JPH01300897A - Production of delta6,9,12,15-octadecatetraenoic acid-containing lipid - Google Patents

Abstract

PURPOSE:To stably obtain the title compound useful for drugs, etc., through a fermentation process, by making a culture of octadecatetraenoic acid- productive microorganisms in a culture medium with, as a carbon source, a fatty acid to be a precursor of octadecatetraenoic acid. CONSTITUTION: <6>,<9>,<12>,<15>-octadecatetraenoic acid-productive microorganisms (e.g., Mortiella elongata IFO 8570) is put to culture in a culture medium with, as a carbon source, a fatty acid (pref. linoleic acid) to be precursor of <6>,<9>,<12>,<15>- octadecatetraenoic acid to obtain the objective lipid. It is suggested that the culture be, normally, made in a liquid medium through a shaking culture process. Also the culture temperature is pref. 20-30 deg.C, and the culture time 4-8 days.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for producing a lipid containing Δ6,9,12,15-octadecatetraenoic acid (hereinafter abbreviated as Cl11+4), and specifically relates to a fermentation method. This invention relates to a method for efficiently producing Cl64-containing lipids.

[Conventional technology 3 Problems to be solved by the invention] CI8+
4 is useful as a medicine, biochemical reagent, etc. This substance is naturally found in the family Baraginaceae.
Its location is known in seeds, plankton, aquatic organisms, etc., and conventionally it has only been obtained by extraction from these natural products.

Therefore, it has not been possible to obtain it in a stable quantity, and its use has been severely restricted.

[Means for Solving the Problem] As a result of repeated studies to develop a method for stably producing C184, the present inventor discovered that 0184 could be produced by a fermentation method using a specific microorganism, and Based on this knowledge, we have completed the present invention.

That is, the present invention provides a method for producing a C16+4-containing lipid, which comprises culturing a microorganism capable of producing C184 as a carbon source in a medium containing a fatty acid that is a precursor of C114, and collecting a C164-containing lipid from the culture. It provides:

The microorganism used in the present invention is a microorganism capable of producing C384, such as Mucor circinelloides (
Mucor circinelloides)HUT
1123゜ Same HU T1121 (FERM P-935
9), Mukor Zuyavanix (lid, Java
icus) HllT 1162 (FERM P-936
0), + = Dioborus heterosporus (Cont
dioborusheterosporus) 8TCC
12586, Mortierella elongata (Morti)
ella elongata) IPo 8570.

The medium for culturing the above microorganisms uses C3 as a carbon source.
It is essential to contain a fatty acid that is a precursor of 84. Such fatty acids have 18 carbon atoms and 1 to 1 double bond.
The unsaturated fatty acids of No. 3 are suitable, and specific examples thereof include linoleic acid, α-lylunic acid, and the like. Linseed oil, safflower oil, sunflower oil, perilla oil, etc. can be used as substances containing such fatty acids. These fatty acids can be used in the form of derivatives such as bakaesters of the free acids. The above-mentioned fatty acids are preferably added to the medium at a rate of 0.5 to 10 voρ% as fatty acids.

As a carbon source, in addition to the above-mentioned fatty acids, carbohydrates such as glucose, starch, and blackstrap molasses can also be used in combination, and in this case, the appropriate amount of addition is 2 to 5 van%.
.

Further, as nitrogen sources, those containing organic nitrogen such as yeast extract, polypeptone, malt extract, corn steep liquor, etc., and those containing inorganic nitrogen such as (N144) 2504 are used. Furthermore, as inorganic salts, magnesium salts (Mg5o4, 7H20, etc.), potassium phosphate (
KH2PO4, etc.), iron salts (FeS04/7) 120)
.

Zinc salt (ZnSO4) or the like is used, and other trace elements and other nutritional sources can be added as necessary.

