JPS5920348B2 - Method for producing bacterial cell culture - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing bacterial cells and cultures by culturing bacteria in a medium containing methanol as the main carbon source.

More specifically, it is characterized by culturing bacteria belonging to the genus Methylomonas and capable of assimilating methanol in a medium containing methanol as the main carbon source, and separating and collecting the resulting bacterial culture. The purpose is to industrially produce bacterial cultures, especially bacterial proteins, which have high utility value as food and feed, using methanol, which is inexpensive and available in large quantities, as a raw material. It is advantageous to manufacture this product.

In recent years, attempts have been made to use microbial cultures using petroleum, especially normal paraffin, as the main carbon source as feed, but normal paraffin is insoluble in water, and compared to microbial culture methods using sugars as the carbon source, requires large amounts of oxygen,
In addition, there are various problems such as toxicity due to impurities mixed into the produced culture.

The inventors of the present invention selected methanol as a carbon source that can be obtained cheaply and in large quantities and can solve the above problems, and as a result of various studies, they have found that a medium using methanol as a carbon source exhibits extremely good growth. The present invention has been completed, in which the methanol-assimilating bacteria shown below are isolated from activated sludge of a sewage treatment plant, and the bacteria are used to produce a bacterial cell culture.

The methanol-assimilating bacterium isolated by the present inventors is a new bacterial species belonging to the genus Methylomonas, and the strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology (hereinafter referred to as the Institute of Microbiology). It has been deposited as No. 3468.

Its mycological properties are as follows.

1. Mycological properties ■-1 Morphological properties O1% methanol inorganic salts, cultured on agar slant at 30°C for 24 hours, 0.4-0.5X1. Single or double-chain bacilli of 0 to 1.5 microns, motile by polar flagella, negative for Durham staining, non-sporulating, forming capsule.

0 Bacilli, singly or in chains of several to ten or more bacilli, cultured for 24 hours at 37°C in the same medium as above.

1-2 Cultural properties 01 timetanol inorganic salt agar colony, cultured at 37°C for 24 hours, good growth, round, smooth, gold edge, raised, translucent,
White with a slight yellowish tinge.

OI% methanol inorganic salt agar slant culture at 37°C for 24 hours,
Good growth, filamentous, shiny, and sticky.

O1% methanol inorganic salt broth, cultured for 124 hours at 37°C, film-like, slightly cloudy, small amount of sediment, meat juice agar slant, no growth, meat juice, no growth, gelatin puncture culture, no growth, no liquefaction, 1% methanol gelatin Puncture culture, good growth on the top surface, filamentous liquefied Zezu medium General requirements C: Only methanol, preferably 3% or less (growth is very slow at 3% or more) N: Cannot use anything other than NH4 salt.

(NO3 salt grows very slowly and does not grow in urea) Other minerals, vitamin Bl (Other vitamins are not required) Culture conditions 20-40℃, preferably 30-37℃ (It will die at 42℃ or higher, At temperatures below 30°C, it simply grows, but does not proliferate) pH 6.0 to 7.5
, preferably 6.3 to 7.2, more preferably 6.7
~7.0 1-3 Physiological properties (1) Nitrate reduction None 2) Denitrification reaction None 3) MR test (contains methanol) Negative (4) VP test (contains methanol) Negative (5) Indole generation of
Not produced (6) Hydrogen sulfide generation Not produced (Ca) Hydrolysis of starch Negative (8) Use of citric acid Not used (9) Inorganic nitrogen source NO3 <Nitrate, which is used together with NH4, is weaker (10) Pigment production (methanol medium) No water-soluble pigment, bacterial cell pigment, yellow-red (11) Urease negative (12)
Oxidase positive (13) Catalase positive (14) Growth range (pH, temperature) 5.6, 7, 8, 9, 10, 11 pH - 10 + 1 - 1 - 1 -) - + Fine soil 10, 20, 30 , 37, 4
2 °G hygrothermist ox kan kanshi 05) Attitude towards oxygen obligate aerobic (16
) Utilization of carbon source I L-arabinose (-) 2 D-xylose (-) 3 D-gelkol (=) 4 D-mannose (-) 5 D-fructose (-) 6 D-galactose (-) 7 Maltose ( =) 8 Sucrose (-) 9 Lactose (-) 10 Trehalose (-) 11 D-Sorvit (-) 12 D
-Mannit (-)13 Innojit
(-) 14 Glycerin (-) 15 Starch (-) 16 Rhamnose (-) 17 Acetic acid (-Na) 0 18 Formic acid (-Na) 19 Ethanol (-) 20 Methylamine ■ 21 Mekun (-) 22 Formalin (- ) αD Auxotrophic Vitamin B1 The composition of the above-mentioned methanol inorganic salt medium is as follows.

4 g of disodium phosphate dodecahydrate 1 2 g of potassium phosphate,
Diammonium phosphate 3g, magnesium sulfate heptahydrate 0.
Fll, calcium chloride dihydrate 50 ~, ferric chloride hexahydrate 5 mtg, manganese sulfate 4-6 hydrate 2 my, thiamine hydrochloride 100 γ, tap water 1 t.

pP 6.8 to 7.0, methanol 10 ml For carbon source assimilation, add 1.5% agar and 1% carbon source to be tested (formalin 0.05%) to the above medium, and incubate at 30°C for 14 hours.
Cultured for 1 day.

Regarding the growth range, methanol inorganic salt agar medium was used.

