JPH068324B2 - Mutual separation method of cyclodextrins - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for separating and purifying cyclodextrins, and more specifically to adsorbing cyclodextrins onto a chemically modified silica carrier, and then adsorbing the cyclodextrins. Consisting of fractional elution with warm water,
The present invention relates to a method for mutually separating cyclodextrins.

[Prior Art] Cyclodextrins are degradation products obtained by allowing cyclodextrin-producing enzymes produced by microorganisms such as Bacillus macerans to act on starch or starch degradation products, and utilize its inclusion activity. Therefore, it is expected to have a wide range of applications in the fields of food, pharmaceuticals, cosmetics and the like.

Recently, efforts have been made in various fields to develop an industrial production method of such cyclodextrins, but since these cyclodextrins are produced by causing an enzyme to act on starch or a starch degradation product, The produced liquid contains various cyclodextrins mixed together and also contains a large amount of linear or branched oligosaccharides. Therefore, in order to obtain cyclodextrin with high purity, it is necessary to separate and collect only specific cyclodextrin from those sugar solutions, but with the conventional method, such cyclodextrin product with high purity can be obtained at low cost. Not successful in manufacturing.

The representative methods of purifying cyclodextrins known so far are as follows.

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

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

A method using a hydrophobic synthetic adsorption resin composed of a porous polymer (JP-A-56-805).

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

However, these methods have some drawbacks when separating cyclodextrins from other oligosaccharides, dextrins, etc., but have the drawback that they cannot be used for separating cyclodextrins from each other.

[Problems to be Solved by the Invention] The present inventors have conducted studies to find an efficient and practical method for separating cyclodextrin in consideration of such circumstances, and as a result, they have been chemically modified. It was found that the silica carrier selectively adsorbs only cyclodextrin, and succeeded in separating oligosaccharides and cyclodextrins by utilizing this, and applied for a patent as a "purification method of cyclodextrin" previously. did.

However, since this method usually uses 80 ° C or higher hot water for elution of cyclodextrins, various cyclodextrins are eluted at almost the same time, so it is possible to separate cyclodextrins from other sugars. However, there is a drawback in that cyclodextrin cannot be separated from each other substantially.

As a result of detailed examination of the elution pattern of cyclodextrins by varying the temperature of this hot water, the present inventors have shown that
When the temperature of warm water is below room temperature, only glucose and oligosaccharides are eluted, and cyclodextrins are hardly eluted, but when the temperature of warm water is higher than about 20 ° C, elution occurs gradually, and depending on the type of cyclodextrin, the elution rate is considerably high. I found a difference.

[Means for Solving Problems] The present invention has been completed based on the above new findings, and a cyclodextrin mixture is brought into contact with a chemically modified silica carrier to adsorb cyclodextrins to the silica carrier. Then, the adsorbed cyclodextrins are separated and eluted by using hot water to separate the cyclodextrins from each other.

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 preferred is a silica carrier substituted with a C ₁₈ linear alkylsilyl group so that the carbon content is 7-20%. 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. Things can also be used.

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. However, such a chemically modified silica carrier has already been used in Preparative-C18 (manufactured by Waters), YMC-ODS-ALL, YMC-G.
Since it is marketed under the trade name of EL-C8 (above, product of Yamamura Chemical Laboratory), it is convenient to purchase and use them appropriately.

According to the invention, the contacting of the cyclodextrin mixture with the chemically modified silica support can be carried out in various ways.
The most preferable method is a method in which a solution of cyclodextrins is allowed to flow down a column packed with a chemically modified silica carrier, but a method of adding a silica carrier to the solution and mixing them can also be used.

In the solution of the cyclodextrin mixture which is brought into contact with the chemically modified silica carrier, α-cyclodextrin, β-
Cyclodextrin, γ-cyclodextrin may be contained in any proportion, and also glucose,
It does not matter even if oligosaccharides are mixed. The cyclodextrin content in the solution may be as low as 1% or less, and the solution may contain an enzyme or the like as long as it does not inhibit the adsorption of the cyclodextrin. The concentration of the solution to be brought into contact with the chemically modified silica carrier is from 1% or less to 60% in any of the above methods.
It can be used in a very wide range up to a high concentration of 10% or more.

