JPH0634730B2 - Method for producing palmitoleic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] Palmitoleic acid (hereinafter abbreviated as POA) is one of unsaturated fatty acids widely existing in the living world.
Inhibition of dental caries, relationship with health aging, use in the field of cosmetics, and recently the effect of protecting vascular disorders in hypertensive diseases (Japanese Patent Laid-Open No. 59-175425), etc. have been reported, showing excellent pharmacological and physiological effects. It has been clarified that it is expected to be widely applied to cosmetics and pharmaceuticals in the future.

[Conventional technology]

As an industrial method of POA, P by extraction method from plants such as macadamia nuts and rice bran and fish oil such as whales and sardines
Although oils and the like containing OA are known, little is known about purification techniques for high purification. In general, as a method for extracting and purifying from a plant, a lipid component obtained by a squeezing method or the like is subjected to saponification treatment with an alkali or enzymatic decomposition treatment with a lipase, and the fatty acids released are subjected to a molecular distillation method. -Methods for purifying and isolating a target substance by a countercurrent distribution method, a chromatographic method, etc. are known. On the other hand, regarding the POA fermentation production method using microorganisms such as yeast,
It is not known at all, and there are only a large number of research reports on fatty acid composition, membrane constitution and function as bacterial lipid components under the conditions of various species and various culture conditions as follows. That is, in S. cerevisiae, the influence of the culture temperature on the POA content [Biochem. Biophys. Acta. 260 , 639
(1972)], [J. Biol. Chem. 254 , 12281.
(1979)], transition of POA content under anaerobic and aerobic culture conditions [J. Brew. Soc. Japan. 72 , 735 (197).
7)], [J. Cell. Biol. 37 , 221 (1968)].
[Arch. Microbiol. 66 , 34 (1969)], [Arch.
Microbiol. 117 , 239 (1978)], fatty acid composition of cytoplasmic membrane and cell wall [Chem. Phys. Lipids. 4 , 247].
(1970)], relationship between fatty acid composition and function [Antonie
van Leeuwenhoek. 38 , 129 (1972)], [Exp.
Mycol. 8 , 55 (1984)], cell membrane lipid composition and EEtO.
Relationship between H resistance [J. Inst. Brew. 82 , 218 (197)
6)], and lipid composition under anaerobic and aerobic culture conditions in S. uvarum [J. Inst. Brew. 88 , 367 (198).
2)], (Pro.Con-Eur.Brew. 18 , 285 (198)
1)], Effect of culture temperature on POA content [ASBC
J. 40 , 26 (1982)], Effect of thiamine pyridoxine on fatty acid composition [Biochem. Biophys. Res. Co.
mmun. 59 , 777 (1974)] Furthermore, fatty acid composition [Agricultural 46 , 657 (1972)] and the like have been reported in S. rouxii. As a fatty acid extraction method containing POA, chloroform / methanol mixture extraction, methanol・
After the alkali pretreatment, ether extraction or the like is performed, and the extracted lipid components are subjected to saponification methyl esterification and gas chromatographic analysis. Regarding the method for isolating POA from microbial cells such as yeast on an industrial level, , Not reported at all.

[Problems to be solved by the invention]

Fatty acids existing in microbial cells such as yeast are almost in free form, but are in triglyceride, sterol ester, and ester bond type as complex lipid. Therefore, after microbial cell destruction and ester bond hydrolysis or saponification treatment are performed in advance, contaminants such as glycerin that are by-produced at that time are removed from the aqueous layer, and free fatty acids containing POA are extracted with a solvent. By completing the method of further purification treatment, high-purity POA can be produced from microbial cells on an industrial level, and can be supplied cheaply and easily as compared with natural products, and widely used in cosmetics and pharmaceuticals. It can be used for various purposes.

[Means for solving problems]

As a result of diligent studies on a method for extracting and purifying POA from yeast cells, the present inventors have previously destroyed the cells under acidic conditions and then subjected to saponification treatment, and then used alone or two or more kinds of water under acidic conditions. The present invention has been completed by finding a method of producing a high-purity POA by extracting and separating a lipid component containing POA with a solvent that does not mix, and further performing crystallization and column chromatography.

