JPS595276B2 - Vitamin B12 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides vitamin B1 production using methanol-assimilating bacteria.
□Relates to the method for producing the containing microbial cells.

Specifically, a methanol-assimilating vitamin B□2-producing bacterium belonging to the genus Aeromonas or Achromobactu was cultured in a medium containing methanol as a carbon source and cobalt salt and imidazole added thereto. This is a method for producing microbial cells containing a high percentage of vitamin B□2 by separating and drying the microbial cells.

Vitamin B1□ is a vitamin isolated from the liver as an antiseptic vitamin, and recently, vitamin B1□ has become important as a feed additive.

Vitamin B1□ is produced by propionic acid-fermenting bacteria, butyrate-fermenting bacteria, anaerobic bacteria such as Clostridium bacteria, photosynthetic bacteria such as Rhodopseudomonas bacteria, and n- belonging to Nocardia, Arthrobacter, and Pseudomonas genera.
It has been recognized that it is produced and accumulated in paraffin hydrocarbon-assimilating bacteria, methane-assimilating bacteria, and methanol-assimilating bacteria belonging to the genus Pseudomonas and Protoaminobacter.

The present inventors have conducted a series of studies on cultivating methanol-assimilating bacteria and using the dried bacterial bodies as feed for livestock.

In the process, when cobalt salts and imidazole are added to a medium using methanol as a carbon source and methanol-assimilating bacteria belonging to the genus Aeromonas and Achromobactus are cultured, vitamin B1□ accumulates within the bacterial bodies of the bacteria. They discovered this and completed the present invention.

When a significant amount of vitamin B12 is accumulated in the cells of microorganisms, it is effective to add constituent components or metabolic precursors of vitamin B12 to the culture medium. The present invention is the first to describe the method of adding imidazole.

The microorganism used in the present invention is Aeromonas methanococcus.
Lamb (Aeromonas methanophilu)
mnov, sp, R-1014, Microtechnical Research Institute deposit number 2
808), Aeromonas methanocola (Aeromo
nasmethanocola nov, sp, R
Bacteria of the genus Aeromonas, such as Achromobacter -1332, Microtechnical Institute Deposit No. 2809), Achromobacter methanolo
xidans nov, sp.

These are Achromobacter bacteria such as R-1013, Microtechnical Research Institute deposit number 2693), and these are all entrusted to the Microbial Technology Research Institute (4?
8534 Japanese Patent Application No. 112554/1973).

The mycological properties of these bacteria are as follows.

Aeromonas mechnophyllum R-1014 (A) Morphological properties (1.5% methanol broth agar plate 32°C, 2
4-hour culture) Pattern: short bacilli, single or 2-3 chained, with single-polar flagella.

Size 20, 5 to 0.7 x 0.8 to 1.3 microns Motility: Motile Durham staining: Negative Acid-fast staining: Negative Spores: Not formed Cell pleomorphism: None 5) Culture properties 1 .5% methanol gravy agar Colony growth is good, round, smooth surface, raised, golden edges, glossy, cream color, buttery'; no pigment formation.

Good growth of gravy agar colonies, round, smooth surface, ridges, golden edges, glossy, pale skin color, somewhat viscous, no pigment formation.

1.5% methanol Synthetic agar Colony grows well, round, smooth surface, raised, golden edges, glossy, white cream color, buttery, no pigment formation.

1.5% methanol gravy agar Slope growth is good, filamentous growth, glossy, pale skin color, no pigment formation.

Good growth on gravy agar slope, filamentous growth, glossy, pale skin color, no pigment formation.

Good growth on 1.5% methanol synthetic agar slope, filamentous growth, glossy, pale skin color, no pigment formation.

1.5% methanol synthetic liquid medium Growth is good, white film formation, white precipitate, liquid is cloudy, no pigment formation.

Good growth of meat juice, white film formation, white bacterial ring, sediment, liquid is cloudy, no pigment formation.

Flesh gelatin puncture culture Upper surface ash cream, pale skin-colored growth at the puncture site, gas generation at the puncture site, no liquefaction.

