JP4523909B2 - Acid heteropolysaccharide AX-1 - Google Patents

Description

  The present invention relates to a novel acidic heteropolysaccharide, and more particularly to a novel acidic heteropolysaccharide produced by a kind of acetic acid bacteria.

  Microorganisms produce various polysaccharides, and among them, acetic acid bacteria are known to produce many polysaccharides, for example, those composed of a single constituent sugar such as cellulose, levan and dextran.

On the other hand, it is also known that certain acetic acid bacteria produce acidic water-soluble polysaccharides (hereinafter sometimes referred to as acidic heteropolysaccharides) composed of different types of saccharides and containing organic acids.
For example, an acetic acid bacterium belonging to the genus Acetobacter is glucose: rhamnose: mannose: glucuronic acid = 4: 0.9 to 1.1: 0.9 to 1.1: 0.9 to 1.1 It has been reported to produce an acidic heteropolysaccharide called AM-2 containing about 4-8% acetyl groups in a ratio (see, for example, Patent Document 1), and then the acidic heteropolysaccharide is glucose: The composition sugar ratio of rhamnose: mannose: glucuronic acid: acetyl group≈4: 1: 1: 1: 1, and the primary structure thereof was also revealed (see, for example, Non-Patent Document 3).

  Also, Acetobacter xylinum NRRL B43 strain is referred to as Acetane having a constituent sugar ratio of glucose: rhamnose: mannose: glucuronic acid: acetyl group≈4: 1: 1: 1: 1-2. It has also been reported to produce acidic heteropolysaccharides similar in structure to AM-2 (see, for example, Non-Patent Document 2).

  Furthermore, acetic acid bacteria belonging to the genus Acetobacter produce acidic heteropolysaccharides with a constituent sugar ratio of glucose: galactose: mannose: glucuronic acid = 10: 3-6: 0.5-2: 0.5-2 (See, for example, Patent Document 2), and then the acidic heteropolysaccharide is referred to as AM-1, and the constituent sugar ratio is glucose: galactose: mannose: glucuronic acid = 6: 2: 1. 1 and its primary structure has been clarified (for example, see Non-Patent Document 1).

  The acidic heteropolysaccharides produced by these acetic acid bacteria have been widely studied mainly for use in foods and drinks due to their viscous properties and the like, and more recently, tumor suppressive action (for example, see Patent Document 3) and Applications focusing on pharmacological actions such as allergy suppressing action (see, for example, Patent Document 4) are also being studied.

However, it has been demanded to develop a novel acidic heteropolysaccharide having a further excellent effect.
“Agricultural and Biological Chemistry”, Vol. 48, No. 10, p. 2405-2414, 1984 “Journal of General Microbiology”, 133, p. 2123-2135, 1987 “Agricultural and Biological Chemistry”, Vol. 49, No. 4, p. 959-966, 1985 JP 59-203498 A JP 58-78596 A JP 2004-59549 A JP 2004-59547 A

  As described above, an object of the present invention is to discover a novel acidic heteropolysaccharide and provide a method for producing the same.

  In view of the above problems, intensive studies were made, and in the process, the novel acetic acid strain isolated from the vinegar production process was found to produce a novel acidic heteropolysaccharide, and the present invention was completed.

  The present inventors have extensively searched for the ability to produce microbial polysaccharides involved in the brewing process of various fermented foods that have been used for human consumption since ancient times and whose safety has been confirmed historically.

  As a result, Acetobacter acetic acid bacteria isolated from vinegar fermenters produce polysaccharides mainly composed of glucose, mannose, galactose and glucuronic acid, and this polysaccharide is conventionally known. The present invention was completed based on the discovery of a novel acidic heteropolysaccharide different from the existing polysaccharide.

  That is, this invention of Claim 1 is related with acidic heteropolysaccharide AX-1 which has the following physicochemical property.

(1) An acidic heteropolysaccharide having glucose, galactose, mannose and glucuronic acid as main constituents and a constituent sugar ratio of glucose: galactose: mannose: glucuronic acid = 5: 1: 1: 1.
(2) The primary structure is as shown in Chemical Formula 1.

