JP5270122B2 - Acidic mucopolysaccharide-producing microorganism, method for producing acidic mucopolysaccharide, and whitening agent containing acidic mucopolysaccharide as an active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism producing an acidic mucopolysaccharide, a method for producing an acidic mucopolysaccharide, and a skin whitening agent compounded with an acidic mucopolysaccharide as an active component. <P>SOLUTION: There are provided a microorganism belonging to the genus Cobetia, giving a cultured product by the cultivation under a cultivation condition suitable for the microorganism of the genus Cobetia and enabling the separation and recovery of an acidic mucopolysaccharide from the cultured product without using the selective removal of sulfated polysaccharides; a microorganism belonging to the genus Cobetia wherein the 16S rDNA base sequence has a homology of 99.7% to Cobetia marina DSM 4741 and not 100% homology; a microorganism belonging to the genus Cobetia and named as Cobetia sp. WAK-1A (deposition number: FERM P-21101); a method for producing an acidic mucopolysaccharide, especially an acidic mucopolysaccharide for skin whitening agent by using the microorganism; and a skin whitening agent containing, as an active component, an acidic mucopolysaccharide derived from a microorganism of the genus Cobetia and produced by the production method, or its physiologically allowable salt or derivative. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a novel microorganism producing acidic mucopolysaccharide, an improved method for producing acidic mucopolysaccharide using the same, and a whitening agent containing acidic mucopolysaccharide produced by the microorganism as an active ingredient. Furthermore, the present invention relates to an external whitening agent, a cosmetic containing an external whitening agent, an oral whitening agent, and a food and drink containing an oral whitening agent (a whitening health food).

  The melanin pigment that causes skin coloring is partially unknown, but is generally considered to be caused by the production of melanin in the tissue by sunburn or the like. When the skin is irradiated with ultraviolet rays caused by sunlight, tyrosine present in the skin is enzymatically oxidized by the action of the enzyme tyrosinase, and then changes to black melanin through several steps of reaction. This is the process of pigment production. In the melanin formation process, the amino acid tyrosine is oxidized by tyrosinase to become dopa (dihydroxyphenylalanine; DOPA), and further oxidized by the action of tyrosinase to become dopaquinone. Finally, indole-5,6- The quinone is polymerized and bound to the protein is the melanin pigment. Therefore, by inhibiting the activity of tyrosinase, it is possible to suppress the occurrence of spots and freckles in human skin.

  Conventionally, as a whitening cosmetic composition that suppresses the production of melanin and maintains the whitening state of the skin, hydroquinone derivatives such as arbutin, ellagic acid, ascorbic acid and its glycosides, kojic acid and its derivatives, etc. It has been known. For example, in recent years, cosmetics using vitamin C (L-ascorbic acid) derivatives having excellent reducing ability have been proposed, but vitamin C derivatives have difficulty in stability and are almost effective for external use. Not.

  On the other hand, phosphorylated polysaccharides produced by lactic acid bacteria have a melanoma growth inhibitory action and a melanin production inhibitory action, and the utility as a whitening agent is disclosed (Patent Document 1).

In addition, various marine bacteria are known to produce polysaccharides, and some bacterial species have been reported to produce acidic mucopolysaccharides (Patent Literature 2, Patent Literature 3, Non-Patent Literature). 1). In these acidic mucopolysaccharides, uronic acid residues such as glucuronic acid and amino sugar residues such as N-acetylglucosamine and N-acetylgalactosamine repeat a certain sequence to form a sugar chain. It differs from neutral polysaccharides in that it also contains neutral sugar residues such as glucose and galactose and has a structure in which amino acids and pyruvate groups are bonded as side chains.
However, these cosmetics still have various problems and have problems in practical use.
For example, kojic acid as a whitening component may cause carcinogenesis, and hydroquinone derivatives such as arbutin have safety problems such as rash and rash.
Moreover, although the composition of animal and plant extracts is known, the effect is not satisfactory.

Therefore, the present inventor has the formula of formula (1) produced by marine bacteria in order to provide a whitening agent having an excellent whitening effect and a novel ingredient having high stability and safety to the skin as an active ingredient. A whitening agent comprising an acidic mucopolysaccharide represented by the structural formula (in the following description, simply referred to as “the present acidic mucopolysaccharide”) or a physiologically acceptable salt or derivative thereof as an active ingredient. Developed (Patent Document 4, Non-Patent Document 2).
(1)

(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is a pyruvate, and n is a gel. The average molecular weight measured by filtration chromatography represents the number of repetitions indicating that the average molecular weight is 1 to 1.5 million with pullulan as a standard.)

  With respect to the acidic mucopolysaccharide, physiological activities such as chondrocyte proliferation promoting effect, melanin production inhibiting effect, macrophage activating effect and the like have been reported so far.

  This acidic mucopolysaccharide is produced by a strain of Pseudomonas (Patent Document 2, Non-Patent Documents 2 and 3). However, the strains described in these documents are not desired on a commercial scale. This is because, in shake (or agitation) culture such as jar fermenter or large-scale tank culture, the removal of the selectively produced sulfated polysaccharide is complicated and leads to high costs.

  In particular, in the present acidic mucopolysaccharide production process using the liquid shaking culture (or stirring culture) method, the sulfated polysaccharide selectively produced along with it causes various problems in the separation and purification of the acidic mucopolysaccharide. Therefore, in order to enable production of the present acidic mucopolysaccharide by a microorganism at a practical scale level, a microorganism that does not involve simultaneous production of sulfated polysaccharide is desired.

Shamsuddin et al., Fisheries Science, 64,469-473, 1998. M. Matsuda et al., Fisheries Science, 63, 983-988, 1997. M. Matsuda et al., Nippon Suisan Gakkaishi, 58, 1735-1741, 1992. Japanese Patent No. 2971027 JP 2002-065292 A Japanese Patent No. 3083858 Japanese Patent No. 3802011

  The present invention has been made in view of the above circumstances, and its purpose is to produce the acidic mucopolysaccharide on the industrial scale, which is a compound that sufficiently satisfies both the pigmentation improving (whitening) effect and the safety to the skin. In addition, microorganisms that do not produce sulfated polysaccharides that are not present in conventional microorganisms, a method for efficiently and inexpensively producing acidic mucopolysaccharides using the microorganisms, and the acidic mucopolysaccharides produced by the microorganisms as active ingredients It is to provide a whitening agent. Furthermore, it is providing the food / beverage (whitening health food) containing an external whitening agent, the cosmetics containing an external whitening agent, an oral whitening agent, and an oral whitening agent. Whitening effects such as pigmentation, blotches, freckles, and light spots after sunburn are caused by things that are used daily, such as cosmetics, or those that are taken daily, such as food and drink Therefore, it is desired that prevention be configured, and an object of the present invention is to provide a whitening agent that has no side effects, is highly safe even for long-term continuous use, and can be used for foods and beverages and cosmetics.

  As a result of repeated studies to solve the above-mentioned problems, the present inventors have found that the present acid mucopolysaccharide is not accompanied by the accompanying production of sulfated polysaccharide in microorganisms belonging to the genus Cobetia [conventional of Patent Document 2]. It is the same substance (same structural formula and the same functionality as whitening function) as acidic mucopolysaccharide (registered trademark: Uminoglycan) made from the strain. The present invention has been completed.

  That is, surprisingly, in the inventor's screening research process for this acidic mucopolysaccharide-producing microbial strain, the present inventor was accompanied by the accompanying production of sulfated polysaccharides from marine microorganisms collected from seaweeds that inhabit the coast of the Seto Inland Sea. And succeeded in obtaining a novel strain producing this acidic mucopolysaccharide.

  The novel strain of the present invention differs from the strain of Pseudomonas described in Patent Document 2, Non-Patent Documents 2 and 3 in essential characteristics. That is, the novel strain of the present invention does not have the accompanying production of sulfated polysaccharide, which is a basic characteristic of the strains described in Patent Document 2 and Non-Patent Documents 2 and 3.

