JPH0223877A - Production of eicosapentaenoic acid - Google Patents

Abstract

PURPOSE:To obtain the subject eicosapentaenoic acid having high purity and useful as a pharmaceutical, etc., in an easily purifiable state in a short time by producing and accumulating an eicosapentaenoic acid-containing lipid in a microorganism of genus Pseudomonas, etc., and separating the acid from the lipid. CONSTITUTION:A microbial strain capable of producing lipid containing eicosapentaenoic acid (abbreviated as EPA) and belonging to genus Pseudomonas [e.g., Pseudomonas putrefaciens (SCRC-2181)], Alteromonas [e.g., Alteromonas putrefaciens (SCRC-2871)], Shewanella [e.g., Schiwanella putrefaciens (SCRC-2874)], etc., is cultured to produce and accumulate the EPA-containing lipid and, as necessary, EPA is separated from the lipid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a lipid containing eicosapentaenoic acid (hereinafter referred to as EPA) and a method for producing eicosapentaenoic acid by a fermentation method.

[Conventional technology]

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 ■ Lowering effect on VLDL-cholesterol, LDL-cholesterol, and HDL-cholesterol increasing effect in the blood (antiarteriosclerosis 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 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 prostaglandin, has an inhibitory effect on platelet aggregation, and is being considered for use as a therapeutic and preventive agent for thrombosis. Furthermore, EPA has particularly high activity among polyunsaturated fatty acids that contribute to lowering plasma cholesterol levels, and is much 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 effects has been demonstrated by the Danish company Diamond Velvet (Yam, J, C1in,
Nutr., 28, 958.1975), but as is clear from its chemical structure, it is extremely difficult to chemically synthesize it. For this reason, it has become recommended in Japan to consume blue-backed fish such as sardines, mackerel, and saury, which contain a large amount of EPA.

Most of the EPA commercially available as health foods today are fractionated fish oils obtained by the point method, and their EPA content is about 10 to 30%. Fish oil extracted by the point 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 EPA has all cis-type double bonds Because it is a linear polyunsaturated fatty acid with 20 carbon atoms, it is an unstable fatty acid that is extremely easily oxidized, so when concentrating EPA from fish oil, it is necessary to avoid oxygen, light, heat, etc. There is. Further, the various organic solvents used for fractionating these fish oil EPAs are usually removed under reduced pressure, but their complete removal poses 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 converting the free acids into methyl or ethyl esters, which are then subjected to low-temperature treatment. In many cases, it is further purified to an EpA concentration of 90% or more by a fractional crystallization method, a urea addition method, a vacuum distillation method, a reversed phase chromatography method, or the like.

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 alga Monodus, Euglena, or diatoms have been used. EPA production methods are becoming increasingly common, and EPA production using microorganisms is attracting attention.

Recently, Gellerman and Schlenk (J.L.Gell)
ermanand H, 5chlenk, BBA, 57
323.1979) and Yamada et al. (1986 Japan Fermentation Engineering Society Conference, 1986) reported on EPA-producing Zuruka mold (filamentous fungus). 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 through culture control. However, compared to bacteria, etc., the culture time is long (about 7 days to 1 month), and improvement in productivity remains a major problem.

[Problems to be solved by the present invention]

As mentioned above, there are several problems in producing EPA, which is considered as a health food or medicine, by extracting it from fish oil or by culturing algae, mold, etc. 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 problems]

The present inventors conducted extensive research searching for bacteria capable of producing EPA in the ocean, and as a result, discovered that Pseudomonas (
It was discovered that bacteria belonging to the genus Pseudos+onas), the genus Alteromonas, or the genus Shewanella produce EPA, and the present invention was completed based on this knowledge.

Therefore, the present invention produces and accumulates eicosapentaenoic acid-containing lipids by culturing microorganisms that belong to the genus Pseudomonas, Alteromonas, or Shewanella and are capable of producing eicosapentaenoic acid-containing lipids,
A lipid containing eicosapentaenoic acid or a method for producing eicosapentaenoic acid, which is characterized by further isolating eicosapentaenoic acid if desired, and a lipid belonging to the genus Pseudomonas, Alteromonas, or Shewanella that can produce the lipid containing eicosapentaenoic acid. It provides microorganisms.

[Specific explanation]

(1) Microorganisms The microorganisms used in the present invention may belong to the genus Pseudomonas, Alteromonas, or Shewanella and can produce EPA or a lipid containing EPA, and such microorganisms may be new from the natural world. separated into
Or it can be selected from preserved bacteria.

Examples of microorganisms belonging to Pseudomonas include Pseudomonas butrefaciens-$ and n+onas.
Pseudomonas pyurefaciens SCRC--2181 and SCRC--2201°S isolated by the present inventors as new strains.
CRC--2271, SCRC--2341, SCRC
--2451, SCRC-2642, SCRC--27
92, SCRC--2878, SCRC--3011.

SCRC--3022 may be mentioned.

As an example of a microorganism belonging to the Alteromonas family, Alteromonas pyurefaciens
as utrefaciens), and Alteromonas pyurefaciens SCRC--2871, which the present inventors isolated as a new strain, and Alteromonas butrefaciens subsp.
e-faciens 5ubspecies
sa amifaciens) SCRC-1162,
Alteromonas pyurefaciens (A1ter)
oa+onas utrefaciens SCR
Examples include C--2871. On the other hand, examples of preserved strains belonging to this species include Alteromonas pyutrifacens IAM-1510 and Hyalteromonas pyutrifaciens TAM-12425, and these preserved strains have their respective deposit numbers. In addition, IAM can be used freely.

An example of a microorganism belonging to Alteromonas is Alteromonas 7. An example of a preserved microorganism belonging to this species is Alteromonas IAM-12641, which is named after its deposit number. It can be freely obtained from IAM.

As an example of microorganisms belonging to Alteromonas, EP
The present inventors have newly isolated a microorganism that has A-producing ability, Alteromonas rumensus endan anus 1u.
SCRC--6444 identified and named as
, and Alteromonas xylosus
One example is SCRC-2517, which was identified and named as onas 7.

As an example of a microorganism belonging to Shewanella, Shewanella pyurefaciens can be mentioned, and Shewanella pyurefaciens SCR, which was isolated by the present inventors as a new strain, can be mentioned.
C--2874 can be mentioned.

Also, as preserved strains belonging to this species, Shewanella buterifaciens IAM-1510, Shewanella pieutrifaciens IAM-12425, etc. have EPA-producing ability.

The above new bacterial strain was isolated as follows. A culture medium having the composition shown in Table 1 below was prepared.

Table 1: Meat extract 1% Peptone 1% NaC1O, 5% Agar 1.5% Tap water pH 7.0 On this agar plate, marine organism samples collected from various oceans were diluted appropriately with sterilized physiological saline. 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 various oceans. Next, the agar removed from the medium shown in the table was dispensed into test tubes in 5 d portions 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, E
PA production ability was assayed. In this way, the following strain was obtained which significantly produced EPA. The sources of isolation of these bacterial strains are shown in the table below.

SCRC--3011, SCRC--3022SCRC
-2517 Sanriku Coast Bungosui Bay These strains each have the mycological properties shown in Table 3 below.

Based on the above mycological properties, these strains were classified into Bergey's Manual of Determinative Bacteriology.
erminative Bacteriolity)
8th edition, 1974 [Reference 1] iManual of Clinical Microbiology
of C11nical Micr.

biology) 3rd edition, 1980 [Reference 2]
;Bergey's Manual of Systematic Bacteriology
f Systematic Bacteriology)
Volume 1, page 352, 1984 [Reference 3]; and Journal of General Microbiology.
-biology) LiL3057 3074 (198
3) It was identified as follows according to (Reference document 4).

SCRC--2181, SCRC--2201, SCR
C--2271, SCRC--2341, SCRC--
2451, SCRC--2642, SCRC--279
2.

SCRC--2878, SCRC--3011, and S
CRC--3022 is a Durham-negative bacillus that is aerobic, motile, has one polar flagellum, and has catalase and oxidase activities, so according to References 1 and 2, it belongs to the genus Pseudomonas. belongs to Pseudomonas piuterifaciens is known as a species of the genus Pseudomonas, and the above-mentioned properties almost match those described in the literature, so the above 10 strains are identified as Pseudomonas piutrifaciens. Note that some of the test items for acid and gas production from sugars do not necessarily match those described in the literature, but these are not very important items from a taxonomy perspective, and they generally vary widely even within the same species. However, it is not necessarily defined by these descriptions.

In addition, reference document 4 states that Pseudomonas pyurefaciens is close to the genus Alteromonas according to the classification based on the G+C content of DNA, so it is classified as Pseudomonas peurefaciens
romonas utrefaciens). This is also described in Reference Document 3 mentioned above.

Furthermore, in recent years, systematic and applied microbiology
pplied Microbiology) 6.1
71 182 (1985) (reference document 5), the genus Sheevanella was newly proposed, and the above bacterial species was classified as Sheevanella piutrifaciens (Sheevanella).
Shewanella utrefaciens)
It is proposed that

SCRC-2871 is an aerobic, motile, and one polar flagellum, and is a Durum-negative rod that has catalase and oxidase activities, and the G+C content of its DNA is 47.1%. , and belongs to the genus Alteromonas according to the above-mentioned documents 1, 2.3, and 4. Alteromonas pyutrifascens is known as a species of the genus Alteromonas, and since the above properties are nearly identical to those described in the literature, this strain is identified as Alteromonas pyutrifascens.

SCRC--2874 is an aerobic, motile, and one polar flagellum, and is a Durum-negative rod that has catalase and oxidase activities, and exhibits the mycological properties shown in the table above. The above documents 1 and 2! J [According to the criteria, it belongs to Pseudomonas buterifaciens. However, in the reference document 4, Pseudomonas pyurefaciens has G+ of DNA.
From the classification based on C content, Alteromonas
Due to its closeness to the genus Alteromonas utr
efaciens), and this is also described in the above-mentioned document 3. Furthermore, in the above-mentioned document 5, the genus Shewanella was newly proposed based on the base sequence of 5S rRNA,
The above bacterial species were Shiwanella piutrifaciens (Sh
It is proposed that it should be changed to ``esvanella''. Based on the above, this strain is identified as Shewanella pyutrifascens, which belongs to the genus Shewanella.

SCRC--1162 has the following characteristics.

■ Durham negative ■ Motile ■ Non-spore-forming aerobic bacterium ■ O-F test negative ■ Catalase and oxidase positive ■ Has nitrate reducing ability ■ Has hydrogen sulfide producing ability ■ Has gelatin/DNA degrading ability ■ Glucose Acid production (+), gas production [phase]
Quinone type: Ubiquinone 7 and 8, menaquinone, methylmenaquinone■ Has peritrichous flagella @ Na”requiring This strain SCRC--1162 has these properties
The taxonomic position of the strain is identified as follows according to references 1.2, 3 and 4 above. According to the items from ■ to [Phase] above, Alteromonas butrefaciens] Teromonas utrefaciens
It is considered to be a strain related to s). however,
From the morphology at the electron microscopic level, it has pericytary flagella,
In addition, since it does not have ornithine-degrading ability and is Na-requiring, this strain was isolated from Alteromonas pyurefaciens subsp. sagamifaciens (Alte.
romonas utrefaciens subsp
It was identified and named as Ecies sa an+1 faciens-.

SCR-6444 and SCR-2517 have the following main properties.

■ Durham negative ■ Motile ■ Non-spore-forming aerobic bacterium ■ O-F test negative ■ Catalase, oxidase positive ■ Urease negative ■ Gelatin, DNA degrading ability ■ Acid production (+) from glucose, gas production ■Na
(J! or seawater requirement @l G+C content: SCRC--6444; 41
．． 4%, SCRC--2517, 41.1%, this strain SCRC--6444 having these properties
The taxonomic position of the SCRC-2517 strain is identified as follows according to the classification criteria of References 12.3 and 4 above. According to the items from ■ to [phase] above, Alteromonas butrefaciens
It is considered to be a strain related to Alteromonas faciens or Alteromonas aurantia. However, SCR
C--6444 has the ability to reduce nitrate and utilize D-mannose, and does not have the ability to decompose ornithine, so it does not belong to any conventional species, and this strain has been classified as Alteromonas.
Lumensus (＾lteromonas lumen)
sas).

On the other hand, SCRC-2517 has the ability to reduce nitrate and utilize D-mannose, D-xylose, and D-galactose, but does not have the ability to degrade ornithine or hydrogen sulfide. Because of this, it does not belong to any conventional species and is classified as Alteromonas xylosus (Alteromonas xylosus).
It was identified and named ``x Gensindansen Ω-''.

Among the new strains of the present invention described above, the following strains have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and have been further transferred to the International Deposit under the Butapept Treaty.

Pseudomonas pyutrifaciens (Pseud)
osonas utrefacience)SCRC
-2878 Deposited on December 26, 1988, Microtechnology Research Institute Article No. 9114 (FERM P-9114) Transferred to international deposit on December 17, 1988.
No. 23 (FUR-BP-1623) Alteromonas
Pyutrifacens (Alteromonas)
utrefacience) SCRC-2871 Showa 6
Deposited on January 28, 1982, FERM Pi15B No. 9158 (FERM Pi15B) Transferred to international deposit on December 17, 1988 Deposit No. 162 of FERM
No. 4 (FERM BP-1624) Shewanella utr
effacience SCRC--2874 Deposited on January 28, 1988 FERMS deposit No. 9159 (FERM P-9159) Transferred to international deposit on December 17, 1988 FERMS deposit No. 16
No. 25 (FERM BP, 1625) Alteromonas
Alteromonasutrefaciens subspecies sagamifaciens
ns 5ubspecia sa a+wifa
Science) SCRC-1162 Deposited on February 20, 1988 FERMS deposit No. 9210 (FERM P-9210) Transferred to international deposit on December 17, 1988 FERMS deposit No. 16
No. 26 (FERM BP-1626) Alteromonas
Lumensa A1tero+wonasLumensa
s) SCRC--6444 Deposited on February 20, 1986, Microtechnical Research Institute Bacteria No. 9667 (FERM P-9667) Alteromonas xylosus (A 1 teromon
as■Hudan SCRC-2517 Deposited on October 20, 1988, Fiber Science and Technology Research Institute Bacteria Collection No. 9668 (FERM P-9668) Above, we have described in detail the bacterial strains isolated from the natural world. It is also possible to obtain strains with 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 slant medium, a medium commonly used for preserving Pseudomonas bacteria, such as the medium described above for bacterial isolation, can be used. Furthermore, freeze-drying preservation can also be carried out according to conventional methods.

(2) Method for producing EPA When attempting to produce the EPA of the present invention by culturing the microorganism in the first stage, any medium that can grow the microorganism of the present invention may be used as the basic nutrient medium. This medium contains one or more nitrogen sources, such as yeast extract, peptone, meat extract, and the like. Furthermore, various sugars can be added to this medium as a carbon source, if necessary. It is preferable to add sodium chloride, natural seawater, or artificial seawater to this medium.

Cultivation may be carried out using either a solid medium or a liquid medium, but in order to obtain a large amount of the desired EPA, it is preferable to use a liquid medium and use static culture, shaking culture, aeration/agitation culture, etc. It is preferable to culture under atmospheric conditions.

The culture temperature may be any temperature within the temperature range at which bacteria grow and EPA is produced, preferably from 5 to 30".
C, more preferably 15 to 25°C. The pH ranges from 6 to 9, preferably from 7 to 8. The culture time may be selected so as to produce a collectable amount of EPA-containing lipid, and is preferably 8 to 48 hours.

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

A normal lipid purification method can be used as a purification method.

For example, bacterial cells are collected from the culture solution by conventional bacterial collection methods such as centrifugation and filtration. Next, the bacterial cells are washed with water, saline, or a buffer solution such as a phosphate buffer solution, if desired, and then resuspended in these solutions. This suspension is
Extraction is carried out with a solvent commonly used for the extraction of lipids, for example a chloroform/methanol mixture, and the phases are separated to obtain a chloroform phase. Next, the chloroform phase is removed by evaporation to obtain a material containing EPA-containing lipids. Free EP can be obtained by saponifying this using a conventional method.
A or a salt thereof can be obtained.

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

Next, specific examples of the method for producing EPA-containing lipids of the present invention are shown in Examples.

Meat extract 1.0%, peptone 1.0%, NaCj! 0
．． 5% of medium 201 adjusted to pH 7.0.
After heat sterilization at 21°C for 15 minutes, Pseudomonas pyutrifaciens SCRC--28713 (Sokoken Bacterial Serial No. 9114) was inoculated and cultured aerobically at 25°C for 24 hours. After culturing, the cells were collected using a centrifuge to obtain cells with a wet weight of approximately 150 g (16.5 g in dry weight).

After washing the bacterial cells once with 0.85% saline, they were suspended in 11. This bacterial suspension is II! After shaking well and extracting with a chloroform-methanol solution (2:1 v/v), the mixture was centrifuged to obtain a chloroform phase.

Furthermore, the aqueous phase and the bacterial cells were extracted by shaking with chloroform at 600 mZ, and then centrifuged to obtain a chloroform phase.

The lipid fraction obtained by concentrating the chloroform extracted fraction to dryness was 1.16 g (7.03% per dry bacterial cell). This lipid fraction contains EPA. This fraction was 0.
80 in 95% ethanol containing 3 N NaOH.
Saponification was carried out at ℃ for 1 hour to obtain a product containing the sodium salt of EPA. This was neutralized with 6NH (J) to release free E
A product containing PA was obtained. Next, after methyl esterification with diazomethane etc., the content was measured by gas chromatography analysis, and it was found that 0.114g (9.8% of lipid fraction, 0.69% of dry bacterial cells). It was found that EPA was included.

Meat extract 1.0%, peptone 1.0%, Na (J!Q
, 5% of medium 201 adjusted to pH 7.0.
After heat sterilizing at 21℃ for 15 minutes, Alteromonas
Buterifaciens SCRC--2871 (Feikoken Bacteria No. 9158) was inoculated and cultured aerobically at 25° C. for 24 hours. After culturing, the cells were collected using a centrifuge to obtain cells with a wet weight of approximately 135 g (15.0 g in dry weight).

After washing the bacterial cells once with 0.85% saline, they were suspended in 11. This cell suspension was extracted by shaking well with a chloroform-methanol solution (2:1 v/v) of 11, and then centrifuged to obtain a chloroform phase.

Furthermore, the aqueous phase and bacterial cells were extracted by shaking with chloroform 600- and then centrifuged to obtain a chloroform phase.

The lipid fraction obtained by concentrating the chloroform extracted fraction to dryness was 1.05 g (7.00% per dry cell body). This lipid fraction contains EPA. Both parts are 0
．． Saponification was carried out in 95% ethanol containing 3N-NaOH at 80° C. for 1 hour to obtain a product containing the sodium salt of EPA. This is 6N-HC7! to obtain a product containing free EPA. Next, a part of this product was methyl esterified with diazomethane, and then analyzed by gas chromatography to determine its content. It was found that 0.74% of dry bacterial cells) EPA was contained.

The product containing free and %lt EP A was purified by silica gel chromatography [eluent: n-hexane/ethyl ether (9:1)] to obtain both fractions containing only EPA, which were evaporated to dryness. 0.10 by doing
g of EPA was obtained.

Meat extract 1.0%, peptone 1.0%, NaCj! 0.
Medium 201 containing 5% and adjusted to pH 7.0 was added for 12
After heat sterilization at 1°C for 15 minutes, the seedlings were inoculated with Shewanella butrefaciens SCRC--2874 (Feikoken Bacterium No. 9159) and cultured aerobically at 25°C for 24 hours. After culturing, collect the bacterial cells using a centrifuge and reduce the wet weight to approximately 20
0 g (22.5 g in dry weight) of bacterial cells was obtained.

After washing the bacterial cells once with 0.85% saline, they were suspended in 11. This cell suspension was extracted by shaking well with a chloroform-methanol solution (2:1 v/v) of 11, and then centrifuged to obtain a chloroform phase.

Furthermore, the aqueous phase and bacterial cells were extracted by shaking with chloroform 600- and then centrifuged to obtain a chloroform phase.

Chloroform extracted fraction? The lipid fraction obtained by condensation to dryness was 1.78 g (7.91% per dry bacterial cell). This lipid fraction contains EPA. Both parts are 0
．． Saponification was carried out in 95% ethanol containing 3N-NaOH at 80° C. for 1 hour to obtain a product containing the sodium salt of EPA. This is 6N-)1cJ! neutralized by
A product containing free EPA was obtained. Next, a part of this product was methyl esterified with diazomethane, and the content was measured by gas chromatograph analysis. ,
It was found that 1.07% of the dry bacterial cells contained EPA.

The product containing free EPA was purified by silica gel column chromatography (eluent: n-hexane/ethyl ether (9:1)) to obtain two fractions containing only EPA, which were evaporated to dryness. 22g EP
I got an A.

Meat extract 1.0%, peptone 1.0%, NaCl 0.
Medium 301 containing 5% and adjusted to pH 7.0 was added for 12
After heat sterilizing at 1°C for 15 minutes, inoculation with Alteromonas pyutrifaciens subsp. Sagamifaciens SCRC--1162 (Feikoken Bacteria No. 9210) was carried out aerobically at 25°C for 24 hours. Cultured.

After culturing, the bacterial cells were collected using a centrifuge and weighed approximately 205 g (
A bacterial cell weighing 25.6 g (dry weight) was obtained. 0.
After washing once with 85% saline, it was suspended in 1.51. After shaking and extracting this bacterial cell suspension with a 1.51 chloroform-methanol solution (2:1 v/v),
Centrifugation was performed to obtain a chloroform phase. Furthermore, the aqueous phase and bacterial cells were treated with chloroform at 900 rn! After shaking and extraction, centrifugation was performed to obtain a chloroform phase.

The lipid fraction obtained by concentrating and drying the chloroform extracted fraction was 2.13 g (8.32% per dry cell body).
It was saponified in ethanol at 80°C for 1 hour, and this was diluted with 6N-
Neutralization with H(J) gave a product containing free EPA.

Next, after methyl esterification with diazomethane, it was analyzed with a gas chromatograph, and the measurement result was 0.205
g (9.6% of lipid fraction, 0.80% of dry bacterial cells) was found to contain EPA.

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.

Meat extract 1.0%, peptone 1.0%, Na (40,5
% and adjusted to pH 7.0 medium 20/121
After heat sterilization at 15°C for 15 minutes, Alteromonas hanedai IAM-12641 was inoculated and cultured aerobically at 15°C for 24 hours. After culturing, the cells were collected using a centrifuge to obtain cells weighing about 150 g wet (14.9 g dry). After washing the bacterial cells once with 0.85% saline,
1. This bacterial cell suspension was mixed with 1/1 chloroform-
After thorough shaking and extraction with a methanol solution (2:1 v/v), the mixture was centrifuged to obtain a chloroform phase. Furthermore, the aqueous phase and bacterial cells were extracted by shaking with chloroform 600- and then centrifuged to obtain a chloroform phase. The lipid fraction obtained by concentrating the chloroform extracted fraction to dryness was 1.18 g.
(7.92% per dry bacterial body). Both fractions were saponified in 95% ethanol containing 0.3 N-NaOH at 80° C. for 1 hour, and this was neutralized with 6N-H (J) to obtain a product containing free EPA. Next, after methyl esterification with diazomethane, it was analyzed by gas chromatography and found to contain 0.145 g (12.3% of lipid fraction, 0.97% of dry bacterial cells) of EPA. This free fatty acid mixture containing EPA was subjected to column reverse phase partition chromatography using a silica gel column with methanol as the eluent to obtain 0.126 g of purified EPA.

Meat extract 1.0%, peptone 1.0%, NaC7! 0
．． 101 containing 5% and adjusted to pH 7.0
After heat sterilizing at 21℃ for 15 minutes, Alteromonas
Alteromonas lumen
sas) SCRC-6444 (Micro Engineering Naeyo No. 9667
No.) was inoculated and cultured aerobically at 25°C for 24 hours. After culturing, the bacterial cells were collected using a centrifuge and weighed approximately 160 g (
A bacterial cell weighing 20.4 g (dry weight) was obtained. 0.85 bacterial cells
After washing once with 0.51% saline, it was suspended in 0.51% saline. This cell suspension was extracted by shaking well with a 0.51 chloroform-methanol solution (2:1 v/v), and then centrifuged to obtain a chloroform phase. Furthermore, the aqueous phase and the bacterial cells were shaken and extracted with 300% chloroform, and then centrifuged to obtain a chloroform phase. The lipid fraction obtained by concentrating the chloroform extracted fraction to dryness was 1.05 g.
(5.13% per dry bacterial body). Both parts are 0
．． 8 in 95% ethanol containing 3 N-NaOH.
The mixture was saponified at 0°C for 1 hour and neutralized with 6N-HCl to obtain a product containing free EPA.

Next, after methyl esterification with diazomethane, it was analyzed with a gas chromatograph, and the measurement result was 0.198.
g (18.9% of lipid fraction, 0.97% of dry bacterial cells)
) was found to contain EPA.

Using a silica gel column, this free fatty acid mixture containing EPA was mixed with methanol. EPA purified by column reverse phase partition chromatography as eluate
0.177g was obtained.

Meat extract 1.0%, peptone 1.0%, NaC10.5
% and adjusted to pH 7.0 to 121
After heat sterilizing at ℃ for 15 minutes, Alteromonas xylosus (Alteromonas dscRc-2
517 (Feikoken Bacteria No. 9668) was inoculated and cultured aerobically at 25°C for 24 hours. After culturing, the bacterial cells were collected using a centrifuge and weighed approximately 145 g (dry weight: 19.6 g).
) were obtained. The bacterial cells were washed once with 0.85% saline and then suspended in 0.51% saline. This bacterial cell suspension was mixed with a 0.51 chloroform-methanol solution (2:
1 v/v) and then centrifuged to obtain a chloroform phase. Furthermore, the aqueous phase and the bacterial cells were shaken and extracted with 300% chloroform, and then centrifuged to obtain a chloroform phase. The lipid fraction obtained by concentrating and drying the chloroform extracted fraction was 1.01 g (5.0 g per dry bacterial cell).
15%). This fraction was dissolved in 0.3 N-NaOH
was saponified for 1 hour at 80° C. in 95% ethanol containing 95% ethanol, which was neutralized with 6N-H (J) to obtain a product containing free EPA.

Next, after methyl esterification with diazomethane, it was analyzed with a gas chromatograph, and the measurement result was 0.173.
g (17.1% of lipid fraction, 0.88% of dry bacterial cells)
It was found that it contains EPA.

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.
0.159g was obtained.

Claims (1)

[Scope of Claims] 1. Eicosapentaenoic acid-containing lipids by culturing a microorganism that belongs to the genus Pseudomonas, Alteromonas, or Shewanella and is capable of producing eicosapentaenoic acid-containing lipids. A method for producing eicosapentaenoic acid-containing lipids or eicosapentaenoic acid, which comprises producing and accumulating eicosapentaenoic acid, and further isolating eicosapentaenoic acid if desired. 2. A microorganism belonging to the genus Pseudomonas, Alteromonas, or Shewanella that can produce eicosapentaenoic acid-containing lipids. 3. The Pseudomonas microorganism is Pseudomonas putrifaciens.
trefaciens) SCRC-2181, SCRC
-2201, SCRC-2271, SCRC-2341
, SCRC-2451, SCRC-2642, SCRC
-2792, SCRC-2878, SCRC-3011
or SCRC-3022, the microorganism according to claim 2. 4. The microorganism of the genus Alteromonas is Alteromonas putrifaciens (\Alteromonas\
¥putrefaciens¥) SCRC-2871,
Alteromonas putrifaciens subspecies sagamifaciens (￥Alteromonas
￥ ￥putrefaciens￥ subspeci
es￥sagamifaciens￥)SCRC-1
162, Alteromonas rumensus (￥Alter
omonas￥￥lumensas￥)SCRC-6
444, or Alteromonas xylosus (￥Alt
eromonas￥￥xylosus￥)SCRC-
The microorganism according to claim 2, which is No. 2517. 5. The microorganism of the genus Shewanella is Shewanella putrifaciens (￥Shewanella￥￥putr
The microorganism according to claim 2, which is SCRC-2874.