RU2626568C1 - Dense nutrient medium for cultivation and collection of biomass of plague microbe of ypestis ev vaccine strain - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium for cultivation and growth of plague microbial biomass of the Y. pestis EV vaccine strain contains the enzymatic hydrolyzate of corn extract as a nutrient base, condensed, sodium chloride, 2-substituted 12-aqueous sodium phosphate, Mohr's salt, microbiological agar and drinking water at a specified ratio of ingredients.

EFFECT: invention allows to increase the yield of biomass of the vaccine strain of plague EB microbe.

3 ex

Description

The invention relates to microbiology, namely to the preparation of microbiological culture media for cultivating a plague microbe and growing biomass of Y. pestis EV and can be used to obtain a sufficient amount of biomass with a certain percentage of living microbial cells.

Nutrient medium for cultivation of the vaccine strain of the plague microbe Y. pestis EV including, g / l: hydrolyzed beef meat containing amine nitrogen 0.12%; sodium chloride 4.0; sodium phosphate 2-substituted 12-aqueous 4.0; sodium sulfide 0.04; hydrochloric acid (1: 1) 1.6; microbiological agar 24.0; drinking water up to 1 liter (Industrial regulations for production, plague live vaccines, lyophilisate for the preparation of suspension for injection, cutaneous scarification application and inhalation. PR 01897080-09-09. Registration No. 2134-09. 2009, 253 p.).

The disadvantage of this environment is its high cost.

Closest to the proposed invention relates to a nutrient medium consisting of a mixture of two hydrolysates in a ratio of 1: 2 for the cultivation of a vaccine strain of the plague microbe Y. pestis EV including, g / l: hydrolyzed corn extract containing 0.45% amine nitrogen; casein hydrolyzate containing amine nitrogen of 0.45%, - 85; sodium chloride 3.0; sodium phosphate 2-substituted 12-aqueous 2.0; sodium sulfide 0.4; molybdenum ammonium 0.5; microbiological agar 30.0; pH 7.0 ± 0.2 drinking water up to 1 liter. The mixture is thoroughly mixed and poured into the loading door of the installation for drying protein hydrolysates. Drying is carried out at a temperature of (80 ± 2) ° C, under a vacuum of at least 0.6 atm (NPO Allergen, Stavropol). Dried agar is crushed with ceramic balls in a drying chamber for 2.5-3 hours, periodically (every 15-20 minutes), changing its position (from horizontal to extreme left and right) for stirring. (The dissertation for the degree of candidate of biological sciences., Saratov 1994, Gyulushanyan KS)

The disadvantage of this environment is its complexity and bulkiness.

The aim of the invention is to obtain a high-quality nutrient medium dense for cultivation and cultivation of biomass of the vaccine strain of the plague microbe Y. pestis EV on a protein basis from plant material - enzymatic hydrolyzate of corn extract (condensed), which provides a sufficient amount of biomass with a certain percentage of living microbial cells, as well as significant economic effect.

The essence of the invention lies in the fact that the nutrient medium is dense as a nutrient base, contains an enzymatic hydrolyzate of corn extract (condensed), sodium chloride, sodium phosphate 2-substituted 12-water, microbiological agar, drinking water, with the addition of a stimulating additive - salt Pestilence in the following ratio of ingredients, g / l:

Enzymatic Corn Extract Hydrolyzate (condensed) 40.0-56.0 Sodium Chloride 4.0-6.0 Sodium phosphate 2-substituted 12-aqueous 3.0-5.0 Salt Mora 0.08-0.12 Microbiological agar 15.0-19.0 Drinking water up to 1 l

