JP2003274992A - Method for producing chromanol glycoside composition having high purity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a nearly colorless chromanol glycoside composition having high purity and high water solubility. <P>SOLUTION: The method for the production of the chromanol glycoside composition comprises the reaction of a 2-substituted alcohol with a sugar in the presence of an enzyme catalyzing the corresponding sugar transfer reaction in combination with an antioxidation agent. The chromanol glycoside composition produced by the method is almost colorless and has high purity and, accordingly, the composition is preferably usable in various fields without purification, etc. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing a chromanol glycoside composition having high purity and extremely high water solubility.

[0002]

2. Description of the Related Art General formula (I):

[0003]

[Chemical 3]

The chromanol glycoside represented by is obtained by substituting an alcohol for the phytyl group at the 2-position of the chroman ring of α-tocopherol, which is a typical vitamin E, and further binding a sugar. It is a compound having water solubility and excellent antioxidant properties. Such compounds are known in JP-A-7-118287.

As a method for industrially and inexpensively producing such a chromanol glycoside as a highly pure product, a method using an enzyme has been developed. As such a method, a 2-substituted alcohol and a sugar are efficiently transglycosylated by an enzymatic reaction, and a chromanol glycoside having a m of 1 to 6 in the general formula (I) as a main component is used as a main component and is highly pure. Japanese Patent Application Laid-Open No. 2000-213892 discloses a technique for producing a chromanol glycoside composition having extremely high water solubility. Specifically, this method is represented by the following formula (II):

[0006]

[Chemical 4]

The 2-substituted alcohol and sugar represented by
It is characterized in that the reaction is carried out in the presence of cyclomaltodextrin glucanotransferase in the presence of an organic solvent. Furthermore, the same reaction is performed by using an immobilized enzyme in which cyclomaltodextrin glucanotransferase is immobilized on porous chitosan beads or weakly basic anion exchange resin, and the enzyme is reused to realize low cost. This method is also disclosed (Japanese Patent Laid-Open No. Hei 20)
02-000290).

Originally, chromanol glycosides are colorless, and naturally chromanol glycoside compositions should also be colorless.
However, the chromanol glycoside composition produced by the enzymatic method has a problem that it is undesirably colored. For example, the compositions obtained by the methods described in JP-A 2000-213892 and JP-A 2002-000290 may have a pale reddish brown color. Such coloring is not only inappropriate when the composition is directly manufactured into a product, but also has a problem in terms of purity because there is a high possibility that some by-product is produced. It is technically difficult to selectively remove only such by-products from the chromanol glycoside composition, and it causes high cost, and therefore some measures are required.

[0009]

DISCLOSURE OF THE INVENTION The present invention has been conceived in view of the above problems, and an object thereof is to provide a method for obtaining a high-purity chromanol glycoside composition which is nearly colorless and has high water solubility. It is to be.

[0010]

As a result of pursuing the cause of coloring, the present inventors have made an antioxidant coexist in the reaction solution,
Further, it was found that by carrying out the reaction in a state where dissolved oxygen in the reaction solution is removed, a chromanol glycoside having high purity and close to colorless can be efficiently produced.

That is, the present invention provides the following general formula (1)

[0012]

[Chemical 5]

(Wherein R ¹ , R ² , R ³ and R ⁴
Are the same or different and each represents a hydrogen atom or a lower alkyl group, R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, and n represents an integer of 0 to 4).
In a method comprising reacting a substituted alcohol with a sugar in the presence of an enzyme that catalyzes a corresponding transglycosylation action, the reaction is carried out in the presence of an antioxidant, and the following general formula (2)

[0014]

[Chemical 6]

(Wherein R ¹ , R ² , R ³ and R ⁴
Are the same or different and each represents a hydrogen atom or a lower alkyl group, R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group, and X represents a hydrogen atom of a hydroxyl group in the sugar residue which is a lower alkyl group or a lower acyl group. The production of a chromanol glycoside composition represented by a monosaccharide residue or an oligosaccharide residue which may be substituted, n is an integer of 0 to 4, and m is an integer of 1 to 10. Is the way.

Further, the present invention is the above-mentioned production method, wherein the concentration of dissolved oxygen in the reaction solution is further reduced.

Further, the present invention is the above production method, wherein the antioxidant is water-soluble.

Further, the present invention is the above production method, wherein the antioxidant is ascorbic acid.

Further, in the present invention, the enzyme which catalyzes the corresponding glycosyl transfer activity is an immobilized enzyme in which cyclomaltodextrin glucanotransferase is immobilized on porous chitosan beads or a weakly basic anion exchange resin. It is the manufacturing method.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The features of the present invention will be described in detail below.

The above-mentioned Japanese Patent Laid-Open No. 2002-0
When the cause of the coloration of the resulting product in the enzymatic production method described in Japanese Patent No. 00290 was investigated, the chromanol derivative produced during the preparation of the chromanol glycoside composition, particularly during the reaction of the 2-substituted alcohol and the sugar was investigated. It was found that the glycoside was decomposed by the oxidative action to produce a by-product.
It is considered that this oxidative decomposition reaction is due to a reaction represented by the following chemical formula.

