JPH0549497A - Method for producing zeaxanthin - Google Patents

Abstract

PURPOSE:To obtain the title compound useful for coloring cultured fishes, being readily purified, in high yield without admixture of chlorophyll by culturing a bacterium belonging to the genus Alteromonas, capable of producing zeaxanthin in a medium and collecting a product from the culture mixture. CONSTITUTION:A bacterium [e.g. Alteromonas sp. KK10,203C (FERM P-12,434)] belonging to the genus Alteromonas, capable of producing zeaxanthin is inoculated into a seed medium, subjected to shaking culture at 25 deg.C for 48 hours, the prepared seed culture solution is transferred to a culture tank in a ratio of 2%V/V, subjected to aerated spinner culture at 25 deg.C for 120 hours, the culture solution is filtered off to collect cells, which are ground, extracted with acetone, the extracted solution is concentrated, passed through a polystyrene-based adsorbent column, an adsorbed substance is eluted with methanol, concentrated, extracted with benzene and purified by silica gel chromatography and high- performance liquid chromatography to give zeaxanthin in high yield.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing zeaxanthin produced by microorganisms. Zeaxanthin is useful for deep-fried farmed fish, improving yolk quality of chicken eggs, and is also used as an antioxidant.

[0002]

2. Description of the Related Art Conventionally, zeaxanthin has been widely used for the purpose of raising the color of cultured ayu and improving the yolk quality of chicken eggs, and most of them are prepared by mass-culturing microalga Spirulina. However, in culturing microalgae, light that is indispensable for photosynthesis must be supplied, and site conditions for collecting sunlight and equipment 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 Erwiniauredovora
Fermentative production by lactic acid has also been studied (J.
704, 1990), but the yield is extremely poor, and a problem remains in practical use.
From the above, development of a simple method for producing zeaxanthin is desired.

[0003]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide zeaxanthin more easily than the conventional production method using microalgae.

[0004]

The present invention provides a method for producing zeaxanthin, which comprises culturing in a medium a microorganism belonging to the genus Alteromonas and having the ability to produce zeaxanthin, and collecting zeaxanthin from the culture. The present invention will be described in detail below.

As the zeaxanthin-producing strain, any strain can be used as long as it belongs to the genus Alteromonas and has a zeaxanthin-producing ability. In addition, any artificial mutation method of these strains, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagen or the like, or a naturally occurring mutant, or a mutant by genetic engineering or cell fusion, as long as it produces zeaxanthin, is used in the present invention. Can be used for. A representative strain is the KK10203C strain.

The bacteriological properties of the KK10203C strain will be described below. The properties are determined by the method of Shimizu et al. [Gen Kadota, Nobuo Taga: Marine Microbial Research Method, Academic Publishing Center pp.229 (198).
5)] was followed. The morphological examination was performed by using an optical microscope, and particularly by using a scanning electron microscope for the morphology of the spore surface. The mycological properties of the KK10203C strain are as follows. (1) Gram stain Negative (2) Morphology Shape / size of bacterium: rod-like, 2.2 μm × 0.25 μm Motility: Yes Flagella / length: Extremely monochrysian, 2.0 μm (3) Bacterial pigment: Yellow ( 4) Physiological properties Oxidase: Negative Glucose degradability: Negative Gelatin degradability: Positive DNA degradability: Negative (5) OF test negative Based on the above findings of mycological properties, strain KK10203C was identified as a microorganism belonging to the genus Alteromonas. Identified. In identification of species in this genus, N. K. League (N.
R. Krieg), edited by JG Holt, Bergey's Manual of Systematic Bacteriology
It searched based on.

[0007] As a result of the search, it was difficult to identify a species that matches the characteristics of the KK10203C strain, and the KK10203C strain was designated as Alteromonas sp. And this strain was sent to the Institute of Microbial Technology, Institute of Industrial Technology
No. (FERM -12434). (Date of original deposit: Heisei 3
August 19th). The above microorganisms are generally cultured in a medium used for culturing microorganisms, and the zeaxanthin produced can be collected by a conventional method.

