JPS63216490A - Production of eicosapentaenoic acid by microorganism and microorganism using therefor - Google Patents

Abstract

PURPOSE:To produce eicosapentaenoic acid (EPA) and EPA-containing lipid useful as health foods or pharmaceuticals by fermentation process, by culturing a specific microbial strain belonging to Vibrio genus in a nutrient medium. CONSTITUTION:Vibrio splendius SCRC-1226 (FERM P-9212) is cultured under aerobic condition in a liquid medium containing a nitrogen source consisting of one or more yeast extract, peptone, meat extract, etc., and sodium chloride or natural or artificial seawater and, if necessary, added with various kinds of saccharides as carbon sources at 5-30 deg.C, preferably 15-25 deg.C and pH 6-9, preferably pH 7-8 for 8-48hr by standing culture, shaking culture or culture under aeration and agitation. After the completion of culture, EPA-containing lipid is separated from the microbial cell by solvent extraction and the lipid is saponified to separate EPA.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to eicosapentaenoic acid (hereinafter referred to as EPA)
The present invention relates to a method for producing contained lipids and eicosapentaenoic acid, and microorganisms for producing them.

(Prior Art) Polyunsaturated fatty acids represented by EPA play an important role as a constituent of biological membranes. Furthermore, the following pharmacological actions of EPA are known so far. ■ Platelet aggregation inhibitory effect (thrombolytic effect) ■
Neutral fat lowering effect in the blood ■For VLDL-cholesterol, LDL-cholesterol m manipulation in the blood, HD
L-cholesterol increasing effect (anti-arteriosclerotic effect) ■Blood viscosity lowering effect ■Blood pressure lowering effect ■Anti-inflammatory effect■ Anti-tumor effect.Furthermore, it serves as a substrate for the production of prostaglandin family, and is used in mammals including humans. Performs essential functions in the body. In particular, EPA is important as a substrate for the production of type 3 prostaglandin, has an inhibitory effect on platelet aggregation, and is being considered for use as a therapeutic and preventive agent for thrombospasm. Furthermore, EPA has particularly high activity among polyunsaturated fatty acids that contribute to lowering blood cholesterol levels, and is more effective than linoleic acid and the like contained in ordinary vegetable oils. It is also known as an essential nutrient for fish, etc.

In this way, the possibility of EPA being used as a health food or medicine based on its antithrombotic or lipid-lowering effect was demonstrated by the Danish company Diavelve (Am, J, C).
1in, Nutr,, 28. 958°197
5), but as is clear from its chemical structure, it is extremely difficult to chemically synthesize it. For this reason, in Japan, EPA
It has become recommended to eat blue-backed fish such as sardines, mackerel, and saury, which contain a large amount of

Most of the EPA commercially available as health foods today is fractionated fish oil obtained by the boiling method, and its EPA content is about 10 to 30%. Fish oil extracted by the boiling method is a mixed glyceride containing many types of fatty acids as constituent fatty acids, and not only is it difficult to isolate and purify each component, but all EPA contains cis double bonds. Because it is a linear polyunsaturated fatty acid with 5 carbon atoms and 20 carbon atoms, it is an unstable fatty acid that is extremely easily oxidized, so when concentrating EPA from fish oil, avoid oxygen, light, heat, etc. There is a need. Furthermore, various organic solvents such as acetone and methyl ethyl ketone used in the fractionation of fish oil EPA are usually removed under reduced pressure.
Complete removal involves many technical and cost problems.

EPA as a pharmaceutical product is produced by treating fish oil extracted by various methods enzymatically or under alkaline conditions to hydrolyze it to free fatty acids, or by converting the free acids into methyl or ethyl esters and then converting them into methyl or ethyl esters. In many cases, it is further purified by low-temperature fractional crystallization, urea addition, vacuum distillation, reversed phase chromatography, etc. to reach an EPAt degree of 90% or more.

