JPS6123993B2 - - 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, Flavobacterium tosaensis. We discovered this and established a method to economically produce large quantities of microorganisms using this discovery. The gist of the present invention resides in a method for producing microbial cells, which comprises culturing bacteria belonging to Flavobacterium tosaensis 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 Flavobacterium based on their mycological properties, but they differ in various respects from known bacterial species belonging to the genus Flavobacterium. was determined to be a new bacterial species and named Flavobacterium tosaensis. Typical microbial cells in the present invention are:
Flavobacterium tosaensis DS-1
(Microorganism Accession No. 4058), and the mycological properties of this bacterium are shown below. (a) Morphology 3 at 37℃ in broth liquid medium and broth agar medium
Cultured for 1 day. Cell shape and size: Bacillus, (0.5~
0.75) × (1.6-2.2) μ Cell population: unicellular or bicellular Motile presence: non-motile. Presence or absence of spores: None Gram staining: Gram negative Acid-fastness: Negative (b) Growth status in each of the following media Juice agar plate culture: Vigorous growth after 3 days of culture at 37°C. The colony is circular in shape and 2 to 3 mm in size. The ridge is semi-lenticular or convex in the center. The structure is homogeneous, the surface is smooth, and the periphery is completely green. Milky yellow or milky white, glossy and opaque. The hardness is viscous. Methanol-containing agar plate culture: Vigorous growth after 3 days of culture at 37°C. The colony has a circular shape and a size of 2 to 2.5 mm. The ridge is semi-lenticular or convex in the center. The surface is smooth and the periphery is complete.
White or milky, glossy and opaque. The hardness is viscous. Broth agar slant culture: Uniform growth after 3 days of culture at 37°C. The ridges are moderate, the surface is smooth, milky yellow or milky white, glossy and opaque. The hardness is viscous. Methanol agar slant culture: Grows uniformly along the inoculation line after 3 days of culture at 37°C. The bump is moderate;
The surface is smooth. White or milky white, glossy and opaque. The hardness is viscous. Broth liquid medium: Grows vigorously and becomes cloudy when cultured at 37℃ for 3 days. There is precipitation, and a white bacterial ring is formed on the surface. Peptone water liquid culture: When cultured at 37℃ for 3 days, it grows vigorously and becomes cloudy. There is precipitation, forming a bacterial ring on the surface. Methanol-containing liquid culture: When cultured at 37℃ for 3 days, it grows vigorously and becomes cloudy. There is precipitation, forming a bacterial ring on the surface. Juice puncture culture: When cultured at 37°C for 3 days, it grows on the surface or to a depth of 2 to 3 mm from the surface. Meat juice gelatin puncture culture: After culturing at 30°C for 5 days, there is growth and precipitation on the surface, and the gelatin is liquefied. Litmus milk: cultured at 37°C. Produces acid and turns red. solidify. (c) The following physiological properties Nitrate reduction: positive Denitrification reaction: positive MR test: negative VP test: positive Indole formation: negative Hydrogen sulfide formation: negative Starch hydrolysis: negative Utilization of citric acid: Koser's Negative in culture medium Inorganic nitrogen sources: Use ammonium salts and nitrates, respectively. Pigment production: No colored pigments are produced in King AB medium. Urease: Positive Oxidase: Positive Catalase: Positive Growth range: Temperature and PH were varied in methanol-containing liquid medium. It grows in the pH range of 5 to 9, but pH 6 to 8 is preferred. It does not grow at PH4 and 10. It grows at a temperature of 5 to 44 degrees Celsius, but 25 to 42 degrees Celsius is suitable. It does not grow at 45℃. Attitude towards oxygen: Aerobic O-F test (according to Hugh Leifson method): Metabolizes glucose oxidatively. (Acid production is weak.) Whether acid and gas are produced from the following sugars:
Using peptone water at a sugar concentration of 1% and a temperature of 37℃
Cultured for 10 days.

