RU2292213C2 - METHOD FOR PROBIOTIC PRODUCTION BASED ON 3 COLIFORM BACTERIUM STRAINS Echerichia coli AND FECAL STREPTOCOCCUS STRAIN Enterococcus faecalis - Google Patents

Abstract

FIELD: biotechnology, in particular drug production.

SUBSTANCE: claimed method includes join deep cultivation of three coliform bacterium strains Echerichia coli and fecal streptococcus strain Enterococcus faecalis in casein broth with gelatin for 7-9 h under continuous agitation and aeration and feeding of glucose solution.

EFFECT: multicomponent probiotic with improved specific characteristics such as increased antagonistic activity; decreased cultivation time; increase biomass yield.

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Description

The invention relates to the medical industry, in particular biotechnology, and can be used in the manufacture of medicines.

Currently, drugs consisting of living microorganisms (probiotic preparations) are widely used in medicine and veterinary medicine to normalize the intestinal microflora and increase the general resistance of the body. (Sheveleva SA Probiotics, prebiotics and probiotic products. Current status of the issue. // Nutrition issues. - 1999. - Volume 68. - No. 2. - p. 32-40)

The works of O. Karapetyan and A. Karapetyan are known, as a result of which a biologically active food supplement - bacterial dry Okarin concentrate (TU 550 MU 0583467001-93), designed to normalize the microflora of the gastrointestinal tract, was developed. It included three strains of Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35-3 / 61, and one strain of fecal enterococcus Enterococcus faecalis G-35-4 / 62, registered at the State Research Institute for Standardization and Control of Medical Biological Preparations. L.A. Tarasevich 03.22.83 (passport No. 01-07 / 89) and in the collection of US cultures (1992) under deposit numbers ATCC No. 55343, ATCC No. 55344, ATCC No. 55345, ATCC No. 55346. These strains were isolated from the body of a healthy person, have high adhesive and antagonistic activity against a wide range of pathogenic and opportunistic microorganisms, are resistant to antibiotic therapy and radiation exposure, and have carcinolytic properties.

Closest to the claimed technical essence and the achieved result, selected as a prototype, is a method of obtaining a bacterial dry concentrate Okarin described by Karapetyan OG, Karapetyan A.O., Fanardzhan B.A., Ter-Kazaryan S.Sh. in the article “Improving the uncomfortable state in chronic gastrointestinal diseases by restoring the intestinal microflora using a dairy product” // Sb.auch.tr. Modern cheese making technology and non-waste milk processing. Yerevan, 1989, pp. 310-311 //, approved for use as a biologically active food supplement in 1993

The method includes obtaining uterine cultures of strains in a nutrient medium based on corn extract and stationary separate cultivation of microorganisms in milk, followed by mixing the grown cultures, adding sucrose and gelatin as a protection medium and freeze drying.

The disadvantage of this method is the low quality of the final product: i.e. it was not possible to create an equal number of living microbial cells of each of the used strains, while enterococcus prevailed significantly; the indicator of antagonistic activity of the concentrate with delayed antagonism, expressed in zones of growth inhibition of the test strains, was not more than 10 mm This is due to the fact that with stationary cultivation it is impossible to create standard cultivation conditions in each series: there is no possibility to control the cultivation process with instrumentation; Milk, which is one of the components of the nutrient medium, has a number of disadvantages: non-standard composition in different regions, the presence of antimicrobial agents in milk, lactose content, and contamination by extraneous microflora. In addition, the nutrient medium on the extract of corn is not recommended in the technology for medical immunobiological preparations. The selected conditions of the method of stationary cultivation are not economical, because with a cultivation duration of (18 ± 1) h, the biomass yield from 1 ml of the nutrient medium is not more than 6 · 10 ⁶ live microbial cells of Escherichia coli and fecal enterococcus strains. The index of antagonistic activity of the concentrate with delayed antagonism, expressed in zones of growth inhibition of test strains, is not more than 10 mm.

The problem solved by the invention is the creation of an industrial method of producing a probiotic based on Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35-3 / 61 and fecal enterococcus Enterococcus faecalis G-35-4 / 62.

The technical result from the use of the invention is to obtain a standard preparation with an equal number of living microbial cells of each of the studied strains and to increase the antagonistic activity of the probiotic preparation.

This result is achieved by the fact that in the method for producing a probiotic based on E. coli strains Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35-3 / 61 and fecal enterococcus Enterococcus faecalis G -35-4 / 62 by the method of deep cultivation of uterine cultures in a nutrient medium, the addition of a protective environment and lyophilic drying, the deep cultivation of uterine cultures is carried out together in casein broth with gelatin for 7-9 hours at a pH of 7.4-7.8, at temperature of 37-38 ° С, constant stirring, aeration and supply of a 40% glucose solution .

