RU2526577C1 - BACTERIAL STRAIN Lactobacillus acidophilus HAVING HIGH RESISTANCE TO T-2 TOXIN - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention can be used for development of antitoxic preparations and feed additives for prevention of mycotoxicosis in farm animals and poultry. The bacterial strain Lactobacillus acidophilus is deposited in the Russian Collection of Microorganisms under the registration number RCM B-2794D. The strain has resistance to T-2 toxin and capacity to destroy it metabolically in the habitat of bacteria. The resistance to the strain T-2 toxin is up to 77%, and the ability to destroy T-2 toxin is 1.8Ч10^-12 nmol/CFU.

EFFECT: increased resistance of the strain.

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Description

The invention relates to the field of microbiology, biotechnology and agriculture, in particular to the prevention of mycotoxicosis in farm animals and poultry, and can be used to develop new antitoxic drugs and feed additives.

One of the methods for the prevention of mycotoxicosis is the use of live probiotic microorganisms, the action of which is based on the ability of symbiotic bacteria to biochemically destroy mycotoxins in the gastrointestinal tract (GIT) and weaken their negative effect, which helps to improve the physiological state of the body. At the same time, the level of beneficial bacteria in the gastrointestinal tract of animals consuming contaminated feed is significantly reduced, which, accordingly, not only reduces their ability to degrade hazardous substances (mycotoxins) coming from the feed, but also provokes the manifestation of related pathologies (dysbacterioses), aggravating the general toxic Effect. In contrast, the use of biological products that contribute to the colonization of the intestines with useful forms of symbionts can increase the body's resistance to the negative effects of feed factors.

However, at elevated concentrations of mycotoxins in compound feeds, probiotic preparations are not able to fully manifest their detoxifying properties, which is due to the individual properties of a particular type and strain of microorganisms, many of which die in the aggressive toxic environment of the gastrointestinal tract without having a positive effect. The use of specially selected strains of bacteria resistant to the action of mycotoxins can solve this problem.

Recently, selection has been actively conducted in various areas. So, for example, there is a known method for detecting cultures of microorganisms that decompose pesticides ("A method for detecting microorganisms - xenobiotic destructors" / Patent for invention No. 2051961-1996). Also known are strains of E. coli that have been selected for resistance to L-leucine ("The strain of bacteria Escherichia coli producer of L-leucine (options)" / Patent for invention No. 2140450 - 1999).

However, according to the results of a patent search, no strains oriented towards the destruction of mycotoxins and characterized by high ability to grow in environments with a high content of bacteria were found from the entire variety of bacteria. In some of the closest patents, the object of biodegradation is pesticides (patent No. 2051961) or 2,4,5-trichlorophenoxyacetic acid (patents Nos. 2129605 and 2130067), which are a source of nutrients (nitrogen, energy) for the growth of certain microorganisms. However, these synthetic xenobiotics obtained by chemical synthesis to control weeds and phytopathogenic fungal microflora or as products of industrial pollution, although they can have a negative effect on animals and humans, are fundamentally different from mycotoxins in their properties. So, for example, T-2 toxin cannot be used by bacteria as nutrients, but it actively inhibits metabolic processes in a microbial cell, contributing to its death (bactericidal effect).

On the other hand, there is a known method for the prevention of mycotoxicosis (patent No. 2297842), based on the use of the antagonistic activity of strains of bacteria Bacillus subtilis TNP-3-DEPT and TNP-5-DEPT against toxin-forming molds. However, in this patent it is only a question of inhibiting the growth of potentially dangerous fungal microflora by bacteria, but not of their ability to destroy mycotoxins formed or already existing in fodder raw materials.

However, certain strains of Brevibacterium flavum bacteria obtained by selection, resistant to sulfaguanidin and azaserin ("Brevibacterium flavum bacterial strain - producer of L-glutamine (options)" / Patent for invention No. 2084520 - 1997) are of particular interest. Despite the differences in the species composition of bacteria and the type of chemical compounds that are affected by microorganisms, the principle proposed in this patent for isolating new bacterial strains by cumulative selection is closer in essence to the proposed one and was used by us as a prototype. According to the prototype, new bacterial strains were obtained from wild as a result of several stages of selection: in the known method, isolated cultures of microorganisms were sown on a nutrient medium containing sulfaguanidin or azaserin, cultivated, and selected microorganisms resistant to the studied substances selected during the first stage of selection were used for the next one.

The objective of the present invention is the selection of a bacterial strain Lactobacillus acidophilus, which has useful properties - resistance and ability to oxidative destruction of T-2 toxin to less dangerous products. The new strain of Lactobacillus acidophilus is deposited in the All-Russian collection of microorganisms with the identification symbol “GE3-294” assigned by the authors and has registration number VKM B-2794D.

The new strain was obtained from field Lactobacillus acidophilus isolated from the contents of the gastrointestinal tract of broiler chickens suffering from a moderate form of chronic T-2 mycotoxicosis, which was experimentally caused under vivarium conditions.

