JPH0761271B2 - Method for producing lipid containing eicosapentaenoic acid using microorganism - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a lipid containing eicosapentaenoic acid (hereinafter referred to as EPA).

(Prior Art) Polyunsaturated fatty acids represented by EPA play an important role as constituents of biological membranes. Further, the following are known as the pharmacological actions of EPA known so far. Platelet aggregation inhibitory action (thrombolytic action) Neutral fat lowering action in blood VLDL-cholesterol, LD in blood
L-cholesterol lowering action, HDL-cholesterol increasing action (anti-arteriosclerotic action) Blood viscosity lowering action Blood pressure lowering action Anti-inflammatory action Anti-tumor action. Furthermore, it serves as a substrate for the production of the prostaglandin family, and exerts an essential function in the body of higher mammals including humans. In particular, EPA is important as a substrate for the production of type 3 prostaglandins, has an inhibitory action on platelet aggregation, and its application as a therapeutic and prophylactic agent for thrombosis is being investigated. Further EP
Among the polyunsaturated fatty acids that contribute to the lowering of plasma cholesterol level, A has a particularly high activity and is far more effective than linoleic acid contained in ordinary vegetable oils.
It is also known as an essential nutrient for fish and the like.

Thus, EPA is likely Denmark Tire Berg as health foods or medicines based on the thrombus inhibiting action or lipid-lowering action (Am.J.Clin.Nutr., 28, 95
8,1975), but it is extremely difficult to carry out chemical synthesis as is clear from its chemical structure. Because of this, EP also in Japan
Ingestion of blue-backed fish such as sardines, mackerel, saury, etc., which are high in A, has been recommended.

Most of the EPAs marketed today as health foods are fish oil fractions obtained by the boiling method,
Its EPA content is about 10-30%. The fish oil extracted by the boil-off method is a mixed glyceride containing many kinds of fatty acids as constituent fatty acids, and not only is it difficult to isolate and purify each component, but EPA has all five cis-type double bonds. Since it is a linear polyunsaturated fatty acid with 20 carbon atoms, it is an unstable fatty acid that is extremely easily oxidized. When concentrating EPA from fish oil, it is necessary to avoid oxygen, light, heat, etc. There is. Furthermore, various organic solvents such as acetone and methyl ethyl ketone used for the separation of these fish oil EPAs are usually removed under reduced pressure, but their complete removal has many technical and cost problems.

EPA as a pharmaceutical agent treats fish oils extracted by various methods enzymatically or under alkaline conditions to hydrolyze them into free fatty acids, or after converting the free acids into methyl or ethyl esters, these are treated at low temperature. In many cases, the EPA concentration is 90% or more after further purification by a fractional crystallization method, a urea addition method, a vacuum distillation method or a reverse phase chromatography method. However, EPA concentrates obtained using these methods may use various organic solvents during the process, or
Since high heat of near 0 ° C is applied, there is concern about deterioration due to residual organic solvent and polymerization, isomerization or oxidation of EPA. Furthermore, when fish oil or the like is used as a raw material for EPA, it is difficult to remove docosenoic acid, which is suspected as one of the causes of heart disease, so there is a problem in using it in health foods, pharmaceuticals, etc. ing.

On the other hand, recently, in incomplete purification / concentration, microorganisms such as Chlorella, unicellular alga Monodas, Euglena or diatom were used for the purpose of improving the extraction method from fish oil, which had the drawback that fish odor remained. As EPA production methods are becoming more and more popular, EPA production using microorganisms is drawing attention. Recently, Gellerman and Schlenk (JLGe
llerman and H.Schlenk, BBA, 573, 23,1979) and, Hitoshi Yamada (1986 Japan fermentation Engineering Society convention, in the announcement of 1986),
A report has been made on molds (molds) that produce EPA.

EPA production by these microorganisms is due to its fatty acid composition,
Separation and purification is relatively easy, and due to culture control,
It has advantages such as easy control of EPA production. However, when these filamentous fungi are used, the culture time is longer than that of bacteria (7 days to 1 month),
Improving productivity remains a major problem.

(Problems to be Solved by the Present Invention) As described above, extraction production from fish oil of EPA, which is considered as a health food or a drug, or
There are some problems in producing algae, molds and the like by culture. From these, an object of the present invention is to find a method of fermentative production of EPA-containing lipids using bacteria, which is easy to purify and can be obtained with high purity, and whose culture time is short and culture control is easy. .

