RU2360962C2 - Nutrient base production method and nutrient base for yersinia and vibrio microorganisms fermentation - Google Patents

Abstract

FIELD: food products.

SUBSTANCE: method provides for protein raw material hydrolysis. Baker's yeasts and caseine are used as protein raw material. Hydrolysis is performed by enzymic complex, obtained from choleric vaccine production wastes - ultrafiltrate culture liquid - cholera vibrio producing strain M 41 at 45°C and pH 7.4±0.2 during 20-22 hours with nutrient base production. Baker's yeasts enzymic hydrolyzate or caseine enzymic hydrolyzate are used as nutrient base in mentioned nutritional mediums. Nutritional mediums contain baker's yeasts enzymic hydrolyzate or caseine enzymic hydrolyzate, chloride natrium, tapwater and agar. Nutritional medium additionally contains yeast autolysate if there is nutritional medium as nutrient base of caseine enzymic hydrolyzate.

EFFECT: reduced time of raw materials hydrolyzate while keeping nutritional medium growing properties, reduced quantity of digesting agent and expanded range of nutritional mediums.

5 cl, 1 tbl, 6 ex

Description

The invention relates to microbiology and biotechnology, and in particular to a method for producing protein hydrolysates and a nutrient medium intended for growing microorganisms.

Background to the invention.

Currently, in the construction of high-grade nutrient media, meat bases are mainly giving way to casein, hydrolysates of fish raw materials and yeast are also widely used. Raw materials for the manufacture of nutrients should be affordable, standard and cheap. New methods of hydrolysis of the feedstock are also being sought.

Nutrient media are known for the cultivation of various microorganisms, including pathogens, prepared by tryptic hydrolysis of meat according to Hottinger, from pancreatic hydrolyzate of casein, soybeans and peas, Caspian sprat, baker's yeast (Kozlov Yu.A. Nutrient media in medical microbiology. - M .: Medgiz., 1950, p. 51-111).

Known nutrient medium for growing microorganisms containing pancreatic hydrolyzate of casein, baker's yeast extract, lactose, salts of manganese and magnesium, ascorbic acid (patent RU No. 2027754, C12N 1/20).

A known monocomponent, protein-based, plague microbe cultivation medium based on pancreatic digest of baker’s yeast obtained by the Hottinger method (Nutrient plague microbial culture medium at 37 ° C based on pancreatic digest of baker’s yeast: R&D report: 01.200.2 12994: Rostov -on-Don scientific - research anti-plague institute, North Caucasian anti-plague station; performed by: Mazrukho AB, Krivachenko KB, Kaminsky DK, [et al.] / Methodical documents and reports on a dignity - epidemiological protection of the territory of the Russian Federation: Verat, collection / Under the editorship of Doctor of Medical Sciences, Professor V.V. Kutyrev. - Saratov: OJSC "Volga Book Publishing House", 2005. - p.39, ISBN 5-7633-1061-6).

A known nutrient medium for deep cultivation of the plague microbe vaccine strain based on the enzymatic hydrolyzate of skim milk proteins with an amine nitrogen content of 120 mg% and a concentration of hydrogen ions of pH 6.8-7.2. The composition of this medium includes a nutrient base, monosubstituted potassium phosphate, disubstituted potassium phosphate, magnesium sulfate, sodium chloride (patent RU No. 2270856, C12N 1/20, C12QN 1/10).

A known nutrient medium for cultivating a plague microbe vaccine strain of the following composition is: pancreatic soybean hydrolyzate, sodium chloride, disubstituted sodium phosphate, sodium sulfate, distilled water (RU patent No. 2245362, C12N 1/20, C12QN 1/04).

The disadvantage of microbiological media prepared on the basis of pancreatic hydrolysates is the increased cost of the pancreas, its non-standard, as well as the duration (5 days or more) of the process of enzymatic hydrolysis, which also increases the final cost of the medium. Moreover, the use of raw materials of animal origin can lead to the contamination of nutrient media with biological agents (mycoplasmas, prions, etc.).

The closest analogue is a medium for the cultivation of microorganisms containing an enzymatic hydrolyzate of soy, an autolysate of baker's yeast, sodium chloride, and tap water (Dyachenko Yu.V., Levitsky A.P. Soybean bacteriological growth media and the prospects for their use in clinical bacteriology // Clinical laboratory diagnostics, 2001, No. 3. - p. 49-50).

Known methods for producing protein hydrolysates based on the enzymatic hydrolysis of protein raw materials with proteolytic preparations isolated from certain bacteria or molds.

