RU2091075C1 - Complex bacterial preparation for treatment and prophylaxis of gastroenteric disease in animals - Google Patents

Abstract

FIELD: biotechnology, veterinary science. SUBSTANCE: preparation has new strain Lactobacillus acidophilus VKPM B-6535 (140-160 million live cells/g) and strain Enterococcus faecium VKPM B-2990 (190-210 million live cells/g). Preparation shows the enhanced antagonistic activity with respect to some microorganisms and good adaptivity of microbes in gastroenteric tract of fur animals, agriculture animals and poultry. EFFECT: enhanced effectiveness of preparation. 3 tbl

Description

 The invention relates to the microbiological industry and, in particular, to the production of a bacterial preparation used for feeding fur animals, domestic animals (dogs), farm animals and poultry for the prevention and treatment of gastrointestinal diseases.

 Various bacterial preparations (probiotics) are known for the prevention and treatment of diarrhea and diobacteriosis, as well as stimulating the growth and development of young farm animals. Probiotics can contain only one bacterial strain, or several, up to eight different strains and types of bacteria, can be included in their composition. The trend towards the creation of multi-strain preparations is explained by the fact that they are active in a wider range of conditions and animal species. Currently, probiotics mainly include a strain of intestinal origin of species such as Lactobacillus acidophilus, L. casei, L. helveticus, L. lactis, L. salivarius, L. plantarum, Enterococcus faecalis, E. faecium and Bifidobacterium spp. In addition, cultures isolated from fermented milk products, for example, L. delbrueckii ss, can also be used. bulgaricus and Streptococcus thermophilus. Almost all the probiotics currently in demand contain lactobacilli and / or streptococci; few contain bifidobacteria.

An example of a probiotic preparation containing one strain is acidophilus, which contains at least 200 million acidophilic bacteria Lactobacillus acidophilus in 1 g [1] Among the complex probiotic preparations, propacid, which contains acidophilic lactobacilli and propionic acid bacteria [2], describes the drug, which contains antibiotic-resistant strains of lactic acid bacteria L. acidophilus, L. delbrueckii ss. bulgaricus and S. lactis [3] It is designed to restore and maintain the balance of the intestinal flora of animals, disturbed by the use of antibacterial agents. Several probiotic preparations are known that include cultures of L. delbrueckii ss. bulgaricus and S. thermophilus [4 5]
Also known is the complex microbial preparation SBA for the treatment and prevention of gastrointestinal diseases in animals, which we have chosen as the closest analogue [7] According to the description, it contains L. acidophilus, S. faecium and Bifidobacterium bifidum. The drug is used to treat farm animals and poultry. The disadvantage of the SBA drug is that it does not have a wide enough range of antimicrobial activity and has a low antagonistic activity (Table 1). In addition, when it is developed, the yields of its constituent cultures are not high enough, and it is also impossible to use molasses, which is a cheap source of carbon in industrial environments when receiving the drug.

 An object of the present invention is to provide a bacterial preparation for fur animals, domestic animals (dogs), farm animals and poultry with a wider range of antimicrobial activity and higher antagonistic activity.

 The problem is solved by obtaining a complex bacterial preparation Entericide P containing a new strain of Lactobacillus acidophilus 495 and Enterococcus faecium strain VKPM B-2990 (the strain was obtained from the Museum of Culture of Industrial Microorganisms GNI Genetics).

 Strain Lactobacillus acidophilus 495 was isolated from the intestinal contents of the arctic fox.

The identification of the strain was carried out according to the main morphological, cultural and physiological-biochemical properties, according to the Bergey determinant [8]
The strain Lactobacillus acidophilus 495 is deposited in the All-Russian collection of cultures of industrial microorganisms GNI genetics under the number VKPM B-6535 and is characterized by the following features.

 Morphological signs.

 Cells in the form of thin straight sticks with blunt ends, located singly or in chains. Fixed, gram-positive, do not form spores and capsules, do not have flagella.

 Cultural signs.

 It grows on rich nutrient media containing organic components. When growing on hydrolyzed milk or MPC medium after 12 hours, uniform turbidity is observed. On dense MPC medium, colonies are convex, opaque, whitish, usually rough, but become smooth and dense in the presence of Tween-80. On whey agar form surface colonies with a diameter of 2 to 3 mm and deep colonies in the form of cotton balls.

 Physiological and biochemical characteristics.

Catalase-negative. Optional anaerobic. With the breakdown of sugars, at least half of the final carbon products are lactic acid. It does not form pigment. Grows at pH 5.0 5.8. The optimum growth temperature of the culture is 37 39 ^o C, maximum 45 ^o C, minimum 20 ^o C. It has complex nutritional needs for amino acids, peptides, nucleic acid derivatives, vitamins, salts of fatty acids or their esters and fermented carbohydrates. Does not thin the gelatin. Does not form gas from glucose or gluconate.

