JPH07236496A - Production of kojic acid glucoside and production of kojic acid monoglucoside - Google Patents

Abstract

PURPOSE:To obtain a kojic acid glucoside having antibacterial activity, oxidation preventing function and discoloration preventing function, exhibiting high stability and useful for foods, cosmetics, chemicals, etc., by treating kojic acid and a glucan having alpha-1,4 bond with an amylase X-23. CONSTITUTION:This kojic acid glucoside having excellent solubility in water and stability to heat, light and alkali compared with kojic acid, exhibiting antibacterial activity, oxidation preventing function, discoloration preventing function, chelating action and polyphenyl oxidase inhibiting activity, useful for foods, cosmetics, chemicals, etc., and expressed by formula can be produced in high glucosylation ratio by dissolving kojic acid and a glucan having alpha-1,4 bond (e.g. maltopentaose) in 20mM acetic acid buffer solution (pH4.0), adding amylase X-23 to the solution and reacting at 40 deg.C for 16hr.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing kojic acid glycoside and a method for producing monoglucoside kojic acid.

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION Kojic acid has useful properties such as antibacterial activity, antioxidant function, chelating action and polyphenol oxidase inhibitory activity. In recent years, the discoloration preventing function of kojic acid has been utilized. Further, the function of suppressing the production of melanin is also used in cosmetics, and is commercialized as a whitening cosmetic.

However, the use of kojic acid is limited because of its very low stability to light, heat and alkali, and its low solubility in water. Therefore, it has been desired to improve these properties.

It has been reported that kojic acid glycosides can be obtained by synthesis by an organic chemical method, but for application to foods, cosmetics, pharmaceuticals, etc., glycosides with high safety and high purity The production by a biochemical method (enzymatic reaction) capable of obtaining is desirable. However, only one example of the enzymatic glycosylation method of kojic acid has been reported. According to the report (Japanese Patent Application Laid-Open No. 5-39298), Streptomyces griseus (Streptt)
omyces griseus), maltotriose-producing enzyme derived from Bacillus subtilis, Pseudomonas stutzeri,
Pseudomonas
as saccharophila), Bacillus circulans
And other maltohexaose-forming enzymes, Bacillus licheniformis
rmis), Bacillus subtilis
subtilis), Bacillus cereus (Baci)
Klebsie, a maltopentaose-forming enzyme from L. cereus
Lla pneumoniae), Bacillus subtilis, Bacillus circulans F-2 (Bacillus cir)
culans F-2), Bacillus circulans G
-6 (Bacillus circulans G-
6) The maltohexaose synthase derived from 6) was used. According to the same report (Japanese Patent Application Laid-Open No. 5-39298), the saccharification rate (percentage of kojic acid glycosidated in the added kojic acid) was about several percent, which was not practical.

The object of the present invention is to produce a kojic acid glycoside by a biochemical method with a higher glycosylation rate than the conventional techniques.

[0005]

[Means for Solving the Problems]

In order to solve this problem, the inventor of the present invention used amylase X-23 or glycated α-amylase derived from bacteria (hereinafter, abbreviated as BSA) as an enzyme (hereinafter, amylase X-23 and The glycated α-amylase derived from bacteria is collectively called the enzyme group of the present invention).
Amylase X-23 is, for example, Bacillus subtilis (B
and those produced by A. subtilis) X-23 (Deposit No. P-13560 of the Institute of Microbiology, Japan). The BSA is, for example, one produced by the IFO 14140. Kojic acid was used as an acceptor for acting the enzyme group of the present invention, and glucan having an α-1,4 bond was used as a donor. In the present specification, the glucan having α-1,4 bond refers to maltooligosaccharide, amylose, amylopectin, various starches and the like.

Kojic acid (preferably having a concentration of 1 in the reaction solution)
Amylase X-23 or BSA was added to a solution containing glucan having an α-1.4 bond (preferably the concentration in the reaction solution was 5 to 30%), and the reaction was carried out at normal temperature and time. Let The reaction pH may be pH 3 to 11 at which the enzymatic reaction is carried out, and preferably pH 3.5 to pH 5 at which coloring of the reaction solution due to oxidation of kojic acid during the reaction and decomposition of kojic acid itself are less likely to occur. The reaction solution after the reaction contains kojic acid glycoside. By purifying this reaction solution by chromatography, a purified preparation of kojic acid glycoside can be obtained. The kojic acid glycoside mentioned here is composed of kojic acid monoglucoside and kojic acid oligoglucoside (kojic acid diglucoside, kojic acid triglucoside, kojic acid tetraglucoside, etc.).

