WO2015152145A1

WO2015152145A1 - Process for producing crystalline 2-o-α-d-glucosyl-l-ascorbic acid powder

Info

Publication number: WO2015152145A1
Application number: PCT/JP2015/059900
Authority: WO
Inventors: 敦史長井; 晃典梅山
Original assignee: 東洋精糖株式会社
Priority date: 2014-03-31
Filing date: 2015-03-30
Publication date: 2015-10-08
Also published as: JPWO2015152145A1; TW201620922A

Abstract

The present invention provides a process for producing a crystalline 2-O-α-D-glucosyl-L-ascorbic acid (AG) powder, the process making it possible to more improve the recovery than in conventional processes, shorten the time required for the steps, and make dissolution operation of the obtained powder easier. This process for producing a crystalline AG powder comprises the following steps (a) to (d): step (a) in which a glycosyltransferase is made to act on a solution containing both L-ascorbic acid and an α-glucosyl-sugar compound and a glucoamylase is subsequently made to act thereon, thereby preparing an AG-containing solution; step (b) in which the AG-containing solution is purified to obtain an aqueous solution of a purification product having an AG content of 90 mass% or higher in terms of anhydride amount; step (c) in which the aqueous solution of a purification product is mixed with an organic solvent and thereafter the temperature of the mixed solution is lowered to cause crystals of AG to separate out; and step (d) in which the AG crystals that have separated out are recovered and the recovered crystals are dried to obtain a crystalline AG powder.

Description

Method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder

The present invention relates to a method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder, and more specifically, compared to the conventional method, 2-O-α-D-glucosyl-L-ascorbic acid crystal powder The present invention relates to a production method excellent in recovery rate and dissolution workability of the recovered product.

L-ascorbic acid has been conventionally used for various uses including foods and drinks and cosmetics because of its excellent physiological activity and antioxidant action. On the other hand, L-ascorbic acid is unstable because of its direct reduction property, and has a great disadvantage that it is susceptible to oxidative degradation and easily loses its physiological activity. In order to eliminate the disadvantages of L-ascorbic acid, 2-O-α-D-glucosyl-L-ascorbic acid in which one molecule of D-glucose is bonded to the hydroxyl group at the 2-position of L-ascorbic acid (in the present specification, "Ascorbic acid 2-glucoside" is sometimes abbreviated). This 2-O-α-D-glucosyl-L-ascorbic acid does not show direct reducing properties, is stable, and in vivo, L-ascorbic acid and D-glucose are produced by enzymes originally present in the living body. It has an epoch-making characteristic that it is decomposed into L-ascorbic acid and exhibits its original physiological activity. In addition, many uses of 2-O-α-D-glucosyl-L-ascorbic acid have been developed. Conventionally, as a food material, food additive material, cosmetic material, quasi drug material, or pharmaceutical material, In addition to the use of L-ascorbic acid, L-ascorbic acid is unstable, so that it has been widely used in other applications where L-ascorbic acid could not be used conventionally.

2-O-α-D-glucosyl-L-ascorbic acid is generally added to a solution containing L-ascorbic acid and an α-glucosyl sugar compound in a cyclomaltodextrin glucanotransferase (hereinafter referred to as “CGTase”). Or a glycosyltransferase such as α-glucosidase, or by reacting such a glycosyltransferase and glucoamylase (Patent Document 1). In addition, crystals of 2-O-α-D-glucosyl-L-ascorbic acid can be crystallized from a supersaturated aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid. α-D-glucosyl-L-ascorbic acid and a powder containing 2-O-α-D-glucosyl-L-ascorbic acid crystals containing the same have also been developed (Patent Document 2).

Furthermore, various technologies related to ascorbic acid 2-glucoside and its production method are still under development. For example, Patent Document 3 discloses 2-O-α-D-glucosyl-L-ascorbic acid hydrous crystals that can coexist with 2-O-α-D-glucosyl-L-ascorbic anhydride crystals and a method for producing the same. It is disclosed. Patent Document 4 discloses a specific controlled cooling method (controlled cooling method or pseudo control) from a solution containing more than 86% by mass of 2-O-α-D-glucosyl-L-ascorbic acid in terms of anhydride. A manufacturing method is disclosed in which anhydrous crystals are precipitated by a cooling method) to obtain 2-O-α-D-glucosyl-L-ascorbic acid anhydrous crystal-containing powder that is harder to solidify than conventional products.

Japanese Patent Laid-Open No. 3-139288 Japanese Unexamined Patent Publication No. 3-135992 International Publication WO2012 / 033218 Pamphlet JP 2013-55932 A

Various methods for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder based on the enzymatic method have been developed, but there is room for improvement in improving the recovery rate and shortening the process time. was there. There is also room for improvement in improving the workability of dissolving 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the enzymatic method.

The present invention can improve the recovery rate compared to the prior art, reduce the process time, and improve the workability of dissolving the resulting powder. 2-O-α-D-glucosyl-L- It is an object of the present invention to provide a method for producing ascorbic acid crystal powder.

The inventors have mixed an aqueous solution of purified 2-O-α-D-glucosyl-L-ascorbic acid having a purity of a specific value or more with an organic solvent such as ethanol having compatibility with water, By lowering the crystallization, 2-O-α-D-glucosyl-L-ascorbic acid crystals can be obtained at a higher recovery rate and in a shorter process time than the conventional method using only water for crystallization. I found out that I can. In addition, the obtained 2-O-α-D-glucosyl-L-ascorbic acid crystal powder has a higher workability for dissolution than conventional products, and as a factor, the jet properties and specific surface area defined by specific physical properties are improved. It has also been found that 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the method of the present invention can be distinguished from conventional products as substances.

