JPS6022913B2 - 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 aichensis. Using this, we established a method for economically producing large quantities of microbial cells.

The gist of the present invention resides in a method for producing microbial cells, which comprises culturing bacteria belonging to Pseudomonas aichensis in a medium containing methanol as a main carbon source, and collecting cells from the culture.

The microbial cells of the present invention appear to belong to the genus Pseudomonas based on their mycological properties, but they are different in various respects compared to known bacterial species belonging to the genus Pseudomonas. It was determined to be a new bacterial species, and Pseudomonas aichensis (Pseudomonas aichensis).
iensis).

A typical microbial cell in the present invention is Pseudomonas aichensis DS-26 (Choken Bacteria Deposit No. 4107), and the mycological properties of this bacterium are shown below. [a} Form: Cultivated at 37q0 for 3 days in broth and broth agar medium.

■ Cell shape and size: Kajibacterium, (0.3-0.4)
×(0.75-1.0)mu ■ Population of cells: unicellular or bicellular.

■ Presence or absence of motility: Motile.

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

■ Gram staining: Gram negative.

■ Anti-acidity: Negative.

'b} Growth status in each of the following media■ Broth agar plate culture: Vigorous growth after 3 days of culture at 37q0.

The colony is circular in shape and has 4 to 6 legs in size. The protuberance is navel-shaped (concave in the center). The surface is smooth and the surrounding area is completely green.
is fissured and wavy. Milky white, glossy and opaque. The hardness is sticky. ■ Methanol-containing agar plate culture: Vigorous growth after 5 days of culture at 370°C. The colony has a circular shape and a size of 2~
3 side. The surface is smooth and the surrounding area is completely green. Milky white, shiny and opaque. The hardness is buttery. ■ Meat juice agar slant culture: 3
700 for 3 days and grew uniformly along the inoculation line.

The ridges are moderate, the surface is smooth, and the edges are entirely green or wavy. Milky white, glossy and opaque.

The hardness is buttery. ■ Methanol agar slant culture: 370
After 5 days of culture, the seeds grew uniformly along the inoculation line.

The ridges are moderate, the surface is smooth, and the margins are entire. Milky white,
Shiny and opaque. The hardness is buttery. ■ Meat juice liquid static pupil culture: Vigorous growth after 3 days of culture at 370.

There is precipitation. Opacity. Forms a bacterial film on the surface. ■ Meat juice liquid shake culture: When cultured at 37q0 for 3 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. ■ Static culture in methanol-containing liquid: After 3 days of culture at 370°C, it grows vigorously and becomes cloudy. There is precipitation. Forms a thin bacterial ring. Opacity. ■ Methanol-containing liquid shaking culture: When cultured at 370℃ for 3 days, it grows vigorously and becomes cloudy. There is precipitation. Opacity. ■ Juice puncture culture: Cultured at 370 for 3 days, growing to the surface or to a depth of 2 to 3 sides from the surface.

The surface is milky white. ■ Meat juice gelatin puncture culture: After 5 days of culture at 30'0, there was growth on the surface and precipitation.

Liquefy gelatin. ■ Litmus milk: Coagulates by culturing at 370°C.

'c - The following physiological properties ■ Reduction of nitrate: negative ■ Denitrification reaction: negative ■ Old M test: negative ■ VP test: positive ■ Formation of indole: negative ■ Formation of hydrogen sulfide: negative ■ Hydrolysis of starch: Negative■ Use of citric acid: Negative with Koser's medium■ Inorganic nitrogen source: Uses ammonium salts but does not use nitrates.

■ Pigment production: Colored pigments are not produced in King A and B media.

■ Urease: enteric ■ Oxidase: positive ■ Catalase: positive ■ Range of growth: Using the methanol-containing liquid tower described later, the temperature and fence were varied (adjusted by adding HCI or NaOH aqueous solution).

It grows in the pH range of 5 to 9, but does not grow at pH 4 or 10. Grows at a temperature of 5 to 45 pm, but 25 to 43 q.
o is preferred. It will not grow at 50qo.

■ Attitude towards oxygen: aerobic ■ ○-F test (by Hu scale ifson method):
Glucose does not produce acids or gases either aerobically or anaerobically.

(2) Whether or not acids and gases are produced from the following sugars: Cultivation was carried out using bepton water at a sugar concentration of 1% by weight and a temperature of 3700 for 10 days.

Acid Cas (1) L-arabinose 10 (weak) (2) D-xylose 1 (3) D-curcose + (weak) (4) D-mannose + (weak) (5) D-fructose + (weak) ) (6) D-galactose - maltose (8) sucrose + (weak) (9) lactose (10) trehalose + (weak) (11) D-solpit (12) D-mannit + (13) inosyt ○4) Chrycerin + (15 starch) Assimilation of sugars: In the methanol-containing liquid medium described later, sugar was used instead of methanol, and cultured at a sugar concentration of 0.5% by weight and a temperature of 370 for 10 days.

[11L-arabinose + (very weak) [2] D
-Xylose 10 (very weak) {3} D-glucose + (very weak) {4) D-mannose +
(Very weak) '5' D-Fructose + (Very weak) ■ D-Galactose 10 (Very weak) {7}
Maltose + (very weak) [8} Sucrose + (very weak) '9) Lactose action trehalose + (very weak) (11) D-solpit - (12) D-mannit + (very weak) (13)
Inosit + (weak) (1 heart glycerin
10 (very weak) (15) Starch'd} Separation source:
In the above culture test, the methanol-containing agar base and methanol-containing liquid medium were as follows.

'1} Methanol-containing agar medium KQP04 1.5 evening, Na2HP04 3.2
Evening, (N is) 04 3 Evening, M is 04/7th 20
0.5 evening, CaC12, 2nd 200.1 evening, FeS04
・7 ratio 0 0.01 evening, ZnS.

