JPS6022912B2 - Method for producing microbial cells - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing microbial cells.

In recent years, methods for effectively utilizing microbial cells as feed, food, medicine, industrial raw materials, etc. have been explored.

Furthermore, in order to produce microbial cells industrially and effectively, it is expected to use methanol as a carbon source, which is likely to be supplied in large quantities at low cost in the chemical industry. In view of these circumstances, the present inventors conducted a widespread search in nature for microorganisms that can be efficiently cultivated by aerobically assimilating methanol as a main carbon source, and as a result, discovered a new bacterial species, Pseudomonas huacahamensis. Using this, we established a method for economically producing large quantities of microbial cells.

The gist of the present invention is that bacteria belonging to Pseudomonas wakayamaensis are cultured in a medium containing methanol as the main carbon source, and bacterial bodies are collected from the culture.
It consists in a method for producing microbial cells. The microbial cells of the present invention appear to belong to the genus Pseudomonas based on their mycological properties, but since they differ in various respects from known bacterial species belonging to the genus Pseudomonas, they are considered to be a new bacterial species. It was concluded that Pseudomonas huacahamensis (
It was named PseMomonaswakarat maensis).

A typical microbial cell in the present invention is Pseudomonas wakayamaensis DS-25 (microbial accession number: Chokoken Bacteria No. 410), and the boxy properties of this bacterium are shown below. Culture was carried out at 370℃ for 3 days in 'a' form broth liquid medium and broth agar plate.

■ Cell shape and size: Birch mold, (0.3-0.4)
×(2.0-2.2)mu ■ Population of cells: single cells or twin cells.

■ Motility: Motility present.

It has polar flagella. ■ Presence of spores: None.

■ Gram staining: Gram negative ■ Acid-fast: negative 'b} Growth status in each of the following media ■ Juice agar plate culture: Vigorous growth after 3 days of culture at 370.

The colony is circular in shape and has 3 to 4 ribs in size. The surface is rough and the periphery is fissured. Milky white, matte and opaque. The hardness is clayey. ■ Methanol-containing agar plate culture: Vigorous growth after 5 days of culture at 37°C. The colony is circular in shape and 3 to 4 sided in size. The protuberance is navel-shaped (concave in the center). The surface is rough, and the entire periphery is full. Milky white, glossy and opaque. The hardness is sticky. ■ Broth agar slant culture: Grows uniformly along the inoculation line after 3 days of culture at 370.

The ridges are moderate and the surface is smooth. The margins are wavy or fissured.
It is milky yellow white in color and has a bright luster and is opaque.The hardness is that of viscous steel.■
Methanol agar slant culture: Grows uniformly on the inoculation line after 5 days of culture at 37q0.

The ridges are moderate, the surface is smooth, and the margins are entire. Milky white,
Shiny and opaque. The hardness is sticky steel. ■ Meat juice liquid static culture: Growth was moderate after 3 days of culture at 370. No precipitation. Forms a bacterial film on the surface. ■ Meat juice liquid shaking culture: 3 at 370
When cultured for days, it grows vigorously and becomes cloudy.

There is precipitation. Opacity. ■ Beptone water liquid shake culture: 37
After culturing at 0 for 3 days, the cells grow vigorously and become cloudy. There is precipitation. ■ Methanol-containing liquid static tatami culture: When cultured at 370℃ for 3 days, it grows vigorously and becomes cloudy. There is precipitation. Forms a thin bacterial ring. Opacity. ■ Methanol-containing liquid shaker culture: 370
After 3 days of culture, it grows vigorously and becomes cloudy.

There is precipitation. Opacity. ■ Juice puncture culture: When cultured at 370 for 3 days, it grows on the surface or to a depth of 1 to 3 willows from the surface.

The surface is milky white. ■ Meat juice gelatin puncture culture: After 5 days of culture at 370, there was growth and precipitation on the surface.

Liquefy gelatin. ■ Litmus milk: Cultured at 370.

solidify. 'c' The following physiological properties ■ Reduction of nitrate:
Intestinal ■ Denitrification reaction: Negative ■ M eaves test: Negative ■ VP test: Positive/Positive ■ Indole production: Negative ■ Hydrogen sulfide production: Negative ■ Starch hydrolysis: Enteric ■ Citric acid utilization: Koser's Positive in culture medium ■ Inorganic nitrogen source: Use ammonium salts and nitrates, respectively ■ Pigment production: No colored pigments are produced in King A and B medium.

