JPH09224693A - Production of 1-menthyl-alpha-d-glucopyranoside by enzyme reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the above safe less-expensive compound which is water soluble and capable of expressing mint flavor characteristics to menthol by an enzymatic transfer reaction of a saccharide using an enzyme, which hydrolyzes an α-1,4-glucoside bonding at a nonreducing terminal of saccharide. SOLUTION: This 1-menthyl-α-D-glucopyranoside having high water solubility is easily produced by a transfer reaction of a saccharide with an enzyme as follows. 1-Menthol is placed into a test tube, an enzyme (e.g., α-glucosidase) hydrolyzing an α-1,4-glucoside bond at a nonreducing terminal of saccharide and a substrate (e.g., maltose) of the enzyme are added to the menthol, and these compounds are made to react with each other in ethanol at 40 deg.C for 12hr under stirring. Although the obtained 1-menthyl-α-D-glucopyranoside itself does not have a mint flavor characteristic to menthol, it is hydrolyzed by various kinds of carbohydrase, acid, etc., to express fresh mint flavor characteristic to menthol and useful as a sustaining and stabilizing agent of fresh feeling in various kinds of foods, mouth refrigerants, fragrance inhaling taste-improving agents for tobacco, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to l-menthyl-α.
-A method for producing D-glucopyranoside.

[0002]

2. Description of the Related Art Menthol is widely used for medicinal purposes, food applications, toothpaste, and other oral fresheners because of its light flavor with a unique refreshing sensation. However, menthol has a problem that its sublimation property causes a decrease in flavor of light load with the passage of time, and since it is poorly soluble in water, it is conventionally mixed as solid particles or suspended using an emulsifier. It had to be carried out either by making it cloudy or by dissolving it in an organic solvent such as alcohol.

In order to overcome these problems, the use of menthol glycoside has been proposed [Japanese Patent Publication No. 51-105]. This menthol glycoside has a structure in which oligosaccharides are added to menthol, is highly water-soluble, and does not exhibit the light-tasting flavor peculiar to menthol itself, but is hydrolyzed by various carbohydrases or acids. Separates into menthol and sugar, and develops a light flavor with a cool sensation peculiar to menthol [Agric. Biol. Chem., 43, 307 (1979),
And "Fragrance" No. 130, 79 (1981)].

Since such menthol glycoside is water-soluble and non-sublimable, it does not need to be sealed and stored like menthol, and is an extremely stable substance. Does not change at all. Furthermore, the refreshing sensation and the pharmacological action peculiar to menthol can be sustained or can be exerted at once by appropriately adjusting the decomposition rate.

The menthol glycoside having these characteristics can be used for the purpose of maintaining a refreshing sensation and as a stabilizer in various foods, mouth refreshers, tobacco flavor improving agents, and the like. . In fact, JP-A-62-1
Japanese Patent Application No. 61716 discloses an application in a dentifrice composition, Japanese Patent Application Laid-Open No. 6-329528 discloses application to cosmetics for keeping a refreshing feeling for a long time, and Japanese Patent Application Laid-Open No. 5-219929 discloses. Discloses application to tobacco flavor improver.

Regarding the method for producing menthol glycoside,
Japanese Patent Publication No. 51-105 discloses a method of organically synthesizing menthyl glucoside from glucose and menthol, an organic synthetic method using acetyl glucose or acetobromoglucose, and the like. Also, the above-mentioned Agric. Biol. C
hem., 43, 307 (1979), and "Fragrance" No. 130, 79 (19)
81), a method of isomerizing menthyl-tetraacetyl-β-glucopyranoside to an α-type to synthesize menthyl-α-D-glucopyranoside and an organic synthesis method of various menthol glycosides have been reported.

However, these methods have a problem that the manufacturing cost is high because an expensive catalyst is used and a multi-step reaction process is required. Further, in order to use the obtained menthol glycoside in food, it was necessary to completely remove the organic synthetic reagent for safety.

[0008]

Therefore, an object of the present invention is to provide the menthol glycoside, l-menthyl-α-, without using an expensive catalyst or requiring a multi-step reaction process. An object of the present invention is to provide a method for synthesizing D-glucopyranoside at low cost.

[0009]

Means for Solving the Problems and Embodiments of the Invention
As a result of earnest research and study, the inventors of the present invention have used 1-menthyl-α- by an enzymatic reaction using α-glucosidase, a sugar such as sucrose or maltose as a substrate, and 1-menthol as a sugar acceptor. A method for synthesizing D-glucopyranoside was developed.