4th, 7th 201, 4 skin evening, CuS.

4th and 9th 200.25 evening, Na2MOO4, 2nd 200.24 skin evening, C.

CI2・Mother LOO. 24 Hadayu, MnS04 Nagi 20 1
．． 2 hours of skin, 20 hours of agar, 1 level of distilled water at 120qo for 15 minutes
After sterilizing for 20 minutes, 20 minutes of methanol was added aseptically. [2] In a methanol-containing liquid base methanol-containing agar medium, no agar was used, and the amount of methanol was strained by 5.

The experimental method is ``Bergey's Manual Determinative Bacteriology.''
8th edition (1974), ``Summary of Bacteriology Practice'' (edited by the Institute of Medical Science Alumni Association, revised edition published by Maruzen Co., Ltd. in 1975), and ``Classification and Identification of Microorganisms'' (Hasegawa). Edited by Takeji, published by Hitsuji University of Tokyo Press, 1973).

As a result of examining the mycological properties of this bacterium in accordance with the classification criteria in the above-mentioned Purges Manual, this bacterium was recognized to belong to the genus Pseudomonas.

This bacterium was compared with the two main descendants of the genus Pseudomonas 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, PseMomonas alcaligenes has flagella, grows at 4100, liquefies gelatin, and is negative for starch hydrolysis.
alcali sella nes) is considered to be the closest.

However, when comparing this bacterium and Pseudomonas alcaligenes in all properties including assimilation, it is found that in addition to the methanol assimilation characteristic of this bacterium, growth temperature, glucose,
The assimilation properties of trehalose and inosit differ.
In other known documents, JP-A-4
Pseudomonas methanol oxidans (Pseudomonas methane) in Publication No.
oro phantom dans), Pseudomonas methanolitica (Pseudomonas methanolytica) in Japanese Patent Publication No. 51-4149.
nasmethanolitica).

However, this bacterium differs from Pseudomonas methanoloxidasus in urease, growth temperature, acid production from a large number of various sugars, lactose assimilation ability, D-sorbitum assimilation ability, and starch assimilation ability. Also Pseudomonas
It differs from M. methanolytica in nitrate reduction, hydrogen sulfide production, growth temperature, acid production from a large number of various sugars, lactose assimilation ability, D-solpit assimilation ability, and starch assimilation ability. As mentioned above, it was confirmed that this bacterium is significantly different from any known Pseudomonas species.

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 45Qo, preferably 25Qo.
~43qo, and the pH is 5-9. Culture may be batch culture or continuous culture. Methanol is used as the main carbon source for the beach, but the amount of methanol is preferably less than 50 m/s.

Nitrogen sources include inorganic substances such as ammonium salt 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. 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.

The medium may be a natural medium as long as it contains appropriate amounts of substances essential for growth or growth-promoting substances such as methanol, nitrogen sources, inorganic substances, and vitamin t-amic acids. The pH of the medium is conveniently adjusted by phosphate and ammonia. To collect bacterial cells from culture,
Furnace filtration, far-D separation, etc. may be carried out according to conventional methods, and washing and drying may also be carried out.

According to the present invention, methanol, which is abundantly supplied from the chemical industry, can be used as the main carbon source, and Pseudomonas
Microorganisms belonging to Aichensis 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 1.5taN
a2HP04 3.2 evening (
NA) 2S04 3T MgS04・
7 days 20 0.5ta CaC12・
2LO 0.1taFeS04/7
Day 20 0.02ta ZnS04・
7th 20 2.8m Evening CuS〇4・
3L 〇 〇-5m Shio Na2NL.

4,2 days 20 0.48 kota CoC12・
Mother LO O-48 no TaMnS04/9
Day 20: Put 100 liters of culture medium consisting of 2.4 of the above components into the Sakaguchi flask with a capacity of 0.5, sterilize it at 120 pm for 15 minutes, then add 0.2 ml of methanol aseptically. Added.

Pseudomonas aichiensis DS-2 was precultured at 370 for 2 hours in a medium with the same composition as above except that the amount of methanol was 0.5% by weight.
The preculture solution containing 6 cells was diluted to 0. The cells were plated to obtain % of the acceptable amount, and shake culture was performed at 3700 for 2 hours. The culture was centrifuged to separate the bacterial cells, further washed with water, and then dried at 100° C. for 2 hours to obtain dried bacterial cells at a rate of 0.88 bacterial cells per 1 night of culture. The generation change time in the logarithmic growth phase of this culture is 1. It was a patchy time. The crude protein content of the bacterial cells was 79% by weight. Example 2 500 mL of a medium having the same composition as in Example 1 except that the amount of methanol added was 5 mL was placed in a mini jar with a capacity of 1, and after sterilizing each powder at 12 mL, the above amount of methanol was added. To this, a pre-culture solution containing Pseudomonas aichensis DS-26 cells obtained by pre-cultivating at 370℃ for 2 hours in a medium with the same composition as in Example 1 was added to the solution to give a concentration of 2% by volume. The bacteria were shielded as described above, and cultured under aeration conditions at 370°C for 2 hours while maintaining the pH at 7.0 with 1% by weight ammonia water.

The culture was centrifuged to separate the bacterial cells, further washed with water, and then dried at 100 qo for 2 feeding hours, yielding 4.1 dry bacterial cells per culture.

Claims (1)

[Scope of Claims] 1. A method for producing microbial cells, which comprises culturing bacteria belonging to Pseudomonas aitiensis in a medium containing methanol as a main carbon source, and collecting cells from the culture. 2. The method for producing microorganism cells according to claim 1, wherein the bacteria belonging to Pseudomonas aichiensis is Pseudomonas aichiensis DS-26.