JP2009201369A - New astaxanthin dirhamnoside and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an astaxanthin dirhamnoside of an astaxanthin-like coloring matter having both of antioxidant activity and water solubility, and to provide a method for producing the same. <P>SOLUTION: The new astaxanthin dirhamnoside of the formula is obtained by culturing an astaxanthin dirhamnoside-producing bacteria belonging to the genus Sphingomonas, and collecting the compound from the resulted product. The method for producing the astaxanthin dirhamnoside is also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

      The present invention relates to a novel astaxanthin dirhamnoside and a process for producing the same.

      Carotenoids are naturally occurring pigments that have been applied to various uses because of their high antioxidant activity and bright yellow-orange hue. Among them, astaxanthin, which is a red pigment that is a molecular species having high antioxidant activity, has been increasingly used as a health food (Patent Document 1) and a food additive (Patent Document 2), and also colored livestock meat (Patent Document 3). ) Is expanding its use.

Astaxanthin has a high fat solubility, so its utilization rate is low even when taken. For this reason, astaxanthins in which two glucose molecules are bound have been reported for the purpose of improving solubility (Patent Documents 4 and 5).
JP 2005-34135 A JP 2002-262824 A JP-A-7-99925 JP-A-10-327865 JP 2007-330105 A

However, astaxanthin is expected to have a certain degree of bias in the efficiency and distribution of uptake into the living body due to its high fat solubility. Therefore, development of more useful astaxanthin is demanded.
That is, in order to further expand the application of astaxanthin and its related substances, it is desired to develop molecular species that exhibit higher antioxidant activity and have different physical properties.

Therefore, the present inventors have searched for a new microorganism to develop a related compound having physical properties different from those of astaxanthin. As a result, the inventors have isolated a microorganism that produces a new astaxanthin-like substance from Misasa Onsen, Tottori Prefecture.
This new microorganism was confirmed to be Sphingomonas astaxanthinifaciens classified into the genus Sphingomonas.
As a result of culturing this new microorganism and analyzing the culture, it was found that it was a new astaxanthin dirhamnoside having an astaxanthin-like pigment, and the present invention was completed.

[式（１）中、Ｒは、α−ラムノピラノシル基を表す。]で表されるアスタキサンチンジラムノシドを提供するものである。[(3S,3'S)-3,3'-bis(a-D-rhamnopyranosyloxy)-b,b-carotene-4,4'-dione]。
（２）スフィンゴモナス属（Ｓｐｈｉｎｇｏｍｏｎａｓ）に属するアスタキサンチンジラムノシド物質生産菌を培養し、その培養物よりアスタキサンチンジラムノシドを採取することを特徴とするアスタキサンチンジラムノシドの製造法を提供するものである。
（３）スフィンゴモナス属（Ｓｐｈｉｎｇｏｍｏｎａｓ）に属する微生物がスフィンゴモナス・アスタキサンチニファシエンス（Ｓｐｈｉｎｇｏｍｏｎａｓ ａｓｔａｘａｎｔｈｉｎｉｆａｃｉｅｎｓ）である上記（２）記載のアスタキサンチンジラムノシドの製造法を提供するものである。
（４）スフィンゴモナス・アスタキサンチニファシエンス（Ｓｐｈｉｎｇｏｍｏｎａｓ ａｓｔａｘａｎｔｈｉｎｉｆａｃｉｅｎｓ）がスフィンゴモナス・アスタキサンチニファシエンスＴＤＭＡ−１７（ＮＢＲＣ番号：１０２１４６）である上記（３）記載のアスタキサンチンジラムノシドの製造法を提供するものである。
（５）上記（１）記載のアスタキサンチンジラムノシドを含有する組成物を提供するものである。 That is, the present invention as described below is provided.
(1) The following formula (1)

[In the formula (1), R represents an α-rhamnopyranosyl group. Astaxanthin dirhamnoside represented by the formula: [(3S, 3 ′S) -3,3′-bis (aD-rhamnopyranosyloxy) -b, b-carotene-4,4′-dione].
(2) A method for producing astaxanthin dirhamnoside, comprising culturing an astaxanthin dirhamnoside substance-producing bacterium belonging to the genus Sphingomonas and collecting astaxanthin dirhamnoside from the culture. is there.
(3) The present invention provides the method for producing astaxanthin dirhamnoside according to the above (2), wherein the microorganism belonging to the genus Sphingomonas is Sphingomonas astaxanthinifaciens.
(4) A method for producing an astaxanthin diramnoside as described in (3) above, wherein Sphingomonas astaxanthinifaciens is Sphingomonas astaxanthinifaciens TDMA-17 (NBRC number: 102146). To do.
(5) A composition containing the astaxanthin dirhamnoside described in (1) above is provided.

