JP4224267B2 - Purification method of glycoside - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for purifying glycosides, and more particularly to a method for purifying glycosides, which comprises treating a mixture of glycoside and glucose with glucose oxidase.
[0002]
[Prior art]
Ethyl glucoside is a taste component contained in sake, wine, mirin, and vinegar, and has a refreshing glucose-like sweetness and unique bitterness. As its use, for example, a flavor enhancer / improver for foods and beverages made of β-D-ethylglucoside (Patent No. 2744595), an alcohol-stimulated odor mitigator containing ethyl-α-D-glucopyranoside as an active ingredient (patent) No. 2873408), skin conditioning action of α-ethyl glucoside (Japan Cosmetics Engineers Journal, 32 (1), 10-16 (1998)) has been proposed. As a method for producing ethyl glucoside, for example, a method for chemically synthesizing glucose and ethanol as raw materials (Rocznikichi, 49 (12), 2113-2115 (1975)), a method for producing biochemically, For example, a method of allowing Schizosaccharomyces pombe to act on a sugar-containing ethanol solution (Japanese Patent Publication No. 5-56958), a method of causing Aspergillus niger-derived α-glucosidase to act on a sugar-containing ethanol solution (Japanese Patent Publication No. 6-30608), a method of allowing an enzyme having α-1,6-glucosyl transfer activity to act on a carbohydrate in the presence of a microorganism having alcohol-fermenting ability (Japanese Patent Laid-Open No. 11-243987), maltose Due to microorganisms belonging to the genus Mortierella A conventional method of allowing α-glucosidase to act (Japanese Patent Laid-Open No. 2001-61490), a method of causing Talaromyces duponti-derived transglucosidase to act on a carbohydrate in the presence of ethanol (Japanese Patent Laid-Open No. 2002-126992), etc. Proposed.
[0003]
In addition, as other methods for producing glycosides, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group, a compound having a hydroxyl group of a flavonoid-related compound and a glucan having an α-1,4 bond are derived from Bacillus stearothermophilus. A method for producing a glycoside that allows α-glucosidase to act (JP-A-9-87294) has been proposed.
[0004]
[Problems to be solved by the invention]
In the conventionally proposed methods for producing glycosides such as ethyl glucoside, the purity of ethyl glucoside is low, and in order to achieve high purity, it is necessary to remove the saccharides that are present using complicated separation operations. An industrial purification method has been demanded. For example, when biochemically producing ethyl glucoside using transglucosidase with maltose as a substrate, relatively high purity ethyl glucoside is obtained, but even in that case, the content relative to the sugar composition is 50% or less. . Also, in the production of glycosides of compounds having an alcoholic hydroxyl group, a phenolic hydroxyl group, or a flavonoid-related compound, glucose is mixed, and a high-purity glycoside cannot be obtained.
[0005]
Accordingly, an object of the present invention is to provide a method for industrially purifying glycosides such as ethyl glucoside with high purity and efficiency.
[0006]
[Means for Solving the Problems]
As a result of intensive studies to solve the problems as described above, the present inventors have now obtained a high-purity glycoside by adding glucose oxidase to a system in which glycoside and glucose are mixed. The present invention has been completed.
[0007]
Thus, according to the present invention, there is provided a method for purifying a glycoside characterized in that glucose is removed by treating a mixture of glycoside and glucose with glucose oxidase, preferably in the presence of salts.
[0008]
In addition, the present invention provides a glycoside by producing α-glucosidase on a saccharide, starch or a degradation product thereof in the presence of a compound having a hydroxyl group, and then remaining glucose, preferably glucose oxidase in the presence of salts. The present invention provides a method for purifying a glycoside, characterized in that the glycoside is removed by treatment with the above.
[0009]
Hereinafter, the present invention will be described in more detail.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The gist of the present invention is that glucose oxidase is allowed to act on a mixture of glycoside and glucose to remove the mixed glucose. In the present invention, the glycoside includes any of α-form, β-form or a mixture thereof, for example, ethyl glucoside is any of ethyl-α-glucoside, ethyl-β-glucoside or a mixture thereof. Is also included.