  By examining the 16S rDNA base sequence (SEQ ID NO: 1) of the obtained new strain, and further cultivating and biochemical characteristics, an attempt was made to identify the new strain, and a novel microorganism belonging to the genus Cobetia (Cobetia) It was revealed that. Subsequently, the culture solution of the novel strain was analyzed to confirm that the acidic mucopolysaccharide was produced and the production of sulfated polysaccharide was lacking, and an improved method for producing the acidic mucopolysaccharide was invented.

  As will be described later, it was determined through taxonomic surveys such as 16S rDNA nucleotide sequence analysis that the new strain belongs to the genus Cobetia, so that the new strain was designated as Cobetia sp. WAK-1A. Named and deposited at the National Institute of Advanced Industrial Science and Technology Patent Biology Depositary Center (1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan 305-8565), deposit number FERM on November 20, 2006 Commissioned as P-21101.

That is, the gist of the present invention is the Covetia microorganism described in the following (1) to ( 4 ).
(1) The microorganism of the genus Covetia is Cobetia sp. WAK-1A (deposit number FERM)
P-21101), a culture is obtained by culturing under a culture condition suitable for the microorganism of the genus Covetia , and then an acidic mucopolysaccharide represented by the structural formula of the following formula (1) is selected from the culture as a sulfated polysaccharide. Microbe of the genus Covetia, characterized in that it can be separated and recovered without selective removal.

(1)
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)
(2) The culture is carried out for 2 days to 7 days in a range of 22 ° C. to 28 ° C. in a solid medium, liquid standing, and shaking (or stirring) conditions using a culture medium containing a carbon source, a nitrogen source and a mineral salt. The microorganism belonging to the genus Covetia according to (1), which is used for culture.
(3) The Covetia microorganism described in the above (1) or (2) having the following mycological properties.
<Form>
Cell morphology: Aspergillus motility: Yes Spore formation: None <Growth state>
Colony morphology: Circular colony color: Cream color Surface of colony: Smooth growth temperature: Grows at 10 ° C, 30 ° C, 37 ° C and 45 ° C.
Growth pH: 5.5 to 9.5 (optimum growth pH: 6.5 to 7.5)
Oxygen demand: Aerobic <Physiological properties>
Gram staining: Negative catalase reaction: Positive oxidase reaction: Acid / gas production from negative glucose: Negative / negative
O / F test (oxidation / fermentation): negative / negative β-galactosidase activity: positive indole production: negative nitrate reduction: negative starch hydrolysis: negative gelatin hydrolysis: negative esculin hydrolysis: negative urease: negative arginine dihydrolase: negative Assimilation (D-glucose, lactose, saccharose, sodium acetate, sodium citrate, sodium gluconate, sodium succinate): Yes (L-arabinose, maltose, D-mannose, D-mannitol, N-acetyl-D- Glucosamine, n-capric acid, adipic acid, dl-malic acid, phenyl acetate, L-alanine, L-histidine, L-serine): None (4) 16S rDNA nucleotide sequence is Cobetia marina ( Cobetia marina )
The microorganism of the genus Covetia according to the above (1), (2) or (3) having a base sequence of SEQ ID NO: 1 having a homology rate of 99.7% with that of DSM 4741 and 100% not matching.

In addition, the gist of the present invention is the method for producing acidic mucopolysaccharides described in the following ( 5 ) or ( 6 ).
( 5 ) A step of culturing the Covetia microorganism according to any one of (1) to ( 4 ) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And a method for producing acidic mucopolysaccharide comprising isolating acidic mucopolysaccharide from a culture solution without selective removal of sulfate polysaccharide.
( 6 ) The method for producing acidic mucopolysaccharide according to ( 5 ), wherein the acidic mucopolysaccharide is an acidic mucopolysaccharide for whitening agents.

Moreover, this invention makes a summary the manufacturing method of the whitening agent as described in the following (7) and (8).
(7) A step of culturing the Covetia microorganism according to any one of (1) to (4) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And acidic mucopolysaccharides are produced by a production method comprising a step of isolating acidic mucopolysaccharides from a culture solution without selective removal of sulfate polysaccharides, and the obtained acidic mucopolysaccharides are intact or physiologically acceptable. and acylated by salt or fatty acid is, the production method of the acidic mucopolysaccharides, or whitening agent characterized by producing a whitening agent containing the acylated product as an active ingredient according to their physiologically acceptable salts or fatty acids .
(8) The method for producing a whitening agent according to the above (7), wherein the whitening agent is an external preparation for skin.

Moreover, this invention makes the summary the manufacturing method of cosmetics as described in the following (9).
(9) A step of culturing the Covetia microorganism according to any one of (1) to (4) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And acidic mucopolysaccharides are produced by a production method comprising a step of isolating acidic mucopolysaccharides from a culture solution without selective removal of sulfate polysaccharides, and the obtained acidic mucopolysaccharides are intact or physiologically acceptable. is an acylated by salts or fatty acids, to produce a topical lightening agent comprising acidic mucopolysaccharides, or acylated by their physiologically acceptable salts or fatty acids as an active ingredient, from the obtained external lightening agent, A cosmetic production method comprising producing a cosmetic comprising the external whitening agent.

Moreover, this invention makes a summary the manufacturing method of the food and drink as described in the following (10).
(10) A step of culturing the Covetia microorganism according to any one of (1) to (4) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And acidic mucopolysaccharides are produced by a production method comprising a step of isolating acidic mucopolysaccharides from a culture solution without selective removal of sulfate polysaccharides, and the obtained acidic mucopolysaccharides are intact or physiologically acceptable. is an acylated by salts or fatty acids, method for producing the acidic mucopolysaccharides, or food, which comprises producing a food containing an acylated by their physiologically acceptable salts or fatty acids.

ADVANTAGE OF THE INVENTION According to this invention, the novel microorganisms which can produce this acidic mucopolysaccharide during culture | cultivation, without entraining and producing sulfated polysaccharide can be provided. More specifically, according to the present invention, it was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba City, Ibaraki 305-8586, Japan). November 20, Year An isolated Cobetia sp. WAK-1A, deposited under accession number FERM P-21101, is provided.
By using this microorganism, the acidic mucopolysaccharide can be safely and mass-produced with stirring in a jar fermenter or tank, and the acidic mucopolysaccharide can be easily and easily separated and purified in large quantities at a simple process. Enables economical industrial production scale.
In addition, according to the method for producing a whitening agent according to the present invention, it has a melanin production inhibitory action, and has an excellent effect on lightening whitening, such as pigmentation, blotches, freckles, and liver spots after sunburn, A whitening agent having excellent safety can be produced.
Furthermore, according to the present invention, it has an inhibitory action on melanin production, and has excellent effects on lightening and whitening of pigmentation, stains, freckles, and liver spots after sunburn, and also has excellent safety. Whitening agents can be provided.
The whitening effect is desired to be prevented by what is used daily such as cosmetics, or by what is taken daily such as food and drink. According to the present invention, Therefore, it is possible to provide a whitening agent that has no side effects, is highly safe even for long-term continuous use, and can be used for food and drink and cosmetics.

  Cobetia sp. WAK-1A (FERM P-21101), which is a novel strain of the present invention, is characterized in that it is characterized by the presence of sulfated polysaccharides in the culture as distinctive from Pseudomonas strains that produce this acidic mucopolysaccharide. It is to accumulate the acidic mucopolysaccharide represented by the structural formula of the formula (1) without accumulating and accumulating it. This property was confirmed by analyzing the polysaccharide fraction obtained from the novel strain culture solution as described in the following Examples.

(1)

(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)

  This isolated strain (FERM P-21101) is a Gram-negative gonococcus, has motor ability, is non-sporing and is heterotrophic. Moreover, it grew sufficiently on MB2216 agar (Becton Dickinson, MD, USA), and produced viscous colonies. The isolated strain did not liquefy gelatin, did not hydrolyze starch, oxidized carbohydrates such as lactose with the production of relatively small amounts of acid, but did not generate gas. This strain was catalase positive but oxidase and indole negative.