[0022]

[Chemical 7]

Since the by-products produced by such an oxidative decomposition reaction are colored, the chromanol glycoside composition is colored brown. However, it is technically difficult and costly to remove these by-products from the chromanol glycoside.

Therefore, when a method for obtaining a near colorless chromanol glycoside composition by suppressing the formation of by-products by suppressing the oxidizing action during the reaction was sought, an antioxidant was added to the reaction solution to carry out the oxidation reaction. It was found that by suppressing the formation of by-products, the production of by-products can be effectively suppressed and a high-purity chromanol glycoside composition can be produced.

That is, in the method for producing the chromanol glycoside composition represented by the general formula (2) of the present invention, the 2-substituted alcohol represented by the general formula (1) and the sugar are subjected to the corresponding glycosyl transfer. In the method comprising reacting in the presence of an enzyme that catalyzes the action, the reaction is carried out in the coexistence of an antioxidant to suppress the oxidation reaction and suppress the coloration due to by-product formation.

The method of adding the antioxidant used in the method of the present invention is characterized in that, for example, the antioxidant used is water-soluble or water-insoluble, and the reaction system is water-based or organic solvent-based. Based on the above, it is preferable to select an appropriate addition method to uniformly disperse in the reaction system, and it is not particularly limited, but for example, (1) a method of adding it to a mixture of a substrate (sugar and 2-substituted alcohol), (2) A method of adding it to one of the substrates and then mixing it with the other substrate, and (3) a method of mixing each component in the form of a solution or as it is. For example, when the antioxidant used is water-soluble and the reaction system is water-based, any of the above methods (1) to (3) can be applied.
Further, for example, when the antioxidant to be used is water-insoluble and the reaction system is water-based, a method capable of dissolving the antioxidant in any one of the substrates is used, and the method of (2) above is used. It is possible to apply the method, or to dissolve the antioxidant in a solvent that can dissolve the antioxidant in advance and apply the methods (1) to (3).

As the liquid for dissolving the substrate, the enzyme, the antioxidant, etc., any liquid can be used as long as it does not affect the enzymatic reaction according to the present invention. For example, water or
Conventional well-known buffers such as acetate buffer, Tris buffer, phosphate buffer, citrate buffer and the like can be mentioned. On the other hand, the 2-substituted alcohol, which is the substrate, is poorly soluble in water, so it is preferably dissolved in an organic solvent. The organic solvent used to dissolve the 2-substituted alcohol will be described later.

As the antioxidant used in the method of the present invention, any substance can be used as long as it is a substance having an antioxidant action and does not affect the enzymatic reaction. For example, carotenoids and vitamins A , Vitamin B ₂
Ascorbic acid, monodehydroascorbic acid, ascorbic acid stearic acid ester, ascorbic acid palmitic acid ester, 2,3-diketo-L-gulonic acid, vitamin E, tocopherols, phenolcarboxylic acids such as gallic acid and their esters, flavonoids, Erythorbic acid, sodium erythorbate, BHA, BHT, nordihydroguaiaretic acid (NDGA), dilauryl thiodipropionate and the like are used. Of these, ascorbic acid, which has high water solubility, is preferable.

The concentration of the antioxidant is preferably 0.01 to 0.5 (mass / volume)% in the reaction solution, more preferably 0.1.
It is 05 to 0.2 (mass / volume)%. If the concentration of the antioxidant is less than 0.01 (mass / volume)%, the suppression of by-product formation may be insufficient, while if it exceeds 0.5 (mass / volume)%, the amount added will be There is a risk that the appropriate effect may not be obtained.

In addition to such antioxidants, for example, ED
It is also useful to add a chelating agent such as TA and trisodium ethylenediamine hydroxyethyl triacetate, sodium citrate, and gluconic acid to the reaction solution to enhance the inhibitory effect on the decomposition reaction of chromanol glycoside. These substances have an effect of suppressing the chromanol glycoside decomposition reaction by the metal ions in the reaction solution, particularly the transition metal ions. Therefore, by further adding these substances to the reaction solution, it is possible to further enhance the antioxidant and the effect of suppressing the oxidative decomposition reaction by reducing the dissolved oxygen. The amount of these substances to be used varies depending on the substance to be used and is not particularly limited, but it is sufficient that the reaction liquid contains an amount capable of suppressing the oxidation reaction to the extent that it does not affect the enzymatic reaction. For example, the concentration when using a chelating agent is preferably 0.1 to 5 m in the reaction solution.
M, more preferably 0.2 to 2 mM.