That is, the present invention includes the following inventions.

[1]
A process for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder, comprising the following steps (a) to (d):
(A) 2-O-α-D-glucosyl-L-ascorbine obtained by allowing a glycosyltransferase to act on a solution containing L-ascorbic acid and an α-glucosyl sugar compound and then causing glucoamylase to act Preparing an acid-containing solution;
(B) The 2-O-α-D-glucosyl-L-ascorbic acid-containing solution is purified so that the content of 2-O-α-D-glucosyl-L-ascorbic acid is 90% by mass in terms of anhydride. A step of obtaining an aqueous solution of the purified product as described above;
(C) a step of crystallizing 2-O-α-D-glucosyl-L-ascorbic acid crystals by mixing an aqueous solution of the purified product and an organic solvent and then lowering the temperature of the solution;
(D) The crystallized 2-O-α-D-glucosyl-L-ascorbic acid crystal is recovered, and the recovered crystal is dried to give 2-O-α-D-glucosyl-L- A step of obtaining ascorbic acid crystal powder.

[2]
The method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder according to [1], wherein the organic solvent in the step (c) is an alcohol solvent or a ketone solvent.

[3]
In the step (c), 2-O-α-D-glucosyl-L according to [1] or [2], wherein 10 to 500 mass% of the organic solvent is mixed with the aqueous solution of the purified product. -Method for producing ascorbic acid crystal powder.

[4]
2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the production method according to any one of [1] to [3].

According to the present invention, 2-O-α-D-glucosyl-L-ascorbic acid is obtained by crystallization after mixing an organic solvent with an aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid. Rather than crystallizing from an aqueous solution, the recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals can be increased and the process time can be shortened, thereby producing a production method with excellent mass productivity. Can be provided. Further, according to the present invention, it is possible to provide 2-O-α-D-glucosyl-L-ascorbic acid crystal powder having improved jetting properties and specific surface area, and thereby excellent workability in dissolution.

<Method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder>
The method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention basically includes the following steps (a) to (d):
A process for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder, comprising the following steps (a) to (d):
(A) 2-O-α-D-glucosyl-L-ascorbine obtained by allowing a glycosyltransferase to act on a solution containing L-ascorbic acid and an α-glucosyl sugar compound and then causing glucoamylase to act A step of preparing an acid-containing solution (enzyme treatment step);
(B) The 2-O-α-D-glucosyl-L-ascorbic acid-containing solution is purified so that the content of 2-O-α-D-glucosyl-L-ascorbic acid is 90% by mass in terms of anhydride. A step of obtaining an aqueous solution of the purified product as described above (purification step);
(C) A step of crystallizing 2-O-α-D-glucosyl-L-ascorbic acid crystals by mixing an aqueous solution of the purified product and an organic solvent and then lowering the temperature of the solution (crystallization) Process);
(D) The crystallized 2-O-α-D-glucosyl-L-ascorbic acid crystal is recovered, and the recovered crystal is dried to give 2-O-α-D-glucosyl-L- A step of obtaining ascorbic acid crystal powder (drying step).

(A) Enzyme treatment step The enzyme treatment step (a) was obtained by allowing a glycosyltransferase to act on a solution containing L-ascorbic acid and an α-glucosyl sugar compound, and then allowing glucoamylase to act. This is a step of preparing a solution containing —O-α-D-glucosyl-L-ascorbic acid. A method for preparing such a solution containing 2-O-α-D-glucosyl-L-ascorbic acid is known, and various manufacturing methods such as an improved method further including preferable requirements other than the above basic requirements are used. can do.

“L-ascorbic acid” used as one of the raw materials of 2-O-α-D-glucosyl-L-ascorbic acid (substrate for enzyme reaction) may be an alkali metal salt even if it is in the form of a hydroxy acid. They may be in the form of a metal salt such as an alkaline earth metal salt or a mixture thereof.

The “α-glucosyl sugar compound” used as the other raw material of 2-O-α-D-glucosyl-L-ascorbic acid can be converted from L-ascorbic acid to 2-O-α by simultaneously acting on a glycosyltransferase. There is no particular limitation as long as it can generate -D-glucosyl-L-ascorbic acid. Examples of such α-glucosyl sugar compounds include malto-oligosaccharides such as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, dextrin, cyclodextrin, and amylose. Starch hydrolyzate, liquefied starch (potato starch, sweet potato starch, tapioca starch, corn starch, wheat starch and other liquid starch obtained by the action of heat-resistant α-amylase), gelatinized starch (starch) , Starch obtained by suspending particles in water and heating them, and starch that has been changed into a gel by disintegrating particles), soluble starch (starch obtained by solubilizing starch in water by acid treatment and lowering the viscosity of the solution) Etc.

“Glycosyltransferase” used in the first stage enzyme reaction (hereinafter referred to as “first enzyme reaction”) converts a sugar residue derived from an α-glucosyl sugar compound into a hydroxyl group at the 2-position of L-ascorbic acid. It is an enzyme that has the effect of transferring and binding. Examples of such glycosyltransferases include α-glucosidase (EC 3.2.1.20), cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), α-amylase (EC 3.2.1.1) and the like. As CGTase, those derived from various organisms, particularly microorganisms, are known, and appropriate ones can be selected and used. CGTase may have a natural amino acid sequence derived from such an organism, or may have an amino acid sequence obtained by modifying the natural amino acid sequence such as substitution, addition (insertion), or deletion. Thing (mutant) may be sufficient.