(Chemical formula 1)
→ 4) -β-D-Glu- (1 → 4) -β-D-Glu- (1 →
3
↑
1
α-D-Man
2
↑
1
β-D-GlucA
4
↑
1
α-D-Glu
6
↑
1
β-D-Glu
6
↑
1
β-D-Gal
6
↑
1
β-D-Glu

  The present invention according to claim 2 is characterized in that an acidic heteropolysaccharide AX-1 producing bacterium belonging to the genus Acetobacter is cultured, and the acidic heteropolysaccharide AX-1 is collected from the culture. It is related with the manufacturing method of acidic heteropolysaccharide AX-1 of Claim 1.

  Furthermore, the present invention described in claim 3 is characterized in that the acidic heteropolysaccharide AX-1 producing bacterium belonging to the genus Acetobacter is Acetobacter xylinum. Relates to the described method.

  The present invention according to claim 4 is characterized in that the Acetobacter xylinum is Acetobacter xylinum NCI1005 strain (FERM BP-10415). Concerning the method.

  According to the present invention, a novel acidic heteropolysaccharide is provided, and a producing bacterium capable of efficiently producing the acidic heteropolysaccharide can be obtained. By using the producing bacterium, a novel acidic heteropolysaccharide is obtained. It is possible to provide a method for efficiently producing the. The polysaccharide according to the present invention is a polysaccharide produced by acetic acid bacteria that have been used for vinegar brewing since ancient times and whose safety has been confirmed historically. In addition to the food industry, it can be widely used as a thickening / emulsification stabilizer in the cosmetics industry, the pharmaceutical industry, and other various industries.

The present invention relates to a novel acidic heteropolysaccharide AX-1, and enables its production by using an Acetobacter bacterium.
Hereinafter, the present invention will be described in detail.

(1) Producing bacteria of the present invention Acetic acid bacteria that produce the novel acidic heteropolysaccharide of the present invention are present in the vinegar production process, so that vinegar raw materials, vinegar fermented liquor, etc. are used as separation sources, It can be obtained by setting culture conditions, performing enrichment culture, and selecting.

  A novel acid heteropolysaccharide AX-1 producing bacterium selected in this manner is an acetic acid bacterium belonging to the genus Acetobacter, and is preferably Acetobacter xylinum. Among them, Acetobacter zylinum is preferable. (Acetobacter xylinum) NCI1005 strain is exemplified as the most excellent one.

  Acetobacter zylinum NCI1005 strain has been deposited as FERM BP-10415 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1-6 Higashi 1-chome, 1-chome Tsukuba, Ibaraki 305-8586) Yes.

(2) Production of acidic heteropolysaccharide of the present invention The acidic heteropolysaccharide of the present invention can be produced, for example, by the following method. That is, it can be produced by culturing Acetobacter zylinum NCI1005 strain (FERM BP-10415) in an appropriate medium and collecting the acidic heteropolysaccharide from the culture.

  Moreover, since the polysaccharide is an acidic substance, it can be recovered by adding cetyltrimethylammonium bromide or the like to the culture solution excluding the cells to precipitate the polysaccharide.

  The crudely purified polysaccharide can be purified according to the polysaccharide purification method. For example, the above-mentioned crudely purified polysaccharide is redissolved in water, and after heat treatment, it is centrifuged to completely remove insoluble matters, and reprecipitation with a precipitating agent such as acetone is repeated to obtain a high-purity white cotton-like product. The above polysaccharide is obtained.

  A highly purified product can also be obtained by using precipitation (CTAB treatment) with cetyltrimethylammonium bromide, dialysis, ion exchange resin, and the like in combination.

(3) Acidic heteropolysaccharide of the present invention The acidic heteropolysaccharide of the present invention comprises glucose, galactose, mannose, and glucuronic acid as main constituent components, and the constituent sugar ratio is glucose: galactose: mannose: glucuronic acid = 5: 1. An acidic heteropolysaccharide that is 1: 1.

  After adding 2N trifluoroacetic acid, hydrolyzing the polysaccharide of the present invention by treating at 120 ° C. for 5 hours, removing the trifluoroacetic acid by drying under reduced pressure, and then analyzing the dried product by liquid chromatography, It is confirmed that glucose, galactose, mannose, and glucuronic acid are main constituent sugars.