Here, the α-glucosidase in the present invention is an enzyme capable of hydrolyzing an α-1,4-glucoside bond at the non-reducing end to produce α-glucose, and dextrin, maltose, and sucrose. In addition to so-called maltase having a wide range of substrate specificities such as claose, and α-amylase type enzymes capable of cleaving α-1,4-bonds in starch are also included.

The origin of the α-glucosidase used in the method of the present invention is not particularly limited, and it can be derived from animals, plants, microorganisms and the like, but yeast-derived α-glucosidase is preferred.

In the method of the present invention, α-glucosidase can be used by appropriately immobilizing it on silica gel or various carriers, coating it with a surfactant or the like, or chemically modifying it.

Menthol has four stereoisomers and three optical isomers for each, and there are a total of 12 isomers. Menthol in the present invention has a refreshing flavor. Refers to l-type menthol.

In the enzymatic reaction in the method of the present invention,
The substrate solution is preferably an aqueous solution of maltose or sucrose, but is not limited to these and α-
Any saccharide having a glucosidic bond can be used as a substrate. And, it is preferable that the sugar concentration in these aqueous solutions is as high as possible in order to increase the amount of the product.

In the enzymatic reaction in the method of the present invention,
The sugar-accepting l-menthol takes the form of a solid, a partially solid solution (semi-solution), or a solution depending on the temperature of the reaction system, and the reaction is possible in any form. It is preferable to react in a two-layer system of an aqueous substrate solution and 1-menthol. As shown in the examples below, the optimum temperature of the enzymatic reaction in the method of the present invention is about 30 to about 3.
The temperature is 5 ° C., and 1-menthol is in a semi-solution state in this temperature range. In addition, by adjusting the amount of 1-menthol added in the enzyme reaction system to about 3.75% (w / v) or less, l-menthyl-α-D-glucopyranoside relative to 1-menthol used. The production rate of can be increased.

The enzyme reaction in the method of the present invention is preferably carried out in a two-layer system of an aqueous substrate solution and semi-solution 1-menthol as described above, but 1-menthol is dissolved in a suitable organic solvent. It is also possible to use it for an enzymatic reaction with a substrate aqueous solution. As the organic solvent, hexane,
It is possible to use a solvent which is incompatible with water such as ether, ethyl acetate, oil and fat, a solvent which is miscible with water such as acetone and ethanol, or a solvent which is a combination thereof.

The amount of 1-menthyl-α-D-glucopyranoside produced is increased by adding a surfactant, particularly Tween 20 (polyoxyethylene sorbitan monolaurate) or sucrose monolaurate, to the enzyme reaction system. In such a case, it is appropriate that the amount of the surfactant added is about 0.1 to 1.0%.
% Is preferable.

The 1-menthyl-α-D-glucopyranoside produced by the enzymatic reaction of the present invention can be used as it is in the reaction solution, and can be appropriately separated and purified, or the solvent can be distilled off from the reaction solution. It can be appropriately concentrated before use.

The 1-menthyl-α-D-glucopyranoside and the mixture thereof produced by the method of the present invention are
As a safe and stable menthol agent, or a food material that gradually decomposes in the mouth to give a menthol-specific refreshing feeling,
It can be widely used as a cosmetic material.

The present invention will be described below with reference to examples.
This is for explaining the present invention and does not limit the technical scope of the present invention.

[0021]

【Example】

[1] Synthesis of menthol glycoside by various enzymes 10 mg of l-menthol was put in a small test tube, and 5
100 units of various glycolytic enzymes and 50 to 200 mg of substrates corresponding to the enzymes are added, and the total amount is 0.5 ml,
The ethanol concentration was adjusted to 2% (v / v), and the mixture was reacted at 40 ° C. for 12 hours with stirring. The reaction product was detected by thin layer chromatography.

Table 1 below shows various saccharide-degrading enzymes used, reaction conditions, and presence / absence of menthol glycoside formation.
As shown in Table 1, production of menthol glycoside was confirmed only in the system using α-glucosidase as the enzyme.

[0023]

[Table 1]

[2] Identification of Enzymatic Reaction Product The following test was conducted to identify the menthol glycoside produced in the α-glucosidase, maltose and 1-menthol system.