A known method of producing a hydrolyzate of yeast biomass. The method involves the hydrolysis of yeast biomass with a complex enzyme preparation obtained on the basis of the Aspergillus oryzae VKPM F-683 micromycete strain or its culture fluid at pH 4.5-6.0, followed by pasteurization at 80-90 ° C for 15-20 minutes and drying (patent RU No. 2104300, C12N 1/06, A23J 1/18, A23L 1/28).

A search by patents and scientific and technical sources of information showed that at present, among the bacterial enzyme preparations of domestic and foreign production, the most commonly used drug is protosubtiline, which has protease activity.

Closest to the claimed is a method of obtaining protein bases for the preparation of bacteriological culture media (Dyachenko Yu.V., Levitsky A.P. Soy bacteriological culture media and the prospects for their use in clinical bacteriology // Clinical laboratory diagnostics, 2001, No. 3. - .49-50). The method suggests the use of soybean extract as a raw material and enzymatic hydrolysis with protosubtilin at 47-50 ° C and pH 7.3 for 5-8 hours or pronase at 37 ° C, pH 7.2 for 2 hours.

However, the use of such preparations of a bacterial nature has not yet received wide distribution due to their lower availability and insufficient knowledge.

The invention is aimed at achieving a nutrient medium that is not inferior in its growth properties to prototypes and at the same time uses a by-product of the production of cholera vaccine as a source of enzyme complex for digestion of the nutrient base in the preparation technology, which solves the problem of recycling production waste.

The technical result of the invention is: to replace expensive non-standard digesting agents with cheaper and standard; in reducing the time of the hydrolysis process (5-7 times), while maintaining the growth properties of the nutrient medium; a significant reduction in the amount of digesting agent; in the rational utilization of industrial wastes and the expansion of a set of high-grade environments, including for the industrial cultivation of bacteria.

The problem is solved by using a cholera vibrio low molecular weight enzyme complex isolated from the waste product of the cholera vaccine - ultrafiltrate of the culture fluid of production strain M 41 and called proteovibrin as an agent for digesting the nutrient base. For the preparation of a nutrient base, protein raw materials of various origins can be used as feedstock: baker's yeast and casein are chosen as such.

Pre-prepared protein raw materials are subjected to hydrolysis with the enzyme complex of cholera vibrio - proteovibrin. The ratio of the enzyme complex to protein is 1: 400-1: 500, hydrolysis is carried out at a temperature of 45 ° C, pH 7.4 ± 0.2 for 20-22 hours

On the basis of this, it is possible to prepare a complete and affordable liquid or dense nutrient medium for growing microorganisms. If necessary, other components can be added to the nutrient base to expand the range of cultivated microorganisms depending on their nutritional needs.

The enzymatic complex of proteovibrin is obtained from the ultrafiltrate of a detoxified culture fluid of the production strain of cholera vibrio of serovar Ogawa M 41. During one cultivation in the reactor, after concentrating the O-antigen-containing component of the vaccine, ultrafiltrate (more than 200 l) accumulates on the UVA-PS-20-1040 ultrafiltration apparatus. , which is a waste of production. It is concentrated 15-17 times on a similar apparatus PS-17-1040 with a lower fiber throughput and precipitated with ammonium sulfate to 80% of saturation, followed by centrifugation and dialysis against distilled water. The drug in liquid form can be stored for a long time (up to 1 year) in a frozen state without loss of activity. The drug can be lyophilized and stored for a long time (more than 2 years), the yield of dry product is 0.25 ± 0.05 g per 1 liter. The planned release of the cholera vaccine provides the required amount of the enzyme complex. The highly active proteovibrin enzyme complex obtained in the described manner from the waste products of the production of cholera chemical vaccines has not yet had further industrial use.

Proteovibrin is a dark brown liquid or loose powder of a dark brown color, readily soluble in water. Liquid proteovibrin contains 18 ± 2 mg / ml protein; in a dry preparation, the protein content is 55 ± 77%. Proteovibrin in its composition contains 16000 ± 2000 conv. units / mg of neutral protease (test medium with milk pH 7.2), 20,000 ± 4000 conv. units / mg protein of alkaline protease (test medium with milk pH 8.5), 8000 ± 400 conv. units / mg of acidic protease protein (test medium with milk pH 5.6), 12500 ± 2000 conv. units / mg of protein phospholipase A; (test medium with yolk pH 7.5), 770 ± 10 srvc. units / mg of protein phospholipase C (test medium with yolk pH 7.5).