In addition to the use of such an antioxidant, it is preferable to further suppress the oxidative decomposition reaction of the chromanol glycoside by reducing the dissolved oxygen concentration in the reaction solution. If the dissolved oxygen in the reaction solution is reduced, the probability that the chromanol glycoside is naturally oxidized will be lowered, so that a higher effect of suppressing the oxidative decomposition reaction can be obtained together with the use of the antioxidant.

As a method for reducing dissolved oxygen, various known techniques can be used and are not particularly limited. For example,
Examples of the method include bubbling an inert gas such as nitrogen, helium, and argon into the reaction solution to expel dissolved oxygen, depressurizing by decompressing with an aspirator, and removing dissolved oxygen through a membrane such as a hollow fiber membrane. To be Above all, a method of bubbling an inert gas to expel dissolved oxygen is preferable because the apparatus is simple and inexpensive.

A method for bubbling an inert gas into the reaction solution to expel dissolved oxygen will be described below. The inert gas used is, for example, nitrogen, helium, argon or the like, preferably nitrogen gas. The inert gas is
Using a regulator, etc., generally a container volume of 1 ml
The reaction solution is aerated for 0.5 to 5 minutes. The ventilation amount is not particularly limited, but is generally 100 to 500.
ml / min, preferably 150-300 ml / min.
By bubbling an inert gas into the reaction solution within the range of the aeration time and the aeration amount as described above, the dissolved oxygen can be effectively expelled.

Next, the chromanol glycoside represented by the general formula (2) produced by the method of the present invention will be described.

In the general formula (2), R ¹ , R ² , R
The lower alkyl group of ³ , R ⁴ and R ⁵ is preferably a lower alkyl group having 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group and a butyl group. , Isobutyl group, pentyl group, isopentyl group, hexyl group, heptyl group, octyl group and the like. Of these, a methyl group or an ethyl group is preferable. The lower acyl group for R ⁵ is preferably a lower acyl group having 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, and examples thereof include a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group and a valeryl group. , Isovaleryl group, pivaloyl group, hexanoyl group, heptanoyl group, octanoyl and the like. Among these,
An acetyl group, a propionyl group or a butyryl group is preferred. The monosaccharide residue of X includes glucose, galactose, fucose, xylose, mannose, rhamnose, fructose, arabinose, lyxose, ribose, allose, altrose, idose, talose, deoxyribose, 2-deoxyribose, quinovose,
Examples thereof include sugar residues such as abequeose. Examples of the oligosaccharide residue of X include those in which 2 to 4 of the above-mentioned monosaccharides are linked, for example, sugar residues of maltose, lactose, cellobiose, raffinose, xylobiose, sucrose and the like. Of these, monosaccharide residues such as glucose, galactose, fucose, xylose, rhamnose, mannose and fructose are preferable. The hydrogen atom of the hydroxyl group in the sugar residue of X is a lower alkyl group, preferably a lower alkyl group having 1 to 8 carbon atoms, or a lower acyl group, preferably a lower acyl group having 1 to 10 carbon atoms. May be replaced with. Furthermore, n is 0-4, preferably 1-3.
Is an integer of 1, more preferably 1, and m is an integer of 1 to 6, preferably 1 to 3. A preferable example of the chromanol glycoside represented by the general formula (1) is 2- (α-D-
Glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, 2- (β-D-galactopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, 2- (β-L-fucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, 2- (α-L-rhamnopyranosyl) methyl-2,
5,7,8-Tetramethylchroman-6-ol, 2-
(Β-D-xylopyranosyl) methyl-2,5,7,8
-Tetramethylchroman-6-ol, 2- (β-D-
Glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, 2- (β-D-fructofuranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol, 2- (α-D-mannopyranosyl)
Methyl-2,5,7,8-tetramethylchroman-6-
Examples include oars.

Next, the 2-substituted alcohol of the general formula (1) used in the present invention will be described. In the general formula (1), R ¹ , R ² , R ³ , R ⁴ , R ⁵ and n are the same as the definition of the general formula (2), and therefore the description thereof is omitted. Further, such a 2-substituted alcohol is disclosed, for example, in JP-A-7-1.
It can be prepared by the method disclosed in 18287.

The concentration of the 2-substituted alcohol of the general formula (1) used in the present invention in the reaction solution is 0.3 to 1.5 (mass / weight).
%), Preferably 0.5 to 1 (mass / volume)%. The concentration of 2-substituted alcohol is 0.3 (mass / volume)
If it is less than 1.5%, the synthesis efficiency may decrease, while if it exceeds 1.5 (mass / volume)%, 2 in the reaction system may occur.
-Substituted alcohol may be precipitated and the reaction efficiency may be reduced.

