JPH068326B2 - Method for separating and purifying glucosyl-cyclodextrin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for separating and purifying glycosyl-cyclodextrin, more specifically, a sugar solution containing glycosyl-cyclodextrin, cyclodextrin and oligosaccharide is chemically modified. The present invention relates to a method for separating and purifying glycosyl-cyclodextrins, which comprises adsorbing the adsorbed cyclodextrins on a silica carrier and then fractionally eluting the adsorbed cyclodextrins with an aqueous ethanol solution having a specific concentration.

[Conventional technology]

Cyclodextrin is a decomposition product obtained by reacting starch or a starch degradation product with a cyclodextrin-producing enzyme produced by a Bacillus macerans sugar microorganism, and by utilizing its inclusion action, food, pharmaceuticals, cosmetics. It is a substance that is expected to be used in a wide range of fields such as. Among them, a branch of cyclodextrin in which a branch such as glucose is bound to the nucleus of the cyclodextrin, a so-called branched cyclodextrin, which has extremely high solubility in water, is not limited to the above-mentioned fields, and has a wider application in general industrial fields and the like. Is expected.

Under such circumstances, efforts have recently been made in various fields to develop an industrial production method of glucosyl-cyclodextrin. However, since these glucosyl-cyclodextrins are produced directly from starch or a starch degradation product by the action of an enzyme, or by reacting cyclodextrin and an oligosaccharide with an enzyme, a large amount of direct glucosyl-cyclodextrin is present in the resulting solution. Mixtures of chain or branched oligosaccharides and unbranched cyclodextrins. Therefore, since it is extremely difficult to separate and collect only glucosyl-cyclodextrin from the sugar solution, it is a fact that industrial production of a purified product having a high glucosyl-cyclodextrin content has not yet been successful.

The typical purification methods of known cyclodextrins are as follows.

(1) A method in which an organic solvent such as acetone is added to a sugar solution to precipitate cyclodextrin (see Japanese Patent Publication No. 52-8385).

(2) Method of purification using anion exchange resin (Japanese Patent Publication No. 46)
-9223 reference).

(3) A method using a hydrophobic synthetic adsorption resin made of a porous polymer (see Japanese Patent Laid-Open No. 56-805).

(4) A method of fractionating a strongly acidic ion exchange resin with an alkali metal salt (see JP-A-57-30702).

However, all of these methods have some effects when separating cyclodextrins from other oligosaccharides, dextrins, etc., but are not sufficiently effective for industrial use. In addition, it has the drawback that it can hardly be used to separate cyclodextrins from each other.

[Problems to be solved by the invention]

In view of the above-mentioned conventional state of the art, the present inventors have conducted research to find an efficient and practical method for separating cyclodextrin, and as a result, the chemically modified silica carrier selectively selects cyclodextrin. We found that it was adsorbed, succeeded in separating oligosaccharides and cyclodextrins using this, and previously filed a patent application as a "purification method of cyclodextrins" in Japanese Patent Application No. 61-14.
4781 specification).

However, since 10% (wt%, the same applies hereinafter) ethanol aqueous solution is used for elution of cyclodextrins in this method, various cyclodextrins are eluted at almost the same time. Therefore, cyclodextrin and other saccharides are separated from each other. However, it has the disadvantage that the separation of cyclodextrin and glucosyl-cyclodextrin is virtually impossible.

According to the research conducted by the present inventors, when cyclodextrins adsorbed on a chemically modified silica carrier are eluted with a 10% aqueous ethanol solution, various cyclodextrins are eluted at almost the same time as shown in FIG. Is very difficult to separate from each other. That is, FIG. 1 shows a sugar solution containing cyclodextrins (composition of cyclodextrin: diglucosyl-α-cyclodextrin.
13.4%, glucosyl-α-cyclodextrin 14.6
%, Α-cyclodextrin 21.9%) is passed through a column of a chemically modified silica carrier to be adsorbed, and then coexisting oligosaccharides are removed by washing with water, and then the adsorbed cyclodextrin is eluted with a 10% aqueous ethanol solution. As shown in Fig. 1, the elution pattern of glucosyl-α-cyclodextrin and the elution classification of glucosyl-α-cyclodextrin, and the elution classification of α-cyclodextrin are complete. Since they overlap, it is almost impossible to separate each component from each other.

