical industry of the Russian Federation, approved July 19, 1994). At the same time, antagonistically active strains of Bifidum bacterium bifidum N 1,791 or LVA-3 are used to obtain the drug.

 For the cultivation of bacteria using synthetic nutrient media Blaurock, nutrient agar with glucose, fish peptone agar, a medium based on blood hydrolyzate. At the same time, bacteria strains are grown on one of these media, which are then tested for biochemical properties, acid formation ability, antagonistic activity, harmlessness, and the number of bacteria in 1 ml. Subject to their compliance with the requirements, the bacteria are placed in a stabilizing drying environment and lyophilized. The resulting preparation is a light yellow powder of dry bacteria with the ingredients of a stabilizing drying environment (sucrose, gelatin, skim milk) and is used as a prophylactic and therapeutic agent for gastrointestinal diseases in a diluted state with water.

The disadvantages of the method include:
- the use of artificial nutrient media, including synthetic components with side effects (peptone, sodium chloride, sodium phosphate disubstituted, iron sulfate, microbiological agar, etc.)
- low therapeutic effect due to the fact that the bacteria are in a biologically and biochemically inactive form, have a low colonizing ability, the introduction of a bacterial preparation into the body does not occur in the natural environment of bacteria, which reduces their activity.

 A known method of producing lactobacterin (Pharmacopoeia article 42-252BC-89, approved 12.02.1989, Ministry of Health of the USSR).

 To obtain dry lactobacterin, antagonistically active strains of L. fermentum 90T-C4 or L. plantarum BP-A3 are used.

 Bacteria are also grown on synthetic media MPC-1, MPC-4, including magnesium sulfate, cysteine hydrochloride, disubstituted potassium phosphate, tween-80, peptone, etc. The technology for obtaining the final product is similar to that described above. As stabilizing media, edible gelatin, sucrose, and nonfat milk are introduced into the product. After drying, the resulting product is a dry powder of light yellow color, which is also used after dilution in water as a prophylactic and therapeutic agent for gastrointestinal diseases.

 The disadvantages of this analogue are the disadvantages characteristic of the above analogue.

 For the prototype, we have adopted a method for producing bacterial compositions for the preparation of Bifilact dietary-nutritional food (Instructions for the preparation of sour-milk Bifilact in dairy kitchens, M, 1987. Approved by the Main Directorate of Medical Treatment of Children and Mothers of the Ministry of Health of the USSR 03/27/87, N 11-14 / 6-33, hereinafter referred to as Bifilact).

 For the preparation of bacterial compositions in the manufacture of Bifilact, dry bifidumbacterin or liquid sourdough from one of the strains of bifidobacteria, dry lactobacterin or liquid sourdough, hydrolyzate-milk medium, milk-yeast medium are used.

 The starter cultures of bifidobacteria and lactobacilli are obtained as follows. For this, a bottle with a lyophilized strain of bifidobacteria is cultured in 75 ml of hydrolyzate-milk medium or Blaurock liver medium for 24 hours. Sourdough from lactobacilli is obtained from dry lactobacterin, which is seeded in a milky-yeast medium, incubated for 20-22 hours.

 Blaurock's nutrient medium for growing bifidobacteria is prepared by combining hepatic broth, dry peptone, sodium chloride, L-cysteine, microbiological agar, subsequent boiling, autoclaving.

Nutrient hydrolyzate-milk medium is prepared on the basis of a skim milk hydrolyzate. To do this, skim milk is brought to a boil, boiled for 2 minutes, cooled to 45 ^o C, set the pH to 8.0-10.0 with 20% NaOH solution, add pancreatin, stir for 4 hours in an oven, constantly adjust the pH. Chloroform is added and left in the oven until morning. After 24 hours, adjust the pH with a 30% solution of acetic acid to 4.5; boil, filter, determine the content of amine nitrogen and chlorides. The cooled hydrolyzate is diluted with water in a 1: 1 ratio, cast and agar-agar is added, and NaCl and peptone are added to the remaining part. Heated, combined with molten agar. Bring to a certain pH, boil, filter, add lactose and hydrochloride cysteine, sterilize by autoclaving.

 The medium for lactobacilli is prepared on the basis of a milk hydrolyzate in the manner described above, yeast autolysate and glucose are added to it.

 The composition of bacteria for the preparation of Bifilact is obtained by mixing lactobacilli and bifidobacteria in a ratio of 2: 1, respectively.

The disadvantages of the prototype:
-the complexity of the implementation of the method in connection with a large number of technological operations;
- non-compliance with environmental cleanliness in connection with the use of synthetic additives in the cultivation of bacteria;
- the high cost of the resulting composition due to the complexity of the technology and the large number of ingredients used;
- the unreliability of the quality indicators of the resulting product due to the lack of control over the maturation of biomass;
- low activity of the resulting biomass
The aim of the invention is to improve the quality of microbial compositions.

