JPS6225989A - Production of phosphatidylcholine having high content of gamma-linolenic acid - Google Patents

Abstract

PURPOSE:To make it possible to collect efficiently gamma-linolenic acid, by separating phosphatidylcholine having high content of gamma-linolenic acid from lipid collected from a specific mold and concentrating the phosphatidylcholine. CONSTITUTION:A strain such as Mortierella isabellina, Mortierella vinacea Mortierella ramaniana, Mortierella ramaniana var. anglispora, or Mortierella nana, belonging to the genus Mortierella is cultivated in a medium containing a highly concentrated carbohydrate as a carbon source, a mold with high lipid content is obtained and gamma-linolenic acid is produced from lipid collected from the mold. In producing gamma-linoleic acid, phosphatidylcholine having high content of gamma-linolenic acid is separated from the lipid, concentrated and gamma-linolenic acid is obtained. To separate the phosphatidylcholine, the lipid collected from the mold is subjected to alcohol extraction at the first step of multi-stage extraction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing phosphatidylcholine with a high content of γ-linolenic acid using a filamentous fungus of the genus Mortierella.

[Prior art]

, Isabelina, Vinacea, belonging to the genus Pamoltierella;
By culturing filamentous fungi such as L. Lamaniana, L. Methods of production have already been proposed. (Patent Application No. 59-22394).

By the way, phosphatidylcholine, which is a type of glycerophospholipid, is known to have a structure as shown in the following formula.

CHrOCOP.

CH-OCOR' Its structural fatty acid is RCOOH or R,'C0O
There is no prior art in which phosphatidylcholine containing 20% or more of γ-linolenic acid in H is produced, isolated and concentrated from microorganisms.

〔the purpose〕

The present invention provides phosphati-2= The purpose of the present invention is to provide a method for producing zircoline.

〔composition〕

That is, according to the present invention, in producing phosphatidylcholine with a high content of γ-linolenic acid, a filamentous fungal strain of Isabelina, Vinacea, Nana, Lamaniana, Lamaniana anglispora or Nana belonging to the genus Mortierella is used to use a high concentration of carbohydrates as a carbon source. Especially multi-stage extraction (patent application A method for efficiently separating and concentrating phosphatidylcholine having a high γ-linolenic acid content from the first stage alcohol extraction section A in No. 60-1.0283) by, for example, chromatography. A method for producing phosphatidylcholine is provided.

[Bacterial cell culture method]

In the present invention, the bacteria used is Mortierella (M
ortierella) genus Isabelina
-anniana) (IFO8287), Ramaniana angrispora (ramaniana var, ang
lispora) (IFO8187:]. All of the above-mentioned bacteria are filamentous fungi that are kept at the Hatsu Hatsu Research Institute and are listed in the IFO Catalog (Strain Catalog).

Examples of carbohydrates used as the charcoal source of the medium for culturing the filamentous fungi include glucose, fructose, saccharose, molasses, starch, and wood syrup. Preferably, 20 to 400 g of carbohydrates are used in the medium 11. Examples of nitrogen sources include inorganic nitrogen sources such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, etc., or urea, peptone,
Organic nitrogen sources such as yeast extract, corn, and stave liquor can be used. Examples of inorganic salts include KH2PO4;
K2HPO4, NaC7, FeSO4,7H□0,
Maso4'7H20, ZnSO4.7H20, 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 shaking culture, aerated agitation culture, or the like. The pH of the medium is 3.0-6
．． 0 is good, and culture is usually carried out for about 2 to 10 days. Furthermore, acetic acid or acetate salts (alkali metal salts such as sodium salts and potassium salts) are added to the initial culture medium, in the case of aerated agitation culture, to the preculture medium, or in the intermediate stage of culture to increase the concentration of carbon sources, etc. 0 depending on conditions.
Culture of bacterial cells is carried out by adding 1 to 20 g/l of the medium (Japanese Patent Application No. 1982-1-5162). In this way, bacterial cells with a high content of lipids with a high content of unsaturated fatty acids such as γ-linolenic acid are produced with high productivity.
Since the cells are separated from the culture and lipids are contained in the filamentous cells, it is preferable to collect lipids with a high content of unsaturated fatty acids such as γ-linolenic acid from the cells. It has become clear that when separating the bacterial cells from the culture, the hyphae do not spread too much and can be cultured in extremely small units (1 to 10 cells) with a rate of about 60 cells.