Depending on the type of glycosyltransferase, the type of α-glucosyl sugar compound that can transfer sugar efficiently varies. When α-glucosidase is used as the glycosyltransferase, the α-glucosyl sugar compound includes malto-oligosaccharides such as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, or the like. DE value (Dextrose Equivalent: A value obtained by setting the reducing power of a sugar solution such as starch per solid when the glucose is 100, and the larger the value, the higher the degree of degradation and the lower the molecular weight. ) About 5 to 60 dextrins, partial hydrolysates of starch and the like are preferred. When CGTase is used as the glycosyltransferase, the α-glucosyl sugar compound is preferably cyclodextrin or a partially hydrolyzed starch from starch gelatinized product having a DE value of less than 1 to dextrin having a DE value of about 60. When α-amylase is used as the glycosyltransferase, the α-glucosyl sugar compound is preferably a starch hydrolyzate from starch gelatinized product having a DE value of less than 1 to dextrin having a DE value of about 30.

“Glucoamylase” (EC 3.2.1.3) used in the second stage enzyme reaction (hereinafter referred to as “second enzyme reaction”) is a by-product produced by the action of the glycosyltransferase used in the first enzyme reaction. A compound in which two or more -D-glucosyl groups are linked to a sugar residue bonded to the hydroxyl group at the 2-position of L-ascorbic acid is converted to a 2-O- with only one α-D-glucosyl group bonded An enzyme for converting to α-D-glucosyl-L-ascorbic acid, which has an action of hydrolyzing the sugar residue of the by-product to reduce the number of α-D-glucosyl groups to one It is. At the same time, the glucoamylase also has an action of hydrolyzing the unreacted α-glucosyl sugar compound remaining in the reaction solution to convert it into D-glucose, whereby the subsequent purification step (b ) Can be easily removed. As glucoamylase, those derived from various organisms, particularly microorganisms, are known, and appropriate ones can be selected and used.

When CGTase or α-amylase is used as a glycosyltransferase, the number of α-D-glucosyl groups bound to the hydroxyl group at the 2-position of L-ascorbic acid tends to be larger than when α-glucosidase is used. is there. For example, when CGTase is used, the number of α-D-glucosyl groups to be bound is distributed to about 1 to 7, that is, the desired 2-O-α-D-glucosyl-L-ascorbic acid (bound α- The number of D-glucosyl groups is not only 1) but also 2-O-α-D-maltosyl-L-ascorbic acid (same as 2), 2-O-α-D-maltotriosyl-L-ascorbic acid (same as above) 3), 2-O-α-D-maltotetraosyl-L-ascorbic acid (same 4), 2-O-α-D-maltopentaosyl-L-ascorbic acid (same 5), 2-O A mixture containing by-products such as -α-D-maltohexaosyl-L-ascorbic acid (same as 6) and 2-O-α-D-maltoheptaosyl-L-ascorbic acid (same as 7) is produced. . When α-amylase is used, a similar mixture is produced although the distribution is somewhat narrower than when CGTase is used. Therefore, even when an enzyme that produces such a by-product is used as a glycosyltransferase, 2-O-α-D-glucosyl-L-ascorbic acid intended for the by-product can be obtained by using glucoamylase in combination. The purity of 2-O-α-D-glucosyl-L-ascorbic acid can be improved. It is obtained in the subsequent step (b) that the by-product is converted to 2-O-α-D-glucosyl-L-ascorbic acid as much as possible, that is, the residual amount of the by-product is kept as low as possible. It is suitable for increasing the purity of the purified product to a specific value or higher.

The concentration of a solution containing L-ascorbic acid and an α-glucosyl sugar compound (usually an aqueous solution) in which the first enzyme reaction is performed is usually the sum of L-ascorbic acid and α-glucosyl sugar compound as substrates. Is 1 to 40% by mass. The mass ratio of L-ascorbic acid and α-glucosyl sugar compound is usually from 2: 8 to 7: 3. When the amount of α-glucosyl sugar compound increases beyond the above range, sugar transfer to L-ascorbic acid proceeds efficiently, but is limited by the low initial concentration of L-ascorbic acid. The production rate of —O-α-D-glucosyl-L-ascorbic acid remains low. On the contrary, if the amount of L-ascorbic acid increases beyond the above range, the amount of unreacted L-ascorbic acid increases, which is inefficient for industrial production.

The amount of glycosyltransferase used for the first enzyme reaction is usually 1 to 500 units per 1 g of the total amount of L-ascorbic acid and α-glucosyl sugar compound as substrates. Such an amount of glycosyltransferase is added to a solution containing L-ascorbic acid and an α-glucosyl sugar compound. Usually, the pH of the solution is kept at 3 to 10 and the temperature is kept at 30 to 70 ° C. The first enzyme reaction can be carried out by allowing the reaction to proceed for at least hours, preferably 12 to 96 hours.

The amount of glucoamylase used for the second enzyme reaction can be adjusted according to the amount of by-products generated by the first enzyme reaction as described above. When carried out, the amount is usually 100 to 5000 units per gram of the total amount of α-glucosyl sugar compound as a substrate. Glucoamylase is added to the reaction solution after heating the reaction solution of the first enzyme reaction to deactivate the glycosyltransferase to stop the first enzyme reaction. To save energy and time required for cooling the heated enzyme reaction solution, use a glucoamylase that can exhibit practical enzyme activity even at a relatively high temperature, for example, 40 to 60 ° C. Is preferred. Usually, the second enzyme is prepared by maintaining the pH of the reaction solution to which glucoamylase has been added at 2.5 to 6.5 and the temperature at 20 to 75 ° C., and allowing the reaction to proceed for 2 hours or more, preferably 4 to 12 hours. The reaction can be performed.