The microorganisms are usually cultured in a liquid medium by shaking culture with aeration and agitation, and the culture temperature for 1 hour is set under conditions that will increase the production of the desired lipid, taking into consideration the properties of the microorganisms. , usually 15 to 40
C., preferably 20 to 30.degree. C., for 2 to 10 days, preferably 4 to 8 days. In addition, the microorganism can be cultured for about 2 to 4 days in a medium containing carbohydrates or fats and oils as a carbon source, and the culture can be continued by adding the fatty acid to this preculture. Furthermore, increasing the dissolved oxygen concentration during culture (for example, increasing the pressure inside the culture vessel to 2 atm) is also a C1
Effective for 84 production.

After completion of the culture, C1♂4 is collected from the culture.

et84 may be collected directly from the culture, but since the culture also contains fats and oils added as a carbon source,
It is preferable to separate bacterial cells from the culture and collect C184 from the bacterial cells. CI8+4 can be collected by conventional methods such as solvent extraction and chromatography.

According to the present invention, C184 per microbial culture solution Ik is 0.
It can be stably produced at a ratio of about 1 to 0.4 g.

[Examples] Next, the present invention will be explained using Examples, but the present invention is not limited thereto.

Example I In order to search for C464-producing strains, linseed oil (15% oleic acid, 15% oleic acid,
Contains 16% linoleic acid and 60% α-linolic acid) in medium 1
2 voρ% per base was added. This medium 10
0mj+ was placed in a 5001 Erlenmeyer flask, and predetermined microorganisms were inoculated and cultured with shaking at 28°C for 2 days.

Table 1 Polypeptone 0.3g Yeast extract 0.3g (N)+4)2504 1.5' gK
LPL 1. OgMgSO4・78
20 0.5 gFeSO41H200,
01g CII3COONa 5. Og distilled water 1. After the completion of OIl culture, the culture solution was separated into bacterial cells and supernatant by centrifugation, and the obtained bacterial cells were added to 0.1 M phosphate buffer (pH 7,
After washing with 0), the bacterial cells were separated by suction filtration.

The oils and fats in the bacterial cells were extracted by adding methanol and chloroform to a mixture of the bacterial cells and glass peas, and homogenizing the mixture. After converting the extracted oils into methyl esters, the fatty acid composition was examined using gas chromatography. .

Table 2 shows the measurement results of the C1B+4 production ability of each microorganism. Note that C184 was identified by the following method.

(2) C384 contained in seaweed was separated by silica gel column chromatography impregnated with silver nitrate. This C184
When the oils and fats extracted from the bacterial cells as described above were analyzed using capillary gas chromatography (Column 2 PE020M), the retention times of the two were found to match. Also, these 2f! When these oils and fats were mixed and analyzed by capillary gas chromatography, the peak of the Cll1J fraction became large.

(2) A toluene fraction and a tetraene fraction were separated by thin layer chromatography impregnated with silver nitrate, each fraction was oxidized by osmium oxidation method, and further converted into trimethylsilyl ether, and identified by capillary gas mass spectrum.

Example 2 In order to examine the oil concentration in a medium suitable for C164 production, the above-mentioned linseed oil was added in various proportions to medium 11 having the composition shown in Table 1, and each medium was treated with Mucor circinelloides) ItlT 1121. (FEBMP-93
59) and cultured with shaking at 28°C for 2 days.

After the culture was completed, lipids within the bacterial cells were analyzed by the method shown in Example 1, and the results are shown in Table 3.

10 8.61 39.74 3.42 1.97
0.06320 15.43 55.45 8.55
1.0B 0.08930 20.13 60.
08 12.10 0.79 0.09450 19
．． 24 58.25 11.21 0.81 0.09
1 Example 3 Safflower oil (containing 12% oleic acid and 77% linoleic acid) was added as a carbon source to a medium having the composition shown in Table 1.
A medium was prepared by adding 2 voj% or 4 vo1% per jZ. 500% of this medium 1001111'
It was placed in a 15J Erlenmeyer flask, inoculated with Mucor circinelloides HUT 1121 (FERM P-9359), and cultured with shaking at 28°C for 2 days.