As a result of detailed examination of the elution pattern of cyclodextrins by varying the ethanol concentration of the eluate, the present inventors have found that only oligosaccharides are eluted and the cyclodextrin Although it was hardly eluted, it was found that when the ethanol concentration was 2 to 7%, elution gradually occurred, and as shown in FIG. 2, there was a considerable difference in the elution rate depending on the type of cyclodextrin. That is, FIG. 2 shows a case where a sugar solution having the same composition as that of FIG. 1 is passed through a chemically modified silica carrier to be adsorbed, washed with water, and then the adsorbed cyclodextrins are eluted with a 5% ethanol aqueous solution. It shows the elution pattern, because there is a large gap between the elution category of glucosyl-α-cyclodextrin and the elution category of diglucosyl-α-cyclodextrin, the elution category of α-cyclodextrin, each Mutual separation of components is possible.

[Means for solving problems]

The present invention has been completed based on the new findings as described above, and a sugar solution containing glucosyl-cyclodextrin, cyclodextrin and oligosaccharide is brought into contact with a chemically modified silica carrier to allow cyclodextrins to react with the silica. In a method of separating and purifying glucosyl-cyclodextrin, which comprises adsorbing to a carrier, and then adsorbing the adsorbed cyclodextrin by fractional elution with an aqueous ethanol solution, first, the oligosaccharides are eluted and removed with water, and then the ethanol of the eluate is removed. A method for separating and purifying glucosyl-cyclodextrin, which comprises performing fractional elution of cyclodextrins by increasing the concentration stepwise or continuously.

Chemically modified silica carrier used in the method of the present invention are those having a silanol group of silica gel substituted with straight alkyl silyl group C ₈ -C ₁₈ structure, particularly preferably, carbon content of the silica support It is substituted with a C ₁₈ linear alkylsilyl group so that the ratio becomes 7 to 20%. Further, the silica gel (silica gel substituted with an octadecylsilyl group) has a residual silanol group further substituted with a trimethylsilyl group (endcapping), and further has a structure substituted with an octylsilyl group instead of the octadecylsilyl group. Those can also be used in the present invention.

These chemically modified silica carriers can be produced by reacting silica gel with an alkylchlorosilane and, if desired, by reacting the reaction product with trimethylchlorosilane. Such a chemically modified silica carrier is commercially available under the trade name of, for example, Preparative-C18 (manufactured by Waters Co., Ltd.), YMC-ODS-ALL, YMS-GEL-C8 (manufactured by Yamamura Chemical Research Laboratories). Because
It is convenient to use these.

According to the separation and purification method of the present invention, the sugar solution and the chemically modified silica carrier can be brought into contact with each other in various conventionally known forms. The most preferred method is a method in which the sugar solution is made to flow down through a column packed with a chemically modified silica carrier, but in addition to this, a method in which the silica carrier is added to the sugar solution and mixed is also suitably used.

In the sugar solution to be brought into contact with the chemically modified silica carrier, in addition to glucosyl-cyclodextrin and unbranched cyclodextrin, other sugar solutions may be used alone or in combination of two or more, and as a cyclodextrin, glucosyl is used. -Cyclodextrin and unbranched α-
Cyclodextrin, β-cyclodextrin, γ
-It does not matter in what proportion cyclodextrin is contained. The content of glucosyl-cyclodextrin in these sugar solutions may be as low as 1% or less, and enzymatic sugar may be contained in the sugar solution as long as it does not inhibit the adsorption of cyclodextrins. The concentration of the sugar solution to be brought into contact with the silica carrier can be used in a very wide range from a low concentration of 1% or less to a high concentration of 60% or more in any of the above methods.

According to the separation and purification method of the present invention, elution of cyclodextrins adsorbed on the chemically modified silica carrier is carried out as follows. That is, first, a silica carrier is washed with a predetermined amount (usually 2 to 3 times the carrier volume) of water to remove oligosaccharides, and then a 2 to 7% ethanol aqueous solution is used to fractionate and elute cyclodextrins. Then, cyclodextrin adsorbed on the silica carrier is eluted and recovered with a 10% ethanol aqueous solution.

According to the present invention, when cyclodextrins adsorbed on a chemically modified silica carrier are eluted with an aqueous ethanol solution, those having the branch of glucosyl are rapidly eluted in the same α-cyclodextrin, and cyclodextrins having no branching. Is eluted later. Therefore, the fractional elution of the present invention is carried out by using an aqueous ethanol solution of 2 to 7% in combination with the cyclodextrin composition of the raw material liquid, and if necessary, the cyclodextrin adsorbed on the silica carrier is eluted with an aqueous 10% ethanol solution. To do.

