RU2190015C1 - Strain lactobacillus brevis ba-13 used for probiotic preparations and foodstuffs preparing - Google Patents

Abstract

FIELD: biotechnology, medicine. SUBSTANCE: invention relates to the production of probiotic preparation. The strain of lactobacilli Lactobacillus brevis BA-13 (collection of The State scientific-research institute of standardization and control of medicinal biological preparations named for L.A. Tarasevitch, number 261) is isolated from stomach mucosa scrape of health adult man. The strain shows good expressed antagonistic activity with respect to pathogenic microorganisms, for example, staphylococci, klebsiellas, chelicobacteria and fungi of genus Candida. The strain Lactobacillus brevis BA-13 shows sufficient activity of acid formation (120 T), resistance to antibiotics: cefoxitin, cefotetane, tetracycline, metronidazol, teicoplanin, valcomycin. The strain Lactobacillus brevis BA-13 shows high technological indices. Culturing the strain in fermenter in casein- yeast medium and the following concentration of culture and lyophilic drying with addition of sucrose-gelatin medium ensures to obtain biomass with concentration of cells up to 1 x 10¹¹ CFU/g. EFFECT: strain indicated above, valuable properties. 2 tbl

Description

 The invention relates to biotechnology and can be used in medicine in the manufacture of medications to normalize the intestinal microflora and vagina in dysbiotic conditions of various etiologies in children and adults, as well as in the food industry in food production.

 Currently, probiotic - lactobacterin is prepared from the microbial mass of live, antagonistically active strains of Lactobacillus plantarum 8P-A3 and Lactobacillus fermentum 90T-S4, Lactobacillus fermentum 39, Lactobacillus plantarum 38 (see FS 42-3241-95, tablets Lactobacillus.

 However, it is known that other species of lactobacilli, in particular, Lactobacillus brevis, also take part in the realization of the positive role of human normoflora. In addition, the mechanism of the protective effect of certain types of lactobacilli is not the same, which is why it is advisable to use other types of lactobacilli, which are representatives of normoflora, for the production of a drug from lactobacterin.

 The objective of the invention is to find a strain of microorganisms of the species Lactobacillus brevis, which has high adhesive properties to vaginal and gastric epithelium, high antibiotic and proteolytic activity, as well as the necessary activity of acid formation.

 A new strain of Lactobacillus brevis BA-13 isolated from scraping the gastric mucosa of a healthy adult.

 The strain is deposited in the collection of the State Research Institute of Standardization and Control of Medical Biological Preparations. L.A. Tarasovich at number 261.

 Cultural and morphological characters.

 Polymorphic gram-positive sticks with a length of 3.5 to 6.0 μm with a thickness of 1.2 μm, thickened, straight, short, sometimes approaching a cocciform shape or slightly curved with rounded ends. They are motionless, do not form a spore, can be arranged in pairs or form short chains.

It grows on a Blaurock liquid medium containing glucose at 37 ^° C (incubation for 24 hours) in the lower half of the medium column with the formation of a flocculent precipitate without gas formation.

On plates with a dense nutrient medium, after 18-24 hours, the colonies form convex, slightly brilliant, with a diameter of 0.8-1.6 mm. At 15 ^o With there is a steady growth of colonies. At 45 ^o With the growth of colonies is practically absent. The optimal temperature of cultivation (37 ± 1) ^o C. The method of biomass accumulation: hepatic medium Blaurock.

 The drying medium is sucrose-gelatin.

It is stored in a lyophilized form at a temperature of 0-8 ^o C.

 Physiological and biochemical characteristics.

 Anaerobic (optional anaerobic).

 Data on enzymatic activity are shown in table 1.

 Catalase does not form, gelatin does not thin, indole and hydrogen sulfide does not form.

Ferments milk after 12 hours (at 37 ^o C) with the formation of a dense, even clot without syneresis.

The activity of acid formation of the strain is 120 ^o T.

Biotechnological characteristics
The strain produces organic acids, (mainly lactic acid) and antibiotic-like substances (bacteriocins), which provide manifestations of antagonistic activity against pathogenic and opportunistic microorganisms. Growing the strain and freeze drying provides biomass with a concentration of 1 • 10 ¹¹ microbial bodies / g

 Antagonistic properties.

 Antagonistic activity was studied in relation to staphylococci, hemolysing Escherichia coli, cytrobacteria, salmonella, helicobacteria and fungi of the genus Candida. As indicator strains, reference strains of the above conditionally pathogenic microorganisms were used, as well as strains isolated from children and adults during examination for dysbiosis and from patients with various pathologies of the gastrointestinal tract.

 The antagonistic activity of the strain of lactobacilli Lactobacillus brevis BA-13 is shown in table 2.

 Antibiotic resistance of the strain.

 Using a test to determine the sensitivity of microorganisms to antibiotics ATV ANA 14 260 of Bio Merieux company containing 25 antibiotics, it was found that the strain is resistant to cefoxitin, cefotetan, tetracycline, metronidazole, teicoplanin, vancomycin.

 Acute and chronic toxicity.

 In the study of acute and chronic toxicity in mice weighing 15-18 g, it was shown that the strain does not have toxic properties.