<Microorganisms that produce this acid mucopolysaccharide without the production of sulfated polysaccharides>
The microorganism of the present invention is characterized in that a culture solution is obtained by culturing under a culture condition suitable for a microorganism of the genus Covetia, and then acidic mucopolysaccharide can be separated and recovered from the culture solution without selectively removing the sulfated polysaccharide. It is a microorganism of the genus Covetia. More specifically, the microorganisms of the present invention can be obtained from Cobetia marina DSM 4741 (VALIDATION LIST No 88. Int. J. Syst. Evol. Microbiol., 52, 1915-1916, 2002., DRArahal et al .: System. Appl. Microbiol., 25, 207-211, 2002) and 99.7% homologous 16S rDNA base sequence (SEQ ID NO: 1) and 100% homologous microorganism, sulfated polysaccharide It is a microorganism that produces this acidic mucopolysaccharide without accompanying production.
The Cobetia marina DSM4741 strain is positioned as a standard strain of Cobetia marina. Deposit numbers and public deposit numbers are as follows.
1. ATCC253741 (American Type Culture Collection, Manassas, VA, USA)
2. DSM4741 (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany)
3. NCIMB1877 (National Collection of Industrial and Marine Bacteria, Aberdeen, UK)
The genus Cobetia is also a marine bacterium and is one of the interests in the development of marine resources, but there is no one that produces this acid mucopolysaccharide. The DSM4741 strain was first named and approved by Arahal et al. (Above reference 2) (above reference 1).

As an example of a microorganism that produces the present acid mucopolysaccharide without accompanying the production of such sulfate polysaccharide, the WAK-1A strain isolated from the seaweed sample collected in the Seto Inland Sea by the present inventors can be mentioned.
<Isolation of a novel strain producing this acidic mucopolysaccharide>
In order to isolate a novel strain that produces this acid mucopolysaccharide, it has been confirmed that it is isolated from wakame and cultured in a seawater medium consisting of peptone, yeast extract and sucrose to produce this acid mucopolysaccharide. As a result of screening for strains lacking the ability to produce sulfate polysaccharides, it was found that the WAK-1A strain has properties suitable for its purpose.

  Based on the results of homology searches against the 16S rDNA nucleotide sequence database of bacterial reference strains using BLAST, the WAK-1A strain was found to belong to the genus Cobetia and was named Cobetia sp. WAK-1A . This strain has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan 305-8586). Deposit number: FERM on November 20, 2006 Commissioned as P-21101.

<Preparation of acidic mucopolysaccharide>
Various methods are known as methods for preparing this acidic mucopolysaccharide, but those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals. For example, according to the method of Mazda et al. (M. Matsuda et al .: Nippon Suisan Gakkaishi, 58, 1735-1741, 1992), the novel strain is used as sucrose as a carbon source, peptone as a nitrogen source, seawater containing yeast extract and / or Polysaccharides can be produced by culturing artificial seawater in a medium hardened with agar and collected and purified. More specifically, for example, in agar plate culture, sucrose as a carbon source, peptone as a nitrogen source, a polysaccharide-producing seawater and / or artificial seawater medium containing yeast extract in a medium solidified with agar and the strain or its mutation The acidic mucopolysaccharide can be obtained by culturing the strain and separating and purifying the acidic mucopolysaccharide from the mucilage produced on the agar plate.

  The basic medium for cultivating microorganisms that produce the acidic mucopolysaccharide can grow microorganisms that can produce polysaccharides, and contain at least a carbon source, a nitrogen source, various inorganic salts, and trace elements. What is contained is used. More preferably, a culture medium capable of growing microorganisms belonging to the genus Cobetia is used as the basic medium. Examples of the carbon source include sugars such as glucose and saccharose, molasses and waste molasses. One or two or more carbon sources can be used alone or in combination. Nitrogen sources include compounds such as nitrates and ammonium salts, and natural products such as peptone, yeast extract and amino acids. One or two or more nitrogen sources can be used alone or in combination. Examples of the inorganic salt include phosphate, magnesium salt, potassium salt and the like. One or two or more inorganic salts can be used alone or in combination. In the case of a solid medium, agar is used.

  The culture conditions used can be the same as those normally used for culturing microorganisms, such as the medium to be used, the pH of the medium, the additives to the medium, and the culture temperature.

  As the microorganism used for production of the acidic mucopolysaccharide, a microorganism capable of producing the acidic mucopolysaccharide without accompanying the production of sulfated polysaccharide is used. Specific examples include bacteria belonging to the genus Covetia, and more specific examples include Cobetia sp. WAK-1A (FERM P-21101) or a mutant thereof. This strain is a marine bacterium isolated from seaweed in the Seto Inland Sea by the inventor, and its taxonomic characteristics are described in the following examples.

  The culture conditions are not limited as long as the pH during the culture is a range in which microorganisms can grow and produce polysaccharides (pH 5.5 to 9.5), but usually a pH in the range of 6.5 to 7.5. Is preferred. The culture temperature is not limited as long as microorganisms grow and produce polysaccharides, but a range of 22 ° C. to 28 ° C. is good for producing polysaccharides. The culture period varies depending on the culture pH and temperature, but usually 2 to 7 days is appropriate.

  By culturing a microorganism using the above-described medium and microorganism, the target acidic mucopolysaccharide can be efficiently produced.

  The chemical structure of the acid mucopolysaccharide is described in detail in the literature (M. Matsuda et al .: Fisheries Science, 63, 983-988, 1997). The molar ratio of the components is N-acetyl-D-galactosamine: D-glucuronic acid: N-acetyl-L-galactosamine: pyruvic acid is 2: 1: 1: 1, and the average molecular weight measured by gel filtration chromatography is pullulan. The standard is 1 to 1.5 million. Specifically, high performance liquid chromatography (manufactured by Shimadzu) using Asahipak GFA-7M (manufactured by Showa Denko) as a column, 0.1N NaCl as the mobile phase, pullulan having a known molecular weight (Shodex STANDARD KIT P-82, It can be measured using a molecular weight retention time standard curve prepared as a standard sample. Cellulose acetate membrane electrophoresis or high performance liquid chromatography can be used for the analysis of the constituent components. For analysis of this component, the polysaccharide fraction was hydrolyzed with 2M trifluoroacetic acid (TFA) or 4N-HCl at 100 ° C. for 12 hours, and TFA or HCl was removed by a rotary evaporator as a sample. Analysis of neutral sugar, uronic acid, organic acid, sulfate ion and amino sugar. In particular, the analysis of uronic acid is performed by analyzing the neutral sugar by hydrolysis after reduction of the carboxyl group of the polysaccharide. The organic acid and sulfuric acid group are analyzed by ion chromatography analysis, and the organic acid can also be an enzymatic method. In addition, the component and sugar chain binding mode can be analyzed by 1HNMR analysis of the polysaccharide fraction.

  About the production method of the present acidic mucopolysaccharide, the production method of the present acidic mucopolysaccharide as a preferred embodiment includes (a) a step of separating a novel strain that produces the present acidic mucopolysaccharide without the production of sulfated polysaccharide; (B) culturing the novel strain to produce the acidic mucopolysaccharide, and (c) separating and recovering the produced acidic mucopolysaccharide.

Subsequently, the above embodiment will be described in detail for each step.
(a) a step of isolating a novel strain that produces the acidic mucopolysaccharide without producing the sulfated polysaccharide, and a strain that is preserved as a strain that produces the acidic mucopolysaccharide is composed of peptone, yeast extract, sucrose, A polysaccharide fraction is separated and purified by precipitation with ethanol from a culture obtained by shaking culture in a medium prepared with artificial seawater. This is tested for the presence or absence of sulfated polysaccharide production by DEAE-cellulose column chromatography analysis, chemical analysis, nuclear magnetic resonance analysis, etc., according to the methods described in Non-Patent Documents 1 and 2 above.
(b) Step of culturing microorganisms to produce polysaccharides To obtain the present acidic mucopolysaccharide, the WAK-1A strain or a mutant thereof is used as the microorganism. By using the WAK-1A strain or a mutant thereof as the microorganism, the acidic mucopolysaccharide can be produced efficiently. Liquid culture can be performed by stationary, shaking (stirring) culture, and agar plate can be used for solid culture.