As the enzyme used in the present invention, 2-
The catalyst is not particularly limited as long as it catalyzes the transglycosylation action corresponding to the substituted alcohol and the sugar, and those derived from almost all sources can be used. Examples of such an enzyme include Bacillus stearothermophilus (Bacillus stearotherm) manufactured by Hayashibara Institute of Biological Sciences.
Cyclomaltodextrin glucanotransferase derived from O. philus or Bacillus macerans manufactured by Amano Pharmaceutical Co., Ltd.
ans) -derived cyclomaltodextrin glucanotransferase, and preferably Bacillus stearothermophilus-derived cyclomaltodextrin glucanotransferase. The amount of enzyme is preferably 10 to 50 U, more preferably 12 to 10 U per 1 ml of the reaction solution.
It is 40U. If the amount of enzyme is less than 10 U, the reaction efficiency may be low, and if it exceeds 50 U, the reaction efficiency corresponding to the amount added may not be obtained. In this case, 1U is the same as defined in the examples below.

The types of sugars used in the present invention are preferably maltooligosaccharides having a degree of polymerization of maltotriose or higher, soluble starch, starch and cyclodextrin (α, β, γ). The concentration of sugar in the reaction solution is preferably 3 to 20 (mass / volume)%, and particularly preferably 5 to 15 (mass / volume)%. When the sugar concentration is less than 3 (mass / volume)%,
There is a possibility that the synthesis efficiency may decrease, and on the other hand, if it exceeds 20 (mass / volume)%, the proportion of the chromanol glycoside (m is 7 or more) that is poorly soluble in water may increase.

In the present invention, since the 2-substituted alcohol is poorly soluble in water, an organic solvent is added to the reaction system to
It is preferable to appropriately disperse the substituted alcohol in the reaction solution. The organic solvent used is preferably a water-soluble organic solvent, specifically, dimethyl sulfoxide, N, N
-Dimethylformamide, methanol, ethanol, acetone, acetonitrile and the like can be mentioned, but dimethyl sulfoxide is particularly preferable. The organic solvent does not need to include an amount that completely dissolves the 2-substituted alcohol in the sugar solution, and the concentration of the organic solvent in the reaction solution is 1 to 20 (volume / volume)%, preferably 2 to 15 (volume / volume). Volume)%.
If it is less than 1 (volume / volume)%, the reaction liquid of the 2-substituted alcohol may be poorly dispersed and the synthesis rate may be lowered.
If it exceeds 0 (volume / volume)%, the proportion of chromanol glycoside (m is 7 or more), which is poorly soluble in water, may increase, and the enzyme may be inactivated to lower the synthesis rate.

The reaction temperature in the production method of the present invention is 3
It is 0 to 50 ° C, preferably 35 to 45 ° C. If the reaction temperature is lower than 30 ° C, the reaction efficiency may be deteriorated,
If the temperature exceeds 50 ° C, the decomposition of the chromanol glycoside is promoted, which may make it impossible to obtain a high-purity chromanol glycoside mixture.

The reaction time in the production method of the present invention is 1
It is 5 to 40 hours, preferably 20 to 35 hours. If the reaction time is less than 15 hours, the reaction rate may be insufficient, and if it exceeds 40 hours, the decomposition of chromanol glycosides may be promoted and a high-purity chromanol glycoside mixture may not be obtained. There is.

As other conditions in the production method of the present invention, the pH is generally 4.5 to 8.5, preferably 5.0 to 7.5. However, since the optimum pH differs depending on the type of enzyme used, it is preferable to adjust the reaction solution appropriately within the optimum pH range.

In the method described above, it is possible to reuse the enzyme after the reaction by using an immobilized enzyme in which cyclomaltodextrin glucanotransferase is immobilized on porous chitosan beads or weakly basic anion exchange resin. Therefore, it is preferable.

The immobilized enzyme will be described in detail below. Porous chitosan beads used in the present invention, crustacean shells such as crabs and shrimps, the skin of insects such as silkworms,
Chitin derived from shells and bone marrow of molluscs such as krill and squid in 100 to 20% sodium hydroxide solution in 100 to 20
The chitin is N-deacetylated by treating at 0 ° C. for 4 to 5 hours, the N-deacetylated product is washed with water and dried to obtain chitosan, and the chitosan is made granular and porous to increase the surface area. Can be obtained by increasing the adsorption capacity. Specific examples of such porous chitosan beads include Chitopearl BCW-2510 and BCW-26.
10, BCW-2610, BCW-3010, BCW-
3510 (both manufactured by Fuji Spinning Co., Ltd.) and the like.

The particle size of the chitosan beads used in the present invention is not particularly limited, but considering the pressure loss and the particle surface area, it is 0.1 to 3.0 mm, preferably 0.5 to 2. 0 mm, more preferably 0.5 to
It is 1.5 mm.

The weakly basic anion exchange resin used in the present invention is, for example, a styrene type anion exchange resin or a phenol type anion exchange resin. Examples of the styrene-based anion exchange resin include a styrene / divinylbenzene copolymer and a weakly basic anion exchange resin having a quaternary ammonium group as a functional group. The styrene / divinylbenzene copolymer may contain a polymerization component other than these components as long as it does not impair the adsorptivity to enzymes, thermal stability and the like. Further, the particle size, particle size distribution, specific surface area, pore volume, pore size, degree of basicity and the like of the ion exchange resin can be appropriately determined. Such styrenic anion exchange resin is widely commercially available for preparing immobilized enzyme and is easily available, and as a specific example, Amberlite IRA-93Z.
U (manufactured by Organo Corporation), Diaion WA-30
(Manufactured by Mitsubishi Chemical Co., Ltd.) and the like can be used.

