JPH05328979A - Production of zeaxanthin and novel flexibacter microorganism used therefor - Google Patents

Abstract

PURPOSE:To industrially advantageously obtain the subject compound useful for redyeing culturing fishes, improving the yokes of eggs, antioxidants, etc., by culturing a Flexibacter sp.DK30223 strain in a medium and subsequently collecting the product from the culture solution. CONSTITUTION:A Flexibacter sp.DK30223 strain as a spawn is inoculated on a medium containing peptone, a yeast extract, ferric phosphate, sodium acetate, purified water, sea water, etc., and then vibration-cultured at 25 deg.C for 48hr. The obtained culture solution is transferred to a medium having the same composition as the medium used in the culture tank, cultured at 25 deg.C for 72hr by an aeration-stirring method and then centrifuged into the cells and the medium. The cells are mixed with acetone and extracted with stirring. The extraction solution is collected, concentrated and subsequently subjected to an ethyl acetate/ water-distributing treatment. The ethyl acetate layer is separated, subjected to the evaporation of the solvent, again dissolved in benzene, and subsequently purified by silica gel chromatography and by high performance liquid chromatography to readily provide the objective zeaxanthin in high yield with an extremely general culture device.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing zeaxanthin, which is useful for deep-fried farmed fish, improving yolk quality of chicken eggs, etc., and which is also used as an antioxidant, and a novel flexi used therefor. It relates to microorganisms of the genus Bacterium.

[0002]

2. Description of the Prior Art Zeaxanthin has been widely used for the purpose of fried cultured ayu and improving yolk quality of chicken eggs, and most of them are prepared by mass-culturing spirulina which is a kind of microalgae. .. However, in culturing microalgae, light that is indispensable for photosynthesis must be supplied, and site conditions for collecting sunlight and facilities such as a culture device for supplying artificial light are required. In addition, microalgae generally have a slow growth rate, and it takes 1 to several weeks to obtain a sufficient culture density, which causes a problem in productivity. Furthermore, in the purification of zeaxanthin derived from microalgae, it is difficult to separate it from chlorophyll, and the purification process is complicated. To overcome these points, the land bacterium Erwinia
Fermentative production by uredovora has also been studied (J. Bacter
iol. 172 , 6704, 1990), but the yield is extremely poor and there remains a practical problem. From the above, development of a simple method for producing zeaxanthin has been desired.

In order to achieve such a subject, the present inventors have
Previously, we succeeded in easily producing zeaxanthin by using Alteromonas spp.
A patent application was filed as Japanese Patent Application No. 3-212454 on March 23, but as a result of further research, it is possible to produce zeaxanthin more simply and efficiently by using a microorganism that coexists with Daidai isokaimen. And succeeded in completing the present invention.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide zeaxanthin more simply and efficiently than conventional methods for producing microalgae and bacteria.

[0005]

Means for Solving the Problems The method for producing zeaxanthin of the present invention is characterized by culturing Flexiobacter sp. DK30223 strain in a medium and collecting zeaxanthin from the culture. Hereinafter, the present invention will be described in detail.

As the zeaxanthin-producing strain, any strain can be used as long as it is a strain belonging to marine Flexibacter sp. DK30223 and having a zeaxanthin-producing ability. Further, artificial mutation methods of these strains, for example, ultraviolet irradiation, X-ray irradiation, mutagen treatment, etc. or naturally occurring mutants, or genetically engineered or cell fusion mutants as long as they produce zeaxanthin, It can be used in the present invention.
In addition, Flexiobacter sp. DK30223 strain is
Since zeaxanthin is produced with higher purity than the Alteromonas sp. KK10203C strain used in No. 2454, the purification process for obtaining the target substance can be omitted.

The bacteriological properties of the Flexiobacter sp. DK30223 strain are described below. The properties are determined by the method of Shimizu et al. [Gen Kadota, Nobuo Taga: Marine Microbial Research Methods, Academic Publishing Center pp.229 (1985). )] Morphological examination
An optical microscope was used, and a scanning electron microscope was used for the morphology of the spore surface. Flexibactor sp.DK30223
The mycological properties of the strain are as follows.

(1) Gram stain negative (2) Morphology Form and size of bacteria: Rod-like, 10.0 μm × 0.2 μm Motility: Yes Flagella: None (3) Cell pigment: Yellow (4) Physiological properties Oxidase: Positive Glucose degradability: Negative Gelatin degradability: Negative DNA degradability: Negative (5) OF test Negative From the above findings of mycological properties, strain DK30223 was identified as a microorganism belonging to the genus Flexibacter. In the identification of species in this genus
(NR Krieg), JG Holt
Hen, The Vergies Manual of Systematic
Bacteriology (Bergey's Manual of Systematic Bac
teriology).

