JPH0484892A - Production of eicosapentaenoic acid - Google Patents

Abstract

PURPOSE:To readily obtain eicosapentaenoic acid in a short time by cultivating a specific bacterium in a medium containing pyruvic acid and/or lactic acid. CONSTITUTION:(A) A bacterium such as Pseudomonas putrefaciens SCRC-2,181, SCRC-2,201, SCRC-2,271, SCRC-2,341, etc., Alteromonas putrefaciens SCRC-2,871, etc., Shewanella putrefaciens SCRC-2,874, etc., is obtained by selection form marine bacteria capable of producing eicosapentaenoic acid. Then 0.1-5wt.% high-purity substance (B) such as pyruvic acid and/or lactic acid or composition containing >=10wt.% component B and about 1.0wt.% peptone are blended with sea water at pH about 7.0 to give a medium (C). Then the bacterium A is inoculated into the medium C and aerobically culture at about 25 deg.C for about 24 hours to give a culture mixture (D). Then the component D is centrifuged to give cells, which are extracted to give eicosapentaenoic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial application field The present invention is directed to the production of eicosapentaenoic acid (hereinafter referred to as Eicosapentaenoic acid) from microorganisms.
The present invention relates to a method for producing PA (referred to as PA). The E
PA is useful in fields such as food, cosmetics, medicine, agriculture, fisheries, and chemical products.

(b) Conventional Technology Polyunsaturated fatty acids represented by EPA are essential fatty acids for higher animals and play an important role in living organisms. In addition, the natural effects of highly unsaturated fatty acids are known, and in particular, EPA is being considered as a pharmaceutical agent for thrombosis treatment due to its pharmacological effects such as serum lipid improving effect and antithrombotic effect. Demand for refined products is expected to expand.

Conventional methods for producing high-purity EPA include extraction and purification from fish oil or certain types of microorganisms. EPA can be purified using low-temperature fractional crystallization, urea addition, vacuum distillation, chromatography, etc.
PA contains EPA, arachidonic acid, and docosahexaenoic acid in fish oil, and requires complicated operations and expensive equipment in the refining process.

On the other hand, the present inventors previously proposed a method for producing EPA from microorganisms (JP-A-63-216488, 63-2
16489.64-2587. Japanese Patent Publication No. 1-191694
．． 2-23877), however, there were problems in terms of cost when mass production was performed.

(c) Problems to be Solved by the Invention An object of the present invention is to provide a manufacturing method that can easily produce EPA industrially and efficiently in a short time.

(=) Means for Solving the Problems As a result of intensive research to find a production method that meets the above objectives, the present inventors discovered that microorganisms were cultured by adding pyruvic acid and/or lactic acid to the medium. , we obtained the following findings.

That is, (1) the production of ammonia, which is released as a metabolic product by microorganisms and suppresses the growth of the microorganisms, was suppressed, (2) the growth of bacteria was increased, and (2) the production amount of EPA-containing lipids was increased.

The present invention was completed based on the above findings, and is a method for producing EPA in which EPA is extracted from microorganisms cultured by adding pyruvic acid and/or lactic acid to a medium.

The pyruvic acid and/or lactic acid used in the present invention may be of high purity, and a composition containing them may be used, preferably a composition containing 10% or more,
A more preferable example is a composition containing 50% or more. Such a composition is usually added to a medium at a concentration of 0.1
~5%, more preferably 0.2-1.0%.

The microorganisms that can be used in the present invention are not particularly limited in genus, species, or strain, but usually include Pseudomonas genus, Alteromonas (A
l terosonas) If or Sh wanela (Sh
Marine microorganisms classified into the genus Ewanella are used. The properties of these microorganisms are described in detail in European Patent Application No. 87311372.4, which was previously proposed by the present inventors as a microorganism for producing EPA. As an example of the above-mentioned microorganisms belonging to Pseudomonas, Pseudomonas piutrifaciens (
Peudomanas utrefaciens)S
CRC-2181 (FERMBP-2917), 5CR
C-2201 (FER ABP-2916), 5CRC-
2271 (FER ABP-2915), SCRC-23
41 (FERMBP-2918), 5CRC-2451
(FERMBP-2919) SCRC-2642 (FE
R1'1BP-2920), 5CRC-2792 (FE
RMBP-2921), SCI? C-2878 (FER
MBP-1623), SCRC-3011 (FERMB
P-2913>, 5CRC-3022 (FEIIMBP
-2914).