After completion of the culture, intracellular lipids were analyzed by the method shown in Example 1, and the results are shown in Table 4.

2G 17.29 51.92 8.9B Q,
IQ 0.0'0B40 27.75 51.84
14.33 0.12 0.016 Example 4 A medium was prepared by adding α-lylunic acid ethyl ester as a carbon source to a medium having the composition shown in Table 1 at a rate of 1 voR% per medium tjZ. This medium is 100mj! 50
It was placed in a 0 ml Erlenmeyer flask, inoculated with Mucor circinelloides H11T 1121 (FERM P-9359), and cultured with shaking at 28°C for 3 days.

After completion of the culture, intracellular lipids were analyzed by the method shown in Example 1, and the results are shown in Table 5.

Table 5 10 8.63 35.22 3.04 3.64
0.111 Example 5 Five volumes of the culture medium having the composition shown in Table 6 were placed in a 102 jar fermenter, sterilized at 121 t for 15 minutes, and then precultured using 600++ l of the same medium. Mucor circinelloides HUT 1123 and incubated at 28℃ for 4 hours.
Aerated agitation culture was performed for days.

After the culture was completed, the cells were collected and freeze-dried, and the lipids inside the cells were extracted with hexane using a ball mill. The obtained fats and oils were analyzed by the method shown in Example 1, and the results are shown in Table 7.

Example 6 The same procedure as in Example 5 was carried out, except that the pressure in the culture vessel was set to 2 atm, thereby increasing the amount of dissolved oxygen in the culture solution to 1100 pp or more. The results are shown in Table 7.

As is clear from the table, the CIa:4 yield increased about 1.8 times compared to when dissolved oxygen was not controlled (Example 5).

Table 6 Linseed oil 50.0 g Polypeptone
0.3g Yeast extract 0.3g (NH4) 2504 7.5 gKH
2PO41,O8 MgSO4・7H200,5g FeSO4'7820 0.01g Distilled water 1. On No. 7 Table 5 20.583!1.30 B, Q! l 2. Q3
(1,16462B, 0431.928.313.63
0.302 Comparative Example 1 Two types of media were prepared by adding 30 g/R of glucose to the culture medium shown in Table 1 and 30 g/R of linseed oil, and adding Mucor circinelloides HUT 112 to these.
1 (FERM P-9359) and incubated at 28℃ for 2 hours.
Culture was performed for 1 day.

After completion of the culture, intracellular lipids were analyzed by the method shown in Example 1, and the productivity of C184 was compared. The results are shown in Table 8.

As is clear from the table, no production of C284 was observed when glucose was used as the carbon source.

Table 8 Curcose 13.52 35.05 4.7
4--7 Seed oil 20.13
60.08 12.10 0.79 0.0
94 Example 7 The same procedure as in Example 6 was carried out except that the test strain was changed to Mucor circinelloides HUT 1121 (FERM P-9359) and the culture period was changed to 5 days. The results are shown in Table 9.

As is clear from the table, when using the HllT 1121 strain, the CI8+4 was the same as when using the OUT' 1123 strain.
Productivity was seen.

Table 9 30.07 34.47 10.37 3.00 0.
311 [Effects of the Invention] According to the present invention, (
:la4 can be stably produced by fermentation production using microorganisms.

Patent applicant: Idemitsu Petrochemical Co., Ltd.

Claims (1)

[Claims] Δ^6^, ^9^, ^1^2^, ^1^5 Using microorganisms capable of producing octadecatetraenoic acid as a carbon source Δ^
6^,^9^,^1^2^,^1^5- Cultivate in a medium containing fatty acids that are precursors of octadecatetraenoic acid, and extract Δ^6^,^9^,^1 from the culture. Δ^6^,^9^,^1^2^,^1^5-octadecatetraenoic acid characterized by collecting lipids containing ^2^,^1^5-octadecatetraenoic acid Method for producing contained lipids.