(Means for Solving Problems) The inventors of the present invention have extensively researched bacteria having an EPA-producing ability widely in the ocean, and as a result, Pasteurella (Pasteure)
It was found that bacteria belonging to the genus lla) produce EPA, and the present invention was completed based on this finding.

Therefore, the present invention provides EP by culturing a microorganism belonging to the genus Pasteurella and capable of producing an EPA-containing lipid.
The present invention provides a method for producing an eicosapentaenoic acid-containing lipid, which comprises producing and accumulating an A-containing lipid, and then collecting the lipid.

(Specific Description) (1) Microorganisms The microorganisms used in the present invention may be those belonging to the genus Pasteurella and capable of producing EPA or a lipid containing the same, and such microorganisms are new from the natural world. Can be separated into

Examples of microorganisms belonging to Pasteurella include Pasteurella haemorytica, and Pasteurella haemorytica SCRC-2288 isolated by the present inventors as a new strain.

This new strain was isolated as follows.

A medium having the composition shown in Table 1 below was prepared.

This agar plate medium was inoculated with a sample of marine organisms collected from various oceans, diluted appropriately with sterilized physiological saline, and cultured at 25 ° C for 1 to 2 days. The colonies appearing on this agar plate medium were picked up on a slant medium having the same medium composition.

In this way, a large number of strains were isolated from marine organism samples collected from the oceans of various regions. Next, 5 ml of the medium obtained by removing agar from the medium in the table was dispensed into test tubes and sterilized in the same manner. Each strain was cultured in this medium at 25 ° C for 1 to 2 days. From the obtained culture solution, the EPA production performance was tested by the method described below. In this way, the following strains that prominently produce EPA were obtained from marine organism samples collected from off the coast of Yoichi, Hokkaido.

This strain has the following mycological properties.

Observation items a) Morphology 1 Cell shape Short bacillus Size 0.6 × 1.2 μm 2 Presence or absence of polymorphism 3 Presence or absence of motility Absence of flagella Single / polar flagella 4 Presence of spores None 5 Gram stain Negative 6 Anti-acid negative b) Growth state in each medium 1 Meat broth agar plate culture (25 ° C, 2 days) a) Colony shape (diameter) 1.5 × 1.5 mm b) Colony shape Circular c) Shape of colony surface Smooth ) Protrusion of colonies Trapezoidal e) Periphery of colonies f) Color of colonies Yellowish brown) Transparency of colonies Translucency of colonies G) Luster of colonies Re) Soluble pigment formation None 2 Beef agar slope culture (25 ℃, 2 days) b) Good or bad growth b) Luster of colony Luster 3 Broth liquid medium (25 ° C, 2 days) b) Growth of the surface No b) Turbidity Had turbid Ha) Precipitating powder d) No gas generation 4 Meat gelatin (25 ℃, 2 days) Gelatin liquefaction liquefaction 5 Mass milk reduction c) Physiological properties 1 Reduction of sulfate + 2 Denitrification − 3 MR − 4 VP − 5 Indole production − 6 Hydrogen sulfide production + 7 Starch hydrolysis − 8 Citric acid utilization a) Koser − Ro) Christensen − 9 Utilization of nitrate − 10 Pigment formation a) King A medium − b) King B medium − 11 Urease − 12 Oxidase + 13 Catalase + 14 Growth range a) pH 5 to 9 b) Temperature 5 ° C to 30 ° C 15 Oxygen Attitude toward facultative anaerobic 16 O-F test (glucose) + (F) 17 Acid and gas production from sugar 1.L-arabinose-2.D-xylose + 3.D-glucose + 4.D-mannose-5 .D-fructose-6.D-galactose + 7 Maltose + 8 Sucrose + 9 Lactose-10.trehalose-11.D-sorbit-12.D-mannite + 13.inosit-14.glycerin-15 . Starch- ( However, gas generation All items-) d) Other properties Growth on SS agar-Growth on MacConkey agar-6.5% NaCl salt tolerance + DNase + ornithine decomposition-Arginine decomposition-Na ⁺ requirement-above Due to such properties as described above, this strain SCRC-2288 has the following major features.

Gram-negative non-spore forming facultative anaerobic bacillus OF test positive (fermenting sugar) catalase, oxidase positive nitrate-reducing capacity positive Strain SCRC-2288 with these properties The taxonomic position is Bergey's Manual of System Bacteriology.
tematic Bacteriology) Volume 1, 550 pages, 1984 classification criteria Pasteurella hemolytica ( Pasteure
lla haemolytica ).