However, the EPA concentrate obtained using these methods is difficult to obtain because various organic solvents are used during the process or
Because high heat close to 0°C is applied, there are concerns about deterioration due to residual organic solvents, polymerization of EPA, isomerization, or oxidation.

Furthermore, when fish oil is used as a raw material for EPA, it is difficult to remove tocosenoic acid, which is suspected to be one of the causes of heart disease, which poses problems when used in health foods, medicines, etc. ing.

On the other hand, recently, with the aim of improving the extraction method from fish oil, which has disadvantages such as incomplete purification and concentration, leaving a fishy odor, microorganisms such as Chlorella, unicellular algae Monodus, Euglena, or Kei 1 have been used. As production methods for F and PA have been defeated, EPA production using microorganisms is attracting attention.

Recently, Gellerman and Schelenk (J, L.

Gellerman and H, 5chlenk
.

BBA, 573, 23.1979) and Yamada et al. (1986 Japan Fermentation Engineering Society Conference, 1986), E.
A report was made on Zurchin mold (filamentous fungus) that produces PA.

EPA production by these microorganisms has advantages such as relatively easy separation and purification due to its fatty acid composition, and easy control of EPA production by culturing txm. However, when these filamentous fungi are used, the cultivation time is longer (about 7 days to 1 month) than with bacteria, and improvement in productivity remains a major problem.

C) Problems to be Solved by the Present Invention) As mentioned above, there are no problems with the production of EPA, which is considered as a health food or medicine, by extraction from fish oil or by culturing algae or mold. , there are some problems. Based on these facts, the purpose of the present invention is to discover a method for fermentation production of EPA-containing lipids using bacteria, which is easy to purify and obtain highly pure products, and which has a short culture time and easy culture control. .

(Means for Solving the Problems) As a result of extensive research searching for bacteria capable of producing EPA in the ocean, the present inventors discovered that Vibrio (Vibr.
We discovered that bacteria belonging to the genus io) produce EPA, and based on this finding, we completed the present invention.

Therefore, the present invention produces and accumulates eicosapentaenoic acid-containing lipids by culturing microorganisms that belong to the genus Vibrio and can produce eicosapentaenoic acid-containing lipids, and further isolates eicosapentaenoic acid if desired. The present invention provides a characteristic eicosapentaenoic acid-containing lipid or a method for producing eicosapentaenoic acid, and a microorganism of the genus Vibrio that can produce the eicosapentaenoic acid-containing lipid.

(Specific explanation) +11 Microorganisms The microorganisms used in the present invention belong to the genus Vibrio,
Any microorganism can be used as long as it can produce EPA or a lipid containing it, and such microorganisms can be newly isolated from nature.

An example of a microorganism belonging to the Vibrio family is Vibrio subrendidus (Vibrio subrendidus).
Vibrio spren ≠ didus 5CRC-122, which the inventor isolated as a new strain.
I can name 6.

This strain was isolated as follows.

A culture medium having the composition shown in the following table was prepared.

Part - Surface meat extract 1.0% Peptone 1.0% NaC 10.5M Tap water pH 7.0 On this agar plate medium, marine organism samples collected from various oceans were diluted appropriately with physiological saline containing i1i bacteria. The cells were inoculated and cultured at 25°C for 1 to 2 days. Colonies that appeared on this agar plate were plated onto a slanted medium with the same medium composition.

In this way, a large number of bacterial strains were isolated from marine organism samples collected from the oceans of various places.Next, the agar was removed from the culture medium shown in the table above, and 5 microorganisms were dispensed into test tubes and sterilized in the same manner. Each strain was isolated. In this way, E
The above-mentioned bacterial strain that significantly produces PA was obtained from a marine organism sample collected from Sagami Bay.

This new strain has the following mycological properties.