(B) Purification step The purification step (b) is performed by purifying the 2-O-α-D-glucosyl-L-ascorbic acid-containing solution prepared in step (a) to obtain 2-O-α-D-glucosyl. This is a step of obtaining an aqueous solution of a purified product having a content of L-ascorbic acid of 90% by mass or more in terms of anhydride.

In addition to the produced 2-O-α-D-glucosyl-L-ascorbic acid, the reaction solution after the completion of the first and second enzyme reactions was used as a raw material and remained unreacted L-ascorbic acid And α-glucosyl sugar compounds and by-products as described above are mixed. By removing such unreacted products and by-products from the reaction solution, a purified product having a content of 2-O-α-D-glucosyl-L-ascorbic acid of 90% by mass or more in terms of anhydride is obtained. An aqueous solution can be prepared.

The purity of the purified 2-O-α-D-glucosyl-L-ascorbic acid in the solution containing 2-O-α-D-glucosyl-L-ascorbic acid (that is, 2-O— in the purified product). The content of α-D-glucosyl-L-ascorbic acid is calculated from quantitative values based on the peak areas of each component drawn in the chromatogram, which is created, for example, in column chromatography for the purification process described below. can do. Purity [%] = (peak area of anhydride of 2-O-α-D-glucosyl-L-ascorbic acid) / (sum of peak areas of all components). At this time, regarding the peak area representing the hydrate of 2-O-α-D-glucosyl-L-ascorbic acid, the molecular weight of (2-O-α-D-glucosyl-L-ascorbic anhydride) / By multiplying by (molecular weight of 2-O-α-D-glucosyl-L-ascorbic acid hydrate) and converting, it is regarded as an anhydride of 2-O-α-D-glucosyl-L-ascorbic acid. Include in purity calculation.

The method used for the purification treatment is not particularly limited, but in general, the reaction solution after the completion of the first and second enzyme reactions is first decolorized and filtered with activated carbon or the like, and then the filtrate is dehydrated with a cation exchange resin. After salting, an aqueous solution of a purified product having a predetermined purity or higher is obtained by a purification treatment using column chromatography for removing the unreacted materials and the like.

Examples of the column chromatography include column chromatography using an anion exchange resin for removing an α-glucosyl sugar compound such as D-glucose among unreacted substances, followed by L-ascorbic acid among unreacted substances. It is preferable to perform column chromatography using a cation exchange resin or a porous resin for removal. This order may be reversed, followed by column chromatography using a cation exchange resin or porous resin to remove L-ascorbic acid, followed by an anion to remove an α-glucosyl sugar compound such as D-glucose. Column chromatography using an exchange resin can also be performed. Methods for purifying 2-O-α-D-glucosyl-L-ascorbic acid by column chromatography using such anion exchange resin, cation exchange resin or porous resin are known, and various methods relating to these column chromatography are known. Conditions such as the concentration of the reaction solution loaded on the column (in terms of solid content), the loading amount of the reaction solution with respect to the resin (volume ratio), the linear velocity and amount of purified water that passes as the eluent, and the like can be adjusted as appropriate. .

(C) Crystallization step The crystallization step (c) is a mixture of the purified aqueous solution obtained in the purification step (b) and an organic solvent, and then lowering the temperature of the solution to give 2-O- This is a step of crystallizing α-D-glucosyl-L-ascorbic acid crystals. By including this step in the method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder, high-purity 2-O-α-D-glucosyl-L-ascorbic acid crystals are highly recovered. The reason is that the organic solvent vaporizes more easily than water, so that the time required for the subsequent drying step (d) can be shortened.

The aqueous solution of the purified product obtained in the purification step (b) is usually concentrated by reducing water before mixing with an organic solvent, and 2-O-α-D-glucosyl-L-ascorbic acid is in a supersaturated state. To be. This concentration treatment can be performed by heating and / or decompression according to a conventional method. The degree of supersaturation of 2-O-α-D-glucosyl-L-ascorbic acid depends on the concentration and temperature of the 2-O-α-D-glucosyl-L-ascorbic acid aqueous solution, but is usually 1.05-1. .50 is adjusted. For example, such supersaturation can be achieved by concentrating an aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid to a concentration of 65 to 85% by mass within a temperature range of 30 to 45 ° C.

The mixing of the supersaturated aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid with the organic solvent may be performed by adding the latter to the former or by adding the former to the latter. Moreover, the addition method may be performed by any method of batch addition and sequential addition. In the case of sequential addition, it may be added by continuous dripping or may be divided and sequentially added.

