JPS5818067B2 - Kobokintaino Seizouhouhou - Google Patents

Description

Detailed Description of the Invention The present invention involves culturing Torulopsis S-189, a novel yeast that belongs to the genus Torulopsis and has the ability to assimilate methanol, in a medium containing methanol as the main carbon source, and extracting yeast cells from the culture solution. The present invention relates to a method for producing yeast cells, which comprises collecting yeast cells.

Conventionally, as raw materials for producing yeast cells, carbon sources have mainly relied on molasses, sulfite waste liquid, and the like.

However, there are problems in terms of supply amount and price for these substances to be used as industrial raw materials.

The present inventors searched widely in nature for microorganisms that can strongly assimilate methanol, which can be obtained in large quantities and at low cost through the organic synthetic chemical industry, as a main carbon source, and found a unique yeast capable of assimilating methanol. was completed.

This yeast is a very specific yeast that can grow using methanol as the only carbon source, and its mycological properties indicate that it belongs to the genus Torulopsis, but it differs from known species belonging to this genus in various ways. Therefore, it was determined that it was a new bacterial species, Torulopsis S-
The present invention was completed by using a new type of yeast named Ko(7) 189.

That is, the present invention is a method for producing yeast cells, which comprises culturing Torulopsis S-189 in a medium containing methanol as the main carbon source, and collecting yeast boxes from the culture solution.

Next, this methanol-assimilating yeast Torulopsis (Toru)
lops is) S-189 (Microtechnical Laboratory No. 3116
) shows the mycological properties of

(1) Growth status in each medium (a) MY liquid medium: Cultured at 26°C for 23 to 7 days.

The cells are (1,7~2.0) x (1,7X2.0
)micron.

Round or oval, singly or in pairs.

Yeast particles form slightly but do not form a film.

It does not form meristems and grows by multipolar budding method.

(b) MY agar medium: When cultured at 25° C. for 5 days, the colonies are grayish white, the ridges are semi-lenticular, the surface is rough, and the periphery is round or wavy.

(c) Slide culture on corn extract agar medium: Cultivated at 25°C, without forming hyphae or pseudohyphae.

(d) Methanol-containing agar medium: When cultured at 35°C for 3 days, the colonies are white, the ridges are semi-lenticular, the surface is rough, and the periphery is wavy.

(2) Formation of ascospores: Gorodkow medium: No ascospore formation was observed in any of the sodium acetate medium, malt extract agar medium, vegetable juice agar medium, carrot piece medium, etc.

(3) Formation of extruded spores No extruded spore formation was observed in the two-MY agar flat medium.

(4) Physiological properties (a) Optimal growth conditions Grows well at 20° to 35°C.

Good growth was observed at pH 3.5 to 6.0.

(b) Range of growth Grows at 7-38°C.

However, when the temperature exceeds 38°C, growth becomes poor.

Growth is poor when the pH is below 3.0 or above 8.0.

(c) Nitrate assimilability: Yes (d) Fat degradability: None e) Degradability of urea: None f) Liquefiability of gelatin: None g) Salt tolerance: None h) Carotenoid production: None i) Significant organic acid production: None j) Formation of starch-like substances: None k) Vitamin requirement: Requires biotin.

(1) Methanol assimilation Grows well using dimethane as the sole carbon source.

(m) Production of esters: None 5) Fermentability of sugars D-glucose + D-galactose - sucrose + maltose - lactose - raffinose + trehalose + melibiose - cellobiose - (6) Assimilation of various carbon sources D -arabinose + L-arabinose + D-ribose + D-xylose + D-glucose + D-mannose + D-galactose + (weak) L-rhamnose + D-fructose + L-sorbose + maltose ゛ - sucrose + lactose - Melibiose - Cellobiose - Trehalose + Raffinose + Melezitose - α-Methyl-D-glucoside - Arbutin - ・Dextrin Single-soluble starch - Inulin + Ethanol + Atonit + Erythrit + Inosit - D-Mannit + D-Sorbit + Dulcit - D -Gluconic acid + glycerin + 2-keto-logluconic acid 10DL-lactic acid + (weak) succinic acid
- Citric acid + (weak) Separation source: soil The yeast according to the present invention does not form ascospores, extrusions, or fisspores, the vegetative cells are round or oval, and multiply by multipolar budding method, and hyphae. and not forming pseudohyphae,
and Lodar (I, od
der) Author: [The East Ataxonomics Study (
The Yeasts, ATaxonomic 5tu
dy 1970) J, it is considered to belong to the genus Torulopsis.

Comparing the physiological properties of this yeast with the known strains described by Mr. Loder, it is found that it assimilates nitrate, assimilates D-glucose, sucrose, D-mannite, and D-sorbitol, and utilizes maltose and lactose. Torulopsis magnoliae (Torulopsis magnoliae)
1ae).