■ Urease: enteric ■ Oxidase: positive ■ Catalase: intestinal
Adjustment was made by adding an aqueous H solution.

) Grows in the pH range of 6-9. It does not grow at pH 5 and 10. The temperature is 5-40 oo, but it grows at 25-40 oo.
℃ is suitable. It does not grow at 41°C.

■ Attitude towards oxygen: aerobic ■ ○One F test (based on the Hu Guifson method);
Metabolizes glucose oxidatively.

■ Whether or not acid and gas are produced from the following brans: Cultivation was carried out using bepton water at a sugar concentration of 1% by weight and a temperature of 37° C. for 10 days.

Acid Gas ■ Saccharide assimilation ability: In the methanol-containing liquid medium described below, sugar was used instead of methanol, and the culture was carried out at a sugar concentration of 0.5% by weight and a temperature of 370 for 10 days.

[11L-arapinose + (2'D-xylose 10'31 D-glucose + (weak) {4}
D-mannose +'5} D-fructose + (weak) (61D-galactose
+ (weak) '71 Maltose +
Z'81 Sucrose 10 (weak)
(9} Lactose '10 trehalose + (11) D-sorbit + (12) D-mannite + (13) Inosite + (1Q glycerin 10 (15) starch + 'd' Isolation source: soil. Methanol-containing in the above culture test. The agar base and methanol-containing liquid medium were as follows.

○} Methanol-containing agar medium KH2P04 1.5 evening, Na2HP04 3.2
Evening, (N ratio) 2S04 3 evening, MgS04・7th 2
0 0.5 evening, CaC12・2nd 20 0.1 evening, Fe
S04・7&0 0.012・ZnS.

4th and 7th 201.4 females, CuS.

4.9 too.

〇． 9 of 25, Na2MOO4・2 days 200.9 of 24
,C. C12・Mother LOO. 24-2, MnS04・Spot 2
0 1.2-9, agar 20 evenings, distilled water 1 evening 12,000 yen
After sterilizing for 15 minutes, 20 minutes of methanol was added aseptically. '21 Methanol-containing liquid medium Methanol-containing agar medium without using agar and with 5 terts of methanol strained.

Experimental methods are described in the Berges Manual of Deterministic Bacteriology.
y1 s Manual of Detennjnat
ive mother cteriolo intuition) 3rd edition (1974),
“Bacteriology Practice Summary” (edited by the Institute of Medical Science Alumni Association, revised edition 1)
973 published by Ohmaruzen Co., Ltd.) and "Classification and Identification of Microorganisms" (edited by Takeji Hasegawa, published by the University of Tokyo Press in 1975).

When the above mycological properties were examined according to the classification criteria in the Purges Manual, it was confirmed that it belonged to the genus Pseudomonas.

This bacterium was compared with the two major Pseudomonas fleas classified in the above literature in terms of the properties listed in the table in the same book.

Comparing properties other than carbon source assimilation, Pseudomonas facilis (Pseudomonas facilis)
domonas facilis) is considered to be the closest.

However, when comparing this bacterium and Pseudomonas facilis in all properties including assimilation, it is found that in addition to methanol assimilation, which is a characteristic of this bacterium, starch hydrolysis, lactose assimilation, and sucrose They differ in their assimilation ability. Also, from other known documents, JP-A-49-6192 is found to be a relatively similar bacterial species.
Pseudomonas methanolo phantom in the publication No.
ans), Pseudomonas aeruginosa in Tokukan Sho 50-1154480 Publication.
aem bonito dish sa), Pseudomonas methanolytica (PseMomon
asmethanolitica).

However, this bacterium, Pseudomonas methanol oxidans, has the ability to reduce nitrate, the fact that ureatase is enteric, the growth temperature and pH range, the production of acid from a large number of various brans, and the ability to assimilate lactose. There are differences in In addition, Pseudomonas aeruginosa is characterized by a negative MR test, a positive VP test, a growth temperature and pH range, a growth rate in liquid static calendar culture without meat, and the absence of precipitation. They differ in that they do not change. Furthermore, Pseudomonas methanolytica does not produce hydrogen sulfide, hydrolyzes starch,
They differ in the use of citric acid, the temperature and pH range of growth, the generation of acid from a large number of various sugars, and the fact that they do not assimilate lactose. As mentioned above, it was confirmed that this bacterium is significantly different from any known Pseudomonas species. 0 Therefore, this bacterium was determined to be a new bacterial species of the genus Pseudomonas.

