JPS6314696A - Production of bishomo gamma-linolenic acid - Google Patents

Abstract

PURPOSE:To obtain bishomo-gamma-linolenic acid (BLA) by a simple process in high yield and inexpensively, by cultivating a specific microorganism in a medium. CONSTITUTION:Mortierella elongata SAM 0219 strain belonging to the genus Mortierella, capable of producing BLA, is inoculated into a medium containing 0.1-30wt% carbon source (e.g. glucose), 0.01-5wt% nitrogen source (e.g. peptone) and, if necessary, hydrocarbon, fatty acid (salt), fats and oils, etc., and cultivated by aerated spinner culture method, etc., at 5-40 deg.C at 4-10pH for 2-10 days to form and accumulate BLA or lipid containing BLA in the mold. Then the mold is separated from the medium, the prepared mold is extracted with methanol, etc., to give a lipid compound of BLA. Then the compound is esterified with methanol, prepared BLA methyl ester is hydrolyzed with an alkali, extracted with an ether, etc., and purified.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing bishomo-γ-linolenic acid by a fermentation method.

[Prior art]

Bishomo-γ-linolenic acid (eicosatrienoic acid) is known to exist as one of the constituent fatty acids of fish oil and seaweed. However, since its content is small, isolated and purified products are very expensive.
There is a strong desire to develop a manufacturing method with high production efficiency. In addition,
There is no known method for producing bishomo-gamma-linolenic acid by fermentation.

[Problem that the invention seeks to solve]

The present invention uses a microorganism of the genus Mortierella, which has not been known to have the ability to produce bishomo-gamma-linolenic acid, to produce high yield and simple production using an inexpensive and commonly used culture medium. It is an object of the present invention to provide a method capable of producing bishomo-gamma-linolenic acid in a step.

[Means for solving problems]

The above purpose was to cultivate a microorganism belonging to the genus Mortierella and capable of producing bishomo-γ-linolenic acid.
- A method for producing bishomo-γ-linolenic acid characterized by producing a lipid containing linolenic acid or bishomo-γ-linolenic acid and collecting bishomo-γ-linolenic acid.

[Specific explanation]

In the present invention, any microorganism that belongs to the genus Rutierella and has the ability to produce bishomo-γ-linolenic acid can be used. As such microorganisms,
For example; [Rutierella elongata (Mortier)
pjl-a mountain+n-Ba4+13 IPO8570
, Mortierella exigua Qj42r-1jarL
! 1laJ fee and needle IFO8571, Morticorella・
Mortierella h33ro
pj+i-j-g) IPo 5941 and the like. All of these strains can be obtained manually without any restrictions from the Fermentation Research Institute.

In addition, the present inventors isolated the bacterial strain Mortierella elongata SAM 0219 (m.
No. 03) can also be used.

Next, the above-mentioned bacterial strain SAM 0219 (Feikoken Bacteria No. 8
703) is described.

Growth status in each medium Culture conditions: 25°C, darkness 1, malt extract agar medium Colony growth is good, colonies on the 2nd day of culture have a diameter of 2
8-31 mm, colonies on the 5th day of culture have a diameter of 65-7 mm.
2n+n+, Colony exhibits a ruptured shape, development of aerial mycelium is poor, formation of sporangia spores is good, sporangium stalks are produced from aerial mycelium, and has an odor similar to garlic.

2. Growth of colonies on potato/glucose agar medium is good. Colonies on the second day of culture have a diameter of 2.
7-31 mm, colonies on the 5th day of culture have a diameter of 75-8 mm.
0 mm, the colony exhibits the shape of a flower, aerial mycelia are significantly developed in the center of the colony, the back side of the colony is yellowish-white or yellow, the formation of sporangia is poor, there is an odor similar to garlic, and the odor is somewhat strong.

3. The growth of the colony on Tsahesok agar medium is relatively good, the diameter of the colony on the second day of culture is 22-24mm, the diameter of the colony on the fifth day of culture is 50-53mm, the development of aerial mycelium is poor, aerial mycelium is may be closely intertwined.

The formation of sporangium spores is very good, and the sporangium stalks arise from aerial hyphae. Has an odor similar to garlic.

4. LCA agar medium (medium preparation method is provided by Hiroshi Miura,
(According to Mitsuyo Kudo, "Agar Medium for Aquatic Deuteromycetes," Bulletin of the Japanese Painting Society, Vol. 11, pp. 11'6-118, 1970) The growth of Shironi is good, and the diameter of the colony on the second day of culture is 2727-24), culture a: Colony number 51-1 has a diameter of 6464-6 (i, double-), exhibits a ruptured shape, aerial hyphae are scarce except in the center of the colony, and sporangia. Formation number: 1. Good. The sporangium stalks are stronger than the aerial mycelia. There is an odor similar to garlic.