That is, the cyclodextrin of the raw material liquid is mainly α-
When it is composed of cyclodextrins, use an aqueous ethanol solution with an ethanol content of 5 to 7%.
When it is composed of cyclodextrins, an aqueous ethanol solution having an ethanol content of 3 to 4% is used, and when it is composed of γ-cyclodextrin, an aqueous ethanol solution having an ethanol content of 2 to 3% is used. Is preferred. In addition, since the elution speed of cyclodextrins from the silica carrier is slightly different depending on the type of silica carrier used, it is preferable to increase or decrease the ethanol concentration of the eluate by 1 to 2% depending on the silica carrier used. preferable. Therefore, in the present invention, while appropriately considering the above points, the ethanol concentration of the eluate is appropriately adjusted according to the cyclodextrin composition in the raw material liquid,
It is necessary to be able to obtain a favorable elution pattern.

Further, in the present invention, as described above, in addition to the method of gradually increasing the ethanol concentration of the aqueous ethanol solution to perform the elution, the ethanol concentration is continuously increased between 0% and 10% to perform the elution. It is also possible to use a so-called gradient elution method.

The amount of eluent used for elution is the same as the volume of the chemically modified silica carrier or several tens of times, but preferably 5 to 30 times.

In the present invention, the flow rate of the eluate has almost no effect on the separation efficiency and can be performed at an arbitrary rate, but normally, in consideration of work efficiency and the like, the range of SV = 3 to 30 is set. preferable.

The eluate from the column is collected in a fraction collector in the order of elution. As described above, in the elution with the aqueous ethanol solution, glucosyl-cyclodextrin is eluted early, and unbranched cyclodextrin is eluted later, so the cyclodextrin composition of the eluate collected in each fraction collector is analyzed by HPLC, etc. It is possible to collect each of the same components, concentrate, and, if necessary, dry to obtain a powder. Further, in the case of large-scale processing, it is possible to perform the same fractionation / fractionation from the peak fractionation or the amount of eluate using the RI detector by the fractionation device.

Further, by repeating the above-mentioned adsorption / separation / elution operation for the cyclodextrins thus separated, it is possible to obtain glucosyl-cyclodextrin of higher purity.

For applications other than food, for example, glucosyl-cyclodextrin used for general industry, it is possible to use methanol instead of ethanol and carry out the same procedure as above.

〔The invention's effect〕

According to the present invention, not only glucosyl-cyclodextrin and unbranched cyclodextrin can be separated, but also extremely high-purity glucosyl-cyclodextrin can be obtained. Therefore, the present invention is extremely useful for industrial production of glucosyl-cyclodextrin.

〔Example〕

Next, the present invention will be described in more detail and specifically with reference to Examples, but it goes without saying that the technical scope of the present invention is not limited to these Examples.

Example 1 Mixed solution of cyclodextrin containing glucosyl-α-cyclodextrin and oligosaccharide (B × 36.0%, composition: branched α-cyclodextrin 18.2%, α-cyclodextrin 19.1%, saccharides such as glucose 62.5% 6) 6 .
4 ml of octadecylsilyl (ODS) carrier column (diameter
It was loaded onto 2.0 cm × length 7.0 cm, carrier amount 10 g), and then 60 m of water was passed through the column at SV = 47 to elute and remove coexisting oligosaccharides. Next, 200 ml of a 5% ethanol aqueous solution was passed through the column at SV = 4.8 to elute the branched cyclodextrin adsorbed on the carrier, and the eluate was fractionated every 3.3 m by a fraction collector. After completing the collection, pass 60 ml of 10% aqueous ethanol solution,
The cyclodextrins remaining in the column were eluted and collected.

The sugar composition of each fraction collected as described above was confirmed (measured) by HPLC, and the elution pattern shown in FIG. 3 was obtained. In the figure,
Curve represents glucosyl-α-cyclodextrin, curve represents diglucosyl-α-cyclodextrin, and curve represents α-cyclodextrin.