According to the present invention, the elution of cyclodextrins adsorbed on the chemically modified silica carrier is performed as follows.
First, if necessary, the chemically modified silica carrier on which cyclodextrins are adsorbed is washed with a predetermined amount (2 to 3 times the carrier volume) of cold water to remove coexisting glucose and oligosaccharides, and then the temperature of the eluate. Is continuously raised from about 20 ° C to 80 ° C, usually about 30 ° C to 60 ° C, and is passed through the column to elute cyclodextrins.

According to the present invention, the cyclodextrins adsorbed on the chemically modified silica carrier show γ with increasing temperature of the eluate.
-Cyclodextrin is eluted first, followed by β-
Cyclodextrin and α-cyclodextrin are eluted in this order.

The speed of elution of cyclodextrins from the chemically modified silica carrier varies slightly depending on the type of silica carrier used, so the temperature of the eluate should be 10-20 ° C higher or lower depending on the chemically modified silica carrier used. There is a need to. Therefore, in the present invention, in consideration of the above points, the temperature of the eluate (warm water) is appropriately adjusted according to the cyclodextrin composition in the raw material liquid so that a preferable elution pattern can be obtained. is necessary.

Further, in the present invention, as described above, the eluate (warm water)
In addition to the so-called gradient elution method in which the temperature is continuously raised between about 20-80 ° C to perform elution, a method in which the temperature of the eluate (warm water) is increased stepwise to elute is used. It is also possible.

The amount of the eluent used for elution is usually several tens of times the volume of the chemically modified silica carrier.

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 the range of SV = 3-30 is selected in consideration of work efficiency and the like. .

The eluate from the column is collected in a fraction collector in the order of elution. In the elution of the present invention, γ-cyclodextrin is eluted earliest as described above, and then β-cyclodextrin and α-cyclodextrin are eluted in this order, so the cyclone of the eluate collected in each fraction collector. Confirm the dextrin composition by HPLC etc., collect and concentrate each same component,
Further, if necessary, it can be dried 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 further repeating the above-mentioned adsorption / separation / elution operation for the cyclodextrins thus separated, it is possible to obtain cyclodextrins of higher purity.

EFFECTS OF THE INVENTION According to the present invention, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin can be efficiently separated from each other without using any organic solvent, and the purity is extremely high. It is characterized by being able to obtain cyclodextrins of. Therefore, the present invention is extremely useful for industrially producing high-purity cyclodextrins.

EXAMPLES Next, the present invention will be described in more detail and specifically with reference to examples.

Example 1 Commercially available cyclodextrin powder candy (Shimizu Port refined sugar KK
Manufactured by Dexey Pearl KM-50) was treated with glucoamylase to prepare a cyclodextrin mixture solution (sugar composition: α-cyclodextrin 27.8%, β-cyclodextrin 14.3%, γ-cyclodextrin.
3.9%, glucose and other sugars 54.0%) to a concentration of 21.0%.

Next, 8 g of this sugar solution was added to octadecylsilyl (ODS).
The column (diameter 2.0 cm × length 6.0 cm, carrier amount 8 g) is loaded onto the carrier to adsorb cyclodextrins to the carrier. Next, 40 ml of water is passed through the column at SV = 10 to elute and remove coexisting sugars such as glucose. Next, while passing the solution at the same rate, the temperature of the eluate in the column is gradually raised to around 60 ° C, and the cyclodextrins adsorbed on the carrier are fractionally eluted (the eluate is a fraction collector every 2.7 ml). To collect.).

The sugar composition of each fraction collected as above was analyzed by HPLC.
The elution pattern of FIG. 1 was obtained (in the figure, the solid line represents α-cyclodextrin, the dotted line represents β-cyclodextrin, and the chain line represents γ-.
It represents cyclodextrin, and the solid line represents the temperature of the eluate passing through the column. ).

Next, each of these fractions was divided according to its cyclodextrin composition, concentrated and dried to obtain 5 kinds of powders. The yield and composition of each of these powders is
Shown in the table. From the P3 powder in Table 1, 111.6 mg (purity 98.5%) of β-cyclodextrin was obtained by the crystallization method.

[Brief description of drawings]

FIG. 1 is a curve diagram showing an elution pattern of cyclodextrins obtained in Example 1.

Claims (1)

[Claims] 1. A method of contacting a cyclodextrin mixture with a chemically modified silica carrier to adsorb cyclodextrin in a liquid to the silica carrier, and then adsorbing the adsorbed cyclodextrin separately by warm water. Mutual separation method of cyclodextrins.