JPS6244917B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a method for producing phosphatidylethanolamine having a high content of γ-linolenic acid using a filamentous fungus of the genus Morteierella. [Prior art] Isabelina, Vinacea, Lamaniana, Lamaniana anglispora, belonging to the genus Morteierella,
A method has already been proposed for producing microbial cells with a high content of γ-linolenic acid-containing lipids at a high density by culturing filamentous microbial cells such as N. and Nana in a medium containing a high concentration of carbohydrate as a carbon source. (Unexamined Japanese Patent Publication 1986-
Publication No. 168391 (Patent Application No. 59-22394)). By the way, phosphatidylethanolamine, which is a type of glycerophospholipid, is known to have a structure as shown in the following formula. Its structural fatty acid RCOOH or
There is no prior art in which phosphatidylethanolamine containing 20% or more of γ-linolenic acid in R'COOH is produced, isolated and concentrated from microorganisms. [Objective] An object of the present invention is to provide a method for producing phosphatidylethanolamine having a high content of γ-linolenic acid. [Structure] That is, according to the present invention, in producing phosphatidylethanolamine with a high content of γ-linolenic acid, a filamentous fungal strain of Isabelina, Vinacea, Nana, Lamaniana, Lamaniana angrispora or Nana belonging to the genus Morteierella is used. From the alcohol extraction fraction of γ-linolenic acid-containing lipids extracted from bacterial cells with a high content of γ-linolenic acid-containing lipids obtained at high density in the medium by culturing in a medium with a high concentration of carbohydrates as a carbon source. γ characterized by efficiently separating and concentrating phosphatidylethanolamine with a high content of γ-linolenic acid, for example, by an adsorption method.
- A method for producing phosphatidylethanolamine with high linolenic acid content is provided. [Bacteria cell culture method] In the present invention, the bacteria used is isabellina of the genus Mortierella.
ina) [IFO7824, 7884, 7873, 8183, 8309] Vinacea [IFO6738], nana
[IFO8794], Ramani-ana
[IFO8287] and various strains of Ramaniana var. anglispora [IFO8187]. All of the above-mentioned bacteria are filamentous bacteria that are preserved at the Fermentation Research Institute and are listed in the IFO catalog (inventory of bacterial strains). Examples of carbohydrates used as carbon sources for the medium for culturing the filamentous fungi include glucose, fructose, succalose, molasses, starch, and wood saccharification liquor. Carbohydrates range from 20 to 1 in medium 1
Preferably 400g is used. As the nitrogen source, for example, an inorganic nitrogen source such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, etc., or an organic nitrogen source such as urea, peptone, yeast extract, corn, or Steve liquor can be used. Examples of inorganic salts include
KH ₂ PO ₄ , K ₂ HPO ₄ , NaCl, FeSO ₄・7H ₂ O,
MgSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ O, 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, aeration stirring culture, or the like. PH of medium
A value of 3.0 to 6.0 is good, and culture is usually carried out for about 2 to 10 days. Furthermore, at the beginning of culture, that is, in the initial medium,
In addition, in the case of aerated agitation culture, acetic acid or acetate (alkali metal salts such as sodium salts and potassium salts) is added to the preculture medium or at an intermediate stage of culture at 0.1 to 20 g/min depending on culture conditions such as carbon source concentration.
Culture of bacterial cells is carried out by adding the same amount to the culture medium (Japanese Patent Application No. 115162/1982). In this way, the culture contains unsaturated fatty acids such as γ-linolenic acid, and bacterial cells with high lipid content are produced with high productivity. Therefore, it is preferable to collect lipids with a high content of unsaturated fatty acids such as γ-linolenic acid from the bacterial cells. When separating the bacterial cells from the culture, the hyphae do not spread very much and are cultured in extremely small units (1 to 10 cells). It has been revealed that this product has the advantage of becoming a bacterial cell (with a water content of approximately 60%). [Extraction method] Collection of lipids with a high content of γ-linolenic acid is performed by a multi-stage extraction method. That is, in the present invention, filamentous fungi of the genus Morteierella are first extracted in the first extraction step using alcohol in the presence of water. In this case, the bacterial cells used as the raw material for treatment can be 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. From the point of view of economy, 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 by crushing the bacterial cells, efficient extraction treatment can be achieved. achieved. 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. With such a crusher, the bacterial cells are subjected to mechanical forces such as compressive force and crushing force, and some of the bacterial cells are damaged or crushed. In this case, it is not preferable to crush the bacterial cells too finely, and 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 propanol are usually used, but from the viewpoint of safety for the human body, it is preferable to use ethanol. The alcohol solvent is used in an amount of 2 to 7 parts by weight, preferably 3 to 6 parts by weight, per 1 part by weight of bacterial cells (on a dry matter basis). 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 0.2 to 0.2 to 1 weight of alcohol solvent.
