JPH06284896A - Production of polyphenol glycoside - Google Patents

Abstract

(57) [Summary] [Objective] To provide a method for producing an enzyme of polyphenol glycoside, which is useful as an intermediate or a compounding ingredient of medicines and cosmetics. A method for producing a polyphenol glycoside, which comprises reacting a sugar substrate with a polyphenol receptor in the presence of a saccharified amylase having no cyclodextrin synthesizing ability and capable of degrading maltooligosaccharide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a polyphenol glycoside using a saccharified amylase having no cyclodextrin synthesizing ability and malto-oligosaccharide degrading ability.

[0002]

BACKGROUND OF THE INVENTION Polyphenol glycosides have hitherto been used not only as sweeteners, analgesics, laxatives, antimalarials and tonics, but also as a blending component of cosmetics which exhibit an excellent whitening effect. Available (Japanese Patent Application No. 3-341)
The present applicant has previously provided a method for producing a polyphenol glycoside using a novel enzyme that does not have cyclodextrin synthesizing ability and maltose decomposing ability (Japanese Patent Application No. Hei 4). -27926 specification).

The present invention has been made to provide a method for producing a polyphenol glycoside using a different enzyme.

[0004]

[Means for Solving the Problems] That is, according to the present invention, among saccharified amylases which have not been industrially used in the past, an enzyme having a cyclodextrin synthesizing ability and a maltooligosaccharide degrading ability is a polyphenol derivative. It was made based on the finding that it has a glycosynthetic ability, and the gist thereof is, in the presence of a glycosylated amylase that does not have a cyclodextrin synthetic ability and has a maltooligosaccharide degrading ability, a sugar substrate and a polyphenol receptor. In a method for producing a polyphenol glycoside.

The enzyme used in the present invention, that is, the saccharified amylase having no cyclodextrin synthesizing ability and malto-oligosaccharide degrading ability is an enzyme known per se, but it is said that the enzyme has polyphenol glycoside synthesizing ability. That was not known at all. Examples of this type of saccharified amylase include Bacillus subtilis, Streptococcus bovis, Streptomyces hygroscopicus, Streptomyces plecox, and Rhizopus.
Examples are enzymes having the above-mentioned properties, which are produced from bacteria such as Dermer and Aspergillus niger.

The preparation of the saccharified amylase using the above-mentioned bacteria is known per se, for example, the method of Fukumoto et al. [Proceedings of Japan Academy (Proc. J.
apan Academy), 27, 352-358 (1)
951) and Amylase Symposium (Japan Applied Enzymes Association, Amylase Section), pp. 47-53 (1965).
Year))] etc.

Glycated amylase having the above characteristics is
It hydrolyzes a wide range of sugar substrates and transfers glucose to various polyphenols, resulting in a wide variety of polyphenol glycosides. The sugar substrate and polyphenols receptors of this kind are given below: sugar substrate: starches, amylopectin, amylose, maltooligosaccharides (G ₂ ~G _7). Polyphenol receptors : catechin, caffeic acid, kojic acid, hydroquinone, catechol, resorcinol, protocatechuic acid, α-resorcylic acid, phloroglucinol, gallic acid.

The method of reacting a sugar substrate such as starch and a polyphenol acceptor such as catechin in the presence of the above-mentioned enzyme is usually a p-type reaction system using a buffer such as an acetate buffer.
H is adjusted to about 4 to 9 and is performed at about 10 to 60 ° C. for about 3 to 70 hours. Water, methanol / water as the reaction solvent
(5 to 50% by volume), ethanol / water (5 to 50% by volume),
Ethyl acetate / water (10 to 80% by volume) and the like are exemplified. The step of removing the enzyme from the produced polyphenol glycoside can be omitted by immobilizing the enzyme to be used on an insoluble carrier.

[0009]

EXAMPLES The present invention will be described below with reference to examples. Example 1 The enzyme was prepared according to the method of Fukumoto et al. Soluble starch 10 w / v%, polypeptone 2 w / v
%, An aqueous solution containing 2% w / v meat extract at 121 ° C.
The medium was prepared by autoclaving for 5 minutes. 100 ml of the medium was put into a Sakaguchi flask (500 ml), and then 1 platinum loop of Bacillus subtilis 1FO 14140 was inoculated, and the mixture was shake-cultured at 30 ° C. for 24 hours (seed culture). The culture solution was mixed with 3 liters of the medium newly prepared as described above, and the mixture was stirred in a mini jar (5 liters) at 200 rpm.
And under the condition of aeration rate of 3 liter / min, 3
The cells were cultured with aeration and stirring at 7 ° C. for 120 hours (main culture). Centrifuge 2.5 liters of culture solution (9000r
pm: 15 minutes), ammonium sulfate was added to 2.45 liters of the culture supernatant obtained by
A 0.6-saturated fraction was obtained according to a conventional method. The fraction was dialyzed against water and the pH was adjusted to 5.0 with 1M acetic acid.
It was injected into a Duolite C-10 column (15.9 cm ² × 27 cm) equilibrated with 0 mM acetate buffer (pH 5.0), and the column was washed with 2 liters of the acetate buffer. An ultrafiltration membrane having a molecular weight cut off of 3000 (Pencil type module SEP-0013 manufactured by Asahi Kasei Kogyo Co., Ltd.)
Was concentrated to 150 ml to obtain a partially purified enzyme.
The results of the above purification process are summarized in Table 1.