Tasks:
- increase the biochemical activity of biomass;
- obtaining morphologically mature biomass;
- an exception to the technology for the production of synthetic additives and preservatives;
- reduction in cost and labor.

 The essence of the invention is to obtain a composition for the production of therapeutic and dietetic food by using Bifilact as a precipitant of milk proteins to obtain bacteria growth media, provided it is introduced into skim milk, followed by filtration, separate cultivation in a filtrate with glucose in a concentration of 0.2-0, 4% of lactobacilli and bifidobacteria, mixing them in a ratio of 2: 1 (to a concentration of 1.0-1.2% of lactobacilli and 0.5-0.6% of bifidobacteria in milk upon receipt of the product - Bifilact TU 9222-001-34960875- 97) for microbial composition.

 The resulting composition, the authors assign the name "Enterogs".

The method of obtaining the composition Enterogs is as follows:
A) Prepare a nutrient medium for growing lactobacilli and bifidobacteria, for this:
1. prepare Bifilact milk protein precipitant according to the prototype, for this it is necessary:
- cow's milk is harvested in accordance with GOST 13277-79 not less than 1 grade, withstanding alcohol test with ethyl alcohol 72% concentration;
- dry bifidumbacterin or liquid sourdough from one of the strains of bifidobacteria;
- dry lactobacterin or liquid yeast from one of the strains of lactobacilli;
- hydrolyzate-milk environment;
- milky yeast medium.

 A vial or ampoule with a lyophilized strain of bifidumbacterin is sterilely opened and 3-5 ml of hydrolyzate-milk medium or liver medium of Blaurock are poured.

The contents of the ampoule or vial are shaken vigorously until a uniform suspension is obtained and poured into a vial containing 70 ml of hydrolyzate-milk medium (or liver medium), kept in an thermostat at 37-38 ^o C for 24 hours.

To prepare a starter culture from lactobacilli, use an ampoule with lactobacterin, usually containing 3 doses, dissolve with sterile physiological saline at the rate of 1 ml per 1 dose of the drug, sow in a milky-yeast medium (milk hydrolyzate + 5% yeast autolysate + 0.2% glucose) from calculation of 1:10 lactobacilli per 10 l of medium at a temperature of 37-38 ^o C.

Fermented milk from B. Bifidum 791 in an amount of 1-1.5% is added to milk sterilized at 0.5 atm (110-112 ^o C) for 30 min or boiled for 1 hour and cooled to 37-38 ^o C. simultaneously with the fermentation of lactobacilli in the amount of 2-5% by volume of the product. Fermented milk is thoroughly mixed, poured into sterile bottles, corked and placed in a thermostat at 37-38 ^o C. Hold for 10-16 hours until a clot appears. Get Bifilact.

Bifilact is added to 750 ^o C skim milk in an amount of 750 ml per 2 l of milk as a protein precipitant.

The curdled milk proteins are removed by filtration, and the remaining filtrate is re-pasteurized, a glucose solution is added to a final concentration of 0.2-0.4%. The resulting medium is cooled to a temperature of 35-37 ^o C. In the resulting medium, bifidum and lactobacilli are separately grown. Bifidumbacterin ampoule is added to 250 ml of the obtained medium for growing B.bifidum 791. For L.plantarum cultivation, 8 times, lactobacterin ampoule is added to 250 ml of the obtained medium. Place the medium with bacteria for cultivation at 37 ^o C. After 18 to 20 hours of cultivation, the growth of cultures is monitored microscopically. To do this, 0.2 ml of the bacterial suspension is placed on glass, a smear is made, the smear is stained according to Gram. After 18-20 hours of cultivation in the culture of B. bifidum 791, coccal forms of B. bifidum 791 are observed, and in the culture of L. plantarum 8 TIMs, rod and coccal forms of L. plantarum 791 are observed in equal proportions. The culture of L. plantarum 8 PA3 is left to ripen for another 6 hours, and the culture of B. bifidum 791 is 22 hours. During this time, with the next morphological control in the L.plantarum culture 8 TIMES, all lactobacilli of the culture are mainly rod-shaped, the bacteria in the B. bifidum 791 culture also have the appearance of mainly polar-colored polymorphic rods. The cultures are considered morphologically mature and are used to prepare Enterogs compositions by mixing them (in the ratio: 1 part of bifidumbacterium culture and 2 parts of lactobacillus culture; final concentration of bifidumbacterium culture in milk is 0.5-0.6%, and lactobacillus culture is 1.0- 1.2%), then the Enterogs composition is used either for therapeutic purposes or for the preparation of the Bifilact-TU 9222-001- 34960875-97 therapeutic food product.

 To prove the objectives using the proposed composition Enterogs, we obtained a preparation of dietetic food Bifilact TU 9222-001- 34960875-97.

