JPH07274983A - Production of oil and fat containing long chain highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To obtain an oil and fat containing a long chain highly unsaturated fatty acid having a pharmacological effect, in a high concentration efficiently, by culturing a microorganism belonging to Candida, etc., having the activity of hydrolyzing the oil and fat, together with the oil and fat, and collecting the product by chemical treatment. CONSTITUTION:A microorganism belonging to Candida, Penicillium, Aspergillus, Pseudomonas or Alcaligenes, having the hydrolyzing activity of an oil and fat (e.g. Candida rugosa ATCC14830, etc.) is inoculated to a medium containing the oil and fat (e.g. fish oil, etc.), and the oil and fat are extracted by liquid - liquid partition with hexane after culturing at 28 deg.C for 60hrs. Then, by subjecting the product to a deacidification treatment with an alkali, the objective oil and fat which contains a long chain highly unsaturated fatty acid having physiological activity and a pharmacological effect to human in a high concentration.

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 a long-chain highly unsaturated fatty acid-containing oil or fat, and particularly to a high-concentration long-chain highly unsaturated fatty acid from an oil or fat containing a low concentration of long-chain highly unsaturated fatty acid. The present invention relates to a method for producing fats and oils containing.

[0002]

2. Description of the Related Art Recently, long-chain polyunsaturated fatty acids (hereinafter abbreviated as PUFA) have been attracting attention for their physiological activity and pharmacological effect on humans, and their utilization has been actively studied. Enzyme treatment with lipase has been mainly used for the preparation of this PUFA-rich product. For example, it is prepared through treatments such as esterification of docosahexaenoic acid (DHA), a free fatty acid derivative, using lipase, hydrolysis of fish oil triglyceride, and the like.

The above-mentioned enzyme is usually obtained from microorganisms, but when used as an enzyme as described above, there is a problem that an extraction and purification step is required and the production cost is high. Also, purchasing an enzyme was expensive. As a means for solving these problems, it is possible to utilize fermentation using the microorganism as it is. Fermentation does not require the step of extracting and purifying the enzyme as described above, and facilitates PU
High FA content products can be prepared.

However, to date, there has been no report of a method for preparing a PUFA-rich product by fermentation. This is because PUFA is a lipophilic substance. The process of fermentation is generally applied to water soluble substances and not lipophilic substances. Exceptionally, the sterol fermentation method is known as a fermentation method for lipophilic substances, but the substrate concentration is 5
% Was the limit, and it was difficult to call it an efficient fermentation method (Attila Szentirmai, J of Industrial Microbiolo
gy Vol. 6 (1990) 101-115). In addition, as an example of using microorganisms for fats and oils, in the production of cocoa substitute fats, there is an example in which lipase is accumulated in the cells of Rhizopus quinensis and the dried cells are used to treat the oils and fats. However, this was a chemical reaction that utilized accumulated lipase as a catalyst (Tsuneo Yamane, Oil Chemistry Vol.40, No.10, 1
93-201 (1991)).

[0005] Recently, since organic solvent-resistant bacteria have been isolated by Inoue and Higoshi, the momentum of using the fermentation method for lipophilic substances has been increasing, and especially as a countermeasure for environmental pollution by petroleum. Interest in (K.
Moriya and K. Horikoshi, Jof Fermentation and Bioe
ngineering Vol. 76, No. 5, 397-399 (1993)), still PUF
No attention is paid to the use of A for the preparation of high content products.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently producing fats and oils containing a high concentration of long-chain highly unsaturated fatty acids using microorganisms.

[0007]

Means for Solving the Problems As a result of intensive research in view of the above problems, the present inventors have been able to produce fats and oils containing a high concentration of PUFA by fermentation by culturing microorganisms together with fats and oils, and The present invention has been completed by finding a microorganism suitable for producing a high-concentration PUFA-containing fat or oil by fermentation.

That is, according to the present invention, a microorganism belonging to the genus Candida, the genus Penicillium, the genus Aspergillus, the genus Pseudomonas or the genus Alcaligenes, which has a hydrolyzing ability for fats and oils, is cultivated with the fats and oils, and a long-chain highly-adhesive protein is obtained from the obtained culture solution. A method for producing a long-chain highly unsaturated fatty acid-containing oil or fat, which comprises preparing a saturated fatty acid-containing oil or fat.