As an “organic solvent” for mixing with an aqueous solution of a purified product, miscibility (compatibility) with a supersaturated aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid is good, and 2-O-α Those which are hardly soluble in -D-glucosyl-L-ascorbic acid crystals are preferred. Examples of organic solvents having such properties include alcohol solvents such as methanol, ethanol, propanol, isopropanol, and butanol; ketone solvents such as acetone and methyl ethyl ketone; ethylene glycol, propylene glycol, butylene glycol, polyethylene glycol, and polypropylene. Glycol solvents such as glycol and polyoxyethylene polyoxypropylene copolymers; Ether alcohol solvents such as monomethyl ether, monoethyl ether, monopropyl ether, monoisopropyl ether and monobutyl ether of the glycol solvents; Dimethyl ether, diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, methyl ethyl ether, Le propyl ether, methyl isopropyl ether, methyl butyl ether, ethyl propyl ether, ethyl isopropyl ether, polyether solvents such as ethyl ether; and acetonitrile, and a nitrile-based solvent such as propionitrile. Among these, organic solvents such as alcohol solvents and ketone solvents are easily available, and the recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals is improved and the crystallization time is shortened. Since it is excellent in the effect of this invention, it is preferable. Of these, methanol and ethanol, particularly ethanol, are not only excellent in the action and effect of the present invention, but also 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the production method of the present invention. From the viewpoint of safety when used in foods, cosmetics, quasi drugs, pharmaceuticals and the like, it can be said to be a suitable organic solvent.

The amount of the organic solvent used depends on the effects of the present invention, that is, the recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals, the time of this step, and finally 2-O-α- It can be set in consideration of the type of organic solvent and other conditions so that the effect of improving the dissolution workability of D-glucosyl-L-ascorbic acid crystal powder can be obtained, but 2-O-α- The amount is preferably 10 to 500% by mass, more preferably 50 to 300% by mass, based on the (supersaturated) aqueous solution of a purified product of D-glucosyl-L-ascorbic acid.

The temperature at which 2-O-α-D-glucosyl-L-ascorbic acid crystals are crystallized by mixing a supersaturated aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid and an organic solvent is particularly limited. However, since the thermal stability of 2-O-α-D-glucosyl-L-ascorbic acid crystals is poor, it is preferably 60 ° C. or lower, more preferably 50 ° C. or lower. For example, a mixed solution of 2-O-α-D-glucosyl-L-ascorbine supersaturated aqueous solution and an organic solvent is preferably heated in the range of 30 to 60 ° C., more preferably in the range of 30 to 50 ° C. The 2-O-α-D-glucosyl-L-ascorbic acid crystal may be crystallized while the mixed solution is cooled.

The cooling method of the mixed solution of 2-O-α-D-glucosyl-L-ascorbic acid supersaturated aqueous solution and organic solvent is not particularly limited. For example, the temperature of the mixed solution is slowly lowered by natural cooling. What should I do? The necessary crystallization time is usually 12 hours or less, preferably 24 hours or less, more preferably 36 hours or less.

Further, when the 2-O-α-D-glucosyl-L-ascorbic acid supersaturated aqueous solution and the organic solvent are mixed, a seed crystal of 2-O-α-D-glucosyl-L-ascorbic acid may be added. Good. For example, before mixing with an organic solvent, a seed crystal is added to a supersaturated aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid to partially crystallize it, and then mixed with the organic solvent. Good.

The addition amount of the seed crystal is not particularly limited, but is preferably 0.01 to 5.0% by mass with respect to the 2-O-α-D-glucosyl-L-ascorbic acid supersaturated aqueous solution. The content is preferably 0.1 to 1.0% by mass.

(D) Drying step In the drying step (d), the crystallized 2-O-α-D-glucosyl-L-ascorbic acid crystal obtained in the crystallization step (c) was recovered and recovered. This is a step of obtaining 2-O-α-D-glucosyl-L-ascorbic acid crystal powder by drying the crystal.

The 2-O-α-D-glucosyl-L-ascorbic acid crystals are obtained by centrifuging the mass kit (mixture of crystals and molasses) obtained in the crystallization step (c) according to a conventional honeydness method. The —O-α-D-glucosyl-L-ascorbic acid crystals can be recovered by separating from the mass kit. The recovered 2-O-α-D-glucosyl-L-ascorbic acid crystals are usually used to remove a small amount of purified water or ethanol, for example, recovered crystals, in order to remove amorphous molasses adhering to the surface. It is cleaned by spraying (showering) 30% by mass or less of purified water.

The collected and washed 2-O-α-D-glucosyl-L-ascorbic acid crystals are kept in a predetermined temperature and humidity atmosphere for a certain period of time and dried to give 2-O-α-D-glucosyl- L-ascorbic acid crystal powder is obtained. The temperature, humidity and time conditions for this drying treatment are such that a 2-O-α-D-glucosyl-L-ascorbic acid crystal powder having a quality (for example, crystallinity) according to the application can be obtained. It can adjust suitably, considering the kind of organic solvent used at the crystallization process (c). Preferred conditions for the drying treatment include a temperature of 20 to 55 ° C., a humidity (relative humidity) of 60 to 90%, and a time of 3 to 20 hours. Moreover, you may reduce pressure as needed for a drying process.

The concentration of the organic solvent remaining in the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the production method of the present invention is usually 0.1 to 10,000 ppm, preferably 1 to 5000 ppm, More preferably, it is 10 to 1000 ppm. The concentration of the organic solvent remaining in the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder can be measured by a gas chromatography method (measurement conditions are described in the Examples).

The 2-O-α-D-glucosyl-L-ascorbic acid crystal powder that has undergone the drying treatment is then allowed to naturally cool to room temperature or forcedly cooled by blowing clean air at about room temperature.

<2-O-α-D-glucosyl-L-ascorbic acid crystal powder and its use>
2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the production method of the present invention (hereinafter referred to as “2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention”). ) Is a 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by a conventional production method (hereinafter referred to as “conventional 2-O-α-D-glucosyl-L-ascorbic acid crystal powder”. ”(When the conditions are the same except that the organic solvent is not added in the crystallization process), the dissolution workability is excellent. For example, when the crystal powder is added to a solvent such as water and mixed. It can be dissolved in a short time.