Note that some of the items of the acid and gas generation test from sugar do not necessarily match those described in the literature,
These are not so important taxonomic items, and they generally change often even in the same species, and are not necessarily specified in these descriptions.

Although the strains isolated from the natural world have been described in detail above, mutations can be generated in these strains to obtain strains with higher productivity.

The strain of this invention can be stored according to a conventional method,
For example, it can be stored on an agar slant medium or by a freeze-drying method. As the agar slant medium, it is possible to use the medium commonly used for the preservation of Pasteurella spp.
Also, freeze-drying can be preserved according to a conventional method.

The above-mentioned strain Pasteurella hemolytica SCRC-2288 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microindustry Research Institute No. 9425 (FERM P-9425).

(2) Method for Producing EPA-Containing Lipid When an EPA-containing lipid is produced by culturing the above-mentioned microorganism, any basal nutrient medium may be used as long as the microorganism of the present invention can grow therein. .

This medium contains, as a nitrogen source, one or more kinds of yeast extract, peptone, meat extract and the like. If necessary, various sugars can be added to this medium as a carbon source.

For the culture, either a solid medium or a liquid medium may be used, but in order to obtain a large amount of the desired EPA-containing lipid, it is preferable to use a liquid medium for the culture. The culture temperature may be any temperature within the temperature range in which the bacteria grow and EPA is produced, preferably 5 to 30 ° C, more preferably 15 to 25 ° C. pH is 5-9, preferably 7-8
Is the range. The culture time may be selected such that a harvestable amount of EPA-containing lipid is produced, and preferably 8 to 48 hours.

Next, the EPA-containing lipid is collected from the obtained culture. As a purification method, a usual lipid purification method can be used. For example, the cells are collected from the culture solution by a conventional cell-collecting means such as centrifugation or filtration. Next, if desired, the bacterial cells are treated with water,
After washing with a saline solution or a buffer solution such as a phosphate buffer solution, the cells are resuspended in these solutions. The suspension is extracted with a solvent commonly used for lipid extraction, for example, a chloroform / methanol mixture, and the phases are separated to obtain a chloroform phase. Then, the chloroform phase is removed by evaporation to obtain a material containing EPA-containing lipid.
Free EPA or its salt can be obtained by saponifying it by a conventional method.

Thus, according to the present invention, a large amount of EPA-containing lipid and EPA can be obtained in a short period of time, which is easy to purify by fermentation production using the above bacteria.

Next, a specific example of the method for producing the EPA-containing lipid of the present invention will be shown.

Example 1 Production of EPA-containing lipids from Pasteurella hemolytica SCRC-2288 Meat extract 1.0%, peptone 1.0%, NaCl 0.5%, pH
After 20 liters of the medium adjusted to 7.0 was sterilized by heating at 121 ° C. for 15 minutes, Pasteurella hemolytica SCRC-2288 (Microtechnology Research Institute No. 9425) was inoculated and cultured at 25 ° C. for 24 hours. After culturing, the bacterial cells were collected by a centrifuge to obtain a bacterial cell having a wet weight of about 195 g (dry weight of 17.6 g). The cells were washed once with 0.85% saline and then suspended in 1. The cell suspension was well shake-extracted with a chloroform-methanol solution of 1 (2: 1 v / v) and then centrifuged to obtain a chloroform phase. Further, the aqueous phase and the bacterial cells were extracted by shaking with 600 ml of chloroform and then centrifuged to obtain a chloroform phase. 1.54 g of lipid fraction obtained by concentrating the chloroform extraction fraction to dryness
(8.75% per dry cell).

This fraction was added to 80% in 95% ethanol containing 0.3N NaOH.
Saponification was carried out at ℃ for 1 hour, and this was neutralized with 6N-HCl to obtain a free fatty acid mixture.

This free fatty acid mixture was methyl esterified with diazomethane, then analyzed by gas chromatography and measured to find 0.166 g (10.8% of lipid fraction, 0.94% of dried cells).
It turns out that it contains EPA.

0.152 g of EPA purified by subjecting this mixture of free fatty acid containing EPA to column reverse phase partition chromatography using a silica gel column with methanol as an eluent.
Got

Claims (2)

[Claims] 1. A method of culturing a microorganism belonging to the genus Pasteurella and capable of producing a lipid containing eicosapentaenoic acid, thereby producing and accumulating the lipid containing eicosapentaenoic acid, and then collecting the lipid. And a method for producing a lipid containing eicosapentaenoic acid. 2. The microorganism is Pasteurella hemolytica (Past
The production method according to claim 1, which is eurella haemolytica.