RU2105562C1 - Method of preparing base-bacillus subtilis bacterial preparation - Google Patents

Abstract

FIELD: microbiological industry, agriculture, food industry. SUBSTANCE: method involves culturing bacterium Bacillus subtilis on semipermeable diaphragm surface placed on solid nutrient medium to maximal biomass accumulation and transfer of bacteria to spore form by cell cooling to temperature 10 C, not above, and keeping at this temperature for 24 h, not less, followed by bacterium spores mixing with filling agent. Before mixing with filling agent spores are separated from semipermeable diaphragm. Filling agent: a mixture of sugar and starch at ratio bacterium spore biomass and starch 1:1, not above, and bacterium spore biomass and sugar 1:50, not above. Method can be used for preparing dry bacterial preparations as a spore form. EFFECT: enhanced purity, simplified technology. 5 cl, 1 tbl

Description

 The invention relates to a technology for the production of dry bacterial preparations for agriculture, medicine and the food industry.

A known method of producing a bacterial preparation based on a strain of Bacillus subtilis c-3102 (application EPO N 0287699, class C 12 N 1/20, publ. 26.10.88). The method includes suspension cultivation of a bacterial strain in a nutrient medium containing a carbon source, a nitrogen source, inorganic substances, vitamins, amino acids, at a pH of 6 8 and a temperature of 35 40 ^o C for from 0.5 to 7 days. The bacterial culture is separated from the nutrient medium, washed and concentrated. The resulting product is dried and mixed with filler additives to obtain pharmaceutical or veterinary preparations.

 The disadvantage of this method is the complexity of the technology for producing the drug, because the method includes washing the bacterial cells from the components of the nutrient medium, and then concentrating the bacteria by centrifugation. The drug has a short shelf life, because It contains live bacteria in the form of vegetative sticks, which cannot retain their biologically active properties for a long time.

 There is another method for producing a bacterial preparation based on Bacillus subtilis (Japanese application No. 13-31171, class A 61 K 35/74, publ. 02.05.91), including cultivating a bacterial strain on grain or a product of its grinding, followed by drying and additional grinding received product. The drug is used as a feed additive for pets and birds.

 The disadvantage of this method is the low purity of the resulting preparation, as a result of which it has a narrow scope (only as a feed additive for animals). In addition, the drug also has a short shelf life, because contains live bacteria that cannot retain their biologically active properties for a long time.

A known method of producing a preparation for protecting plants from diseases (Pat. RF N 2019966, class A 01 N 63/00, publ. 1994), including suspension cultivation of the bacteria strain You. subtilis IPM-215 to the maximum accumulation of biomass, followed by a decrease in the concentration of the main components of the nutrient medium for the conversion of bacteria into a spore form. The resulting culture fluid with bacterial spores is dried and mixed with an excipient. As a filler, diammonium phosphate, surfactants (OP-7 or OP-10) and sulphite-yeast mash are used. The drug has the following ratio of components, wt. dry concentrate of culture fluid (spore titer no less than 40 • 10 ⁹ spores / g) 40 65, surfactant 4 7, sulphite-alcohol mash the rest.

The disadvantage is the low purity of the drug, because it contains components of the cultivation medium, which limits its scope (only as a means of plant protection). This technology for producing bacterial spores does not allow to obtain a high titer of these spores (not more than 4 • 10 ¹⁰ spores / g), that is, bacterial cells are under different conditions in the process of spore formation, and therefore some bacteria die before they form support . In addition, the technology for producing the drug is complex and has high energy consumption, because provides for the drying of drugs.

 A known method for producing a bacterial preparation based on Bacillus subtilis by culturing a bacterial strain of Bacillus subtilis VKM B-1666 on a solid nutrient medium, followed by separation of cells from the medium and freeze-drying them (ed. Certificate of the USSR N 1708350, class A 61 K 35/66 , 1992).

 This method has the same disadvantages as described above.

 The objective of the invention is the creation of such a method for producing a bacterial preparation based on Bacillus subtilis, which would ensure an increase in the titer of bacterial spores and the purity of the drug, as well as simplifying the technology for its preparation.

 This problem is solved in that in a method for producing a bacterial preparation, comprising cultivating Bac.subtilis bacteria on a nutrient medium to the maximum biomass accumulation, converting the bacteria into a spore form and then mixing them with a filler, according to the invention, the Bac.subtilis bacteria spores are separated before mixing with the filler from a nutrient medium, and as a filler, a mixture of sugar and starch is used with a ratio of biomass of bacterial and starch spores of not more than 1: 1 and biomass of bacterial and sugar spores of not more than 1:50.

 With an increase in the ratio of bacterial starch spores to more than 1: 1, not all spores will be immobilized on starch, which will reduce the quality of the preparation.

 With an increase in the ratio of bacterial spores and sugar more than 1:50, the required conditions for the conservation of bacterial spores and optimal moisture redistribution in the mass of the drug are not provided, which reduces its storage time.