Amylase X-23 has the following physicochemical properties.

1. Action: Amylase X-23 is α-1.
Glucans having 4 bonds are decomposed (hydrolyzed), and maltooligosaccharides (glucose, maltose, and maltotriose) below maltotriose are accumulated. When the reaction is continued for a longer time, maltotriose is decomposed into glucose and maltose. Phenol related compounds or flavonoid related compounds, kojic acid and α-
If glucan with 1.4 bond is present at the same time, α-
It has the property of decomposing glucan having a 1.4 bond and performing glycosylation by α bond to the OH group in the acceptor of phenol analog, flavonoid analog, or kojic acid. When compared with BSA, amylase X-23 has a similar action, but is greatly different in various stability (stability against pH and heat).

2. Receptor specificity Phenolic OH group or flavone in phenol related compounds such as hydroquinone and dimethoxyphenol,
Glycosylation is carried out by α-bonding to the OH group in flavonoid-related compounds such as isoflavone, flavonol, flavanone, flavanonol, catechin, aurone, anthocyanidin, chalcone and dihydrochalcone, or to the alcoholic OH group in kojic acid.

3. Optimum pH and stable pH When the soluble starch is hydrolyzed, the optimum pH is pH6.
-7 and the stable pH is pH 5-8 (Fig. 1).

4. Titer assay method (α-amylase activity assay method) To 50 μl of enzyme solution incubated at 40 ° C., 200 μl of 1.5% soluble starch (pH adjusted to 5.5 with 40 mM acetic acid-Na buffer solution) incubated at 40 ° C. was added. Four
Incubate for 10 minutes at 0 ° C. The amylase reaction is stopped by adding 2.0 ml of a 5: 1 mixture of 0.5N acetic acid and 0.5N hydrochloric acid to the reaction mixture and stirring. 0.1 ml of this solution is sampled, 5 ml of a solution containing 0.005% iodine and 0.05% potassium iodide is added, and the mixture is stirred and allowed to stand at room temperature for 20 minutes. 6 of this solution
The absorbance at 60 nm is measured, and this absorbance is designated as C. Separately, as a blank, the absorbance obtained by adjusting the reaction system using purified water instead of the above enzyme solution and performing the same operation is designated as B. From C and B thus obtained, the amylase activity A is obtained by the following formula. A = (B−C) / B × 10 However, 1 unit of amylase activity is assumed to be 10% of B value.

(Method for measuring amount of glycoside produced) Donor 10 such as soluble starch or maltopentaose
%, And a substrate containing 2% of kojic acid as an acceptor, and the enzyme solution were mixed at 1: 1 and reacted at 40 ° C. for 16 to 24 hours. The reaction solution was diluted 15-fold and subjected to HPLC (TSK
-GELG-2500PW Tosoh, 0.2M
Analysis was carried out with an eluent containing NaCl at pH 4.0). Detection is by absorbance at 280 nm. As a control, a reaction system using purified water instead of the enzyme solution is prepared and the same operation as above is performed.

(Method of measuring glycosylation rate) As described above, the percentage of kojic acid glycosylated in the kojic acid added to the reaction solution is hereinafter referred to as the glycosylation rate. The glycosidation rate is calculated by the following calculation formula from the results obtained by the above HPLC analysis. Glycosylation rate = peak area of glycoside /
Control receptor peak area x 100

5. Optimum temperature range for action Stable glycosylation at 30 ° C to 70 ° C at pH 5.5. (Fig. 2)

6. Inhibitors Copper ion and zinc ion are inhibited, but EDTA, calcium ion, manganese ion, barium ion, magnesium ion and cobalt ion are hardly inhibited respectively (FIG. 3).