  The polysaccharide of the present invention is acidic because white precipitate is formed when cetyltrimethylammonium bromide or cetylpyridinium chloride is added.

  That is, the polysaccharide of the present invention is an acidic heteropolysaccharide having glucose, galactose, mannose, and glucuronic acid as main components.

  Hereinafter, the present invention will be specifically described with reference to examples and the like.

Example 1
Isolation and identification of producing bacteria

(1) Separation of production bacteria After sampling from the vinegar fermenter, the cells were cultured at 30 ° C. for 24 hours in a test tube containing 5 ml of YPG medium. Thereafter, it was diluted to 1/10 with a YPG medium, and further cultured in a test tube for 24 hours. This culture solution was inoculated on an agar medium containing 6% glucose, 1% yeast extract, 0.4% polypeptone, 2% calcium carbonate and 1.5% agar, and statically cultured at 30 ° C. for 5 days. A colony showing viscosity was obtained. This operation was repeated several times until a single colony was obtained.

  The finally obtained colonies were picked and transferred to a slant agar medium having the above composition, cultured at 30 ° C. for 3 days, and stored at 4 ° C.

(2) Identification of producing bacteria The bacteriological properties of the producing bacteria obtained as described above were examined as follows.
Note that mycological properties include various identification documents (for example, published by the University of Tokyo Press on June 20, 1975, edited by Takeharu Hasegawa, “Classification and Identification of Microorganisms”, The Journal of General and Applied Microbio. The Journal of General and Applied Microbiology, Vol. 10, No. 2, p. 95-126, 1964, and The Society for Applied Bacteriology Technical Series No. 2 (The Society) for Applied Bacteriology Technical Series No.2) Identification Methods for Microbiology (Identification Methods for Microbiology) olologists), pp. 1-8, 1968).

  Yeast extract-glucose agar was prepared by dissolving 5 g of yeast extract, 30 g of glucose, 3 g of polypeptone and 15 g of agar in 1 liter of distilled water and adjusting the pH to 6.5. The yeast extract-glucose liquid medium was 5 g of yeast extract. 30 g of glucose and 3 g of polypeptone were dissolved in 1 liter of distilled water, pH was adjusted to 6.5, sterilized, ethanol was added aseptically 3% (V / V), MY plate medium was 10 g of glucose, Polypeptone 5g, yeast extract 3g, malt extract 3g, agar 15g dissolved in 1 liter of distilled water, pH adjusted to 6.5, gravy liquid medium dissolved in 10 g of meat extract and 10 g of polypeptone dissolved in 1 liter of distilled water. pH adjusted to 6.5, sweetened broth liquid medium is 10g glucose, 10g meat extract, 10g polypeptone in distilled water The pH was dissolved in ter is obtained by adjusting to 6.5.

  The identification of ubiquinone was performed by filter paper chromatography, thin layer chromatography, infrared and ultraviolet absorption spectra, and mass spectrometry.

I. Morphological findings Shape Short bowl Size 0.5 ~ 0.7 × 1.0 ~ 1.2μm
Population Single or linked Motility None Sporulation Not formed Gram stain Negative Antioxidant Negative

II. Culture findings Yeast extract-glucose agar plate culture (4 days at 30 ° C)
Shape Round Edge Smooth and full edge Raised Raised
1. Glossy Surface Smooth Color tone Light yellow and glossy Calcium carbonate-containing yeast extract-glucose slope culture (3 days at 30 ° C)
Growth Good / Good Good Ridge Medium Surface Smooth Edge Smooth and full edge Color tone Light yellow and glossy Ethanol-containing yeast extract-glucose liquid stationary culture (cultured at 30 ° C for 4 days)
It grows well. Forms a fragile fungus film even when wet. It becomes turbid and some settles to the bottom. A thick film made of cellulose is not formed.
4). Meat juice stationary culture (cultured at 30 ° C for 7 days)
Poor growth. A thick film made of cellulose is not formed. Grows in a ring shape.
5). Glucose-containing meat extract liquid static culture (cultured at 30 ° C for 7 days)
Good growth. A thick film made of cellulose is not formed. The culture solution becomes turbid and some precipitates. A thin fungus film is formed.
6). MY gelatin high-layer culture (cultured at 20 ° C for 7 days)
Good growth. No liquefaction.
7). Litmus milk (7 days at 30 ° C)
No solidification.

