JPH0591889A - Production of oil and fat and microorganism therefor - Google Patents

Abstract

PURPOSE:To facilitate the extraction process and reduce the cost by cultivating a specific yeast in the presence of a fatty acid (alkyl ester), thereby effecting extracellular production of an oil and fat, especially an oil and fat usable as a substitute for cacao fat. CONSTITUTION:A microbial strain belonging to the genus Trichosporon, Saccharomycopsis, Candida or (Cryptococcus and capable of producing an oil and fat out of the microbial cell by the cultivation in the presence of a fatty acid (alkyl ester) [e.g. new microbial strain, Trichosporon sp. SH45Y (FERM BP-1236)] is cultivated in a medium containing a fatty acid (alkyl ester), and the produced and accumulated oil and fat is separated from the cultivation liquid. The fatty acid alkyl ester is preferably ethyl palmitate, etc., and the fatty acid is preferably oleic acid, capric acid, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an oil and fat which is produced and accumulated in an extracellular culture solution by a fermentation method.
The present invention also relates to a method for producing an oil or fat which is a cacao butter-like oil or fat in the above production method, or an oil or fat which contains palmitoleic acid as a constituent fatty acid in a high proportion.
The present invention also relates to a method for producing fats and oils from fatty acids, which are oil by-products. The present invention also relates to a microorganism having a high oil and fat production capacity.

[0002]

2. Description of the Related Art As a method for producing intracellular fats and oils by fermentation, Endomyce (Oidium) genus, Endomyce
s), Candida, Rhodotoru
la), Cryptococcus genus or Rhodosporidium genus microorganisms for producing a cocoa butter substitute fat (JP-A-51-12386)
8); A method for producing a cocoa butter substitute fat by a microorganism belonging to the genus Endomyces, Rhodotorula or Lipomyces (JP-A-52-122672); Saccharomyces (Sa
A method for producing a lipid component (including triglyceride) having a high content of palmitoleic acid by a microorganism belonging to the genus ccharomyces (JP-A-63-287491); Mortierell
a) A method for producing lipids (including fats and oils) having a high γ-linolenic acid content by microorganisms belonging to the genus (JP-A-59-130191); and lipids having a high palmitoleic acid content due to microorganisms belonging to the genus Kloeckera A method for producing (including neutral lipid) is known (JP-A-1-108991), and production of fats and oils by microorganisms belonging to the genus Trychosporon has been reported [NJ Moon et al., J. Am. . Oil Che
m.Soc., 55 , 683-688 (1978) and H. Kaneko et al., Li
pids, 11 (No. 12), 837-844 (1976)]. In addition, it is known to produce palmitoleic acid in Saccharomyces microorganisms (JP-A-62-289191).

As a method for extracellular production of lipids by fermentation, a method of culturing molds or algae in the presence of a surfactant is known (Japanese Patent Laid-Open No. 62-3791). In addition, Candida lipolytica (lypolytica), which has the ability to produce fatty acids in vitro
Has been reported on the extracellular production of fatty acids by a mutant strain belonging to [T. Miyakawa et al., Agric. Biol. Chem., 48 ,
499 (1984)]. Palmitoleic acid is a kind of monounsaturated fatty acid having 16 carbon atoms and is contained in mink oil and macadamian nut oil in an amount of 15 to 20%. Palmitoleic acid has a useful pharmacological action [antitumor action, tooth decay inhibitory action, and protective action against vascular disorders in hypertensive diseases (JP-A-62-289191), etc.]
In addition, oils and fats having palmitoleic acid as a constituent component have good affinity with the skin and are useful as cosmetic materials. Cocoa butter mainly contains a triglyceride having a saturated fatty acid group at the α and α'positions and an oleic acid group which is a monoene fatty acid group at the β position.