The present invention will be described in detail below. [1] Fats and oils The fats and oils used in the present invention are long-chain highly unsaturated fatty acids (PUFA).
Specifically, fish fat, whale fat, krill oil, marine chlorella oil and mixed fats and oils containing these fats, conjugated isomerized oils or partially hydrogenated oils may be used. It is preferable to use fish oil from the viewpoint of the content of PUFA. These fats and oils typically contain 15-55% by weight of PUFA.
It is included. In the present invention, the long-chain highly unsaturated fatty acid means a fatty acid having 18 or more carbon atoms per molecule and 3 or more double bonds.

[2] Microorganism The microorganism used in the present invention may be any microorganism as long as it can produce a high-concentration PUFA-containing fat or oil from a low-concentration PUFA-containing fat or oil by fermentation. Such microorganisms include, for example, Candida (Candi).
da), Aspergillus (Aspergillus) genus, Pseudomonas (Pseudomonas) genus, Penicillium (Penicillum) genus, Alcaligenes (Alcaligenes) genus and the like microorganisms, specifically, Candida rugosa ATCC1483
0, Candida Eronobi ATCC20000, Aspergillus Orisee ATCC14605, Aspergillus Orisee
Viridis ATCC26850, Pseudomonas ATCC21808, Penicillium roquehorti IFO4622, Penicillium caseicolum ATCC24936, Alcaligenes ATCC31371, etc. are used.

[3] Method for Producing Long-Chain Polyunsaturated Fatty Acid-Containing Glyceride In the present invention, an oil or fat containing a high concentration of PUFA is produced by culturing the above-mentioned microorganism in a medium together with the oil or fat. Hereinafter, an example of the manufacturing method will be described. First, the microorganism to be used is cultivated in a medium, and when the microorganism has undergone logarithmic growth, PUFA-containing fats and oils are added, followed by culturing. The amount of fat or oil added is preferably 1 to 500% by weight of the culture solution, and particularly preferably 10 to 100% by weight. Examples of nutrients of the medium include polypeptone, potato extract, malt extract, yeast extract, bactopeptone, soybean flour, starch, phytonpeptone, meat extract and the like. As the inorganic salts, ammonium sulfate, magnesium sulfate, calcium chloride, dipotassium phosphate, sodium chloride, etc. are used, but magnesium sulfate, calcium chloride is included, the growth is good, and fats and oils containing high concentrations of PUFA are produced. can do.

The pH of the culture broth varies depending on the bacterium, but for example, in the case of the genus Candida, it is preferably about pH 5 to 9, particularly pH 7.
The vicinity is preferable. The culturing temperature also varies depending on the fungus, but for example, in the genus Candida, it is preferably 20 to 30 ° C, particularly 28 ° C.
Before and after is preferable. The culture / fermentation is 24 to 72 after adding oil and fat.
It is preferably carried out for a period of time, preferably with stirring.

After culturing, the culture solution is treated with an alkali by a conventional method, the oil and fat are extracted with hexane, and the hexane extract is washed with water to prepare a PUFA-containing oil and fat. The PUFA content of the constituent fatty acids in the oil / fat thus obtained is higher than the initial concentration.

[0014]

The present invention will be described in more detail below with reference to examples, but these examples do not limit the scope of the present invention. (Example 1) Candida rugosa ATCC14830 cultured for 12 hours
0.88 g of fish oil (DHA content: 29%) was added to 10 ml, and the mixture was cultured at 28 ° C. for 60 hours while shaking. The medium contained malt extract 0.3%, yeast extract 0.3%, bactopeptone 0.5%, calcium chloride 0.1% and magnesium sulfate 0.025% and had a pH of 6.5. This medium is hereinafter referred to as YMCM medium. After culturing for 60 hours, fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. The PUFA glyceride yield after treatment was 24%, and the constituent fatty acids in the fish oil were measured by gas chromatography to find that the DHA content was 52%.

Example 2 Aspergillus oryzae ATCC14605 was cultured in 10 ml of YMCM medium for 12 hours to give 0.88.
g of fish oil (DHA content: 37%) was added, and the mixture was further cultivated at 28 ° C. for 48 hours with shaking. After culturing, the fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. The yield of PUFA glyceride after treatment is 42%,
When the constituent fatty acids in the fish oil were measured by gas chromatography, the DHA content was 42%.

(Example 3) In Example 2, the fish oil (EP
PUFA-containing fats and oils were produced in the same manner except that A content: 19%) was used. The EPA content of constituent fatty acids in the obtained fish oil was 23%.

