Bacterial strain lactobacillus acidophilus having high resistance to t-2 toxin

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.

1 ex

 

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

One of the ways to prevent mycotoxicosis is the use of living probiotic microorganisms, which are based on the ability of symbiotic bacteria biochemically to destroy mycotoxins in the gastrointestinal tract (GIT) and to mitigate their negative effects, which improves the physiological state of the organism. At the same time, the level of beneficial bacteria in the digestive tract of animals consuming contaminated feed is significantly reduced, which, accordingly, not only reduces their ability to degrade coming from food hazardous substances (mycotoxins), but also provokes a manifestation related pathologies (dysbacteriosis), contributing to a General toxic effect. In contrast, the use of biological products, contributing to the colonization of the intestine useful forms of symbionts, can increase the body's resistance to the negative effects of feeding factors.

However, at higher concentrations of mycotoxins in animal feed probiotic preparations are not able to fully exercise their de is oxycarbide properties, because of the individual characteristics of a certain species and strains of microorganisms, many of which die in the aggressive-toxic environment of the gastrointestinal tract, before it can have a positive action. To solve this problem is the use of specially selected strains of bacteria that are resistant to the action of mycotoxins.

Recently, the selection actively in various directions. For example, there is a method of identifying cultures of microorganisms decomposing pesticides ("Method for detection of microorganisms - destructors of xenobiotics" / Patent for the invention №2051961 - 1996). Also known strains of E. coli that have passed the selection for resistance to L-Latino ("Strain of E. coli bacteria producing L-leucine (options)" / Patent for the invention №2140450 - 1999).

However, according to the results of a patent search on the diversity of bacteria was not detected strains, focused on the destruction of mycotoxins and high ability to grow in environments with high content. In a number of the most similar patents subject to biodegradation are pesticides (patent No. 2051961) or 2,4,5-trichlorophenoxyacetic acid (patent No. 2129605 and 2130067), which is the source of nutrients (nitrogen, energy) for the growth of some microorganisms. However, these synthetic xenobiotics, obtained by chemical Shin is ESA for weed control and phytopathogenic fungal microflora or which are products of industrial pollution, although they can have a negative effect on animals and humans, but their properties are fundamentally different from mycotoxins. So, for example, T-2 toxin cannot be used by bacteria as a nutrient, but actively suppresses the metabolic processes in microbial cell, leading to its destruction (bactericidal effect).

On the other hand, there is a method of prevention of mycotoxicoses (patent No. 2297842), based on the use of antagonistic activity of bacterial strains Bacillus subtilis TNP-3-DEPT and consumer goods-5-DEPT regarding toxinogenic fungi. However, this patent only refers to suppressing the growth of bacteria potentially dangerous fungal microflora, but not about their ability to destroy the formed or already existing in the raw feed material mycotoxins.

However, of particular interest to new strains of bacteria Brevibacterium flavum received selection by resistant sulfaguanidine and azaserine ("bacterial Strain Brevibacterium flavum producing L-glutamine (options)" / Patent for the invention №2084520 - 1997). Despite the differences in the species composition of bacteria and the type of chemical compounds, which target microorganisms proposed in this patent the principle of the allocation of new strains of bacteria by cumulative selection is more similar in its essence to the pre is proposed and has been used by us as a prototype. According to prototype new strains of bacteria were obtained from the wild as a result of several stages of selection: in a known way on a nutrient medium containing sulfaguanidine or azaserine, seeded cultures of the microorganisms were cultivated crops, and selected during the first phase of selection of microorganisms that are resistant to the investigated substances were used for the next.

The present invention is the selection of a strain of bacteria Lactobacillus acidophilus, possessing useful properties - stability and ability to oxidative degradation of T-2 toxin to less hazardous products. A new strain of Lactobacillus acidophilus deposited in the all-Russian collection of microorganisms assigned by the authors of the identification symbol "GE-294 and has a registration number VKM B-2794D.

A new strain obtained from field Lactobacillus acidophilus isolated from the contents of the digestive tract of broiler chickens suffering from a mild form of chronic T-2-mycotoxicosis induced experimentally in vivarium conditions.

At the initial stage of the original lactobacilli were typed and inoculated on nutrient medium "Lactobacilos" (Wednesday "s-56 composition (g/l): pancreatic hydrolysate fish meal - 20,0; Baker's yeast extract - 5,0; meat extract - 5,0; glucose - 20,0; potassium phosphate 1-substituted - 2,0; sodium acetate - 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. In the future a few (1-5) colonies of L. acidophilus were isolated, propagated and grown in the laboratory on similar environments with artificial amendment T-2 toxin in increasing concentrations (0.1 to 0.5 μg/ml) by repeated passage (4-8 times per month). Periodically (1-2 times a month with exposure time 1-3 days) on Wednesday added a number of mutagenic factors (sodium arsenite [5-65 µg/l], nitroso compounds [100-380 µg/l]) and subjected to UV irradiation in the medium (270-310 nm) and shortwave (120-250 nm) spectrum with an exposure 10-40 min to increase the frequency of recombination and mutational changes, allowing later to get the colony (forms) with higher (20-30% higher than the previous generation) tolerance to T-2 toxin.

