JPH02268690A - Production of lipid containing bishomo-gamma-linolenic acid - Google Patents

Abstract

PURPOSE:To produce the title lipid in a large amount inexpensively from the culture mixture by adding sesame oil to the culture medium in the cultivation of a microorganism producing a lipid containing bishomo-gamma-linolenic acid in Conidiobolus. CONSTITUTION:A microorganism in Conidiobolus, capable of producing lipid Containing bishomo-gamma-linolenic acid, preferably Conidiobolus nanodes CBS 183/62) is cultured in a medium containing sesame oil and the subject lipid is collected from the culture mixture. The cultivation is carried out usually in a liquid medium aerobically under stirring. The temperature is preferably 20 to 30 deg.C and the time is 3 to 5 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for inexpensively mass-producing a lipid containing bishomo-γ-linolenic acid (8-11-14 eicosatrienoic acid).

[Prior Art and Problems to be Solved by the Invention] Microorganisms that produce bishomo γ-linolenic acid include the genus Mortinilla, Entomofusora, and Conidiobolus. However, when these microorganisms are cultured using glucose as a carbon source, there is a drawback that the production amount of bishomo-γ-linolenic acid is low.

[Means for Solving the Problems] Therefore, the present inventors investigated ways to improve the productivity of bishomo-γ-linolenic acid, and found that by adding sesame oil or components in sesame oil to the culture medium, bishomo-γ-linolenic acid - It was discovered that linolenic acid can be efficiently mass-produced, and the present invention was completed based on this knowledge.

That is, the present invention uses a microorganism that belongs to the genus Conidiobolus and has the ability to produce bishomo-γ-linolenic acid-containing lipids.
Cultured in a medium containing sesame oil, bishomo-
The present invention provides a method for producing a bishomo-γ-linolenic acid-containing lipid, which comprises collecting the γ-linolenic acid-containing lipid.

Examples of microorganisms that belong to the genus Conidiobolus and have the ability to produce bishomo-γ-linolenic acid-containing lipids used in the present invention include Conidiopolus nanodes (Conidiopolus nanodes).
idiobolus nanodes) CB5183
/62. Conidiobolus lamblausinis (Con
diaboIus lamprauges) 8TCC
12585, Conideiobolus heterosporus (Co
Mouth 1dioboIusheterosporus) A
Examples include TCC12941. Among these, Conidiobolus nanodes CB5183/62 is preferred.

In the present invention, it is essential that the medium for culturing the above-mentioned microorganism to produce bishomo-gamma-linolenic acid-containing lipid contains sesame oil or components in sesame oil. Here, sesame oil refers to the oil obtained from sesame seeds, and can be used for both edible and medicinal purposes, particularly preferably those with pressed ends, and further extracted from sesame seeds or ground sesame seeds with dichloromethane-methanol, etc. Sesame oil, an extract obtained by steam distilling a press-extracted product, and an extract obtained by removing triglycerides in sesame oil by silica gel column chromatography can also be used. The amount of sesame oil added is 0.1-1
5 voR%. It is preferable to add sesame oil to the medium before the initial cultivation, but it may be added during the cultivation.

As a medium for culturing the above-mentioned microorganisms, a medium containing a carbon source, a nitrogen source, inorganic salts, etc. is used. In addition to the above-mentioned sesame oil, carbohydrates such as glucose, olive oil, safflower oil, and γ-linolenic acid-containing fats and oils, as well as fats and oils, are used as carbon sources. Among these carbon sources, γ-linolenic acid-containing fats and oils are preferred. Here, as the γ-linolenic acid-containing oil and fat, evening primrose oil; Mortierella fMort
Examples include microbial oils extracted from filamentous fungi belonging to the genus Ierella J, the genus Mucor, CunniBhamella X, and the like.

A plurality of types of these carbon sources may be used, or they may be added during the culture. Usually, these carbon sources may be added to the culture medium at 2 to 15 voR%.

In addition, yeast extract and peptone are nitrogen sources.

Organic nitrogen sources such as soybean meal are preferred, and inorganic salts include potassium phosphate (KH2PO4) and iron salts (FeSO4.
71120), magnesium salt (MgSO4・7N, 0
), zinc salt (ZnSO4), etc. are used. In addition, trace elements and nutritional sources can be added as necessary.

The above-mentioned microorganisms are usually cultured in a liquid medium by shaking culture 1 aerated agitation culture, etc., and the culture temperature, time, etc. are set in such a way that the desired amount of lipid produced is high, taking into account the properties of the microorganism, etc. You just need to set the conditions, usually 15 to 4
It is carried out at 0°C, preferably 20-30°C, for 2-7 days, preferably 3-5 days.

