JPS6013679B2 - Production method of vitamin B↓1↓2 by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses vitamin B2-producing bacteria belonging to the genera Micrococcus, Arthrobacter, and Paracoccus isolated from the ocean or marine-related materials in a nutrient medium containing 2 to 5% salt, singly or in combination. The present invention relates to a method for producing vitamin B2 using a novel fermentation method by collecting vitamin B2 from a culture obtained by mixing and culturing the above.

Vitamin B2 is an essential factor in the in vivo metabolism of post-acids, fats, proteins, carbohydrates, etc., and is used as a medicine or feed additive for humans, poultry, and/or livestock, such as as a treatment for pernicious anemia. It is widely used in practical applications.

Conventionally, the fermentation method for producing vitamins B and 2 is as follows:
Extraction of vitamin B2 from the residual liquid after extraction of antibiotics such as streptomycin and chlorotetracycline, extraction of vitamin B2 from a culture using Propionibacterium bran as the medium carbon source, and other methods such as Bacillus, Butylibacterium, Flavo A method for extracting and collecting vitamin B2 from cells such as bacteria and their culture fluids is known.

On the other hand, the present inventors assimilated methanol as a carbon source for the purpose of fermentative production of microbial proteins or physiologically active substances, and proliferated them on beaches using seawater or artificial seawater with a salt concentration of 2% or more, preferably 2 to 5%. As a result of searching for microorganisms that
Among the bacterial strains collected from seawater, coastal soil, or marine products along the Seto Inland Sea coast, strains belonging to the genus Paracoccus, Micrococcus, and Arthrobacter assimilate methanol at a high conversion rate of approximately 50% into bacterial bodies. They discovered that vitamins B and 2 can be accumulated by culturing in a nutrient tower using seawater or artificial seawater, and completed the present invention.

The microorganisms used in the method of the present invention are obtained by inoculating 1 part of a suspension of seawater, seabed soil, or seaweed from the coast of Hiroshima Bay into 500 Sakaguchi flasks at 28°C and 40°C. 4%
After repeating enrichment culture twice in a medium containing 0.02% sodium chloride and 0.02% methanol as main components, the cells were isolated by ordinary plate culture in a medium prepared by adding agar to the above medium.

Representative strains of these include 1-secretor 2-A bacteria:
Paracoccus sp. 112L-A (ParaCoC
C female Sp, 12L-A), 12A2-X bacteria: Micrococcus luteus (Micr ■ McSlute ship), 1
2A2 OY bacteria: Micrococcus variens (Mic
rococcus varians) and young A fungi: Arthrobacter sp.
rsp. Sha-A) can be mentioned.

The advantages of producing vitamins B and 2 using the above strains are that methanol can be used as a carbon source, that is, methanol is cheaper than other petroleum-based raw materials, and it is also water-soluble. Due to the low oxygen demand and low calorific value during fermentation, and the fact that seawater or artificial seawater can be used, this production can be carried out even in locations where fresh water is not easily available. Has many advantages.

All of these exemplified bacterial strains are new bacterial species or unique bacterial strains that can utilize methanol among known bacterial species, and their main mycological properties are as follows.

1 Morphological properties (1) Broth agar medium (cultured at 30°C for 1 to 2 days) 0 Cultural properties (1) Broth agar plate culture (2 Flesh agar slant culture Other properties such as color, gloss, surrounding area, etc. of fungal moss The properties were almost similar to those of plate culture.

■ Meat juice culture (4) Meat juice gelatin puncture culture (culture at 20°C for about 1 month) (5) Litmus milk culture m Physiological properties The influence of salt concentration on the growth of the 4 bacterial strains is as shown in the following table.

Basal medium composition: liquid medium containing 1% beptone and 1% meat extract Culture conditions: 3000°C, cultured for 5 days All of the four strains listed above have 2-5% salt content compared to the salt-free medium. Better growth was observed.

This is also related to the fact that 4 bacterial strains were isolated from seabed soil, and their growth is greatly promoted by containing 2 to 5% salt.

Furthermore, as shown in the inorganic nitrogen source utilization test, these strains do not grow well in media that do not contain yeast extract, etc., and it is thought that some growth factors other than vitamins B and 2 are required for their growth. It will be done. The above properties of the four bacterial strains are explained in Pursey's Manual of Determinative Bacteriology No. 8.
When searching with reference to the description in the edition (1974), 12A2
- Strain A belongs to the genus Paracoccus (GenusPara).
coccus).

This genus includes Paracoccus denitrificans (P. d
P. enimfica) and P. halodenitrificans are described in the said book. A difference was observed between the 12A21A strain and Paracoccus denitrificans in the ability to assimilate carbon sources as described below. Therefore, it is considered to be a different bacterial species from Paracoccus denitrificans.

