RU2605543C1 - METHOD FOR OBTAINING SPORE CULTURE ON BASIS OF BACTERIAL STRAIN Bacillus species 1839 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, microbiology and can be used for production of spore material of bacteria strain Bacillus sp. 1,839. Method involves inoculation of nutrient medium with bacteria of strain Bacillus sp. 1,839 with further cultivation on it. Biomass is separated by centrifugation at 1,000-1,500 rpm. In obtained bacteria biomass hypertonic phosphate buffer is added, containing potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride at given ratio. Bacillus sp. 1,839 incubation is performed until formation of spores. For removal of remained vegetative cells thermal treatment is performed by heating obtained precipitate of spores on water bath at least for 10 minutes at 70-90 °C, supernatant is centrifuged and removed.

EFFECT: invention simplifies method and allows obtaining spore culture in any bacterial or veterinary laboratories.

3 cl, 4 ex

Description

The invention relates to biotechnology, microbiology and can be used for the production of spore material from bacteria Bacillus sp. 1839.

A known method for producing spores from bacteria Bacillus weihenstephanensis KBAB4 (Baril E., Coroller L., Postollec F. et al. The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history. International Journal of Food Microbiology. v. 146. 2011. P. 57-62). The method includes inoculating a liquid nutrient medium with 0.1 ml of bacteria in 100 ml of Bacillus weihenstephanensis KBAB4 medium and cultivating the vegetative bacteria cells in a liquid nutrient medium for 6 to 100 hours at one of the indicated temperatures: 10 °, 20 ° or 30 ° C until the concentration is reached 6 × 10 ⁷ cells / ml, then the bacteria are centrifuged at 6000 × g, 10 min, 12 ° C, and the bacterial mass is transferred to phosphate buffer to add spore formation with the addition of mineral salts of the following composition: K ₂ HPO ₄ · 3H ₂ O: 4.5 g / l; anhydrous KH ₂ PO ₄ : 1.8 g / l, with the addition of CaCl ₂ · 2H ₂ O 8.0 mg / l, MnSO ₄ · H ₂ O 1.5 mg / l; pH 7.2, which can withstand from 4 to 20 days with constant stirring at 100 rpm at 10, 20 or 30 ° C until spores form. Spores are separated from the mineral buffer by centrifugation at 6000 × g for 10 min, 12 ° C.

The disadvantages of this method include:

1) the transition of the culture to a spore state takes up to 20 days, prolonged cultivation leads to a rise in the cost of the production process, including increasing energy costs for electricity, maintaining equipment, and increasing the risk of contamination with concomitant microflora;

2) the use of phosphate buffer with the addition of mineral salts of the specified composition does not allow to obtain spores from the marine strain of Bacillus sp. 1839;

3) the centrifugation rate for separating bacterial biomass (6000 × g) is a strict condition and is not suitable for the marine strain of Bacillus sp. 1839, since spore destruction may occur at the indicated rate;

4) the need to maintain the proposed temperatures during cultivation (10 ° C or 30 ° C), in the end, not only complicates the method of obtaining a spore culture, but also requires additional labor costs.

The objective of the proposed technical solution is to develop a method for producing spore material based on the marine strain of microorganisms Bacillus sp. 1839, a decrease in the duration of the process and labor costs.

The problem is achieved in a new way to obtain spore material based on a strain of Bacillus sp. 1839, involving the cultivation of bacteria, the separation of bacterial biomass from the liquid nutrient medium by centrifugation, the addition of a phosphate buffer to the resulting biomass of bacteria, which is used as a hypertonic phosphate buffer solution of the following composition: potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride from 46.334 to 56.334 g / l; incubation of bacteria in it until spores form for 15-60 minutes at room temperature and sedimentation of the resulting spores by centrifugation.

Use as a source for the production of spore material of bacteria of the strain Bacillus sp. 1839 provides a tetrodotoxin producer culture with a high content of endospores and free spores. It has been established that endospores accumulate large amounts of toxin, which makes it possible to concentrate TTX in spore-bearing cells and in free spores. Due to the accumulation of tetrodotoxin in endospores and free spores, when it is obtained from endospores and free spores, there is no need for additional purification of the toxin from the nutrient medium.