[0010]

[Table 1]

The activity amount (unit) in Table 1 is a value calculated by the following method. An active amount of soluble starch was added to 50 mM sodium acetate buffer (pH 5.
3). 0.45 solution dissolved at a concentration of 0.5 w / v%
After adding 0.05 ml of enzyme solution to ml and reacting at 40 ° C. for 10 minutes, the reaction was stopped by adding 1.0 ml of 0.5N hydrochloric acid, and then 5 mg of iodine and 50 mg of potassium iodide were added to 100 ml of water. 2.5 ml of the solution dissolved in was added and left at room temperature for 20 minutes, then 660n
The absorbance at m was measured, and the amount of enzyme that reduced the absorbance by 1% per minute was defined as 1 unit (a blank test was performed by the same operation as above except that the buffer solution was used instead of the enzyme).

Example 2 (Method for measuring polyphenol glycoside synthesis activity) 0.5 ml of a solution of hydroquinone in ethyl acetate (0.5 M)
And soluble starch 5 w / v%, sodium acetate buffer
(pH 5.3) 50 mM and enzyme 1 produced in Example 1
Test tube with a lid containing 0.5 ml of an aqueous solution containing 0 unit / ml
(5 ml), and the test tube was shaken at 40 ° C. for 18 hours under the condition of a rotation speed of 280 rpm. After standing, the aqueous layer was subjected to thin layer chromatography analysis under the following conditions to confirm the polyphenol glycoside synthesis activity of the enzyme. Thin layer: Silica gel 60F254 glass plate manufactured by Merck & Co., Inc. Developing solvent: ethyl acetate / acetic acid / water = 3/2/2 (volume ratio) Detection: 33 v / v% sulfuric acid / methanol mixed solution is sprayed on the thin layer, and then the thin layer Is heated at 120 ° C. for 10 minutes. Rf value: 0.61 to 0.86 (Rf value of the decomposed product of starch is 0.58 or less)

Examples 3 and 4 The polyphenol glycoside synthesizing activity of the enzyme of the present invention was confirmed according to the procedure of Example 2 except that amylopectin, amylose or maltooligosaccharide was used instead of starch.

Examples 5 to 13 (Receptor Specificity) Catechin, caffeic acid, kojic acid, catechol, resorcinol, protocatechuic acid, α-resorcylic acid, phloroglucinol or gallic acid is used instead of hydroquinone. Other than the above, according to the procedure of Example 2,
The activity of the enzyme according to the present invention to synthesize polyphenol glycoside was confirmed.

Example 14 (Preparation of hydroquinone glycoside) 1,000 units of enzyme solution prepared in Example 1, 2.5 w / v% hydroquinone, 2 w / v% starch and acetate buffer
A hydroquinone glycoside was synthesized by putting 100 ml of a mixed solution of 10 mM (pH 5.3) in an Erlenmeyer flask (200 ml) and performing a reaction at 40 ° C. for 90 hours. After 200 ml of ethanol was added to the reaction mixture to stop the reaction, the precipitated insoluble matter was removed by centrifugation. The residual liquid was subjected to vacuum concentration and drying treatment using a rotary evaporator, then the residue was dissolved in 10 ml of water, and the aqueous solution was subjected to column chromatography treatment under the following conditions to obtain a reaction product with an eluent volume of 440 to 480 ml. 2, the reaction product 1 was eluted with the same volume of 480 to 540 ml: column: inner diameter 2.6 cm x length 91 cm packing material: biogel p-2 (400 mesh) eluent: 5% (v / v) ethanol Aqueous solution The obtained fractions were concentrated to dryness under reduced pressure using a rotary evaporator, and then dried using a vacuum dryer to obtain product 1 as a white powder.
10 mg and 51 mg of the product 2 were obtained. The product was single in thin layer chromatography under the following conditions. (Rf value of product 1: 0.61, Rf of product 2
Value: 0.52): Thin layer: Silica gel 60F254 Developing solvent: Ethyl acetate / acetic acid / water (3: 1: 1) Detection: UV irradiation or 110-120 ° C after spraying nitric acid / methanol (1: 2) mixture Heat to develop color. As a result of ¹ H-NMR analysis, the products 1 and 2 were identified as hydroquinone-O-α-D-glucopyranoside and hydroquinone-O-α-maltoside, respectively.

[0016]

INDUSTRIAL APPLICABILITY According to the present invention, a wide range of sugar substrates and polyphenol acceptors are used as raw materials by utilizing a specific saccharified amylase which has not been used industrially in the past. It is possible to efficiently produce a wide variety of polyphenol glycosides useful as a compounding ingredient of

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Toyokazu Nishino 14-5 Shimokita Town, Neyagawa City, Osaka Kurashiki Spinning Co., Ltd.

Claims (4)

[Claims] 1. No cyclodextrin synthesizing ability,
A method for producing a polyphenol glycoside, which comprises reacting a sugar substrate with a polyphenol acceptor in the presence of a saccharified amylase capable of degrading maltooligosaccharides. 2. The saccharified amylase is an enzyme produced by Bacillus subtilis, Streptococcus bovis, Streptomyces hygroscopicus, Streptomyces plecox, Rhizopus dermer or Aspergillus niger. the method of. 3. The method according to claim 1, wherein the sugar substrate is starch, amylose, amylopectin or maltooligosaccharide (G _{2 to} G ₇ ). 4. The polyphenol receptor is catechin, caffeic acid, kojic acid, hydroquinone, catechol, resorcinol, protocatechuic acid, α-resorcylic acid,
The method according to claim 1, which is phloroglucinol or gallic acid.