JPS5817587B2 - Kobo Kintaino Seizou Hohou - Google Patents

Description

[Detailed description of the invention] The present invention is directed to a culture medium containing methanol as the main carbon source.

The present invention relates to a method for producing yeast cells, which comprises culturing Torulopsis R-14, a novel yeast that belongs to the genus Torulopsis and has the ability to assimilate methanol, and collecting yeast cells from the culture solution.

Traditionally, carbon has been used as a raw material for producing yeast cells.

The sources have mainly relied on molasses, sulfite pulp waste liquid, etc.

However, these substances have problems in terms of supply amount and price for use 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 discovered a unique yeast that has the ability to assimilate methanol. I was able to get it.

This yeast is a very unique 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 is different from known species belonging to the genus Torulopsis in terms of species. Because of the differences, it was concluded that it was a new bacterial species and it was classified as Torulopsis.
s) The present invention was completed by using this new type of yeast, named R-14.

That is, the present invention is a method for producing yeast cells, which comprises overtones of Torulopsis R-14 in a medium containing methanol as the main carbon source, and collecting yeast cells from the culture solution.

Next, this methanol-assimilating yeast Torulopsis (Tor)
ulopsis) R-14 (Feikoken Bacillus No. 3114
The mycological properties of FERM-PA3114) are shown.

(a) Growth status in each medium■ ■Liquid medium: 26℃, cultured for 3 to 7 days, cells (1,
2-1.5) x (1.7-2.0) microns.

Round or oval, singly or in pairs.

A few yeast rings form, but no capsule.

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

■ MY agar medium: When cultured at 25°C for 5 days, the colonies were pale yellowish white, the ridges were semi-lenticular, and the surface was smooth.
The periphery is rounded or wavy.

■ Slide culture on corn extract agar medium:
Culture at 25°C.

Does not form hyphae or pseudohyphae.

■ Methanol-containing agar medium: When cultured at 35℃ for 3 days,
The colony is pale yellow in color, with semi-lenticular ridges, smooth surface, and wavy edges.

(b) Formation of ascospores: Gorodkow medium, acetic acid solution
No ascospore formation was observed in any of the Da medium, malt extract agar medium, vegetable juice agar medium, and carrot piece medium.

(c) Formation of extruded spores: No extruded spore formation was observed on the MY agar flat medium.

(d) Physiological properties ■ Optimal growth conditions Grows well at 20-35°C.

Good growth was observed at pH 3.0 to 6.0.

■ Growth range Growth at 38°C is poor.

Growth is poor when the pH is below 2.5 or above 8.0.

It was killed by heating at 60°C for 30 minutes.

■ Nitrate assimilability: Yes ■ Fat degradability: None ■ Degradability of urea: None ■ Liquefiability of gelatin: None ■ Salt tolerance: 4-5 ■ Carotenoid production: none, ■ Significant organic acid formation: None [Phase] Formation of starch-like substances: None 0 Vitamin requirement: Requires biotin.

@ Methanol assimilation ability: Grows well with methanol as the only carbon source.

0 Production of esters: None e) Fermentability of sugars: None f) Fermentation of various carbon sources D-arabinose + L-arabinose + D-ribose + D-xylose + D-glucose + D-mannose + D-galactose - L-rhamnose + D-fructose + L-sorbose + maltose - sucrose - lactose - melipiose - cellobiose + trehalose - raffinose - melezitose - α-methyl-D-glucoside - arbutin + soluble starch - dextrin - inulin - ethanol + Atonite + Erythrite + Inosit - D-Mannit + D-Sorbit + Dulcit - D-Gluconic acid Decoglycerin + 2-Keto D-Gluconic acid - DL-Lactic acid - Succinic acid - Citric acid ~ Isolation source: Soil The yeast according to the present invention does not form ascospores, extrusions, or fisspores, the vegetative cells are circular or oval, and multiply by a multipolar budding method, and do not form hyphae or pseudohyphae;
Lodd
er) Author [-The East A Taxonomy]
Study (The Yeasts, A Taxonom
ic 5tudy 1970), 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 and cellobiose, and does not assimilate sucrose, maltose, lactose, or D-galactose. This approximates Torulopsis norvegica.

