JPH0634707B2 - Method for producing yeast cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention uses marine yeast as a fermentation raw material, which is ethanol that can be produced in large quantities from reproducible biomass resources.
The present invention relates to a method for producing yeast cells which accumulate lipids having a high linoleic acid (18: 2) and linolenic acid (18: 3) content in the cells.

More specifically, the present invention relates to a method for producing a yeast cell containing a lipid having a high linoleic acid and linolenic acid content, by culturing a marine yeast strain belonging to the genus Candida and having ethanol-assimilating ability in a medium containing ethanol as a carbon source. .

[Conventional technology]

Conventionally, it is known that microbial protein (SCP) can be used as feed for fish farming and feed for livestock. And these SCPs can be produced from raw materials such as molasses, pulp waste liquor, n-paraffin, methane, methanol and ethanol (Yoshiro Masuda, Development of Microbial Protein, Kodansha, 1978).

It is also known that marine yeast serves as a feed for zooplankton (Rhinoceros rotifer), which is indispensable for fish feed and seed production of marine fish (Japanese Patent Publication No. 60-40808).

Recently, fish have been found to require 18: 2, 18: 3 and ω3 polyunsaturated fatty acids as essential fatty acids, although they differ depending on the fish species as in mammals. For example, coho salmon demand 18: 3, and chum salmon require 18: 2 and 18: 3, whether raised in seawater or freshwater (Jissui, 48,
1745, 1982).

However, conventionally developed baker's yeast, pulp yeast,
When SCP such as petroleum yeast is used as a feed for fish farming, these yeast cells are poorly dispersed in water and the feed efficiency is poor, and even live yeast is high in hypertonic solution with high ion concentration like seawater. It is unstable and causes the active ingredient in the cells to flow out, and also has the drawback of causing contamination of breeding water (Akihiko Shirota, Fisheries Biology, p. 303, Seiseisha Koseikaku, 1
975).

Marine yeasts have been developed in order to solve the above-mentioned drawbacks, but glucose and alcohol yeast-producing liquid (Nissui, 49, 1015, 1983) are used as fermentation raw materials.
In addition, although there are research reports on the ethanol-utilizing ability of marine yeast, ethanol-utilizing ability is only known as a part of the physiological properties of the fungus (Nissui, 49, 1015).
P., 1983).

There have been no attempts to use ethanol, which can be supplied in large quantities, to produce SCP from marine yeast.

Also, research on the production of fats and oils by microorganisms has been conducted for a long time, but there is little research on the production of fats and oils with special uses and functions, for example, fats and oils containing a large amount of linoleic acid, which is an essential fatty acid, The desired result has not been obtained (Oil Chemistry, Vol. 31, p. 431, 1982). A method for producing a yeast cell that forms a lipid having a high linoleic acid content is described in Japanese Examined Patent Publication No. 57-2314, but when ethanol is used as a carbon source, 18: 2 is 11.2 in the fatty acid composition of the neutral lipid. %, 1
8: 3 is 1.3%, that of polar lipids 18: 2 is 13.8%, 18: 3
The content is 1.6%, which is not high, and especially the content of 18: 3 is low. When methanol was used as the carbon source, the 18: 2 content of the fatty acid composition of neutral lipids doubled to 22.8%.
The 8: 3 content is as low as 1.7%. By microorganisms as above 1
Attempts have been made to produce fats and oils containing a large amount of 8: 2, but none of them was satisfactory.

Nothing is known about the production of marine yeast that accumulates lipids with a high linoleic acid (18: 2) and linolenic acid (18: 3) content in cells using ethanol as a fermentation raw material.

[Problems to be Solved by the Invention]

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide linol, which is an essential fatty acid, which is stable in seawater, has good feed efficiency, from ethanol which is supplied in large quantities from biomass resources. It is intended to provide a method for producing a marine yeast that accumulates lipids having a high content of acid (18: 2) and linolenic acid (18: 3) in cells.

The present inventors have conducted extensive studies to achieve this object, and as a result, have ethanol assimilation, grow well in a medium containing ethanol as a carbon source, and develop linoleic acid (18: 2) and linolenic acid (18: 2). : 3) A marine yeast that accumulates a high content of lipids in the microbial cells has been found from seawater in coastal waters, and the present invention has been completed.

[Means for Solving the Problems]

The gist of the present invention is that belonging to the genus Candida, having ethanol assimilation, and capable of forming a lipid having a high linoleic acid and linolenic acid content in the cells, culturing in a medium containing ethanol as a carbon source. And a method for producing a yeast cell containing a lipid having a high linoleic acid and linolenic acid content.

In the present invention, any microorganism can be used as long as it is a marine yeast belonging to the genus Candida, having ethanol assimilation, and forming lipids containing 18: 2 and 18: 3 in the cells. As such a microorganism, for example, the Candida sp. S30W2 strain (FERM P-11023) isolated from coastal seawater by the present inventors can be exemplified.