7. Conditions of deactivation: Complete deactivation when treated at 100 ° C for 15 minutes.

8. Purification method Bacillus subtilis
After removing the cells from the culture solution of is) X-23 (Deposit P-13560, Microtechnology Research Institute), salting out with ammonium sulfate (80% saturation) is performed to dissolve the resulting precipitate, and dialysis is performed. This was subjected to Q-Sepharose column chromatography (pH
Dialysis is carried out after a 0.5 to 0.5 M NaCl gradient of 5.5). Then, Phenyl Sepharose column chromatography (0.8M-0M ammonium sulfate concentration gradient)
After dialysis, dialysis is performed. The dialyzed solution is subjected to gel filtration with Superose 12 and freeze-dried to obtain a purified sample. The purified preparation shows a single band on SDS-polyacrylamide gel electrophoresis.

9. Molecular weight: Approximately 65,000 when measured by SDS-polyacrylamide gel electrophoresis.

10. Ultraviolet absorption spectrum The maximum absorption is 267 nm and the minimum absorption is 252 nm (FIG. 4).

11. Amino acid analysis The amino acid composition is shown in FIG.

BSA has the following physicochemical properties.

1. Action: The action of BSA is HAND B
OOK OF AMYLASE AND RELATE
D ENZYMES; Edited by The A
mylize Research Society o
f Japan. That is, α-1.
Glucans having 4 bonds are decomposed (hydrolyzed), and maltooligosaccharides (glucose, maltose, and maltotriose) below maltotriose are accumulated. When the reaction is continued for a longer time, maltotriose is decomposed into glucose and maltose. The transfer reaction is further described. Phenol-related compounds or flavonoid-related compounds, substances that can serve as receptors such as kojic acid and α-
If glucan with 1,4 bond is present at the same time, α-
1.4 Decomposes a glucan having a bond, and performs glycosylation by α bond to the OH group of a phenol analog compound, a flavonoid analog compound and kojic acid which are acceptors.

2. Receptor specificity The OH group in the phenolic analog compound such as hydroquinone and dimethoxyphenol or the OH group in the flavonoid analog compound such as flavone, isoflavone, flavonol, flavanone, flavanonol, catechin, aurone, anthocyanidin, chalcone and dihydrochalcone is α Glycosylation is performed by binding.

3. Optimum pH and stable pH When the soluble starch is hydrolyzed, the optimum pH is pH 5.
3 and the stable pH is pH 4.5 to 9.2. When glycosylating hydroquinone, the optimum pH is pH 6 and the stable pH is pH 5-8.

4. Titer assay The same as the titer assay for amylase X-23.

5. Optimum temperature range for action Stable glycosylation at 30 to 60 ° C at pH 5.5.

6. Inhibitors Copper and zinc ions were inhibited, but EDTA, calcium ion, manganese ion, barium ion, magnesium ion and cobalt ion were hardly inhibited. Thus, it was stable against various inhibitors.

7. Conditions of deactivation: Complete deactivation when treated at 100 ° C for 15 minutes.

9. It has a molecular weight of about 47,000. 10. Purification method The same as the purification method for amylase X-23.

The means for obtaining kojic acid monoglucoside from kojic acid glycoside is as follows. When a reaction solution containing kojic acid oligoglucoside and kojic acid monoglucoside produced by reacting the enzyme group of the present invention with a solution containing kojic acid and a donor is allowed to act with glucoamylase by a conventional method, only kojic acid monoglucoside is accumulated. To do.

[0032]

[Action]

1, the enzyme group of the present application is an enzyme group reported in JP-A-5-39298, namely, maltotriose-producing enzyme (enzyme that hydrolyzes a substrate to an exo type to produce maltotriose, H
AND BOOK OF AMYLASE AND R
ELATED ENZYMES; Edited by T
he Amylase Research Society
ty of Japan), maltohexaose-forming enzyme (HANDBOOK OF AMY, an enzyme that hydrolyzes a substrate into an exo form to produce maltohexaose)
LASE AND RELATED ENZYMES;
Edited by The Amylase Res
(See "Each Society of Japan") and maltopentaose synthase (soluble starch,
It does not act on maltooligosaccharides that have a lower degree of polymerization than maltopentaose, by hydrolyzing amylose and the like to form maltopentaose. HAND BOOK OF AM
YLASE AND RELATED ENZYMES;
Edited by The Amylase Res
(See "Each Society of Japan")
Is a type of α-amylase having a completely different action.
The enzyme group of the present invention decomposes (hydrolyzes) glucan having an α-1.4 bond, and accumulates maltooligosaccharides (glucose, maltose, and maltotriose) below maltotriose. When the reaction is continued for a longer time, maltotriose is decomposed into glucose and maltose.