The phenolic anion exchange resin is, for example, a phenol / formalin condensation polymer or a phenol / formalin / polyamine compound condensation polymer, and a weakly basic anion exchange resin having a tertiary amine as a functional group. Resin can be mentioned. The phenol / formalin condensation polymer and the phenol / formalin polyamine compound condensation polymer may contain a polymerization component other than these components as long as the adsorption property to an enzyme and the thermal stability are not impaired. Further, the particle size, particle size distribution, specific surface area, pore volume, pore size, degree of basicity and the like of the ion exchange resin can be appropriately determined. Such a phenolic anion exchange resin is widely commercially available for preparing an immobilized enzyme and is easily available, and specific examples include Duolite No. 500 series (manufactured by Sumitomo Chemical Co., Ltd.) and the like. Is.

In the above-mentioned porous chitosan beads and weakly basic anion exchange resins, their mass is referred to as the wet mass. The wet mass is a mass measured in a state where the porous chitosan beads in the dry state or the weakly basic anion exchange resin is allowed to absorb water or a buffer solution to sufficiently remove excess water, that is, in an actual use state.

The particle size of the weakly basic anion exchange resin used in the present invention is not particularly limited, but considering pressure loss, particle surface area and the like, it is 0.1 to 3.0 mm.
And preferably 0.3 to 1.5 mm.

As a method for producing the immobilized enzyme according to the present invention, the aforementioned cyclomaltodextrin glucanotransferase is contacted with porous chitosan beads or weakly basic anion exchange resin in water or an appropriate buffer solution. There is a way. An embodiment of an immobilization method when using Chitopearl BCW-3510 as a carrier for porous chitosan beads is shown below. After fully equilibrating 1 g (wet mass) of Chitopearl BCW-3510 with 10 to 100 mM of each buffer solution (pH 5.0 to 7.0), 1 g of carrier (wet mass) was added to cyclomaltodextrin lucer. 0.01 to 100 mg, preferably 1 to 100 mg, more preferably 5 to 50 mg
What was melt | dissolved in 1 ml of the same buffer is added, and it mixes well. Then 4 to 60 ° C, preferably 10 to 40 ° C
Then, it is left standing for 1 to 24 hours, preferably 2 to 15 hours, or subjected to reciprocal shaking treatment for 0.5 to 10 hours, preferably 1 to 5 hours. The rotation speed of the shaking process is 50 to 1
It is 80 rpm, preferably 100 to 140 rpm.
Then, the treatment liquid is filtered with a filter paper or a glass filter,
Then, wash with water or a buffer solution until the enzyme is not eluted.

In the above embodiment, when cyclomaltodextrin glucanotransferase derived from Bacillus stearothermophilus is used as the enzyme, the amount of the transferase added is 1 g of carrier (wet mass), 0.01-20 mg (specific activity 175 U
/ Mg), preferably 0.1-10 mg.

The immobilized enzyme according to the present invention can be obtained by the above method. At this time, in order to further improve the immobilized enzyme, a porous chitosan bead or a weakly basic anion exchange resin is used as a crosslinking agent. The carrier that has been pretreated with can be used as an immobilized carrier. The use of such a carrier has an advantage that an immobilized enzyme having a higher glycosyl transfer activity in the presence of an organic solvent can be obtained.

The cross-linking agent used in the present invention is not particularly limited, but in the case of cross-linking the porous chitosan beads, a cross-linking agent having the ability to bond with the amino group of the chitosan beads when contacting with the chitosan beads is preferable. When the weakly basic anion exchange resin is crosslinked, it is preferable that the weakly basic anion exchange resin has the ability to bond with the amino group of the weakly basic anion exchange resin when contacted. In this case, examples of the cross-linking agent that can be used for the porous chitosan beads and the weakly basic anion exchange resin include, for example, glutaraldehyde, epichlorohydrin, bisdiazobenzidine, hexamethylene diisocyanate, toluene diisocyanate, and hexamethylene diisoate. Examples thereof include thiocyanate and N, N-ethylene bismaleimide. These crosslinking agents can be used alone or in combination of two or more kinds.
Glutaraldehyde and epichlorohydrin are preferred.