Here, this acidic mucopolysaccharide has a structural unit of the following formula (1).
(1)

(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)
And it becomes possible to obtain this acidic mucopolysaccharide of high purity by a high yield by passing through the extraction and collection | recovery process demonstrated below.

(c) Step of separating and recovering the produced predetermined polysaccharide As a method of extracting the acidic mucopolysaccharide from the culture obtained by the above production method, a conventionally known method used for separating and recovering polysaccharides is used. be able to. For example, after sterilizing the culture as it is or at a high temperature, the cells are removed by centrifugation, and this is concentrated as it is or after adding 2-3 times amount of ethanol, isopropanol, acetone, or the like, This causes precipitation. This precipitate is dissolved again in water or 1 to 15% by weight sodium chloride solution, and then precipitation with alcohol or the like is repeated 2-3 times, dialyzed with water, and using a spray dryer or a freeze dryer. The acidic mucopolysaccharide is obtained by drying. In addition, electrodialysis and ultrafiltration can be used. For further purification, various chromatographies such as ion exchange and gel filtration, precipitation or salting out with a quaternary ammonium salt, and the like can be used.

  When removing the cells from the culture solution, a membrane separation apparatus including a hollow fiber membrane module having a pore diameter of 0.1 to 0.45 μm can be used.

The acidic mucopolysaccharide is thus produced and recovered.
Acidic mucopolysaccharides produced by Cobetia sp. WAK-1A (deposit number FERM P-21101) obtained by the present invention can experimentally support that melanin production is strongly suppressed and cytotoxicity is extremely weak. It was. Therefore, the acidic mucopolysaccharide according to the present invention is useful as an active ingredient of a whitening agent. The whitening agent is applied to various wide fields such as cosmetics including quasi-drugs, pharmaceuticals, and foods.
The acidic mucopolysaccharides according to the present invention may be formulated in the form of salts or derivatives thereof such as physiologically acceptable salts, sodium salts, potassium salts, etc., for example, acylated products with fatty acids, preferably acetylated products. .
The acidic mucopolysaccharide O-acyl derivative according to the present invention can be obtained, for example, by treating acidic mucopolysaccharide with acetic anhydride or acid chloride in a dilute aqueous acid solution in a homogeneous system. Alternatively, when soluble in an organic solvent such as formamide, an O-acetyl derivative of acidic mucopolysaccharide can be obtained by treatment with pyridine and acetic anhydride. In addition, for example, it can also be prepared by the method described in JP-A-3-143540 and the method described in JP-A-6-9707. Although the O-acetylation rate varies depending on the acylation method, 1 or more, preferably 3 or more, more preferably 4 or more O-acetyl groups are introduced into each repeating unit of the acidic mucopolysaccharide. Yes.

  Furthermore, the acidic mucopolysaccharide according to the present invention is blended as an active ingredient in the composition and prepared as a whitening agent as the composition.

  In the whitening agent as the composition, the acidic mucopolysaccharide contains an effective amount for exerting a whitening action. Generally, these compounds are used as active ingredients in an amount of at least 0.001% by weight, preferably 0%. What is necessary is just to add so that it may contain 0.01 to 20.0 weight%, Preferably 0.05 to 10.0 weight%. If it is less than 0.001% by weight, the effect referred to in the present invention is not sufficiently exerted, and if it exceeds 20.0% by weight, it is difficult to make a preparation. Moreover, even if it mix | blends 10.0 weight% or more, the improvement of the big effect is not seen. It is not always necessary to isolate and use the active ingredient, and a crude product containing the compound of the present invention can be used as long as the effects of the present invention are not impaired.

In addition to the acidic mucopolysaccharide as an essential component, the whitening agent as the composition requires other components that are usually used in cosmetics, quasi-drugs, pharmaceuticals, etc., as long as the effects of the present invention are not impaired. Accordingly, it can be appropriately blended and manufactured.
Examples of the other components include oils, surfactants, powders, colorants, water, alcohols, thickeners, antioxidants, ultraviolet absorbers, and humectants. In addition, metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine , Hot water extract of karin fruit, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, 3-O-ethylascorbic acid, arbutin Whitening agents such as kojic acid, saccharides such as glucose, fructose, mannose, sucrose, trehalose, vitamin A derivatives such as retinoic acid, retinol, retinol acetic acid, retinol palmitic acid, and the like can be appropriately blended.

  The administration route in the case of administering the whitening agent as the composition is not particularly limited, and it can be administered by any method such as oral, parenteral and topical. The dosage is not particularly limited because it is appropriately adjusted depending on the subject (mammal, particularly human), age, sex, individual differences, symptoms, etc. For example, 0.1 to 500 mg / kg as the acidic mucopolysaccharide of the present invention. Preferably, a dose of 0.5 to 200 mg / kg can be administered orally or parenterally once or divided into several times a day.

  The whitening agent as the composition is preferably used in the form of a topical skin preparation externally applied to the skin, and is applied by a method of direct application or spraying to the skin. For example, it is excellent when applied to the skin for the face and body. Demonstrate whitening effect.

  The dosage form of the external preparation for skin that is a suitable form of the whitening agent as the composition is not particularly limited as long as it can exert the effects of the present invention, and includes, for example, a solution system, a solubilization system, an emulsification system, Arbitrary dosage forms such as a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, a solid, an ointment, a gel, an aerosol, and a mousse can be used.

  The skin external preparation, which is a suitable form of the whitening agent as the composition, is applied, for example, in the field of cosmetics and pharmaceuticals including quasi-drugs, and the product forms include, for example, ointments, emulsions, creams, lotions. , Gels, packs, etc. can take any form. The topical skin preparations are quasi-drugs such as lotion, milky lotion, cream, essence (beauty serum), basic cosmetics such as lotion, makeup cosmetics such as foundation and lipstick, aromatic cosmetics, cosmetics for hair, body cosmetics, etc. Can take the form of cosmetics.

  The acidic mucopolysaccharide according to the present invention can be incorporated into an external preparation for skin, and is particularly effective in preventing and improving pigmentation, stains, freckles, liver spots, etc. after sunburn, and has an excellent effect on skin whitening. It is useful as an external preparation for skin whitening.

  In the above-mentioned external preparation for skin, the acidic mucopolysaccharide contains an effective amount for exerting a whitening effect. Generally, at least 0.001% by weight or more, preferably 0.01-% or more of the total amount of these compounds as active ingredients. What is necessary is just to add so that it may contain 20.0 weight%, Preferably 0.05-10.0 weight%. If it is less than 0.001% by weight, the effect referred to in the present invention is not sufficiently exerted, and if it exceeds 20.0% by weight, it is difficult to make a preparation. Moreover, even if it mix | blends 10.0 weight% or more, the improvement of the big effect is not seen. It is not always necessary to isolate and use the active ingredient, and a crude product containing the compound of the present invention can be used as long as the effects of the present invention are not impaired.

In addition to the above-mentioned essential component acidic mucopolysaccharide, the external preparation for skin includes other components that are usually used in external preparations such as cosmetics, quasi-drugs, and pharmaceuticals, as long as the effects of the present invention are not impaired. Other whitening agents, moisturizers, antioxidants, oils, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc. as required It can mix | blend suitably.
In addition, metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine , Hot water extract of karin fruit, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, 3-O-ethylascorbic acid, arbutin Whitening agents such as kojic acid, saccharides such as glucose, fructose, mannose, sucrose, trehalose, vitamin A derivatives such as retinoic acid, retinol, retinol acetic acid, retinol palmitic acid, and the like can be appropriately blended.