2, Decomposition of Starch The enzyme group of the present invention hydrolyzes starch to produce glucose, maltose, maltotriose, and branched oligosaccharides.

3, Sugar transfer The enzyme group of the present invention decomposes α-1.4 glucan in the reaction solution, and at the same time, the phenolic OH group in the reaction solution, the OH group in the flavonoid-related compound and the OH group of kojic acid. To sugar α
Transfer by binding.

4. PH for Carrying out Glycosylation Reaction In neutral and alkaline conditions, it is desirable to carry out the reaction in an acidic region because kojic acid is oxidized and colored. Considering the transfer activity of the enzymes of the present invention and the influence of pH (Fig. 7), pH 3.5
Thus, if the reaction is carried out at pH 5, the glycosylation rate is high and the coloring due to the oxidation of kojic acid can be effectively prevented.

4, When glucoamylase is allowed to act on kojic acid oligoglucoside, it completely hydrolyzes the oligosaccharide transferred to kojic acid to glucose to produce only kojic acid monoglucoside.

5. Properties of kojic acid monoglucoside (1) Structure of kojic acid monoglucoside The purified sample of kojic acid monoglucoside obtained by the present invention is treated with α-glucosidase (manufactured by TOYOBO). When the HPLC analysis shown in the method for measuring the glycosidation rate was performed, the peak of kojic acid monoglucoside disappeared completely, and the peak of kojic acid newly appeared. It was found that glucose is an α-linked glycoside. Furthermore, the structure of kojic acid monoglucoside was analyzed by NMR.
(JEOL JNM-GX270), the same results as above were confirmed. The results of NMR analysis and the structure of kojic acid monoglucoside are shown in FIGS. 8 and 9.

(2) Solubility in water Monoglucoside kojic acid was added to 2.5 ml of 1.0 ml of water.
× 10 ⁻³ mol was dissolved. Kojic acid is 2.9 × 10
^{Only -4} mol dissolved. That is, it was about 10 times larger than that of kojic acid.

(3) Stability to light When the 0.12 molar solution was allowed to stand in the sunlight for 5 days, almost no coloration was observed. This coloring degree is 400n
When it was measured by the absorbance of m, it was reduced to about 25% as compared with the case of kojic acid. This effect was even more remarkable in the alkaline solution.

(4) Absorption in the ultraviolet region As a result of measuring the total absorption in the ultraviolet region, it was effective as an ultraviolet absorber like kojic acid (FIG. 10).

(5) Inhibitory effect on polyphenol oxidase (tyrosinase) It was effectively inhibited as much as kojic acid (Fig. 11).

[0042]

Example 1 Kojic acid was dissolved in 20 mM acetate buffer (pH 4.0) so as to be 5.0% (weight / weight) and maltopentaose 10.0% (weight / weight). Amylase X-containing 60 units of amylase activity was added to 10 ml of this solution.
10 ml of 23 solution was added. The reaction solution was reacted at 40 ° C. for 16 hours. The glycosidation rate when the reaction was carried out under these conditions was 32%. If necessary, this reaction solution is hydrolyzed to oligosaccharides transferred to kojic acid by glucoamylase treatment and oligosaccharides as hydrolysis products to give glucose, and then Sephadex G-10
Unreacted kojic acid and kojic acid glycosides were fractionated by gel filtration using a resin, and the glycosides were adsorbed on an activated carbon column equilibrated with purified water, and then adsorbed on the activated carbon column with 10% methanol. After the unreacted sugar adsorbed was eluted, the glycoside was eluted with 60% methanol. 60%
The methanol fraction was dried under reduced pressure, dissolved again in purified water and freeze-dried. By the above operation, about 270 mg of monoglucoside kojic acid could be obtained.

Further, after 6 to 15 hours from the start of the reaction, maltopentaose as a new donor was added to the above reaction solution to further increase the glycosidation rate. When 6 hours after the start of the reaction, maltopentaose was added so as to be 5%, the glycosylation rate was about 43%.

