RU2415922C1 - Nutrient medium for lactic acid bacilli cultivation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: nutrient medium contains an enzymatic hydrolyzate of beef containing 0.7-0.9 % of amine nitrogen, an enzymatic hydrolyzate of Basketplant, glucose, microbiological agar and distilled water in the preset amounts.

EFFECT: higher lactic acid bacilli yield.

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Description

FIELD OF THE INVENTION

The invention relates to microbiology, in particular to the production of culture media for the cultivation of lactobacilli, and can be used in the industrial production of medicines.

State of the art

Known nutrient medium for the cultivation of microorganisms, including an amino acid base, sodium chloride, while the amino acid base is used for the ammonia-enzymatic hydrolyzate of waste from vaccine-whey production and incubator production in the form of globulins, or chicken embryos, or blood clots, or fibrin, obtained by ammonia hydrolyzate of crushed waste mixed with demineralized water and subsequent hydrolysis of the cattle’s native pancreas to soda the holding of amine nitrogen 500-700 mg% in the following ratio of chemical compounds, wt.%:

Peptone 1-2

Sodium Chloride 1.0-1.2

Ammonium salts 0.03-0.1

as well, mg%

Total Nitrogen 1100-1400

Amine Nitrogen 200-220

Tryptophan 80-200

(see Pat. RU No. 2103345, class C12N 1/00, publ. 01/27/1998).

The disadvantage of this nutrient medium is the high cost.

A known nutrient medium, the basis of which is hydrolyzed milk, yeast autolysate, agar, pH 6, in the following ratio of chemical compounds:

Hydrolyzed Milk 1000 ml

Yeast autolysate 3%

Agar 3%

(Bannikova L.A. Selection of lactic acid bacteria and their use in the dairy industry. Moscow "Food Industry", 1975 - S. - 224).

The disadvantage of this environment is its low productivity.

The closest in technical essence and the achieved positive effect and adopted by the authors for the prototype is a nutrient medium for surface and deep cultivation of lactobacilli, containing a nutrient base, glucose, agar-agar, growth stimulator and distilled water (RU 2002113739 A1, 02.27.2004).

The disadvantage of this environment is the lack of nutritional value for lactobacilli.

Disclosure of invention

The objective of the invention is to obtain a high-quality nutrient medium based on an enzymatic hydrolyzate of beef meat with the addition of an enzymatic hydrolyzate of callis aromatic as a growth promoter of lactobacilli.

The technical result that can be obtained using the present invention is to increase the number of colonies of lactobacilli.

The technical result is achieved using a nutrient medium for the cultivation of lactobacilli, containing a nutrient base, glucose, microbiological agar, growth stimulator and distilled water, and as a nutrient base, the medium contains an enzymatic hydrolyzed beef meat with an amine nitrogen content of 0.7-0.9%, and as a growth promoter is an enzymatic hydrolyzate of fragrant callisia in the following ingredients, wt.%:

Enzymatic hydrolyzate beef meat with an amine nitrogen content of 0.7-0.9% 28.0-30.0 Enzymatic Hydrolyzate 0.9-1.0 Glucose 18.0-20.0 Microbiological agar 0.9-1.0 Distilled water rest.

The essence of obtaining a nutrient medium for the cultivation of lactobacilli is as follows.

To prepare the nutrient base, beef meat is used as a feedstock, containing on average 12.7% to 21.71% nitrogenous substances, meat minerals consist of sodium, potassium, calcium, phosphorus and iron salts; their total number reaches 1%. The meat also contains vitamins, minerals. Beef meat contains more extractives than fresh frozen meat (Yu.A. Kozlov. Nutrient media in medical microbiology. Medgiz. 1950, p. 46).

The following amino acids, g / l, are formed from protein substances in beef meat during enzymatic hydrolysis: lysine, methionine, tryptophan, cystine, threonine, series, phenylalanine, proline, alanine, glycine, valine, leucine, tyrosine, histidine.

Glucose is a carbon source for the growth of lactobacilli.

In order to stimulate the effect, an enzymatic hydrolyzate from fragrant callisia is added to the nutrient medium.

Kallizia is one of the few indoor plants that can rely on the universality of the product of its hydrolysis (hydrolyzate) due to the content of numerous unique elements necessary for the growth and development of lactobacilli.