[Extraction method]

Collection of lipids with a high content of γ-linolenic acid is carried out using a multi-stage extraction method. That is, in the present invention, Mortierella filamentous fungi are first extracted in the first extraction step using alcohol in the presence of water. In this case, as the bacterial cells used as the raw material for treatment, it is possible to use a hydrated bacterial cake with a water content of about 50 to 80, separated from the culture medium by centrifugation or filtration, or a dried product thereof, but it is not economical. From this point of view, it is advantageous to use a water-containing bacterial cell cake. In addition, in this first extraction treatment step, it is necessary to crush the bacterial cells by applying mechanical force in the presence of water and in an alcohol solvent, and efficient extraction treatment is achieved by crushing the bacterial cells. Ru. As such an extraction device that involves crushing the bacterial cells, a conventionally known wet grinder such as a ball mill, a Masatsu disk mill, a Hensel mixer, etc. can be used.

1: With such a crusher, the bacterial cells are subjected to compressive force or mechanical force such as mechanical force, and some of them are damaged or crushed.In this case, the bacterial cells are crushed too finely. From the viewpoint of filterability, it is preferable to apply mechanical force to such an extent that the particle size of the bacterial cells does not substantially change.As the alcohol solvent, lower alcohols such as methanol, ethanol, and propatool are usually used. However, from the point of view of safety for the human body, it is preferable to use ethanol.The proportion of alcohol solvent used is 1 part by weight of bacterial cells (dry basis).
The ratio is 2 to 7 parts by weight, preferably 3 to 6 parts by weight. In this first extraction process, in order to elute polar lipids, it is necessary to perform the extraction process in the presence of water, and the amount of water present is 0 per 1 weight of alcohol solvent.
．． The amount is 2 to 0.7 parts by weight, and the suitable amount is 0.3 to 0.6 parts by weight. Addition of water to this first extraction treatment system can be carried out using microbial cells containing water, or can be carried out by adding it to an alcohol solvent. The extraction tank lipid recovery rate is usually 5 to 30 weight percent, preferably 8 to 25 weight percent, relative to the total lipid recovery rate.

Next, the first extraction product obtained above is separated into a crushed bacterial cell component and an alcohol solvent component containing polar lipids in a first solid-liquid separation step. In this case, the solid-liquid separation method is
Conventional methods such as centrifugation and filtration separation methods are employed. The lipid component can be obtained by distilling off the solvent from the alcohol solvent component containing the obtained polar lipid under reduced pressure in accordance with a conventional method.

[Adsorption separation method]

γ-
Phosphatidylco-11, which has a high content of linolenic acid, is separated,
Refined. mV, 50 to 300 mV, preferably lOO to 200 mV, silicic acid (silica gel),
Hexane, cyclohexane, carbon tetrachloride, benzene, chloroform,
Diethyl ether, ethyl acetate, acetone, acetonitrile, methanol, and aqueous acetic acid are used as solvents, depending on the polarity of the solvent, and a mixture of one or two or three solvents is used, for example, chloroform and methanol 4:1.3:2. 1:
1. By sequentially flowing a 1:2 mixed solvent, etc., separation and elution is possible due to the difference in adsorption power of each polar lipid, and γ
- An eluate containing phosphatidylcholine with a high linolenic content is obtained. It has been found that phosphatidylcholine with a high content of γ-linolenic acid can be concentrated by distilling off the solvent from this eluate under reduced pressure using a conventional method.

Thus, according to the present invention, it is possible to produce phosphatidylcholine with a high content of γ-linolenic acid from lipids collected from bacterial cells with a high lipid content cultured at high density in a medium containing a high concentration of carbohydrates as a carbon source. It is also an essential fatty acid required by mammals as part of their diet. This means that γ-linolenic acid is a precursor that becomes Tehishomo-γ-linolenic acid and then arachidonic acid, and bishomo-γ-linolenic acid.
- This is because linolenic acid and arachidonic acid become prostaglandins, E, F, a, and E2 + F2a, and play extremely important physiological roles in living organisms. Therefore, it is clear that γ-linolenic acid-containing lipids can be used as pharmaceuticals.

In particular, it is known that γ-linolenic acid is stored as a phospholipid in living organisms, and it is thought that the phospholipid state acts more effectively as a precursor for turning into prostaglandins. Therefore, phosphatidylcholine, which has a high content of γ-linolenic acid of 2° or more, can be fully expected to be used as a pharmaceutical.