III. Physiological properties Reduction of nitrate None Denitrification reaction None VP test negative 4. Generation of indole None 5. Generation of hydrogen sulfide None 6. 6. Starch hydrolysis None. Use of citric acid (Christensen's medium) None Use of inorganic nitrogen source None (nitrate) None (ammonium salt)
9. No pigment formation in the medium None 10. Urease activity None Catalase activity Yes 12. Growth pH range 3.0-7.5 (optimal pH range 4.0-5.5)
13. Growth temperature range 15-35 ° C (optimal temperature range 20-28 ° C)
14 Attitude toward oxygen Aerobic 15.5-Ketogluconic acid production Yes 16. Formation of dihydroxyacetone Yes 17. Utilization of ethanol Weakly utilize ethanol to produce acetic acid 18. 18. Acetic acid assimilation None. Lactic acid assimilation None. Vitamin requirement Yes 21. Degradability of acetic acid Yes 22. Degradability of lactic acid Yes 23. Ferric chloride reaction negative (glucose medium)

IV. Utilization of carbon source and generation of acid and gas are shown in Table 1. The meanings of the symbols in the table are as follows. That is, +: assimilate or generate, ++: well assimilate or generate well, +++: very well assimilate or generate very well, −: not assimilate or not generate, ±: slightly Assimilate or generate slightly.

V. Types of coenzymes in electron transport system Main components of coenzymes: Ubiquinone-10

  As a result of the above, this production bacterium is a gram-negative aerobic gonococcus that oxidizes ethanol to produce acetic acid, and can grow even at pH 3.0, so it belongs to the genus Acetobacter or Gluconobacter generally called acetic acid bacteria Is clear.

In addition, this production bacterium has the main ubiquinone type Q ₁₀ and is essential for the growth of vitamins, and has the ability to produce dihydroxyacetone as a gluconobacter, but on the other hand, acetic acid and lactic acid. In terms of degradability, it exhibits properties as an Acetobacter genus and is difficult to determine with either Acetobacter or Gluconobacter genus, but exhibits degradability of acetic acid and lactic acid, and glucose, The ability to accumulate the above-mentioned novel acidic heteropolysaccharides containing galactose, mannose, and glucuronic acid as main components is considered appropriate to certify that the producing bacteria of the present invention belong to the genus Acetobacter. It was.

Furthermore, examples of the genus Acetobacter having Q ₁₀ as a major ubiquinone include Acetobacter xylinum and Acetobacter aceti subspecies liquefaciens.

Among these, Acetobacter aceti subspecies liquefaciens is different in terms of FeCl ₃ reaction in a glucose medium, growth in the presence of ethanol, pigment formation, and the like.

  Acetobacter xylinum is different depending on the presence or absence of cellulose-producing ability, but taking into account that the ability to produce cellulose is very lacking, this producing bacterium is cellulose of Acetobacter xylinum. It was considered that the bacteria lacked the ability to produce.

  From the above results, it was identified that it was appropriate to recognize that this production strain belongs to Acetobacter ylinum, and as a result, this bacterium was named Acetobacter xylinum NCI1005. FERM BP-10415 has been deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba City, Ibaraki 305-8586).

(Example 2)
Preparation of acidic heteropolysaccharides of the present invention

(1) Preparation of acidic heteropolysaccharide Acetobacter zylinum NCI1005 strain (FERM BP-10415) was used as the acetic acid strain used, and acidic heteropolysaccharide was produced by the following method.

  First, inoculate the strain on an agar medium containing 3% glucose, 0.5% yeast extract, 0.3% polypeptone, 2% calcium carbonate and 1.5% agar, and leave it at 30 ° C. for 3 days. To form colonies.

  Next, PP medium (3% glucose, 3% mannitol, 0.2% yeast extract, 0.009% dipotassium hydrogen phosphate, 0.01% potassium dihydrogen phosphate, 0.025% magnesium sulfate, 0.0. A test tube containing 7 ml of (0009% ferric chloride, 0.5% sodium citrate) was inoculated with the above colony collected with a platinum loop and cultured at 30 ° C. for 24 hours with shaking at 120 rpm. 1 ml of this culture solution was inoculated into a 500 ml Sakaguchi flask containing 100 ml of PP medium and cultured with shaking at 120 rpm at 30 ° C. for 48 hours.