In addition, Paracoccus halodenitrificans is affected by many factors such as the concentration of salt contained in the medium and the growth state of the bacteria, the production of acid oxidatively from glucose, the optimum temperature for growth, and the temperature range for growth. Differences were observed in terms of Therefore 1
2A2-A is considered to be closely related to Paracoccus halodenitrificans, but there is a large difference in salt tolerance, which is a characteristic property, and the 1-secretor 2-A strain is more sensitive to methanol. Considering its ability to assimilate, it is judged to be a new bacterial species that differs from Paracoccus halodenitrificans by 8U. Therefore, 12A21A is Parakotsukasu Spy.
12A2-A (Parac ratio cusp.12A2-A
) was named. The strain 1 and 2-X is considered to belong to the genus Micrococcus from the above-mentioned properties.

The aforementioned book, Virgie's Manual, states that this genus includes Micrococcus luteus, Micrococcus variens, and Micrococcus variens.
Three species of Micrococcus roseus have been described, but none of them are known to utilize methanol, a compound. The 12L-X strain was most similar to Micrococcus luteus among the three bacterial species of the genus Micrococcus.

All four strains used in this application are classified as methanol-utilizing bacteria, so-called obligate methanol-utilizing bacteria group (facu).
ltsive methanol-utilizing
It is clear that it belongs to Bacteria.

The 12mm-× strain is Micrococcus luteus (Macro
It was identified as a unique strain that belongs to the coccus lutea (L. coccus lutea) and has the ability to transform methanol.

Next, the 12A2-OY strain was also considered to belong to the genus Micrococcus due to its various properties, and among the three known bacterial species, it most closely resembled Micrococcus variens.

There, Microcococcus variens (Mjcrococ
It was identified as a unique strain belonging to C. varians) and having the ability to assimilate methanol.

Finally, the Water-A strain was bent into a dogleg shape under the microscope.
A large number of bending type cells were observed, and Gram staining showed that young cells were mostly stained in the shadows, while older cells were irregular in shape, with many round cells being observed, indicating Gramvariable cells.
ble). Because of this morphology, it was thought to be a bacterium called Coryneform bacterium (Co form cteda).

The Sha-A strain moves by flagella.

Observation using flagellum* color and electron microscopy shows that most of the flagella are monoflagellates, and their arrangement is mostly polar or slightly offset polar. Hoko nenetenagellu
m) was also recognized. Considering the above morphological observations and the fact that the source of this bacterium was seabed soil, it would be appropriate to place it in the genus Arthrobacter within the coryneform bacteria. The GC content of the DNA of the Sha-A strain was 69.3%. In addition, the result of analysis of bacterial cell fatty acid composition shows that the number of carbon atoms is 16.
The main constituent fatty acids were 18 linear chain sum fatty acids and 18 carbon atoms, and this discordant fatty acid was contained in the largest amount. It is known that many obligate methanol-assimilating bacteria exhibit such a bacterial cell fatty acid composition. Among the coryneform bacteria known so far, the one that assimilates methanol is Corynebacterium methanophilum (Corynebacterium methane).
anophj】skin m*1), Arthrobacter rufescens*2
) has been reported. References *1 Journal of the Fermentation Engineering Society (
J. Fermention TeCh side logic) 56
・4.243 (1978) *2 Fermentation Engineering Society Examination
48, 323 (1970) It is thought that both of these strains and the A-A strain exhibit morphologically similar properties.

However, Corynebacterium menophilum is positive in Gram staining and is not motile, and its physiological properties are positive for the production of hydrogen sulfide and indole, clearly distinguishing it from the 2b-A strain. The strain is clearly different from the Teisha-A bacterium, which is red in color and non-motile. Based on the above, the young-A strain was recognized as a new strain belonging to the genus Arthrobacter, and Arthrobacter sp.
named it -A (~thro-gandersp. Shaichi A).

Both strains have been deposited at the Microbial Technology Research Institute,
Parakotsukas sp. 12L-A (Par
acocc ship sp. 1st Sha 2-A) The strain is Chokoken Bacterium No. 5
No. 205, Micrococcus luteus (Mjcroco
cc female lu us 12L-x) is 520th
No. 7, Micrococcus variens
c-spot vanami is 20Y) is the collection number 5206 and Arthrobacter sp. 2b-A (~th
rotorsp. Sha-A) has obtained the deposit number No. 5204. In the method of the present invention, the above-mentioned box strain is used alone or in a mixture in a culture medium using seawater or artificial seawater containing methanol as a carbon source and other nutrients necessary for the growth of the bacteria. Mixed culture is suitable for B2 accumulation.

In general, it is advantageous for the medium to be in the form of a liquid broth, and the culture is preferably carried out under aerobic conditions. Cultivation in a liquid medium may be carried out by either shaking culture or condensation culture, but it is industrially advantageous to use so-called deep aerated light cultivation. Although the culture medium composition can be selected as appropriate, all strains use alcohols and alcohols as carbon sources.
For example, methanol, ethanol, ethylene glycol,
Propylene glycol and organic acids such as succinic acid, malic acid, etc. and glucose or carbohydrates such as maltose, sucrose are used, but methanol or glucose is advantageously used.