As the medium, assimilable carbon source, nitrogen source,
Either a synthetic medium or a natural medium can be used as long as it is a medium containing an inorganic substance and necessary growth and production promoting substances in an appropriate amount. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. may be used alone or in combination. Further, depending on the assimilation ability of the bacterium, hydrocarbons, alcohols, organic acids, etc. are also used. As the nitrogen source, ammonium chloride, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, casamino acid, etc. may be 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. Furthermore, a trace amount 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, particularly a deep agitation culturing method is most suitable. A suitable culture temperature is 16 to 37 ° C, especially 22 to 30 ° C. The pH of the culture medium during the culture may be maintained at 4 to 10, especially 6 to 8 by adding aqueous ammonia or ammonium carbonate solution. desirable. When liquid culture is generally 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
It is carried out according to a method commonly used for isolating and purifying a microbial metabolite from the culture. For example, microbial cells are ground and hexane, benzene, chloroform, acetone,
Extract with ether, ethyl acetate, etc. Then, the extract is concentrated and then subjected to silica gel column chromatography, gel filtration (Sephadex LH-20), etc. to separate and purify zeaxanthin. 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.

[0011]

INDUSTRIAL APPLICABILITY According to the present invention, zeaxanthin conventionally obtained from a culture of microalgae can be easily obtained in a high yield by an extremely general culture device. In addition, there was no mixing of chlorophyll peculiar to microalgae, which was greatly improved in the purification process. The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited by these examples.

[0012]

[Example] An Alteromonas sp.kk10203C strain is used as an inoculum. Seed medium with the composition of peptone 5 g / L, yeast extract 1 g / L, glucose 2 g / L, ferric phosphate 0.04 g / L, sodium acetate 0.01 g / L, purified water 250 ml, seawater 750 ml (before sterilization (pH 7.7) 100 ml was made into a 200 ml Erlenmeyer flask, the strain was inoculated, and shaken at 25 ° C for 48 hours.
(100 rpm) Culture was performed. The seed culture thus obtained was transferred at a rate of 2% v / v to 5 L of a medium having the same composition as the above in a 10 L culture tank, and aerated and agitated at 25 ° C. (rotation speed 100 rpm, / The culture was performed at an aeration rate of 1 L / min).

During the culture, the pH of the medium is not particularly controlled,
Cultured for 120 hours. After the culture solution was filtered and the cells were ground, 2 L of acetone was added and stirred, and the precipitate was filtered off to obtain 7 L of extract. The extract was concentrated to 3 L and passed through a column of polystyrene adsorbent Diaion pH 20 (1 L) to adsorb the active substance. The active substance was eluted with methanol after eluting impurities with deionized water and 30% methanol. The active fraction was concentrated and extracted with benzene. The extract was dehydrated with sodium sulfate and then concentrated. This was further developed with hexane: acetone = 8: 2 using a silica gel column (silica gel 60 manufactured by Nacalai Tesque). Fraction A eluted
Was concentrated to give 2 ml of a yellow concentrate. Fraction A is 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). Fraction B eluted
Was concentrated to obtain 8 mg of zeaxanthin. The zeaxanthin thus obtained was in agreement with a known one in hydrogen nucleus NMR: visible light absorption spectrometry, mass spectrometry.

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[Procedure amendment]

[Submission date] September 24, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

During the culture, the pH of the medium is not particularly controlled,
Cultured for 120 hours. After the culture solution was filtered and the bacterial cells were ground, 2 L of acetone was added and stirred, and the precipitate was filtered off to obtain 2 L of extract. The extract was concentrated to 100 ml and passed through a column of polystyrene adsorbent Diaion pH 20 (1 L) to adsorb the active substance. The active substance was eluted with methanol after eluting impurities with deionized water and 30% methanol. The active fraction was concentrated and extracted with benzene. The extract was dehydrated with sodium sulfate and then concentrated. This was further developed with hexane: acetone = 8: 2 using a silica gel column (silica gel 60 manufactured by Nacalai Tesque). The eluted fraction A was concentrated to obtain 2 ml of a yellow concentrated liquid. Fraction A was purified by high performance liquid chromatography (Cosmosil 5SL manufactured by Nacalai Tesque, Inc., inner diameter 8 mm, length 250 mm, hexane: acetone = 8: 2, flow rate 1.2 ml / min). The eluted fraction B was concentrated to obtain 8 mg of zeaxanthin. The zeaxanthin thus obtained was in agreement with a known one in hydrogen nucleus NMR: visible light absorption spectrometry, mass spectrometry.

Claims (2)

[Claims] 1. A method for producing zeaxanthin, which comprises culturing in a medium a microorganism belonging to the genus Alteromonas and having the ability to produce zeaxanthin, and collecting zeaxanthin from the culture. 2. The method for producing zeaxanthin according to claim 1, wherein the microorganism belonging to the genus Alteromonas is Alteromonas sp.