JPH04108393A - Production of oleic acid-containing lipid and microorganism - Google Patents

Abstract

PURPOSE:To obtain a readily purifiable oleic acid-containing lipid of high purity by culturing a microorganism capable of producing the oleic acid-containing lipid and producing the oleic acid-containing lipid. CONSTITUTION:A new microorganism capable of producing a lipid containing oleic acid in high purity is cultured to produce the oleic acid-containing lipid and the oleic acid or phospholipid oleate or both, as desired, are isolated to produce the oleic acid-containing lipid, oleic acid and/or phospholipid oleate. SCRC-S007 (FERM P-11671) can be cited as an example of the aforementioned microorganism. Culturing may be carried out by using any of a solid and a liquid culture media. In order to obtain the objective oleic acid in a large amount, the liquid culture medium is preferably used to preform the culturing under aerobic conditions by a stationary culture, shaking culture or aeration.spinner culture method. The phospholipid oleate can be obtained by treating the oleic acid-containing lipid by precipitation with acetone.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention provides an oleic acid-containing
Or, it relates to a method for producing oleic acid phospholipid and a new species of microorganism for producing the same.

(Prior art) The types of lipids and fatty acid composition that constitute the cell membranes of living organisms are
It is becoming clear from research on cell membranes that it has a major influence on the affinity of membrane proteins and contributes greatly to their stability and activity. On the other hand, from research on physiologically active components in living organisms, it has been known that long-chain saturated or unsaturated fatty acids, or straight-chain or branched fatty acids, in particular, have anti-ulcer and anti-inflammatory effects. Although there are many unknowns regarding the physiologically active effects of these fatty acids,
It has also been reported that the activity differs depending on whether it is in the free or ester form. When such fatty acids are used as therapeutic agents, not only the chemical type at the time of expression of activity, but also absorption efficiency upon administration and stability in the body are major factors that influence the overall efficacy. Fatty acids are not accumulated in free or ester forms in living organisms, and in many cases exist as phospholipids and glycolipids in biological membranes or glycerides in adipose tissue. Free fatty acids and fatty acid esters: Everyone knows how toxic they are. Therefore, fatty acids with physiological activity (1): 2, 1. When it comes to lipids and glycolipids, it is thought that they are superior to the free and ester forms in terms of in vivo stability and absorption efficiency, and as a result, high physiological activity can be expected.

Currently available naturally occurring phospholipids: It is difficult to limit the fatty acid composition of the anol group. That is, the effect can only be expected in the presence of fatty acids other than the desired fatty acids.

(Problem B to be solved by the invention) In order to more effectively utilize the physiologically active effects of long-chain fatty acids, or to assay unknown physiologically active effects, and to be able to utilize them in a wide range, fatty acids are freed. Phospholipids, which are constituents of biological cell membranes rather than esters or esters, are a promising chemical form.

However, it is necessary to limit the constituent fatty acids, and at present we have no choice but to turn to chemical synthesis, which is difficult to produce in large quantities at low cost. The object of the present invention is to find a method for producing a lipid containing the first constituent fatty acid f1 using a microorganism that can be obtained in a short culture time and that is easy to control the culture.

(One step to solving the problem) The present inventors discovered a new type of microorganism in the ocean that produces lipid containing almost only oleic acid as a constituent fatty acid, and based on this knowledge22, the present inventors Completed the invention.

Therefore, the present invention produces lipids containing oleic acid by culturing a new type of microorganism capable of producing lipids containing highly purified oleic acid, and optionally produces monophospholipids containing oleic acid and/or oleic acid. The present invention provides a method for producing oleic acid-containing lipids, oleic acid and/or oleic acid phospholipids, and a new species of microorganism capable of producing oleic acid-containing lipids.

(Specific explanation) ■ Microorganisms The new species of microorganisms used in the present invention are capable of producing oleic acid or lipids containing oleic acid, and such microorganisms can be newly isolated in nature. .

An example of such a microorganism is 5CRC-5゜07 (
1) No. 671).

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

Table 1 Peptone 1% Yeast Extract
0.3% meat extract
0.3% NaC 13% Distilled water pH 7.0 This agar plate medium was inoculated with a seawater sample collected from the Southern Ocean, moderately diluted with sterilized physiological saline, and incubated at 25°C for 1~
It was cultured for 2 days. Colonies that appeared on this agar plate medium were plated onto a slanted medium having the same medium composition.

