Strain of microorganism escherichia coli used in producing probiotic microcicol b 5/98

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes the strain Escherichia coli S5/98 producing microcine B 5/98 showing the broad spectrum of antagonistic activity against microorganisms of genus Escherichia, Salmonella and Klebsiella that is isolated from adult pig feces. The strain inhibits 69-99% of escherichia and 42.6-80% of salmonella of wild type and shows activity against colicinogenic strains of intestinal bacillus and 10 different species of salmonella. The strain has no pathogenicity and doesn't elicit toxic, toxigenic and allergic properties. The strain is deposited in All-Russian collection of industrial microorganisms at № B-8564. The strain is used in preparing probiotic microcicol B 5/98 in liquid and dry formulation. Using the invention shows regulatory effect on intestine microflora and enhances nonspecific resistance of animals.

EFFECT: valuable properties of strain and probiotic.

1 ex

 

The invention relates to the microbiological industry, and the production of probiotics, in particular.

At present, there are over 10 types of microtines, studied their structure and mechanisms of action, are working on the genetic control of the synthesis of microtines and investigated their role in the ecology of the intestinal bacteria. A wide spectrum of antagonistic activity of these compounds suggests about the prospects of their application producers for the prevention and treatment of intestinal infections in young animals and birds. But so far offered only one probiotic romaka developed on the basis of genetically engineered strain of Escherichia coli M17(p74), producing microcyn s [Sokolova N.A., Hops I.A., Sagalevich E.A. the Use of Ramalla in veterinary medicine. Veterinary science. 2001. No. 11. P.46-49]. The drug is recommended for the prevention of neonatal colibacillosis of calves, pigs and chickens. Its use is more than twice reduces the incidence of calves septic form of colibacillosis, and test ramkola in pigs have shown that it reduces the incidence of diarrhoea in piglets 2-5 times and 11-46% reduces the mortality of young animals [Kavruk PS, Kononenko A.B., Britov SV Directed regulation of microbiocenosis in reproducing the premises of pig-breeding farms and nonspecific prophylaxis of bacterial intestinal infections in osat. In the book: Environmental problems of pathology, pharmacology and therapy animals. Proceedings of the International coordination meeting, 19-23 may 1997. Voronezh, 1997. S-316]. However, it should be noted that the produced strain of E. coli M17(R) microcyn s has on the cells of bacteria only bacteriostatic action, which is a significant drawback. Other disadvantages should indicate that genes determining products microcina and immunity to him, localized on plasmids, which in principle may be lost. Therefore, the search for new producers of microtines providing a bactericidal effect on opportunistic and pathogenic bacteria of the digestive tract of animals and birds, it seems necessary.

The purpose of the invention to provide a new strain, producing microcyn type and which have a bactericidal effect on sensitive cells of Enterobacteriaceae with a broad spectrum of antagonistic activity against potential enteric pathogens that cause disease in farm animals and humans carrying the genes products microcina and immunity to him in the composition of the bacterial chromosome.

A new strain of Escherichia coli S 5/98 selected using traditional microbiological methods from the feces of adult pigs. The strain is deposited in PMBC under the In-8564 and is characterized by the following features.

Cultural and morphological characteristics.

Cells of strain are represented by sticks with rounded ends, negatively stainable gram. Smears are placed in separate cells and disordered clusters. Spores and capsules do not form.

On the environment Endo formed dark red with a characteristic metallic sheen colonies.

On double tryptose agar (1/4 of nourishing ingredients from the official recipe) grow round, convex, with smooth edge, up to 2 mm in diameter, shiny, translucent, gray-white colonies.

In nutrient broth with glucose blurred and gray-white precipitate on the bottom of the tube.

Physiological and biochemical characteristics.

The optimum temperature 37-39°C. Growing at 15°and 45°C. Dies after 30 min exposure at 60°C.

Strain tolerant phenol (0,6%), ethanol (2%), grows well in medium with 20% bile. Produces gas from glucose, does not form hydrogen sulfide, which gives a positive reaction with methyl red and negative Voges-Proskauer. Growing on citrate agar Simmons, but does not use acetate and malonate as an energy source; phenylalanine and urea not hydrolyses.

Sprayway with the formation of acid, the arabinose, lactose, maltose, mannitol, glucose, xylose, trehalose, sorbitol and not fermented sucrose, Inositol, dulcet and salicin.

Ø the AMM sensitive to oxacillin, to chloramphenicol, streptomycin, gentamicin, rifampicin, neomycin, erythromycin, tetracycline, kanamycin, vancomycin, clindamycin, clarithromycin, amikacin, and also to nalidixic acid fusidin.

The strain produces microcyn In 5/98.

Microcyn In 5/98 has a molecular weight less than 10 kDa. Its production is reduced or absent when the cultivation of the producer in nutrient-rich environments and increases lean-burn (minimum) nutrient media. It is inactivated by subtilisin E, metalloproteinases of .mesentericus, protoliterate and pronasol, but resistant to thermolysin and trypsin.

Plasmids in strain is not detected, i.e. genes determining products microcina and immunity to him, localized on the bacterial chromosome.

The strain has a broad spectrum of antagonistic activity and suppresses 69-99% of Escherichia and 42.6-80% of Salmonella wild-type, isolated from the intestines of cattle, pigs, chickens and cats. Active against colicinogenic strains of E. coli. Among Salmonella inhibits Salmonella species give, S. bovis morbipicans 988, S. dublin 42, S. 1446 london, S. gaminare, S. derby, S. amager 2399, S. rostock, S. readiry, S. enteritidis 41997. Shows antagonism against bacteria of the genus Klebsiella.

The strain is not pathogenic for mice and rabbits, no toxic and allergic effects on the animal organism.

Example. For the preparation of probiotic who prepare nutrient broth or RM-broth, to which is added 0.2 percent glucose. the pH of the medium was adjusted to 6.8 to 7.2, poured into test tubes, flasks or heat-resistant bottles and sterilized at 0.5 ATM - 30 minutes

As inoculum used daily culture of E.coli S 5/98, which is added in amounts of 5-10% by volume of the environment. Crops incubated at 38±1°C for 24 h, then the microbial biomass is separated from the culture fluid (separation, filtration or centrifugation) and suspensorium her in a protective environment (1/20 part of the original volume)containing phosphates, sucrose and skim milk, bring a suspension of biomass in a protective atmosphere to a creamy consistency by adding nonfat dry skim milk, mix thoroughly, cooled to +2-4°and conduct L-drying in a vacuum dryer directly from the liquid state.

The dried mass is crushed, standardizes the addition of fillers (starch, sucrose, lactose, dry skim milk) and Packed in plastic bags.

Received probiotic has a high antagonistic activity against several pathogenic and conditionally pathogenic microorganisms (Escherichia, Salmonella, Klebsiella), stimulates the multiplication in the intestinal microbiocenosis of bifido - and follow, i.e. supports in the digestive tract for optimal microbial balance and increases non-specific re istinnosti animals.

The bacterial strain Escherichia coli VKPM B-8564 used for the production of probiotic.



 

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