Fragrant callisia has the following biochemical composition: carotenoids, ascorbic acid, flavonoids, pectins, tannins, catechins. Callizia also has vitamin C, vitamin B2 (riboflavin) and B15 (pantothenic acid), vitamin PP (nicotinic acid), micro and macro elements: potassium, calcium, iron, manganese, vanadium, nickel, copper, zinc, gallium, bromine , strontium, rubidium, zirconium, lead, uranium, thorium (Zolotoy Us./M.N. Kedrova, 2006, p. 7-80).

Preparation of the nutrient basis of the proposed nutrient medium for growing microorganisms is as follows.

1.5 l of tap water is poured into the digester and brought to a boil. Pre-chopped beef meat in the amount of 1.00 kg is loaded into boiling water and boiled for 15 minutes, fat is removed during boiling. Then the heating is stopped and left to settle for 20 minutes. The meat is minced with a meat grinder, the meat water is cooled to a temperature of 45 ± 1 ° C, the pH value is adjusted to a value of 8.5 ± 0.1 with sodium carbonate. Minced meat in the amount of 2.00 kg, meat water 5.00 l and the pancreas of cattle in the amount of 0.20 kg are placed in 10 liter bottles, covered with cotton gauze plugs, covered with parchment paper, strengthened with rubber rings. Chloroform is added to the bottle as a preservative to a final concentration of 1%.

The hydrolysis process is carried out in a heat chamber at a temperature of 37 ° C. The mixture was stirred manually every 15 minutes for 14 days. The dynamics of the hydrolysis process is controlled by determining amine nitrogen, and upon reaching 0.7-0.9%, the heating is stopped, the digest is defended. Then manually filtered through fabric filters. The filtrate is collected in 10 liter glass containers. For preservation, chloroform is added in an amount of 1% to the volume of the filtrate, closed with rubber stoppers, labeled and transportation is carried out in a cold chamber, where it is stored at a temperature of 2-8 ° C.

The method of preparation of a growth stimulator, which is used as an enzymatic hydrolyzate of callis aromatic for the cultivation of lactobacilli, is as follows. The fragrant callisia shoot in an amount of 200 g is thoroughly washed in running tap water (previously placed in gauze). The washed substrate is ground, placed in a glass container and 600 ml (1: 3) of tap water heated to a temperature of 45 + 1 ° C are added. The mixture was alkalized with Na ₂ CO ₃ to a pH of 8.2-8.3 in phenolphthalein. 2% chloroform and 10 tablets of a digestive enzyme agent, pancreatin, are added to the total volume of the contents, they are covered with a tight cotton-gauze swab with parchment and placed in a heat chamber at a temperature of 45 + 1 ° С. Stand for 10 days, shaking during the first day every 15 minutes for 5 minutes, and on subsequent days every two hours for 5 minutes. The dynamics of the enzymatic process is controlled by the increase in amine nitrogen. On 9-10 days, the increase in this indicator stops, the heat chamber is turned off, the hydrolyzate is left for a day. Then filtered through a filter cloth. 2% chloroform is added to the filtrate, plugged with a rubber stopper, stored at a temperature of 2 to 8 ° C. Use as needed. The resulting hydrolyzate has the color of blood serum, the indicator of amine nitrogen is 0.075%; dry residue - 1.5%.

Examples of specific production of a nutrient medium for the cultivation of lactobacilli.

The effectiveness of the studied environments was evaluated in accordance with the methodological recommendations “To the control of nutrient media according to biological indicators. Moscow, 1980, using L.plantarum 8P-A3, L.acidophilus EP 317-402 as test strains of lactobacilli.

The test cultures were grown on solid agar plates with a pH of 6.8 ± 0.1 at a temperature of 37 ° C for 2 days. Of the two-day crops prepare 1 billion suspension of MT test strains equal to 10 units according to the optical turbidity standard OSO GISK im. L.A. Tarasovich, then serial ten-fold dilutions in physiological saline in a volume of 4.5 ml are adjusted to a content of 1 ml 1000 m.k. From this dilution, suspension of cultures is sown in 0.1 ml per 3 Petri dishes of spilled dried agar. Crops are incubated at 37 ° C for 5 days with daily viewing. After 24-120 hours, the counts of grown colonies are taken into account.

