JPS60168391A - Production of gamma-linolenic acid-containing lipid and gamma- linolenic acid - Google Patents

Abstract

PURPOSE:To produce the titled compound in high productivity, by culturing a specific microbial strain belonging to Mortierella genus in a medium containing carbohydrate in high concentration as a carbon source, thereby producing the microbial cell containing a large amount of a gamma-linolenic acid-containing lipid in high density. CONSTITUTION:A microbial strain belonging to Mortierella genus, e.g. isabellina (IPO7824, 7884, 8183, 8308, 8309), vinacea (IPO6738), ramaniana (IPO8287), ramaniana var.anglispora (IPO6744, 8187) or nana (IPO8794) is cultured in a medium containing a carbohydrate in high concentration as a carbon source. The microbial cells containing large amount of a gamma-linolenic acid-containing lipid can be produced in high density, and a gamma-linolenic acid-containing lipid and gamma-linolenic acid can be produced in high productivity therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention cultivates filamentous fungi of the genus Morteierella in a medium containing highly concentrated carbohydrates as a carbon source, thereby cultivating γ-lylunic acid-containing lipids [neutral lipids (fats and oils, etc.), polar lipids ( Phospholipids, glycolipids)] are highly productive for producing γ-phospholipid-containing lipids and γ-phospholipids at high density and collecting γ-phospholipid-containing lipids or γ-phosphoric acid from cultured bacterial cells. The present invention relates to a method for producing lylunic acid.

Currently, γ-lylunic acid or its containing lipids are
09nOt'bera biennis TJ,), but the productivity is extremely low, and the search for alternative plant oil (R, B, Wo], t,
R, klej, man.

R, K, England, J, Amer, Oil
Chem, Soc, ,F) Q l 358 (1
983)) has been attempted. All plants that produce γ-lyrinnic acid-containing lipids are special, and it is difficult to increase their productivity, including by collecting seeds. On the other hand, production by microorganisms does not require solar energy;
It is known to have the following advantages: it is not affected by the weather, it can be produced on a factory scale without requiring much land, it has high productivity and efficiency, and the production needle can be controlled by free labor. To date, the microorganisms containing γ-lylunic acid in their lipids include Mucor globosus, Mucor bu'/
Rus (R, O, Mllmma eta], , Ll
pld, 8.6.

584 (1,971,) ), Yuanehora cucurbitarum [H, B, Whj, te, Jr, , S,
S, Rowell, Biochim, Bj, oph
ys.

ACta, ,] 16, 388 (] 966)
), Hythium tehallianum, Zabrolegnia littoralis, Rhizopas stronipha, Rhizopus alhizas, Pumyces frachesreanus, Mucor javanicus, Helysteilum pilforme (R, Shaw
.

Biochim, Biophys, Acta, 98
, 230 (] 965)), Entomophthora coronata (R, O, Mumma, T, K, Brus
-zewsKj, , Lipid, e, 5 , 9 ] 5
(1970)), but when growing bacterial cells in a flask scale or in a small culture tank, the carbohydrate concentration of the raw carbon source is 20 to 6.
The amount of cells grown was small, and the content of lipids produced was only 3 to 30 g, indicating extremely low productivity of γ-lylunic acid-containing lipids.

Bacterial cells obtained by culturing in a medium contain lipids containing 20 to 12 γ-lylunic acid in total lipid fatty acids at a ratio of 3% to 3% of dry bacterial cells.
It was found that the content is 0 to 60%. Moreover, in this case, unlike bacteria and yeast, filamentous fungi that proliferate in hyphae are generally difficult to culture at high density in aerated agitation culture, whereas high-density culture of the filamentous fungi belonging to the genus Morteierella is found that it is possible. Specifically, a specific filamentous fungus belonging to the genus Morteierella grows in small units without elongating the hyphae by increasing the agitation speed during aerated agitation culture using a medium containing high-concentration carbohydrates as a carbon source. High-density culture of bacterial cells is possible in a state where the content of lipids including IJ phosphoric acid is high, for example, when the raw carbohydrate (glucose 270 so/l medium) is kept at 30°C.
Completely consumed after 172 hours of bacterial culture, the amount of proliferated bacterial cells (dry weight) is 3-'101# medium or more, the lipid production amount is approximately 50 g/11 medium containing 4 or more γ-lylunic acid, and the lipid content is approximately As a result, the amount of γ-lylunic acid produced per liter of medium was approximately 2.
．． 0g, productivity of γ-lylunic acid for dry bacterial cells 20%
The above values are obtained.

The present invention has been developed by culturing strains of Isabelina, Vinacea, Lamaniana, Lamaniana anglispora and Nana belonging to the genus Morteierella in a medium containing a high concentration of carbohydrate as a carbon source. γ- is a highly productive γ- phospholipid that produces γ-lylunic acid-containing lipids or γ-lylunic acid from cultured bacterial cells. This is a method for producing lylunic acid-containing lipid and γ-lylunic acid.