Over the next 1.5 years with a frequency of up to 2-3 weeks counting of grown colonies of lactic acid bacteria and selection of the most resistant to mycotoxin microorganisms. In subsequent selected bacteria were perseval on the same Cup, but with a greater concentration of T-2 toxin. Upon reaching the mycotoxin concentration in a nutrient medium at 0.5 µg/ml produced cultivar is done on the media with this concentration of toxin in order to consolidate the sign of the high level of strains.

It is known that mycotoxins are extremely hazardous class of organic compounds that have a negative impact on human health and productivity of C. farm animals. High prevalence of problems mycotoxicosis suggests several possible ways of using this strain with the preventive purpose: 1) the inclusion of antitoxic food supplements (drugs) during the forced feeding contaminated with mycotoxins feed; 2) use as starter cultures in the production of dairy products, which will differ elevated curative properties against T-2 toxin; 3) the inclusion of feed raw materials (silage, haylage) to prevent the accumulation therein of mycotoxins or their treatment during storage. Thus, the final effect may consist in the application of the proposed strain in animal husbandry and fodder production for the prevention of chronic and acute T-2 toxicosis.

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

The morphology of cells: gram still sticks with rounded ends of 0.7×to 1.0 μm, spores do not form.

Cultural characteristics: after 48 hours of growth at 37°C in an atmosphere of normal air in a nutrient medium "Lactobacilos" about aboutsa white colonies with a diameter of 1-3 mm, the surface colonies are smooth, convex, edge smooth, opaque, non-pigmented, the structure is homogeneous, pasty consistency.

Physiological and biochemical characteristics: the microorganism is optional gone anaerobic. Gelatine does not dilute. For growth, a acetate, Riboflavin, calcium Pantothenate, Niacin, folic acid. Grows at 45°C, no growth at 15°C, the optimum temperature of the cultivation of 35-39°C.

The selection of the bacterial strain L. acidophilus "HE-294"with pre-selected him properties: stability and capacity for destruction T-2 mycotoxin, produced as follows.

Example. Before sowing, nutrient medium was poured into sterile Petri dishes with a layer of 4-5 mm and was dried in a thermostat at 37°C for 40 minutes In the composition of the medium was made by T-2 toxin in an amount of 0.1 µg/ml of medium and seeded selected cultures of microorganisms Lactobacillus acidophilus. Because of its physico-chemical properties of mycotoxins in General (and T-2 toxin in particular) are water-insoluble hydrophobic compounds with the aim of ensuring a uniform distribution in the environment added surfactant "tween 80 (polyoxyethylene(20)sorbitan monooleate) - based 8-30×10-4M/l environment. The specified concentration of the emulsifier had no negative impact on the growth of microorganisms.

Crops were incubated with thermostat at 37°C. After 48 hours were fixed to the grown colonies, thereby evaluating the resistance of microorganisms to this mycotoxin. An additional selection criterion was the ability of lactobacilli to destroy the mycotoxin, which was estimated by the change of the absolute concentrations of T-2 toxin in the environment before and after incubation, expressed per 1 grown colony. Thus, the main selection criteria were the growth of the colonies and the ability of microorganisms to destructivity T-2 toxin in a nutrient medium.

Selected the most promising microorganisms resulting from the first passage, suspensively in physiological solution of sodium chloride at a concentration of 1-5 billion microbial cells in 1 ml of the Suspension was used as inoculum for the second passage, where the mycotoxin concentration in the medium was increased to 0.2 µg/ml While lactobacilli on Petri dishes or in suspension periodically (1-2 times per month) was subjected to mutagenic factors that increase the frequency of occurrence of more sustainable forms. Further, similarly reseeding and the subsequent selection of resistant to mycotoxin microorganisms was carried out on nutrient medium with a concentration of T-2 toxin of 0.3, 0.4, and 0.5 μg/ml

Looping listed manipulations allowed us to identify a new strain of Lactobacillus acidophilus "GEZ-294, sustainable the th high mycotoxin concentrations in the environment. In the multi-stage cumulative selection was possible to increase the resistance of lactobacilli to T-2 toxin: the resistance of the microorganisms obtained in the first passage, was 12-16%, a new strain, it significantly increased to 77% (or 6-7 times); the ability to destructivity T-2 toxin in microorganisms of the first passage was $ 5.2×10-12and the new strain Lactobacillus acidophilus "HE-294" it increased to 1.8×10-9nmol / CFU, respectively (or 346 times). The resistance characteristic is 98-99%.

The bacterial strain Lactobacillus acidophilus, deposited in the all-Russian collection of microorganisms under the registration number VKM B-2794D highly resistant to T-2 toxin and metabolic ability to destroy it in the habitat of the bacteria.



 

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