A large number of bacterial strains were thus isolated from the seawater samples collected from the ocean, and the agar was removed from the culture medium shown in Table 1, which was then placed under partial pressure of 5 parts in test tubes and sterilized in the same manner. The new bacterial species was cultured in this medium at 25°C for 1 to 2 days. The oleic acid production ability of the obtained culture solution was assayed by the method described below. In this way, a new bacterial strain 5CRC-8007 which significantly produces oleic acid was obtained. This new bacterial species has the following mycological properties.

Observation items a) Form l Cell shape size 2 Presence or absence of polymorphism 3 Presence or absence of motility Epiphytic state of flagella 4 Presence or absence of spores 5 Gram staining 6 Anti-acidity Nagabu bacterium 1, OX 4.0μm Nothing Yes One, 8if [2 hairs] Nothing negative negative b’7@Growing conditions in a busy area l Meat juice agar plate culture (25°C Ino colony shape (diameter) C) Colony shape C) Shape of colony surface 2) Prominence of colony e) Colony periphery f) Colony color tone g) Colony transparency H) Colony gloss ) Generation of soluble pigments 2. Broth agar slant culture (25℃).

b) Quality of growth mouth) Colony gloss 3. Meat juice liquid medium (25℃).

b) Growth opening on the surface) Turbidity C) Sedimentation 2) Gas generation 2 days) 2, OX 2.4 m Round, smooth platform-like gold edge Pale yellow opaque No dull light for 2 days) Good dull light for 2 days) Non-turbid powder No liquefaction for 2 days (Coagulation 2, 904 Meat Juice Sera Senshi (25℃ Gelatin (liquefaction 5 Ridmus Milk C) Physiological properties 1 Reduction of nitrate 2 Denitrification MR 4■P 5 Indole formation 6 Hydrogen sulfide Production of starch 7 Hydrolysis of starch 8 Utilization of citric acid a) Koser 9 Christensen 9 Utilization of nitrate 10 Pigment production a) King A Medium inlet)) (ing B Medium 1) Urease 12 Oxidase 13 Catalase 14 Growth range a) pH Mouth) Temperature 15 Attitude towards M elements 160-F test (glucose) 17 Acid and gas production from $IR 1, L-arabinose 2, D-xylose 3, D-glucose 4, D-mannose 5.0-fructose 6, D-galactose 7, maltose 8, sucrose 9, lactose 10° trehalose 1), D-sorbitol 12, D-mannitol 13, Innoshift 6-10 1°C-35°C Aerobic 14, Glycerin 15, Den'bun d ) Other properties Growth on SS agar medium Growth on Juman Conkey agar medium 6.5% Na Cl salt tolerance DNAse Decomposition of ornithine Decomposition of arginine Decomposition of gelatin G-C content 44.2-44.5 Based on the above mycological properties, Harsey's Manual of Determinative Bacteriology 8th Edition, 1
This bacterium was identified as a new species of bacterium according to the 989 classification criteria.

■ Method for producing oleic acid-containing lipids, oleic acid, and oleic acid phospholipids When attempting to produce the oleic acid of the present invention by culturing the above-mentioned microorganisms, the microorganisms of the invention of Jichiney are grown in a basal nutrient medium.''7 Use whatever you get, −(
・. The medium contains a nitrogen source and one or more of, for example, yeast extract, peptone, meat extract.

In addition, various sugars can be added to this medium as a carbon source as required. 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 oleic acid, a liquid medium is used, and aerobic culture is performed using static culture, shaking culture, or aeration/agitation culture. It is preferable to carry out the culturing under these conditions.

The culture temperature may be any temperature within the temperature range at which bacteria grow and oleic acid is produced, preferably from 1 to 35
It is ℃. The pH ranges from 6 to 10. The culture time may be selected such that a collectable amount of oleic acid-containing lipid is produced, and is preferably 10 to 120 hours.

Next, the oleic acid of the present invention is collected from the resulting culture. As a purification method, a conventional lipid purification method can be used. For example, two bacterial cells can be collected from a T8 nutrient solution using conventional bacterial collection methods such as L-separation filtration.