Microscope observation Sporangial stalks, branching methods of sporangial stalks, sporangia, sporangial spores, etc. were observed using microscopic specimens of each culture medium and direct microscopic examination of colonies.

Sporangium stalk length 87. '5-320 μm, width 3-7.5 μm at the base, tapering towards the tip, 1.0-2.5
It becomes μm. Sporangial stalks are often branched at the base. The spores are spherical, 15-30 μm in diameter, contain many sporangiospores, and leave a somewhat indistinct color after detachment. Sporangium spores oval, rarely kidney-shaped, smooth surface, 7.5
-12.5x 5-7.5 μm, relatively large number of chlamydospores are formed. May occur singly or in rare cases in combination. Sometimes several hyphae are exposed around it. Oval or subspherical, 1
2.5-30x 7.5-15μm0 or diameter 12.
No 5-158m0 zygospores are observed.

3. Physiological properties Optimal growth conditions pH: 6-9 Temperature = 20-30°C Growth range pH: 4-10 Temperature: 5-40°C According to the above mycological properties, the strain of the present invention (SAM-02
19) Taxonomic I! '7. Search for iF/, J, A
, von Arx.

”The Genera of Fun14i sp
orulatingin PureCulture,”
3rd ed., ,1. Cramer+l91(L
and H.

Domsch, W., Gam5. &'I', 11
．． Anderson, “Compendium of
Soil Fungi,”＾cad0mic l're
SS, 1980, the columnar axis, which forms a single sporangium at the tip of the sporangial stalk, is 1.1.
The present fungal strain is considered to be a fungus belonging to the genus J9, since the cysts do not have accessory filaments and the cultured mycelia emit an odor similar to that of corn.

Therefore, W, Gam5. ”＾flay to tlu
+ 5 pieces of Mortierella,”
r'crsoonia 5-, 38]-391,
Mort, known according to 1977, i3;
Comparing the species of the genus and mycological properties, this strain has a non-velvety texture, cultured mycelium emits an odor similar to garlic, and the sporangial stalk is 87.5-320μ in length.
In m, branching occurs only at the bottom and does not branch into clusters of grapes.
Mortierella genus Mortierell because it contains many sporangiospores inside the spore
Subgenus a (Sugen, Mortiere trout-↓Iy
-grop, h i Ia section (Sect.

It is considered to be included in Hyzop h i I a ).

There are 22 types of anal beating in 1 ha section. Comparing the various mycological properties of this strain with these 22 species, the present strain is considered to be similar to the three species: ``Ella Zy3 Findings'', ``Vagina Nakayoku'', and ``RuA''. Therefore, K, 11
．． Domsch, W., Gam5. &T,-H,8n
derson.

Compendium of 5 oil Fungi,
” Academic Press.

1980, W. Gam5. ”Some new or
note 5 pieces of Mo
rtierella,” Persoonia9, 1
11-140.1977), and G. Linnema.
nn, “Mortierella Coemans 1
863. “H, Zycha & R, Siepmann
, ”Mucorales Eine Besch-r
eibung Alter Gattungen
and Arten Dieser Pilz
-gruppe,”pp, 155-140. J, Cr
Amer, 1969, the present strain was compared with these three species in terms of mycological properties. This strain clearly differs from LH Akinami in the length of the sporangium, the width of the base, and the size of the sporangium.

L1 Note: Tula refers to the shape and size of sporangium spores.
clearly different. The sporangial stalk is slightly shorter than the L-inch scale.
The chlamydospores differ in that they are oval or spherical in shape and are sometimes chained, and the chlamydospores sometimes produce several hyphae around them. The difference is that this strain is Mortierella, elo'
! It was judged that this was not sufficient to classify it as a different species from 8a and ja. Therefore, the present inventors developed this strain using Mortiere.
It was identified as lla cl-pgHata SAM 0219. SAM 0219 stock is 1985 3 Jl 1.
On the 9th, the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (F)
RI) under accession number FERM P-8703.