  The dosage form of the external preparation for skin is not particularly limited, and is not particularly limited as long as the effect of the present invention can be exhibited. For example, a solution system, a solubilization system, an emulsification system, a powder dispersion system, water- Any dosage form such as oil two-layer system, water-oil-powder three-layer system, solid, ointment, gel, aerosol, mousse and the like can be taken.

  Further, the external preparation for skin is applied in the fields of cosmetics and pharmaceuticals including quasi-drugs, for example, and the product forms include any forms such as ointments, emulsions, creams, lotions, gels, packs, etc. Can take. The topical skin preparations are quasi-drugs such as lotion, milky lotion, cream, essence (beauty serum), basic cosmetics such as lotion, makeup cosmetics such as foundation and lipstick, aromatic cosmetics, cosmetics for hair, body cosmetics, etc. Can take the form of cosmetics.

  The present invention relates to a composition in the form of a food or drink or a medicine containing a whitening agent associated with prevention and improvement of pigmentation / stain / freckle / liver spots after sunburn comprising the acidic mucopolysaccharide as an active ingredient The present invention relates to foods and drinks that contain the present acidic mucopolysaccharide as an active ingredient and that are labeled for the prevention and improvement of pigmentation, stains, freckles, and liver spots after sunburn. Therefore, the medicinal product or food for specified health use according to the present invention is characterized in that the blended acidic mucopolysaccharide is contained as an active ingredient.

When it is in the form of a pharmaceutical, it is a composition for preventing and improving post-tanning pigmentation / stain / freckle / liver plaque etc. containing this acidic mucopolysaccharide together with one or more pharmaceutically acceptable carriers . The pharmaceutical composition comprises a therapeutically effective amount of a pharmacologically active compound of the present invention alone or in combination with one or more pharmaceutically acceptable carriers.
When this acidic mucopolysaccharide is used as the above-mentioned preparation, it can be carried out by a known method. Specifically, for example, it can be carried out as described below.

  The preparation of the present invention comprises tablets, pills, capsules (including hard capsules, soft capsules, microcapsules), powders, granules, fine granules, troches, liquids (syrups) with sugar coating or coating as necessary , Emulsions, suspensions, etc.).

  The preparation of the present invention can be mixed with a physiologically acceptable carrier or the like as long as the effects of the present invention are not inhibited. As physiologically acceptable carriers, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, binders, disintegrants, lubricants in solid formulations; solvents in liquid formulations, dissolution aids Agents, suspending agents, buffering agents, thickeners, emulsifiers and the like. In addition, formulation additives such as coloring agents, sweetening agents, and antioxidants can be used as necessary. Furthermore, you may coat the formulation of this invention.

  Examples of the excipient include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose (for example, microcrystalline cellulose), low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, Examples include gum arabic, dextrin, pullulan, light anhydrous silicic acid, synthetic aluminum silicate and magnesium aluminate metasilicate. Examples of the binder include pregelatinized starch, sucrose, gelatin, macrogol, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose ( HPC), hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone (PVP), and the like. Examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, crosslinked polyvinylpyrrolidone, carmellose sodium, croscarmellose sodium, sodium carboxymethyl starch, light anhydrous silicic acid, low-substituted hydroxypropylcellulose, Examples include cation exchange resin, partially pregelatinized starch, and corn starch. Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, talc, waxes, colloidal silica, DL-leucine, sodium lauryl sulfate, magnesium lauryl sulfate, macrogol, and aerosil.

  Examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, medium chain fatty acid triglyceride (MCT), vegetable oil (for example, safflower oil, sesame oil, corn oil, olive oil, cottonseed oil, soybean Lecithin, etc.). Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, and the like. can give. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, Hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate, and the like. Examples of the thickener include natural gums and cellulose derivatives. Examples of emulsifiers include fatty acid esters (eg, sucrose fatty acid ester, glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester), wax (eg, beeswax, rapeseed hydrogenated oil, safflower hydrogenated oil, palm Examples thereof include hydrogenated oil, sitosterol, stigmasterol, campesterol, brush casterol, cacao butter powder, carnauba wax, rice wax, molasses, paraffin, and the like, and lecithin (for example, egg yolk lecithin and soybean lecithin).

  Examples of the colorant include water-soluble edible tar dyes (eg, edible red Nos. 2 and 3, edible yellow Nos. 4 and 5, edible blue Nos. 1 and 2 and the like, water-insoluble lake dyes (eg, Examples of the water-soluble edible tar pigment include aluminum salts, natural pigments (eg, β-carotene, chlorophyll, bengara, etc.) Examples of the sweetener include sucrose, lactose, sodium saccharin, dipotassium glycyrrhizinate, Examples include aspartame, stevia, etc. Examples of the antioxidant include sulfite, ascorbic acid, and alkali metal salts and alkaline earth metal salts thereof.

  Tablets, granules, fine granules and the like may be coated by a usual method using a coating substrate for the purpose of masking taste, improving light stability, improving appearance or enteric properties. Examples of the coating base include sugar coating base, water-soluble film coating substrate, enteric film coating substrate and the like.

  Examples of the sugar coating base include sucrose, and one or more kinds selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.

  Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer Synthetic polymers such as E (Eudragit E (trade name), Rohm Pharma) polyvinylpyrrolidone; and polysaccharides such as pullulan. Examples of enteric film coating bases include cellulose polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L (Eudragit L (trade name)) Acrylic polymers such as Rohm Pharma), methacrylic acid copolymer LD (Eudragit L-30D55 (trade name) Rohm Pharma), methacrylic acid copolymer S (Eudragit S (trade name) Rohm Pharma), etc. Examples include natural products. These coating bases may be coated singly or in combination of two or more at an appropriate ratio, or two or more may be coated sequentially.

  In the case of a composition in the form of a food or drink, or a food or drink with an indication of function or efficacy, the food composition or food or drink of the present invention is a functional food, health food, food for specified health use, or for the sick It may be prepared as a food, supplement or the like, and is preferably prepared as a functional food. Examples of the shape of the food composition or food or drink of the present invention include a tablet shape, a round shape, a capsule shape (including hard capsules, soft capsules, and microcapsules), a powder shape, a granular shape, a fine granular shape, a troche shape, Liquids (including syrups, milks, and suspensions) are included, and tablets and capsules are preferred.

  The food composition of the present invention is particularly preferably tablet-like or capsule-like, and particularly preferably a tablet-like functional food or a capsule-like functional food.

  The food composition of the present invention can be produced, for example, by incorporating the acidic mucopolysaccharide into the food by a known method. Specifically, for example, the tablet-shaped food composition includes, for example, the present acidic mucopolysaccharide and raw materials such as excipients (for example, lactose, sucrose, mannitol, etc.), sweeteners, flavoring agents, It can manufacture by mixing and shape | molding in a tablet shape by applying a pressure with a tableting machine etc. Other materials (for example, vitamins such as vitamin C, minerals such as iron, dietary fiber, etc.) can be added as necessary. Capsule-shaped food composition is, for example, filled with a liquid, suspension, paste, powder or granule-shaped food composition containing the acidic mucopolysaccharide, or encapsulated with a capsule base. Can be manufactured.

  As long as the effects of the present invention are not impaired, the food composition of the present invention can be used in addition to commonly used food materials, food additives, various nutrients, vitamins, flavor substances (for example, cheese and chocolate), and physiological May be blended with an acceptable carrier. As physiologically acceptable carriers, various conventional organic or inorganic carrier materials are used, and excipients, binders, disintegrants, lubricants, coloring agents, sweeteners, preservatives, antioxidants, Examples include thickeners and emulsifiers. Examples of food additives include colorants, sweeteners, preservatives, antioxidants, and flavoring agents. Furthermore, other materials, for example, minerals such as iron, dietary fibers such as pectin, carrageenan, and mannan may be contained.

  Excipients, binders, disintegrants, lubricants, solvents, solubilizers, suspending agents, buffers, thickeners, colorants, sweeteners, preservatives, and antioxidants are described above. The thing similar to what is used for the formulation of this invention is mention | raise | lifted.