(Q Physiological properties (test medium contains 1.5% methanol) Grows at a growth temperature of 219°C to 42°C.

It does not grow at 45°C.

Growth pH: 5-9 Oxygen requirement: Aerobic when methanol is used as a carbon source.

Facultatively anaerobic when using glucose as the carbon source.

OF test (Huleifson medium): Acid is also produced anaerobically through fermentation.

Gas generation (glucose medium) Lidomus milk with gas generation: Good growth, forming white bacterial rings.

Pep) to convert. Lidmus is bleached.

No milk curdling. Gelatin liquefaction: Does not liquefy.

Formation of hydrogen sulfide: Produces.

Starch decomposition and two decomposition.

Indole generation: Not generated.

Nitrate reduction: produces nitrite.

Catalase activity: Positive Oxidase activity: Positive Urease activity Two positive vP reactions Two negative MR test: Negative Ammonia production: Positive Nitrogen availability: Utilizes ammonium salts, nitrates, urea.

Denitrification reaction: Yes.

Use of citric acid: Use Koser medium.

Christensenj @ Use.

Salt tolerance: Grows up to 6%.

Availability of carbon sources, production of acids and gases Isolation source: Seawater Aeromonas methanocola R-1332 people Morphological properties (cultured on 1.5% methanol broth agar plate at 32°C for 24 hours Image shape: short bacilli, mono- Or there are flagella with 2-3 chains and 2-4 polar flagella.

Size = 0.4-0.7 x O, 8-1.2 microns Mobility: Motile.

Durham stain: negative Acid-fast staining: negative Spores: Not formed.

Cell pleomorphism: None.

(B) Culture properties: 1.5% methanol broth agar Colony grows well, round, smooth surface, ridged center, glossy gold edge, cream color, buttery, no pigment formation.

Good growth of gravy agar colonies, round, smooth surface, raised center, golden edges, glossy, cream color, buttery, pigment, no formation.

1.5% methanol Synthetic agar Colony grows well, round, surface smooth, flat with no bulges, slightly wavy around, gray cream color, glossy, buttery, no pigment formation.

1.5% methanol gravy agar Slope growth is good, filamentous growth, glossy, gray cream color, no pigment formation.

Good growth on gravy agar slope, filamentous growth, glossy, sunrise cream color, no pigment formation.

Good growth on 1.5% methanol synthetic agar slope, filamentous growth, glossy, gray-cream color, no pigment formation.

1.5% methanol synthetic liquid medium Growth is good, white film formation, white precipitate, liquid cloudy, no pigment formation.

Good growth of meat juice, slightly white fungal ring, film formation, liquid cloudy,
Pigment production and meat juice gelatin puncture culture. Gray cream-colored cotton-like growth at the puncture site, with liquefaction.

C) Physiological properties (test medium contains 1.5% methanol) Growth temperature: Grows at 13°C to 39°C.

It does not grow at 43°C.

Growth pH: 5-9 Oxygen requirement: aerobic when using methanol as a carbon source;
Facultatively anaerobic when using glucose as the carbon source.

OF test (Huleinoson medium): Acid is also produced anaerobically through fermentation.

Gas generation (glucose medium): No gas generation.

Litmus milk cultivar grows well, forms a white fungal ring, and converts into peptonate.

Lidmus is bleached. Milk curdles.

Gelatin liquefaction: liquefy.

Formation of hydrogen sulfide: Produces.

Starch decomposition and two decomposition.

Indole generation: Not generated.

Reduction of nitrate produces dinitrite.

Catalase activity: Positive oxidase activity: Positive urease activity: Positive vp reaction: Positive MR test: Negative Ammonia production: Positive Nitrogen availability: Utilizes ammonium salts, nitrates, urea.

Denitrification reaction: Yes.

Use of citric acid: Use Koser medium.

Use Christensenf@ground.

Grows to food tolerance = 8%.

Carbon source availability, acid and gas production Note: Although the production of diacid is observed, the culture As the process progresses, the pn indicator is decolored. Ru.