As the pretreatment method, a method of bringing a cross-linking agent into contact with the carrier in water or the above buffer solution can also be used. The concentration of the cross-linking agent is usually 0.0 with respect to the carrier.
The range of 1 to 5 (volume / volume)% is preferable. Chitopearl BCW-3510 as a carrier for porous chitosan beads
And an embodiment of a method of pretreatment when glutaraldehyde is used as a cross-linking agent. Chitopearl BCW-3510 1g (wet mass) 2 (volume / volume)
% Glutaraldehyde (hereinafter, abbreviated as "GLA") solution, and the solution is allowed to penetrate into 2 ml, and 4 to 40 ° C, preferably 10 to 3
It is left standing at 0 ° C. for 0.5 to 3 hours, preferably 1 to 2 hours, or is subjected to reciprocal shaking treatment (50 to 180 rpm, preferably 10 hours) for 0.5 to 3 hours, preferably 1 to 2 hours.
After 0 to 140 rpm), filtration with a filter paper or a glass filter is performed to remove excess GLA and washing, whereby the desired crosslinked carrier can be obtained.

The amount of cyclomaltodextrin glucanotransferase immobilized on the porous chitosan beads in the above-mentioned embodiment varies depending on the treatment conditions and the type of carrier used. For example, GLA-treated chitopearl BCW-3510 is used. When is used as an immobilizing carrier, 0.01 g to 100 m of Chitopearl BCW-3510 (wet mass) in a buffer solution as described above is used.
g, preferably 1 to 100 mg, more preferably 5 to 5
It is 0 mg. If the immobilization amount is less than 0.01 mg, the treatment efficiency may be insufficient.
If the amount exceeds mg, the treatment efficiency corresponding to the immobilized amount may not be obtained.

Similarly, the amount of cyclomaltodextrin glucanotransferase immobilized on the weakly basic anion exchange resin in the above-mentioned embodiment varies depending on the treatment conditions, the type of carrier used, etc. With respect to 1 g (wet mass) of the exchange resin, 0.01 to 100 mg, preferably 1 to 100 m in the buffer solution as described above.
g, more preferably 5 to 50 mg.

In particular, in the above-described embodiment, when cyclomaltodextrin glucanotransferase derived from Bacillus stearothermophilus is used, the immobilization amount is Chitopearl BCW-3510 1 g.
It is 0.01 to 20 mg, preferably 0.1 to 10 mg, based on (wet mass). The immobilization treatment of the enzyme on the immobilization carrier can be performed by standing or shaking treatment under the same conditions as the crosslinking treatment with GLA. At this time, the unadsorbed enzyme can be removed by washing with a filter paper or a glass filter until the enzyme is not eluted.

As an enzyme immobilization method, in addition to the method described above, a porous chitosan bead carrier is packed in a column, and then a 2 (volume / volume)% GLA solution is passed through for 2-3 hours, It is possible to employ a method in which the enzyme solution is immobilized after being thoroughly washed with sterile distilled water and then passed through an enzyme solution.

The immobilized enzyme according to the present invention obtained as described above has almost the same optimum temperature curve as compared with the enzyme before immobilization, but the optimum pH curve should be immobilized. Showed that the activity on the slightly acidic side tended to increase.

The transglycosylation reaction in the method for producing the chromanol glycoside composition of the present invention is carried out, for example, by packing the immobilized enzyme in a column, adjusting the pH and temperature to the above-mentioned ranges, and carrying out the 2-substitution as the substrate. It can be carried out by continuously passing a mixed solution of alcohol, the sugar and the antioxidant through the column. Alternatively, the immobilized enzyme may be directly added to the reaction solution, and the reaction may be performed while dispersing the immobilized enzyme in the reaction solution by using a method such as shaking.

The chromanol-glycoside composition produced by this method is said to be almost colorless because it hardly produces colored by-products because the oxidative decomposition reaction of chromanol-glycoside is effectively suppressed. It has characteristics. Further, in the method of the present invention, the chromanol glycoside composition is a chromanol glycoside in which m is 1 to 6 in the general formula (2), by appropriately adjusting the reaction conditions, the substrate, and the amount of the antioxidant. Contains any of the compositions,
It is characterized in that m is 7 or more and the content ratio of the chromanol glycoside is 3 (mass / volume)% or less. Since chromanol glycosides having m of 7 or more are poorly soluble in water, it is preferable that they are not contained in the composition as much as possible. That point,
The chromanol-glycoside composition produced by the present invention has a high solubility in water because the chromanol-glycoside composition having m of 1 to 6 is the main component. Therefore, according to the production method of the present invention, it is possible to obtain a highly pure chromanol glycoside composition which is nearly colorless and has high water solubility.

[0063]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

<Method for measuring the activity of cyclomaltodextrin glucanotransferase> 5 ml of 0.3 (vol / vol)% soluble starch solution prepared with 0.02 M acetate buffer (pH 5.5) containing 2 mM calcium chloride Then, 0.2 ml of an appropriately diluted enzyme solution was added and reacted at 40 ° C. for 10 minutes. 0.5 ml was taken from the obtained reaction solution,
The reaction was stopped by mixing 15 ml of a 0.02 N sulfuric acid aqueous solution. Further, 0.2 ml of 0.1N potassium iodide solution was added to the reaction solution to develop color, and the absorbance at 660 nm was measured. Under these conditions, soluble starch 15
The amount of enzyme that completely eliminates the coloration of mg of iodine was set to 1 unit (U).