[0004]

In the case of intracellular production of fats and oils, the process of extracting fats and oils is complicated. That is, the bacterial cells are first recovered by centrifugation or filtration, and then the wet bacterial cells are crushed (mechanical crushing with a colloid mill, ball mill, homogenizer, etc., crushing with ultrasonic waves), and oils and fats are extracted with n-hexane etc. Or dry the wet cells (freeze drying,
Spray drying etc.), chloroform: methanol mixed solvent or n-
A step of extracting fats and oils with a hexane: isopropanol mixed solvent or the like is required. In addition, in the production of fats and oils by fungi, generally, the cultured bacterial cells are apt to agglomerate, so that the bacterial cells are entangled with the stirring blades, are destroyed thereby, and the reproducibility is not sufficient. In addition, in the production of fats and oils by algae, the culture time is generally 1 week to 1
It takes as long as a month and requires light irradiation equipment as ancillary equipment.
In addition, in the extracellular production of fats and oils in the presence of a surfactant, after the culture is completed, the produced fats and oils are generally extracted with an organic solvent. The problem of insufficient separation is likely to occur. An object of the present invention is to provide a method for facilitating the production of fats and oils outside yeast cells by using yeast to facilitate the extraction process and reduce costs. Another object of the present invention is to provide an extracellular fermentation production method of fats and oils which can avoid the problems of fats and oils production by molds and algae. Another object of the present invention is to provide an extracellular fermentation production method of fats and oils, which is advantageous in that extraction of fats and oils after fermentation production with an organic solvent facilitates separation of an aqueous phase and an organic solvent phase. Especially. It is a further object of the present invention to provide a method for fermentatively producing a cocoa butter-like oil and / or an oil and fat containing palmitoleic acid as a constituent component outside the cell. A further object of the present invention is to provide a microorganism having a high oil and fat production capacity, which is useful in carrying out the above method. A further object of the present invention is to provide a means for recovering vegetable oil fatty acids contained in distillate oil, soda oil syrup, etc., which are by-produced in the vegetable oil production process, and which have not been effectively used conventionally, as fats and oils.

[0005]

[Means for Solving the Problems] As a result of repeated studies aimed at the extracellular fermentation production of fats and oils, the present inventors have found that the culture is carried out in an ordinary fermentation medium, for example, a fermentation medium containing glucose as a main carbon source. The present invention has been found to find that fat-producing microorganisms of a specific genus that do not exhibit extracorporeal fat and oil production, produce and accumulate a large amount of fats and oils outside the cells by culturing in the presence of fatty acid alkyl ester or fatty acid. .. That is, the present invention all belong to the genus Trichosporone, Saccharomycopsis, Candida or Cryptococcus in yeast, and have the ability to produce fats and oils extracellularly by culturing in the presence of fatty acid alkyl ester or fatty acid. A method for producing fats and oils, which comprises culturing a microorganism in a medium containing a fatty acid alkyl ester or a fatty acid to cause the fats and oils to be produced and accumulated outside the cells in the culture liquid, and collecting the fats and oils produced and accumulated from the culture liquid. ..

The microorganism used in the present invention belongs to the genus Trichosporone, the genus Saccharomycopsis, the genus Candida or the genus Cryptococcus, and produces oils and fats outside the cells by culturing in the presence of fatty acid alkyl ester or fatty acid. Any microorganism can be used as long as it has the ability. For example, a mutant strain of a microorganism having the above properties can be used in the present invention as long as it retains the above properties. Such mutagenesis is carried out by usual mutagenesis treatments such as irradiation with ultraviolet rays and X-rays, N-methyl-N'-nitro-N.
-Can be performed by treatment with a drug such as nitrosoguanidine. Specific strains that can be used include Trichosporon species (Tric isolated from soil in Kagoshima City).
hosporon sp.) SH45Y (Ministry of Industrial Science, No. 12362)
, And its mutants, Trichosporon species SH45Y-E1 (Microtechnological Research Institute No. 12363) and Trichosporon species SH45Y-E2 (Microtechnical Research Institute No. 12364), Saccharomycopsis lipolytica IFO -1659, Candida saitoana IFO-0380, C. tropicalis IFO-1647,
And Cryptococcus albidas
idus) IFO-1530.