Then, each of these fractions glucose fractions, branched cyclodextrin fractions, cyclodextrin and branched cyclodextrin mixed fractions, after dividing into cyclodextrin fractions, fractions containing branched cyclodextrin were collected, After concentration and drying, 248 mg of branched α-cyclodextrin powder and 338 mg of mixed powder of branched cyclodextrin and cyclodextrin were obtained. The cyclodextrin composition of this branched cyclodextrin powder was 74.8% for glucosyl-α-cyclodextrin.
%, Diglucosyl-α-cyclodextrin 20.8
%, Α-cyclodextrin was 3.9%, and the cyclodextrin composition of the mixed powder of branched cyclodextrin and cyclodextrin was 39.7% branched cyclodextrin and 60.3% cyclodextrin. The recovery rate of branched α-cyclodextrin is almost 100%.
%Met. In addition to this, 215 mg of α-cyclodextrin (purity 98.9%, α-cyclodextrin recovery rate 49.9%) was separated and collected.

Example 2 Mixed solution of cyclodextrin containing glucosyl-β-cyclodextrin and oligosaccharide (B × 17.0%, composition: branched β-cyclodextrin 10.9%, β-cyclodextrin 8.3%, sugars such as glucose 80.2%) 30 .
0 ml of octadecylsilyl (ODS) carrier column (diameter
It was loaded onto 2.0 cm × length 7.0 cm, carrier amount 10 g), and then 60 ml of water was passed through the column at SV = 48 to elute and remove coexisting oligosaccharides. Next, 120 ml of 5% aqueous ethanol solution was passed through the column at SV = 4.8 to elute the cyclodextrins adsorbed on the carrier, and the eluate was 3.3 m.
Each l was collected with a fraction collector. After completion of the fractionation, 60 ml of a 10% aqueous ethanol solution was passed through to elute and collect cyclodextrins remaining in the column.

The sugar composition of each fraction collected as described above was confirmed (measured) by HPLC, and each of these fractions was analyzed according to the sugar composition as a glucose fraction, a branched cyclodextrin fraction,
Divided into a mixed fraction of cyclodextrin and branched cyclodextrin and a fraction of cyclodextrin, the fractions containing branched cyclodextrin were collected, concentrated and dried to give branched β-cyclodextrin powder 566m.
318 mg of a mixed powder of g and branched β-cyclodextrin and β-cyclodextrin was obtained. The cyclodextrin composition of this branched cyclodextrin powder is glucosyl-β-cyclodextrin 63.6%, diglucosyl-β-cyclodextrin is 25.1%, the cyclodextrin composition of the mixed powder of branched cyclodextrin and cyclodextrin is glucosyl-β. -Cyclodextrin 30.2%, β-Cyclodextrin
It was 69.8%. The recovery rate of branched β-cyclodextrin was almost 100%. In addition to this, 183 mg of β-cyclodextrin (purity 99.7%, recovery rate of β-cyclodextrin 42.4%) was separated and collected.

Example 3 Solid content concentration of 39.8 containing branched cyclodextrins
%, Sugar composition; glucosyl-α-cyclodextrin 4.
3%, α-cyclodextrin 6.8%, other cyclodextrins 1.1%, reducing sugar of glucose sugar 87.8% sugar solution (after applying a cyclodextrin-producing enzyme of Bacillus macerans to the branched dextons, Taka amylase and glucoamylase (Obtained by the action of the above) 144 g on an octadecylsilyl (ODS) carrier column (diameter 36 mm x length 115 mm, carrier amount 50 g).
Cyclodextrin was adsorbed on the carrier and washed with 420 ml of water to elute and remove coexisting oligosaccharides.

Next, prepare 1000 ml of water in the mixing tank and 1000 ml of 10% ethanol aqueous solution in the replenishment tank, and adjust the flow rate from both to gradually change the ethanol concentration of the eluate from 0% to 10%. Gradient elution of 0 to 10% ethanol was performed to elute cyclodextrins adsorbed on the carrier (SV = 6.0, the eluate was fractionated every 10 ml by a fraction collector).

The sugar composition of each fraction collected as described above was confirmed (measured) by HPLC, and the elution pattern shown in FIG. 4 was obtained. In the figure,
Showing the curve glucosyl-α-cyclodextrin of
Curve represents diglucosyl-α-cyclodextrin, curve represents glucosyl-β-cyclodextrin, and curve represents α-cyclodextrin.