Bac.subtilis bacteria spores are obtained by cooling the biomass to a temperature not exceeding 10 ^o C, and keeping it at this temperature for at least 1 day.

As a nutrient medium for the cultivation of bacteria, dense nutrient agar is used, including peptone-yeast or meat and peptone extract. Moreover, the surface of the nutrient medium is covered with a semipermeable membrane, followed by the application of a seed dose of Bac.subtilis bacteria to it in a concentration not exceeding 10 ⁸ cells / ml. The separation of the accumulated biomass of bacteria in spore form from the nutrient medium is carried out by removing the biomass of bacteria from the surface of a semipermeable membrane using a glass spatula.

 To ensure immobilization of bacterial spores on starch particles, a mixture of sugar and starch in a ratio of 1: 1 is preliminarily prepared, which is mixed with bacterial biomass at a ratio of biomass and a mixture of these filler components not more than 1: 3, followed by the rest of sugar added to the resulting mixture.

 With preliminary mixing of starch with sugar in a ratio of 1: 1, the need for saline is reduced several times, i.e. the process of immobilization of bacterial spores on starch particles is carried out with minimal dilution of the mixture. With an increase in the ratio of biomass of bacterial spores and a mixture of sugar with starch more than 1: 3, not all bacterial spores can be immobilized on starch particles.

Upon receipt of the drug, Bac.subtilis bacterial spore concentrate is used (after removal from a semipermeable membrane) with a titer of at least 3 • 10 ¹⁰ spores / g in the following ratio of components, wt.

Bac.subtilis bacteria spores 0.1 1.0
Starch 1.0 3.0
Sugar Else
An increase in the purity of the preparation is ensured by the cultivation of bacteria on the surface of a semipermeable membrane, as a result of which the components of the nutrient medium practically do not mix with the biomass of bacteria and do not pollute it, and the latter is a ready-made concentrate that does not require filtration or centrifugation.

A high titer of bacterial spores (up to 10 ¹⁰ spores / g) is ensured by creating the same conditions for spore formation for each bacterial cell.

 The immobilization of bacterial spores on starch provides them with additional mechanical protection, prevents the adhesion of bacterial spores and ensures a more even distribution in the mass of the filler.

 Sugar is not only a sorbent-filler, but also has a stabilizing and preserving effect on the preparation, creates milder conditions for storing bacterial spores.

 In addition, the proposed method greatly simplifies the technology of obtaining the drug both at the stages of cultivation (the technology does not use expensive fermenters, cleaning and concentration of the drug are not required), and at the stages of obtaining its finished form (removal or redistribution of moisture combined with obtaining the finished form of the drug) .

 Example 1.

 The method of cultivation of bacteria Bac. subtilis.

 To obtain the drug, a bacterial strain Bacillus subtilis VKPM B-4759 is used, which has antiviral and antibacterial activity, which is capable of producing α-2 human interferon along with suppression of pathogenic and conditionally pathogenic microflora and is harmless to warm-blooded animals.

The cultivation of the strain of bacteria is carried out on a solid nutrient medium of the following composition:
Agar agar 20 g / l
Peptone 12 g / l
Yeast extract 5 g / l
NaCl 5 g / l
Water Up to 1l
Kanamycin 50 mg / L
25 ml of bacteria with a titer of about 10 ⁸ cells / ml are required per 1 liter of agar medium. The nutrient medium is formed in the container in the form of a layer on which a semipermeable membrane, for example a cellophane film, is laid. A seed dose of the bacterial strain is applied on top of the film. Cultivation is carried out in a thermostat at a temperature of 34 37 ^o C for 20 24 hours. Next, the tank is transferred to a thermostat with a temperature of 6 10 ^o C and kept at this temperature for 1 day. before the formation of bacterial spores. With this cultivation method, a spore titer of 3x10 ¹⁰ spores / g can be obtained. Bacterial spores are removed with a glass spatula from the surface of the cellophane film and collected in a sterile container. These bacterial spores are pure biomass concentrate from which the preparation is prepared.

 Example 2

 The cultivation method of the strain of bacteria Bac. subtilis N 534 deposited in the All-Union Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms under N 1666 D.

The cultivation of a strain of bacteria is carried out in a dense nutrient medium. To do this, 3% sterile meat-peptone agar with a volume of 250 ml is placed in a container. A cellophane film is placed on a solid nutrient medium. On top of the film, aseptic is applied, 10 ml of suspension of the production strain of B. subtilis VKM N B-1666 D containing 10 ⁸ bacterial cells in 1 ml of 0.9% sodium chloride solution. The container is placed in a thermostat with a temperature of 36 37 ^o C. After 24 hours, subject to asepsis, the container with the grown culture is transferred to a thermostat with a temperature of 7 ^o C and kept at this temperature for 1 day. before the formation of bacterial spores. Bacterial spores are removed with a glass spatula from the surface of the cellophane film and pure biomass is collected in a glass container. With this cultivation method, a spore titer of 3x10 ¹⁰ spores / g can be obtained.