As a feedstock, corn extract- (condensed) containing an average of 9.4% protein is used. Due to the high solids content of nitrogenous substances (up to 45%) and carbohydrates (up to 25%), the extract is a good nutrient medium in the production of penicillin and other antibiotics and vitamins. In addition, it is rich in trace elements, mg / kg: zinc 22; manganese 5; copper 5; cobalt 0.02; iodine 0.30, macrocells,%: phosphorus 0.25; sodium 0.04; calcium 0.59; potassium 0.36; magnesium 0.12; sulfur 0.11; chlorine 0.04; amino acids, in%: arginine 90.0; histidine 72.0; leucine 72.0; isoleucine 89.0; phenylalanine 59.0; threonine 95.0; valine 12.7; lysine 0.96; methionine 0.56; tryptophan 0.22; methionine + cystine 0.27, and vitamins, mg / kg: carotene (vitamin A) 8.0; B ₁ 4.0; B ₂ 1.0; B ₃ (pantothenic acid) 6.5; B ₄ (choline) 400; B ₅ 17; B ₆ 2.9. As is known (I.V. Petrukhin, 1989), corn proteins are characterized by an increased content of threonine, histidine, leucine, valine, which supply macroergic bonds, corn proteins also contain a large amount of such essential amino acids as isoleucine, leucine, arginine, phenylalanine. (M.F. Nesterin, I.M. Skurikhin. Chemical composition of food products. Moscow. "Food Industry". 1979. 247 p.). There is also information about the ability of leucine, as branched chain amino acids, to initiate protein synthesis (E. A. Gazov, X. S. Morgan, / USA / 1989).

Sodium chloride, hch, GOST 4233-77 batch 24, shelf life 12.2015. Bacteria need a minimal amount of salt. Being dissolved in a nutrient medium and being in a state of electrolytic decay, ionized mineral compounds are simultaneously catalysts of various chemical processes occurring in a microbial cell.

The inclusion in the medium of sodium phosphate 2 substituted 12-water is justified by the widespread use of phosphates in the preparation of culture media, because these are the only inorganic compounds that have a buffering effect in a physiologically important pH range, they are low toxic (Guidelines for the manufacture and use of culture media and solutions for microbiological purposes, the cultivation of cells and viruses, M. 1989). (M.S. Polyak; V.I. Sukharevich; M.E. Sukharevich. Nutrient media for medical and sanitary microbiology. St. Petersburg: ELBI-St. Petersburg. - 2008. - P. 37-38).

Salt of Mora GOST 4208-72. Double sulfate salt of iron oxide and ammonium. Not combustible, not toxic. Lot 14, production date October 2014. Shelf life 3 years. Mora salt is supplied by NPF Nevsky Chemist LLC St. Petersburg (Neva Reagent).

Microbiological agar - bacteriological agar (European type). Producer: "Pronadisa" Spain. Lot LF 15130184. Shelf life until 10.2017. Production date: October 2013.

Drinking water-GOST R 51232-98 meets all hygiene requirements.

Preparation of enzymatic hydrolyzate from corn extract (condensed).

A method of preparing a nutrient base for growing the plague microbe of the vaccine strain Y. pestis EV is as follows: corn extract (condensed) in an amount of 4.0 kg is placed in a digester, then 24 l of drinking water is added, boiled for 5 minutes, the pH is adjusted to 8 , 2 40% sodium hydroxide solution in an amount of 52 ml, then the infusion is poured into 10 l containers, cooled to 46 ° C. In the cooled infusion, add the pancreas of cattle (cattle) at a rate of 30.0 per 1 liter, set the pH to 8.2-8.5, add 1.5% chloroform. Hydrolysis is carried out in a heat chamber at a temperature of 46 ° C for 10 days. For the first 24 hours, the mixture is stirred every 15 minutes, then the mixture is stirred every 2 hours. The level of amine nitrogen is measured daily. On day 10, amine nitrogen amounted to 1120 mg%, and the dry residue, respectively, 25.5%. With the cessation of the growth of amine nitrogen, which happens on the 7-10th day, stop mixing and heating. Allowed to stand for 2 days, then the liquid is filtered off from the precipitate on a press filter, bottled with 1% chloroform, poured with sterile rubber stoppers and stored at 4-6 ° C.