Isolation source: Seawater Achromobacter methanoloxidans R-013 (8) Morphological properties (1.5% methanol broth agar plate, cultured at 32°C for 24 hours) Image shape: Short bacillus, long rods are not observed. do not have.

Single or in chains of two.

No pleomorphism. No flagella.

Size 2 0.5-0.8 microns x 0.9-1.2 microns Motile: Non-motile Duram staining Nigeram negative Acid-fast staining: Negative Spores 2 No B) Cultural properties 1.5% methanol gravy agar Good colony growth, round, smooth surface, flat ridges, all bound, grayish tan, glossy, buttery.

1.5% methanol Synthetic agar Colony growth is good, round, surface smooth, raised, all bound, slightly skin-colored gray cream color, glossy, buttery.

Good growth on 1.5% methanol gravy agar slope, filamentous, glossy, grayish brown. Good growth on 1.5% methanol synthetic agar slope, filamentous, glossy, slightly skin-colored, gray cream color.

1.5% methanol synthetic liquid medium No film formation was observed.

The liquid becomes cloudy. There is a slightly skin-colored sediment.

gravy No film is formed.

The liquid is not cloudy. There is a gray cream colored sediment.

Gravy agar slope Good growth, grayish-brown cream color, filamentous growth.

:Q Physiological properties (test medium contains 1.5% methanol) Growth is good at growth temperature = 20°C to 40°C.

It does not grow at 43°C.

Growth pH: Grows at pH 6-9.

Oxygen requirement: aerobic Lidomus Milk: Forms a slightly white bacterial ring.

No peptonization occurs, but milk coagulation is observed.

The liquid is purple but not alkaline. Gelatin liquefaction: Does not liquefy.

Formation of hydrogen sulfide: Produces.

Starch decomposition: not decomposed Indole generation: Not generated.

Nitrate reduction: No nitrite formation.

Catalase activity: positive Urease activity: negative vp reaction: negative MR test: negative Ammonia production: negative Availability of nitrogen sources: Use ammonium salts and nitrates.

Usability of citric acid: Not used.

Salt tolerance: Grows up to 4%.

Utilization of carbon sources, acid production Growth Acid production Methanol 10 - Ethanol - Glucose - Fructose - Glycerin - Galactose - Inositol - Sorbi 1-L - Manitol - Arabinose -- Xylose -- Sucrose -- Trehalose -- Lactose -- Maltose -- Starch -- Separation source: soil The fermentation medium used in the present invention contains methanol as the main carbon source, as well as conventional nitrogen sources and inorganic A medium supplemented with salts and optionally trace organic nutrients is used.

Methanol is used as a carbon source, but if the concentration of methanol is too high, it will inhibit the growth of microorganisms, so it is better not to keep the methanol concentration during culture too high.

Therefore, although it is possible to terminate the culture using only methanol added at the beginning, it is generally better to culture while maintaining a low concentration, so methanol is added sequentially to reduce the methanol concentration in the culture solution to 0.05. ~1.5 (wt%).

As the nitrogen source, 77monium sulfate, ammonium chloride, ammonia, ammonium phosphate, urea, ammonium nitrate, sodium nitrate, etc. used in normal fermentation are used.

Inorganic salts include potassium lysate, magnesium sulfate, phosphoric acid,
In addition to potassium chloride and sodium chloride, iron, manganese,
Addition of salts such as zinc and calcium is effective.

As a trace organic nutrient source, addition of soybean protein hydrolyzate, cane molasses, cornstarch liquor, yeast, extract, etc. is effective for producing and accumulating vitamin B12.

Furthermore, cobalt salt and imidazole, which are characteristics of the present invention, are added.

As the cobalt salt, cobalt sulfate, hydrochloride, nitrate, carbonate, etc. can be used.

These may be added at the initial stage of culture. Culture conditions include good aeration and stirring, culture temperature 25-38℃, and pH 6.0-8.
．． It is necessary to adjust it to 0.

Cultivation is usually completed in 2 to 5 days.