.,

С.,

R.,

С.М. Tetrodotoxin (ТТХ) as a Therapeutic Agent for Pain. Marine Drugs. 2012; 10(2):281-305;

C.O., Bredeloux P., Findlay I., Maupoil V. A TTX-Sensitive Resting Na(+) Permeability Contributes to the Catecholaminergic Automatic Activity in Rat Pulmonary Vein. J Cardiovasc Electrophysiol. 2014. Oct. 27). Установлено, что накопление токсина у Bacillus sp. 1839 происходит главным образом в эндоспорах и свободных спорах, т.е. количество токсина напрямую зависит от бактериальной биомассы (Magarlamov T.Yu., Beleneva I.A., Chernyshev A.V., Kukhlevsky A.D. Tetrodotoxin-producing Bacillus sp. from the ribbon worm (Nemertea) Cephalothrix simula (Iwata, 1952) // Toxicon. 2014. Vol. 85. P. 46-51).Bacillus sp. 1839 - tetrodotoxin-producing bacteria isolated from marine worms (Nemertea). Strain Bacillus sp. 1839 is an aerobic spore-forming bacterium. Vegetative cells have the form of straight single sticks, 2.48-2.78 microns long and 0.42-0.44 microns wide. Under the influence of unfavorable factors, as well as with the depletion of nutrients in the environment, vegetative cells become spores. The resulting spores have a spherical shape 0.78-1.27 μm long and 0.37-0.75 μm wide. There are a large number of toxins secreted by spore-forming bacteria, including substances that act on cells (phospholipases, hemolysins) and tissues (collagenases, elastases) in animals and humans. It has been shown that Bacillus sp. 1839 produces tetrodotoxin, which is the strongest poison of neuroparalytic action and can be used in medicine as an anesthetic and antiarrhythmic (Nieto FR, Cobos EJ, Tejada M.

.,

FROM.,

R.,

CM. Tetrodotoxin (TTX) as a Therapeutic Agent for Pain. Marine Drugs. 2012; 10 (2): 281-305;

CO, Bredeloux P., Findlay I., Maupoil V. A. TTX-Sensitive Resting Na (+) Permeability Contributes to the Catecholaminergic Automatic Activity in Rat Pulmonary Vein. J Cardiovasc Electrophysiol. 2014. Oct. 27). It has been established that the accumulation of toxin in Bacillus sp. 1839 occurs mainly in endospores and free spores, i.e. the amount of toxin directly depends on the bacterial biomass (Magarlamov T.Yu., Beleneva IA, Chernyshev AV, Kukhlevsky AD Tetrodotoxin-producing Bacillus sp. from the ribbon worm (Nemertea) Cephalothrix simula (Iwata, 1952) // Toxicon. 2014. Vol. 85. P. 46-51).

The use of the proposed hypertonic phosphate buffer solution stimulates spore formation in a short period of time, which significantly accelerates the process of obtaining spores and at the same time allows them to be further washed from the nutrient medium, which not only increases the purity of the product, but also reduces the duration of the process of obtaining the target product. To achieve this goal, it is advisable to use hypertonic phosphate buffer: potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride from 46.334 to 56.334 g / l. The prepared phosphate buffer has a pH of 7.8, which corresponds to the optimum growth for marine bacteria of the strain Bacillus sp. 1839.

The time of incubation (incubation) of bacteria in hypertonic phosphate buffer for 15-60 minutes also contributes to the achievement of the claimed technical result, as a result of which there is a significant reduction in the process of obtaining the target product. A decrease in the amount of aging time of less than 15 minutes in the buffer solution does not cause spore formation, and an increase in time of more than 60 minutes leads to the death of vegetative forms of bacteria without transition to the spore form.

In order to clean the remaining vegetative cells, they are additionally heat treated using one of the known methods, in particular, the obtained spore precipitate is heated for at least 10 min at 70-90 ° С in a water bath, a buffer solution is added, centrifuged and the supernatant containing the remaining vegetative cells is removed.

It is advisable both to separate the biomass of bacteria from the liquid medium and to precipitate spores, centrifuging for no more than 5 minutes at 1000-1500 rpm, this allows not only to obtain high-quality spore material, but also to reduce labor and energy costs.

The method is as follows.