In addition, these may be used alone or in combination. As the nitrogen source, for example, inorganic nitrogen compounds such as ammonia salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, yeast extract, and meat extract are used.

In addition, as for inorganic salts, calcium salts, magnesium salts, potassium salts, phosphates, etc. may be added among seawater composition salts, or vitamin B2 precursors such as cobalt chloride, choline chloride, betaine, or pendimidazole may be added as necessary. may be added. The above-mentioned bacterial strains are inoculated into the medium prepared in this way, and they may be inoculated alone or with two or more strains at the same time, or they may be pre-cultured separately and then inoculated at the same time. It is desirable to use them in combination to accumulate large quantities of vitamins B and 2.

In any case, the ratio of inoculation of these strains can be changed as appropriate depending on the purpose. When culturing, the temperature is 25-40°C, preferably 25-35°C, and 3
The culture period is chosen to be from 1 to about 15 days.

Vitamins B and 2 produced in the culture obtained as described above can be extracted using a known solvent extraction method using phenol, phthanol, etc., or a column chromatography method using activated carbon, silica gel, ion exchange resin, etc. It can be collected by using appropriate combinations.

For example, when a culture is first centrifuged to obtain microbial cells and then separated from the culture as Cia/type vitamins B and 2, cyanide ions are added to the box, adjusted to axis 5 with an acid such as sulfuric acid, and then boiled. (For example, U.S. Patent
t2621144). The amount of vitamin B2 to be produced was determined by measuring the amount of culture solution 30. p. m. After centrifugation for 18 minutes, 0.01% KCN aqueous solution was added to the bacterial cells for 10 minutes.
The pH was adjusted to 5.0 with IN sulfuric acid, and the dish b's. j.
After pressurized extraction for 15 minutes, the temperature was returned to 30°C with deionized water, and the amount of vitamin B2 during fermentation and purification was confirmed by microbiological quantitative method using Escherichia co1j215. The present invention will be explained in more detail with reference to Examples below.

Example 1 Arthrobacter sp.
No. 204) as below, artificial seawater alteration tower site (ASW tower site)
Inoculate the culture solution 12, which was cultured 3 days ago, into the customer's mini jar fermenter containing the following sterilized artificial seawater base material 1.2. /
The cells were cultured for 5 days at a rotational speed of 70 rpm.

The phosphate in the medium is added aseptically after being sterilized separately.
In the early stage of culture, keep the rotation speed at 20 pm and adjust the pH of the medium.
After a decrease was observed, the rotation speed was increased to the above-mentioned culture conditions. At this time, the concentration of vitamin B2 produced was 4.2 mountains/so. Medium composition (ASW medium) * Day 3B○30
．． 152 so〃, MnC12・4 days 200.42 terz,
Z, C of ZnC1232.

CI2.6 days 201.43 multi-Z, CuC13・2 days 2
00.02 Shiosugi, Na2Moo4・2nd 207.26
Pillow disaster ** (monojime Z thiamin 1.0, riboflavin 1
．． 0, Pyritoxine 1.0, Bantothenic acid or Renwam 1
．． 0, nicotinic acid 1.0, fluoric acid 0.1, P-aminobenzoic acid 0.1 Artificial seawater basic medium * Rare metals are the same as ASW medium.

Example 2 Paracoccus sp.
205), Micrococcus luteus (Choken Bacteria No. 5207), and Micrococcus variens (Chococcus Bacteria No. 5206) were precultured individually in ASW medium in the same machine as Example 1. In the same artificial seawater basic medium,
The culture was carried out for 7 days.

The culture temperature was 30:00 for Paracoccus sp. 12L-A, and 27°C for the other two strains. At this time, the concentration of vitamin B, 2 produced by each strain was 4. Paracoccus sp. 12L-A. Monoyu/
Micrococcus luteus: 3.6
Micrococcus variance: 6.2.

Example 3 Paracoccus sp. 12A21A (Choken Bacteria No. 5205), Micrococcus luteus (Chococcus Bacteria No. 5207), and Micrococcus variens (Chococcus Bacteria No. 5206) ) was inoculated into a 500 M flask containing 100 volumes of ASW medium using an agar slant supplemented with ASW medium, and cultured at 28 qo for one day and night at 118 pm' with a trowel.

Claims (1)

[Claims] 1. Single or two or more types of vitamin B_1_2-producing bacteria belonging to the genus Micrococcus, Arthrobacter and Paracoccus isolated from the ocean, coastal soil or marine products, containing 2% or more of salt, other nitrogen sources, carbon sources, other inorganic salts, 25-40°C in a nutrient medium containing trace elements;
Cultivate aerobically at pH 6-8 and extract vitamin B from the culture solution.
A method for producing vitamin B_1_2 by a fermentation method, which is characterized by collecting vitamin B_1_2.