Deep cultivation is carried out separately or together, on any standard nutrient media used in the industrial method of cultivating these microorganisms, with constant stirring and aeration, and the supply of nutrient solutions. As the protection medium use any medium used in the preparation of bacterial preparations. The protection medium is either prepared in advance and introduced into the reactor after cultivation, or the individual components of the protective medium are introduced into the nutrient medium.

The advantage of the proposed method is that the cultivation process is carried out under standard conditions, the cultivation time is reduced to 8 hours, while the biomass yield is increased 10 times, which allows to obtain a high economic effect. Antagonistic activity, which is one of the main qualitative characteristics of the drug, increases by 1.5-2 times.

The method is as follows:

One of the prepared culture media is used, for example casein-yeast, casein broth, casein broth with gelatin, casein broth with gelatin and sodium alginate. During separate cultivation, the uterine culture of one of the strains is introduced into the nutrient medium, while the uterine cultures of Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35-3 / 61 and fecal enterococcus Enterococcus faecalis G-35-4 / 62.

Cultivation is carried out with constant monitoring of pH, temperature, constant or periodic mixing and aeration, the supply of a nutrient solution. After the cultivation process is completed, one of the previously prepared protection media is introduced into the reactor, for example, sucrose-gelatin-milk, sucrose-milk. The resulting biomass is poured and then dried.

The following examples illustrate the invention.

Example 1

The uterine cultures of each of the four strains are introduced sequentially into the bioreactor with a nutritious casein-yeast medium (production schedule PR 732-98). The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and supply of sterile air and 40% glucose solution. After the cultivation process is completed, a previously prepared sterile sugar-gelatin-milk protection medium (PR 732-98) is introduced into the reactor. The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 2 · 10 ⁷ to 4 · 10 ⁷ live microbial cells of strains of E. coli and fecal enterococcus. The antagonistic activity of the drug, expressed in the zone of growth inhibition of the test strains, is from 14 to 16 mm.

Example 2

Cultivation is carried out in four bioreactors. In each of the four bioreactors with nutritious casein-yeast medium (PR 732-98), one uterine culture of Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35 is introduced into the uterine culture -3/61 and fecal enterococcus Enterococcus faecalis G-35-4 / 62. The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and the supply of sterile air and 40% glucose solution. After the cultivation process is completed, the contents of the four bioreactors are combined into one and the previously prepared sterile milky sucrose-milk protection medium is introduced (PR 732-98). The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring into either bottles or metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 10 ⁷ to 3 · 10 ⁷ live microbial cells of strains of Escherichia coli and fecal enterococcus. The antagonistic activity of the drug, expressed in the zone of growth retardation of the test strains, is from 10 to 13 mm.

Example 3

In a bioreactor with casein broth (PR 730-98), uterine cultures of each of the four strains are added sequentially. The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and supply of sterile air and 40% glucose solution. After the cultivation process is completed, a previously prepared sterile sugar-gelatin-milk protection medium (PR 732-98) is introduced into the reactor. The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 2 · 10 ⁸ to 6 · 10 ⁸ live microbial cells of E. coli and fecal enterococcus strains. The antagonistic activity of the drug, expressed in the zone of growth retardation of the test strains, is from 17 to 19 mm.

Example 4

Cultivation is carried out in four bioreactors. In each of the four casein broth bioreactors (PR 730-98), uterine cultures of each E. coli strain Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35-3 / 61 are introduced and fecal enterococcus Enterococcus faecalis G-35-4 / 62. The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and supply of sterile air and 40% glucose solution. After the cultivation process is completed, the contents of the four bioreactors are combined into one and the previously prepared sterile milky sucrose-milk protection medium is introduced (PR 732-98). The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 10 ⁸ to 2 · 10 ⁸ live microbial cells of strains of Escherichia coli and fecal enterococcus. The antagonistic activity of the drug, expressed in the zone of growth retardation of the test strains, is from 15 to 17 mm.

Example 5

In the bioreactor with casein broth with gelatin (PR 730-98), uterine cultures of each of the four strains are added sequentially. The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and supply of sterile air and 40% glucose solution. After the cultivation process is completed, a previously prepared sterile sucrose-milk protection medium (PR 732-98) is introduced into the reactor. The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The output of biomass from 1 ml of nutrient medium is from 4 · 10 ⁸ to 7 · 10 ⁸ living microbial cells of E. coli and fecal enterococcus strains. The antagonistic activity of the drug, expressed in the zone of growth inhibition of the test strains, is from 18 to 20 mm.