However, this strain and Torulopsis magnoliae have the ability to ferment raffinose and trehalose, trehalose, D-xylose, L-rhamnose, L-arabinose, D-arabinose, erythritol, attonite,
The assimilation properties of DL-lactic acid are different.

From the above points, it does not match the known yeast described in Mr. Loder's book.

Furthermore, the methanol assimilation ability of this strain did not match any known yeast, and it was concluded that it belonged to a new bacterial species.

The experimental method followed the above-mentioned book by Mr. Roder and ``Classification and Identification Method of Yeast'' by Shoji Iizuka Hirogoto.

The methanol-containing agar medium used had the following composition.

That is, (NH4)280,2.9゜KH2PO40
,5g, M, @5O47H200,5g.

NaC1O,05,9, yeast extract 0.1g, biotin 10μg1 thiamine hydrochloride 400μg1 agar 20g1
The mixture was made of 8 g of methanol and 11 g of water, the pH was adjusted to 5.5, and the mixture was sterilized with IKg/G1'It for 10 minutes.

In addition, for the methanol assimilation test, a liquid medium obtained by removing agar from the above medium was used.

As the medium used for culturing this yeast, either a synthetic medium or a natural medium can be used as long as it contains methanol as the main carbon source, a nitrogen source, inorganic substances, vitamins, and other growth-promoting substances in appropriate amounts.

This yeast can grow even if the methanol concentration in the medium is 5% by weight, but in order to reduce the evaporation loss of methanol due to aeration, the initial concentration of methanol in the medium is kept as low as possible and methanol is added in accordance with methanol consumption. However, it is preferable to adopt a culture method that maintains a low concentration of methanol in the culture solution. Nitrogen sources for this yeast include inorganic nitrogen compounds such as ammonium salts and nitrates, urea, corn stapley, yeast extract, peptone, etc. organic nitrogen compounds are used.

In addition, when using ammonium salt as a nitrogen source, the pH of the culture solution decreases when ammonium ions are consumed for bacterial growth, so in order to maintain a suitable pH, add ammonia, caustic soda, etc. to caustic soda. need to be added.

In addition, other inorganic salts such as magnesium salts, phosphates, potassium salts, calcium salts, and iron salts, as well as substances essential for growth or growth-promoting substances such as vitamins and amino acids, may be added to the medium of this yeast. 710 is also preferable.

During culturing, the temperature is kept at 20-38°C, preferably 2.
9-35°C and pH 3.0-8.0, preferably 3.
．． 0 to 6.0 and perform aerobic culture.

In general, the temperature at which methanol assimilating yeast can grow is 3.
The temperature is 0 to 33℃, but this yeast is grown at a higher temperature (35℃).
Since good culture can be achieved, the cost of removing heat generated during culture is low, and the present invention has high practical value.

Further, the culture method may be either batch culture or continuous culture.

In order to collect yeast cells from the culture solution, filtration, centrifugation, etc. may be performed.

Clean if necessary.

The present invention will be explained below with reference to Examples.

Example 1 i (NH4)2SO42g, KH2PO40.5g
.

MgSO47H2O0.5g, NaCl o,o
5 g, yeast extract 0.1. ? 1 biotin 10μg 1 thiamine hydrochloride 400t4. Water 17. 100ml of a medium consisting of pH 4.5 was placed in a 5007IL Meyer flask at a rate of 1 kg/cyit.

After 10 minutes of heating, methanol was added to 1 (V/V)%, and Torulopsis S-189 (
FERM P/163116) and culture temperature 3.
Shaking culture was performed at 5°C.

After culturing for 36 hours, the culture solution was centrifuged to separate the bacterial cells.

Vacuum drying was performed at 50° C. for 4 hours to obtain 2.6 g of bacterial cells per 11 culture fluids.

Example 2 (NH')2SO410g, KH,, PO42,5,
? .

Mg5047H202,5g, NaClO,25j
! , Yeast: Kiss 0.1g, Biotin 10μg, Thiamine Hydrochloride 400μg, Water 11. Put 15A of medium consisting of pH 4.5 into a 31-volume jar fermentor, and add ]kg/
ffl for 20 minutes, and 75.9 g of methanol was added thereto to prepare a medium.

A culture solution of Torulopsis S-189, which had been previously cultured for 36 hours in a medium with the same composition, was inoculated at a concentration of 2% by volume, and aerated culture was performed at 35°C.

The pH was automatically maintained at 4.5 with aqueous ammonia, and the methanol concentration in the culture solution was adjusted to 0.2 to 0.5 (V
The cells were cultured while sequentially adding □ so that the concentration of the cells was □/V)%°.

After 40 hours of culture, the amount of methanol added was 8.5 (V/
V)% was reached.

Culture was stopped and bacteria were collected by centrifugation.

This was freeze-dried to obtain 21 bacterial cells per 11 culture fluids.

Claims (1)

[Claims] 1. A method for producing yeast cells, which comprises culturing Torulopsis S-189 in a medium containing methanol as the main carbon source, and collecting yeast cells from the culture solution.