Nutrient medium for the accumulation of biomass vaccine strain of the plague microbe

 

(57) Abstract:

The invention relates to biotechnology and sanitary Microbiology, in particular, is concerned with the obtaining of a nutrient medium for the accumulation of biomass vaccine strain of the plague microbe. Nutrient medium contains as the basis of equal volumes of enzymatic hydrolysates of beef and soy beans, microbiological agar, sodium chloride, sodium phosphate 2-substituted, sodium sulphate, distilled water. The invention improves the cumulative properties of the medium, and also to make it cheaper.

The invention relates to biotechnology and can be used in medical and industrial Microbiology.

Known nutrient medium of the following composition, g/l: agar-agar 300,0; water soy (beans), 200,0; peptone 10.0 g; sodium chloride 5,0 (Kozlov, Y. A. Nutrient medium in medical Microbiology. 1950, S. 111).

The disadvantage of this environment is that when growing vaccine strain of the plague microbe biomass harvesting is 27 billion, M. K.

Closest to the proposed invention relates nutrient medium, including g/l: agar microbiological 18,0; hydrolyzed beef Maya water to 1 l; pH 7,2±0,l (Birger M. O. Handbook of microbiological and virological methods, 1982, S. 53).

The aim of the invention is to increase the cumulative properties of the medium, and its cheaper.

The essence of the invention lies in the fact that the nutrient medium contains equal amounts of enzymatic hydrolysate of beef and soy (beans) and stimulating additive, and as a stimulating additives used sodium sulfite in the following ratio of ingredients, g/l:

Agar microbiological 16,0-18,0

Enzymic hydrolysate beef 100,0-150,0

Enzymatic hydrolyzed soy 100,0-150,0

Sodium chloride 4,0-6,0

Sodium phosphate 2-substituted 0,3-0,5

Sodium sanitarily 0,0003-0,0005

Distilled water the Rest

Preparation of enzymatic hydrolysate of beef in the reactor: pour tap water from the calculation of available meat (1 kg of meat 1.5 liters of water). Meat without fat and suhaila cut into pieces of size 2.5×2,5 see Cook the meat for 15 minutes, then removed, cooled and passed through a meat grinder. The broth has cooled to 50&#aleso based 80,0-100.0 g of the pancreas in 1 l water, depending on the activity of the pancreas. The activity must be at least 5 thousand units of Fold-gross. Add chloroform to 2% of the total volume of liquid. Digestion is at 37°C. the First day of the hydrolysate interfere with a mechanical stirrer every 15 min for 5 minutes In the future translate into an automatic mixer that turns on every 2 hours for 5 minutes Daily to determine the amine nitrogen in the hydrolysate. With halting amine nitrogen, which is for 7-9 days, the stirrer is switched off, the heating is stopped. Give ecstatica for 2 days, then the liquid is filtered from the precipitate at a press filter, poured into bottles with addition of 2% chloroform, aprobability and stored at a temperature of 4-6°C.

Preparation of enzymatic hydrolysate of soybean seeds: seeds in quantities of 0.5 kg, five-soaked in 5 l of hot water during the day, and last infusion of beans boiled for 40 minutes Then the infusion is cast in a 10-litre container is cooled, the beans are crushed by a grinder and placed in a flask. Add the pancreas of cattle, minced in a meat grinder, activity 5 thousand units of Fold-gross based 20,0 g / l, set pH to 8.2 20% solution of sodium hydroxide in the amount of 0.003, dobas every 15 minutes manually the next day stir every 2 hours manually. Daily measure the level of amino nitrogen, which is 3 days of hydrolysis reaches 0,6±0,05%.

Characterization of the enzymatic hydrolysate beef: pH 7,2; total nitrogen 902 mg%; amine nitrogen 600 mg%; the percentage cleavage of 74.0%; sodium chloride 0,086%; peptone 1,2%; calcium 4,2 mg%; magnesium 5,16 mg%; phosphorus total 96.8 mg%; inorganic phosphorus of 56.4 mg%; dry residue 10,0+1,5%; reducing agents 364 mg%.