Next, add the second bacterial cell to water, saline, or water as desired. ■Liquid,
For example, after washing with phosphate buffer, etc., re-inject into these solutions. , become cloudy. This suspension/gJ/fi is converted into a solvent commonly used for lipid extraction, such as chloroform/gJ/fi.
Extract with a methanol mixture and phase split to obtain a chloroform phase. Next, the chloroform phase is removed by evaporation to obtain a material containing an oleic acid-containing lipid.

Free oleic acid or its salt can be obtained by saponifying it by a conventional method.

Oleic acid phospholipids can be produced by treating oleic acid-containing lipids by acetone precipitation.

Thus, by using the above-mentioned bacteria, the present invention can easily purify and obtain a large amount of oleic acid-containing lipids, oleic acid and/or oleic acid phospholipids in a short period of time.

Next, specific examples of the present invention will be shown.

Oleic acid-containing M from Example 1.5cRc-soo7
1% production peptone, 0.3% yeast extract, 03% meat extract,
After sterilizing the medium containing 14.5% NaC and adjusting the pH to 7.0 for 15 minutes, 5CRC-8
007 was inoculated and cultured aerobically at 25°C for 12 hours.

After culturing, the bacterial cells were collected using a centrifugal dispensing machine, and the dry weight was approximately 160.
■ Bacterial cells were obtained. After washing the bacterial cells once with 1/2 concentration artificial seawater, they were suspended in 10 1/2 concentration artificial seawater. This bacterial cell suspension was mixed with lO chloroform/methanol solution (
2:I v/v) and then centrifuged to obtain a chloroform phase. Furthermore, the same solution was extracted twice, the chloroform phase was combined, and the extracted fractions were concentrated to dryness. The lipid fraction obtained was 6.5 (3.8% per dry bacterial cell). This lipid fraction contains oleic acid. This lipid fraction was heated with 8% hydrochloric acid and methanol at 80° C. for 1 hour to methyl esterify it, and then analyzed by gas chromatography, and it was found that it contained 4.3 μ of oleic acid. Note that the proportion of oleic acid in the total fatty acids was 90.5%.

Example 2.5 Production of oleic acid phospholipids from CRC-5007 Peptone 1%, yeast extract 0.3%, meat extract 0.3%
, medium 1 containing 3% NaCl and adjusted to pH 7.0
) was heat sterilized for 15 minutes, then inoculated with 5CRC-3OO7 and cultured aerobically at 25°C for 12 hours. After culturing, the bacterial cells were collected using a centrifuge, and about 12 g of bacterial cells were obtained using a wet heavy lid. The cells were washed once with artificial seawater of J/2 concentration and then suspended in artificial seawater of 1/2 concentration of 100. The 235 bacterial cells were extracted by shaking thoroughly with a 100% chloroform/methanol solution (2:1 v/v), and then centrifuged to obtain a chloroform phase. Furthermore, extract twice with the same solution, combine the chloroform phases, and reduce the pressure! Shrunk. This concentrated solution was added to 50 ml of cold acetone to obtain phospholipids as a precipitated fraction. Remove the supernatant by filtration! After eluting the phospholipid precipitate remaining on the filter paper with chloroform, the chloroform was removed under reduced pressure to obtain about 60 μg of phospholipid. The fatty acid composition of this phospholipid was analyzed by gas chromatography in the same manner as in Example 1, and it was found that it contained about 42 oleic acid. The proportion of oleic acid in the total fatty acids was 88.7%.

Procedural text (spontaneous) July 1, 1991 Director General of Patent Persons Wataru Fukasawa 1.Display of the incident 1990 Patent Application No. 224434 2. Name of the invention

Claims (3)

[Claims] (1) A method for producing an oleic acid-containing lipid, which comprises producing an oleic acid-containing lipid by culturing a microorganism capable of producing an oleic acid-containing lipid. (2) A method for producing oleic acid and/or oleic acid phospholipid, which comprises isolating oleic acid and/or oleic acid phospholipid from the oleic acid-containing lipid according to claim (1). (3) SCRC-S007, a microorganism capable of producing oleic acid-containing lipids.