Example 6

Cultivation is carried out in four bioreactors. Uterine cultures of each strain, Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35- are introduced into each of the four casein broth bioreactors (PR 730-98) with gelatin. 3/61 and fecal enterococcus Enterococcus faecalis G-35-4 / 62.

The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and the supply of sterile air and 40% glucose solution. After the cultivation process is completed, the contents of the four bioreactors are combined into one and the previously prepared sterile milky sucrose protection medium is introduced (PR 732-98). The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 2 · 10 ⁸ to 4 · 10 ⁸ live microbial cells of E. coli and fecal enterococcus strains. The antagonistic activity of the drug, expressed in the zone of growth inhibition of the test strains, is from 14 to 16 mm.

Example 7

In the bioreactor with casein broth (PR 730-98) with gelatin and sodium alginate, uterine cultures of each strain are introduced sequentially. The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and the supply of sterile air and 40% glucose solution. After the cultivation process is completed, a previously prepared sterile sucrose-milk protection medium (PR 732-98) is introduced into the reactor. The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 6 · 10 ⁸ to 8 · 10 ⁸ live microbial cells of E. coli and fecal enterococcus strains. The antagonistic activity of the drug, expressed in the zone of growth retardation of test strains, is from 19 to 22 mm.

Example 8

Cultivation is carried out in four bioreactors. Uterine cultures of each strain, Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G are introduced into each of the four casein broth bioreactors (PR 730-98) with gelatin and sodium alginate. -35-3 / 61 and fecal enterococcus Enterococcus faecalis G-35-4 / 62.

The cultivation process is carried out for (8 ± 1) hours at pH 7.4-7.8, temperature (37.5 ± 0.5) ° C, constant stirring and supply of sterile air and 40% glucose solution. After the cultivation process is completed, the resulting cultures of each strain are combined in one reactor and a pre-prepared sterile sugar-milk protection medium is introduced (PR 732-98). The resulting biomass is cooled to a temperature of (27 ± 3) ° C and poured with constant stirring, or in bottles or in metal containers and then lyophilized. The biomass yield from 1 ml of culture medium is from 3 · 10 ⁸ to 4 · 10 ⁸ live microbial cells of strains of Escherichia coli and fecal enterococcus. The antagonistic activity of the drug, expressed in the zone of growth retardation of test strains, is from 16 to 18 mm.

A study of the effectiveness of the probiotic obtained by the claimed method was carried out in the Gastroenterological Center of the Road Clinical Hospital (Nizhny Novgorod) with the permission of the Ministry of Health of the Russian Federation (No. 42 dated February 13, 2003). As a result of clinical trials for 80 patients, the drug was shown to be highly effective for the prevention and treatment of intestinal dysbiosis in patients with chronic diseases of the gastrointestinal tract, expressed in a reduction in the term of relief of the main symptoms of the disease by 2-3 days, and the normalization of the microbial landscape of the intestine in 95 % of patients.

According to the decision of the Committee of Medical Immunobiological Preparations (Minutes No. 4 dated 06/25/2003), probiotic okarin was recommended for registration in the Russian Federation.

The effectiveness of the probiotic obtained by the claimed method was also studied on the basis of the Nizhny Novgorod Veterinary Institute of the Non-Chernozem Zone of the Russian Federation and the Nizhny Novgorod State Agricultural Academy in a number of livestock farms in the Nizhny Novgorod Region. Tests were conducted on two types of farm animals: calves and chickens. As a result of clinical trials, the drug was shown to be highly effective for the prevention and treatment of gastrointestinal diseases of calves, expressed in the positive effect of ocarin on the safety of the number of chickens and calves, as well as in a greater increase in live weight of experimental animals compared to the control.

The advantage of the proposed method is that the cultivation process is carried out under standard conditions, the cultivation time is reduced to 8 hours, while the biomass yield is increased 10 times, which allows to obtain a high economic effect. The proposed method allows to obtain a standard preparation with an equal number of living microbial cells of each of the used strains. Antagonistic activity, which is one of the main qualitative characteristics of the drug, increases by 1.5-2 times.

Claims (1)

A method of obtaining a probiotic based on E. coli strains Escherichia coli G-35-1 / 59, Escherichia coli G-35-2 / 60, Escherichia coli G-35-3 / 61 and fecal enterococcus Enterococcus faecalis G-35-4 / 62 by deep cultivation of uterine cultures in a nutrient medium, the addition of a protective environment and freeze drying, characterized in that the deep cultivation of uterine cultures is carried out together in casein broth with gelatin for 7-9 hours, at a pH of 7.4-7.8, at a temperature of 37 -38 ° С, constant stirring, aeration and supply of a 40% glucose solution.