【table】

[Table] (14) Glycerin + (weak) -
(15) Starch + (weak) −
Assimilation of sugars: using sugars instead of methanol in a methanol-containing liquid medium,
The cells were cultured for 10 days at a sugar concentration of 1% and a temperature of 37°C. (1) L-arabinose + (weak) (2) D-xylose + (weak) (3) D-glucose + (weak) (4) D-mannose + (weak) (5) D-fructose + (weak) (6) D-galactose + (weak) (7) Maltose + (weak) (8) Sucrose + (weak) (9) Lactose + (weak) (10) Trehalose + (weak) (11) D-sorbitol − (12) D-Mannite + (weak) (13) Inosite + (14) Glycerin + (weak) (15) Starch + (weak) (d) Isolation source: soil In addition, methanol-containing agar medium and The methanol-containing liquid medium was as follows. (1) Methanol-containing agar medium KH ₂ PO ₄ 1.5g, Na ₂ HPO ₄ 3.2g,
(NH ₄ ) ₂ SO ₄ 3g, MgSO ₄・7H ₂ O 0.5g,
Cacl ₂・2H ₂ O 0.1g, FeSO ₄・7H ₂ O 0.01
g, ZnSO ₄・7H ₂ O 1.4 mg, CuSO ₄・5H ₂ O
0.25mg, Na ₂ M ₀ O ₄・2H ₂ O 0.24mg, C ₀ cl ₂・
6H ₂ O 0.24mg, MnSO ₄・5H ₂ O 1.2mg, agar
After sterilizing 20g of distilled water at 120℃ for 15 minutes, 20g of methanol was added aseptically. (2) Methanol-containing liquid medium: 20g of agar in a methanol-containing agar medium.
without using methanol and with 5g of methanol. The experimental method is described in the Verge Ace Manual below, "Bacteriology Practice Summary" edited by the Institute of Medical Science Alumni Association.
(revised edition 1975) and “Classification and Fixation of Microorganisms” (1975) edited by Takeji Hasegawa. The above-mentioned mycological properties are described in "Bergey's Manual of Determinative Bacteriology".
Bacteriology) 8th edition (1974), they are bacilli, gram-negative, grow in broth, aerobic, do not form spores, and produce acid from glucose. It is most similar to the genus Flavobacterium because it produces , lacks motility, and does not ferment lactose. The difference from Flavobacterium lies in that the colonies of this bacterium are not yellow but milky yellow or milky white. However, if it is assumed that it is a genus other than Flavobacterium due to differences in the colonies, the Key
If you refer to the section to Genera of Manual,
The properties do not match any of the genera listed in Section -101, 102, and 103. Therefore, this bacterium was determined to be a bacterium of the genus Flavobacterium. When comparing this bacterium with the 12 main species of the genus Flavobacterium described in the above-mentioned Verge Ace manual in terms of the properties listed in the table in the same book, Flavobacterium
Flavobacterium
ferrugineum) and Flavobacterium rigense. However, when comparing this bacterium and these bacteria in more detail, including the properties described in the text of the same manual,
This bacterium differs from Flavobacterium faluginium in terms of starch hydrolysis and growth on meat juice agar, and from Flavobacterium ligens in terms of motility, growth in litmus milk, and attitude toward oxygen. . Moreover, there is no other known literature that has properties similar to this bacterium. Therefore, this bacterium was determined to be a new bacterial species of the genus Flavobacterium. 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 44°C, preferably 25 to 42°C, and the pH is 5 to 44°C.
-9, preferably 6-8. Culture may be batch culture or continuous culture. Methanol is used as the main carbon source in the culture medium, and the amount of methanol is preferably 50 g/or less. As nitrogen sources, inorganic substances such as ammonium salts and nitrates, and organic substances such as urea, casein,
Cornstap liquor, peptone, yeast extract, meat extract, etc., as a phosphorus source, phosphates, etc.