At the initial stage, the initial lactobacilli were typed and plated on Lactobacagar nutrient medium (C-56 medium, composition (g / l): pancreatic hydrolyzate of fish meal - 20.0; baker's yeast extract - 5.0; meat extract - 5.0; glucose - 20.0; potassium phosphate 1-substituted - 2.0; sodium acetic acid - 5.0; Tween-80 - 1 ml; ammonium citrate 1-substituted - 2.0; magnesium sulfate - 0 , 1; manganese chloride - 0.05; agar - 13.0 ± 2.0, pH 5.7 ± 0.3; FSUE SSC PM), intended for the isolation and cultivation of lactobacilli. Subsequently, several (1-5) colonies of L. acidophilus were isolated, propagated and grown in laboratory conditions on similar media with the artificial introduction of T-2 toxin in them in increasing concentrations (0.1-0.5 μg / ml) by repeated passivation (4-8 times a month). A number of mutagenic factors (sodium arsenite [5-65 μg / l], nitroso compounds [100-380 μg / l]) were added periodically (1-2 times a month with an exposure of 1-3 days) and subjected to UV irradiation in medium - (270-310 nm) and short-wavelength (120-250 nm) spectrum with an exposure of 10-40 min to increase the frequency of recombination and mutation changes, which subsequently allow to obtain colonies (forms) with a higher (20-30% higher than that of previous generation) tolerance to T-2 toxin.

Over the next 1.5 years, with a frequency of up to 2-3 weeks, the grown colonies of lactic acid bacteria were counted and the microorganisms most resistant to mycotoxin were selected. Subsequently, the selected bacteria were subcultured on similar plates, but with a higher concentration of T-2 toxin. When the concentration of mycotoxin in the nutrient medium was reached at a level of 0.5 μg / ml, cultivation was performed on media with a given concentration of toxin in order to fix the trait in highly active strains.

Mycotoxins are known to be an extremely dangerous class of organic compounds that have a negative effect on human health and agricultural productivity. animals. The high prevalence of the mycotoxicosis problem suggests several possible ways of using this strain for prophylactic purposes: 1) inclusion of antitoxic feed additives (preparations) in the case of forced feeding of feed contaminated with mycotoxins; 2) use as starter cultures in the production of fermented milk products, which will have enhanced therapeutic and prophylactic properties with respect to T-2 toxin; 3) inclusion in the composition of feed raw materials (silage, haylage) to prevent the accumulation of mycotoxins in it or their neutralization during storage. Thus, the final technical result may consist in the use of the inventive strain in animal husbandry and feed production for the prevention of acute and chronic T-2 toxicosis.

Description of the cultural-morphological and physiological-biochemical characteristics of the strain.

Cell morphology: gram-positive fixed rods with rounded ends 0.7 × 1.0 μm, do not form spores.

Cultural signs: after 48 hours of growth at 37 ° C in an atmosphere of ordinary air on a Laktobakagar nutrient medium, white colonies with a diameter of 1-3 mm are formed, the surface of the colonies is smooth, convex in shape, the edges are even, opaque, non-pigmented, the structure is homogeneous, and the texture is pasty.

Physiological and biochemical characteristics: the microorganism is an optional anaerobic. It does not dilute gelatin. Growth requires acetate, riboflavin, calcium pantothenate, niacin, folic acid. Grows at 45 ° C, no growth at 15 ° C, the optimal cultivation temperature is 35-39 ° C.

The selection of the bacterial strain L. acidophilus "GE3-294", with its predetermined properties: resistance and the ability to destruction of T-2 mycotoxin, was performed as follows.

Example. Before sowing, the nutrient medium was poured into sterile Petri dishes with a layer of 4-5 mm and dried in an thermostat at 37 ° C for 40 min. T-2 toxin was added to the medium in an amount of 0.1 μg / ml of medium and inoculated with isolated cultures of microorganisms L. acidophilus. Due to their physicochemical properties, mycotoxins in general (and T-2 toxin in particular) are water-insoluble hydrophobic compounds in order to ensure conditions for their uniform distribution, Tween 80 surfactant (polyoxyethylene (20) sorbitan monooleate was added to the medium) ) - from the calculation of 8-30 × 10 ^-4 M / l of medium. The indicated concentration of emulsifier did not adversely affect the growth of microflora.

Crops were incubated in an incubator at 37 ° C. After 48 hours, the grown colonies were recorded, thereby assessing the resistance of microorganisms to this mycotoxin. An additional selection criterion was the ability of lactobacilli to destroy mycotoxin, which was assessed by the change in the absolute concentration of T-2 toxin in the medium before and after incubation, expressed as per 1 grown colony. Thus, the main selection criteria were colony growth and the ability of microorganisms to degrade the T-2 toxin in the culture medium.

The selected most promising microorganisms obtained as a result of the first passage were suspended in a physiological solution of sodium chloride at a concentration of 1-5 billion microbial cells in 1 ml. The suspension was used as seed for the second passage, where the concentration of mycotoxin in the medium was increased to 0.2 μg / ml. At the same time, lactobacilli on Petri dishes or in suspension were periodically (1-2 times per month) exposed to mutagenic factors that increase the frequency of appearance of more stable forms. Further, in a similar way, reseeding and subsequent selection of mycotoxin-resistant microorganisms was carried out on nutrient media with a T-2 concentration of 0.3 toxin; 0.4 and 0.5 μg / ml.

Performing the cycle of these manipulations made it possible to isolate a new strain of Lactobacillus acidophilus "GEZ-294", resistant to high concentrations of mycotoxin in the medium. As a result of multi-stage cumulative selection, it was possible to increase the resistance of lactobacilli to T-2 toxin: the resistance of microorganisms obtained in the first passage was 12-16%, in the new strain it increased significantly - up to 77% (or 6-7 times); the ability to degrade the T-2 toxin in the first passage microorganisms was 5.2 × 10 ^-12 , and in the new strain - Lactobacillus acidophilus "ГЕ3-294" it increased to 1.8 × 10 ^-9 nmol / CFU, respectively (or 346 times ) Sign stability - 98-99%.

Claims (1)

The bacterial strain Lactobacillus acidophilus, deposited in the All-Russian collection of microorganisms under registration number VKM B-2794D, has increased resistance to T-2 toxin and the ability to metabolically destroy it in the habitat of bacteria.