  25 ml of a 5 L jar fermenter containing 2.5 L of PP medium was inoculated with the above preculture solution, and main culture was carried out at 30 ° C., 350 rpm, 0.5 vvm for 120 hours to produce acidic heteropolysaccharides. .

  From this culture solution, a freeze-dried preparation of acidic heteropolysaccharide was obtained by the method described in a previous report (for example, see Non-Patent Document 3).

  That is, the culture solution was centrifuged (10,000 × g, 30 minutes), and the cultured cells were completely removed from the supernatant by Celite filtration. This was dissolved in distilled water, 5% cetyltrimethylammonium bromide was added, and the precipitated acidic polysaccharide fraction was recovered.

  The collected acidic heteropolysaccharide fraction was dissolved in 20% NaCl and dialyzed overnight in running water. After completion of dialysis, ethanol was added to collect the resulting precipitate, followed by further dialysis against distilled water overnight, followed by lyophilization to prepare a polysaccharide preparation, which was named AX-1.

(Example 3)
Analysis of AX-1

(1) Physical properties The physical properties were investigated in accordance with previous reports (for example, see Patent Document 1), and the following results were obtained.
1. Color reaction Anthrone reaction: positive, carbazole reaction: positive, Elson-Morgan reaction: negative, iodine reaction: positive Solubility in solvents Soluble in water, insoluble in ethanol, ether, acetone, etc.
3. Color and shape The purified product is white cotton or fiber.
4). Viscosity The aqueous solution is colorless and transparent and has a viscosity. The viscosity of the 1% aqueous solution is 1200 to 2000 (25 ° C., 30 rpm).
5). Elemental analysis value C = 34.3 ± 1%: H = 4.8 ± 1%: N = 0%: Ash content = 3.0 ± 1.0%
6). Specific rotation The specific rotation [α] of this substance at 27 ° C. with respect to the sodium D line: 0 to + 20 ° (C = 0.33, aqueous solution)
7). Melting point Brown changes at 100 ° C, black brown changes at 180 ° C, and decomposes at 250 ° C.
8). Infrared absorption spectrum As shown in FIG.

(2) Determination of Constituent Sugar The sugar composition of AX-1 prepared by the above method was analyzed with reference to the method described in a previous report (for example, see Non-Patent Document 3).

  That is, 100 μl of 2N trifluoroacetic acid was added to 1 mg of lyophilized AX-1 and heated at 120 ° C. for 5 hours for hydrolysis. The obtained hydrolyzate was dried under reduced pressure to remove trifluoroacetic acid, and then subjected to sugar analysis. The sugar analysis was performed using HPLC (High Performance Ion Exchange Chromatography) (manufactured by Shimadzu Corporation). The chromatogram of sugar analysis is as shown in FIG.

  The sugar type was identified from the peak retention time of the chromatogram obtained as a result. From the results of the above analysis, it was found that the main components of AX-1 were glucose, galactose, mannose, and glucuronic acid.

(3) Molecular weight analysis The molecular weight of AX-1 was analyzed by the following GPC (Gel permeation chromatography) analysis method. The GPC analysis conditions were as follows.

[GPC analysis conditions]
・ Apparatus: HLC-8220 (manufactured by Tosoh Corporation)
-Column; TSKgel Super AWM-H (6 mm x 15 cm) x 2 (made by Tosoh Corporation)
Eluent: 5 mM lithium bromide / dimethyl sulfoxide Flow rate: 0.6 ml / min
Sample concentration: 1 mg / ml
・ Injection volume: 10 μl
Column temperature: 40 ° C
・ Reference material: Pullulan

  The molecular weight of AX-1 analyzed by the above method was about 2.5 million.

(4) Structural analysis The structural analysis of AX-1 was analyzed by ¹ H-NMR (Nuclear Magnetic Resonance) method and methylation analysis method.

