RU2844477C1 - Method for producing an accumulation liquid culture medium for culturing a vaccine strain of yersinia pestis ev plague microbe - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method for producing an accumulation liquid culture medium for culturing a vaccine strain of plague microbe Y. pestis EV, comprising diluting an enzymatic hydrolysate of casein and an enzymatic hydrolysate of soya beans with distilled water to an amine nitrogen content of 120 mg %; then following is mixed: 89.0 g of enzymatic hydrolysate of casein, 89.0 g of enzymatic hydrolysate of soya beans, 10.0 g of dry enzymatic peptone, 0.2 g of magnesium sulphate, 3.0 g of sodium chloride, 4.0 g of disubstituted sodium phosphate dodecahydrate, 20.0 g of activated carbon, distilled water is added to 1 l, pH is set to 8.3±0.1 using 20 % solution of sodium hydroxide; medium is boiled for 3-5 minutes, then filtered through belting fabric filter and filter paper; adding to filtrate growth-stimulating additives – sodium sulphite 0.3 g and ammonium molybdate tetrahydrate 0.5 g, intensively stirring; adding boiled distilled water to initial volume, establishing pH 7.2±0.1 with hydrochloric acid solution in dilution of 1:1; sterilized for 20 minutes at temperature (115±1) °C.

EFFECT: invention enables to obtain a high concentration of microbial cells.

1 cl, 3 ex

Description

The invention relates to biotechnology and microbiology, namely to the preparation of microbiological storage liquid nutrient media for cultivating the vaccine strain of the plague microbe Y. pestis EV, and can be used in obtaining a nutrient medium that ensures the yield of biomass with a high concentration of microbial cells.

A liquid nutrient medium is known for cultivating the vaccine strain of the plague microbe Yersinia pestis EV, where the enzymatic hydrolysate of beef meat is used as the nutrient base - 170.0-190.0 ml. The nutrient medium also contains: sodium chloride 3.0-5.0 g; sodium sulfite 3.0-5.0 g; disodium phosphate 3.0-5.0 g; Karpuzidi stimulator 0.006-0.010 ml; purified water - up to 1 l (Patent of the Russian Federation No. 2433170 "Liquid nutrient medium for cultivating the plague microbe of the vaccine strain EV". Published. 11/10/2011. Bulletin No. 31).

The disadvantage of this environment is its low productivity.

The closest to the proposed invention is a liquid nutrient medium for cultivating the vaccine strain of the plague microbe Yersinia pestis EV, wherein the enzymatic hydrolysate of soybeans (beans) - 320.0-420.0 ml and the following ingredients are used as the nutrient base: sodium chloride 4.0-6.0 g; dibasic sodium phosphate 3.0-5.0 g; sodium sulfite 0.3-0.5 g; lipoic acid 0.4-0.6 g; 20% sodium hydroxide solution 2.5-3.5 ml; distilled water - up to 1 l. (Patent of the Russian Federation No. 2260620 “Nutrient medium (liquid) for cultivating the plague microbe of the EB vaccine strain.” Published on September 20, 2005. Bulletin No. 26).

The disadvantage of this environment is the difficulty of its preparation.

The aim of the invention is to obtain a liquid nutrient medium for cultivating the vaccine strain of the plague microbe Y. pestis EV.

The technical result of the invention consists in that the enzymatic hydrolysate of casein together with the enzymatic hydrolysate of soybeans (beans) and dry enzymatic peptone, as well as sodium chloride, sodium phosphate dibasic 12-aqueous, magnesium sulfate, activated carbon, distilled water and growth-stimulating additives - ammonium molybdate 4-aqueous and sodium sulfite - are used as the main source of nitrogen nutrition in the preparation of the medium.

The ingredients used together increase the cumulative properties of the nutrient medium in the following ratio:

Enzymatic hydrolysate of casein 89.0 ml

Enzymatic hydrolysate of soybeans (beans) 89.0 ml

Dry enzymatic peptone 10.0 g

Sodium chloride 3.0 g

Sodium phosphate dibasic 12-hydrate 4.0 g

Ammonium molybdate 4-aqueous 0.5 g

Sodium sulfite 0.3 g

Magnesium sulfate 0.2 g

Activated carbon 20.0 g

Distilled water up to 1 l

Casein (TU 8-5344-113607) is a phosphorus-containing protein obtained from skim milk and is the most complete type of raw material for nutrient media. Its content ranges from 23 to 90% of the total content of milk proteins. The elemental average composition of unfractionated casein (in %) is as follows: carbon - 53; hydrogen - 7.1; nitrogen - 15.63; oxygen - 22.6; sulfur - 0.82; phosphorus - 0.85. Pancreatic hydrolysate of casein is obtained using pancreatic enzymes. The enzymatic base of casein contains low-molecular peptides and a full set of amino acids: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine + cystine, methionine, tryptophan, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, tyrosine, which provides the biological need of microorganisms for nitrogen nutrition.