(Example 4) In Example 2, fish oil (DH
PUFA-containing fats and oils were produced in the same manner except that A content: 16%) was used. The DHA content of the constituent fatty acids in the obtained fish oil was 24%.

(Example 5) Aspergillus oryzae viridis ATCC26850 was cultured in 10 ml of YMCM medium for 12 hours, 0.88 g of fish oil (DHA content: 37%) was added, and the mixture was further shaken at 28 ° C for 48 hours. Cultured. After culturing, liquid
The fish oil was extracted with hexane by liquid distribution, and then deoxidized with alkali. When the constituent fatty acids in the fish oil were measured by gas chromatography, the DHA content was 42%.
Met.

Example 6 In Example 5, fish oil (DH
PUFA-containing fats and oils were produced in the same manner except that A content: 16%) was used. The DHA content of the constituent fatty acids in the obtained fish oil was 25%.

(Example 7) Alcaligenes ATCC31371
Fish oil (DHA content: 29%) 0.88 in 10 ml of liquid that was cultured for 15 hours
g was added, and the mixture was cultured at 28 ° C. for 48 hours with shaking. The medium contained 0.7% meat extract, 1.0% bactopeptone, 0.3% sodium chloride, 0.1% calcium chloride and 0.025% magnesium sulfate and had a pH of 7.0. This medium is hereinafter referred to as NB medium. After culturing, the fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali.
The PUFA glyceride yield after treatment was 38%, and the constituent fatty acids in the fish oil were measured by gas chromatography to find that the DHA content was 43%.

(Example 8) Candida eronovi ATCC 20000 was cultured in 10 ml of YMCM medium for 12 hours to give 0.88 g.
Fish oil (DHA content: 29%) was added, and while shaking 2
It was cultured at 8 ° C for 42 hours. After culturing, the fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. The PUFA glyceride yield after the treatment was 49%, and the constituent fatty acids in the fish oil were measured by gas chromatography to find that the DHA content was 40%.

(Example 9) Pseudomonas ATCC21808 was cultured in 10 ml of NB medium for 12 hours, and 0.88 g of fish oil (DH
A content: 37%), and at 42 ° C under shaking at 42
Incubated for hours. After culturing, the fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. When the constituent fatty acids in the fish oil were measured by gas chromatography, the DHA content was 52%.

(Example 10) In Example 9, fish oil (EP
PUFA-containing fats and oils were produced in the same manner except that A content: 19%) was used. The EPA content of constituent fatty acids in the obtained fish oil was 23%.

(Example 11) In Example 9, the fish oil (EP
PUFA-containing fats and oils were produced in the same manner except that A content: 16%) was used. The yield of the obtained PUFA glyceride was 22%, and the EPA content of the constituent fatty acids in the fish oil was 25%.

(Example 12) Penicillium loquehorti IFO4622 was cultured in 10 ml of YMCM medium for 12 hours to give 0.88
g of fish oil (DHA content: 29%) was added, and the mixture was further cultured at 28 ° C. for 60 hours while shaking. After culturing, the fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. The yield of PUFA glyceride after treatment is 78%,
When the constituent fatty acids in the fish oil were measured by gas chromatography, the DHA content was 35%.

(Example 13) Penicillium casei column ATCC24936 was cultured in 10 ml of YMCM medium for 12 hours, and
88 g of fish oil (DHA content: 16%) was added, and the mixture was further cultured with shaking at 28 ° C for 48 hours. After culturing, the fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. When the constituent fatty acids in the fish oil were measured by gas chromatography, the DHA content was 29%.

Example 14 In Example 13, fish oil (EP
PUFA-containing fats and oils were produced in the same manner except that A content: 19%) was used. The EPA content of the constituent fatty acids in the obtained fish oil was 24%.

(Example 15) In Example 13, fish oil (DH
PUFA-containing fats and oils were produced in the same manner except that A content: 37%) was used. The DHA content of the constituent fatty acids in the obtained fish oil was 49%.

(Example 16) Candida rugosa ATCC14830 was cultured in 50 ml of YMCM medium for 12 hours to obtain 4.4 g.
Fish oil (DHA content: 29%) was added and shaken at 28 ℃
It was cultured under 60 hours. After culturing, extract fish oil with hexane,
Then, deoxidation treatment with alkali was performed. The PUFA glyceride yield after the treatment was 24%, and the constituent fatty acids in the fish oil were measured by gas chromatography to find that the DHA content was 57%.