Sulfate and the like are used as the sulfur source. Also magnesium, potassium, calcium, sodium, iron, manganese, copper, zinc, molybdenum,
Appropriate amounts of metal salts of cobalt, boron, and other metal sources are added, and, if necessary, substances or growth-promoting substances such as vitamins and amino acids that are essential for the growth of bacterial cells 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, vitamins, and amino acids. Adjustment of the pH of the medium is conveniently done by phosphate and alumia. To collect bacterial cells from a culture, conventional methods such as filtration and centrifugation may be performed, and washing and drying may also be performed. 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 Flavobacterium tosaensis 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 KH ₂ PO ₄ 1.5g Na ₂ HPO ₄ 3.2g (NH ₄ ) ₂ SO ₄ 3g MgSO ₄・7H ₂ O 0.5g CaCl ₂・2H ₂ O 0.1g FeSO ₄・7H ₂ O 0.02g ZnSO ₄・7H ₂ O 2.8mg CuSO ₄・5H ₂ O 0.5mg Na ₂ M ₀ O ₄・2H ₂ O 0.48mg Cocl ₂・6H ₂ O 0.48mg MnSO ₄・5H ₂ O 2.4mg Distilled water 1 Consists of each of the above components Volume of 500ml medium is 1
After sterilization at 120°C for 15 minutes, 5 g of methanol was added aseptically. Flavobacterium tosaensis was precultured at 37°C for 24 hours in a medium with the same composition as above.
A preculture solution containing DS-1 cells was inoculated at 2% by volume, and cultured at 37°C for 20 hours under aeration and stirring conditions while maintaining the pH at 7.0 with 13% by weight ammonia water. The culture was centrifuged to separate the bacterial cells, further washed with water, and then dried at 100° C. for 24 hours to obtain 4.5 g of dried bacterial cells per culture. The generation change time in the logarithmic growth phase of this culture was 1.4 hours. In addition, the crude protein content of bacterial cells is
It was 80% by weight. Example 2 Culture medium 1.2 with the same composition as in Example 1 was placed in a jar fermenter with a capacity of 2.5, and heated at 120°C.
After sterilizing for 20 minutes, add 12g of methanol aseptically.
This was incubated at 37°C with a medium having the same composition as in Example 1.
Flavobacterium obtained by pre-culture for 24 hours
The preculture solution containing Tosaensis DS-1 cells was added to
Inoculate by volume% and adjust pH to 7.0 with 13% by weight ammonia water.
Culture was carried out at 37°C under aeration and agitation conditions. The bacterial cell concentration after 20 hours from the start of culture was 4.5g/
It was hot. The following metal salt aqueous solutions were added per culture. 18ml of 10wt% KH ₂ PO ₄ aqueous solution, 5wt%
77 ml of Na ₂ HPo ₄ aqueous solution, 18 ml of 10 wt% MgSO ₄ 7H ₂ O aqueous solution, 3.5 ml of 10 wt % Cacl _{2 2} H ₂ O aqueous solution,
5 ml of 1 wt% FeSO ₄ 7H ₂ O aqueous solution, 1 wt%
_ZnSO4・_7H2O aqueous solution 4ml, 1wt% _CuSO4・
0.7 ml of 5H ₂ O aqueous solution, 1% by weight MnSO ₄ 5H ₂ O aqueous solution
1.1ml, 0.1% by weight _H3BO3 aqueous solution _2ml , 0.1% by weight
0.5 ml _{of CoCl 2.6H 2 O} aqueous solution, and 20 hours after the start of culture, the concentration in the methanol culture was 1 to 5 g/
was automatically added to maintain the 29 years since the start of culture
The amount of methanol added after 96 hours per culture
reached g. 29 hours after the start of culture, the culture was centrifuged to separate the bacterial cells, and after further washing with water,
After drying at 100°C for 24 hours, dried bacterial cells were obtained at a ratio of 32 g per culture. The crude protein content of this bacterial cell was 77% by weight.

Claims (1)

[Scope of Claims] 1. A method for producing microbial cells, which comprises culturing bacteria belonging to Flavobacterium tosaensis in a medium containing methanol as a main carbon source, and collecting cells from the culture. 2 Bacteria belonging to Flavobacterium tosaensis are Flavobacterium tosaensis DS.
-1, the method for producing microorganism cells according to claim 1.