For its preparation apply:
a) cow's skim milk according to GOST 13277 of not less than 1 grade, withstanding alcohol test with ethyl alcohol 72% concentration;
b) the Enterogs composition obtained from lactobacilli and bifidobacteria.

In skim milk pasteurized at 0.5 ATM (110-112 ^o C) for 30 minutes and cooled to 37-38 ^o C make the composition Enterogs (concentration of the culture of B. bifidum 791 - 0.5-0.6%, the concentration of the culture of lactobacilli - 1.0-1.2%). After keeping the fermented milk at 37-38 ^o C receive the drug Bifilact-TU 9222-001-34960875-97.

 Bifilact TU 9222-001-34960875-97 can be used as a protein precipitant instead of Bifilact in the preparation of lacto- and bifidumbacterium growth media in the preparation of Enterogs composition. The resulting preparation Bifilact TU 9222-001-34960875 has the following indicators compared with Bifilact according to the prototype (see table 1).

 In the process of preparing batches of the Bifilact TU 9222-001-34960875-97 dietetic food preparation, we developed optimal glucose concentrations in the growing medium of bifidum and lactobacilli and optimal ratios of suspensions of grown bifidum and lactobacilli to obtain Enterogs composition (Tables 2 and 3) .

 The method of obtaining the Enterogs composition for the production of Bifilact TU 9222-001-34960875-97 was tested under production conditions at AOZT PK Kubanitekhnoservis for 3 months, in over 100 batches.

 Example 1

Bifilact was prepared as described above as a precipitant of milk proteins. 750 ml of Bifilact were introduced into 2 l of pasteurized skim milk heated to 95 ^o C and after 2 minutes the curd protein was filtered, glucose solution was added to the filtrate to a concentration of 0.2%. Got a nutrient medium for growing lactobacilli and bifidobacteria (B. bifidum 791, L.plantarum 8 times). The nutrient medium was divided and a lyophilized culture of bifidobacteria was introduced into one part of the medium, and lactobacilli into the other. Cultivated plants under morphological control to their full maturity. Then, lactobacilli were introduced into skim milk to a concentration of 1.0% and bifidobacteria to a concentration of 0.5% (Enterogs composition), observing a 2: 1 ratio at a temperature of 37 ^o C, fermented milk was kept for 5 hours. Received Bifilact TU 9222-001-34960875-97 was evaluated by the following quality characteristics:
Consistency, appearance - uniform clot, serum 1/10 volume.

 Taste and smell - sour-milk, without extraneous smacks and smells.

 Color - milky white throughout the mass.

Acidity - 60 ^o T.

The content of live microbial cells in 1 ml of the product is 10 ¹⁵ .

 Example 2

Bifilact was obtained as described above as a precipitant of milk proteins. Then 750 ml of Bifilact were introduced into 2 l of pasteurized skim milk heated to 95 ^° C and after 2 minutes the curd protein was filtered, glucose solution was added to the filtrate to a concentration of 0.4%. Got a nutrient medium for growing lactobacilli and bifidobacteria (B. bifidum, L. plantarum). Cultivated plants under morphological control to their full maturity. Then, lactobacilli were introduced into skim milk to a concentration of 1.2% and bifidobacteria to a concentration of 0.6 (Enterogs composition), observing a 2: 1 ratio, at a temperature of 37 ^o C, the fermented milk was kept for 5 hours. Received Bifilact TU 9222-001-34960875-97 was evaluated by the following quality characteristics:
Consistency, appearance - uniform clot, serum 1/10 volume.

 Taste and smell - sour-milk, without extraneous smacks and smells.

 Color - milky white throughout the mass.

Acidity - 60 ^o T.

The content of live microbial cells in 1 ml of the product is 10 ¹⁵ .

 The proposed method for preparing the Enterogs composition does not involve the use of synthetic additives in bacterial growth media; in this regard, the Enterogs composition and Bifilact TU 9222-001-34960875-97 obtained on its basis are environmentally friendly. The maturation rate of Bifilact TU 9222-001-34960875, obtained on the basis of the bacterial composition Enterogs is 2-3 times higher than the Bifilact according to the prototype. The cost of the Enterogs composition is 5-6 times lower than the cost of the starter cultures obtained for the production of Bifilact using synthetic media.

Claims (1)

 A method of producing a composition for the production of therapeutic and dietary food Bifilact, which provides the preparation of a nutrient medium, the separate cultivation of cultures of Bifidobacterium bifidum bifidum 791 and Lactobacillus Lactobacillus plantarum 8 times and subsequent mixing of the obtained starter cultures in a ratio of 1: 2, characterized in that the separate cultivation of bacteria and Bifid lactobacilli are maintained on a nutrient medium obtained by precipitation of skim milk proteins by Bifilact, filtering them and introducing hl into the obtained filtrate goats to a concentration of 0.2 - 0.4%, obtained leaven Enterogs mixed to obtain a composition.

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