RU2084233C1 - Method of probiotic preparing for veterinary science - Google Patents

Abstract

FIELD: biotechnology, veterinary science. SUBSTANCE: method involves culturing pure cultures of Bacillus subtilis, Rumenococcus albus, Lactobacillus plantarum, their the following separate culturing on the liquid soybean-liver medium for 72 h, mixing cultural fluids containing microorganism biomass at an equal volumes. Obtained probiotic is used for treatment endometritis, mastitis and diarrheas in cattle. EFFECT: improved method of preparing, enhanced effectiveness of probiotic. 2 tbl

Description

 The invention relates to biotechnology and can be used to obtain probiotic preparations for the treatment of endometritis, mastitis and dyspepsia in cattle.

A known method of producing dry lactobacterin, including obtaining pure cultures of Lactobacillus fermenti and Lactobacillus plantarum, applying them to the prepared culture medium, growing 8 10 hours at T = 37 ^o C and freeze drying of the drug [1] Lactobacterin is used in the treatment of gastrointestinal diseases.

 A known method of producing bificol dry, including obtaining pure cultures of bifidobacteria and Escherichia coli, applying them to a prepared nutrient medium, growing by the deep method, packing the suspension in bottles, freezing and drying [2] Used in the treatment of gastrointestinal diseases.

The disadvantages of these methods are:
reduction in the concentration of viable microbes up to 50% in the process of lyophilization;
the need to mix the drug with liquid before use;
use for the treatment of only gastrointestinal diseases.

The closest technical solution is a method of obtaining a biosan preparation, including growing pure cultures of Lactobacillus plantarum and Lactobacillus buchneri, adding them to a flask on a prepared soy-hepatic nutrient medium and culturing in a thermostat for 48 hours at T = 36-38 ^o C. In 1 ml of biosan there should be 2.5 3.5 billion microbial bodies. In veterinary practice, biosan is used to treat endometritis and uterine sanitation with artificial insemination of cows [3]
During the joint cultivation of any microorganisms, including the preparation of biosan, their spontaneous growth occurs and the advantage in biomass growth is for those species that have a high growth rate, which leads to loss of control over the concentration of individual cultures of microorganisms and their percentage ratio . In addition, the drug, consisting of microorganisms of the same kind, has one-sided tropism, which consists in the manifestation of antagonism only to part of the conditionally pathogenic microflora, therefore, localized exposure.

 The problem that needs to be solved is as follows: to develop such a method of obtaining a probiotic for use in veterinary practice, which will allow for reliable control over the percentage ratio and concentration of microorganisms in the dose of the drug and to obtain a drug with a wide spectrum of action.

In the process of solving the problem, a method has been developed to obtain an LCL probiotic (based on the initial letters of the names of probiotic preparations containing these types of microorganisms: bactisubtil, cellobacterin and lactobacterin), which includes growing pure cultures of microorganisms and cultivating them on a nutrient medium at T = 36 38 ^o C moreover, the cultivation of microorganisms Bacillus subtilis, Rumenococcus albus, Lactobacillus plantarum is carried out separately for 72 hours, and then the liquids containing the biomass of microorganisms are mixed in equal volumes.

To obtain the probiotic BCL, pure cultures of the microorganisms Bacillus subtilis, Rumenococcus albus, Lactobacillus plantarum are grown on beveled meat-peton agar (MPA) with the addition of 1% glucose in an incubator at T = 37 ^° C ± 1 for 48 hours. To obtain a liquid nutrient medium take fresh cattle liver, cut into pieces no larger than 5x5x5 cm, weigh, fill with equal weight by the amount of tap water and cook for an hour. The extract obtained is filtered through a cotton-gauze filter, mixed with soy hydrolyzate in a ratio of 1: 3, 0.5% NaCl, 2% glucose is added, boiled for 15 minutes, filtered through a cotton-gauze filter, poured into a container, closed with cotton-gauze plugs and sterilized at 0.5 atm for 30 minutes. Pure cultures of Bacillus subtilis, Rumenococcus albus, Lactobacillus plantarum, each in a separate container, are introduced into a liquid nutrient medium with an MPA bacteriological loop cooled to room temperature and cultured in an incubator at T = 37 ^° C ± 1 for 72 hours.

 The number of microorganisms grown is determined by comparison with the turbidity standard. The concentration of microorganisms in each case should be at least 2.5 billion in 1 ml. Culture liquids containing the biomass of microorganisms are mixed in equal volumes and the BCL preparation is packaged in containers intended for use.

 When carrying out work on the manufacture of the drug, it is necessary to observe the established rules of asepsis and antiseptics.

Storage of the drug is carried out in the refrigerator at T = + 5. + 10 ^o C for six months.

 Example 1

To obtain the probiotic BCL, pure cultures of microorganisms Bacillus subtilis 534, Rumenococcus albus 1-33, Lactobacillus plantarum 8p-A3 are prepared. For this, each culture is grown in a thermostat at T = 37 ^o C on mowed meat-peptone agar with the addition of 1% glucose.