[0044]

Example 2 Kojic acid was dissolved in 20 mM acetate buffer (pH 4.0) so as to be 5.0% (weight / weight) and maltopentaose 10.0% (weight / weight). To 10 ml of this solution, 10 ml of a BSA solution having 60 units of amylase activity was added. The above reaction solution was subjected to 16 at 40 ° C.
Reacted for hours. The glycosidation rate when the reaction was carried out under these conditions was 31%. If necessary, this reaction solution is completely hydrolyzed to oligosaccharides transferred to kojic acid by a glucoamylase treatment and oligosaccharides of hydrolysis products to glucose, and then subjected to gel filtration using Sephadex G-10 resin, Unreacted kojic acid and kojic acid glycoside were fractionated. Further, the glycoside was adsorbed on the activated carbon column equilibrated with purified water, and then the unreacted sugar adsorbed on the activated carbon column was eluted with 10% methanol.
The glycoside was eluted with methanol. The 60% methanol fraction was dried under reduced pressure, then dissolved again in purified water and freeze-dried. About 240 mg of monoglucoside kojic acid could be obtained by the above operation.

Further, after 6 to 15 hours from the start of the reaction, maltopentaose as a donor was added to the above reaction solution to further increase the glycosidation rate. When 6 hours after the start of the reaction, maltopentaose was added so as to be 5%, the glycosidation rate was about 42%.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, it was possible to obtain a kojic acid glycoside with a glycosylation rate of 30% or more in a usual batch reaction. It was also possible to further increase the glycosidation rate by adding a donor during the reaction. According to the present invention, since kojic acid can be glycosylated with a highly safe enzymatic reaction at a high glycosylation rate, a large amount of kojic acid glycoside can be industrially obtained at a low cost. The kojic acid glycoside obtained as described above is superior to kojic acid in solubility in water and stability to heat, light, or alkali. Therefore, by using the kojic acid glycoside obtained by the present invention alone or in combination with other components, cosmetics, food and drink, luxury items, pharmaceuticals, antioxidants, stabilizers, preservatives, browning inhibitors, chelates. It can be advantageously used as an agent or the like. Among them, when used in cosmetics due to the ultraviolet absorption, polyphenol oxidase inhibitory and chelating effects of kojic acid glycoside, it is advantageously used as a whitening agent due to its effects of preventing ultraviolet rays, sunburn, and preventing the formation of spots and freckles. can do. In addition, since it has the above-mentioned action, when applied to food and drink,
It can be advantageously used to prevent browning of food during the manufacturing, storage and transportation stages.

[Brief description of drawings]

FIG. 1 shows the optimum pH and stable pH of amylase X-23 when soluble starch was used as a substrate.

FIG. 2 shows the optimum temperature and thermostability of amylase X-23 at pH 5.5.

FIG. 3 shows the effect of amylase X-23 on various inhibitors.

FIG. 4 shows an ultraviolet absorption spectrum of amylase X-23.

FIG. 5 shows the results of amino acid analysis of amylase X-23.

FIG. 6 shows Amylase X-23 Superose 1
The elution curve of 2 column chromatography is shown.

FIG. 7 shows the relationship between the transglycosylation activity of amylase X-23 and BSA and pH.

FIG. 7 shows ¹³ C-, ¹ H-NMR analysis of kojic acid glycoside obtained by the action of amylase X-23.

FIG. 8 shows the structure of kojic acid glycoside obtained by the action of amylase X-23 or amylase BSA.

FIG. 9 shows ultraviolet absorption of kojic acid glycoside and kojic acid.

FIG. 10 shows the inhibitory effect of kojic acid glycoside and kojic acid on polyphenol oxidase (tyrosinase).

Claims (4)

[Claims] 1. A method for producing a kojic acid glycoside, which comprises reacting amylase X-23 with a glucan having an α-1.4 bond with kojic acid. 2. A method for producing a kojic acid glycoside, which comprises allowing a glycated α-amylase derived from a bacteria to act on a glucan having an α-1.4 bond with kojic acid. 3. The method for producing kojic acid glycoside according to claim 1 or 2, wherein the method is operated at pH 3.5 to pH 5. 4. A process for producing monoglucoside of kojic acid, which comprises reacting kojic acid glycosides obtained in claim 1 and claim 2 with glucoamylase and then purifying by chromatography.