RU2180001C2 - STRAIN OF BACTERIUM PSEUDOMONAS FLUORESCENCE VKM V-2224D AS PRODUCER OF VITAMIN B12. FIELD: microbiological industry, vitamins production - Google Patents

Abstract

SUBSTANCE: invention relates to the new strain producing vitamin B₁₂. Strain of bacterium Pseudomonas fluorescence VKM V-2224D is the producer of vitamin B₁₂, it grows at 28-32 C and pH 7-7.2 at constant stirring. Culture growth occurs parallel with vitamin B₁₂ biosynthesis under aerobic conditions. Productivity of culture is 130-150 mcg/ml for 150 h of fermentation growth. EFFECT: new strain indicated above, enhanced productivity. 2 tbl

Description

The invention relates to the microbiological industry and relates to a new strain of bacteria Pseudomonas fluorescens BKM B-2224D producing vitamin B ₁₂ , which is used as an anti-anemic drug in medicine and in the production of feed additives for farm animals and poultry.

Currently, the industry uses only microbiological synthesis to produce vitamin B ₁₂ .

Known methods for microbiological production of vitamin B ₁₂ using methane-forming bacteria (Methanobacteria) US patent 3961971 (1976), propionic acid bacteria (US patent 4210720 (1980), owned by the Japanese company Nippon Oil Company. In Japan, the company Daiseru kagakue koge .k .. (EP 647717, 1994) vitamin A is produced using Acenetobacterium bacteria, Takeda Chemical Industries LTD (US Pat. No. 5,545,538) obtains Vitamin B ₁₂ using bacteria Rhizobium Colalaminogenum FERMBP-4429, Nippon Sekiyu KK ( Japan patent 5-6996) as a producer of vitamin B ₁₂ applies Propionic acid resistant Propionibacterium shermani or frendenreiichii bacteria strain With these methods for producing vitamin B _{12, the} concentration of vitamin B ₁₂ in the culture fluid does not exceed 60-70 μg / ml.

In Russia, the microbiological synthesis of vitamin B ₁₂ using bacteria Propionibacyerium shermani (patent 1737915) allows to achieve a concentration of vitamin B ₁₂ in the culture fluid at the level of 40-50 mg / l (prototype).

The aim of the invention is to obtain a strain of bacteria capable of synthesizing vitamin B ₁₂ in a higher concentration than known microbial producers.

The inventive bacterial strain Pseudomonas fluorescens BKM B-2224D - producer of vitamin B ₁₂ obtained by multistage selection and selection of biosynthesis active vitamin B ₁₂ options from the collection strain of Pseudomonas fluorescens B98.

 The strain was deposited in the collection of microorganisms of the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences under the number BKM B-2224D.

When a producer is grown on a fermentation medium with molasses, inorganic salts and vitamin formation stimulants, the concentration of vitamin B ₁₂ in the culture fluid reaches 120-150 μg / ml.

The strain Pseudomonas fluorescens VKM B-2224D is characterized by the following features:
Cultural and morphological signs:
Bacteria are gram-negative motile bacilli 0.45-0.85 • 0.6 • 15 microns in size, non-spore-forming. On agar medium forms rounded colonies of 1-2 mm white. Cells are found mainly in clusters.

 The strain is resistant to reseeding on agar media.

Culture growth in liquid culture media occurs in parallel with the biosynthesis of vitamin B ₁₂ .

The temperature optimum of growth is 30 ^o C. pH 7-7.2.

 On a solid nutrient medium with molasses, white-gray shiny colonies form, by the end of 7 days the center is expressed, the size of the colonies is 2-3 mm.

 Optional aerob.

The concentration of vitamin B ₁₂ is determined by agar diffusion using an E. Coli culture. 113-3 and spectrophotometric - the principle of which is the transformation of coenzyme B ₁₂ into cobalamin nitrite. The concentration of cobalamin is determined on a spectrophotometer at a wavelength of 361 nm.

To maintain the strain using agar medium with molasses of the following composition, g / l:
Molasses - 100.0
Disubstituted Ammonium Phosphate - 1.5
Ammonium sulfate - 0.3
Microbiological agar - 20.0
Water - Up to 1 L
pH - 7.0-7.2 units

Physiological and biochemical signs:
Does not hydrolyze gelatin, starch, hydrolyzes lecithin. As a carbon source, it absorbs sucrose, betaine, does not absorb glucose, assimilates glutamic, propionic acid. Growth at 42 ^o C varies. Denitrification varies.