[0009] As a result of the search, it is difficult to identify a species that matches the characteristics of the DK30223 strain, and the DK30223 strain was designated as Flexiobacter sp. No. (FERM P-12889) (Original Deposit Date: March 19, 1992). The above-mentioned microorganism is generally cultured in a medium used for culture of the microorganism,
The zeaxanthin produced can be collected by a conventional method.

As the medium, either synthetic medium or natural medium can be used, as long as it is a medium containing assimilable carbon sources, nitrogen sources, inorganic substances and necessary growth and production promoting substances. Carbon sources include glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol,
Molasses and the like are used alone or in combination. Further, depending on the assimilation ability of the bacterium, hydrocarbons, alcohols, organic acids and the like are also used. As a nitrogen source, ammonium chloride,
Ammonium nitrate, sodium nitrate, urea, peptone,
Meat extract, yeast extract, dry yeast, corn steep
Liquor, soybean flour, casamino acid and the like are used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate,
Dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride,
Inorganic salts such as manganese sulfate, zinc sulfate, and copper sulfate and seawater are added as needed. In addition, a trace component that promotes the growth of the bacterium used and the production of zeaxanthin can be appropriately added.

As the culturing method, a general culturing method is used, but a liquid culturing method, especially a deep agitation culturing method is most suitable. A culture temperature of 16 to 37 ° C, particularly 22 to 30 ° C is suitable,
It is desirable to maintain the pH of the medium during culturing at 4 to 10, especially 6 to 8 by adding aqueous ammonia or ammonium carbonate solution. When liquid culture is normally performed for 1 to 7 days, the target substance zeaxanthin is produced and accumulated in the cells. The culture is stopped when the maximum amount of production in the culture is reached.

Isolation and purification of zeaxanthin from the culture is carried out according to a conventional method for isolating and purifying microbial metabolites from the culture. For example, the culture is separated by filtration into a culture filtrate and cells, and the cells are mixed with hexane,
Extract with an organic solvent such as benzene, chloroform, acetone, ether, and ethyl acetate. Next, after concentrating this extract, zeaxanthin is separated and purified by silica gel column chromatography, gel filtration (Sephadex LH-20) and the like. The trend of zeaxanthin during the culture and purification operations can be traced by using the yellow color of zeaxanthin by thin layer chromatography as a guide.

[0013]

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the scope of the present invention is not limited thereto. (Example 1) Flexiobacter sp. DK30223 as an inoculum
Use strains. Peptone 5 g / L, yeast extract 1 g / L,
Ferric phosphate 0.04 g / L, sodium acetate 0.01 g / L,
After preparing 500 ml of seed medium (pH 7.7 before sterilization) having a composition of 250 ml of purified water and 750 ml of seawater in a 1 L Erlenmeyer flask, 1 colony of the strain was inoculated and shaken at 25 ° C for 48 hours ( (100 rpm)
Precultured. 10% of the seed culture thus obtained was added to 5 L of medium having the same composition as the above in a 10 L culture tank.
Transfer at v / v ratio and aeration stirring method at 25 ° C (rotation speed 100rp
Culture was performed at m, aeration rate of 1 L / min).

During the culturing, the pH of the medium is not particularly controlled.
Incubated for hours. The culture broth was separated into cells and medium by centrifugation, the medium was removed, 200 ml of acetone was added to the cells and the mixture was stirred, and then the precipitate was separated by filtration to obtain 200 ml of an extract. The extract was concentrated, and the ethyl acetate layer was separated by partitioning with ethyl acetate / water. Next, the ethyl acetate layer was dehydrated with sodium sulfate, the solvent was distilled off, and the residue was redissolved in benzene. This was further purified by high performance liquid chromatography (Nacalai Tesque Cosmosil 5SL, inner diameter 8 mm length 250 mm, hexane: acetone = 8: 2, flow rate 1.2 ml / min), and the eluted fraction was concentrated to give a yellow dye. 8 mg was obtained. The yellow dye thus obtained was identified as zeaxanthin because its retention time in high performance liquid chromatography, UV-visible absorption spectrum and various instrumental analysis data were in agreement with known ones.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, zeaxanthin conventionally obtained from a culture of microalgae can be easily obtained in a high yield with a very general culture device. In addition, there was no mixing of chlorophyll peculiar to microalgae, which was greatly improved in the purification process.

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Claims (2)

[Claims] 1. A method for producing zeaxanthin, which comprises culturing Flexiobacter sp. DK30223 strain in a medium and collecting zeaxanthin from the culture. 2. A flexiobacter sp. DK30223 strain having the ability to produce zeaxanthin.