It ferments milk well, forming a dense, even clot with a pleasant sour-milk smell and taste. The titratable acidity of the daily culture is 110 ^o T, the maximum titratable acidity at 37 ^o C is 300 ^o T.

 Attitude to carbohydrates.

 Ferments esculin, fructose, galactose, glucose, lactose, maltose, mannose, raffinose, salicin, sucrose, trehalose; does not ferment xylose, ribose, sorbitol, rhamnose, melibiosis, melesitosis, mannitol, cellobiose, arabinose, amygdalin.

 The strain grows in broth in the presence of 2% NaCl, but not 4% NaCl. The strain is stable in bile at a concentration of 40% in the medium. Coagulates milk with 0.5% phenol.

 The antagonistic activity of the culture fluid of the strain (CL) obtained by culturing it in MRS broth [8] for 24 hours was detected both against gram-positive and gram-negative bacteria: Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium, Salmonella abortus-bovis, Salmonella dublin, Salmonella gallinarum, Enterococcus faecalis, Streptococcus sanguis and Pseudomonas aeruginosa. Comparative characteristics of the antimicrobial activity of the strain of Lactobacillus acidophilus 495 and the strain of Lactobacillus acidophilus isolated from the SBA preparation (Lactobacillus acidophilus SBA), in relation to some test cultures are shown in table 1.

 Strain productivity.

 When the strain is cultivated in skim milk for 14 hours, the cell titer is 2 4 billion / ml, and in a fermentation medium 3 6 billion / ml.

 When testing the strain of Lactobacillus acidophilus 495 on white mice, its pathogenicity was established.

The strain is stored in a freeze-dried state or by periodic reseeding (1 time in 30 days) in sterile skim milk. In this case, the crops are incubated at 37 39 ^o C for 18 24 hours

 The proposed strain of Lactobacillus acidophilus 495 has a higher antagonistic activity against certain gram-positive and gram-negative microorganisms (Table 1) and gives higher titers, i.e. has a higher productivity compared to the previously used strain of Lactobacillus acidophilus from the drug SBA (table. 2). The strain Lactobacillus acidophilus 495 also has such an advantage as the ability to ferment sucrose, which is absent in the Lactobacillus acidophilus strain from SBA. Due to this, beet molasses, which is much cheaper than glucose, can be effectively used in the composition of a fermentation nutrient medium for its cultivation as a carbon source.

 The complex bacterial preparation obtained using the proposed strain is characterized by a good survival of lactobacilli in the gastrointestinal tract of fur animals and dogs, apparently, due to the species-specificity of the strain Lactobacillus acidophilus 495 (Table 3).

 Obtaining a complex bacterial preparation enteric P includes the following steps: preparation of a nutrient medium for separate cultivation of its constituent cultures, preparation of an inoculum, preparation of seed, accumulation of bacterial mass, separation of the bacterial mass from the culture fluid, preparation of a protective medium, mixing of the bacterial mass with a protective medium, freezing drying the suspension by sublimation (or spray drying the culture fluid), grinding dry biomass powders, mixing of component cultures, drug standardization, packaging, labeling.

Example 1. In order to obtain a combined bacterial preparation Entericide P, separate cultivation of cultures of Lactobacillus acidophilus 495 and Enterococcus faecium VKPM B-2990 is carried out on a seed culture medium of the following composition,
Cornmeal 3
Corn Extract 0.9
Glucose 1.5 (or molasses beet 1.5 according to RV)
Chemically precipitated chalk 0.5
Potassium phosphate disubstituted 3-water 0.02
Potassium phosphate monosubstituted 0.02
Sodium nitrate 0.03
Cobalt chloride 6-water 0.0005
5-aqueous manganese sulfate 0,0005
Molybdenum Sodium 0.0005
Zinc Sulphate 7-Aqueous 0.0005
Tap water up to 100.

 Glucose and molasses are sterilized separately in an autoclave: glucose in the form of a solution with conc. 400 g / l at a pressure of 0.05 MPa with holding for 30 minutes; molasses in the form of a solution with conc. 500 g / l at 0.08 MPa with an exposure of 40 min. The remaining components of the medium are sterilized together. The pH of the nutrient medium before sterilization is set to 7.2-7.3; after sterilization, 7.0-7.2. Inoculation of the medium is carried out under aseptic conditions.