The SH45Y strain has the following mycological properties. a. Ascospore formation No ascospore formation is observed in Gorodkova medium, Adams medium, malt medium, V-8 medium, potato medium and YM medium. b. Growth condition in each medium (25 ° C., 4 days culture) YM liquid medium-sphere to ellipse 2.5 × 2.5, 2.5 × 2.5 to 3.5 (μ) YM agar medium-sphere to ellipse 3.5 × 3.5, 3.5 × 6 ~ 9 (μ) Pseudohyphal formation (+) Film formation (-) Potato extract agar medium-Mitotic formation (+) c. Physiological properties Vitamin requirement (-) (4 days culture at 25 ℃) Biotin Riboflavin Pyridoxine hydrochloride Inositol folic acid Calcium pantothenate nicotinic acid Thiamine hydrochloride p-aminobenzoic acid Nitrate assimilation (−) Pigment formation (−) Growth temperature (YM medium, 7 days culture) 13 ° C 18 ° C 23 ° C 28 ° C 33 ℃ 37 ℃ − ＋ ＋ ＋ ＋ ＋ ＋ ＋ − − Growth pH (25 ℃, YM medium, 10 days culture) 3.5 4.0 4.5 6.5 7.5 8.2 9.0 9.5 ± ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ±

D. Assimilation of carbon source and fermentability test method After growing and culturing the microbial cells grown in PSA agar medium at 25 ° C. for 4 days once with sterilized water and diluting to 10 ⁵ cells / ml did. Each test medium in which various carbon sources (sugars) were added to yeast nitrogen base (Difco) 5
ml (φ18 mm test tube) was inoculated with 0.1 ml each and statically cultured. The stirring was performed once a day. Assimilation of carbon source (25 ° C, 10-28 days culture) D-glucose (+) ethanol (+) D-galactose (+) mannitol (+) maltose (+) inositol (+) sucrose (+) D-sorbitol (+) Lactose (+) Glycerol (+) D-Raffinose (-) D-Mannose (+) D-Xylose (+) D-Fructose (+) D-Arabinose (+) Soluble starch (+) L-Arabinose ( +) Sodium lactate (+) L-rhamnose (+) Sodium citrate (-) Fermentability of sugars (25 ° C, 7-day culture) D-glucose (-) Lactose (-) D-galactose (-) Maltose (-) ) Sucrose (-) D-raffinose (-)

The Yeasts 3rd edition, edited by NJMKreger-van Rij, Else, based on the following mycological properties
vier Science Publishers S in the light of BV Publishing (1984)
The H45Y strain was identified as a strain belonging to the genus Trichosporone. As mentioned above, this strain has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 12362. This SH45Y strain was mutated by a conventional method to obtain Trichosporone species SH45Y-E1 and SH45Y-E2 strains having improved triglyceride productivity and / or palmitoleic acid productivity as compared to the SH45Y strain, as shown in Examples described later. It was As mentioned above, these are the 1st
Deposited as 2363 and 12364.

Next, the fatty acid alkyl ester used in the present invention will be described. The acid component constituting the fatty acid alkyl ester is usually a saturated or unsaturated fatty acid having 8 to 22 carbon atoms, particularly 12 to 18 carbon atoms, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid. , Linoleic acid, linolenic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid,
Examples include erucic acid. As the alcohol component of the fatty acid alkyl ester, lower alcohols having 1 to 6 carbon atoms such as methanol, ethanol, n-propanol, i-propanol, n-butanol and the like are usually mentioned, but ethanol is preferable. The fatty acid alkyl ester may be used alone or in combination of two or more kinds. Next, as the fatty acid used in the present invention, an unsaturated fatty acid having usually 8 to 22 carbon atoms, particularly 16 to 22 carbon atoms (for example, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid), Medium-chain saturated fatty acids having 8 to 12 carbon atoms (such as caprylic acid, capric acid, and lauric acid), or the above unsaturated fatty acids or medium-chain saturated fatty acids and long-chain saturated fatty acids having 14 to 22 carbon atoms, particularly 14 to 20 carbon atoms (for example, Myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid).
The proportion of unsaturated fatty acids or medium chain saturated fatty acids in the mixture of unsaturated fatty acids or medium chain saturated fatty acids and long chain saturated fatty acids is 10% by weight or more, preferably 25% by weight or more. In addition, as the mixture, vegetable oil and fat fatty acids, such as soybean fatty acid and rapeseed fatty acid, which are contained in distillate oil, soda oil syrup, and the like that are by-produced in the vegetable oil production process and have not been effectively used, can be used. When a mixture of unsaturated fatty acids and saturated fatty acids is used as described above, not only unsaturated fatty acids but also saturated fatty acids are assimilated by the yeast used in the present invention, for example, when palmitic acid or stearic acid is used alone. In these, these are hardly utilized. Further, a fatty acid and a fatty acid alkyl ester may be used in combination, but when the long-chain saturated fatty acid is used as the fatty acid, the proportion thereof is less than 90% by weight, preferably less than 75% by weight.