[0011]
Examples of the compound having a hydroxyl group used in the present invention include methanol, ethanol, 1-propanol, 1-phenylethanol, geraniol, ascorbic acid, kojic acid, 1-butanol, benzyl alcohol, phenoxyethanol, 2-propanol, 1- Compounds having alcoholic hydroxyl groups such as octanol, 3-octanol, citronellol, menthol, furaneol, maltol, cycloten, β-phenylethyl alcohol, cinnamic alcohol, dimethoxyphenol, catechol, resorcinol, hydroquinone, vanillin, catechol, piganol, eugenol And compounds having a phenolic hydroxyl group, such as rutin, flavonol, flavanone, flavone and the like flavonoid-related compounds.
[0012]
First, as a method for producing a glycoside biochemically, ethyl glucoside will be described in detail as an example. As a method for biochemically producing ethyl glucoside, any conventionally known method can be employed, and examples thereof include a method in which α-glucosidase is allowed to act on sugars, starch, or a degradation product thereof in the presence of ethanol. be able to. Glucose, maltose, isomaltose, panose, other oligosaccharides or a mixture thereof, starch syrup or the like can be used as the saccharide, starch or decomposition product thereof. When oligosaccharides having a relatively high degree of polymerization, for example, saccharides of 5 or more sugars, the presence of α-amylase increases the content of disaccharides and 3 saccharides serving as a substrate for α-glucosidase, This is preferable because the yield of ethyl-α-glucoside increases. For example, in the case of maltose, the concentration of these substrates increases the amount of ethyl-α-glucoside generated as the substrate concentration increases. Usually, the maltose concentration in the reaction solution is in the range of about 5 to about 50 w / v%. The inside can be illustrated. The concentration of ethanol in the reaction solution is not particularly limited, and the enzyme reaction is usually performed by adding about 10 to about 30 V / V%, preferably about 20 to about 25 V / V%. Alternatively, ethanol can be produced by coexisting with a microorganism having alcohol-fermenting ability and performing alcoholic fermentation in the reaction solution.
[0013]
The α-glucosidase used in the reaction is an enzyme having α-1,4-glucosyl transfer activity, and a microorganism-derived enzyme can be used. Examples of such microorganisms include molds belonging to the genus Aspergillus, Penicillium, Mucor, Rhizopus, Thermoscus; yeasts belonging to the genus Saccharomyces, Candida; enzymes derived from bacteria belonging to the genus Bacillus be able to. Among them, α-glucosidase derived from mold belonging to the genus Aspergillus is preferable in terms of alcohol resistance and stability. Such an enzyme can be obtained by liquid culture or solid culture of the above-mentioned microorganism, and can be used as either a crude enzyme or a purified enzyme. A commercially available α-glucosidase enzyme agent (trade name: α- Glucosidase “Amano” (manufactured by Amano Enzyme) can also be used. The amount of α-glucosidase added varies depending on the type of enzyme used, the degree of purification, the reaction conditions, etc., and cannot generally be described, but it is usually 100 to 100,000 U per gram of maltose which is a substrate.
[0014]
The enzyme reaction conditions are not particularly limited and vary depending on the type of α-glucosidase used and cannot be generally specified. However, in general, in the temperature range of 20 to 50 ° C., at pH 3 to 8 and for about 5 to 48 hours. By reacting, ethyl-α-glucoside can be produced. In the reaction solution, depending on the type of α-glucosidase to be used and the reaction conditions, it cannot be generally stated, but usually about 0.5 to 1 part by weight of glucose per 1 part by weight of ethyl-α-glucoside is mixed. High-purity ethyl-α-glucoside can be obtained by removing glucose from the reaction solution by treating the mixed glucose with glucose oxidase shown below.