    The novel astaxanthin dirhamnoside of the present invention can be widely used for applications such as a color tone improving agent, an antioxidant, a food coloring material, and a feed additive, like the red pigment astaxanthin.

    As an example of a novel astaxanthin dirhamnoside-producing bacterium used in the present invention, it was newly isolated in Tottori Prefecture, Misasa Onsen, and on September 7, 2006, the National Institute of Technology and Evaluation (NITE) Sphingomonas astaxanthinifaciens TDMA-17 strain (hereinafter referred to as “the present strain”) deposited as NBRC 102146 (December 15 December 2006) at the Patent Microorganism Deposit Center ).

The bacteriological properties of this strain are as follows.
(1) Properties of colonies 20 to 45 ° C. (optimum temperature: 40 ° C.), pH 5.5 to 11.0 (optimum pH: 7.0 to 8.0), about 48 on a nutrient broth agar medium Depending on the culture conditions of time, it shows good growth and forms a smooth colony in red with a diameter of 0.5 to 1.0 mm. However, if 0.25% or more sodium chloride is present, growth is inhibited.

(2) Morphological properties Oval-shaped or rod-shaped cells having a width of 0.3 to 0.5 μm and a length of 0.5 to 0.9 μm, and have motility. Spores are not observed. The cells forming the colony may form a fibrous structure outside the cell.

(3) Physiological properties Gram staining-
Indole generation −
Production of hydrogen sulfide −
Catalase activity −
Oxidase activity −
β-galactosidase activity −
Urease activity −
Tryptophan deaminase activity −
Urease activity −
Lysine decarboxylase activity −
Ornithine decarboxylase activity −
Degradation of esculin ＋
Tween 80 decomposition +
Degradation of gelatin +
Casein degradation ＋
Starch degradation ＋
KINTEN −
DNA-
Cellulose −
Chitin −
Glycosphingolipid +
(Utilization test)
Glucose −
L-arabinose-
D-Mannose-
D-mannitol-
Maltose −
N-acetyl-D-glucosamine −
Gluconic acid −
Capric acid −
Adipic acid −
Malic acid −
Sodium citrate −
Phenylacetic acid −

(4) Chemical taxonomic properties DNA base composition (GC content: mol%) 67.7%
Type of ubiquinone (Coenzyme Q) Q-10

Moreover, as a result of phylogenetic analysis of the 16S rRNA gene, the base sequence (1396 bp) of the 16S rRNA gene of this strain was decoded and a database search was conducted, and as a result, there was 94.5% homology with Sphingomonas aerolata NW12 (AJ429240), It was confirmed to be included in the genus Sphingomonas.
Based on the above results, this strain was identified as a gram-negative aerobic bacterium and belonging to the genus Sphingomonas.
In addition, about Sphingomonas astaxanthinifaciens TDMA-17 which is this strain, about more details, such as a bacteriological property, FEMS Microbiology Letters 273 No. 2 pp. 140-148.

(culture)
The astaxanthin dirhamnoside substance-producing bacterium of the present invention can be cultured in a medium containing nutrients that can be used by ordinary microorganisms. Therefore, any synthetic medium or natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and a substance that helps or promotes growth.
As the medium composition, those usually used for bacterial culture can be used. For example, as the carbon source, glucose, sucrose, maltose, glycerin and the like can be used. As the nitrogen source, meat extract, peptone, yeast extract, malt extract and the like can be used. In addition, inorganic salts capable of generating ions such as sodium, potassium, calcium, and magnesium can be added as necessary. However, sodium chloride cannot be used because it inhibits the growth of this strain.
The culture method can be carried out in accordance with the culture method of general microorganisms depending on the culture conditions of the above-mentioned mycological properties of the strain, and any method such as standing, shaking or aeration stirring can be used. Usually, shaking culture in a liquid medium is suitable.

(Extraction / Purification)
Since the astaxanthin dirhamnoside substance of the present invention has the physicochemical properties described in the examples, it can be extracted and purified from the culture according to the general method based on the properties.
In order to obtain the substance of the present invention from the culture, usual separation means can be used. In particular, since it is often accumulated in the bacterial cells in the culture solution, it is preferable to separate them by centrifugation or filtration. As other extraction / purification methods, for example, solvent extraction, ion exchange resin, adsorption or distribution column chromatography, gel filtration, dialysis, precipitation, etc. may be used alone or in appropriate combination of these methods. it can.
Moreover, the extract obtained from the culture can also be concentrated in the process. The concentration conditions are not particularly limited.
For example, a culture containing an astaxanthin dirhamnoside substance is pulverized and then extracted with an organic solvent. As the organic solvent, methanol, ethanol, hexane, acetone, chloroform, ether, ethyl acetate and the like can be used. The extract is concentrated, and the extract is dissolved in a small amount of chloroform and chromatographed with a chloroform / methanol solvent using a silica gel column equilibrated with chloroform and methanol. The eluate is concentrated to dryness, and an astaxanthin dirhamnoside substance can be obtained by performing thin layer chromatography with a solvent of chloroform / methanol.