In culturing the yeast used in the present invention,
The amount of the fatty acid alkyl ester or fatty acid contained in the medium is generally 0.1 to 50 g / dl, preferably 1
It is ~ 10g / dl. The fatty acid alkyl ester or fatty acid may be added from the beginning of the culture, may be added during the culture in which the bacterium has grown considerably, or may be a complex addition type thereof. That is, the above concentration does not need to be maintained over the entire period of culture, and specific control of the concentration may be appropriately determined in relation to the extracellular accumulation of oil and fat and its amount. Thus, by culturing in the above concentration range, fats and oils are produced and accumulated outside the cells. Further, as will be described later, the constituent fatty acid composition of the oil or fat changes depending on the type of fatty acid of the fatty acid alkyl ester to be added or the type of fatty acid to be added.

Regarding the medium used in the method of the present invention,
The medium usually used for fermentative production of fats and oils is used. That is, as shown in the examples, both a synthetic medium and a natural medium can be used as long as the medium contains a main carbon source, a nitrogen source, an inorganic substance and other nutrients. As the carbon source, the above-mentioned fatty acid alkyl ester or fatty acid may be used alone, and a small amount, for example, 20% by weight or less, preferably 2% by weight or less, of another carbon source relative to the total amount thereof may be added and used. May be. Such other carbon sources include glucose, sucrose, fructose, starch,
Various carbohydrates such as starch hydrolysates and molasses, alcohols such as ethanol, methanol, glycerol and polyalcohols, amino acids such as glutamic acid and aspartic acid, and hydrocarbons such as n-paraffin, etc., depending on the assimilation capacity of the bacteria used. Can be used.

As the nitrogen source, inorganic organic nitrogen compounds such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, sodium nitrate and urea can be used. Further, as the nitrogen source, nitrogen-containing natural products such as peptone, meat extract, yeast extract, corn steve liquor, casein hydrolyzate, fish meal or its digest, defatted soybean meal or its digest can also be used. Inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, ferrous chloride, manganese sulfate, calcium chloride, calcium carbonate, zinc sulfate, copper sulfate, boric acid / molybdic acid. Ammonium, potassium iodide, etc. can be used. If the microorganism used requires a specific nutrient (for example, vitamins such as biotin and thiamine) for growth, the nutrient must be added to the medium in an appropriate amount. When these nutrients are added by being included in the nitrogen-containing natural product used as the nitrogen source, it is of course not necessary to add the nutrients separately.

The culture is carried out under aerobic conditions such as shaking culture or deep agitation culture. The culture temperature is generally 20 to 35 ° C.
Is preferable, but other temperature conditions may be used as long as the temperature allows the bacteria to grow. The pH of the medium during culture is 4.5 to 6.0 when fatty acid ester is used and 4.5 to 6.0 when fatty acid is used.
Maintaining at 7.2 is desirable for high yields. Usually, 3 to 7 days after the start of culture, a large amount of oil and fat is produced and accumulated outside the cells.

After completion of the culture, an extraction solvent is added to the culture medium to extract oils and fats in the extraction solvent. Since some of the oils and fats are attached to the surface of the bacterial cells, the extraction solvent is usually added to the culture-finished liquid itself containing the bacterial cells or a partial concentrate thereof. In addition to the above-mentioned extraction, the fats and oils remaining in the cells may be recovered by a conventional method. As an extraction solvent, an organic solvent that dissolves fats and oils and is liquid with no or little miscibility with water at room temperature, such as a halogenated lower alkane, such as chloroform, methylene chloride, carbon tetrachloride, 1,2-diloroethane;
-Hexane, ethyl ether; ethyl acetate; aromatic hydrocarbons such as benzene, toluene and xylene are preferably used. The amount of the extraction solvent added is not particularly limited as long as it can sufficiently recover the oils and fats produced and accumulated in the culture broth, but generally 50 ml or more, preferably 100 ml to 3 L, particularly preferably 1 liter per 1 liter of the culture broth. Is 100 ml to 1 L
Is.