0.7 parts by weight, preferably 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. This extraction process removes all polar lipids contained in the bacterial cells.
More than 90% of the lipids can be extracted and separated, and a portion of the neutral lipids can also be extracted. Further, in this first extraction step, the lipid recovery rate is usually 5 to 30% by weight, preferably 8 to 25% by weight, based on 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,
As the solid-liquid separation method, a conventional method such as a centrifugal separation method or a filtration separation method is employed. The lipid component can be obtained by distilling off the solvent under reduced pressure from the alcohol solvent component containing the obtained polar lipid in accordance with a conventional method. [Adsorption separation method] By performing a separation operation using adsorption column chromatography on the lipid components obtained by the first stage extraction operation using alcohol as a solvent in the multi-stage extraction method, a phosphorus compound with a high content of γ-linolenic acid is obtained. Atidylethanolamine is separated and purified. That is, 50 to 300 meshes, preferably 100 to
Using a column containing 200 mesh of silicic acid (silica gel), fine porous silica, silica gel H, silica gel G, magnesium silicate, etc.
Using hexane, cyclohexane, carbon tetrachloride, benzene, chloroform, diethyl ether, and aqueous acetic acid as a solvent, depending on the polarity of the solvent, one type or a mixture of two or three types of solvents, such as chloroform and methanol 4:1, 3: 2, 1:1, 1:2
By sequentially flowing a mixed solvent, etc., separation and elution is possible due to the difference in adsorption power of each polar lipid.
An eluate containing phosphatidylethanolamine with a high content of γ-linolenic acid is separated.
It has been found that phosphatidylethanolamine having 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, phosphatidylethanolamine with a high content of γ-linolenic acid can be produced from lipids collected from bacterial cells with a high content of lipids cultured at high density in a medium containing a high concentration of carbohydrates as a carbon source. becomes possible. [Effects] γ-Linolenic acid [18:3(6,9,12)] is an essential fatty acid required in mammals' diet, along with linoleic acid. This is because γ-linolenic acid is a precursor that becomes bishomo-γ-linolenic acid and then arachidonic acid in the body, and bishomo-γ-linolenic acid and arachidonic acid are prostaglandins, E ₁ , F _1a and This is because they form E ₂ and F _2a 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, γ-linolenic acid is known to be stored in the body as a phospholipid, and it is thought that the phospholipid state acts more effectively as a precursor for turning into prostaglandins. As shown above, the γ-linolenic acid content is 20%.
The use of phosphatidylethanolamine as a pharmaceutical with the above-mentioned values is fully expected. Phosphatidylethanolamine is known as a phospholipid mixture (lecithin) containing it as a main component, and is used as a food additive such as an emulsifier in margarine, tyokolate, etc. [Example] Next, the present invention will be explained in detail with reference to Examples. Example Glucose 60g, KH ₂ PO ₄ 2g,
MgSO ₄ 7H ₂ O0.3g, NaCl0.1g, malt extract
0.2g, yeast extract 0.2g, peptone 0.1g,
_FeSO4・_7H2O10mg , CaCl・_2H2O10mg ,
CuSO ₄・5H ₂ O 0.2 mg, MnSO ₄・4H ₂ O 1.0 mg and as a nitrogen source (NH ₄ ) ₂ SO ₄ 3 g [C/N ratio (weight of carbon atoms in carbon source / weight of nitrogen atoms in nitrogen source) ) is approx.
40] mixed with 100 ml of deionized water, if the concentration of carbohydrates (glucose, sugar, etc.) as a carbon source is increased, the medium components are increased according to the concentration, and the nitrogen source is changed to urea. etc., the medium was adjusted so that the C/N ratio remained the same. Thirty culture tanks were filled with 20 of this medium, each strain was inoculated, and cultured at a culture temperature of 30° C. for a predetermined period of time, with an aeration rate of 0.5 to 2.0 vvm, and stirring at 300 to 700 rpm. 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 carbohydrate concentration in the medium, samples of 100 ml were collected at predetermined intervals during the intermediate stage of culture, and the mixture of bacterial cells and medium was filtered using the filtration method. I did the separation.