The NMR measurement conditions were as follows.
・ Apparatus: JNM-A500 (manufactured by JEOL Ltd.)
・ Wavelength: 500MHz
・ Solvent; D ₂ O
Sample concentration: 10 mg / ml (D ₂ O substitution × 2)
・ Measurement temperature: 80 ℃

The ¹ H-NMR obtained by the above method is as shown in FIG. The main peak of the anomeric region was assigned as follows. That is, 5.25 ppm: → 3) -α-Man- (1 →, 4.52 ppm: → 3,4) -β-Glu- (1 → 4.47 ppm: → 4) -β-Glu- (1 → 4.44 ppm: β-GlucA- (1 →. From the above, it was found that the main chain has a structure similar to Acetane (for example, “Carbohydrate Research”). 269, No. 2, p.319-331, 1995).

  In order to analyze the side chain structure, methylation analysis was performed (for example, refer nonpatent literature 1). As a result, β-Glu (1 → at the site of RT13.5 → 6) -β-Glu (1 → at the site of retention time (RT) 9.13 as the main peak → 6 at the site of RT14.9. ) -Β-Gal (1 → was observed at a ratio of approximately 1.6: 2.0: 1.

  As a result, it was found that glucose was present at the end of the side chain, and glucose and galactose were present in a ratio of 2: 1 between glucuronic acid. The primary structure of AX-1 clarified from the above results is as shown in Chemical Formula 1.

In Chemical Formula 1, Glu represents glucose, Man represents mannose, Gal represents galactose, and Gluc represents glucuronic acid.

(Chemical formula 1)
→ 4) -β-D-Glu- (1 → 4) -β-D-Glu- (1 →
3
↑
1
α-D-Man
2
↑
1
β-D-GlucA
4
↑
1
α-D-Glu
6
↑
1
β-D-Glu
6
↑
1
β-D-Gal
6
↑
1
β-D-Glu

  As a result, it was confirmed that the constituent sugar ratio of AX-1 was glucose: galactose: mannose: glucuronic acid = 5: 1: 1: 1. However, the acidic heteropolysaccharide having such a constituent sugar ratio was confirmed. Has not been reported so far, and it was confirmed that the acidic heteropolysaccharide of the present invention is novel from the aspect of the constituent sugar ratio.

Furthermore, there was no previous report on the structure of AX-1 (chemical formula 1) analyzed by ¹ H-NMR, and it was confirmed that AX-1 is a novel acidic heteropolysaccharide.

  The above novel acidic heteropolysaccharide AX-1 can be expected to exhibit excellent effects in application to foods and beverages and applications focusing on pharmacological action.

It is a figure which shows the infrared absorption spectrum of the novel acidic heteropolysaccharide AX-1 of this invention. It is a figure which shows the chromatogram of the ion exchange chromatography which detected the component saccharide | sugar of novel acidic heteropolysaccharide AX-1 of this invention. It is a figure which shows the spectrum of < ¹ > H-NMR which analyzed the structure of novel acidic heteropolysaccharide AX-1 of this invention.

Claims (4)

Acid heteropolysaccharide AX-1 having the following physicochemical properties.
(1) An acidic heteropolysaccharide having glucose, galactose, mannose and glucuronic acid as main constituents and a constituent sugar ratio of glucose: galactose: mannose: glucuronic acid = 5: 1: 1: 1.
(2) The primary structure is as shown in Chemical Formula 1.
(Chemical formula 1)
→ 4) -β-D-Glu- (1 → 4) -β-D-Glu- (1 →
3
↑
1
α-D-Man
2
↑
1
β-D-GlucA
4
↑
1
α-D-Glu
6
↑
1
β-D-Glu
6
↑
1
β-D-Gal
6
↑
1
β-D-Glu   The acidic heteropolysaccharide AX-1 producing bacterium belonging to the genus Acetobacter is cultured, and the acidic heteropolysaccharide AX-1 is collected from the culture, The acidic heteropolysaccharide according to claim 1, A manufacturing method of AX-1.   3. The method according to claim 2, wherein the acid heteropolysaccharide AX-1 producing bacterium belonging to the genus Acetobacter is Acetobacter xylinum.   The method according to claim 3, wherein the Acetobacter xylinum is Acetobacter xylinum NCI1005 strain (FERM BP-10415).

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