The fact that the oil / fat extracted by the extraction solvent is an oil / fat existing outside the bacterial cells means that the phospholipids constituting the cell membrane, which are also extracted when the intracellular oil / fat is extracted, are extracted. It does not substantially exist in the liquid (in contrast, for example, in Example 1 of the above-mentioned JP-A-63-287491, lipids are extracted from a dried bacterial cell with a chloroform-methanol mixture without destroying the bacterial cell. Not only simple lipids such as, but also complex lipids such as phospholipids are extracted together.) Complete (intact) cells are observed after extraction operation. It is clear from the fact that fats and oils are not extracted by such a simple operation (for example, refer to the prior art documents regarding the production of fats and oils described in the section of the prior art).

The oil or fat extracted in the extraction solvent can be directly used as a product by evaporating and removing the extraction solvent according to the purpose, or can be used as a raw material fatty acid ester or its decomposition product by column chromatography, distillation, deoxidation or the like. It can also be purified by removing contaminants such as certain fatty acids or raw material fatty acids (removing fatty acid esters and fatty acids results in almost pure triglycerides), and if necessary, subjecting to treatments such as decolorization and deodorization. You can also These individual treatment operations are conventional methods for producing oils and fats (eg, soybean, corn, rapeseed extract and purification from plants, Takaaki Miyakawa, “Practice of cooking oil”, Koshobo (1988), fermentation. It can be carried out in accordance with the above-mentioned JP-A-63-28491, JP-A-52-122672, etc.) and a general method for refining a fermented product for refining a produced oil and fat.

Cocoa butter is mainly composed of triglycerides having a stearic acid residue or a palmitic acid residue in the α and α'positions and an oleic acid residue in the β position. In the present invention, a long-chain saturated fatty acid (C: 16 to 20) or an ester thereof or a mixture thereof is used alone or a long-chain monounsaturated fatty acid saturated fatty acid (C: 16 to 20) as the raw material fatty acid alkyl or fatty acid. When used in combination with its ester or a mixture thereof, it generally has a composition similar to that of cocoa butter, that is, it has mainly long-chain saturated fatty acid residues (C: 16-20) at the α and α'positions. A fat or oil mainly composed of triglycerides having a long-chain monounsaturated fatty acid residue (C: 16 to 20) in the β-position can be obtained. However, when the long-chain monounsaturated fatty acid, its ester, or a mixture thereof is used in combination as described above, the proportion thereof should be 40% by weight or less in total. Also, when using a long-chain saturated fatty acid, regardless of the above, it shall be used in combination with the above other components,
The long-chain saturated fatty acid content is less than 90% by weight, preferably
It should be less than 75% by weight.

The composition of oils and fats produced and accumulated is greatly affected by the type of fatty acid alkyl ester or fatty acid added to the medium. That is, when a saturated fatty acid alkyl or a saturated fatty acid is used as a carbon source, the proportion of the corresponding saturated fatty acid residue and the corresponding unsaturated fatty acid (mainly monounsaturated fatty acid) residue in the resulting oil or fat is generally increased ( See Examples 1 to 3 below). In addition, when unsaturated fatty acid alkyl or unsaturated fatty acid is used as the carbon source, the proportion of the corresponding unsaturated fatty acid residue in the resulting fat or oil is generally increased (see Examples 2 and 4).
When a mixture of unsaturated fatty acid and saturated fatty acid is used as the carbon source, the proportion of the corresponding unsaturated fatty acid residue and the corresponding saturated fatty acid residue in the resulting fat or oil is generally increased. (Refer to Example 5) The production amount of fats and oils varies greatly depending on the strain used, but the productivity is particularly remarkable for the strains belonging to the genus Trichosporone of the present invention (for example, Examples 1, 2 and 4).
reference). When an alkyl ester of a fatty acid having 16 carbon atoms is used as a carbon source, palmitoleic acid residues are contained in the fats and oils produced and accumulated in the microorganisms used in any genus of the present invention. The number of bacteria belonging to the genus Trichosporone was higher, and the most prominent was Trichosporon species SH45Y-E2 (see Examples 1 and 2).