The mycological properties of this strain S30W2 (FERM P-11023) are as follows.

1. Morphological properties 1) Vegetative cells ^* 1 size (μm) (2-5)
× (3-12) shape oval or sometimes elongated 2) vegetative reproduction ^* 1 Proliferate well by multipolar budding.

3) Pseudohyphae ^* 2 Form pseudohyphae with spores.

4) No meiosis ^* 2 formation.

5) Ascospores ^* 3 No formation.

6) Ejected spores ^* 4 No formation.

7) Teriospore ^* 5 Not formed.

2. Physiological properties 2) Carotenoid pigment ^* 6 is not produced.

3. Others This strain is a yeast that has the ability to assimilate hydrocarbons The colony morphology of this strain is YM and GPY ^* 7 agar plate medium, but the surface is a smooth colony (S-type colony).
The appearance of rough colonies (R-type colonies) was observed in strains with a large number of subcultures on the agar slant medium containing the n-alkane mixture as a carbon source. As a result of separating the R-type colonies and comparing them with the S-type colonies, no significant difference was observed in the cell yield, the cell lipid content and the fatty acid composition of the lipids in the medium containing ethanol as a carbon source.

In addition, the growth temperature when GPY medium of this strain was used
As shown in the figure. The optimum growth temperature was 10 to 20 ℃, but 5 ℃
It grows well even at low temperatures. However, it was a cold-type yeast that could hardly grow above 30 ° C. The test in Fig. 1 is L
Put 10m of GPY medium into the mold test, inoculate yeast S30W2 strain,
Shaking culture was performed. FIG. 1- (A) shows shaking temperature gradient culture equipment, and FIG. 1- (B) shows shaking culture in a constant temperature culture room or constant temperature water tank. ^* 1: YM liquid medium (yeast extract (Difco) 3 g, malt extract (Difco) 3 g, heptone (Difco) 5 g, glucose 10)
g, distilled water 1, pH 5.5), observed at 25 ° C for 3 days, then observed. ^* 2: Cornmeal agar medium (Difco). 25 ° C, 1-1
7 days culture, slide culture method. ^* 3: YM agar (Difco), vegetable juice V-8 agar, G
Using orodkowa agar medium and Acetate agar medium of McClary et al., cultured at 25 ° C for 3 days and then at 20 ° C for 6 weeks and observed. ^* 4: Cultured on potato-glucose agar medium (Difco) for 11 days at 25 ° C and 4 weeks at 20 ° C for observation. Slide glass method. ^* 5: Observed by culturing on corn meal agar medium (Difco) at 10 ° C for 6 weeks. ^* 6: Using malt extract agar medium (Oxoid) at 20 ℃ and 25 ℃
Cultivate for 16 days and observe. ^* 7: GPY medium (glucose 20 g, peptone (Difco) 10
g, yeast extract (Difco) 5 g, distilled water 250 m, aged seawater 750 m, agar 20 g, pH 6.0, pH 5.5 for liquid medium
And The yeasts a taxonomic study (3rd
ed., NJWKreger-van Rij, ed., 1984). As a result, it was confirmed that this strain belongs to the genus Candida. Therefore, the strain Candida sp.S30W2 (Candida sp.S30W2
Ltd.).

As the medium and culture conditions for culturing the microorganism of the present invention, the medium and culture conditions used for culturing ordinary yeast can be used.

The culture medium for culturing the strain used in the present invention, natural seawater or artificial seawater, or seawater obtained by appropriately diluting them, appropriately contains ethanol, a nitrogen source, inorganic salts and vitamins and other growth factors as main carbon sources. A natural medium or a synthetic medium can be used as long as it is a culture medium. In addition, general yeast medium, for example, Wickerham synthetic medium (LJ Wickerham, Taxon
omy of yeasts, Technical Bulletin No.1029, p.6-9, Un
ited States Department of Agriculture, Washington, 1
951) can also be used.

The ethanol concentration of the carbon source is 0.1 to 5.0% (v / v), preferably 0.5 to 2% (v / v). At high concentrations, ethanol inhibits the growth of microorganisms. In addition, ethanol has a low boiling point and may evaporate during culturing. Therefore, it is preferable to maintain a low concentration of ethanol and feed an appropriate amount of ethanol during the culture. However, it is also possible to terminate the culture only with the ethanol added first.

When ethanol is used as a carbon source, other carbon sources that can be assimilated by the yeast strain, such as hydrocarbon and glucose, may be used in combination.

In recent years, ethanol has been able to be mass-produced by a fermentation method from a biomass resource that can be reproduced as an alternative energy to petroleum (fermentation and industry, 39, 490, 1981.
Year; Bioscience and Industry, 47, 844
P., 1989).