After producing the desired lipid in this manner, the lipid is collected from the culture. Bishomo-γ+ IJ phosphoric acid-containing lipids can be collected directly from the culture, but since the culture contains unused sesame oil and fats and oils added as a carbon source, it is necessary to isolate the bacterial cells from the culture. It is preferable to collect the target lipid from the obtained bacterial cells. In this case, conventional methods for separating and purifying bishomo-gamma-linolenic acid may be used, such as solvent extraction, chromatography, etc.

[Example] Next, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.

Comparative Example 1 Glucose 3 was added as a carbon source to a medium with the composition shown in Table 1.
A culture medium was prepared in which 3 vo JZ% oil or 3 vo C% oil containing γ-lylunic acid was added. 50 mρ of this medium
0mA! Conidiobolus nanodes CB5 183 was placed in an Erlenmeyer flask and sterilized at 121'C for 15 minutes.
156 was inoculated and cultured with shaking at 30°C for 4 days.

Table 1 Kl (2PO4 MgSO4 Peptone yeast extract rough FeSO4 1g 0g 5 g 0, 01 g After culturing, collect bacterial cells by centrifugation and wash with phosphate buffer (p) 17.0) After that, the bacterial cells were collected by suction filtration. The cells were placed in an aluminum cup, Glass Peas methanol chloroform was added, the cells were crushed using a homogenizer, and the lipids inside the cells were extracted. The extracted lipids were methyl esterified and the fatty acid composition was analyzed by gas chromatography. The results are shown in Table 2.

Table 2 (g#) (g#+) Glucose 19.0 3.8 Olive oil
21.6 B, 3 Safflower oil 26.6 1
0.2 (g/R) 0.06 0.21 Rate (ne) 12.0 6.1 (g/R) 0.46 0.50 Bishomo-γ-linolenic acid can be identified using the following method. This was done by A sample of bishomo-γ-linolenic acid and a methyl ester of lipid extracted from bacterial cells were mixed and subjected to capillary gas chromatography (column: PEG).
20M), the peak of the bishomo-γ-linolenic acid fraction became large. In addition, the triene fraction was collected from the methyl esterified lipid extracted from the bacterial cells by thin layer chromatography impregnated with silver nitrate.

This fraction contained γ-linolenic acid and bishomo-γ-linolenic acid. From the separated triene fraction, bishomo-
γ-linolenic acid was fractionated. This bishomo-γ-linolenic acid was derivatized with picolinyl and identified by capillary gas mass spectrum.

Example 1 Sesame oil (37% oleic acid, 44% linoleic acid) was added to the medium shown in Table 1 at 3 to 5 vo, ff% per 1°C of the medium.
A medium was prepared containing the following. This medium 100mA! 500
m! ! Conidiobolus nanodes [:BS 183762] was inoculated into the Erlenmeyer flask, and cultured with shaking at 30°C for 4 days. After the cultivation was completed, lipids within the bacterial cells were analyzed in the same manner as in Comparative Example 1. The results are shown in Table 3.

Table 3 fvog main) (g#) (g/i') (*)
(g#) rate (U (g#)3 26.0
8.0 8.8 0.70 11.1 0
．． 884 2B, 2 9.4 7.9
0.74 7.6 0.715
31.0 10.7 6.7 0.72
6.8 0.73 As is clear from Table 3, 5v
The bacterial cell yield reached almost the maximum at 1% of. Furthermore, the content of bishomo-γ-linolenic acid was highest at 3 vofL%, 8.8%, and the yield was highest, 0.74 g/R, at 4 vofL%.

Example 2 In Example 1, sesame oil was added to 3 vou% per 1°C of the medium, and Conidiobolus heterosporus AT (:(
: The same procedure as in Example 1 was carried out except that 12941 was used.

As a result, the bacterial cell yield was 25.1 g/i', the oil yield was 8.0 g/i', the bishomo-γ-linolenic acid content was 42%, and the yield was 0.34 g/f! Met. In addition, the content of arachidonic acid is 1.4%, and the yield is o
, ttg/a.

Example 3 A γ-linolenic acid-containing oil (40% oleic acid, 10% linoleic acid) was added to the culture medium shown in Table 1 as a carbon source.

γ-linolenic acid 16%) 1vo, Q% and sesame oil 2
A medium containing 1% vof was prepared. 100m of this medium
Conidiobolus nanodes CB5183/62 was inoculated into an Erlenmeyer flask at 500 mA' of N, and cultured at 30°C for 4 days. After the culture was completed, lipids within the bacterial cells were analyzed in the same manner as in Comparative Example 1. The results are shown in Table 4.

Table 4 29.1 9.3 8.0
0.74 Example 4 3 g of sesame oil was fractionated using a silica gel column. First, when extracted with hexane, the hexane extracted fraction was mostly triglyceride, and was approximately 2.7 g. Next, the residue after extraction with hexane was extracted with chloroform, and the residue was further extracted with methanol to obtain an extracted fraction (chloroform/methanol extraction fraction) of approximately 300 mg.
Met.