Characterization of the enzymatic hydrolysate of soybean seeds: pH 7,2; amine nitrogen 450 mg%; protein 7,8 g/l; urea 0.88 g/l; glucose 0,23 g/l; potassium 0,76 g/l; sodium of 5.45 g/l; calcium 0.02 g/l; chlorine 0,53 g/l; iron and 0.61 g/l

Of equal bases of the two enzymatic hydrolysates prepared nutrient medium for growing the vaccine strain of the plague microbe in the following way. Equal volumes of hydrolysates diluted with distilled water to testimony amine nitrogen 0.14% 0,125 l each, of sodium chloride 5,0; sodium phosphate 2-substituted 0,004; adjust pH of 20% solution of sodium hydroxide to the testimony of 7.2, agar microbiological 17,0; distilled water the rest.

The medium is heated in an autoclave at 1 ATM for 30 min to dissolve all ingredientlist example, experiencing the culture vaccine strain of the plague microbe, grown on the plates 2% of agar Hottinger, pH to 7.2 at a temperature of 28°C for 24 hours Of daily culture was prepared 1 billion a suspension of microbial cells of the test strain equal to 10 IU optical turbidity standard of CCA gisk named after. L. A. Tarasevich, then serial tenfold dilutions in physiological solution of 4.5 ml conveyed to the content in 1 ml 500 million M. K., and from this breeding suspension culture were sown at 1 ml three mattress. Crops were incubated at 28°C in terms of thermostat. Analysis was performed after 48 hours

Example 1. The test strain was grown on a nutrient medium, aderasa, g/l: agar microbiological 16,0; enzymic hydrolysate beef 100,0; enzymatic hydrolysate of soya beans 100,0; sodium chloride 4,0; sodium phosphate 2-substituted 3,0; sodium sanitarily 0,0003; distilled water to 1 liter Ready environment adjust to pH 7.2 for 20% solution of sodium hydroxide or hydrochloric acid 1:1. With this ratio of ingredients, the amount of biomass amounted to 49 billion m K. in 1 ml.

Example 2. The test strain was grown on a nutrient medium containing, g/l: microbiological agar - 17,0; enzymic hydrolysate beef - 125,0; enzymatic GID; distilled water to 1 liter Ready environment adjust to pH 7.2 for 20 per cent, solution of sodium hydroxide or hydrochloric acid 1:1. With this ratio of ingredients, the amount of biomass amounted to 103 billion m K. in 1 ml.

Example 3. The test strain was grown on a nutrient medium containing, g/l: agar microbiological 18,0; enzymic hydrolysate beef 150,0; enzymatic hydrolysate of soya beans 150,0; sodium chloride 6,0; sodium phosphate 2-substituted 5,0; sodium sanitarily 0,0005; distilled water to 1 liter Ready environment adjust to pH 7.2 for 20% solution of sodium hydroxide or hydrochloric acid 1:1. With this ratio of ingredients, the amount of biomass amounted to 45 billion m K. in 1 ml.

Thus, the claimed nutrient medium (example 2) prepared on the basis of equal volumes of enzymatic hydrolysates of beef and soy beans, it contributes to greater biomass accumulation vaccine strain of the plague microbe.

Nutrient medium for the accumulation of biomass vaccine strain of the plague microbe containing the enzymatic hydrolysate of raw meat, sodium chloride, microbiological agar, sodium phosphate disubstituted, distilleria the positive hydrolyzed beef, additionally, enzymatic hydrolyzed soy and sanitarily sodium in the following ingredients, g/l:

Agar microbiological 16,0-18,0

Enzymic hydrolysate beef 100,0-150,0

Enzymatic hydrolyzed soy 100,0-150,0

Sodium chloride 4,0-6,0

Sodium phosphate 2-substituted 0,3-0,5

Sodium sanitarily 0,0003-0,0005

Distilled water the Rest



 

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FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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