  Vitamins may be water-soluble or fat-soluble, such as retinol palmitate, tocopherol, bisbenchamine, riboflavin, pyridoxine hydrochloride, cyanocobalamin, sodium ascorbate, cholecalciferol, nicotinamide, pantothene. Examples include calcium acid, folic acid, biotin, and choline bitartrate.

  For tablet-like, granule-like, and fine-grained food compositions, etc., coating is carried out by a known method using a coating substrate for the purpose of masking taste, improving light stability, improving appearance, or enteric properties. May be. Examples of the coating substrate include those similar to those used in the preparation of the present invention described above, and can be carried out in the same manner.

  In the present invention, the present acidic mucopolysaccharide is safe, so that it can be administered and / or fed to any general person as a preventive method for the possibility that a certain person may develop pigmentation, stains, freckles, liver spots, etc. in the future. Also good. In a preferred embodiment of the present invention, the acidic mucopolysaccharide may be administered and / or fed to an individual suspected of being at risk for pigmentation, stains, freckles, liver spots and the like.

  When humans ingest the acid mucopolysaccharides or their physiologically acceptable salts or derivatives thereof, the dosage should be determined according to the age, weight and symptoms of the individual. When the present acidic mucopolysaccharide or a physiologically acceptable salt or derivative thereof is used, for example, the acidic mucopolysaccharide of the present invention is 0.1 to 500 mg / kg, preferably 0.5 to 200 mg / kg. The dose can be administered orally or parenterally once or divided into several times a day. Alternatively, it is appropriate to take once a day or in divided portions before meals, after meals or with meals.

  In preferred embodiments of the invention, the acidic mucopolysaccharide is administered orally and / or fed, but other administrations including subcutaneous, inhalation, intravenous, subarachnoid, rectal, or any other suitable route of administration. Routes can also be used. Formulations containing the present acidic mucopolysaccharides can be varied using standard techniques and compounds, depending on the mode of administration.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all to these Examples. Needless to say, the percentage (%) is based on weight unless otherwise specified.

<Isolation of this acid mucopolysaccharide-producing bacterium without the accompanying production of sulfate polysaccharide>
A medium prepared with seawater and / or artificial seawater having a composition of 0.5% peptone, 0.1% yeast extract and 3% sucrose is sterilized for 15 minutes in an autoclave at a temperature of 121 ° C. From the slant culture for storage as a producing microorganism, inoculate one platinum loop into the above-mentioned sterilized medium (10 ml) in a test tube, perform shaking culture at a temperature of 25 ° C. for 24 hours, and then add 500 ml of this preculture solution. 200 ml (121 ° C., sterilized for 15 minutes) of the medium having the above composition was inoculated into an Erlenmeyer flask and cultured at 25 ° C. for 3 days with shaking. After incubation, the culture solution is filtered using a filter aid (Radiolite # 500), ethanol is added to the supernatant from which the cells have been removed, and the precipitated fraction is collected, dissolved in water, and then 5% quaternary ammonium. Fraction precipitated by addition of salt (Cetavlon) solution was collected by filtration.
This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction.
This polysaccharide fraction has a molar ratio of constituents of N-acetyl-D-galactosamine: D-glucuronic acid: N-acetyl-L-galactosamine: pyruvic acid of 2: 1: 1: 1, and gel filtration chromatography. The measured average molecular weight was 1 to 1.5 million with pullulan as a standard. Specifically, high performance liquid chromatography (manufactured by Shimadzu) using Asahipak GFA-7M (manufactured by Showa Denko) as a column, 0.1N NaCl as the mobile phase, pullulan having a known molecular weight (Shodex STANDARD KIT P-82, It was measured using a molecular weight retention time standard curve created as a standard sample.
Cellulose acetate membrane electrophoresis and high performance liquid chromatography were used to analyze the constituent components. For analysis of this component, the polysaccharide fraction was hydrolyzed with 2M trifluoroacetic acid (TFA) or 4N-HCl at 100 ° C. for 12 hours, and the TFA or HCl was removed using a rotary evaporator as a sample. Neutral sugar, uronic acid, organic acid, sulfate ion and amino sugar were analyzed. In particular, uronic acid was analyzed by analyzing the neutral sugar by hydrolysis after reduction of the carboxyl group of the polysaccharide. Analysis of organic acids was performed by ion chromatography, and in addition to this, enzymatic methods were used. The sulfate group was analyzed by ion chromatography analysis of the sample after hydrolysis.
The ¹ H nuclear magnetic resonance analysis of this polysaccharide fraction (Fig. 1) shows that the chemical shift of sulfate polysaccharide with an anomeric proton signal δ = 4.8 ppm (M. Matsuda et al .: Nippon Suisan Gakkaishi, 59, 535-538, 1993) We searched for strains that could not be recognized and that sulfate groups were not found by ion chromatography analysis.

Anomeric proton signal (500 MHz, internal standard: TSP-d4, solvent: D2O), δ = 4.50 (1H, J = 7.6 Hz), δ = 4.58 (1H, J = 8.3 Hz) in ¹ HNMR of this acidic mucopolysaccharide ), δ = 4.57 (1H, J = 8.1 Hz) and δ = 5.07 (1H, J = 2.9 Hz) are shown in FIG.

  As a result, a strain that produces this acidic mucopolysaccharide without accompanying production of sulfate polysaccharide was obtained and named WAK-1A strain. The WAK-1A strain was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-6 Chuo, 1-chome Higashi 1-chome, Tsukuba City, Ibaraki Prefecture 305-8586. Deposit number FERM P- It has been entrusted as 21101.

<Taxonomic position of new strains>
This acid mucopolysaccharide-producing strain WAK-1A (FERM P-21101) without accompanying production of newly isolated sulfate polysaccharide was identified by analysis of 16S rDNA nucleotide sequence. The 16S rDNA nucleotide sequence of this newly isolated WAK-1A strain was determined to belong to Cobetia, showing 99.7% homology with the Cobetia 16S rDNA nucleotide sequence. However, this strain is clearly distinguished from Covetia marina DSM4741, the reference strain of the genus Covetia, by several culture and biochemical characteristics described below.

  The homology search of the WAK-1A strain using the 16S rDNA-Full nucleotide sequence was performed as follows (Table 1: 16S rDNA (16S rRNA gene) nucleotide sequence analysis). The genome extraction procedure followed the BIO RAD protocol. Using the extracted genomic DNA as a template, a region of the entire base sequence 1500-1600 bp of the 16S ribosomal RNA gene (16S rDNA) was amplified by PCR. Thereafter, the amplified 16S rDNA was sequenced to determine the base sequence (SEQ ID NO: 1) of 16S rDNA.

  As a result of homology search against the bacterial reference strain database using BLAST, the 16S rDNA base sequence (SEQ ID NO: 1) of WAK-1A strain showed high homology to Cobetia-derived 16S rDNA, and the homology rate was 99.7%. It showed the highest homology to 16S rDNA of .marina DSM4741 strain.

  As a result of homology search against GenBank / DDBJ / EMBL, the 16S rDNA of WAK-1A strain showed high homology to 16S rDNA derived from C. marina, and the reference strain compared to 16S rDNA of C. marina DSM4741 strain. The homology was 99.7%. In addition, as a result of simple molecular phylogenetic analysis using 16S rDNA of WAK-1A strain and 16S rDNA of the top 10 homology search against the bacterial reference strain database, WAK-1A strain was found to be C. marina 16S rDNA and phylogenetic branch. Was shown to be closely related. However, there is a difference of 4 bases between the 16S rDNAs, and 3 bases are clearly different and do not match completely. From this, although the WAK-1A strain is closely related to C.marina, it is considered that it is a different strain as a species.From the results of this 16S rDNA nucleotide sequence, the WAK-1A strain was changed to C.marina. It was judged that it was appropriate to use closely related Cobetia sp. FIG. 2 shows a molecular phylogenetic tree in which the WAK-1A strain was positioned by 16S rDNA sequencing.