The bacteria used in the present invention is Morti erella (Mortiθrel).
la) Genus Neucer < IJ f (1 sabellin
a) (Engineering FO7824, 7884, 8183, 830
8,8309], vinacea (IFO
6738), u7, = -7su (ramaniana)
(UFO8287) Lamaniana angrispora (
ramaniana4-var, ang], 1spora) (IFO6744,
8187), Nana (IFOR794)
Various strains of.

In addition, all of the above-mentioned m are filamentous fungi that are preserved at the Research Institute for Developmental Research and are listed in the FO Catalog (Strain Catalog).

Examples of carbohydrates used as carbon sources for the medium for culturing the filamentous fungi include glucose, fructose, saccharose, sugar acids, starch, and wood saccharification liquor. Carbohydrates may be present in the medium 11 at 60% or more, or organic nitrogen sources such as urea, peptone, yeast extract, corn stave liquor, etc. are used. Examples of inorganic salts include KH2PO4, 2HPO, and NaC.

Fe504-7H20, M2SO4-7H20, zns
o4.7H20 etc. are used. Add trace elements and other nutritional sources as necessary.

The above-mentioned filamentous fungi are usually cultured in a liquid medium by aeration and agitation. The pH of the medium is 40 to 6.0. Since lylunic acid-containing lipids are contained in the cells of filamentous fungi, it is preferable to collect γ-lylunic acid-containing lipids and γ-lylunic acid from these cells. . When separating the bacterial cells from the culture, the bacterial cells do not spread very much and are cultured in extremely small units (1 to 10 cells), and the collection of lylunic acid-containing lipids is carried out using conventional methods such as solvent extraction. be exposed. It is concentrated and collected from mixed fatty acids or fatty acid esters of γ-lylunic acid-containing lipids by conventional methods such as the urea addition method and the cooling separation method.

Thus, according to the present invention, 7t produces bacterial cells with a high content of γ-lylunic acid-containing lipids using high-aluminated carbohydrates as a carbon source, and from the produced bacterial cells, γ-lylunic acid-containing lipids and γ-
Rillunic acid can be collected, and the present invention is an excellent method for producing γ-lyllunic acid-containing lipids or r-lyllunic acid with high productivity. This is especially true for the γ-lylunic acid-containing lipids currently collected from plant seeds or the γ-
This provides a method for producing rinnic acid from microorganisms with higher productivity.

Note that γ-lylunic acid (18:3 (6,9,12)) is a fatty acid (essential fatty acid) required in the diet that cannot be synthesized in mammals, together with linoleic acid. This γ-lylphinic acid becomes bishomo-γ-lylinic acid in the body and is a precursor to arachidonic acid, and bishomo-γ-lylinic acid and arachidonic acid are respectively prostaglandin, El + J' +, a and hE
21 F2・, which plays an extremely important physiological role in living organisms. Therefore, it is clear that γ-lylunic acid-containing lipids can be used as pharmaceuticals.

Next, examples of the present invention will be shown. It is not more restricted.

Example 1゜Glucose 60i, KH2PO42,! 9,
Mg5047H200,39, NaC70,1jj,
Malt-1kiss 0.2gIys) -x Ki/<
Q, 2g, Hepton o, 1g, Pe5o4゜7H
2010m9, cac12-21-12010mt
;), CuS04-51-1□00.2m9,
MnSO4,4H201,07n9 and (N
H4) 28043 g, (C/N ratio (weight of carbon atoms in carbon source/nitrogen in nitrogen atoms ~ atomic weight) is approximately 40]
When the concentration of carbohydrates (glucose, sugar, etc.) as a carbon source is increased based on a medium prepared by mixing 10,100 O of deionized water, the medium components are increased according to the concentration, and the nitrogen source is changed to urea, etc. When the C/N ratio was changed, the medium was adjusted so that the C/N ratio remained the same.

1. 61 when culturing in an Ol culture tank
．． In the culture tank 301, 201 strains were inoculated, each was incubated at a culture temperature of 30°C for a predetermined period of time with an aeration rate of 0.5~
2. Stirring L-r culture was performed at 300-700 rpm in OVVm. After culturing, the bacterial cells were collected by centrifugation.