A portion of the isolated bacterial cells was accurately weighed and dried overnight at 120°C in a constant temperature bath to determine the moisture content, and lipids were extracted from the remaining bacterial cells. To extract lipids from bacterial cells, a chloroform-methanol (2:1 V/V) mixture was added to the remaining wet bacterial cells and homogenized in the presence of glass beads, thereby crushing the bacterial cells and extracting lipids at the same time. . In order to perform the extraction completely, this was repeated five times and all the extracts were collected. After the above extract was purified by Floch's partition washing method, the solvent was distilled off under reduced pressure, and the total lipid amount was measured gravimetrically. The concentration of carbohydrates (glucose, fructose, sutucarose) in the medium from which the bacterial cells were removed was measured by high-performance liquid chromatography (HPLC), and the culture was terminated when the concentration reached 0. Strains Morteierella lamaniana anglispora IFO8187, Morteierella vinacea
IFO6738, Morteierella Isabelina IFO7824
The amount of bacterial cell growth (dry weight g/), lipid production amount (g/), lipid content (%), lipid content obtained by culturing in 30 culture tanks using glucose as the carbon source and urea as the nitrogen source. The γ-linolenic acid content of the samples 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.

[Table] Cells containing a high content of γ-linolenic acid-containing lipids obtained by mass culturing the Morteierella filamentous fungi shown in Table 1 in 30 culture tanks were collected using a centrifugal dehydrator.
Dehydrate and separate to obtain a bacterial block (cake) with a moisture content of 50 to 70%. This bacterial block (hereinafter referred to as wet bacterial cells) was heated at 120℃ and 2 atm in an autoclave.
After sterilization for 10 minutes, lipids were extracted as described below. Put 1.0 to 1.7 kg of the wet bacterial cells into a stainless steel ball mill with an internal volume of 6, and add 2 ml of ethanol.
was added as a solvent, and the extraction process was performed while crushing the bacterial cells using a ball mill for 4 hours. After filtering the extract, the obtained bacterial cells (water content 3.4%) were subjected to the same extraction treatment as above for 7 hours using hexane 2 as a solvent again. The first sample for the three bacterial strains obtained by the two-stage extraction method
The table shows the extraction rate of lipids from bacterial cells by ethanol in stages.
2. The ethanol-extracted lipids were subjected to column chromatography using silicic acid (100 to 200 mesh) as a packing material in the following manner to concentrate phosphatidylethanolamine. That is,
After adsorbing the lipids on a column packed with 30 g of silicic acid per 1 g of the extracted lipids, neutral lipids were extracted with chloroform and glycolipids were extracted with acetone, chloroform, methanol 4:1 eluent except for phosphatidylethanolamine. After draining the phospholipids, a section was obtained that was eluted using chloroform and methanol 1:1 as an eluent. Analysis of the obtained fraction by thin layer chromatography revealed that phosphatidylethanolamine was concentrated to approximately 90% purity. As shown in Table 2, the extraction rate relative to the amount of ethanol-extracted lipids for the three strains is relatively high at 8.3 to 11.5%, and the extraction rate relative to dry bacterial cells is 0.45 to 0.56.
It was %. After hydrolyzing this phosphatidylethanolamine, it was methyl esterified and the fatty acid composition was compared, and the γ-linolenic acid content was found to be
An extremely high value of 27.9% or more was observed, which is different from about 7% in the neutral lipid category. The fatty acid composition is illustrated in the margin of Table 2, and it has become clear that unsaturated fatty acids other than γ-linolenic acid have a special fatty acid composition with high contents of linoleic acid and oleic acid.

【table】

Claims (1)

[Claims] 1. γ- from lipids collected from bacterial cells with a high lipid content cultured at high density in a medium with a high concentration of carbohydrates as a carbon source by strains of Isabelina, Vinacea, Lamaniana, Lamaniana anglispora or Nana belonging to the genus Morteierella. Separates phosphatidylethanolamine with high linolenic acid content,
A method for producing phosphatidylethanolamine with a high content of γ-linolenic acid, which comprises concentrating it.