  The culture and biochemical characteristics of the WAK-1A strain were examined by culturing at 25 ° C. for 24 hours using MB2216 medium (Becton Dickinson, MD, USA). Based on observation by light microscope and Barrow et al. (Barrow, GI et al .: Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd ed., Cambridge Press, 1993), catalase reaction, oxidase reaction, acid from glucose / Tested for gas production, glucose oxidation / fermentation (OF). The culture and biochemical characteristics were further determined using a test kit AP120NE (bioMerieux, Lyon, France) according to the protocol of the kit. The results are shown in Table 2 (cultural and biochemical tests) and Table 3 (utilization test).

  This strain was a gram-negative gonococci having motility, did not oxidize glucose, showed a positive catalase reaction, and a negative oxidase reaction. It also showed β-galactosidase activity. The WAK-1A strain grows at 10 ° C and 45 ° C, does not hydrolyze starch, and assimilates D-glucose, lactose, saccharose, etc. in assimilation tests, and uses L-arabinose, maltose, L-alanine, etc. Not assimilated. These properties are considered to be similar to C.marina, which was suggested to be related in the results of 16S rDNA nucleotide sequence analysis. However, the point of growing at 45 ° C and the utilization of lactose and saccharose differed from those of C.marina reference strains. Therefore, from this test result, this strain was presumed to be Cobetia sp. Closely related to C. marina.

A medium prepared with seawater and / or artificial seawater having a composition of 0.5% peptone, 0.1% yeast extract and 3% sucrose was sterilized in an autoclave at 121 ° C for 15 minutes, and strain WAK-1A From the slant culture, inoculate one platinum loop into the above sterilized medium (10 ml) in a test tube, shake culture at a temperature of 25 ° C. for 24 hours, and then place this preculture in the 500 ml Erlenmeyer flask. 200 ml of the medium having the composition (121 ° C., sterilized for 15 minutes) was inoculated, and static culture was performed at a temperature of 25 ° C. for 3 days. After culturing, the culture solution is centrifuged or filtered using a filter aid (Radiolite # 500), ethanol is added to the supernatant from which the cells have been removed, and the precipitated fraction is collected. A fraction that precipitated upon addition of a quaternary ammonium salt (Cetavlon) solution was collected by filtration. This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction.
As a practical purification level, the polymer fraction obtained from the culture filtrate by a membrane filtration apparatus equipped with a hollow fiber UF membrane module (Spectrum) with a molecular weight of 50,000 cut is concentrated in a short time. Desalted and recovered, and powdered by lyophilization. The membrane filtration apparatus used was SYLS-SB04 manufactured by Toyobo Engineering.
The polysaccharide fraction thus obtained was confirmed for homogeneity by using cellulose acetate membrane electrophoresis, and by using DEAE-cellulose ion exchange column chromatography analysis, chemical analysis, and nuclear magnetic resonance analysis, The acid mucopolysaccharide was confirmed. Further, as a result of 1HNMR analysis and ion chromatography analysis of these samples, it was confirmed that the sulfated polysaccharide was not contained.

  Until the preculture, the same treatment as in Example 3 was performed, and then this preculture was put into 200 ml (121 ° C., 15 minutes) of a sterilized medium prepared from seawater having the above composition and / or artificial seawater in a 500 ml Erlenmeyer flask. Inoculated and cultured with shaking at 25 ° C. for 3 days. Next, the culture solution is centrifuged or filtered using a filter aid (Radiolite # 500), ethanol is added to the supernatant, and the precipitated fraction is collected, dissolved in water, and then dissolved in 5% quaternary ammonium salt (Cetavlon ) The fractions that precipitated upon addition of the solution were collected by filtration. This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction. As a practical purification level, a polymer fraction obtained from a culture filtrate by a membrane filtration apparatus equipped with a hollow fiber UF membrane module (Spectrum) having a molecular weight of 50,000 cut is concentrated and removed in a short time. The salt was collected and pulverized by lyophilization. The membrane filtration apparatus used was SYLS-SB04 manufactured by Toyobo Engineering. The polysaccharide fraction thus obtained was tested for homogeneity using a cellulose acetate membrane electrophoresis method, and a known book was obtained by DEAE-cellulose ion exchange column chromatography analysis, chemical analysis, and nuclear magnetic resonance analysis. The acid mucopolysaccharide was confirmed. Further, as a result of 1HNMR analysis and ion chromatography analysis of these samples, it was confirmed that the sulfated polysaccharide was not contained.

Until the pre-culture, the same treatment as in Example 3 was performed, 250 ml of agar plate medium obtained by adding 1.5% of agar to the medium described in Example 3 above was spread on a flat plate (18 × 26 cm) and smeared with the pre-culture solution. After culturing at 25 ° C for 4 days, scrape the sticky material generated on the surface of the agar plate, suspend it in 1% phenol solution, and filter the cells with a filter aid (Radiolite # 500). Fraction precipitated by adding ethanol to the supernatant obtained by removing the solution by collecting the solution, and 5% quaternary ammonium salt (Cetavlon) solution was added after dissolution in water, and the fraction precipitated by filtration was collected by filtration. This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction. As a practical purification level, the polymer fraction obtained from the culture filtrate by a membrane filtration apparatus equipped with a hollow fiber UF membrane module (Spectrum) with a molecular weight of 50,000 cut is concentrated in a short time. Desalted and recovered, and powdered by lyophilization.
About the polysaccharide fraction obtained in this way, while confirming uniformity using the cellulose acetate membrane electrophoresis method, it confirmed that it was this well-known acidic mucopolysaccharide by a chemical analysis and a nuclear magnetic resonance analysis. Further, as a result of 1HNMR analysis and ion chromatography analysis of these samples, it was confirmed that the sulfated polysaccharide was not contained.

  The above strains used in Examples 2 to 5 were deposited in the 1st, 1st, 1st Street, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan 305-8586, National Institute of Advanced Industrial Science and Technology. , November 20, 2006 Deposit No. FERM P-21101.

(Inhibition test of melanin production using mouse cell lines)
Using the acidic mucopolysaccharide obtained in Example 4, cell growth and melanin production inhibitory effects on melanoma cells were examined, and the cell growth rate and melanin production amount in the sample addition system were compared with the case of no sample addition. Asked. As a positive control, a polysaccharide (Okazaki et al .: Fragrance Journal, 8, 61-64, 2004) known to have an inhibitory action on melanin production was used.

(Cell culture)
(1) Precultured B16F10 cells were seeded at a cell number of 1.0 × 10 4 cells / well in each well of a 12-well multiwell plate. 48 hours after sowing, the medium was replaced with 0.5 mM theophylline-containing DMEM + 10% FBS medium containing a test substance at a predetermined concentration. The concentration of the test substance was set to 25 μg / ml, 50 μg / ml, and 100 μg / ml. Three wells for each sample concentration were assigned for the assay (N = 3). A plate for counting cell proliferation and two plates for quantifying melanin were used as a set for assay of each sample.
After the medium change, the culture was continued for 72 hours.
(2) The number of cells in each well (relative value) was measured by measuring absorbance at 450 nm using the WST-1 method using a cell proliferation counting plate, thereby measuring the degree of cell proliferation. The result is shown in FIG.

(Melanin content measurement method)
B16 melanoma cells were cultured, melanin produced in the cells was dissolved in alkali, and the amount of melanin produced was determined by measuring the absorbance.
Cells in each well were treated with a 10% trichloroacetic acid solution using a plate for melanin quantification after 72 hours of culture, and then treated with ethanol / ether (1: 1 (V / V)).
A 1N NaOH / 10% DMSO solution was added thereto to solubilize melanin, and then the absorbance was measured at 475 nm. A solution of synthetic melanin (Sigma) dissolved in 1N NaOH / 10% DMSO solution is diluted to a prescribed concentration and used as a standard solution. By comparing the absorbance of this standard solution and the measurement solution, the melanin content in the measurement solution Was calculated.
The amount of melanin production (relative value) was calculated from the number of cells in the well (relative value) obtained in (2) and the amount of melanin in this well.