Next, each of these fractions was divided into a branched cyclodextrin mixture fraction (P ₁ ), a glucosyl-α-cyclodextrin fraction (P ₂ ), and a glucosyl-α-cyclodextrin / α-cyclodextrin fraction (P ₁ ). P ₃ ), α-cyclodextrin fraction (P ₄ ), respectively, concentrated and dried to 1.11 g powder of branched cyclodextrin mixture, 1.24 g glucosyl-α-cyclodextrin powder, glucosyl-α-cyclodextrin. Powder of a mixture of dextrin and α-cyclodextrin 4.
03 g, 0.57 g of powder of α-cyclodextrin was obtained.
The branched cyclodextrin content of this branched cyclodextrin powder is 98.5%, the purity of glucosyl-α-cyclodextrin powder is 87.8%, and the cyclodextrin composition of the mixed powder of glucosyl-α-cyclodextrin and α-cyclodextrin is Glucosyl-α-cyclodextrin was 22.6% and α-cyclodextrin was 77.4%. Moreover, the purity of the α-cyclodextrin powder was 100%, and the recovery rate of the α-cyclodextrin was 14.6%.

Example 4 88.1 ml of a sugar solution having the same composition as that used in Example 3 was passed through a column of octadecylsilyl (ODS) carrier (the same as in Example 3) at SV = 8 to remove cyclodextrins. It was adsorbed on a carrier and further washed with 300 ml of water.

Next, 250 ml of 2% ethanol aqueous solution was passed through the column at SV = 8 to elute and remove the impurities adsorbed on the carrier, and then 800 ml of 5% ethanol aqueous solution was passed through the column at SV = 8 to remove the carrier. The cyclodextrins adsorbed on the column were eluted (the eluate was collected by a fraction collector every 6.2 ml).

The sugar composition of each fraction collected as described above was measured by HPLC, and each of these fractions was fractionated according to the sugar composition, a fraction of a branched cyclodextrin mixture, a mixed fraction of α-cyclodextrin and a branched cyclodextrin, and α-cyclodextrin. Each of the fractions was concentrated and dried to obtain 2.14 g of a mixed powder of branched cyclodextrin and 1.55 g of a mixed powder of α-cyclodextrin and branched cyclodextrin.

The composition of the mixed powder of the branched cyclodextrin is glucosyl-α-cyclodextrin 69.5%, glucosyl-β-cyclodextrin 15.8%, diglucosyl-.
The cyclodextrin is 7.2%, and the composition of the mixed powder of α-cyclodextrin and branched cyclodextrin is α-cyclodextrin 85.7%, glucosyl-α.
-Cyclodextrin 14.3. The recovery rate of branched cyclodextrin was 88.3%. In addition to this, 1.15 g (purity 99.7%, recovery rate 44.0%) of α-cyclodextrin was separately collected.

[Brief description of drawings]

FIG. 1 shows that a sugar solution containing cyclodextrin is passed through a column of a chemically modified silica carrier to adsorb cyclodextrin on the carrier, and coexisting oligosaccharides are removed by washing with water. It is a curve figure which shows the elution pattern at the time of elution with a% ethanol aqueous solution. FIG. 2 is a curve diagram showing an elution pattern of cyclodextrins when a sugar solution having the same tissue as described above was adsorbed on a chemically modified silica carrier in the same manner as described above and then eluted with a 5% ethanol aqueous solution. FIG. 3 is a curve diagram showing an elution pattern of cyclodextrins obtained in Example 1. FIG. 4 is a curve diagram showing the elution pattern of cyclodextrins obtained in Example 3.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeo Sakai 1-10-19 Honda, Niiza City, Saitama Prefecture (72) Inventor Saku Yoshida 1-4-19-701 (72) invention, Saiwaicho, Kawaguchi City, Saitama Prefecture Person Chiwa Makoto 6-20-12 Katsutadai, Yachiyo-shi, Chiba

Claims (1)

[Claims] 1. A sugar solution containing glucosyl-cyclodextrin, cyclodextrin and oligosaccharide is brought into contact with a chemically modified silica carrier to adsorb cyclodextrin in the solution to the silica carrier, and then the adsorbed cyclodextrin. In a method for separating and purifying glycosyl-cyclodextrin, which comprises separating and eluting with an aqueous ethanol solution, first, oligosaccharides are eluted and removed with water, and then the ethanol concentration of the eluate is increased stepwise or continuously to remove cyclodextrin. A method for separating and purifying glycosyl-cyclodextrin, which comprises performing fractional elution.