The following experiment was conducted using these two parts.

Conideiobolus nanodes (BS 18
3/62 was inoculated and cultured with shaking at 30°C for 4 days. After the culture was completed, lipids within the bacterial cells were analyzed in the same manner as in Comparative Example 1. As a result, the content of bishomo-γ-linolenic acid was 13% and the content of arachidonic acid was 5.7%, and the yield of bishomo-γ-linolenic acid was 1.1 g/f and the yield of arachidonic acid was 0.47 g/! ! Met.

Example 5 Sesame oil was added to the medium shown in Table 5 at a ratio of 9 to 9 for medium IJ2.
A medium containing vou% was prepared. This medium 6.12
Place in a jar fermenter and heat at 121℃ for 15 minutes.
Sterilized for minutes. Here, add 3v of sesame oil to the medium shown in Table 1.
Medium 600+ni with 2% oj added! Conidiobolus nanodes precultured with BS 183/62 was inoculated with BS 183/62, and cultured with aeration and agitation at 30°C for 4 days. After the culture was completed, lipids within the bacterial cells were analyzed in the same manner as in Comparative Example 1. As a result, the total bacterial cell yield was 480g, and the total fat and oil yield was 168g.
The content of total bishomo-γ-linolenic acid was 7.1%, and the yield was 12°Og.6 The yield of bishomo-γ-linolenic acid was 2.0g per medium. Also,
The content of arachidonic acid is 4.8%, and the yield is 1.3
g/x.

Table 5 shows H2PO4 gSO4 Peptone yeast extract rough FeSO4-7) i20 Distilled water g 0g To 15g 0.033 fL Example 6 A mixture of equal amounts of sesame oil, olive oil and safflower oil was added to the medium shown in Table 5. A medium was prepared in which 9 voβ% was added to 11. This medium 6JZ was placed in a 10 fl jar fermenter, and Conideiobolus nanodesces xa3/62 was precultured in the same manner as in Example 5.
was inoculated and cultured with aeration and agitation at 30°C for 4 days. After the culture was completed, lipids within the bacterial cells were analyzed in the same manner as in Comparative Example 1. As a result, the total bacterial cell yield was 420g, and the total fat and oil yield was 132g.
g, total bishomo γ-linolenic acid yield was 789 g. The content of bishomo-gamma-linolenic acid was 6.0%, and the yield was 1.3 g per 1 fl of the medium. The content of arachidonic acid is 4.3%, and the yield is x, og
/p.

Example 7 A culture medium having the composition shown in Table 5 was prepared by adding sesame oil in Qvo°C% based on medium IIl. 6k of this medium
Conideiobolus nanodes CBS 1f was placed in a jar fermenter and precultured in the same manner as in Example 5.
13/62 or Conidiobolus lamblausinis ATCC 12585 was inoculated and cultured with aeration and agitation at 30'C for 4 days.

After the cultivation was completed, lipids within the bacterial cells were analyzed in the same manner as in Comparative Example 1. The results are shown in Table 6.

Table 6 (g#) (g/ff) Seagull) (g#)
(g#) CBS 183/62 ATCC12585 Comparative Example 2 Except for using γ-linolenic acid-containing oil (40% oleic acid, 10% linoleic acid, 16% γ-linolenic acid) instead of sesame oil in Example 7. The same operation as in Example 7 was performed. The results are shown in Table 7.

Table 7 (g#) (Ro/ρ) Import) (g#) (g/++) CBS xa3/a2 Conidiobolus lamblausinis 55 3 18.8
3.5 0,66 6.58 ATCC1258
5 Example 8 The same procedure as in Example 4 was carried out except that 30 m8 of each of sesamin and sesamolin separated from sesame oil was used instead of the methanol/chloroform fraction and γ-linolenic acid-containing oil of Example 4. Ta. The results are shown in Table 8.

Table 8 (g#ri (g#') (home) (g#) Chongyang)
(g/-li sesamin 25.0 B, 6
15.8 136 7.3 0.63 Seven Samorins 27.7 8.7 12.5 1
．． 09 10.0 0.87 [Effect of the invention] According to the present invention, when producing bishomo-γ-linolenic acid using microorganisms, bishomo-γ-linolenic acid can be efficiently produced by adding sesame oil to the culture medium. , can be mass-produced at low cost. The obtained bishomo-γ-linolenic acid is useful as a medicine and a biochemical reagent.

Claims (1)

[Claims] (1) A microorganism belonging to the genus Conidiobolus and capable of producing bishomo-γ-linolenic acid-containing lipids is cultured in a medium containing sesame oil, and bishomo-γ-linolenic acid-containing lipids are collected from the culture. Characterized bishomo-γ-
Method for producing linolenic acid-containing lipid.