In the enzyme complex - proteovibrine, obtained from the waste products of cholera chemical vaccines, there are proteolytic and lipolytic enzymes necessary for hydrolysis of the nutrient base in a wide range of pH and some lipid-containing components in it, which indicates a certain similarity with preparations from the pancreas. Moreover, for effective hydrolysis of protein raw materials, small doses of proteovibrin are required - its ratio to protein is 1: 400-1: 500.

The problem is solved in that the nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio contains a nutrient base, yeast autolysate, sodium chloride, water, and casein hydrolyzate obtained by hydrolysis of casein by the cholera vibrio enzyme complex in the following ratio of components, g / l:

casein enzymatic hydrolyzate 300-330 sodium chloride thirty yeast autolysate fifty tap water rest

Alternatively, the nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio contains a nutrient base, sodium chloride, water, and as a nutrient base an enzymatic hydrolyzate of baker's yeast obtained by hydrolysis of baker's yeast with the cholera vibrio enzyme complex, in the following ratio, g / l:

baker's yeast enzymatic hydrolyzate 350-380 sodium chloride thirty tap water rest

The nutrient medium according to the second embodiment does not require the addition of a yeast autolysate, a growth stimulator.

The amount of nutrient base per 1 liter of medium is calculated depending on the initial content of amine nitrogen in it, bringing its value to 100 ± 10 mg%.

To prepare a dense nutrient medium according to the first and second options, agar-agar is additionally added in an amount of 15-20 g / l.

Example 1. Preparation of enzymatic hydrolyzate of baker's yeast.

200 g of pressed yeast are suspended in 800 ml of tap drinking water and autolysis is carried out for 5-6 hours at 48-50 ° C in the presence of 2% chloroform, an inducing agent with preservative properties. At this stage, the process can be stopped, and the autolysate is used as a stimulating additive to the medium according to example 4 in a ratio of 1:20. To do this, it is inactivated at 90 ° C for 30 min, left to stand for 24 hours in the cold at a temperature of 4-6 ° C, decanted and filtered twice through a belting cloth. Poured into glass vials and sterilized at 110 ° C for 30 minutes. With continued hydrolysis, 5 ml of liquid or 150-160 mg of dry proteovibrin (ratio to yeast protein 1: 450-1: 500) is added to the yeast autolysate, the pH is adjusted to 7.4 ± 0.2, incubated for another 16-18 hours with periodic stirring . At the end of hydrolysis, the fermentolizate is acidified with 6 N. HCl to pH 4.6 ± 0.2 and boiled in a water bath for 10 minutes. The precipitate is removed after settling for 24 hours in the cold by decantation and filtration. The filtrate was adjusted to pH 8.5 ± 0.1 with 5 N NaOH and re-boil for 10 minutes, followed by removal of the precipitate. In a transparent nutrient basis, a pH of 7.4 ± 0.2 is set with 6 N HCl. The base contains 590 ± 20 mg% of total nitrogen, 280 ± 20 mg% of amine nitrogen, the degree of protein breakdown is 47 ± 2%.

Example 2. Nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio, (option 2).

To prepare a nutrient medium, the yeast hydrolyzate, sentenced in accordance with the claimed method, is diluted according to the calculation with tap water to an amine nitrogen content of 100 ± 10 mg%, the pH is adjusted to the required value, 0.3% NaCl and water are added to 1 l. To prepare a dense medium, 15-20 g / l agar-agar is added to it. The medium is poured into glass bottles and autoclaved at 110 ° C for 30 minutes.

The finished medium contains the following components, g / l:

baker's yeast fermentolizate 350 (amine nitrogen content 90 mg%) NaCl thirty tap water rest

The use of this nutrient medium provides the growth needs of the microorganisms of the genera Yersinia and Vibrio with the preservation of their morphological characteristics.

Example 3. Nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio, (option 2).

To prepare a nutrient medium, the yeast hydrolyzate, sentenced in accordance with the claimed method, is bred according to the calculation with tap water, the pH is adjusted to the required value, 0.3% NaCl and water are added to 1 l. To prepare a dense medium, 15-20 g / l agar-agar is added to it. The medium is poured into glass bottles and autoclaved at 110 ° C for 30 minutes.

The finished medium contains the following components, g / l:

baker's yeast fermentolizate 380 (amine nitrogen content 110 mg%) NaCl thirty tap water rest

The use of this nutrient medium provides the growth needs of the microorganisms of the genera Yersinia and Vibrio with the preservation of their morphological characteristics.