Example 1. Test strains are grown on a nutrient medium containing, g / l: enzymatic hydrolyzed beef meat 100.0; aromatic hydrolyzate of callisia aromatic 0.6 ml; glucose 16.0; microbiological agar 6.0; distilled water - the rest. With this ratio of ingredients, the number of colonies grown on dense agar plates for lactobacilli with a sowing dose of 10 mk averaged 15 colonies for each test strain.

Example 2. Test strains are grown on a nutrient medium containing, g / l: enzymatic hydrolyzed beef meat 200.0; fragrant hydrolyzate callisia hydrolyzate 0.8 ml; glucose 18.0; microbiological agar 8.0; distilled water - the rest. With this ratio of ingredients, the number of colonies grown on dense agar plates for lactobacilli with a sowing dose of 10 mk averaged 27 colonies for each test strain of lactobacilli.

Example 3. Test strains are grown on a nutrient medium containing, g / l: enzymatic hydrolyzed beef meat 280.0; fragrant hydrolyzate callisia hydrolyzate 0.9 ml; glucose 18.0; microbiological agar 9.0; distilled water - the rest. With this ratio of ingredients, the number of colonies grown on dense agar plates for lactobacilli with a sowing dose of 10 mk averaged 43 colonies for each test strain of lactobacilli.

Example 4. Test strains are grown on a nutrient medium containing, g / l: enzymatic hydrolyzed beef meat 300.0; fragrant hydrolyzate callisia hydrolyzate 1.0 ml; glucose 20.0; microbiological agar 10.0; distilled water - the rest. With this ratio of ingredients, the number of colonies grown on dense agar plates for lactobacilli with a sowing dose of 10 mk averaged 45 colonies for each test strain of lactobacilli.

Example 5. Test strains are grown on a nutrient medium containing, g / l: enzymatic hydrolyzed beef meat 400.0; fragrant hydrolyzate callisia hydrolyzate 1.2 ml; glucose 22.0; microbiological agar 12.0; distilled water - the rest. With this ratio of ingredients, the number of colonies grown on dense agar plates for lactobacilli with a sowing dose of 10 mk averaged 24 colonies for each test strain of lactobacilli.

Example 6. Test strains are grown on a nutrient medium containing, g / l: enzymatic hydrolyzed beef meat 500.0; fragrant hydrolyzate Callisia hydrolyzate 1.4 ml; glucose 24.0; microbiological agar 14.0; distilled water - the rest. With this ratio of ingredients, the number of colonies grown on dense agar plates for lactobacilli with a sowing dose of 10 mk averaged 14 colonies for each test strain of lactobacilli.

The results obtained allow us to determine the optimal options for a nutrient medium based on an enzymatic hydrolyzate of beef meat with a growth stimulator, which is used as an enzymatic hydrolyzate of fragrant callisia (example 3, 4).

Thus, based on the foregoing, we can say about the obvious advantages of the proposed environment, consisting of an enzymatic hydrolyzed beef meat 280,0-300,0; an enzymatic hydrolyzate of fragrant callisia 0.9-1.0; glucose 18.0-20.0; microbiological agar 9.0-10.0, distilled water - the rest. The proposed environment is of high quality, based on an enzymatic hydrolyzed beef meat prepared in distilled water with a growth stimulator.

The invention in comparison with the prototype and other known technical solutions has the following advantages:

- improving the quality of the nutrient medium;

- the possibility of using for cultivation a wide range of microorganisms.

- can replace dry culture media for the cultivation of lactobacilli.

Claims (1)

Nutrient medium for cultivation of lactobacilli containing a nutrient base, glucose, microbiological agar, growth stimulator and distilled water, characterized in that the medium as a nutrient base contains an enzymatic hydrolyzed beef meat with an amine nitrogen content of 0.7-0.9%, and as a growth promoter is an enzymatic hydrolyzate of fragrant callisia in the following ingredients, wt.%:
enzymatic hydrolyzate beef meat with amine nitrogen content of 0.7-0.9% 28.0-30.0 Enzymatic Callisia hydrolyzate 0.9-1.0 glucose 18.0-20.0 microbiological agar 0.9-1.0 distilled water rest