Also, the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention is different in physical properties from the conventional 2-O-α-D-glucosyl-L-ascorbic acid crystal powder. For example, the difference angle (= rest angle−collapse angle), which is one of the physical properties related to jet properties, is larger in the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention, thereby Dr. is known as a method for evaluating powder physical properties (jet properties, fluidity, etc.). The Carr's index (jetability index) is high, and it tends to be excellent in the evaluation of jet characteristics. Various powder physical properties related to jet properties (and fluidity) can be measured according to the powder test method using “Powder Tester PT-X” (Hosokawa Micron Corporation), and the measured values are used. Dr. Carr's index and rating can be determined.

Further, the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention tends to have a larger specific surface area than the conventional 2-O-α-D-glucosyl-L-ascorbic acid crystal powder. It is in.

The physical properties of the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention are as follows. The range of the angle of repose is usually 58.0 ° to 65.0 °, preferably 59.5 ° to 63.0 °. The range of the difference angle is usually 10.0 ° to 25.0 °, preferably 12.0 to 20.0 °. The range of the jetability index is usually 41 to 56, preferably 43 to 51. The range of the specific surface area is usually 0.50 to 1.00, preferably 0.60 to 0.90.

Such 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention is similar to the conventional 2-O-α-D-glucosyl-L-ascorbic acid crystal powder in foods, cosmetics, Has applications in quasi drugs, pharmaceuticals and other fields. In particular, the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder of the present invention is excellent in dissolution workability when an aqueous solution is prepared and used in the manufacturing process of various products. -Α-D-glucosyl-L-ascorbic acid crystal powder is suitable as a raw material for products produced by adding an aqueous solution.

In the description of the following examples, “part” means “part by mass” and “%” means “% by mass”.

[Example 1]
(A) Enzyme treatment step 2.5 parts of starch hydrolyzate was added to 6 parts of water and dissolved by heating. Further, 1.5 parts of L-ascorbic acid was added to adjust the pH to 5 to obtain a substrate solution. To this was added 150 units of CGTase enzyme solution (NOVOZYMES) per gram of starch hydrolyzate, reacted at 50 ° C. for 40 hours, and 2-O-α-D together with 2-O-α-D-glucosyl-L-ascorbic acid. Producing α-glycosyl-L-ascorbic acid such as maltosyl-L-ascorbic acid, 2-O-α-maltotriosyl-L-ascorbic acid, 2-O-α-maltotetraosyl-L-ascorbic acid, etc. It was. The enzyme reaction of the reaction solution was stopped, and a glucoamylase agent (Amano Enzyme Co., Ltd., trade name “Glucoamylase Amano for Sake Brewing” 250,000 units / g) was added to this 600 units per gram of starch hydrolyzate. After treatment at 4 ° C. for 4 hours, α-glycosyl-L-ascorbic acid was decomposed to 2-O-α-D-glucosyl-L-ascorbic acid, and the mixed carbohydrate was decomposed to D-glucose.

(B) Purification step The reaction solution obtained in the above step (a) after stopping the enzyme reaction was decolorized and filtered with activated carbon. The concentration of the obtained 2-O-α-D-glucosyl-L-ascorbic acid-containing aqueous solution after decolorization was 46.5%. The filtrate was desalted by cation exchange resin (H ⁺ form), then anion exchange resins ^- a L- ascorbic acid (OH-type) was removed thereby preferentially adsorbed, and then 2-O-α-D- glucosyl -L- ascorbate anion exchange resin ^- is adsorbed on (OH type), after removing the washing to D- glucose, and eluted with 0.1N aqueous sodium hydroxide solution. Further, the eluate was concentrated to a solid content of about 20% and desalted with a cation exchange resin (H ⁺ type). The purity of 2-O-α-D-glucosyl-L-ascorbic acid obtained in the above step was 91% in terms of anhydride.

(C) Crystallization step 85 parts of an aqueous solution containing 2-O-α-D-glucosyl-L-ascorbic acid obtained in the above step (b) (including 2-O-α-D-glucosyl-L-ascorbic acid) 39.5 parts in terms of anhydride) was heated and concentrated under reduced pressure, and the water was distilled off to obtain 60 parts of an aqueous solution having a 2-O-α-D-glucosyl-L-ascorbic acid concentration of 66%. It was.

The obtained aqueous solution was heated to 40 ° C. with stirring, and 50 parts of ethanol and 0.15 part of seed crystals were added while stirring. The aqueous solution was stirred at 40 ° C. for 1 hour, at 35 ° C. for 1 hour, at 30 ° C. for 1 hour, and at 25 ° C. for 20 hours to crystallize 2-O-α-D-glucosyl-L-ascorbic acid crystals. Analyzed.

(D) Drying step The mass kit obtained in the above step (c) was subjected to solid-liquid separation using a centrifuge. The separated 2-O-α-D-glucosyl-L-ascorbic acid crystals were washed with ethanol and dried under reduced pressure at 35 ° C. to give 30 parts of 2-O-α-D-glucosyl-L-ascorbic acid crystal powder. Got. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals by the above production method was 75.9%.

<Measurement of ethanol concentration>
The ethanol concentration remaining in the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder can be measured by a gas chromatography method according to the following conditions.
(Measurement conditions for gas chromatography)
Apparatus: Gas chromatography (manufactured by Agilent Technologies, 6890N)
Column: HP50 + manufactured by Agilent Technologies
Injector: 260 ° C
Detector: 300 ° C
Temperature raising condition: held at 50 ° C. for 3 minutes, heated to 50 ° C. at 260 ° C. per minute, and held at 260 ° C. for 10 minutes.