The following margin observation items a) Morphology 1 Cell shape Short bacterium size
0.7×1.6μm2 Presence of polymorphism No 3 Presence of motility Flagellum epiphyte status 1, polar flagella 4 Presence of spores No 5 Gram staining Negative 6 Acid-fast
Negative b) Growth status in each medium 1 Broth agar plate culture (25°C, 2 days) a) Colony shape (diameter) 2.0-2.5 mm M mouth) Colony shape circular c) Colony surface shape Smooth 2) Elevated state of the colony Table-like e) Periphery of the colony To the golden edge) Color tone of the colony Pale yellow g) Transparency of the colony Translucent h) Gloss of the colony
Dull glow) Production of soluble pigments None 2 Broth agar slant culture (25°C, 2 days) A) Quality of growth Good or poor Colony gloss Blunt light 3 Broth liquid medium (25°C, 2 days) A) Surface growth Silent ) Turbidity Turbidity C) Precipitation Powder 2) Gas generation None 4 Meat juice gelatin (25℃, 2 days) Gelatin liquefaction Liquefaction 5 Liquefaction 5 Liquification No change C) Physiological properties 1 Reduction of nitrate 12 Denitrification - 3MR- 4VP -5
Indole production 16. Production of hydrogen sulfide - 7. Hydrolysis of starch - 8. Utilization of citric acid A) Kossr - 口) Chrl
atensen - 9 Utilization of nitrate -' 10 Pigment production a) Klng A Medium - mouth) King
B Medium -11 urease
- 12 Oxidase +13 Catalase +14 Growth range a) pH 6-9 0) Temperature 5℃-30℃15
Attitude towards oxygen Facultative anaerobic 16 0-F
Test (glucose) +17 Acid and gas production from Ws 1, L-arabinose - 2.0-xylose - 3.0-glucose + 4, D-mannose - 5, D-fructose - 6, L)-galactose + 7 , maltose 18, sila sugar -9, lactose
- 10, trehalose - 11, D-sorbitol - 12, D-mannitol + 13, inosift - 14, glycerin + 15, starch - (however, gas production - all items) d) Other properties π In SS agar medium Growth Growth on Ten Pine Conchi agar medium + 6.5% NaCl salt tolerance Ten DNA s s + ornithine decomposition - Arginine decomposition - Na requirement Growth on Ten TCBS agar medium C, -C content of Ten DNA 45 .3% or more, this strain 5CRC-1226 has the following major characteristics.

■ Durham negative ■ Has one polar flagellum and is motile ■ Catalase and oxidase positive ■ Requires Na'' G-C content of DNA: 45.3% ■ Grows on TCBS agar medium and has these properties This strain 5CRC-1226
The taxonomic position of strains can be found in Bergey's Manual of Systematic Bacteriology (Bergyo's Manual of Systematic Bacteriology).
s Manual of Systematic
Bacteriology) No. 1@, page 352, 198
4 year classification criteria and the International Journal of Systematic Bacteriology (In
Ler-national Journal o
f Sys-Lematic Bacter
36 (4), 531-543 (19
86) in West et al.
, p, R.

Brayton, T.N., Bryant, andR.
, R. Co Iwe 11), some of the test items for acid and gas production from tongs do not necessarily match those described in the literature, but these are important items from a taxonomic perspective. Rather, they generally vary widely even within the same species, and are not necessarily defined by these descriptions.

Although this new bacterial strain isolated from nature has been described in detail above, it is also possible to generate mutations in these bacteria to obtain strains with even higher productivity.

The strain of this invention can be stored according to conventional methods,
For example, it can be stored on an agar slant medium or by freeze-drying. As the agar suran and the medium, it is possible to use a medium commonly used for preserving Vibrio bacteria, such as the medium described above for the isolation of bacteria. In addition, the freeze-dried 5CRC-1226 was sent to the Institute of Microbiology, Agency of Industrial Science and Technology, and was donated to the Institute of Microbiology, No. q″'L (No. 2 (FERM)).
It has been deposited as P-'11 (z>).

(2) Method for producing EPA When attempting to produce the EPA of the present invention by culturing the above-mentioned microorganisms, any medium may be used as the basal nutrient medium as long as it allows the microorganisms of the present invention to proliferate.