<Preparation of Immobilized Enzyme> (1) GLA Treatment of Carrier BCW, which is porous chitosan beads as an immobilized carrier
-3510 (Fuji Spinning Co., Ltd.) 100 g (wet weight)
2 (vol / vol)% glutaraldehyde (hereinafter G
200 ml of LA) solution was added, and the mixture was shaken (120 rpm) at 30 ° C. for 2 hours. After processing, GL on filter paper
The carrier treated with A was collected, and this filter paper was washed with 3000 ml of 50 mM acetate buffer (pH 5.5) three times with shaking.

(2) Immobilization of cyclomaltodextrin glucanotransferase 100 g of the GLA-treated carrier obtained by the above (1)
(Wet weight), Bacillus stearothermophilus-derived cyclomaltodextrin glucanotransferase, 600 U / ml (protein concentration 8 mg /, dissolved in 50 mM acetate buffer (pH 5.5)) (100 ml) was added, and the mixture was shaken (120 rpm) at 30 ° C. for 2 hours for immobilization. The unadsorbed enzyme was washed by shaking until the enzyme was not eluted with the buffer solution. The immobilized enzyme thus obtained is
600 U of cyclomaltodextrin glucanotransferase was apparently immobilized per 1 g (wet weight) of the carrier.

<Example 1: Chromanol glycoside composition prepared by adding antioxidant to reaction solution> 10 g of dextrin (trade name: Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) and 400 mg of ascorbic acid 50 mM acetate buffer containing 2 mM calcium chloride (p
It was dissolved in H5.5) and the volume was adjusted to 180 ml to obtain a sugar solution. In addition, the following formula (3):

[0068]

[Chemical 8]

1.6 g of 2-substituted alcohol represented by is dissolved in dimethylsulfoxide to make a liquid volume of 20 ml.
A substituted alcohol solution. These sugar solutions and 2
-To the mixed solution of the substituted alcohol solution, 12 g (wet weight) of the immobilized enzyme obtained in the above "Preparation of immobilized enzyme" was added, and the reaction was carried out by shaking at 40 ° C for 20 hours (120 rpm). It was At this time, the transfer rate of the 2-substituted alcohol to the chromanol glycoside mixture was 85.
%Met.

Next, the immobilized enzyme was recovered by filtering the reaction solution, and then the reaction solution was equilibrated with water and filled with a porous synthetic adsorbent (trade name: Diaion HP21, Mitsubishi Chemical Corporation). The solution was passed through the column. The column was washed with 3 column volumes of water. As a result, the mixture of 2-substituted alcohol and chromanol glycoside was adsorbed on the porous synthetic adsorbent, and sugar, ascorbic acid, salts and the like were discharged without being adsorbed. Then, the chromanol glycoside alone was eluted by washing the column with 3 times the column volume of 60 (vol / vol)% methanol. Then, 2.9 g of the chromanol glycoside composition (1) was obtained by concentrating and drying this fraction.

<Example 2: Chromanol glycoside composition prepared by adding an antioxidant to the reaction solution and further reducing dissolved oxygen> Dextrin (trade name: Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) ) 10 g and 400 mg ascorbic acid in 50 m containing 2 mM calcium chloride
Dissolved in M acetate buffer (pH 5.5),
1 to give a sugar solution. Separately, 2 represented by the above formula (3)
-Substituted alcohol (1.6 g) was dissolved in dimethyl sulfoxide to adjust the liquid volume to 20 ml to give a 2-substituted alcohol solution. To the mixed solution of these sugar solution and 2-substituted alcohol solution, 12 g (wet weight) of the immobilized enzyme obtained in the above “Preparation of immobilized enzyme” was added, and further nitrogen gas (100 ml / min) was added to 15 After the dissolved oxygen in the reaction solution was reduced by aeration for minutes, the reaction was carried out by shaking at 40 ° C. for 20 hours (120 rpm). At this time, the transfer rate of the 2-substituted alcohol to the chromanol glycoside mixture was 85%.

Next, after the immobilized enzyme was recovered by filtering this reaction solution, a porous synthetic adsorbent (trade name: Diaion HP21, Mitsubishi Chemical Co., Ltd.) in which the reaction solution was equilibrated with water was filled. The solution was passed through the column. The column was washed with 3 column volumes of water. As a result, the mixture of the 2-substituted alcohol and the chromanol glycoside was adsorbed on the porous synthetic adsorbent, and the sugars and salts flowed out without being adsorbed. Then, the chromanol glycoside alone was eluted by washing the column with 3 times the column volume of 60 (vol / vol)% methanol. Then, the chromanol glycoside composition (2) is obtained by concentrating and drying this fraction.
Was obtained.