That is, after the aqueous suspension of yeast cells containing POA is heat-treated with an acid in advance and then treated with an alkali, the cells are destroyed to further hydrolyze lipid components such as neutral fat, phospholipids and sterol esters. The free fatty acid containing POA is extracted under acidic conditions with a solvent that does not mix alone or with two or more kinds of water, and water-soluble components such as glycerin and phosphate compounds by-produced by hydrolysis are separated and removed into an aqueous layer. . afterwards,
The separated solvent layer is concentrated and recovered under reduced pressure, and sterols /
The present invention was completed by finding that high-purity POA was produced by removing contaminants such as ubiquinone by crystallization and normal phase gel column chromatographic treatment and further purifying by reverse phase gel chromatographic treatment. Hereinafter, the present invention will be described in detail.

As the POA-containing yeast used in the present invention, any yeast can be used as long as it has the ability to accumulate POA in the cells. As a typical example, commercially available baker's yeast, W
ine yeast IFO 2229, Saccharomyces cerevisiae ATCC 18790, NCYC 3
66, also commercially available dry brewer's yeast, and Saccharomyces
Carlsbergensis (Saccharomyces carlsber'gens
is) ATCC 9080, NCYC 74, and Saccharomyces delbruecki
i) NCYC 100, Candida utilis IAM 4815, and Rhodotorula glutilis IFO 1
125 can be indicated.

The concentration of the aqueous suspension may be 1 to 30% by dry weight, preferably 10 to 20%. As the acid for acid treatment,
Sulfuric acid, hydrochloric acid, nitric acid and the like are used, and the initial pH is usually 2 or less, the heating temperature is 60 to 150 ° C., the heating time is 0.5 to 10 hours, preferably pH 1 or less, the temperature is 80 to 120 ° C., and 2 to 6 hours are optimum. The alkali treatment is carried out by adding alkali hydroxide, alkali carbonate, etc., usually at a pH of 10 or higher, and a heating temperature of 60 to 150.
C, heating time 10 minutes-20 hours, PH13 or more, temperature 80-120 degreeC, 10-15 hours are optimal. The pH of the alkaline treatment liquid is adjusted to 7.0 or less, preferably 1.0 to 6.5 using sulfuric acid, hydrochloric acid, nitric acid or the like as an acid for neutralization. As the extraction solvent, an organic solvent that separates from water, such as n-hexane, chloroform, isooctane, petroleum ether, dichloroethane, methyl isobutyl ketone, butanol, ethyl acetate can be used. At this time, a solvent prepared by mixing two or more kinds of hydrophilic solvents such as methanol, ethanol, isopropyl alcohol and acetone in an amount that does not hinder the separation of the extraction solvent and the saponification solution may be used.

The extract is washed with water, dehydrated, concentrated under reduced pressure, the concentrate is dissolved in a small amount of n-hexane, cooled to remove precipitated sterols, and then silica gel, alumina, etc. are used in this order. -Phase column chromatograss treatment removes contaminants such as ubiquinone and sterols, and reverse-phase column chromatograph treatment using ODS gel, HP resin, etc. is performed to remove mixed fatty acids such as palmitic acid and oleic acid. A high-purity POA can be obtained by removing the image.

〔The invention's effect〕

The extraction and purification step of POA by the method of the present invention is simple and it is possible to avoid the use of expensive enzymes such as lipase. Therefore, as a method for producing POA from yeast cells, it is industrially advantageous and has a high purity and a high purity. POA can be recovered.

〔Example〕

 Hereinafter, a specific description will be given with reference to examples.