1-Menthol 2 g, 1.0 M maltose solution (10 mM citrate-phosphate buffer (pH 7.0)) 10
0 ml, yeast-derived α-glucosidase (Biozyme Labora
ToryLimited (UK) made 1000 units in a flask and stirred at 40 ° C with a magnetic stirrer.
For 12 hours.

The solution was freeze-dried and then extracted with ethyl acetate,
After distilling off the solvent of the extract, it was dissolved in n-butanol, 2-propanol and water (10: 5: 4) and applied to a silica gel column (Wakogel C-200, φ25 × 400 mm). Elution was performed with the same solvent, and the eluate was fractionated. After confirming the eluate components by TLC and selecting the fraction in which the reaction product was observed and distilling off the solvent, the fraction was washed with hexane and dried.

The obtained fractionated product (about 100 mg) is
A single spot was shown on TLC. The titer of α-glucosidase was defined as 1 unit of the amount that hydrolyzes 1 µM maltose per minute at 37 ° C.

The fractionated product was subjected to ¹³ C-NMR analysis. The spectrum is shown in FIG. The analysis result is Agri
c. Biol. Chem., 43, 307 (1979), a standard l-menthyl-α-D- which was synthesized and purified by organic chemistry.
It was in agreement with the analysis result of glucopyranoside.

[3] Substrate concentration and production amount The relationship between the substrate concentration and the production amount in the enzyme reaction system was investigated. 4 ml of various aqueous maltose or sucrose solutions (10 mM citrate-phosphate buffer (pH 7.
4)), 50 mg of 1-menthol, and yeast-derived α-
Glucosidase (Biozyme Laboratory Limited (UK)
20 units in a test tube with screw cap
Reciprocal shaking was performed 190 times per minute at 0 ° C., and the reaction was performed for 24 hours.

The reaction product was extracted with ethyl acetate and purified.
The amount of -menthyl-α-D-glucopyranoside was quantified by high performance liquid chromatography (HPLC). HPL
C is an ODS column (TSK-GEL 80TS (φ4.6 ×
250 mm)), the mobile phase is methanol, water (70: 3)
0) and the flow rate was 1 ml / min. A differential refractometer was used for the detector. Under the conditions of the present HPLC, 1-menthyl-α-D-glucopyranoside is eluted in the fraction having a peak retention time of 7.6 min.

The results of this test are shown in FIG. It was confirmed that the production amount of 1-menthyl-α-D-glucopyranoside increases with the substrate concentration up to the maximum amount at which the substrate can be dissolved. When the sucrose concentration was 2M, the yield of 1-menthyl-α-D-glucopyranoside was 5.6 mg.
And the yield based on 1-menthol used was about 11
%Met.

[4] Amount of l-menthol and amount produced In order to examine the influence of the amount of l-menthol in the enzymatic reaction on the yield of l-menthyl-α-D-glucopyranoside, the following test was conducted. Various concentrations of l-menthol and 1.0 M maltose solution (10 mM
4 ml of citric acid-phosphate buffer solution (pH 7.0) and 20 units of α-glucosidase were placed in a test tube with a screw cap, and the reaction was carried out at 40 ° C. for 24 hours by the same operation as described in [2]. -Mentyl-α-D-glucopyranoside was quantified.

The results of this test are shown in FIG. The amount of 1-menthyl-α-D-glucopyranoside produced increased with an increase in the amount of 1-menthol added to the reaction system, but was almost equilibrium at 150 mg (3.75% (w / v)) or more. Reached When 150 mg of 1-menthol was added to the reaction system, the amount of 1-menthyl-α-D-glucopyranoside produced was 3.7 mg, and the yield was 2.5% based on the amount of 1-menthol added. . On the other hand, when 25 mg of 1-menthol was added to the reaction system, the amount of 1-menthyl-α-D-glucopyranoside produced was 1.9 mg,
The yield based on the amount of 1-menthol added was 7.6%, and the smaller the amount of 1-menthol added to the reaction system, the lower the amount of 1-menthyl-α-D- to the amount of 1-menthol.
It was found that the yield of glucopyranoside was high.

[5] Examination of pH of reaction system The following test was conducted to examine the optimum pH of the enzyme reaction system. The same operation as described in [2] above was performed by mixing 4.0 ml of a 1.0 M maltose solution prepared with 10 mM citrate-phosphate buffer having various pHs, 50 mg of 1-menthol, and 20 units of α-glucosidase. The reaction mixture was allowed to react at 40 ° C. for 24 hours to produce 1-menthyl-α-D-
Glucopyranoside was quantified.