In addition, in order to measure the growth rate of bacterial cells, the amount of lipid production, and the concentration of carbohydrates in the medium, 100 ml samples were collected at predetermined intervals during the intermediate stage of culture, and the bacteria were filtered using the oral filtration method. The body and medium were separated. A portion of the isolated bacterial cells was accurately weighed and placed in a constant temperature bath at 120°C to determine the moisture content.
The cells were dried for a day and night, the moisture content was determined, and the remaining bacterial cells were extracted for lipids. To extract lipids from the bacterial cells, add a chloroform-methanol (2:IV/V) mixture to the remaining wet bacterial cells and homogenize in the presence of glass beads to simultaneously crush the bacterial cells and extract the lipids. Ta. In addition,
To ensure complete extraction, this was repeated 5 times and all extracts were collected. After the above-mentioned extract was purified by Folch's partition washing method, the solvent was distilled off under reduced pressure, and the total lipid amount was measured by gravimetric method. Concentrations of carbohydrates (glucose, fructose, sucrose) were measured using high performance liquid chromatography (IPLC) for the culture medium from which bacterial cells were removed, and the culture was terminated when the concentration reached 0.

A portion of the produced lipids was extracted from the bacterial cells and accurately weighed, and after methyl esterification, the fatty acid composition was analyzed by gas chromatography. The remaining lipids were separated into neutral and polar lipids by column cloning using uninlu as a packing material and chloroform and methanol as developing solvents, and the amount of each lipid was determined. Chromatography was performed to determine the fatty acid composition.

The amount of bacterial growth (dry weight) obtained by culturing various Morteierella filamentous fungal strains in 101 and 301 culture tanks using glucose or molasses as a carbon source and varying the nitrogen source and nitrogen source concentration at various initial carbon source concentrations. g/
l), lipid production amount (g/l), lipid content ≠ (correspondence), total neutral and polar lipids (correspondence), total lipids, γ-lylunic acid content in neutral and polar lipids, and γ-lylunic acid content in neutral and polar lipids. As the productivity, the amount produced per liter of medium (Cg/l) and γ in dry bacterial cells
- The results obtained for the lyrinnic acid content are summarized in Table-1. The time shown as the culture time is the time when the carbon source glucose or molasses was completely consumed and disappeared from the medium, and the culture was stopped at that time.

Table 1 shows that even when glucose or molasses used as a carbon source is concentrated at +1100 g/V or higher, with an average of 200 g/l, bacterial cell growth is rapid and the bacterial cell concentration is 40 to 156 g/V.
The amount of lipid produced including L r-lylunic acid is 3 to 83.
The productivity was 32 to 58% in terms of lipid content. In addition, γ-lylunic acid d in the fatty acids of the produced lipids:
Section 35-112 and Tsukimikusa Tanehiko gamma in plant seed oil
- It can be seen that the content of lylunic acid is comparable. The γ-lylunic acid content of the neutral lipid is 33 to 11.0%,
It was observed that the content of polar lipids was quite high, ranging from 121 to 237%. Medium 1 as the productivity of r-lylunic acid
As a result of calculating the production amount per unit, it can be seen that the productivity is high, ranging from 0.9 to 3.4 g.

The γ-lylunic acid content in dry bacterial cells is also 13 to 4.
．． A value of 1% was obtained, which was also found to be approximately the same as the value in plant seeds.

Example 2 The bacterial strain Morteierella lamaniana shown in Example 1
Angris Bora IFO8187 culture temperature 6 at 30°C
Wet bacterial cells 9019 obtained by culturing for 6 hours (dry weight 27
100 g of lipid was extracted according to Example IK. As a result of methyl esterification of 100 g of the obtained lipid by a conventional method, 92 g of fatty acid methyl was obtained. The composition of this fatty acid methyl was determined by gas chromatographic analysis: palmitic acid (313%), cumitooleic acid (1.4%), stearic acid (566%), oleic acid (447%), and linoleic acid (9%).
It was found that 6.4% of γ-lylunic acid was contained in the second column. As a result of repeating the conventional urea addition method three times for this mixed fatty acid methyl, γ-methyl lylunate was found to be 48.2%.
A mixed fatty acid 7.2.9 concentrated at t was obtained. In addition, γ
- The composition of the linoleic acid concentrated mixed fatty acid was 459% linoleic acid, 41% oleic acid, and 1.8% other.

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Claims (2)

[Claims] (1) By culturing both Isabelina, Vinacea, Lamaniana, Lamaniana anglispora and Nana belonging to the genus Morteierella in a medium with a high concentration of carbohydrates as the carbon source, bacterial cells with a high content of γ-lylunic acid-containing lipids were increased. 1. A method for producing a γ-lylunic acid-containing lipid, characterized in that it is produced at a high density. (2) γ-lylunic acid-containing lipids collected from bacterial cells cultured at high density in a medium containing high-concentration carbohydrates as a carbon source of strains of Isabelina, Vinacea, Lamaniana, Lamaniana anglispora, and Nana belonging to the genus Morteierella. 1. A method for producing γ-lyllunic acid, which comprises concentrating γ-lyllunic acid from.