Example 1. Glucose 1000 g, phosphoric acid 65 g, ammonium sulfate 20 g, magnesium sulfate · 7H ₂ O 30 g, zinc sulfate · 7H ₂
O 3g, ferrous sulfate, 7H ₂ O 3g, copper sulfate, 7H
₂ O 150 mg, manganese sulfate 2-4 H ₂ O 300
Aerobic culture of commercially available baker's yeast (trade name: Kaneka Red Yeast) for 60 hours with a jar fermenter in a medium (adjusted to PH 5.0) consisting of mg, 55 g of potassium chloride, 3 g of sodium chloride, 30 g of yeast extract, and 15 tap water. Then, the bacterial cell suspension 2 separated and washed with water (dry cell mass of 30
2g) was prepared. 20 ml of concentrated sulfuric acid is added to this cell suspension (PH1 or less), and the mixture is heated at 100 ° C. for about 5 hours. After cooling, 500 g of sodium hydroxide is added (PH 14 or more) and the mixture is further heated at 100 ° C. for about 10 hours. After cooling
325 ml of concentrated sulfuric acid was added to adjust the pH to 5.5, a mixed solution of n-hexane 5 and isopropyl alcohol 0.5 was added to this, and the mixture was extracted with stirring. After standing, the n-hexane layer was separated. This extract is washed 3 times with water of 5, and only the upper n-hexane layer is concentrated under reduced pressure. This concentrate was dissolved in a small amount of n-hexane and then cooled to 4 ° C. to remove the precipitated sterols, and the solution was applied to a column packed with 130 g of silica gel of Merck & Co., Inc. and mixed with n-hexane-ethyl acetate (10
It was eluted at 0: 0.2). The eluate fraction of POA fractionated with ubiquinone and sterols was concentrated under reduced pressure to obtain 3.2 g of oil having a POA purity of 56% (yield 54%). In order to prepare higher purity POA, 3 g of the obtained oil was dissolved in a small amount of ethanol, and then YMC and ODS gel 4
Apply to a column packed with 5 g of ethanol-water mixture (60:
40) was eluted. The high purity elution fraction of POA fractionated with fatty acids such as palmitic acid and oleic acid was concentrated under reduced pressure to obtain 1.0 g of POA oil having a purity of 98% or more (yield 60%). This product is cis based on the results of gas chromatography, thin layer chromatography and infrared absorption spectrum analysis.
It was confirmed to be -9-hexadecenoic acid (palmitoleic acid). After culturing with the method of the present invention and the same method as described above, extraction from the wound yeast cells with a chloroform-methanol mixture (2: 1), which is a conventional method, and saponification treatment, the same as the method of the present invention The results of preparing high-purity POA by operation are shown in Table 1 for comparison.

Example 2. Chloroform-methanol mixture of extraction solvent (10: 2)
And other conditions are the same as those in the first embodiment.
high purity POA from e yeast IFO 2229
POA oil 1.7 with a purity of 98% or higher
g was obtained. The results are shown in Table 1 together with the results obtained by only the alkali treatment as the extraction pretreatment.

Example 3. 200 g of commercially available dry brewer's yeast cells was adjusted to a cell suspension 2 with tap water. This bacterial cell suspension was treated in the same manner as in Example 1 above to obtain 0.5% POA oil with a purity of 98% or more.
g was obtained, and the result and the result of extraction and purification by a conventional chloroform-methanol mixture (2: 1) from the dried cells were shown in Table 1.
It was shown to.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Tsutomu Ikeda 3-2-19 Ibosaki Takasago, Hyogo Prefecture (72) Inventor Shozo Higuchi 2-14-3 Aoyamadai, Tarumi-ku, Kobe City, Hyogo Prefecture (72) Inventor Kawachi Yoshihiro 1922-323 Nojimachi, Kusatsu City, Shiga Prefecture

Claims (2)

[Claims] 1. Palmitoleic acid (hereinafter abbreviated as POA)
The yeast cell suspension containing yeast is preheated with an acid,
Next, a method for producing palmitoleic acid, which comprises saponifying with an alkali and then extracting under acidic conditions with a solvent that is immiscible with water to collect an oil containing free POA. 2. Crystallization of oil containing free POA and removal of contaminants by normal phase gel chromatography followed by reverse phase gel chromatography to obtain 98% or higher high-purity POA oil. The manufacturing method according to claim 1.