The choice of medium for cultivation is not critical and depends on the availability of ingredients. The media is sterilized by autoclaving.

Bacterial culture of the strain Bacillus sp. 1839 was taken from the Collection of cultures of marine heterotrophic bacteria of the IBM FEB RAS.

Bidistilled or distilled water is used to prepare the phosphate buffer.

Example 1

In compliance with the rules of asepsis, a liquid culture medium is inoculated. For this, a bacterial culture of Bacillus sp. 1839 in the amount of 0.1% of the volume of inoculated medium in the Yoshimitsu-Kimura liquid nutrient medium (Youschimizu and Kimura, 1976) of the following composition: peptone (5.0 g), yeast extract (2.5 g), glucose (1.0 g), K ₂ NRA ₄ (0.2 g), MgSO ₄ · 7H ₂ O (0.1 g), distilled water (500 ml), seawater (500 ml), pH 7.8. Cultivation is carried out at 21 ° C for 7 days. The resulting biomass of bacteria is separated from the liquid nutrient medium by centrifugation: at 1500 rpm for 5 minutes The supernatant is drained and a hypertonic phosphate buffer solution prepared in bidistilled water is added to a pure bacterial precipitate, of the following composition: potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 50 g / l, and bacteria are incubated for 30 minutes at room temperature. The presence of spores is determined in smears of culture stained by the Peshkov method. For these purposes, a smear is prepared, dried. An alkaline solution of methylene blue (1% NaOH) is applied to the glass and heated in a burner flame for 15 s. The preparations are washed with distilled water and stained with a 0.5% solution of neutral red (Sigma, USA). The analysis of drugs is carried out under a light microscope. With this cultivation method, the transition of vegetative cells into spores is 95%. The resulting spore culture does not contain nutrient components.

To clean the remaining vegetative cells, heat treatment is carried out as follows.

The resulting spore precipitate is heated for 10 min at 80 ° C in a water bath, a buffer solution is added (potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 50 g / l) , centrifuged at 1500 rpm for 5 min and remove the supernatant containing the remains of vegetative cells.

Example 2

In compliance with the rules of asepsis, a liquid culture medium is inoculated. For this, 0.1% of the bacterial culture of Bacillus sp. 1839 of the volume of medium in a liquid nutrient medium Yoshimitsu-Kimura (Youschimizu and Kimura, 1976) of the following composition: peptone (5.0 g), yeast extract (2.5 g), glucose (1.0 g), K ₂ NRA ₄ (0.2 g), MgSO ₄ · 7H ₂ O (0.1 g), distilled water (500 ml), seawater (500 ml), pH 7.8. Cultivation is carried out at 22 ° C for 5 days. The resulting biomass of bacteria is separated from the liquid nutrient medium by centrifugation: at 1000 rpm for 5 minutes The supernatant is drained and a hypertonic phosphate buffer solution prepared in bidistilled water is added to a pure bacterial precipitate, of the following composition: potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 46.334 g / l, and the bacteria withstand for 60 minutes at room temperature. The presence of spores is determined in smears of culture stained by the Peshkov method. For these purposes, a smear is prepared, dried. A solution of methylene blue with 1% NaOH is applied to the glass and heated in the burner flame for 15 s. The preparations are washed with distilled water and stained with a 0.5% solution of neutral red (Sigma, USA). The analysis of drugs is carried out under a light microscope. With this cultivation method, the transition of vegetative cells into spores is 80%. The resulting spore culture does not contain nutrient components.

To clean the remaining vegetative cells, heat treatment is carried out as follows.

The resulting precipitate is heated for 10 min at 80 ° C in a water bath, a buffer solution is added (potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 50 g / l), centrifuged at 1500 rpm for 5 min and remove the supernatant containing the remains of vegetative cells.

To clean the remaining vegetative cells, heat treatment is carried out as follows.

The resulting precipitate is heated for 10 min at 90 ° C in a water bath, a buffer solution is added (potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 46.334 g / l), centrifuged at 1000 rpm for 5 min and remove the supernatant containing the remains of vegetative cells.