0.5 parts of 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was added to 2 ml of water and dissolved to obtain an aqueous solution of 2-O-α-D-glucosyl-L-ascorbic acid. Using this aqueous solution, the ethanol concentration was measured under the above measurement conditions. The concentration of ethanol remaining in the 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained in Example 1 was 500 ppm.

<Dissolution workability test>
One part of 2-O-α-D-glucosyl-L-ascorbic acid crystal was added to 10 parts of water at 25 ° C. and stirred to evaluate the dissolution workability. It was considered good when dissolved in less than 5 minutes, and unsatisfactory when it took more than 5 minutes to dissolve. The dissolution workability of the 2-O-α-D-glucosyl-L-ascorbic acid crystals obtained in Example 1 was good.

[Example 2]
A 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the organic solvent in the step (b) was changed from ethanol to methanol. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 74.7%, and the dissolution workability was good.

[Example 3]
A 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the organic solvent in the step (b) was changed from ethanol to acetone. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 72.1%, and the dissolution workability was good.

[Example 4]
A 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the organic solvent in the step (b) was changed from ethanol to methyl ethyl ketone. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 70.6%, and the dissolution workability was good.

[Comparative Example 1]
In the step (b), 2-O-α-D-glucosyl-L-ascorbic acid crystals were obtained in the same manner as in Example 1 except that ethanol was not used as the organic solvent and crystallization was performed only from water. A powder was obtained. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 45.3%, and the dissolution workability was not good.

[Example 5]
In Example 1, (c) 60 parts of an aqueous solution having a 66% concentration of 2-O-α-D-glucosyl-L-ascorbic acid in the crystallization step was changed to “2-O-α-D-glucosyl- A 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the concentration was changed to 55 parts of an aqueous solution having a concentration of L-ascorbic acid of 73%. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 92.1%, and the dissolution workability was good.

[Example 6]
In Example 5, 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the organic solvent in step (b) was changed from ethanol to methanol. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 89.5%, and the dissolution workability was good.

[Example 7]
In Example 5, 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the organic solvent in step (b) was changed from ethanol to acetone. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 80.1%, and the dissolution workability was good.

[Example 8]
In Example 5, 2-O-α-D-glucosyl-L-ascorbic acid crystal powder was obtained in the same manner as in Example 1 except that the organic solvent in step (b) was changed from ethanol to methyl ethyl ketone. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 78.5%, and the dissolution workability was good.

[Comparative Example 2]
In Example 5, 2-O-α-D-glucosyl-L-ascorbine was obtained in the same manner as in Example 5 except that in step (b), ethanol was not used as the organic solvent and crystallized from water alone. An acid crystal powder was obtained. The recovery rate of 2-O-α-D-glucosyl-L-ascorbic acid crystals was 49.5%, and the dissolution workability was not good.

The results of the above examples and comparative examples are summarized in Tables 1 and 2 below. Compared with the comparative example, Examples 1 to 4, 5 to 8 have a large recovery rate and are excellent in dissolution workability. In particular, it can be seen that those using the alcohol solvents of Examples 1, 2, 5, and 6 have a high recovery rate.

<Measurement of powder properties>
The physical properties of the powder can be evaluated by fluidity and jetting properties. In many cases, the fluidity is evaluated from the viewpoint of the ease of powder flow and the jetability of the powder. A 2-O-α-D-glucosyl-L-ascorbic acid crystal (hereinafter referred to as “ethanol crystallization sample”) obtained by the same procedure as in Example 1 and a procedure similar to that in the comparative example were obtained. With respect to the obtained 2-O-α-D-glucosyl-L-ascorbic acid crystal (hereinafter referred to as “crystallizing sample”), various physical property values relating to fluidity and jetting properties are analyzed by the following methods. Tests were conducted, and the measurement results were digitized by Dr. Carr's index, and the physical properties of the powder were evaluated from the total values. The particle size distribution and the true specific gravity are not affected by the numerical values of the fluidity and jet property, but are important values for knowing the physical properties of the powder, and thus were measured together.

(1) Analysis and test method (1-1) Particle size distribution (center particle size, etc.): A sample was acetone according to a wet laser diffraction method using a microtrack particle size distribution system “MT-3300EX II” (Nikkiso Co., Ltd.). The measurement was performed after ultrasonic dispersion (10 minutes).

(1-2) True specific gravity: Using a dry automatic densimeter “AccuPyc II 1340” (Macromericix Japan), according to the gas volume method, the sample was vacuum-dried at room temperature for 15 hours, and then measured by the He gas replacement method. did.

(1-3) Various physical properties relating to fluidity and jetting properties: The following items were measured using “Powder Tester PT-X” (Hosokawa Micron Corporation) according to the powder test method without drying the sample. The uniformity was calculated from the particle size distribution result.