[0020]

EXAMPLES Next, the method of the present invention will be specifically described by way of examples. Example 1 100 ml of the following medium (containing 1.0 g of ethyl palmitate as shown below) was inoculated with 10 ⁷ yeasts each shown in Table 1 below, and cultured at 28 ° C. for 4 days with shaking. After completion of the culturing, 15 ml of chloroform was added to the culture broth and extraction was performed 3 times. The chloroform extracts were combined to a fixed volume of 50 ml. To 2 ml of the chloroform extract, 1.0 mg of triheptadecanoin as an internal standard substance was added, concentrated, and then applied to TLC. n
-Developing with hexane: diethyl ether: acetic acid (80: 20: 1), a triglyceride fraction was collected. To the triglyceride fraction, add 0.6 ml of 10% KOH methanol solution, and at 80 ℃
Heated for 20 minutes. After cooling, 0.7 ml of boron trifluoride-methanol complex methanol solution was added and heated for 2.5 minutes. After cooling, 1.0 ml of n-hexane was added and further heated for 1.5 minutes. After cooling again, a large amount of saturated sodium chloride aqueous solution was added to collect the upper hexane layer. The hexane layer was dehydrated with anhydrous sodium sulfate, and then fatty acids were quantified by gas chromatography.
The total fatty acid amount was multiplied by 1.05 to obtain the triglyceride amount. The results are shown in Table 1.

Medium NaNO ₃ 2.0 g, (NH ₄ ) SO ₄ 2.0 g, K ₂ HPO ₄ 1.0 g, KH ₂ PO _{4
7.0g, MgSO 4 · 7H 2 O0.3g} , CaCl 2 · 2H 2 O 0.1g, NaCl
0.5g, ethyl palmitate 10.0g, vitamin mixed aqueous solution
^{* 1 1} ml, mixed mineral water ^{* 2} 1 ml, 1% chloramphenicol / ethanol 1 ml, distilled water to 1000 ml. ^{* 1. 2} Vitamin mixed solution shown below Biotin 2mg, Calcium pantothenate 400mg, Folic acid 2mg, Inositol 2000mg, Nicotinic acid 400m
g, n-aminobenzoic acid 200mg, pyridoxine hydrochloride
400mg, Riboflavin 200mg, Thiamine hydrochloride 400m
g, make up to 1000 ml with distilled water. Mineral aqueous mixture _{M n SO 4 · 4~5H 2 O} 60mg, Z n SO 4 · 7H 2 O, C u SO 4
・ 5H ₂ O 40mg, FeCl ₂・ 6H ₂ O 250mg, H ₃ BO ₃ 60mg,
_{(NH 4) 6 Mo 7 O} 24 · 4H 2 0 25mg, KI 100mg, distilled water 100
Make it 0 ml.

[0022]

[Table 1] ──────────────────────────────────── Bacterial strain Triglyceride Fatty acid composition of triglyceride Ceride (wt) %) (Mg) C ₁₆ C _{16: 1} C ₁₈ C _{18: 1} C _{18: 2} ─────────────────────────────── ─────── TRICOSPORON SPECIES 113 63.0 15.9 1.4 16.4 3.3 SH45Y (Microbiology Research Institute No. 12362) Saccharomycopsis 53 66.8 8.2 2.5 19.8 2.7 Repolitica IFO-1659 Candida Cytoana 22 64.0 10.3 1.8 21.0 2.9 IFO-0380 Candida tropicalis 17 61.1 13.4 2.2 21.5 1.8 IF0-1647 Cryptococcus arvidas IFO-1530 10 62.6 9.5 2.0 23.5 2.4 Trichosporon species 130 62.7 26.7 0.6 6.7 3.2 SH45Y-E2 No.) ──────────────────────────────────── C ₁₆ palmitic , C _{16: 1} palmitoleic acid, C ₁₈ stearate, C _{18: 1} oleic acid, C _18: Note ₂ linoleic acid, using the same medium as above but containing glucose 2 g / dL instead of ethyl palmitate, the When operated in the same manner as above, substantially no triglyceride fraction was recovered in any of the strains.