For the cultivation of microorganisms, a soya-hepatic environment is prepared, which consists of three parts of a soy hydrolyzate and one part of a liver broth. Soy hydrolyzate is prepared as follows: 80 g of soybean meal is poured with two liters of tap water, add 3% hydrochloric acid with a specific gravity of 1.19, autoclave at 127 ^o C and 1.5 ATM for three hours. After cooling, the mass is filtered through cotton, and then through a paper filter with activated carbon deposited on it. Caustic soda is added until pH = 6.0 and K ₂ HPO ₄ buffer of 0.18% and KH ₂ PO _{4 of} 0.8% are prepared. Liver broth is prepared in the usual way: 0.5 kg of cattle liver is cut into pieces no more than 5x5x5 cm , placed in an enamel pan, pour one liter of water and boil for 1 h, cool the broth, filter through cotton, add caustic soda until pH = 6.0 is reached. Combine 1.5 l of soy hydrolyzate and 0.5 l of liver broth, add 0.5% NaCl and 2% glucose, which is 10 g and 40 g, respectively, pour into 200 ml bottles and autoclave for 30 min at 0.5 atm . The soya-hepatic environment is cooled to 35–38 ^° C and pure cultures of microorganisms are introduced each into a separate bottle with a bacteriological loop (one scraping with MPA per bottle). Cultivate in a thermostat at T = 37 ^o C ± 1 for 72 hours. The concentration of microorganisms after cultivation in each case is not less than 2.5 billion in 1 ml. Culture liquids containing biomass of Bacillus subtilis 534, Rumenococcus albus 1-33, Lactobacillus plantarum 8p-A3 are mixed in a ratio of 1: 1: 1.

 During storage of the obtained BCL preparation, a change in the concentration of microorganisms occurs, since the microorganisms that compose it have different properties, in particular, the growth rate.

 The change in the concentration of microorganisms depending on the shelf life is presented in table. one.

 A comparative study of the therapeutic effect of the BCL probiotic was carried out by using it for the treatment of endometritis and mastitis in cows and dyspepsia in calves at different periods of the storage period. The drug was administered intrauterine, intracisternally and orally. The research results are given in table. 2.

 Example 2

 Obtaining a probiotic BCL is carried out as described in example 1, except that the mixing of culture liquids containing biomass B. subtilis 534, R. albus 1-33, L. plantarum 8p-A3, is carried out in a ratio of 2: 1: 3.

 The change in the concentration of microorganisms depending on the shelf life is presented in table. one.

 The results of studies of the therapeutic effect of the drug BCL are given in table. 2.

 Example 3

 Obtaining a probiotic BCL is carried out as described in example 1, except that the mixing of culture fluids containing biomass B. subtilis 534, R. albus 1-33, L. plantarum 8p-A3, is carried out in a ratio of 1: 1: 2.

 The change in the concentration of microorganisms depending on the shelf life is presented in table. one.

 The results of studies of the therapeutic effect of the drug BCL are given in table. 2.

 Example 4

Obtaining a probiotic is carried out as described in example 1, except that pure cultures of B. subtilis 534, R. albus 1-33, L. plantarum 8p-A3 bacteriological loop (one scraping from the culture) is introduced into one container with soybean nutrient medium and cultured at T = 37 ^o C ± 1 for 72 hours. The resulting culture fluid with biomass of microorganisms is packaged in bottles intended for use.

 The change in the concentration of microorganisms depending on the shelf life is presented in table. one.

 The results of studies of the therapeutic effect of the drug BCL are given in table. 2.

 In each example, the total concentration of microorganisms during storage for 90 days decreases from 9 10 billion in 1 ml to 4 5 billion in 1 ml.

 As can be seen from the table. 1 and 2, the preparation obtained in example 1 contains the optimal concentration of microorganisms and has the highest overall therapeutic efficacy.

 The therapeutic efficacy in the treatment of acute endometritis in example 2, mastitis in example 3, as well as acute endometritis in example 4 is insufficient.

 Thus, the proposed method for the preparation of BCL has several advantages compared to existing ones. As a result of exercising control over the ratio and concentration of microorganisms, a wide spectrum of drug action and a high therapeutic effect are achieved. In addition, the method provides for the preparation and use of an environmentally friendly therapeutic drug probiotic.

Claims (1)

A method of obtaining a probiotic for veterinary medicine, involving the cultivation of lactobacilli and cultivation on soy-liver medium in a thermostat at a temperature of 36 38 ^o C, characterized in that pure cultures of Bacillus subtilis, Ruminococcus albus are additionally grown, Lactobacillus plantarum is used from lactobacilli, cultivation is carried out separately for 72 h, after which the resulting culture fluids are mixed in equal amounts.

Images