For culturing the bacterial cells in the present invention, an aerobic liquid culture method is suitable.

The culture temperature is in the range of 5 to 400C, preferably 25C.
-40qo, and PH' is 6-9, preferably 6-8. Culture may be batch culture or continuous culture.
Methanol is used as the main carbon source in the culture medium, but
Preferably, the amount of methanol is less than 50 methanol/so.
Nitrogen sources include inorganic substances such as ammonium salts and nitrates, organic substances such as urea, casein, corn staple liquor, veptone, yeast extract, meat extract, etc., phosphorus sources such as phosphates, and sulfur sources such as sulfates. is used. In addition, as a source of metals such as magnesium, potassium, calcium, sodium, iron, manganese, copper, zinc, molybdenum, cobalt, and boron, appropriate amounts of their metal salts are added, and as necessary, vitamins, amino acids, etc. are added to the bacterial growth. Substances that are absolutely necessary or growth promoting substances are added.

In addition, natural soil may be used as long as it contains appropriate amounts of methanol, nitrogen sources, inorganic substances, vitamins, amino acids, etc., or growth-promoting substances. It is convenient to adjust the amount using phosphate and ammonia.To collect the bacterial cells from the culture, conventional methods such as furnace filtration and centrifugal separation can be carried out, and washing and drying can be carried out. You can also do it.

According to the present invention, methanol, which is abundantly supplied from the chemical industry, can be used as a main carbon source, and microorganisms belonging to Pseudomonas huacamaensis can be produced in large quantities with good yield.

In addition, the obtained bacterial cells are rich in protein and can be used as feed, food, etc., and can also be effectively used as pharmaceutical raw materials, industrial raw materials, etc. Examples of the present invention are described below.

Example 1 KH2P04 1.5taNa
2HP04 3.2 evening (N
Sun) 2S04 3ta MgS04・
7 days 20 0.5 evening CaC12・
2LO 0.1 Ta FeS0417 days 20 0.02 Ta ZnS.

4th and 7th 20 2.8.

CuS. 4.8L.

〇． 5 imitation Na2NL. 4/2nd 20
9C of 0.48.

CI2/Mother too 〇 0-48 Ta Mh
S. 4th and 9th days 20 2.4 ta distilled water 1. 100 vessels of culture medium consisting of each of the above ingredients to a volume of 0.
The mixture was placed in a Sakaguchi flask and sterilized at 120°C for 18 minutes, and then 0.2ml of methanol was added aseptically.

Pseudomonas wacahamensista DS-25 was precultured with 35 CO for 2 feeding hours in a medium with the same composition as above except that the amount of methanol added was 0.5% by weight. The preculture solution containing bacterial cells was reduced to 0. The bacteria were grown to % of the mother volume, and cultured by shaking for two hours at 35:00. The culture was centrifuged to separate the bacterial cells, further washed with water, and then dried at 100 oo for 24 hours to obtain 0.9 dried bacterial cells per 1 night of culture. The generation change time in the logarithmic growth phase of this culture was 1.5 hours. The crude protein content of the bacterial cells was 7% by weight. Example 2 500 liters of a medium with the same composition as in Example 1 was placed in a mini jar, and after sterilization at 120 am for 15 minutes,
The preculture solution containing Pseudomonas wakayamaensis DS-25 cells obtained by preculturing in a medium with the same composition for 2 hours at 370°C with the addition of 500 methanol aseptically was added to the plate to give a concentration of 2% by volume. The bacteria were cultivated under aerated conditions at 3700° C. for 2 hours while maintaining the pH at 7.0 with 130% by weight ammonia water.

Claims (1)

[Claims] 1. Bacteria belonging to Pseudomonas swakayamaensis,
A method for producing microbial cells, which comprises culturing in a medium containing methanol as the main carbon source and collecting cells from the culture. 2. Bacteria belonging to Pseudomonas swakayamaensis are
The method for producing a microorganism according to claim 1, characterized in that the microorganism is Pseudomonas swakayamaensis DS-25.