Comparative Example 1 4.4 g of fish oil (DHA content: 29%) was added to 50 ml of a solution of Candida rugosa ATCC14830 cultivated in YMCM medium for 12 hours, and a final concentration of 50 mM was added to kill the bacteria at the same time. Sodium azide was added, and the mixture was cultured at 28 ° C. for 60 hours while shaking. The DHA content of constituent fatty acids in the oil and fat after culturing was 30.2%. Similarly, in YMCM medium, Candida rugosa ATCC14830
After culturing for 72 hours, the fungus was killed with 50 mM sodium azide, and the DHA content of the constituent fatty acids in the fats and oils obtained by reacting with fish oil for 60 hours was 30.6%.

Since there is concern about the effect of 50 mM sodium azide on the enzyme, the effect of sodium azide on the lipase produced by Candida rugosa was examined. Fish oil in phosphate buffer (DHA content: 29%)
The DHA content at the end of the reaction was compared between the cases where the lipase was allowed to act on 0.1% of sodium, and sodium azide was added (final concentration: 50 mM) and no sodium azide was added. The reaction was carried out at 37 ° C for 15 hours. As a result, DHA in fats and oils
The content was 52% in all cases, and no effect of sodium azide was observed.

From the results of Example 16 and Comparative Example 1, under YMCM medium in which no microorganisms exist, the ability to hydrolyze fats and oils is weak,
On the other hand, after adding fats and oils, high-concentration PUFA with growth of the microorganism
It can be seen that the contained fats and oils are produced. That is, it is clear that fermentation can increase the DHA content in fats and oils.

(Example 17) Candida Lugosa ATC
Fish oil (DHA content: 29
%) 39.5 g, and the mixture was cultured at 28 ° C. for 60 hours with shaking. The medium used was YMCM medium, and the bacterial concentration when fish oil was added was about 0.7 at an absorption wavelength of 660 nm. After culturing for 60 hours, fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. The PUFA glyceride yield after treatment was 27%, and the constituent fatty acids in the fish oil were measured by gas chromatography to find that the DHA content was 41.4.
%Met.

Example 18 A PUFA-containing fat or oil was produced in the same manner as in Example 17, except that the amount of fish oil added was 23.0 g. The obtained PUFA glyceride yield was 28%,
The DHA content of the constituent fatty acids in the fish oil was 44.6%.

(Example 19) Candida rugosa ATC
Fish oil (DHA content: 29
%) 4.4 g, and cultured at 28 ° C. for 60 hours with shaking. The medium contained soybean flour 2.0%, starch 1.0%, ammonium sulfate 0.1%, dipotassium phosphate 0.5% and magnesium sulfate 0.1%, and had a pH of 6.5. After culturing for 60 hours,
The fish oil was extracted with hexane by liquid-liquid partition, and then deoxidized with alkali. The yield of PUFA glyceride after treatment was 22.8%, and the constituent fatty acids in the fish oil were measured by gas chromatography to find that the DHA content was 46.1%.
Met.

(Example 20) In Example 19, the medium contained 0.5% fish oil, 0.5% ammonium sulfate, 0.5% dipotassium phosphate and 0.1% magnesium sulfate, and had a pH of
PUFA-containing fats and oils were produced in the same manner except that the one of 6.5 was used. The yield of the obtained PUFA glyceride was 46.9%, and the DHA content of the constituent fatty acids in the fish oil was 40.0%.

(Example 21) In Example 19, as the medium, yeast extract 0.3%, ammonium sulfate
0.5%, dipotassium phosphate 0.5% and magnesium sulfate
PUFA-containing fats and oils were produced in the same manner except that those containing 0.1% and having a pH of 6.5 were used. The yield of PUFA glyceride obtained was 31.7%, and the constituent fatty acid DHA in the fish oil was
The content was 50.1%.

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, oils and fats containing a high concentration of long-chain highly unsaturated fatty acids can be efficiently produced.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication (C12P 7/64 C12R 1:69) (C12P 7/64 C12R 1:05) (C12P 7/64 C12R 1:38) (C12P 7/64 C12R 1:80)

Claims (1)

[Claims] 1. A long-chain highly unsaturated fatty acid-containing fat or oil from a culture solution obtained by culturing a microorganism belonging to the genus Candida, the genus Penicillium, the genus Aspergillus, the genus Pseudomonas or the genus Alcaligenes and having the ability to hydrolyze fats and oils, together with the fats and oils. A method for producing a long-chain polyunsaturated fatty acid-containing oil or fat, comprising: