JPS63216488A - 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 bacterial strain belonging to Deleya genus in a nutrient medium. CONSTITUTION:Deleya sagamarinii SCRC-1432 (FERM P-9211) or its mutant is cultured under aerobic condition in a liquid medium containing a carbon source such as saccharides, nitrogen source such as yeast extract, peptone or meat extract and sodium chloride or natural or artificial seawater 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 bacterial 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 blood ■Blood VLDL-cholesterol, LDL-cholesterol lowering effect, HDL
- 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 members, and is used in the body of mammals including humans. perform essential functions. 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 (it is more effective than linoleic acid, etc. contained in ordinary vegetable oils. Also known as nutrients.

In this way, the possibility of EPA being used as a health food or medicine based on its antithrombotic or lipid-lowering effects was demonstrated by the Danish company Diavelve (Am, J, C1).
1n, Nu tr', , 3 engineering, 958°19
75), but as is clear from its chemical structure, it is extremely difficult to chemically synthesize it. For this reason, in Japan, EP
In order to recommend eating blue-backed fish such as sardines, mackerel, and saury, which contain a large amount of A, most of them are fractionated fish oils obtained by the point method, and their EPA content is 10 to 3.
It is about 0%. Fish oil extracted by the point method is a mixed glyceride containing 11,111 fatty acids as constituent fatty acids, and it is difficult to purify each component in a single layer. Since it is a straight chain polyunsaturated fatty acid with 20 carbon atoms, which is owned by Indonesia, it is an unstable fatty acid that is extremely easily oxidized, so when concentrating EPA from fish oil, avoid exposure to 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, 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 by a separate crystallization method, urea addition method, distillation under reduced pressure, reversed phase chromatography, etc. to achieve an EpAtl 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 drawbacks such as residual fishy odor, microorganisms such as chlorella, unicellular alga Monodus, Euglena, or diatoms have been used in incomplete purification and tlili. EPA production methods are becoming increasingly popular, and EPA production using microorganisms is attracting attention.

Recently, Gellerman and Schrezda (J, L.

Gellerman and H, 5chlenk
.

BBA, 513.23.1979) and Yamada et al. (1986 Japan Fermentation Engineering Society Conference, 1986), E.
A report was made on Zurchin mold (filaform 1) that produces PA.

EPA production by these microorganisms is relatively easy to produce by minute MR due to its fatty acid composition, and due to culture control.
It has advantages such as easy control of SPA production. However, when using these filamentous fungi, the cultivation time is longer (about 7 days to 1 month) compared to bacteria etc.
However, improving productivity remains a major issue.

(Problems to be Solved by the Present Invention) As described above, EPA, which is considered as a health food or medicine, cannot be produced by extracting fish oil or by culturing algae or mold. , there are some problems. Based on these facts, the purpose of the present invention is to find a method for fermentation production of EPA-containing lipids using bacteria, which is easy to purify, obtains highly pure products, and has a short culture time (easy culture control). It is in.

(Means for Solving the Problems) As a result of extensive research searching for bacteria capable of producing EPA in the ocean, the present inventors found that Dereya (1) ele
We discovered that bacteria belonging to the genus Ya) produce EPA, and based on this finding, we completed the present invention.

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

(Specific explanation) (1) Microorganisms The microorganisms used in the present invention belong to the genus Dereya, and
Any microorganism can be used as long as it can produce PA or a lipid containing PA, and such microorganisms can be newly isolated from nature.

As an example of a microorganism belonging to Dereya, there is a new strain of Dereya sagamarinii isolated by the present inventor.
1) SCRC-14
I can list 32.

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 10.5% Tap water PH 7.0 On this agar plate medium, marine organism samples collected from various oceans were diluted appropriately with sterilized physiological saline. 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 cultured in this medium at 25°C for 1 to 2 days. From the obtained culture solution, EP was prepared by the method described below.
A production ability was assayed. In this way, the following bacterial strains that significantly produce EPA were obtained from marine organism samples 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
1, OX 2.0 μm2 Presence or absence of polymorphism No 3 Presence or absence of motility Flagellated epiphyte status Peritrichia 4 Presence or absence of spores No 5 Gram staining Negative 6 Acid-fast
Negative b) Growth status in each medium 1 Broth agar plate 1I (25°C, 2 days) b) Colony shape (diameter) 0.6-0.8 mm 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
Pure light) Production of soluble pigments None 2 Broth agar slant culture (25°C, 2 days) A) Quality of growth Good or bad Colony gloss Renko 3 Broth liquid medium (25°C, 2 days) B) Surface growth Silent ) Turbidity C) Precipitation Powder 2) Gas generation None 4 Meat juice gelatin (25℃, 2 days) Gelatin liquefaction Liquefaction 5 Lidomus milk No change C) Physiological property 1 Reduction of 4q acid salt 12 Denitrification
-3MR+4VP
+5 Indole production - 6 Hydrogen sulfide production 17 Starch hydrolysis - 8 Citric acid utilization A) Koser - 口) Chri
stenssn - 9 Utilization of nitrate Volume 10 Pigment production a) King A Medium - mouth) King
B Medium -11 urease
- 12 Oxidase +13 Catalase +14 Growth range pH 6-9 0) Temperature 5℃-30℃15
Attitude towards oxygen Aerobic 16 0-F
Test (glucose) - Acid and gas production from 17Ws 1, L-arabinose - 2,0-xylose - 3.0-glucose + 4, D-mannose - 5, D-fructose - 6, D-galactose - 7, Maltose - 8, sucrose - 9, lactose - 10, trehalose - 11.0 - sorbitol - 12, D-mannitol - 13, inosift - 14, glycerin - 15, starch - (however, all items that produce gas -) d ) Other properties Growth on SS agar medium Growth on Pine Conchi agar medium + 6.5% NaC1 salt tolerance 10 [) Na se + Ornithine decomposition - Arginine decomposition - Na0 requirement Various properties such as 10 or more Due to its properties, this strain 5CRC-1432
has the following major characteristics.

■ Durham negative ■ Has peritial flagella and motility ■ Non-spore-forming aerobic bacterium ■ O-F test negative ■ Catalase, oxidase positive ■ Na + requirement ■ Nitrate reducing ability positive ■ Grows at 35℃■ Art of Systematic Bacteriology (B) that liquefies gelatin
ergeyls Manual of System
atic Bacteriology) Volume 1, 35
2, 1984, it can be identified as a bacterium related to the genus Alcalgenes from the items 1 to 2 above.

However, the International Journal of Systematics Bacteriology
ational Journalof System
atlc Bacteri-ology) 33 (4
), 793-802 (1983), Bowman et al.
L,Bau-mann,R,D,Bowdl tch;
andP, Baumann) is the Na” requirement (above ■
Delayl alkaline earth metals
It is proposed that it be classified as a genus.

Therefore, based on the above, this strain 5CRC-1432 is considered to belong to the genus DeJeya.

However, as a microorganism belonging to the genus Dereya,
Aesta (aasta) Tereya Chiehita (11th year of earth, 1 and 1 earthworks) Dereya Pacifica (7th day of a year) Dereya
There are Venusta (venusta) and Dereya Marina (marlna), which have the highest nitrate reduction ability, growth at 35℃, and gelatin liquefaction ability (items from ■ to 0 above). Strain 5CRC-143
2 is clearly different from the above-mentioned bacterial species of the genus Dereya.

Based on the above findings, this bacterial strain 5CRC-1432 was used as Dereya sagamarinii (Da, which belongs to the Dereya genus).
e a sa amarlnl
1) and named it.

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 from a taxonomic perspective (although they generally change frequently 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 slant medium, it is possible to use a medium commonly used for the preservation of bacteria belonging to the genus Dereya, such as the medium described above for the isolation of bacteria. Furthermore, freeze-drying preservation can be carried out according to conventional methods.

The above novel microorganism Dereya sagamarinii 5CRC-1
432 is FERM P-'l'111, which was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
) has been deposited as.

(21 Method for Producing EPA) If the EPA of the present invention is to be produced by culturing the microorganisms described above, any medium in which the microorganisms of the present invention can grow 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 necessary to add sodium chloride or natural or artificial seawater to this medium.

For cultivation, either a solid medium or a liquid medium may be used, but in order to obtain a large amount of the desired EPA, a liquid medium should be used, and aerobic conditions such as static culture, 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 grow and EPA is produced, preferably 5 to 30°C;
More preferably it is 15-25°C. The pH ranges from 6 to 9, preferably from 7 to 8. The culture time may be selected such that a collectable amount of EPA-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 collection method such as centrifugation or filtration. Next, the cells are washed with water, saline, or a buffer solution, such as a phosphate buffer solution, as desired. , 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 1.0%, NaCf0
15% of medium 20j adjusted to pH 7.0
21'l: After heat sterilizing for 15 minutes, Delaya Sagamarinii 5CRC-1432
No. 1) was inoculated and cultured aerobically at 25°C for 24 hours. After culturing, collect the bacterial cells using a centrifuge and have a wet weight of approximately 108
g (19.8 g dry weight) of bacterial cells were obtained. 0.
After washing once with 85% saline, it was suspended in 11. This suspension was mixed with chloroform-methanol tS.
After shaking and extracting with a solution (2: IV/V) well, 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 1.65 g (8.33% per dry bacterial body).
The mixture was saponified in 95% ethanol containing OH at 80° C. for 1 hour, and neutralized with 5N-MCI to obtain a free fatty acid mixture.

After methyl esterifying this free fatty acid mixture with diazomethane, it was analyzed using a gas chromatograph, and it was determined that it contained 0.203 g (12.6% of lipid fraction, 1.03% of dry bacterial cells) of EPA. I found out that it 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.
o, 189g was obtained.

5. Contents of correction Procedural amendment (voluntary) Mr. Kunio Kogawa, Commissioner of the Patent Office 1.Display of the incident 1986 Patent Application No. 49929 No. 2, Title of the invention Person who makes 3° correction “Detailed description of the invention” column in the specification Rei-/'X　　　　(44) Corrected to "low temperature fractionation, crystallization method".

(2) Table 1 on page 9 of the same is corrected as follows.

Meat extract 1.0% Peptone 1.0% NaClO, 5% Agar 1.5% (3) rAle-ca 1 i on page 17, lines 1-2
-gene3J is corrected to "rAl-caligenes".

/ff bird/1’ni no

Claims (3)

[Claims] (1) By culturing a microorganism that belongs to the genus Deleya 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. An eicosapentaenoic acid-containing lipid or a method for producing eicosapentaenoic acid, characterized by: (2) A microorganism of the genus Deleya that can produce eicosapentaenoic acid-containing lipids. (3) Deleya Sagamarinii (￥Deleya￥￥s
2. The microorganism according to claim 2, which is a microorganism.