Preparation of a dense culture medium

A nutrient medium based on an enzymatic hydrolyzate of corn extract (condensed) for cultivating a plague microbe and growing a vaccine strain of Y. pestis EV is prepared in the following way: an enzymatic hydrolyzate diluted with drinking water to an amine nitrogen reading of 0.12% 48 ml, sodium chloride 5.0; sodium phosphate 2-substituted 12-aqueous 4.0; microbiological agar 17.0; drinking water up to 1 liter. The medium is boiled until it is completely dissolved, then the pH is adjusted to 7.2 ± 0.1 with a 20% sodium hydroxide solution, a stimulant is added: Mohr's salt at a concentration of 0.1 g / l, respectively, poured into graduated bottles (mattresses) of 50, 0 ml, then sterilized at 0.5 atm. within 30 minutes After sterilization, cool to 56 ° C, then mow.

Before preparing the production culture, the vaccine strain of the plague microbe Y. pestis EV is animated by passage through the body of the guinea pig. Before sterilization, all sterile bacteriological pipettes are stored in a refrigerator at (4 ± 1) ° C. An ampoule with a dry culture of strain EV is opened according to aseptic rules, its contents are diluted with 0.9% sodium chloride solution and a number of test tubes with Hottinger beveled agar pH 7.1 ± 0.1 are seeded with microbial suspension (initial culture or culture of the 1st generation). At the same time, its cultural-morphological and enzymatic properties are monitored, 2 test tubes with Giss medium with sucrose, lactose, rhamnose and glycerin are added, to which Andrede indicator is added, and plated on 3 Petri dishes with Hottinger agar pH 7.3 ± 0.1. The culture is also seeded on 3 Petri dishes with Hottinger agar pH 7.1 ± 0.1, which are subsequently used for sequential sieving in order to obtain isolated colonies. All Petri dishes with crops are placed for (48 ± 2) h for growing in a thermostat at a temperature of (27 ± 1) ° C. In that case, if the culture without dissociation, i.e. colony morphology is typical of a plague microbe and the vaccine strain stays firmly on its biochemical type, the initial culture of the 1st generation is transferred to bacteriological tubes (culture of the 2nd generation). Crops incubated (24 ± 1) h at (27 ± 1) ° C. After a day, test tubes with a culture of the second generation are used to cultivate the plague microbe vaccine strain on beveled surfaces in bottles (mattresses). As an example, test the culture of the vaccine strain of the plague microbe EB grown on plates of 2% Hottinger agar, pH 7.2 ± 0.1 at a temperature of (27 ± 1) ° C. Then prepare a suspension of the I daily culture of the test strain, corresponding to 10 units. according to the optical standard sample turbidity (OCO 42-28-85 P), equivalent to 1.0 × 10 ⁹ MK / ml in a sterile 0.9% sodium chloride solution, then diluting a suspension of 1 × 1 from 1.0 × 10 ⁹ adjust the content in 1 ml of 500 million m.k. from this dilution, culture suspensions are sown in 1.0 ml in 3 vials (mattress), then the suspension is distributed by swaying on the beveled surface of the agar, left on specially mounted supports in a beveled state, and placed in a thermostat. Grown for (48 ± 2) h at (27 ± 1) ° C.

The results are taken into account after (48 ± 2) h. After that, the grown culture is washed off with 5 ml of 0.9% sodium chloride solution from each bottle (mattress) by aspirating biomass into a graduated 25 ml test tube with a 5 ml sterile bacteriological pipette and the concentration is established microbes in 1 ml of suspension for CCA turbidity.