In order to culture the bacterial strain used in the present invention, spores, hyphae, or a preculture solution obtained by culturing the strain in advance are inoculated into a liquid medium or a solid medium and cultured. In the case of a liquid medium, any commonly used carbon source such as glucose, fructose, -1-syl'-sucrose, sucrose, maltose, soluble starch, molasses, glycerol, mannitol, etc. can be used. However, it is not limited to these. As nitrogen sources, in addition to natural nitrogen sources such as peptone, yeast extract, malt extract, meat nits, carimino acid, and cornstabilic acid, organic nitrogen sources such as urea, as well as nitrate, ammonium nitrate, etc. ,
Inorganic nitrogen sources such as ammonium sulfate can be used. In addition, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, copper sulfate, and vitamins can also be used as trace nutrient sources, if necessary. There are no particular limitations on the concentration of these medium components as long as they do not impair the growth of microorganisms. In practice, the carbon source generally ranges from 0.1 to 30% by weight, preferably from 1 to 10% by weight,
The concentration of the nitrogen source is preferably 0.01 to 5% by weight, preferably 0.1 to 2% by weight. In addition, the culture temperature is 5-40℃
, preferably 20-30°C, and the pH of the medium is 4-10.
, preferably 6 to 9, aeration agitation culture, shaking culture,
Or perform static culture. Culture is usually carried out for 2 to 10 days.

When culturing on a solid medium, 50~
Culture is carried out for 3 to 14 days at a temperature of 5 to 40°C, preferably 20 to 30°C, using bran, rice husks, rice bran, etc. to which 100% by weight of water has been added. In this case, a nitrogen source, inorganic salts, and micronutrient sources can be added to the medium as necessary.

By culturing in this manner, lipids containing bishomo-γ-linolenic acid are produced and accumulated within the bacterial cells.

When a liquid medium is used, bispo□mo γ-linolenic acid is collected from the cultured cells as follows.

After completion of the culture, cultured bacterial cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation and #passage. Wash the bacterial cells thoroughly with water,
Preferably dry. Drying can be performed by freeze drying, air drying, etc. The dried bacterial cells are preferably subjected to an extraction treatment using a solvent I1-a under a nitrogen stream. Is there a? Ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. can be used as the ff medium, and alternate extraction of methanol and petroleum ether, or a single layer solvent of chloroform-methanol-water can be used. Good results can also be obtained with -C by extraction. From the extract under reduced pressure? By distilling off the solvent 8, a lipid containing high-temperature bishomo-γ-linolenic acid is obtained.

Furthermore, instead of the above method, extraction can be performed using wet bacterial cells. In this case, a water-compatible solvent such as methanol or ethanol, or a water-compatible mixed solvent consisting of these and water and/or another solvent is used. Other steps are the same as above.

The lipid obtained as described above contains bishomo-γ-
Linolenic acid is included as a constituent of lipid compounds, such as fats. Although these can be separated directly, esters with lower alcohols, such as bishomo-γ-
Preferably, it is separated as methyl linolenate. By forming such an ester, it can be easily separated from other lipid components, and other fatty acids produced during culture, such as palmitic acid, oleic acid, linoleic acid, etc. (these are also bishomo-γ - esterified during esterification of linolenic acid). For example, to obtain the methyl ester of bishomo-gamma-linolenic acid, the above extracted lipid is mixed with anhydrous methanol-hydrochloric acid 5% to 10%, BF, -methanol 10% to 50%, etc. for 1 to 24 hours at room temperature. Preferably, it is treated.

In order to recover hisbomo-gamma-linolenic acid methyl ester from the above-mentioned treated solution, it is preferable to extract it with an organic solvent such as dichloromethane, ether, or ethyl acetate.

Next, this extract is dried with monotrium acetate anhydride or the like, and the organic solvent is distilled off, preferably under reduced pressure, to obtain a mixture mainly consisting of fatty acid esters. In this mixture, in addition to the target bishomo-γ-linolenic acid methyl ester, palmitic acid methyl ester,
Contains stearic acid methyl ester, oleic acid methyl ester, etc. In order to isolate bishomo-γ-linolenic acid methyl ester from these fatty acid methyl ester mixtures, column chromatography, low-temperature crystallization, urea inclusion method, etc. are used individually or in combination. can be used.

Bishomo-γ-linolenic acid can be obtained from the thus isolated bisbomo-γ-linolenic acid methyl, by hydrolyzing it with an alkali and then extracting it with an organic solvent such as ether or ethyl acetate.

In addition, in order to collect bishomo-γ-linolenic acid without converting it to its methyl ester, the above-mentioned extracted lipids can be digested with alkaline solution (e.g., 5% sodium hydroxide at room temperature for 2 to 3 hours).
time), the decomposed liquid can be extracted and purified by a method commonly used for extraction and purification of fatty acids.

Next, the present invention will be explained in more detail with reference to Examples.