Ethanol is easily soluble in water and easy to handle as a fermentation material,
It is also highly safe and has been used as a part of food since ancient times.

As the nitrogen source of the medium, in addition to natural nitrogen sources such as peptone, meat extract, constip liquor, casamino acid, malt extract, defatted soybean, ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate, sodium nitrate, potassium nitrate. Inorganic nitrogen sources such as nitrates,
An organic nitrogen source such as urea can be used. The concentration of nitrogen source in the medium is usually 0.01 to 5.0% (w / v), preferably 0.1.
~ 2.0% (w / v).

Further, if necessary, in addition to seawater components, inorganic salts such as copper sulfate, potassium iodide, iron chloride, sodium molybdate, zinc sulfate and the like are used.

Vitamins, amino acids, nucleic acids and salts thereof can also be used as growth factors. Further, peptone, meat extract, constip liquor, casamino acid, yeast extract and the like containing the above growth factors can also be used as a micronutrient source.

These medium components are not particularly limited as long as they have a concentration that does not inhibit the growth of microorganisms.

The concentration of seawater in the medium is 100% natural seawater or 100% artificial seawater,
Alternatively, they can also be used in appropriately diluted seawater. In addition, as shown in FIG. 2, the culture medium prepared by using diluted seawater was superior to the culture medium prepared with 100% seawater in terms of the bacterial cell yield and lipid content. This strain is a medium for general yeast
The vitamins and amino acids in Wickerham's synthetic medium (medium for carbon compound assimilation test) are replaced with 0.5 g of yeast extract, and the medium also grows in a medium containing 1% (v / v) ethanol as a carbon source.

The culture temperature is 3 to 29 ° C., preferably 5 to 20 ° C.
The pH is 3.5 to 8.0, preferably 4 to 6. The culture of this strain may be shaking culture, aeration-agitation culture or static culture. The culture period varies depending on the temperature, but is usually about 20 hours to 2 weeks.

By culturing as described above, lipids containing 18: 2 and 18: 3 are produced and accumulated in the cells. Collection of cells and lipids from the culture is performed as follows.

After completion of the culture, the culture solution is centrifuged to obtain bacterial cells. Wet cells thoroughly washed with water are mechanically ground in an organic solvent such as chloroform-methanol and stirred to extract lipids from the cells. By removing the organic solvent from this extract under reduced pressure, linoleic acid (1
A lipid containing 8: 2) and linolenic acid (18: 3) is obtained.

The marine yeast obtained by the method of the present invention is particularly useful in the production of fish feed, livestock feed, zooplankton feed and highly functional lipids.

Next, the present invention will be described more specifically by way of examples.

Example 1 Medium 100 excluding ethanol from the basic medium composition in Table 1
m was put in a 500 m Erlenmeyer flask, sterilized by an autoclave, and then 1 m of filter-sterilized ethanol was added. Candida sp. S30W2 strain (FERM P-11023) was inoculated in the above medium and shake-cultured at 20 ° C for 2 days to obtain a preculture liquid. This preculture liquid (1 m) was inoculated into the culture flask and shake-cultured at 20 ° C. for 2 days on a rotary shaker (180 rpm).

The cell yield (dry cell weight DCW) is 15 after 10m of the culture solution.
Transfer to a m-volume sedimentation tube, centrifuge, wash the cells with water,
It was dried overnight at 110 ° C., weighed, and determined as DCW per 101 m of culture solution.

Extraction of lipids from the cells was performed as follows. Depending on the growth of the bacterium, the culture completion liquid 400-1500m (the above flasks 4-15
Cells were collected by centrifugation. Wet cells were washed with water to obtain 3-5 g of wet cells and the method of Itoh et al. (Yukagaku,
23, p. 350, 1974), and extracted by grinding and stirring with glass beads in a mixed solvent of chloroform-methanol. The extracted lipid was dried in a vacuum desiccator and then weighed to determine the lipid amount.
Further, a portion of about 0.6 to 1.0 g of the wet microbial cell for lipid extraction was placed in a weighing bottle and dried at 110 ° C. overnight to obtain the dry microbial cell weight. From the dry cell weight, the ratio of the dry cell amount in the wet cell was determined. From this ratio, the dry bacterial cell amount of the wet bacterial cells for lipid extraction was calculated. The lipid content in the bacterial cells was expressed as the content per dry cell in% by weight.

Analysis of fatty acids in lipids was performed as follows. Above lipid 20
~ 30mg 2 at 80 ° C with 90% methanolic 1N KOH
After alkaline decomposition for 14 hours, 14% BF ₃ methanol complex salt treatment was performed to obtain methyl ester of fatty acid. The composition of the methyl ester of this fatty acid was analyzed by gas chromatography.