Example 2 Each of the medium of Example 1 containing 100 ml of the medium of Example 1 and the carbon source shown in Table 2 in place of ethyl palmitate.
100 ml (each containing 1.0 g of the carbon source in Table 2) was inoculated with 10 < ⁷ > pieces of Trichosporon species SH45Y-E1 (Microtechnical Lab. No. 12363), and then the same procedure as in Example 1 was performed. The results are shown in Table 2.

[Table 2] ──────────────────────────────────── Triglyceride Triglyceride Fatty Acid Composition Ceride (mg) ( wt%) Carbon source C ₁₆ C _{16: 1} C ₁₈ C _{18: 1} C _{18: 2} ───────────────────────────── ──────── Ethyl palmitate 174 67.8 19.8 0.7 10.1 1.6 Ethyl stearate 12 1.3-65.3 28.0 5.4 Ethyl palmitate and 54 50.1-2.3 45.0 2.3 Ethyl oleate (1: 1) ────── ──────────────────────────────

Example 3 The same medium as in Example 1 except that the content of ethyl palmitate was different in 5 L of jar fermenter (manufactured by Mitsuwa Rika) 3 L
(Including 60 g of ethyl palmitate) was added and sterilized. Trichosporon species SH45Y-E1 was inoculated into 200 ml of the same medium (containing 4.0 g of glucose) as in Example 1 except that glucose was used instead of ethyl palmitate, and the whole amount of the culture solution was cultured at 28 ° C. for 4 days. Inoculate the medium in fermentor, 28 ℃, 250rpm, aeration 0.5L
The culture was performed for 7 days under the condition of / min. After culturing, add n
-500 ml of hexane (food grade) was added for extraction.
The extraction operation was repeated 3 times in total, and the obtained extracts were combined and concentrated. The recovered oil was distilled to purify triglyceride. The operation was repeated using KDL1 (manufactured by LEYBOLD) under the conditions of 0.1 mmHg and 140 ° C., and the distillation residue was purified by thin layer chromatography until a single spot of triglyceride was obtained. 12.8 g of triglyceride was obtained.

In the analysis of triglyceride, the total fatty acid composition was analyzed in accordance with Example 1 and the 2-position fatty acid was analyzed. The 2-position fatty acid decomposes triglyceride with pancreatin, and is subjected to thin layer chromatography (boric acid-impregnated plate, solvent system:
2-Monoglyceride was recovered with chloroform: acetone = 96: 4) and analyzed in the same manner as the total fatty acid. The results are shown in Table 3.

[Table 3] ───────────────────────────────────── Triglyceride production Fat Fat Acid Composition (wt %) (G) C ₁₆ C _{16: 1} C ₁₈ C _{18: 1} C _{18: 2} ────────────────────────────── ─────── 12.8 Total fatty acids 63.4 10.4 5.2 18.6 2.4 Second-fatty acids 12.2 29.5 1.0 50.3 7.0 ────────────────────────── It is clear from Table 3 that most of the monoene fatty acid is present in the 2nd position and the saturated acid is present in the 1st and 3rd positions.

Example 4 100 ml each of the following medium [1.0 g of commercially available oleic acid as shown below (Kanto Chemical Co., purity 76.3%, composition weight% C ₁₆ 4.
0, C _{16: 1} 7.0, C ₁₈ 1.3, C _{18: 1} 76.3, C _{18: 2} 7.4 and other 4.0)] were inoculated with 10 ⁷ yeasts each shown in Table 4 below, and 5 Culture was carried out with shaking for one day. Medium NaNO ₃ 2.0 g, (NH ₄ ) SO ₄ 2.0 g, K ₂ HPO ₄ 1.0 g, KH ₂ PO _{4
7.0g, MgSO 4 · 7H 2 O0.3g} , CaCl 2 · 2H 2 O 0.1g, NaCl
0.5g, commercial oleic acid 10.0g, vitamin mixed aqueous solution ^{* 1} 1m
l, Mineral mixed aqueous solution ^{* 2} 1 ml, 1% chloramphenicol / ethanol 1 ml, distilled water to 1000 ml. ^* 1.2 ^. Same as in Example 1 Thereafter, the culture-finished solution was treated in the same manner as in Example 1. The results are shown in Table 4.