As an example of a microorganism belonging to Alteromonas, Alteromonas pyurifaciens (Alteromonas
SCRC-2871 (
FERMBP1624) and Alteromonas utrefaciens subspecies sagamifaciens
5ubs eciessa amifaciens)
Examples include SCRC-1162 (FERMBP-1626).

An example of a microorganism belonging to Shewanella is Shewanella vinylifaciens (Shei+anel 1aut.
5CRC2874 (FERMB
P-1625).

In carrying out the present invention, the method proposed earlier (Japanese Unexamined Patent Publication No. 6
3-216488.63-216489.64-258
7. JP-A-1-191694゜2-23877> may be followed. In this case, the medium composition shown in Table 1 can be prepared.

Table 1 Peptone 1.0% yeast extract 0.5% pyruvic acid and/or
Or lactic acid 0.5% No such effect was observed with other representative carbon sources.

Table 2 Example 1 Marine microorganism (Pseudomonas utrefa)
ciens 5CRC2878 (FE Tou BP-1623
) ) with 0.5% yeast extract, 1% peptone, various carbon sources shown in Table 2, and 0.5% pyruvic acid and DL-lactic acid.
% was added and aerobic culture was performed at 25°C in a seawater medium adjusted to pH 7.

After culturing for 24 hours, the bacterial cells were centrifuged, freeze-dried, and the bacterial weights were compared. In addition, the amount of ammonia accumulated in the culture solution was measured using the Notsler method. Furthermore, EPA in the bacterial body
The content was measured by gas chromatography.

As is clear from Table 2, addition of pyruvic acid or DL-lactic acid suppressed ammonia production and increased bacterial cell yield and EPA production. On the other hand, citrate glucose galactose lactose sorbitol erythritol malate succinate pyruvate DL-lactic acid Example 2 Marine microorganism (AIteromonas utrefa
ciens 5CRC2B71 (FER,'1BP-
1624)) in the same medium as in Example J above with the addition of D-lactic acid or L-lactic acid to obtain bacterial cells, centrifugal collection,
After washing and drying, a marine microorganism powder was obtained.

The weight and amount of EPA accumulated in the dried tooth bodies were measured, and the ammonia concentration in the culture solution was measured. The results are shown in Table 3.

(Effect of phantom invention The effects of the present invention are as follows.

By using microorganisms cultured in a medium supplemented with pyruvic acid and/or lactic acid, it has become possible to efficiently produce EPA in a short time.

Claims (5)

[Claims] (1) In culturing marine microorganisms capable of producing eicosapentaenoic acid and producing eicosapentaenoic acid,
A method for producing eicosapentaenoic acid characterized by using a microorganism cultured by adding pyruvate and/or lactic acid to a medium. (2) Marine microorganisms are Pseudomonas
nas) genus, Alteromonas
) or Shewanella genus, the manufacturing method according to claim (1). (3) The Pseudomonas microorganism is Pseudomonas
Pseudomonas
¥putrefaciens¥) SCRC-2181,
SCRC-2201, SCRC-2271, SCRC-
2341, SCRC-2451, SCRC-2642,
SCRC-2792, SCRC-2878, SCRC-
3011 or SCRC-3022, the manufacturing method according to claim (2). (4) The microorganism of the genus Alteromonas is Alteromonas spp.
Putrifaciens (￥ Alteromonas￥
￥ putrefaciens ￥) SCRC-2871 or Alteromonas putrefaciens subsp.
s putrefaciens￥￥subspecie
s sagamifaciens￥)SCRC-116
2. The manufacturing method according to claim (2). (5) The microorganism of the genus Shewanella is Shewanella putrefaciens.
The manufacturing method according to claim (2), which is SCRC-2874.