Example 4. Preparation of enzymatic casein hydrolyzate.

60 g of dry casein is poured into 1 liter of tap drinking water, 8 g of NaHCO ₃ are added (pH 7.4 ± 0.2) and heated in a water bath to 90-95 ° C for 5 minutes. After cooling to 40-50 ° C, 5 ml of liquid proteovibrin (ratio to casein protein 1: 400-1: 500) or dry proteovibrin in an amount of 150-160 mg, previously dissolved in a minimum amount of water, is added to the molten casein. The enzymatic hydrolysis of casein is carried out for 20-22 hours at 45 ° C with periodic stirring. At the end of the process, the hydrolyzate is acidified to pH 4.5 ± 0.2 using 6 N. HCl for inactivation of the enzyme and precipitation of unreacted proteins and incubated in a water bath for 10 minutes After cooling and removing the precipitate by filtration through a fabric or paper filter, set pH 7.4 ± 0.2 using 5 N. NaOH. The resulting nutrient base contains 640 ± 30 mg% of total nitrogen, 300 ± 20 mg% of amine nitrogen, the degree of protein breakdown is 52 ± 3%.

Example 5. Nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio (option 1).

To prepare a nutrient medium, a casein fermentolizate, which was sentenced in accordance with the claimed method, is diluted with tap water, the pH is adjusted to the required value, 0.3% NaCl, 0.5% yeast autolysate and water up to 1 l are added. To prepare a dense medium, 15-20 g / l agar-agar is added to it. The medium is poured into glass bottles and autoclaved at 110 ° C for 30 minutes.

The finished medium contains the following components, g / l:

casein fermentolizate 300 (amine nitrogen content 90 mg%) yeast autolysate fifty NaCl thirty tap water rest

The use of this nutrient medium provides the growth needs of the microorganisms of the genera Yersinia and Vibrio with the preservation of their morphological characteristics.

Example 6. Nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio (option 1).

To prepare a nutrient medium, a casein fermentolizate, which was sentenced in accordance with the claimed method, is diluted with tap water, the pH is adjusted to the required value, 0.3% NaCl, 0.5% yeast autolysate and water up to 1 l are added. To prepare a dense medium, 15-20 g / l agar-agar is added to it. The medium is poured into glass bottles and autoclaved at 110 ° C for 30 minutes.

The finished medium contains the following components, g / l:

casein fermentolizate 330 (amine nitrogen content 110 mg%) yeast autolysate fifty NaCl thirty tap water rest

The use of this nutrient medium provides the growth needs of the microorganisms of the genera Yersinia and Vibrio with the preservation of their morphological characteristics.

To assess the suitability of the proposed culture media, plague, pseudotuberculosis and cholera vibrio pathogens were grown on liquid and solid culture media. The control was medium prepared on the basis of the hydrolyzate according to Hottinger.

The inventive liquid nutrient media according to examples 2,3 and 5,6 provide a clearly visible typical growth of the plague microbe of strains of the EV strain NIIEG and 708 (K-1) when sowing 1000 mcl / ml - no later than after 24 hours of incubation at 28 ° C in the form of a clear broth with loose sediment at the bottom, I-199 pseudotuberculosis microbe as a uniform turbidity of the medium under the same conditions, eitor 103 biovar cholera vibrio as a uniform turbidity of the medium with a film on the surface when sowing 20 mcl ./ml - not later than after 6 hours of incubation at 37 ° C.

The growth efficiency in liquid media is determined by the increase in optical density, measured on a photocolorimeter photoelectric concentration KFK-2 at 670 nm.

The sensitivity of the claimed media is determined by the number of colonies formed on agar plates at sowing 10 and 100 mcl / ml. On a solid nutrient medium according to examples 2 and 4, the strain of the NIIEG strain of the plague microbe forms after 48 hours of incubation at 28 ° C smoothed, weakly granular, more colored colony than on the Hottinger agar. These changes in the morphology of the colonies are not fundamental. The strain I-199 of the pseudotuberculosis microbe and strain 103 of the cholera vibrio on the medium according to examples 2 and 4 give the growth of colonies with typical morphology. The data are given in the table.