Enzymatic hydrolysate of soybeans (beans) (TU 9385-018-78095326-2006) contains about 40% protein. Soy protein contains about 18 different types of amino acids, including essential ones, including glutamic and aspartic acids, which are closely related to carbohydrate metabolism through the tricarboxylic acid cycle, supplying macroergic bonds, soy is also rich in microelements and vitamins.

Dry enzymatic peptone (GOST 13805-76). Peptone contains various amino acids, products of enzymatic hydrolysis of proteins, inorganic microelements necessary for the cultivation of microorganisms of various systematic groups. It is an easily accessible source of nitrogen for microorganisms.

Sodium chloride (GOST 4233-77, chemically pure, analytical grade) is used to maintain the isotonicity of the medium and the oxidation-reduction potential.

Sodium phosphate disubstituted 12-aqueous (GOST 4172-76, analytical grade). Its use is justified by the widespread use of phosphates in the preparation of nutrient media, since these are the only inorganic compounds that have a buffering effect in the physiologically important pH range.

Sodium sulfite (GOST 195-77, analytical grade) stimulates the growth of the plague microbe, helping to maintain the pH level of the environment in the desired range. This property creates favorable conditions in the environment for the growth and reproduction of the overwhelming majority of microorganisms, including pathogenic ones. The sulfur present in the compound is necessary for the formation of the protein of the cells of microorganisms. In addition, the addition of sodium sulfite increases the transparency of the environment.

Ammonium molybdate tetrahydrate (GOST 3765-78, chemically pure, analytical grade) ensures the opening of peptide bonds of protein molecules and improves the accumulative properties of nutrient media.

Magnesium sulfate (GOST 4523-77, chemically pure) promotes rapid sedimentation of biomass. Magnesium, as an important cation of the cell, is an inorganic cofactor for many enzymatic reactions, including reactions involving ATP; it is involved in the binding of enzymes to substrates. Sulfur is a part of proteins (in the amino acids cysteine and methionine) and some coenzymes (CoA, cocarboxylase, etc.).

Activated carbon OU-A (GOST 4453-74) is a black fine powder without smell or taste, insoluble in water and other solvents, has adsorption capacity. Carbon is added to the environment for the sorption of microbial metabolites that negatively affect highly sensitive pathogenic microorganisms.

Distilled water (GOST R 58144-2018) is the most important component of all nutrient media. Water makes up 80-90% of the cellular mass of microorganisms and plays an important role in the implementation of all physiological processes.

Preparation of enzymatic hydrolysate of casein

For 10-12.5 liters of drinking water, use 1 kg of dry casein. The dry casein sample is soaked in cold water (the amount of water is taken into account).

The first two days the water is changed daily, on the third day the water is drained, the casein is squeezed out well through cheesecloth.

Drinking water (minus that absorbed by casein) is poured into the cooking kettle and brought to a boil. The pH is set to 8.0±0.1 by adding 20% sodium hydroxide solution. The soaked casein is lowered into the boiling alkaline water in small portions while stirring. Boil, stirring and maintaining the pH of 8.0±0.1 by adding small portions of 20% sodium hydroxide solution. When the mass becomes homogeneous, without grains, with a stable pH within 8.0±0.1, stop boiling. Check the volume, if necessary, add hot distilled water to the original volume, and adjust the pH again.

The mixture is cooled to a temperature of (45±1)°C, poured into the reactor, pancreas is added (with an activity of at least 5000 units according to Fould-Gross) at a rate of 100 g per 1 liter of water taken. The mixture is mixed well, chloroform is added to 2% of the volume, the reactor is hermetically sealed.

The hydrolysis process is carried out at a temperature of (37±1)°C or (45±1)°C. On the first day, the mixture is stirred every 15-20 minutes for 5 minutes, and then every 1-2 hours (automatic stirrer) for 5 minutes. The process continues for 7-9 days with daily monitoring of amine nitrogen, when the increase ceases, the hydrolysis is stopped. Allow to settle in a cool place for 1-2 days until the sediment completely falls out. The supernatant liquid is drained, the sediment is filtered through a filter cloth using a press filter. The hydrolysate is poured into bottles with the addition of 1% chloroform by volume, closed with sterile rubber stoppers and stored at a temperature of (4±2)°C.