Used in chocolate, etc.

〔Example〕

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

Example Glucose 60g, KH2PO42,! 9, M, 9'5
047 H2O0.3g, NaC10.1,! i',
-=r Luto Equi, ', 0.2 g, Yeast -
1 kiss 0.2g, Pepto yO, Iji, FeSO4゜7
H7H2O10, Mn5O+ -4H2Oi, omp and 3 g of (NH4)2SO4 as a nitrogen source, (C/N ratio (weight of carbon atoms in carbon source/weight of nitrogen atoms in elementary temperature source) is approximately 40) were mixed in 1000 rnl of deionized water. Carbohydrates (glucose, sugar, etc.) that are carbon sources based on the culture medium
If the concentration of C7 is increased, the medium components are increased accordingly, and the nitrogen source is changed to urea etc.
The medium was adjusted to have the same N ratio.

This medium was charged to 20A in culture tank 301, and each strain was inoculated, and in order to measure the carbohydrate concentration in the specified soil at a culture temperature of 30°C, 100 ml of the sample was added at specified intervals during the intermediate stage of culture. A sample was taken, and the bacterial cells and the medium were separated by the mouth filtration method. A portion of the isolated bacterial cells was accurately weighed and dried in a constant temperature bath at 120° C. for a day and night to determine the moisture content, 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 purification using the above extract 1Floch distribution washing method, the solvent was distilled off under reduced pressure, and the total lipid amount was measured gravimetrically. Carbohydrates (glucose, glucose,
The concentration of fructose, sucrose) was measured and the concentration was 06738, Mortierella Isabelina IFO782.
Regarding 4, the amount of bacterial cell growth (dry weight g/l), lipid production <g/lj), lipid content (
H) The γ-linolenic acid content in lipids is summarized in Table 1. Note that the time shown as the culture time is the time when glucose, which is a carbon source, is completely consumed and disappears in the medium, and the culture was stopped at that time.

Table 1 shows that all three strains produce lipids containing γ-linolenic acid with high productivity.

Dehydrate and separate to obtain a bacterial block (cake) with a moisture content of 50 to 70 cm. This bacterial block (hereinafter referred to as wet bacterial body)
After sterilization in an autoclave at 120° C. and 2 atm for 10 minutes, lipids were extracted as shown below.

1.0 to 1.7 kg of the wet bacterial cells were placed in a stainless steel ball mill with an internal volume of 61 kg, ethanol 21 was added as a solvent, and extraction was performed for 4 hours while crushing the bacterial cells in the ball mill. After screwing off the extract, the obtained bacterial cells (water content 3.4%) were subjected to the same extraction treatment as above for 7 hours using hexane 21 as a solvent again.

Table 2 shows the extraction ratio of lipids to bacterial cells by ethanol in the first stage for the three bacterial strains obtained by the two-stage extraction method.

For this ethanol-extracted lipid, silicic acid (100 to 200 mesh) was adsorbed to the filler by the following method.Then, the neutral lipid was absorbed with chloroform, and the glycolipid was dissolved with acetone, chloroform, and methanol (4:1). After eluting phospholipids other than phosphatidylcholine with the same 1:1 eluent, a section was obtained that was eluted using chloroform and methanol 2:3 as an eluent. Analysis of the obtained fraction by thin layer chromatography revealed that phosphatidylcholine was concentrated to approximately 100% purity.

As shown in Table 2, the extraction rates of the three strains relative to the amount of ethanol-extracted lipids were relatively high, ranging from 6.9 to 80 inches, and the extraction rates relative to dry bacterial bodies were 0.32 to 0.46 inches.

After hydrolyzing this phosphatidylcholine, we performed methyl ester and compared the fatty acid composition.
-It was revealed that the extremely high linolenic acid content of 25.7% or more, which is different from about 7% in the neutral lipid category, has a special fatty acid composition with a high υnolic acid content.

Claims (1)

[Claims] (1) Lipids collected from microbial cells with high lipid content cultured at high density in a medium containing high-concentration carbohydrates as a carbon source by strains of Isabelina, Vinacea, Lamaniana, Lamaniana anglispora, or Nana belonging to the genus Mortierella. 1. A method for producing phosphatidylcholine with a high content of γ-linolenic acid, which comprises separating and concentrating phosphatidylcholine with a high content of γ-linolenic acid.