    Moreover, this invention can be used as a composition containing an astaxanthin dirhamnoside substance. The term “composition” as used herein refers to a color tone improver that improves the color tone of fish, egg yolk color, etc., antioxidant, food coloring, feed additive, etc., and can be used by adding to pharmaceuticals and foods. . The composition can further contain excipients used in pharmaceutical additives, food additives, and the like.

Examples of the present invention will be described below, but the present invention is not limited to these examples.
(Example 1) Solid culture Peptone 1.0%, yeast extract 0.5%, malt extract 0.5%, casamino acid 0.5%, fish extract 0.2% and magnesium sulfate 0.1% in distilled water After dissolution, the powder agar 1.5% was added to the one adjusted to pH 7.2 and sterilized by autoclave. After sterilization, it was poured into a sterilized petri dish and solidified to prepare a solid medium. In this medium, Sphingomonas astaxanthinifaciens TDMA-17 strain was inoculated with a platinum loop and cultured at 37 ° C. for 5 days. The obtained bacterial cells were scraped from the surface of the medium and used for the following extraction.

(Example 2) Liquid culture Peptone 0.4%, yeast extract 0.2%, sodium chloride 0.5%, fish extract 0.2%, magnesium sulfate 0.2% and maltose 1.0% in a volume of 500 ml It was dissolved in distilled water in an Erlenmeyer flask, adjusted to pH 7.2, and then adjusted to 100 ml. An autoclave sterilized product was used as a liquid medium. Sphingomonas astaxanthinifaciens TDMA-17 strain producing astaxanthin dirhamnoside was inoculated into this liquid medium with a platinum loop, and cultured with shaking at 37 ° C. for 2 days. Further, 1% of the culture solution was added to 100 ml of a liquid medium having the same composition as described above, and cultured with shaking at 37 ° C. for 2 days. 100 ml of the culture broth thus obtained was separated from the supernatant by centrifugation to obtain bacterial cells.

(Example 3) Bacterial cell extraction 5 times the amount of methanol was added to the bacterial cell of Example 1 or Example 2, and the pigment extraction was performed three times using a shaker at 180 rpm for 30 minutes. The extract was filtered to remove the cells and concentrated to dryness to obtain a methanol extract fraction.

(Example 4) Purification The methanol fraction of Example 3 was dissolved in chloroform and subjected to medium pressure preparative liquid chromatography packed with Purif-pack silica gel (MORITEX). The column was adsorbed on a column, washed with chloroform, and then eluted with chloroform: methanol (9: 1). The obtained fraction was concentrated to dryness, dissolved in chloroform with appropriate addition of methanol, and developed on reverse phase silica gel TLC with a developing solution of chloroform: methanol (4: 1). A red spot in the vicinity of Rf = 0.3 was collected and used as a purified sample.

(Example 5) Physicochemical properties of astaxanthin dirhamnoside Using the purified sample obtained in Example 5, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured, and the results shown in FIGS. Obtained. In addition, as a result of measuring the FAB mass spectrum, from the measured molecular weight (m / z 911.48781 [M + Na] ⁺ molecular weight C ₅₂ H ₇₂ NaO ₁₂ molecular weight (calculated value) 911.49214), this astaxanthin dirhamnoside The molecular formula was estimated as C ₅₂ H ₇₂ O ₁₂ .

^{1 is} a ¹ H-NMR spectrum of astaxanthin dirhamnoside of the present invention.

It is a ¹³ C-NMR spectrum measured in deuterated dimethyl sulfoxide of astaxanthin dirhamnoside of the present invention.

Claims (5)

The following formula (1)

[In the formula (1), R represents an α-rhamnopyranosyl group. Astaxanthin dirhamnoside represented by       A method for producing astaxanthin dirhamnoside, comprising culturing an astaxanthin dirhamnoside substance-producing bacterium belonging to the genus Sphingomonas and collecting astaxanthin dirhamnoside from the culture.      The method for producing astaxanthin dirhamnoside according to claim 2, wherein the microorganism belonging to the genus Sphingomonas is Sphingomonas astaxanthinifaciens.       4. The method for producing astaxanthin diramnoside according to claim 3, wherein Sphingomonas astaxanthiniffaciens is Sphingomonas astaxanthinifaciens TDMA-17 (NBRC number: 102146).       A composition comprising the astaxanthin dirhamnoside according to claim 1.

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