<Comparative Example 1: Preparation of chromanol glycoside composition> 10 g of dextrin (trade name: Paindex # 1, manufactured by Matsutani Chemical Co., Ltd.) in 50 mM acetate buffer (pH 5.5) containing 2 mM calcium chloride. To 180 ml to obtain a sugar solution. Separately, 1.6 g of the 2-substituted alcohol represented by the above formula (3) was dissolved in dimethyl sulfoxide to adjust the liquid volume to 20 ml to give a 2-substituted alcohol solution. To the mixed solution of these sugar solution and 2-substituted alcohol solution, add 12 g (wet weight) of the immobilized enzyme obtained in the above-mentioned “Preparation of immobilized enzyme” and shake at 40 ° C. for 20 hours (120 rpm). The reaction was carried out. At this time, the transfer rate of the 2-substituted alcohol to the chromanol glycoside mixture was 87%.

Next, the immobilized enzyme was recovered by filtering this reaction solution, and then the reaction solution was equilibrated with water and filled with a porous synthetic adsorbent (trade name: Diaion HP21, Mitsubishi Chemical Corporation). The solution was passed through the column. The column was washed with 3 column volumes of water. As a result, the mixture of 2-substituted alcohol and chromanol glycoside was adsorbed on the porous synthetic adsorbent, and sugars, salts and the like flowed out without being adsorbed. The column is then loaded with three column volumes of 60
Only chromanol glycosides were eluted by washing with (volume / volume)% methanol. Then, 2.9 g of the chromanol glycoside composition (3) was obtained by concentrating and drying this fraction.

<Test of Coloring Degree> Each chromanol glycoside composition prepared in Examples 1 and 2 and Comparative Example 1 was dissolved in distilled water to prepare 10 (mass / volume)% aqueous solutions. In order to evaluate the coloring degree of these aqueous solutions, 38
The absorbance at 0 nm was measured. The results are shown in Table 1.

[0076]

[Table 1]

As is clear from Table 1, Example 1 reacted in the presence of an antioxidant and the chromanol glycoside composition obtained by the method of Example 2 in which the dissolved oxygen concentration was further reduced were The absorbance was lower than that obtained by the method of Comparative Example 1 in which the oxidation reaction was not suppressed.

Furthermore, the chromanol glycoside compositions obtained in Examples 1 and 2 and Comparative Example 1 were subjected to HPLC analysis (column: Asahipak NH2P (4.6 × 250 m).
m), eluent: acetonitrile: water (70:30, (volume / volume)), detection: UV290 nm), and electrospray mass ionization mass spectrometry analysis (ESI / MS) analysis results showed that the above chromanol glycosides were present. The chromanol glycoside contained in the body composition is a chromanol glycoside corresponding to the present invention, and the content of the chromanol glycoside of m = 7 and 8 in the general formula (2) is 3 (mass / volume). )%, And the obtained chromanol glycoside composition was found to be a high-purity chromanol glycoside containing chromanol glycosides with m = 1 to 6 as a main component.

From the above results, it was found that the method of the present invention provides a highly pure chromanol glycoside composition which is nearly colorless and highly soluble in water.

[0080]

EFFECTS OF THE INVENTION According to the method for producing a chromanol glycoside composition of the present invention, the action of an antioxidant suppresses the oxidative decomposition reaction of the chromanol glycoside, prevents coloring due to by-product formation, and is nearly colorless. A chromanol glycoside composition can be obtained. Furthermore, by reducing the dissolved oxygen in the reaction solution in addition to the antioxidant, the oxidative decomposition reaction of the chromanol glycoside can be further suppressed. Therefore, the chromanol-glycoside composition obtained by the production method of the present invention is nearly colorless and has high purity, and therefore can be preferably used for various applications without further purification treatment.

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Claims (5)

[Claims] 1. The following general formula (1): (In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom or a lower alkyl group, and R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group,
n represents an integer of 0 to 4), a 2-substituted alcohol represented by the formula (4) and a sugar are reacted in the presence of an enzyme that catalyzes a corresponding transglycosylation action, and the reaction is carried out in the presence of an antioxidant. The following general formula (2) is characterized by being carried out in the coexistence (In the formula, R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom or a lower alkyl group, and R ⁵ represents a hydrogen atom, a lower alkyl group or a lower acyl group,
X represents a monosaccharide residue or an oligosaccharide residue in which a hydrogen atom of a hydroxyl group in the sugar residue may be substituted with a lower alkyl group or a lower acyl group, n is an integer of 0 to 4, and m Is an integer of 1 to 10). 2. The production method according to claim 1, wherein the dissolved oxygen concentration in the reaction solution is further reduced. 3. The antioxidant is water soluble.
Or the manufacturing method according to 2. 4. The antioxidant is ascorbic acid,
The manufacturing method according to claim 3. 5. The enzyme which catalyzes the corresponding glycosyl transfer activity is an immobilized enzyme in which cyclomaltodextrin glucanotransferase is immobilized on porous chitosan beads or a weakly basic anion exchange resin. ~ 4
The manufacturing method according to any one of 1.