Preparation of enzymatic hydrolysate of soybeans (beans)

0.5 kg of soybeans are soaked in 5 liters of hot water for 24 hours, changing the water five times. The last infusion with beans is boiled for 5 minutes. After cooling, the infusion is poured into a ten-liter bottle, the beans are ground in a meat grinder and placed in the infusion. By adding a 20% sodium hydroxide solution, the pH is set to 8.2 ± 0.1. The pancreas is added (with an activity of at least 5000 units according to Fould-Gross) at the rate of 200 g per 1 liter and chloroform up to 2%.

Hydrolysis is carried out in a thermal chamber at a temperature of (37±1)°C for 3 days. During the first 2 hours, the mixture is stirred for 5 minutes every 15 minutes, then for 5 minutes every 6 hours. The level of amine nitrogen is measured daily; at the end of the process, it should reach 0.6±005%.

After stopping stirring, the hydrolyzate is left to settle in a cool place for 1-2 days until the sediment has completely settled. The supernatant is drained, the sediment is filtered through a filter cloth using a press filter. The hydrolyzate is poured into bottles with the addition of chloroform (1% of the volume), closed with sterile rubber stoppers and stored at a temperature of (4±2)°C.

Preparation of liquid nutrient medium for culturing the vaccine strain of the plague microbe Y. pestis EV

Enzymatic hydrolysate of casein and enzymatic hydrolysate of soybeans are diluted with distilled water to an amine nitrogen content of 120 mg%. Mix 89.0 ml of enzymatic hydrolysate of casein, 89.0 ml of enzymatic hydrolysate of soybeans, 10.0 g of dry enzymatic peptone, 0.2 g of magnesium sulfate, 3.0 g of sodium chloride, 4.0 g of dibasic sodium phosphate 12-aqueous, 20.0 g of activated carbon, add distilled water to 1 l. Adjust the pH to 8.3±0.1 using a 20% sodium hydroxide solution. Boil the medium for 3-5 min, then filter through a Belting fabric filter and filter paper.

Growth-stimulating additives are added to the filtrate: 0.3 g sodium sulfite and 0.5 g ammonium molybdate tetrahydrate, and stirred vigorously. Boiled distilled water is added to the initial volume, the pH is adjusted to 7.2±0.1 using a 1:1 dilution of hydrochloric acid. Sterilize for 20 minutes at a temperature of (115±1)°C.

Quality control of the nutrient medium

Physicochemical indicators - determination is carried out according to MUK 4.2.2316-08 “Methods for control of bacteriological nutrient media”.

Appearance. Transparent broth from light yellow to light brown. The appearance of the nutrient medium is assessed visually.

pH. From 7.1 to 7.3. Determination is carried out by potentiometric method.

Dry residue. From 2.6% to 3.4%.

Amine nitrogen. From 0.09 to 0.15%.

Chlorides. In terms of sodium chloride from 0.5% to 0.7%.

Sterility. The determination is carried out by seeding the broth on Petri dishes with Hottinger agar pH 7.4±0.1 and incubating the cultures at a temperature of (37±1)°C for (48±2) hours. No growth should be observed on the dishes.

Biological parameters. The ampoule with the production culture of the Y. pestis EV strain is opened in compliance with aseptic rules and reconstituted with 1.8 ml of 0.9% sodium chloride solution. The resulting suspension is used to inoculate a series of test tubes with Hottinger slant agar, pH 7.1 ± 0.1 (generation 1 culture). The cultures are incubated at (27 ± 1) ° C for 48 hours. The generation 1 culture is subcultured into mattresses with Hottinger broth, pH 7.1 ± 0.1 (generation 2 culture). The cultures are incubated at (27 ± 1) ° C. After 48 hours, the generation 2 culture is used to inoculate bottles with Hottinger broth, pH 7.1 ± 0.1. The culture is grown for 24 hours at a temperature of (27 ± 1) ° C and continuous aeration in a volume of 1.0-1.5 l of air per 1 l of broth. The resulting seed culture is used to determine the effectiveness of the nutrient medium.

Methodology for determining the efficiency of liquid nutrient medium

The increase in microorganisms relative to the seeded ones (efficiency indicator) is determined according to MUK 4.2.2316-08.

The seed culture is titrated to dilutions of 10 ^-4 , 10 ^-5 and 10 ^-6 . 1.0 ml of each dilution is added to a test tube with 9.0 ml of the test nutrient medium, mixed thoroughly and 0.1 ml is seeded onto three Petri dishes with Hottinger agar pH 7.1 ± 0.1 (“zero seeding”). The crops are incubated at (27 ± 1) ° C for 48 hours.