Thus, the implementation of the invention allows for the first time to use for enzymatic hydrolysis of nutrient bases of nutrient media low molecular weight enzyme complex of cholera vibrio - proteovibrin isolated from the waste product of cholera vaccine - ultrafiltrate of the culture fluid of the production strain of cholera vibrio M 41. By the nature of the action of proteovibrin can serve as a substitute for expensive non-standard component - the pancreas. The inventive nutrient medium prepared by the proposed method from a baker's yeast or casein fermentolysate is distinguished by its simplicity of manufacture, availability of raw materials and growth characteristics close to the Hottinger medium for pathogens of plague, pseudotuberculosis and cholera vibrio. In addition, the time of hydrolysis of protein raw materials is significantly reduced and the risk of contamination of nutrient bases and media with biological agents (mycoplasmas, prions, etc.) is eliminated.

The analysis of the prior art carried out by the applicant has established that there are no analogues characterized by sets of features identical to all the features of the claimed objects, therefore, the invention meets the condition of “novelty”.

Search results for known technical solutions in this and related fields of technology in order to identify features that match the distinctive features of the claimed invention from the prototypes have shown that they do not follow explicitly from the prior art.

From the prior art determined by the applicant, the influence of the transformations provided for by the essential features of the claimed invention on the achievement of the specified technical result has not been revealed, therefore, the claimed invention corresponds to the "inventive step".

The application complied with the requirement of unity of invention, since the claimed objects: a method of obtaining a nutrient base and a nutrient medium form a single inventive concept.

Table Type of pathogen and strain T ° C grown
va-niya Growth indicators Culture medium according to example 2, 3 according to example 5, 6 Hottinger (control) Yersinia pestis EV NIIEG lines 28 ° C Growth pattern in broth, 24 hr incubation Transparent broth with loose sediment Sowing dose at which there is a visible increase in 5 ml of broth 10 ⁴ m.cl 10 ⁴ m.cl 10 ⁴ m.cl The optical density of the suspensions at 670 nm, 24 hours of incubation 0.51-0.58 0.54-0.57 0.61-0.64 The number of colonies when sowing 100 mcl 75-80 92-95 96-97 Morphology, colony diameter after 48 hours Smooth colonies with weak granularity, slightly stained, 1.4-1.6 mm Colonies with lace, light 1.8-2 mm Yersinia pseudo-tuberculosis I-199 28 ° C Growth pattern in broth, 24 hours Evenly muddy broth Sowing dose at which there is a visible increase in 5 ml of broth 10 ³ sq.m. 10 ³ sq.m. 10 ³ sq.m. The optical density of the suspensions at 670 nm, 24 hours of incubation 0.66-0.72 0.61-0.69 0.68-0.75 The number of colonies when sowing 100 mcl 80-82 78-83 83-85 Morphology, colony diameter after 48 hours Round colonies with a smooth edge, with a bluish tint in transmitted light, 0.2-0.5 mm Vibrio cholerae biovar eltor 103 37 ° C Growth pattern in broth, 18 hr incubation Evenly cloudy broth with a film on the surface Sowing dose at which there is a visible increase in 5 ml of broth 50 mcl 50 mcl 50 mcl The optical density of the suspensions at 670 nm, 24 hours of incubation 1.08-1.17 1.14-1.19 1.12-1.22 The number of colonies when sowing 100 mcl 36-38 34-39 40-42 Morphology, colony diameter after 18 hours Round translucent colonies with a smooth edge, 2.0-2.5 mm

Claims (5)

1. A method of obtaining a nutrient base, including enzymatic hydrolysis of protein raw materials, characterized in that the hydrolysis of previously prepared raw materials is carried out by the cholera vibrio enzyme complex isolated from the waste product of cholera vaccine - ultrafiltrate of the culture fluid of the production strain of cholera vibrio M 41, with the ratio of the enzyme complex to protein 1: 400-1: 500, temperature 45 ° C, pH 7.4 ± 0.2 for 20-22 hours 2. The method according to claim 1, characterized in that baking yeast or casein is used as the proteinaceous feed. 3. A nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio, containing a nutrient base, yeast autolysate, sodium chloride, water, characterized in that the nutrient base contains the enzymatic hydrolyzate of casein obtained by the method according to claim 1, in the following ratio of components, g / l:
casein enzymatic hydrolyzate 300-330 sodium chloride thirty yeast autolysate fifty tap water rest 4. Nutrient medium for the cultivation of microorganisms of the genera Yersinia and Vibrio, containing a nutrient base, sodium chloride, water, characterized in that the nutrient base contains an enzymatic hydrolyzate of baker's yeast obtained by the method according to claim 1, in the following ratio of components, g / l :
baker's yeast enzymatic hydrolyzate 350-380 sodium chloride thirty tap water rest 5. Nutrient medium according to claims 3 and 4, characterized in that it additionally contains agar-agar in an amount of 15-20 g / l.