Table 2 shows the results of examining the cell yield, lipid content and lipid fatty acid composition. The highest ratio of linoleic acid (18: 2) and linolenic acid (18: 3) in the lipid fatty acid composition after 28 hours of culture was the highest at 38.3%, the latter at 22.4%, and 18: 2 and 1
The 8: 3 ratio accounted for 60.7% of the fatty acid composition. This value is about 3 times higher at 18: 2 and about 20 times at 18: 3 as compared with the results of using ethanol as a carbon source described in JP-B-57-2341.
Characteristically, it is twice as high, especially with a high 18: 3 content. By the second day of culture, the 18: 2 and 18: 3 contents decreased by about 20%.
As shown in Table 2, the high unsaturated fatty acid content seems to indicate the characteristics of cold yeast.

Figure 2 shows the results of examining the effect of seawater concentration in the medium. A medium in which artificial seawater having the medium composition shown in Table 1 was replaced with artificial seawater diluted to various concentrations was used. The lower the seawater concentration, the higher the cell yield and lipid content. Also said Wick
It also grew well in erham's synthetic medium. This yeast was isolated from coastal waters and did not show the characteristics of marine bacteria in terms of salt requirement. Generally, marine yeast
It is said that many of them are salt-tolerant, but do not show salt-requiring requirements like marine bacteria.

Example 2 Using Candida sp. S30W2 strain (FERM P-11023), the effect of culture temperature on the cell yield, cell lipid content and lipid fatty acid composition was examined.

The medium used was a medium in which the artificial seawater having the medium composition shown in Table 1 was diluted to 1/4 and replaced with artificial seawater. Culture at 5 ℃,
The same procedure as in Example 1 was performed at 10 ° C and 20 ° C. Cell yield,
The analysis of the lipid content in the bacterial cells and the fatty acid composition of the lipid was performed in the same manner as in Example 1. The results are shown in Table 3.

Cell yield was highest at 10 ℃, lipid content was 5 ℃ compared to 20 ℃
The tendency was slightly higher on the low temperature side of ℃ and 10 ℃. Of the fatty acid composition in lipids, 18: 2 and 18: 3 were the highest at the beginning of the culture at any culture temperature, decreased significantly when the lipid content became the highest, and showed no significant fluctuations in the stationary phase thereafter. I couldn't see it. The 18: 3 content showed a high value especially at low temperatures.

From the above, by controlling the growth phase and the culture temperature, lipids with high linoleic acid (18: 2) and linolenic acid (18: 3) contents can be obtained.

The highest cell yield at 10 ° C was due to the growth characteristics of the low-temperature yeast and the low efficiency of carbon source utilization with low volatilization of low boiling ethanol. it is conceivable that. Also, when cultured at low temperature, 18: 2,1
The higher unsaturated fatty acid content, such as 8: 3, seems to be to maintain the fluidity of the cell membrane. From the above, it is speculated that the growth at low temperature and the high content of unsaturated fatty acids such as 18: 2 and 18: 3 in the lipid fatty acid composition are due to the characteristics of the low temperature yeast.

〔The invention's effect〕

According to the present invention, ethanol, which can be supplied in large quantities as an alternative energy to petroleum from renewable biomass resources, is used as a raw material, and by assimilating this into microorganisms, the essential fatty acids 18: 2 and 18: 3 A yeast cell containing a high lipid in the cell can be produced. The yeast cells obtained according to the present invention can be widely used as a feed for highly nutritive fish feed, a feed for livestock, and a feed for zooplankton, which is necessary for producing seedlings of marine fish. The yeast cells can also be used to produce highly functional lipids with high 18: 2 and 18: 3 contents.

Furthermore, the obtained yeast cells are stable in seawater,
The active ingredient in the fungus body does not flow out and pollute the breeding water.

[Brief description of drawings]

FIG. 1 is a diagram showing the influence of culture temperature on growth, and FIG. 2 is a diagram showing the influence of salt concentration in the medium on cell yield and cell lipid content.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12P 7/64 C12R 1:72)

Claims (3)

[Claims] 1. A Candida genus, which has an ethanol-utilizing ability, and has a linoleic acid content of 15.8 to 38.3% in a lipid fatty acid composition,
Linolenic acid content 9.2 ~ 22.4%, both content 25.0 ~ 60.7%
Yeast strains capable of forming lipids with high linoleic acid and linolenic acid content in the cells were cultured in a medium containing ethanol as a carbon source, and yeast strains containing lipids with high linoleic acid and linolenic acid contents were obtained from the culture. A method for producing a yeast cell, which comprises collecting the body. 2. The method for producing a yeast cell according to claim 1, wherein the culture is carried out at a low temperature of 5 to 20 ° C. 3. The yeast used is Candida sp.
The method for producing a yeast cell according to claim 1 or 2, which is S30W2 strain (FERM P-11023).