The seeded test tubes with the nutrient medium are incubated at (27±1)°C for 24 hours. Then, after mixing, 0.1 ml of each dilution is seeded onto three Petri dishes with Hottinger agar, pH 7.1±0.1, and incubated at (27±1)°C for 48 hours.

The increase in the number of microorganisms in the cumulative medium during the incubation process is determined by the formula (%):

Where

E - efficiency indicator (growth);

n _t - average number of colonies on plates after incubation of the culture in a liquid accumulation medium;

K - degree of dilution;

n ₀ - average number of colonies with “zero seeding”.

After 48 hours of incubation, the formed colonies are counted on Petri dishes seeded from the initial culture suspensions (“zero” seeding) and after enrichment in a liquid nutrient medium for 24 hours.

The indicator of the effectiveness of the environment is taken as a value equal to the arithmetic mean of the number of certain indicators from three dilutions.

Example 1. The vaccine strain of the plague microbe Y. pestis EV was grown in a liquid nutrient medium containing: enzymatic hydrolysates of casein and soybeans - 69 ml each; dry enzymatic peptone - 8.0 g; magnesium sulfate - 0.1 g; sodium chloride - 1.0 g; sodium phosphate dibasic 12-hydrate - 2.0 g; activated carbon - 18.0 g; sodium sulfite - 0.1 g; ammonium molybdate tetrahydrate - 0.3 g; distilled water - up to 1 liter. The effectiveness of the liquid nutrient medium was determined by the increase in microorganisms relative to the inoculated amount (effectiveness indicator) according to MUK 4.2.2316-08.

With this ratio of ingredients, the increase in the number of Y. pestis EV microbial cells in the cumulative medium during incubation of the crops for 24 hours was 60%.

Example 2. The vaccine strain of the plague microbe Y. pestis EV was grown in a liquid nutrient medium containing 89 ml of enzymatic hydrolysates of casein and soybeans; 10.0 g of dry enzymatic peptone; 0.2 g of magnesium sulfate; 3.0 g of sodium chloride; 4.0 g of dibasic sodium phosphate 12-hydrate; 20.0 g of activated carbon; 0.3 g of sodium sulfite; 0.5 g of ammonium molybdate 4-hydrate; and up to 1 liter of distilled water. The efficiency of the liquid nutrient medium was determined by the growth of microorganisms relative to those inoculated (efficiency indicator) according to MUK 4.2.2316-08.

With this ratio of ingredients, the increase in the number of Y. pestis EV microbial cells in the cumulative medium during incubation of the crops for 24 hours was 104%.

Example 3. The vaccine strain of the plague microbe Y. pestis EV was grown in a liquid nutrient medium containing 109 ml of enzymatic hydrolysates of casein and soybeans; 12.0 g of dry enzymatic peptone; 0.3 g of magnesium sulfate; 6.0 g of dibasic sodium phosphate dodecahydrate; 22.0 g of activated carbon; 0.3 g of sodium sulfite; 0.5 g of ammonium molybdate tetrahydrate; and up to 1 liter of distilled water. The efficiency of the liquid nutrient medium was determined by the growth of microorganisms relative to those inoculated (efficiency indicator) according to MUK 4.2.2316-08.

With this ratio of ingredients, the increase in the number of Y. pestis EV microbial cells in the cumulative medium during incubation of the crops for 24 hours was 75%.

Thus, the claimed liquid cumulative nutrient medium for cultivating the vaccine strain of the plague microbe Y. pestis EV (example 2) ensures the production of biomass with a high concentration of microbial cells after 24 hours of incubation.

Claims (1)

A method for producing a liquid nutrient medium for culturing a vaccine strain of the plague microbe Y. pestis EV, comprising diluting an enzymatic hydrolysate of casein and an enzymatic hydrolysate of soybeans with distilled water to an amine nitrogen content of 120 mg%; then mixing 89.0 g of casein enzymatic hydrolysate, 89.0 g of soybean enzymatic hydrolysate, 10.0 g of dry enzymatic peptone, 0.2 g of magnesium sulfate, 3.0 g of sodium chloride, 4.0 g of dibasic sodium phosphate 12-aqueous, 20.0 g of activated carbon, adding distilled water to 1 l, adjusting the pH to 8.3±0.1 using a 20% sodium hydroxide solution; the medium is boiled for 3-5 minutes, then filtered through a Belting fabric filter and filter paper; growth-stimulating additives are added to the filtrate - 0.3 g sodium sulfite and 0.5 g ammonium molybdate tetrahydrate, mixed vigorously; boiled distilled water is added to the initial volume, the pH is adjusted to 7.2 ± 0.1 using a 1: 1 dilution of hydrochloric acid; sterilized for 20 minutes at a temperature of (115 ± 1) ° C.