[0027]

[Table 4] ──────────────────────────────────── Bacterial strain Triglyceride Fatty acid composition of triglyceride Ceride (wt) %) (Mg) C ₁₆ C _{16: 1} C ₁₈ C _{18: 1} C _{18: 2} Other ───────────────────────────── ──────── TRICOSPORON 158 2.6 4.1 2.0 81.0 7.6 2.7 Species SH45Y Saccharomycopsis 32 3.1 5.0 2.5 78.4 7.4 3.6 Repolitica IFO-1659 Candida Siteana 36 2.8 4.3 2.3 82.1 7.6 0.9 IFO-0380 Candida・ Tropicalis 28 2.0 4.4 1.9 80.7 8.0 3.0 IF0-1647 Cryptococcus arvidas IFO-1530 21 3.4 4.8 1.8 77.5 7.9 4.6 Trichosporon species SH45Y-E1 161 3.2 4.4 1.9 80.5 8.0 2.0 Trichosporon 183 3.8 3.9 2.2 80.4 8.4 1.3 Species SH45Y -E2 ─────────────────────────── ────────

Example 5 A soybean fatty acid (soda oil syrup (manufactured by Showa Sangyo)) recovered from a soda syrup in a SL jar fermenter (manufactured by Mitsuwa Rika) was neutralized with an acid and then extracted with n-hexane to form a hexane layer. Was thoroughly washed with water and n-hexane was distilled off to obtain soybean fatty acid) [composition (% by weight) C ₁₆ 10.
5, C _{16: 1} −, C ₁₈ 4.2, C _{18: 1} 23.3, C _{18: 2} 53.4 and
C _{18: 3} 8.6) 60 g was added and sterilized. The same medium as in Example 1 except that it contains glucose instead of ethyl palmitate.
200 ml (containing 4.0 g of glucose) was inoculated with Trichosporon species SH45Y-E1 and cultured for 4 days at 28 ° C. The total amount of the culture solution was inoculated into the medium in the jar fermenter at 28 ° C, 250 rpm. The culture was carried out for 7 days under the condition of an amount of 0.5 L / min. After completion of the culture, the culture solution was treated in the same manner as in Example 3.
23.2 g of triglyceride was obtained. The total fatty acid composition of this triglyceride is C ₁₆ 7.0, C _{16: 1} −, C ₁₈ 4.6, C _{18: 1} 24.
It was 7, C _{18: 2} 54.8 and C _{18: 3} 8.9.

[0029]

EFFECTS OF THE INVENTION According to the method of the present invention, fats and oils can be fermented and produced outside the cells from the precursor. Further, according to the method of the present invention, it is possible to ferment and produce an oil and fat that can substitute for cocoa butter from the precursor outside the cells. Furthermore, according to the method of the present invention, fats and oils having a high content of palmitoleic acid can be fermented and produced outside the cells from the precursor.

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Claims (4)

[Claims] 1. A member of the genus Trichosporon, the genus Saccharomycopsis, the genus Candida or the genus Cryptococcus,
A microorganism capable of producing fats and oils outside the cells by culturing in the presence of a fatty acid alkyl ester or fatty acid is cultured in a medium containing a fatty acid alkyl ester or fatty acid to produce fats and oils outside the cells in the culture solution. A method for producing fats and oils, which comprises causing the fats to accumulate and collecting the fats and oils produced and accumulated from the culture solution. 2. The method for producing fats and oils according to claim 1, wherein the fats and oils produced are cocoa butter-like fats and oils. 3. The method for producing an oil or fat according to claim 1, wherein the oil or fat produced contains palmitoleic acid as a constituent fatty acid. 4. Trichosporon species SH45
Y (Microtech Lab. No. 12362), and its mutant Trichosporon species SH45Y-E1 (Microtech Lab. No. 12363) and Trichosporon species SH45Y-E2 (Microtech Lab. No. 12364)
issue).