The lyophilized biomass of lactobacilli of the strain Lactobacillus brevis BA-13 with a titer of 10 ⁶ -10 ¹⁰ CFU / g was used as a probiotic preparation for the correction of vaginal microbiocenosis and treatment of gastric and duodenal ulcer.

 Example 1

Patient D., 58 years old, complained of itching, copious discharge from the genitals. Considers herself a patient during the last 3 years after the onset of menopause. She was treated several times with antibiotics. When smear microscopy: white blood cells up to 60 in the field of view, a moderate amount of desquamated epithelium, many coccal forms. Diagnosis: chronic colpitis. Prescribed vaginal suppositories weighing 1.5 g, each containing Lactobacillus brevis BA-13 in an amount of at least 1 • 10 ⁷ CFU, 3 times a day for 2 days, then 2 times a day for a week. Against the background of the treatment, the symptoms of itching disappeared on the 3rd day, hyperemia and discharge decreased. On the 10th day in the smear: in the field of view the leukocytes are single, the epithelium is a moderate amount, there are a lot of lactobacilli. Recommended for prophylactic purposes to repeat the course after 2-3 months.

 Example 2

Patient G., 23 years old, complained of itching in the vagina, sometimes very intense. I took antibiotics for a long time about the inflammatory process in the sinuses. On examination: on the mucous membranes of the vagina, a white curdled plaque, easily removable; the discharge is scarce. In a smear: budding yeast cells were detected. Diagnosis: mycotic vaginitis. Prescribed treatment: tampons, impregnated with a suspension of the drug Lactobacillus brevis strain BA-13 with a titer of 1 • 10 ⁷ CFU / ml, morning and evening for a week. On the 3rd day of treatment, clinical symptoms regressed. On day 7, smear microscopy did not detect yeast cells.

 Example 3

 Patient L., age 62 years. For the first time, the diagnosis of duodenal ulcer (ULC) was made in 1981. After conservative inpatient treatment, remission was observed for 12-13 years. Subsequently, the annual autumn-winter exacerbations, stopped by taking phosphalugel (almagel), raglan, vikalin.

In the fall of 1998, he complained of heartburn, belching, pronounced pain in the epigastric region, and lack of appetite. Prescribed treatment: oral administration of the drug lactobacilli at a dose of 0.9 g with the content in this number of viable cells of lactobacilli 5 • 10 ⁷ . Freshly prepared suspension of freeze-dried strain VA-13 30-40 minutes after eating 3 times a day and bifidumbacterin 3 packets of 1 g 3 times a day 15 minutes before eating. Within 3 hours after taking the drugs, a decrease in the intensity of dyspeptic phenomena, a restoration of appetite was observed. A weekly intake of drugs completely stopped exacerbation of YABDK.

 Example 4

 Preparation of a fermented milk product for therapeutic purposes using the proposed strain of Lactobacillus brevis BA-13 as a monoculture.

To prepare the product, we used the dry biomass of Lactobacillus brevis strain BA-13 with a concentration of 2 • 10 ⁸ CFU / g. In sterilized milk with a temperature of 36-38 ^o With contribute 5% of the starter culture of this strain, the mixture is stirred and kept at this temperature until a clot forms. Fermentation of milk occurs after 12 hours with the formation of a clot with an acidity of 75 ^o T (pH = 4.6 units). After ripening, the product is cooled to 20 ^o C, bottled, and then cooled to 6 ^o C. The finished product has a uniform, liquid consistency, a clean pleasant sour-milk taste with a slight smack of baked milk, acidity - 80 ^o T, contains 1 cm ³ 5 • 10 ⁸ viable cells of the specified strain.

Manufacturing sourdough based on this strain is prepared in sterilized milk from dry sourdough. The dose of inoculation of dry sourdough is 1 • 10 ⁶ CFU / cm ³ , the temperature of preparation of the sourdough is 37-38 ^o C.

 Example 5

 Preparation of a fermented milk product for therapeutic purposes using the proposed strain of Lactobacillus brevis BA-13 as a component of starter cultures.

In pasteurized milk at a temperature of 37-38 ^o C make 1% starter culture of strain VA-13, containing 8 • 10 ⁸ CFU / cm ³ and 4% activated suspension of bifidobacteria strain 1 with a concentration of 5 • 10 ⁷ CFU / cm ³ . Fermentation of milk occurs after 10 hours, the acidity of the clot at the end of fermentation is 80 ^o T (pH = 4.5 units). The product is cooled to 20 ^o C, poured into a container and further cooled to 6 ^o C. The finished product has a uniform consistency, a clean sour-milk, slightly spicy taste, acidity 85 ^o T, contains 1 cm ³ 8 • 10 ⁸ CFU of sour milk sticks and 5 • l0 ⁷ KOE of bifidobacteria.

 A production starter culture based on the Lactobacillus brevis BA-13 strain is prepared as described in Example 1. The activated biomass of bifidobacteria is prepared according to TU 9222-006-16414608-00.

 Thus, the new strain can be recommended for the preparation of probiotic preparations in the form of suppositories, powder for the preparation of suspensions, in capsule and tablet forms, and therapeutic products.

Claims (1)

 Strain Lactobacillus brevis VA-13 (collection of the State Research Institute for Standardization and Control of Medical Biological Preparations named after L. A. Tarasevich No. 261) used for the preparation of probiotic preparations and food products.

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