Regarding the reducibility of nitric acid, a methanol inorganic salt medium was used in which 3 g of nitric acid sorter was added instead of diammonium chloride.

(Other exams are edited by Yotake Hase and published by the University of Tokyo Press (197
5) "Classification and Identification of Microorganisms", pp. 201-245.

) Purge the above properties (Bergey) Manual of Determinative Bacteriology No. 8
As a result of a comparative study of known bacterial groups according to the classification criteria of this edition, it is thought that it belongs to the genus Methylomonas, but there are no descriptions of bacterial species similar to this bacterium in the genus Methylomonas, and therefore it is considered a new bacterial species belonging to the genus Methylomonas. Hypothetically, Methylomonas 5D-
It was named 11.

Comparing Methylomonas 5D-11 used in the present invention with Methylomonas metanica and Methylomonas methanonitrihuicannu, which belong to the genus Methylomonas, and which are described in the 8th edition of Burgee's Manual of Determinative Bacteriology. The table below shows the remarkable differences in their properties.

The following experimental method was used for the above classification.

Methanol inorganic salt medium: 4g of disodium phosphate decahydrate
, 1 potassium phosphate 1, diammonium phosphate 3y1 magnesium sulfate heptahydrate 0.5. ! 7. Calcium chloride dihydrate 50m7, ferric chloride hexahydrate 5"2?, manganese sulfate 4
~ 2 m7 of hexahydrate salt, 100 g of thiamine hydrochloride, 1 tap water
7 pH 6.8 to 7.0, methanol LO ml, assimilation of carbon source: 1.5% agar and 1 part of the test carbon source were added to the above medium excluding methanol and cultured at 30°C for 7 days.

The medium used in the present invention can be either a synthetic medium or a natural medium as long as it contains methanol as a main carbon source, a nitrogen source, inorganic substances, vitamins, and other growth promoting substances. Here are a few requirements for a more preferable culture medium when carrying out this process.
% or more, growth is extremely slow, and at 6%, growth is completely inhibited.

Furthermore, it is not preferable to use anything other than ammonium salt as a nitrogen source.

For example, the bacteria used here have very different properties from conventional methanol-assimilating bacteria, such as very slow growth when nitrates are used, and almost no growth when urea is used. It can be seen that

Furthermore, the bacteria used in the method of the present invention contain vitamin B1.
It has clear requirements and does not require other vitamins.

However, even if vitamins other than B1 are mixed, there is no problem with separate preparation.

Next, as the culture conditions when carrying out the present invention, the general culture conditions previously described in the section on mycological properties are used.

After culturing under the medium and culture conditions as described above, the bacterial cell culture in the culture solution is collected by the usual centrifugation method, filtration method, agglutination method, etc. alone or in combination. be done.

The obtained bacterial cell culture has extremely high protein and vitamin contents, so it can be used as a feed ingredient.
High quality feed with high nutritional value can be provided at low cost.

It is also conceivable to extract and separate protein nucleic acids, vitamins, etc. from the obtained bacterial cell culture by known means and use them as additives for feeds, foods, etc., and medicines.

Hereinafter, the method of the present invention will be explained in more detail with reference to Examples, but these are merely illustrative and do not limit the present invention in any way.

Example l Ammonium sulfate 2L Diammonium phosphate 3g 1 Phosphoric acid 2
Sodium dodecahydrate 2. i, 1 potassium phosphate 1 g 1 magnesium sulfate heptahydrate 0.2 g, calcium chloride 0.01
．． Ferric chloride hexahydrate 0.0211 Manganese sulfate 4-6 hydrate 2m7, cobalt chloride, zinc sulfate, sulfuric acid preparation 0.57
72＠ was dissolved in distilled water and poured, and 2 was placed in a jar fermenter and sterilized.

To this was added 0.8% w/v of filtered methanol and inoculated with Methylomonas 5I)-11, followed by culturing while maintaining the pH at 6.7 using 3N aqueous ammonia.

The stirring at this time was 700 rI) and the aeration rate was 21/min.

After 7 hours of culture, 0.8% methanol was added and culture was continued for a further 6 hours.

After culturing, the bacterial cells were collected by centrifugation, washed once with water, and dried at 105°C until a constant weight was obtained, yielding 6.79 bacterial cells per 1 culture solution.

The yield based on added methanol was 42%.

Moreover, the maximum specific growth rate (μmax) was 0.6.

The crude protein content of the bacterial cells was 74%.

Example 2 Methylomonanu 5D-11 was cultured under the same conditions as in Example 1, and methanol in the medium was measured by gas chromatography. When the methanol concentration became 1Qppm or less, it was 0.4%.
W/v methanol was added.

The bacterial cell concentration (x) reaches 24 El/l 18 hours after culturing.

From this point on, 5 doses of methanol and the same medium as in Example 1 were continuously injected into the culture solution at a dilution rate (■) of 0.25, and the same amount of culture solution was withdrawn from the culture tank to perform continuous culture.

The productivity DX at this time was 5.75, and the yield based on the consumed methanol was 52%.

The crude protein content of the obtained bacterial cells was 765%, and the nucleic acid content was 8.3%.

Crude fat content 7.2% Amino acid composition [Amino acid composition of crude protein content (76.5%)] (All weight%)

Claims (1)

[Claims] 1. Methylomonas 5, a bacterium belonging to the genus Methylomonas and capable of assimilating methanol, in a medium containing methanol as the main carbon source.
A method for producing a bacterial cell culture, which comprises culturing D-ii and separating and collecting a proliferating bacterial cell culture.