The results of this test are shown in FIG. It was confirmed that 1-menthyl-α-D-glucopyranoside was produced when the pH of the reaction system was in the range of 5 to 8, and that a high production amount was obtained at pH 6 to 7, in particular.

[6] Examination of reaction temperature The following test was conducted to examine the optimum temperature of the enzyme reaction. 4.0 ml of 1.0 M maltose solution (10 mM citrate-phosphate buffer (pH 7.0)) and 1-menthol 50
mg, and mixed with 20 units of α-glucosidase,
1-menthyl-α-D produced by reacting at a temperature of 25 to 50 ° C. for 24 hours by the same operation as described in [2] above
-Glucopyranoside was quantified.

The results of this test are shown in FIG. The formation of 1-menthyl-α-D-glucopyranoside was observed at any reaction temperature tested, but it was confirmed that a high production amount was obtained at 30 to 35 ° C.

[7] Effect of Surfactant in Reaction System Adding a surfactant to the reaction system is l-menthyl-α.
The following test was conducted in order to examine the influence on the formation of -D-glucopyranoside. 1.0 M maltose solution (10 mM citrate-phosphate buffer (pH 7.0)) 4.0
ml, 1-menthol 50 mg, α-glucosidase 20 units, and various surfactants 4 mg to 10
mg and 40 mg were mixed (surfactant concentrations in the reaction system were 0.1%, 0.25%, and 1.0%, respectively), and the same operation as described in [2] above was performed at 40 ° C. for 24 hours. After reacting for a period of time, the produced 1-menthyl-α-D-glucopyranoside was quantified.

The results of this test are shown in Table 2 below. Tween type surfactant and sucrose monolaurate,
In the sucrose fatty acid ester of sucrose octaacetate, an increase in the amount of 1-menthyl-α-D-glucopyranoside produced was observed. In particular, it was confirmed that the production amount when 10 mg of Tween 20 was added increased about 3.3 times as much as when the Tween 20 was not added. L-when using Tween 20
The yield of menthyl-α-D-glucopyranoside was about 18% by weight based on 1-menthol used.

[0040]

[Table 2]

[0041]

INDUSTRIAL APPLICABILITY According to the method of the present invention, l-menthyl-α-D-glucopyranoside can be supplied inexpensively without the need for multi-step reactions and expensive catalysts as in the conventional organic chemical synthesis method. be able to.

Furthermore, according to the method of the present invention, it is possible to use a drug which is not harmful to the human body unlike the organic chemical synthesis method.
It is possible to produce 1-menthyl-α-D-glucopyranoside, and to supply the highly safe substance that can be widely used as food materials, cosmetic materials, and the like. It is also possible to add and use the reaction mixture as it is directly in foods etc. without purifying and separating the product.

[Brief description of drawings]

FIG. 1 is a diagram showing a chemical structure of 1-menthyl-α-D-glucopyranoside and a ¹³ C-NMR spectrum of a menthol glycoside produced in an example of the present invention.

FIG. 2 is a graph showing the relationship between the substrate concentration and the amount of 1-menthyl-α-D-glucopyranoside produced in the enzymatic reaction of the method of the present invention.

FIG. 3 is a graph showing the relationship between 1-menthol and the amount of 1-menthyl-α-D-glucopyranoside produced in the enzymatic reaction of the method of the present invention.

FIG. 4 is a graph showing the relationship between reaction pH and the amount of 1-menthyl-α-D-glucopyranoside produced in the enzymatic reaction of the method of the present invention.

FIG. 5 is a graph showing the relationship between the reaction temperature and the amount of 1-menthyl-α-D-glucopyranoside produced in the enzymatic reaction of the method of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Susumu Shimura 1-23-6 Numakage, Urawa-shi, Saitama Prefecture (72) Masaaki Yoshiyama 226-3 Higashimachi, Takasaki-shi, Gunma Prefecture

Claims (1)

[Claims] 1. An enzyme that hydrolyzes an α-1,4-glucoside bond at the non-reducing end of a saccharide is used, and a sugar transfer reaction by the enzyme is used to utilize l-menthyl-α-. A method for producing D-glucopyranoside.