Cells are separated from the cultured solution by centrifugation, and then dried using a commonly used drum dryer or spray dryer.
If dried using a dryer such as a dryer, powdered bacterial cells containing vitamin B1□ will be obtained.

Table 1 shows an example of the basic medium composition used for culturing methanol-assimilating bacteria.

This medium was inoculated with methanol-assimilating bacteria (-1500r
Under the conditions of 50 ml of culture solution in a shaking flask with a volume of ml, the temperature is 36°C, and the methanol concentration is 1.0 (wt%) at the beginning.
, 1.5 (wt%) at 16th and 24th hours, respectively.
After adding and culturing for 48 hours, the bacterial cells were collected by centrifugation, and vitamin B12 in the bacterial cells was quantified by a microbial quantitative method using Escherichia coli (Escherichia coli 215).

As shown in Table 2, it was found that by culturing the methanol-assimilating bacteria used in the present invention in the presence of cobalt salt and imidazole, a significant amount of vitamin B02 was produced in the bacterial cells.

The present invention will be described in detail below with reference to Examples, but these are merely illustrative and do not limit the present invention in any way.

Example 1 Cobalt chloride) 0.002 (
% by weight, hereinafter the same) and 50 oz of a medium supplemented with 0.15% imidazole were placed in a 10007 culture tank,
Heat sterilization was performed at 129°C for 20 minutes.

After cooling, 1.0% methanol was added to this, and at the same time, the methanol-assimilating bacterium Aeromonas methanonerum R-
1014 seed culture solution 20. /? was inoculated and neutralized to pH 7.0 with ammonia at a temperature of 36°C and a stirring speed of 2 min.
Culture was started under aeration and stirring conditions of 50 rotations and an aeration rate of 500 l/min.

The methanol concentration during the culture was analyzed by a conventional method using gas chromatography, and the concentration in the culture solution was determined to be 0.1 to 0.8%.
The cells were cultured for 20 hours while being added sequentially to maintain the same temperature.

The bacterial cell concentration in the culture solution was 35. It was i/7.

This was a yield of 46.1% based on added methanol.

Cells were collected from the cultured solution 5007 using a Sharpless centrifuge and dried using a spray dryer.
．． 9 kg of powdered bacterial cells were obtained.

After extracting a portion of the bacterial cells with hot water, the amount of vitamin B12 was determined by a conventional microbial assay method using N. coli, and it was found to contain 281 mp/kg of vitamin B12.

This is 4.479 vitamin B1 from 5001 culture solution.
The production amount corresponded to □.

Example 2 Medium 5001, which was prepared by adding 0.002% cobalt nitrate (wt%, same hereinafter) and 0.20% imidazole to the basic medium shown in Table 1, was placed in culture tank 10001,
Sterilized at 0°C for 20 minutes.

After cooling, 1.0% methanol was added thereto, and at the same time, seed culture solution 201 of the methanol-assimilating bacterium Achromobacter methanoloxidans R-1013 was inoculated, and cultured with aeration and stirring in the same manner as in Example 1 for 20 minutes. I did it for an hour.

Is the bacterial cell concentration in the culture solution 37.7? /l, which was a yield of 48.1% based on the added methanol.

Cells were collected from 5001 of the cultured solution using a Sharpless centrifuge, dried using a spray dryer, and dried for 16 hours.
．． 6 kg of powdered bacterial cells were obtained.

When a part of the bacterial cells was taken and the vitamin B1□ in the bacterial cells was quantified in the same manner as in Example 1, it was found to be 182 mI? /ky of vitamin B12.

Is this 3.02 from the 5001 culture solution? Vitamin B1 of
The production amount corresponded to 2.

Claims (1)

[Claims] 1. Genus Aeromona
s ) or Achromobacter spp.
This method is characterized by culturing vitamin B□2-producing methanol-assimilating bacteria belonging to the genus romobacter in a medium containing cobalt salts and imidazole, using the methanol carbon source, and separating and drying the bacterial cells from the culture solution. A method for producing vitamin Bt2-containing bacterial cells.