Example 3

Following aseptic rules, 0.1% of the bacterial culture of Bacillus sp. 1839, obtained as a result of one of the previous cultivations, into flasks with a nutrient medium: meat-peptone broth (0.5% aqueous solution of meat extract, 1.0% peptone, 0.5% sodium chloride, pH 7.6). Bacteria are grown at 23 ° C for 4 days. The resulting biomass of bacteria is separated from the liquid nutrient medium by centrifugation: at 1200 rpm for 5 minutes The supernatant is drained and a hypertonic phosphate buffer solution prepared with distilled water is added to the pure bacterial precipitate, of the following composition: potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 56.334 g / l, and bacteria are incubated for 15 min at room temperature. The presence of spores is determined in smears of culture stained by the Peshkov method. For these purposes, a smear is prepared, dried. A solution of methylene blue with 1.0% NaOH is applied to the glass and heated in a burner flame for 15 s. The preparations are washed with distilled water and stained with a 0.5% solution of neutral red (Sigma, USA). The analysis of drugs is carried out under a light microscope. With this method of cultivation, the transition of vegetative cells into spores is 82%. The resulting spore culture does not contain nutrient components.

To clean the remaining vegetative cells, heat treatment is carried out as follows.

The resulting precipitate is heated for 10 min at 70 ° C in a water bath, a buffer solution is added (potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride 56.334 g / l), centrifuged at 1200 rpm for 5 min and remove the supernatant containing the remains of vegetative cells.

Example 4 by a known method (Baril E. et al., 2011)

Inoculation of liquid culture medium with 0.1 ml of bacteria Bacillus sp. 1839 in 100 ml of liquid nutrient medium and vegetative bacteria cells are cultured for 6 h at 30 ° С, then bacteria are precipitated (6000 × g, 10 min, 12 ° С) and transferred to phosphate buffer with the addition of mineral salts of the following composition: 4, 5 g / l K ₂ HPO ₄ · 3H ₂ O; 1.8 g / l anhydrous KH ₂ PO ₄ , with the addition of 8.0 mg / l CaCl ₂ · 2H ₂ O, 1.5 mg / l MnSO ₄ · H ₂ O; pH 7.2. Incubated for 4 days with constant stirring until spores form. Spores are precipitated by centrifugation to separate the finished product from the mineral buffer (6000 g, 10 min, 12 ° C). On the 4th day of culturing spores in a mineral buffer, the culture is represented mainly by vegetative forms, many cells with a destroyed cell wall, and the spore concentration does not exceed 5%.

To neutralize the activity of vegetative cells, heat treatment is carried out: the precipitate is heated for 10 min at 70 ° C in a water bath.

From the results of the experiment of example 4, conducted under the conditions of the known method for producing spore material, it follows that these conditions are not suitable for the strain of Bacillus sp. 1839, since they practically do not provide a dispute.

At the same time, from examples 1-3 it is seen that the claimed invention provides a spore transition of up to 95% of the population within half an hour after incubation in the proposed hypertonic phosphate buffer, that is, it significantly reduces the duration of the spore formation process. The method is simple to implement, has a simple technology and allows you to get a spore culture in any bacteriological or veterinary laboratory with minimal production costs. The proposed method does not require multicomponent nutrient media with scarce substrates.

The claimed method for producing spores in comparison with the known method is less long and is suitable for a strain of Bacillus sp. 1839, the separation of bacteria from a liquid nutrient medium, unlike the prototype, does not require the use of harsh centrifugation conditions.

The study was supported by FEFU, project No. 14-08-06-19_and.

Claims (3)

1. A method for producing spore material based on a strain of Bacillus sp. 1839, which involves the cultivation of bacteria, the separation of bacterial biomass from the liquid nutrient medium by centrifugation, the addition of a phosphate buffer to the resulting biomass of bacteria, which is used as a hypertonic phosphate buffer solution of the following composition: potassium chloride 0.2 g / l, sodium hydrogen phosphate 3.3 g / l, potassium dihydrogen phosphate 0.166 g / l, sodium chloride from 46.334 to 56.334 g / l; incubation of bacteria in it until spores form for 15-60 minutes at room temperature and sedimentation of the resulting spores by centrifugation. 2. The method according to p. 1, characterized in that the centrifugation is carried out at 1000-1500 rpm for not more than 5 minutes 3. The method according to p. 1, characterized in that the precipitate obtained is subjected to heat treatment by heating in a water bath for at least 10 minutes at 70-90 ° C.