 Example 3

 Preparation of the finished product based on Bac. subtilis VKPM B-4759.

 First, prepare a mixture of sugar with starch in a ratio of 1: 1. Then, to 5 parts of the obtained filler mixture, 1 part of bacterial spore biomass is added and mixed.

 In this case, immobilization of bacterial spores on starch particles occurs. Next, sugar is introduced into the resulting mixture at a ratio of biomass of spores of bacteria and sugar of 1: 300. The result is a finished concentrated product in the following ratio of components, wt.

Bac. subtilis VKPM B-4759 with a titer of 3 • 10 ¹⁰ spores / g 0.33
Starch 0.82
Sugar The rest is up to 100%
The residual moisture of the drug is 2 5%
Example 4

 Preparation of the finished product based on Bac. subtilis VKM NB-1666 D.

 First, prepare a mixture of sugar with starch in a ratio of 1: 1. Then, to 3 parts of the obtained filler mixture, 1 part of bacterial spore biomass is added and mixed. A physiological solution, the volume of which is equal to the volume of the biomass of bacteria, is added to the resulting mixture and intensively mixed. In this case, immobilization of bacterial spores on starch particles occurs. Next, sugar is introduced into the resulting mixture at a ratio of biomass of spores of bacteria and sugar of 1: 200. As a result, a finished concentrated preparation is obtained in the following ratio of components, wt.

Bac. subtilis VKM NB-1666 D with a titer of 3.0x10 ¹⁰ spores / g - 0.49
Starch 0.74
Sugar The rest is up to 100%
The residual moisture content of the drug is not more than 5%
Example 5

Data on the storage of preparations of Bac.subtilis in concentrated form at a temperature of 24 ^o C are given in table 1.

From the table. 1, it follows that during 12 months of storage of Bac.subtilis preparations in a spore form at a temperature of 24 ^o C their titer practically did not change, which confirms the high thermal stability of the preparations in spore form, which significantly simplifies the technology of their use in agriculture and medicine.

 Example 6

 The use of drugs Bac. subtilis strains VKPM B-4759 and VKM B-1666.

 The drug is based on a strain of Bac. subtilis VKPM NB-4759 has antiviral and antibacterial activity and can be used for the prevention and treatment of animals and humans. In addition, experimental data confirm the possibility of using this drug as a means of protecting plant fruits from late blight.

 The drug is based on a strain of Bac. subtilis 534 VKM NB-1666 is used for the prevention and treatment of purulent-inflammatory processes of various localization / purulent wounds, osteomyelitis, sepsis, pneumonia, etc. /, food allergies, dysbiosis in warm-blooded animals and humans.

 When using the drug in the form of a concentrate obtained by the proposed method / Example 3 and 4 /, as a prophylactic and therapeutic agent for humans, it is mixed with a food supplement, for example, glucose, sugar in a ratio of 1: 100-1: 200.

 The concentrate of the drug when used as a feed additive for animals is mixed with a filler, for example flour or bran in a ratio of 1: 200-1: 300. The product increases the weight gain of animals, improves the efficiency of feed use and prevents the incidence of animals.

 Plant protection is obtained by mixing the concentrate with water / before use / or in dry form with peat, chalk or ash in a ratio of 1: 200 -1: 500.

 Industrial applicability. The invention can be used in medicine, veterinary medicine and in agriculture as a means of plant protection.

Claims (5)

 1. A method of obtaining a bacterial preparation based on Bacillus subtilis, including seeding and culturing bacteria on a nutrient medium to the maximum accumulation of bacterial cells and their subsequent drying, characterized in that the bacterial cells are transformed into a spore form, the resulting spores are separated from the nutrient medium, and subjected to drying these spores by mixing them with a filler, which is used as a mixture of sugar and starch with a ratio of spores of bacteria and starch of not more than 1 1 and spores of bacteria and sugar of not more than 1 50. 2. The method according to claim 1, characterized in that the translation of bacteria Bac. subtilis in the spore form is carried out by cooling the cells to a temperature not exceeding 10 ^o C, and keeping them at this temperature for at least a day. 3. The method according to claim 1, characterized in that the inoculation of bacteria Bac. subtilis at a concentration not exceeding 10 ⁸ cells. / ml, carried out on a semipermeable membrane located on a nutrient medium, and the separation of bacterial spores is carried out from the surface of the specified membrane.  4. The method according to claim 1, characterized in that before mixing the spores with the filler, a mixture of starch and sugar is prepared in a ratio of 1 to 1, followed by the introduction of bacteria spores therein, the resulting mixture is mixed and the rest of the sugar is added, to obtain the finished product. 5. The method according to claim 1, characterized in that when receiving a concentrate of the drug, Bac bacteria spores are used. subtilis with a titer of at least 3 • 10 ^{1 0} spores / g in the following ratio of components, wt. Bac. subtilis 0.1 1.0
Starch 1.0 3.0
Sugar Else

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