The inoculum is obtained on the image by culturing monocultures at 37 - 39 ^o C for 18 to 20 hours until a dense, even clot forms. The inoculum is used to obtain seed. To this end, the inoculum in the amount of 3 5% contribute to the nutrient medium. After seeding and mixing, the pH should be 5.5 to 6.0. The cultivation of seed for each strain is carried out under anaerobic conditions at a temperature of (39 ± 1) ^o C and an overpressure of 0.02 0.04 MPa. Growing lead for 10 to 14 hours

Seed is used for inoculation of a fermentation nutrient medium. It is introduced in an amount of 3% of the volume of the medium. After seeding and mixing, the pH should be 5.5 to 6.0. The cultivation of each strain is carried out under anaerobic conditions at a temperature of (39 ± 1) ^o C and an overpressure of 0.02 0.04 MPa. The duration of growing each culture is 10–14 hours. At the end of the growing period, the number of viable cells is: when growing Enterococcus, at least 500 million / ml; when growing Lactobacilli, at least 400 million / ml; the pH of the culture fluid is reduced to 4.0 4.5; solids content is 4.8 - 5.2%
After that, the resulting culture fluid with a pH of 4.0 4.5 is made alkaline to pH 5.8 6.0 with a 40% sodium hydroxide solution, then the components serving as a protective medium for subsequent drying are added to it with stirring; molasses in an amount of 15 g / l and corn flour in an amount of 50 g / l, after which the culture fluid is mixed for 15 to 20 minutes.

Drying of the treated culture fluid of Enterococcus or acidophilus bacteria is carried out in a spray dryer. Drying mode: air inlet temperature 130 135 ^o C, outlet 60 65 ^o C.

The standard preparation Entericocide P is obtained by mixing dry powdered products of Enterococcus faecium and Lactobacillus acidophilus cultures obtained after spray drying with a filler, dried corn flour. Corn flour is dried in an oven at a temperature of 70 80 ^o C for 20 30 minutes to a moisture content of 4 to 6%
The obtained complex microbial preparation Entericid P has the following characteristic: fine dry powder from cream to light brown in color with a specific smell and taste, insoluble in water, containing living cells of the strains:
Lactobacillus aciluphilus 495 140 160 ppm;
Enterococcus faecium VKPM B-2990 190 210 ppm.

 Example 2. Culture liquids of acidophilic bacteria and enterococcus of intestinal origin, obtained by the method described in example 1, are subjected to separation, and the isolated biomass is freeze-dried.

Before separation, the culture fluid is alkalinized to pH 5.8 6.0 with 40% sodium hydroxide solution, then cooled with stirring to a temperature of 18 20 ^o C. During the separation produce constant cooling of the culture fluid.

 At the end of the separation, the resulting biomass sediment, which is a yellow-brown paste, is mixed with a protective solution in the ratio of 1 liter of solution per 1 kg of biomass. To obtain a homogeneous mass, the mixture is stirred for 20 25 minutes. As a protective solution, a solution of trisubstituted 5.5-aqueous sodium citric acid (50 g / l) and molasses (15 g / l) is used. The solution is prepared in tap water, sterilized in an autoclave at a pressure of 0.05 MPa with exposure for 1 h and cooled to room temperature.

The resulting creamy suspension is sent to freeze-drying. The drying time is 25-30 hours; the process ends when the residual pressure in the chamber is reduced to 0.1 mbar, and the material temperature reaches (22 ± 3) ^o C, the residual moisture content of the material does not exceed 5%. The resulting dry products are ground to a powder state in a laboratory mill, in which each the product (separately) is loaded in portions and ground for 15 to 20 minutes.

Standardized preparations are obtained by mixing dry powdered products of Enterococcus faecium and Lactobacillus acidophilus cultures obtained after freeze-drying with a filler — dried corn flour. The content of living cells in standardized preparations is:
Lactobacillus acidophilus 495 140 160 ppm
Enterococcus faecium VKPM B-2990 190 210 ppm.

 References.

 1. USSR author's certificate N 306827, publ. 1971.

 2. Platonov A. V. Production of livestock products based on symbiotic microorganisms of the gastrointestinal tract. M. VNIISENTI, 1985, 43 pp.

 3. Antipov V. A. Subbotin V. M. The effectiveness and prospects of the use of probiotics // Veterinary medicine. 1980, N 12, p. 55-57.

4. US Patent N 3876808 /
5. Patent of Germany N 2329746.

 6. British patent N 1431809.

 7. RF patent N2018313, cl, C 12 N 1/20, 1994.

 8. Kandler O. Weiss N. Genus Lactobacillus Bejerink 1901, 212 AL / Bergey's Manual of Sistemic Bacteriology, v. 2 / eds. Smath P.H.A. Mair N.S. Sharpe M. E. Holf J.G. Baltimore, London, Los Angeles, Sidney: Williams and Wilkins, Ins. 1986, pp. 1209 1234.

Claims (1)

A complex bacterial preparation for the treatment and prevention of gastrointestinal diseases of animals, containing lactic acid bacteria Lactobacillus acidophilus and Enterococcus faecium, characterized in that it is from the species Lactobacillus acidophilus contains a strain of Lactobacillus acidophilus VKPM B-6535, and from the species Enterocococcus faecium faecium enterium VKPM B-2990 in the following ratio of components, million living cells / g of the drug:
Lactobacillus acidophilus VKPM B-6535 140 160
Enterococcus faecium VKPM B-2990 190 210a

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