Culture preservation
The strain is stored in the form of a freeze-dried culture at a temperature of 4 ^o C for 2-3 years or on agarized media at constant transfers.

Vitamin B ₁₂ biosynthesis conditions of the strain Pseudomonas fluorescens BKM B-2224D
The biosynthesis of vitamin B _{12 by the} culture of Pseudomonas fluorescens BKM B-2224D is carried out according to the following scheme: seeded culture on an agar medium -> seeded culture on flasks in a liquid medium -> culture on a liquid medium in a sowing apparatus -> biosynthesis on a liquid medium in a fermenter .

To obtain seed for sowing sowing devices use the following medium:
Molasses - 6-12%
Dibasic ammonium phosphate - 0.01-0.1%
Cobalt chloride - 0.001-0.003%
Magnesium sulfate - 0.1-0.3%
Manganese sulfate - 0.01-0.05%
Zinc sulfate - 0.002-0.02%
5-6-dimethylbenzylimidazole - 0.001-0.003%
Water - Up to 1000 L
pH of the medium is 7.0 ± 0.2 units.

 In a rocking flask with a volume of 750 ml with a volume of medium of 50 ml make culture from a test tube in an amount of 0.1%.

Grow a culture at a temperature of 30 ^o C for 48 hours.

The resulting culture in a dose of not less than 0.1% seeded sterile nutrient medium in the sowing apparatus of the following composition:
Molasses - 6-12%
Dibasic ammonium phosphate - 0.01-0.1%
Cobalt chloride - 0.001-0.003%
Magnesium sulfate - 0.1-0.3%
Manganese sulfate - 0.01-0.05%
Zinc sulfate - 0.002-0.02%
5-6-dimethylbenzylimidazole - 0.001-0.003%
Water - Up to 100%
pH of the medium is 7.0 ± 0.2 units.

Fermentation conditions: continuous stirring, temperature 28-32 ^o C, with air supply 02-05 l / l of medium per min for 40-60 hours to an optical density of more than 0.2.

The resulting seed in a dose inoculated with a sterile fermentation medium of the following composition:
Molasses - 14-20%
Sucrose - 2.0-5.0%
Glycerophosphoric acid - 0.2-0.4%
Cobalt chloride - 0.01-0.03%
Choline chloride - 1.0-2.0%
Magnesium sulfate - 0.1-0.3%
Magnesium Oxide - 0.05-0.07%
Ammonium sulfate - 0.2-0.4%
Zinc sulfate - 0.05-0.1%
Cobalt chloride - 0.01-0.02%
5-6-dimethylbenzylimidazole - 0.01-0.02%
Glycerin - 0.1-0.2%
Tap water - Up to 100%
pH of the medium is 7.0 ± 0.2 units.

Fermentation is carried out at a temperature of the culture fluid of 28-32 ^o C, with constant stirring, an excess pressure of 0.5 MPa, with an air flow rate of 0.2 l / l of medium per minute.

The culture of Pseudomonas fluorescens BKM B-2224D is characterized by the fact that its growth occurs in parallel with the biosynthesis of vitamin B ₁₂ under aerobic conditions throughout the entire fermentation process.

The content of vitamin B ₁₂ in the culture fluid at the end of fermentation is 120-150 μg / ml as determined by the microbiological method using E. coli 113-3 as a test culture or spectrophotometric method. When culturing propionic acid bacteria, the content of vitamin B ₁₂ in the culture fluid is 40-50 μg / ml.

At the end of the fermentation process, the culture fluid is acidified with sulfuric acid to a pH of 5.8 units, the culture is lysed at a temperature of 90 ± 10 ^o C. To obtain the finished product, the culture fluid can be processed in a spray dryer with fillers (chalk, salt, bran and etc.) or sent at the stage of receipt and isolation of vitamin B ₁₂ in its purest form.

The results of checking the level of vitamin B ₁₂ biosynthesis by the Pseudomonas fluorescens VKM B-2224D culture under laboratory conditions are shown in Table 1
The results of checking the level of vitamin B ₁₂ biosynthesis by the Pseudomonas fluorescens VKM V-2224D culture under experimental industrial conditions (in devices with a volume of 2.5 m ³ ) are given in Table 2
Sources of information
Russian patent 1737915, C 12 P 19/42 - prototype.

Claims (1)

The bacterial strain Pseudomonas fluorescens VKM B-2224D is a producer of vitamin B ₁₂ .

Images