[0015]
Next, a method for removing glucose in the reaction solution by causing glucose oxidase to act on the reaction solution described above will be described. The glucose oxidase used in the present invention is an enzyme that oxidizes glucose to produce gluconic acid, and examples thereof include enzymes derived from molds belonging to the genus Aspergillus and Penicillium. Such an enzyme can be obtained by liquid culture or solid culture of the above-described microorganism, and can be used as either a crude enzyme or a purified enzyme. Also, a commercially available glucose oxidase enzyme agent (trade name: Hyderase 15 ( Amano Enzyme Co., Ltd.) and Sumiteam GOP (manufactured by Shin Nippon Chemical Industry Co., Ltd.)) can also be used. The amount of glucose oxidase to be added varies depending on the type of enzyme used, the degree of purification, the reaction conditions, etc., and cannot be generally specified, but usually 10 to 1000 U can be exemplified per 1 g of glucose mixed in the reaction solution described above. .
[0016]
The enzyme reaction conditions are not particularly limited and vary depending on the type of glucose oxidase to be used, etc., and cannot be generally stated. Usually, in the temperature range of 20 to 60 ° C., at pH 4 to 9, with oxygen supply by aeration stirring. , About 5 to 24 hours. In the present invention, the treatment with glucose oxidase described above is preferably performed in the presence of salts. It is preferable to treat with glucose oxidase in the presence of salts because the reaction rate can be increased by suppressing the change in pH of the reaction solution due to the generated gluconic acid as the reaction proceeds. Moreover, the gluconic acid produced | generated becomes a gluconate, and isolation | separation with an ethyl-alpha-glucoside becomes easy. Such salts are not particularly limited, and a wide range of salts can be used, and examples thereof include calcium carbonate, magnesium carbonate, and the like. It is preferable to control the pH by supplying an alkali such as Ca (OH) 2. The amount of the salt used is not particularly limited and varies depending on the reaction conditions and cannot be generally specified.
[0017]
After completion of the reaction, the remaining salts are separated by an appropriate separation operation such as filtration, and then water is removed using an appropriate concentration means such as vacuum concentration, and a solvent that can dissolve ethyl-α-glucoside, for example, Ethanol is added to dissolve ethyl-α-glucoside, and the insolubilized gluconate is separated by an appropriate separation means such as filtration. Then, if desired, an appropriate concentration means such as vacuum concentration is employed to remove the solvent. By collecting, high purity ethyl-α-glucoside can be obtained.
[0018]
Hereinafter, the present invention will be specifically described with reference to Examples and Comparative Examples.
[0019]
【Example】
Reference Example 1: Production of ethyl-α-glucoside with α-glucosidase A 1 L 3-diameter flask was charged with 63 g maltose, 114 g ethanol, 500 g pure water, and 3 ml transglucosidase L (manufactured by Amano Enzyme) and reacted at 40 ° C. for 72 hours. To produce ethyl-α-glucoside. The reaction solution was confirmed to be a mixed solution of ethyl-α-glucoside, glucose and ethanol by HPLC (high performance liquid chromatography).
[0020]
Example 1: Removal of glucose by glucose oxidase treatment Unreacted ethanol was removed from the reaction solution prepared in Reference Example 1 under reduced pressure (45 ° C, 50 mmHg). Further, this reaction solution was charged into a 2 L mini jar (manufactured by Maruhishi Co., Ltd.), 3.0 g of Hyderase 15 (manufactured by Amano Enzyme) and 8 g of calcium carbonate were added, and aerated and stirred at 40 ° C. for 10 hours (aeration rate: 0.2 vvm). 600 rpm). After confirming that no glucose remained, water was removed under reduced pressure (45 ° C., 50 mmHg). Ethanol 300ml was added to this and ethyl-alpha-glucoside was extracted. In addition, it confirmed that the produced | generated gluconic acid was not extracted by ethanol as calcium gluconate. Ethanol was recovered from the ethanol extractor under reduced pressure to obtain 15 g of substantially pure ethyl-α-glucoside.
[0021]
【The invention's effect】
According to the present invention, a method for industrially purifying glycosides with high purity and efficiency can be provided.

Claims (1)

A method for purifying ethyl-α-glucoside, comprising treating a mixture of ethyl-α-glucoside and glucose with glucose oxidase in the presence of a salt selected from calcium carbonate and magnesium carbonate and then extracting with ethanol. .