[Production method of bishomo-γ-linolenic acid] Disaster soil glucose 5%, peptone 0.5%, yeast extract 0.3
% and malt extract 0.3% (pl(6,0)
50mj! into a 500mn Usaka flask, 120m
Sterilized at ℃ for 20 minutes. Mortierella elongata S
One platinum loop of AM 0219 (FEI?MP-8703) was inoculated and cultured with shaking at 28° C. for 5 days using a reciprocating shaker C1C11Orp. After culturing, the bacterial cells were collected by filtration, thoroughly washed with water, and then freeze-dried. As a result, 1.2 g of dried bacterial cells were obtained. From this bacterial cell, Bli using a single layer solvent of chloroform-methanol-water
When the total lipids were extracted by the extraction method of GH & Dyer, 290 μ of lipids were obtained. This lipid was methyl esterified by treatment with anhydrous methanol-hydrochloric acid (95:,5) at 20°C for 3 hours, and extracted with ether to obtain the fatty acid methyl 1801E. This was further separated by chromatography.
The bispho-dried γ-linolenic acid methyl fraction was separated, and the solvent was distilled off using a rotary evaporator.
．． 5.2 μ of purified methyl bishomo-γ-linolenate was obtained. A standard sample of methyl bishomo-gamma linolenic acid prepared by methyl esterification of this specimen and commercially available bishomo-gamma-linolenic acid, isolated and purified by Kahumuku , high-performance liquid lithography I+ -, v l-graphic analysis, mass spectrometry, and NMR fraction JI'+', L, =,
When a comparison was made, the two were in agreement in both analyses.

The amount of bispomo-γ-linolenic acid methyl before and after purification was 0.111 μ/m 12 and 0 per medium, respectively.
, 10mg/m 1 , each per dry bacterial cell, 7
．． It was 5mlr/g and 4.3ml/g.

Culture medium 5β with the same composition as in Example was charged into a 15J jar fermentor, and after sterilized at 120°C for 40 minutes, Mortierella elongata SAM 0219 (FERM P-8
703) preculture solution 200m7! was inoculated. 30℃,
Aeration stirring culture was performed for 3 days at an aeration rate of 0.5 v, v, m, and 370 g (dry weight 120 g) of the obtained wet bacterial cells were extracted, hydrolyzed, and methyl esterified in the same manner as in Example 1. As a result, 31 g of total lipid and 19 g of mixed fatty acid methyl were obtained. The content of methyl bishomo-γ-linolenic acid in this product is 5%, and the production amount is 0.19 g/n per medium.
, 7.9 μ/g per dry bacterial cell.

In addition, after the completion of the culture, the culture filtrate obtained by filtration 4.0
After drying 50 ml, extraction, hydrolysis, and methyl esterification were performed in the same manner as in Example 1, resulting in mixed fatty acid methyl 1 containing 5% methyl bishomo-γ-linolenate.
I got 27■.

■Bonmachi, IFO8571), and Mordierella hygrophila (Mortsato e-rejl+4-h2Brp
7 old j+i, IFO5941) were subjected to the same operation as in Example 1, and fatty acid methyls of 65 and 93w were obtained, respectively.

When the methyl bishomo-γ-linolenic acid contained in these methyl fatty acids was purified in fit ml, 2.7 μm and 4.5 μm were obtained for each strain.

・ Fruit 11't Glucose 2%, Yeast 1υ extract 1%, Tween 20
Culture medium containing 0.2% and 0.5% of various hydrocarbons, sodium fatty acids or fats and oils! ! (pHti, 0)
20ml to 100mI! Put it in Mayer, 120
Sterilized at ℃ for 20 minutes. Mortierella elongata S
One loopful of AM 0219 (FERM P-8703) was inoculated and cultured at 28°C for 5 days using a rotary shaker (200 rpm).

The obtained bacterial cells were extracted, hydrolyzed, and methyl esterified in the same manner as in Example 1. For each of the various hydrocarbons, fatty acid sodium 11, and fats and oils added to the medium, the obtained dry bacterial weight, total lipid amount, total fatty acid methyl amount, bishomo-γ-linolenic acid content, and bishomo-γ per medium -The amount of methyl linolenate produced was as shown in the table below.

When sodium fatty acids or fats and oils were added to the standard medium, the amount of bishomo-γ-linolenic acid produced increased by 2 to 20% compared to the area without the addition.

Procedural amendment (voluntary) 1985 1st 2nd

Claims (1)

[Claims] 1. A microorganism belonging to the genus Mortierella and capable of producing bishomo-γ-linolenic acid is cultured to produce bishomo-γ-linolenic acid or bishomo-linolenic acid.
1. A method for producing bishomo-γ-linolenic acid, which comprises producing a lipid containing γ-linolenic acid and collecting bishomo-γ-linolenic acid.