This medium contains one or more nitrogen sources, such as yeast extract, peptone, meat extract, etc. Moreover, various type II species can be added to this medium as a carbon source, if necessary. This medium contains sodium chloride,
Alternatively, it is necessary to add natural seawater or artificial seawater.

Either a solid medium or a liquid medium may be used for culturing, but in order to obtain a large amount of the desired EPA, a liquid medium should be used, and aerobic conditions such as static or shaking culture, aeration/agitation culture, etc. should be used. The culture is preferably carried out at any temperature within the temperature range in which the bacteria can grow and EPA is produced, preferably between 5 and 30°C, more preferably between 15 and 25°C. be. 1>H ranges from 6 to 9, preferably from 7 to 8. The culture time may be selected so that a collectable amount of BPA-containing lipid is produced, and is preferably 8 to 48 hours.

Next, the EPA of the present invention is collected from the resulting culture.

Ordinary lipid purification methods can be used as purification methods.
For example, bacterial cells are collected from a culture solution by a conventional bacterial collection method such as centrifugation or filtration.Next, the bacterial cells are optionally washed with water, saline, or a buffer solution such as a phosphate buffer. , resuspend in these solutions. This suspension is
Extraction is carried out with a solvent commonly used for lipid extraction, such as a chloroform/methanol mixture, and the chloroform phase is obtained by phase separation.Next, the chloroform phase is removed by evaporation to remove the EPA-containing lipid-containing material. can get. Free EP can be obtained by saponifying this using a conventional method.
A or its salt can be obtained.

Thus, according to the present invention, EPA-containing lipids and EPA can be obtained in large amounts in a short time with ease of purification by fermentation production using the above-mentioned bacteria.

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

Example 1 Meat extract 1.0%, peptone 1.054. Na
Medium 2 containing C10.5% and adjusted to pH 7.0
After heat sterilizing Vibrio 01 at 121℃ for 15 minutes,
Splendidas 5CRC-1226 (Huikoken Bacteria Q.
"L (No. 7) was inoculated and cultured aerobically at 25°C for 24 hours. After culturing, Kokutai was collected using a centrifuge and weighed approximately 25
0 g (22.5 g in dry weight) of bacterial cells was obtained. Bacteria body 0.
After washing once with 85% saline, it was suspended in 11. This bacterial cell suspension was mixed with 1 g of chloroform-methanol solution (
After shaking and extracting the mixture well (2:1 v/v), the mixture was centrifuged to obtain a chloroform phase. Furthermore, the aqueous phase and Kokutai were shaken and extracted with chloroform at 600 m, and then centrifuged to obtain a chloroform phase. The lipid fraction obtained by concentrating the chloroform extracted fraction to dryness was 2.06 g (9 g per dry facepiece).
．． 16%), this fraction was treated with 0.3N-NaOH
The mixture was saponified at 80° C. for 1 hour in 95% ethanol containing 20% ethanol, and neutralized with 6N-HCI to obtain a free fatty acid mixture.

After methyl esterifying this free fatty acid mixture with diazomethane, it was analyzed by gas chromatography and found to contain 0.214 g (1000% of lipid fraction, 0.95% of dry bacterial cells) of EPA. I found out that there is.

This free fatty acid mixture containing EPA was purified by column reverse phase partition chromatography using a silica gel column with methanol as the eluent.
185 g of O was obtained.

Claims (3)

[Claims] (1) By culturing a microorganism that belongs to the genus Vibrio and is capable of producing eicosapentaenoic acid-containing lipids, eicosapentaenoic acid-containing lipids are produced and accumulated, and if desired, eicosapentaenoic acid is further isolated. A method for producing an eicosapentaenoic acid-containing lipid or eicosapentanoic acid, characterized by: (2) A microorganism of the genus Vibrio that can produce eicosapentaenoic acid-containing lipids. (3) Vibrio splendidus (￥Vibrio￥
The microorganism according to claim 2, which is SCRC-1226.