  As is apparent from FIG. 3, the addition of the acidic mucopolysaccharide inhibited the melanin production depending on the concentration, and this value was similar to the positive control (23.21 ± 2.34 at 50 μg / ml). The acidic mucopolysaccharide was not observed to inhibit the proliferation of melanoma cells, ie, cytotoxicity. The addition of the acid mucopolysaccharide at each concentration did not change the cell morphology associated with cytotoxicity. That is, it was confirmed that the acidic mucopolysaccharide exhibits a melanin production inhibitory effect within a concentration range that does not affect the cell growth.

  The whitening agent according to the present invention is used in various dosage forms, but as an example, formulation examples of a skin external preparation will be described as examples. As shown in Table 4.

  The whitening agent according to the present invention is used in various dosage forms, but an example of blending a cosmetic liquid will be described as an example. As shown in Table 5.

By using the microorganism of the present invention, preferably Cobetia sp. WAK-1A (FERM P-21101), the acidic mucopolysaccharide represented by the structural formula of the above formula (1) is stirred in a jar fermenter or tank. It can be safely produced in large quantities, and thus has an inhibitory effect on melanin production, and has excellent effects on lightening and whitening of pigmentation, stains, freckles, and liver spots after sunburn, and safety. In addition, an excellent whitening agent and a skin whitening agent for whitening can be easily separated and purified in a large amount and at a low cost by a simple process, thereby enabling an economical industrial production scale.
The whitening agent of the present invention can be directly diluted with water or concentrated or administered as a skin external preparation. Alternatively, it can be diluted directly with water, concentrated, powdered or granulated, and formulated with a known pharmaceutical carrier to produce aerosols, solutions, extracts, suspensions, emulsions, ointments. It can be provided in the form of an agent, a poultice, a liniment, and a lotion. Or used in known cosmetics, quasi drugs, pharmaceuticals, aqueous ingredients, surfactants, oily ingredients, solubilizers, moisturizers, powder ingredients, alcohols, pH adjusters, preservatives, antioxidants, It is prepared by appropriately selecting a thickener, a dye, a pigment, a fragrance and the like as necessary. As an external preparation for skin, it can be in the form of lotion, gel, emulsion, ointment, etc., lotion such as flexible lotion, astringent lotion, emollient cream, moisture cream, massage cream etc. Makeup cosmetics such as creams, emollient emulsions, nourishing emulsions, cleansing emulsions, face wash, skin cleanser, foundation, eye color, teak color, lipstick, shampoo, rinse, hair treatment agent, hair cream, It can be provided as various forms of cosmetics such as hair cosmetics such as hair styling agents, hair nickings, hair nourishing agents, hair growth agents, etc., and bath oils such as bath oils, bath salts, and foam baths.
The whitening agent of the present invention can be administered orally as it is or after diluting with water or the like. Alternatively, it is prepared by formulating it with a known pharmaceutical carrier. For example, the extract as an oral liquid preparation such as a syrup, or the extract is processed into an extract, powder, etc., and blended with a pharmaceutically acceptable carrier, such as tablets, capsules, granules, powders, etc. It can be administered as an oral solid formulation. As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations, solvents, excipients in liquid preparations, Formulated as a suspending agent, binder, and the like. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners, and the like can be used as necessary.

1H NMR spectrum (INOVA-500, 70 ° C.) of this acidic mucopolysaccharide is shown. The simple molecular phylogenetic tree using the 16S rDNA base sequence of WAK-1A strain (FERM P-21101) is shown. The lower left line indicates the scale bar, the number located at the branch of the phylogenetic branch indicates the bootstrap value, and the T at the end of the strain name indicates that type strain. The influence of this acid mucopolysaccharide on the proliferation and melanin production of mouse melanoma cells is shown.

Claims (10)

The microorganism of the genus Covetia is Cobetia sp. WAK-1A (deposit number FERM P-21101), and a culture is obtained by culturing under a culture condition suitable for the microorganism of the genus Covetia. 1. A Covetia microorganism characterized in that the acidic mucopolysaccharide represented by the structural formula (1) can be separated and recovered without selectively removing the sulfated polysaccharide.
(1)
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)   The culture is performed using a culture medium containing a carbon source, a nitrogen source, and a mineral salt for 2 to 7 days in a range of 22 ° C. to 28 ° C. in a solid medium, liquid standing, shaking or stirring conditions. The Covetia microorganism according to claim 1. The microorganism of the genus Covetia according to claim 1 or 2, having the following mycological properties.
<Form>
Cell morphology: Aspergillus motility: Yes Spore formation: None <Growth state>
Colony morphology: Circular colony color: Cream color Surface of colony: Smooth growth temperature: Grows at 10 ° C, 30 ° C, 37 ° C and 45 ° C.
Growth pH: 5.5 to 9.5 (optimum growth pH: 6.5 to 7.5)
Oxygen demand: Aerobic <Physiological properties>
Gram staining: Negative catalase reaction: Positive oxidase reaction: Acid / gas production from negative glucose: Negative / negative
O / F test (oxidation / fermentation): negative / negative β-galactosidase activity: positive indole production: negative nitrate reduction: negative starch hydrolysis: negative gelatin hydrolysis: negative esculin hydrolysis: negative urease: negative arginine dihydrolase: negative Assimilation (D-glucose, lactose, saccharose, sodium acetate, sodium citrate, sodium gluconate, sodium succinate): Yes (L-arabinose, maltose, D-mannose, D-mannitol, N-acetyl-D- Glucosamine, n-capric acid, adipic acid, dl-malic acid, phenyl acetate, L-alanine, L-histidine, L-serine): None The Covetia genus according to claim 1, 2 or 3, wherein the 16S rDNA base sequence has a base sequence of SEQ ID NO: 1 having 99.7% homology with that of Cobetia marina DSM 4741 and 100% does not match Microorganisms. A step of culturing the Covetia microorganism according to any one of claims 1 to 4 to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, and culturing the acidic mucopolysaccharide A method for producing an acidic mucopolysaccharide comprising a step of isolating a sulfate polysaccharide without selective removal from a liquid. The method for producing acidic mucopolysaccharide according to claim 5 , wherein the acidic mucopolysaccharide is an acidic mucopolysaccharide for whitening agents. A step of culturing the Covetia microorganism according to any one of claims 1 to 4 to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, and culturing the acidic mucopolysaccharide Acidic mucopolysaccharides are produced by a production method including a step of isolating without selective removal of sulfated polysaccharides from the liquid, and the obtained acidic mucopolysaccharides are used as they are or with physiologically acceptable salts or fatty acids thereof. A method for producing a whitening agent, characterized in that it comprises an acylated product and the acidic mucopolysaccharide, or an acylated product of a physiologically acceptable salt or fatty acid thereof as an active ingredient. The method for producing a whitening agent according to claim 7, wherein the whitening agent is an external preparation for skin. A step of culturing the Covetia microorganism according to any one of claims 1 to 4 to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, and culturing the acidic mucopolysaccharide Acidic mucopolysaccharides are produced by a production method including a step of isolating without selective removal of sulfated polysaccharides from the liquid, and the obtained acidic mucopolysaccharides are used as they are or with physiologically acceptable salts or fatty acids thereof. A whitening agent for external use is produced as an acylated product , and the acid mucopolysaccharide, or an acylated product of a physiologically acceptable salt or fatty acid thereof as an active ingredient, and the external whitening agent is contained from the obtained external whitening agent. A method for producing a cosmetic, characterized by producing the cosmetic. A step of culturing the Covetia microorganism according to any one of claims 1 to 4 to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, and culturing the acidic mucopolysaccharide Acidic mucopolysaccharides are produced by a production method including a step of isolating without selective removal of sulfated polysaccharides from the liquid, and the obtained acidic mucopolysaccharides are used as they are or with physiologically acceptable salts or fatty acids thereof. A method for producing a food or drink comprising producing the food and drink as an acylated product and containing the acid mucopolysaccharide, or an acylated product of a physiologically acceptable salt or fatty acid thereof.