However, this strain and Torulopsis norvegica are
Cell size, L-sorbose, D-arabino-[nu,
The assimilation properties of D-ribose, erythritol, attonite, DL-lactic acid, succinic acid, and citric acid are different.

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

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

The experimental method was in accordance with the above-mentioned book by Mr. Roder and ``Classification and Identification Method of Yeast'' by Toko Iizuka and Shoji Goto.

Moreover, as a methanol-containing agar medium, one having the following composition was used.

That is, (NI-L) 2 SO+27, KH2P
040.5 '111Mg 80+・7) (2Q0.
5? , NaC1O,05f! , yeast extract 0.17, biotin 10μg, thiamine hydrochloride 400μ7, agar 20
7, 87 methanol, and 1 t of water, the pH was adjusted to 5.5, and the mixture was sterilized at 1 kf/ctrl for 10 minutes.

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

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

This yeast can grow even when the methanol concentration in the medium is 6% 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 the methanol is adjusted to match the consumption of methanol. It is preferable to adopt a culture method in which methanol is added to the culture solution to maintain a low concentration of methanol in the culture solution.

As nitrogen sources for this yeast, inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen compounds such as urea, cornstap liquor, yeast extract, and peptone are used.

Also, when using ammonium salts as a nitrogen source,
When ammonia and tum ions are consumed due to bacterial cell growth, the pH of the culture solution decreases, so it is necessary to add ammonia, caustic potash, caustic soda, etc. to maintain a suitable pH.

1. In the medium of this yeast, inorganic salts such as magnesium salts, phosphates, potassium salts, calcium salts, iron salts, and as necessary substances essential for growth such as vitamins and amino acids, etc. It is also preferred to add promoter substances.

For culturing, the temperature should be kept at 20-38°C, preferably 1 and 2.
9-35°C, pH 25-8.0, preferably 30
~6.0 and perform aerobic culture.

In general, the temperature at which methanol-valorizing yeast can grow is:
Although the temperature is 30 to 35°C, this yeast can be cultured well at a higher temperature (35°C), so the cost of removing the heat generated during culture is small, and the practical value of the present invention is high.

The culture method may be either batch culture or continuous culture.

To collect yeast cells from the culture solution, filtration, centrifugation, etc. may be performed.

If necessary, wash.

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

Example 1 (NH,)2so, 2f, KH2PO20,5? ,
Mg5O, 7H200,5F, Na1l O,05?
, 0.1 g of yeast extract, 10 μg of biotin, 400 μg of thiamine hydrochloride, 100 me of a medium consisting of LA water, pH 4.5, was placed in a 500 ml Meyer flask, sterilized at 1 kg/cm for 10 minutes, and then methanol was poured into the flask. 1 (V/V)%, Torulopsis R-14 (FERM-PA, 31.14) was inoculated thereto, and cultured with shaking at a culture temperature of 35°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 327 bacterial cells per 11 cells.

Example 2 (NH2)2 So 410 L? , KH2PC)42
．． 5? , Iden 5047H202, 5f, NaC1O
,25P, Fe50+ ・7H200,01g, yeast extract 0.11, biotin 10μ7, thiamine hydrochloride 40
0 μg, 1 t of water, and 1st medium consisting of pH 4.5 was placed in a 301-volume jar fermenter, and 1 kg/cr was added.
Sterilize with A for 20 minutes and add methanol at 0.5 (V/
V) %.

To this medium was added Torulopsis R-14, which had been previously cultured in a Mayer flask for 36 hours in a medium with the same composition.
(FERM PA, 3], 14) culture solution was
The cells were inoculated in volume and aerated culture was carried out at 35°C.

The pH was automatically maintained at 45 using ammonia water, and the methanol concentration in the culture solution was 0.2-0.5 (
V/V)%.

After 45 hours of culture, the amount of methanol added was 15 (V/V
) reached %.

Culture was stopped, and bacteria were collected by far JI and % separation.

This was freeze-dried to obtain 30 g of bacterial cells per 1 ton of culture solution.

Claims (1)

[Claims] 1. A method for producing yeast cells, which comprises culturing Torulopsis R-14 in a medium containing methanol as the main carbon source, and collecting yeast cells from the culture solution.