The method of counting the number of microbial cells

Nutrient media from the enzymatic hydrolyzate of corn extract (condensed) for determining the number of live microbial cells must ensure, without the addition of stimulants, the growth of the microbes of the vaccine strain Y. pestis EV on all Petri dishes with agar seeded with 10 mk by CCA turbidity 42-28-85P 10ME. As a growth promoter, 1 ml / l of hemolyzed blood or 0.25 g / l of freshly prepared boiled sodium sulfite is added to the nutrient media before filling them into Petri dishes. Test tubes with 0.9% sodium chloride solution and pipettes used to determine the content of live microbes in biomass should be cooled to a temperature of (4 ± 2) ° C. From each tube with a liquid nutrient medium, the contents in an amount of 0.1 ml are diluted with 0.9% sodium chloride solution in an amount of 0.9 ml (basic dilution 10 ^-1 ). Saline is added sequentially to obtain 1 billion MK / ml. Then, a ten-fold dilution of the obtained suspension in a 0.9% sodium chloride solution is made with a pipette, starting from 10 ^-1 to (0.5 ± 0.01) ml of suspension with 4.5 ± 0.05 ml of a 0.9% sodium chloride solution ending with (10 ^-7 ) and (10 ^-8 ). The diluted biomass from the last two tubes (10 ^-7 ) and (10 ^-8 ) is inoculated with a (0.1 ± 0.01) ml pipette into 2 Petri dishes with nutrient agar (pH 7.2 ± 0.1) and kept at temperature (27 ± 1) ° C for 2-3 days. (Industrial regulation PR for the production of the Plague live vaccine, lyophilisate for the preparation of a suspension for injectable cutaneous application of scarification and inhalation PR 01897080-09-09) dated 11.12.2009.

Example 1. The test strain was grown on a sloping surface of a nutrient medium containing, g / l: enzymatic hydrolyzate of corn extract (condensed) 40.0; sodium chloride 4.0; sodium phosphate 2- substituted 12-aqueous 3.0; Mohr's salt 0.08; microbiological agar 15.0; drinking water up to 1 liter. With this ratio of ingredients, the collection of biomass of Y. pestis EV is 13 ml of suspension, in 1 ml, which received 59 billion m.k., the percentage of live m.k. after (48 ± 1) h, respectively 51; after drying, 20% of live microbial cells were obtained.

Example 2. The test strain was grown on a sloping surface of a nutrient medium containing, g / l: enzymatic hydrolyzate of corn extract (condensed) 48.0; sodium chloride 5.0; sodium phosphate 2-substituted 12-aqueous 4.0; Mora salt 0.1; microbiological agar 17.0; drinking water up to 1 liter. With this ratio of ingredients, the collection of Y. pestis EV biomass amounted to 15 ml of suspension, in 1 ml, which received 100 billion m.k. after (48 ± 1) h, 86.6 respectively, after drying, 43.8% of live microbial cells were obtained.

Example 3. The test strain was grown on a beveled surface of a nutrient medium containing, g / l; enzymatic hydrolyzate of corn extract (condensed) 56.0; sodium chloride 6.0; sodium phosphate 2-substituted 12-aqueous 5.0; Mohr's salt 0.12; microbiological agar 19.0; drinking water up to 1 liter. With this ratio of ingredients, the collection of Y. pestis EV biomass amounted to 14 ml of suspension, in 0.1 ml, which received 65 billion cu.m., the percentage of live microbial cells after 65 (48 ± 1) h, respectively 65, after drying, 23% of live microbial cells.

Thus, the claimed nutrient medium is dense for cultivating the plague microbe and growing the biomass of the vaccine strain Y. pestis EV (Example 2), prepared on the basis of the enzymatic hydrolyzate of the corn extract (condensed), can be used for cultivating and growing the biomass of the vaccine strain of the plague microbe Y. pestis EV, which provides a sufficient amount of biomass with a certain percentage of living microbial cells and a significant economic effect when replacing meat nutritional bases with corn. The profitability of the environment is 31%.

Claims (2)

Dense nutrient medium for cultivation of the plague microbe and cultivation of the plague microbe biomass of the vaccine strain Y. pestis EV, including the nutrient base, sodium chloride, sodium phosphate 2-substituted 12-water, microbiological agar and drinking water, characterized in that it contains as a nutrient base the enzymatic hydrolyzate of the corn extract is condensed and additionally contains Mora salt as a stimulating additive in the following ratio of ingredients, g / l: Enzymatic Corn Extract Hydrolyzate condensed 40.0-56.0 Sodium Chloride 4.0-6.0 Sodium phosphate 2-substituted 12-aqueous 3.0-5.0 Salt Mora 0.08-0.12 Microbiological agar 15.0-19.0 Drinking water up to 1 l