(1-3-1) Items relating to fluidity ・ Loose bulk density: density when powder is filled in a measurement container in a natural fall state ・ Mass bulk density: After filling powder in a measurement container in a natural fall state, Bulk density when tapping 180 times ・ Dynamic apparent specific gravity: Calculated from the formula [(ba) × c / 100 + a] from loose bulk density (a), hard bulk density (b) and compressibility (c)・ Compression degree: Degree of bulkiness obtained from the difference between loose bulk density (a) and firm bulk density (b) ((ba) / b × 100)
・ Repose angle: Angle of the mountain of powder (elevation angle) formed with the powder falling naturally
-Spatula angle: The limit angle at which a stationary powder can maintain its state.-Uniformity: A fluidity evaluation value obtained by calculation in the case of a powder with small cohesiveness and uniform particle size (narrow particle size distribution) Index indicating the degree of uniformity) Uniformity = (60% under-sieving particle size) / (10% under-sieving particle size)
(1-3-2) Items related to jet properties-Collapse angle: Angle of repose after forming an angle of repose, and collapsing by applying an impact-Difference angle: angle obtained by subtracting the collapse angle from the angle of repose Simple measurement values for estimating jetting properties (flushing properties) ・ Dispersity: An index for evaluating the degree of spread when naturally dropped (the wider the particle size distribution, the larger the particle size distribution, the more fluid the particle size distribution) Dispersity = (1-remaining amount on watch glass / sample input) x 100
(1-4) Specific surface area: Using a flow-type specific surface area automatic measuring apparatus “Flowsorb III 2310” (manufactured by Shimadzu Corporation), according to the BET method, 0.5 g of a sample is placed in a measurement cell and removed at 120 ° C. for 20 minutes. After gassing, measurement was performed using nitrogen as an adsorbed gas.

(2) Results Table 3 shows the measurement results of the above items.

The central particle size obtained from the measurement of the particle size distribution was smaller than that in the case of dry measurement (reference: about 150 μm) because it was data after 10 minutes of ultrasonic crushing in acetone. This is thought to be because it was slightly dissolved in acetone and became smaller with ultrasound. The center particle size of the ethanol crystallized sample is slightly smaller than that of the crystallized sample, but the uniformity (= 60% diameter / 10% diameter) is almost the same value for both. Assuming that it is a true sphere, it is expected that there will be no significant difference in crystals.

The difference between the ethanol crystallized sample and the crystallized sample is apparent specific gravity. The dynamic apparent specific gravity is that the dynamic behavior in the actual process is "from the most coarsely packed state to a slightly compressed state (intermediate between loosening and hardening)" and "the degree of dynamic compression depends on the degree of compression" Based on the idea of “Yes”, it is calculated from the above formula. The loose bulk density, solid bulk density, and dynamic apparent specific gravity are all smaller for the ethanol crystallized sample, indicating that the powder as a whole contains air. Therefore, a difference of about 9% also occurs in the degree of compression, and the ethanol crystallized sample is expected to have a gap between particles.

The angle of repose was slightly larger in the ethanol crystallized sample, but the decay angle was smaller in the ethanol crystallized sample, which showed a large difference in the difference angle (= angle of repose−decay angle). . As the difference in apparent specific gravity shows, this is presumably because the crystals were separated from each other in the ethanol crystallized sample, so that the mountain was easy to collapse.

The spatula angle was smaller for the crystallized sample. Spatula angles and repose angles tend to be similar. This time, the opposite results for both samples may be due to the crystal shape (ethanol crystallized sample is slightly irregular, crystallized sample is relatively regular. ).

As described above, since the repose angle and the spatula angle showed opposite tendencies, the numerical value of fluidity was 39.5 for both samples, which was evaluated as “low”. However, the jet properties were affected by the difference in angle of difference, so the ethanol crystallized sample was “high” and the crystallized sample was “normal”. High jetting properties are generally suitable for pneumatic transportation and are easy to ride on air. Therefore, it is also suitable for airflow drying on air (see the application of powder tester measurement values), and taking into account that ethanol is included, ethanol samples are more dry than air samples when using airflow drying. It can be said that it is easy. However, since quantitative control is difficult, it is preferable to take a method other than pneumatic transportation when obtaining an accurate amount.

Also, the powder containing air is easy to mix with water, although its volume increases when compared with the same weight. Furthermore, if the crystal shape is irregular, it is expected that it will be more likely to mix with water.

Finally, it was shown that the specific surface area of the ethanol crystallized sample was larger than that of the crystallized sample.

Due to the difference in jetting property and specific surface area as described above, it is considered that the ethanol crystallization sample has improved dissolution workability when compared with the crystallization sample.

Claims

A process for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder, comprising the following steps (a) to (d):
(A) 2-O-α-D-glucosyl-L-ascorbine obtained by allowing a glycosyltransferase to act on a solution containing L-ascorbic acid and an α-glucosyl sugar compound and then causing glucoamylase to act Preparing an acid-containing solution;
(B) The 2-O-α-D-glucosyl-L-ascorbic acid-containing solution is purified so that the content of 2-O-α-D-glucosyl-L-ascorbic acid is 90% by mass in terms of anhydride. A step of obtaining an aqueous solution of the purified product as described above;
(C) a step of crystallizing 2-O-α-D-glucosyl-L-ascorbic acid crystals by mixing an aqueous solution of the purified product and an organic solvent and then lowering the temperature of the solution;
(D) The crystallized 2-O-α-D-glucosyl-L-ascorbic acid crystal is recovered, and the recovered crystal is dried to give 2-O-α-D-glucosyl-L- A step of obtaining ascorbic acid crystal powder.
The method for producing 2-O-α-D-glucosyl-L-ascorbic acid crystal powder according to claim 1, wherein the organic solvent in the step (c) is an alcohol solvent or a ketone solvent.
The 2-O-α-D-glucosyl-L-ascorbine according to claim 1 or 2, wherein in the step (c), 10 to 500% by mass of the organic solvent is mixed with the aqueous solution of the purified product. Method for producing acid crystal powder.
A 2-O-α-D-glucosyl-L-ascorbic acid crystal powder obtained by the production method according to any one of claims 1 to 3.