JPS63154703A - Separation of maltosyl-cyclodextrins - Google Patents

Abstract

PURPOSE:To separate efficiently maltosyl-cyclodextrins without the use of an organic solvent, by contacting a specific saccharide solution with a chemically modified silica carrier thereby adsorbing it onto the carrier, and then eluting it fractionally with warm water of which temperature is raised stepwise or continuously. CONSTITUTION:Silica gel is reacted with an alkylchlorosilan and, if desired, further reacted with trimethylchlorosilan to produce a chemically modified silica carrier (B) having a structure wherein the silanol group of the silica gel is substituted by an 8-18C linear alkylsilyl group. A solution (A) of a solution containing maltosyl-cyclodextrins, unbranched cyclodextrins and one or more other saccharides (e.g., an oligosaccharide) in various ratios is contacted with component (B) thereby adsorbing it onto component (B) and, if desired, washed with cold water to remove the oligosaccharide, and then eluted at a liquid velocity of 3-30 with warm water (C) in a volume scores of times as much as the volume of component (B) of which temperature is raised continuously or stepwise in the range of 20-80 deg.C. The eluates are separately collected in fin fraction collectors in order of the elution, concentrated and, if desired, dried.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a method for separating and purifying maltosyl-cyclodextrins. A method for separating maltosyl-cyclodextrins, which comprises adsorbing them onto a modified silica carrier, and then fractionally eluting the adsorbed cyclodextrin with hot water to separate the maltosyl-cyclodextrins from the cyclodextrins. Regarding.

[Prior art 1 Cyclodextrin 1, starch or starch extract, Bacillus macerans
It is a decomposition product obtained by the action of cyclodextrin-producing enzymes produced by microorganisms such as A. . Among these, so-called branched cyclodextrins, in which branches such as maltose are bonded to a cyclodextrin core, have a significantly high solubility in water, and are therefore widely applicable not only in the above fields but also in general industrial fields. is expected.

Against this background, efforts have recently been made in various fields to develop industrial methods for producing maltosyl-cyclodextrins. However, these maltosyl-cyclodextrins are produced directly from starch or starch decomposition products by the action of enzymes! ! ? In the production liquid, large amounts of linear or branched oligosaccharides and unbranched cyclodextrins are mixed. Therefore, it is extremely difficult to separate and collect only maltosyl-cyclodextrins from these sugar solutions, and the reality is that industrial production of purified products with a high content of maltosyl-cyclodextrins has not yet been successful. be.

The following are typical methods for purifying cyclodextrins that have been known so far.

(2) A method in which cyclodextrin is precipitated by adding an organic solvent such as acetate to a sugar solution (Japanese Patent Publication No. 8385/1983).

■ Purification method using anion exchange resin
No. 6-9223).

■ Hydrophobic synthetic adsorption made of porous polymer! (Unexamined Japanese Patent Publication No. 56-805).

■ A method of fractionation using an alkali metal salt of a strongly acidic ion exchange resin (Japanese Patent Application Laid-open No. 30702/1983).

However, although these methods are effective to some extent in separating cyclodextrins from other oligosaccharides, dextrins, etc., they are insufficiently effective for industrial use. It has the disadvantage that it can hardly be used for separating cyclodextrin and branched cyclodextrin.

[Problems to be Solved by the Invention] Taking these circumstances into consideration, the present inventors have conducted extensive research to find an efficient and practical method for separating cyclodextrin, and as a result, they have found a method for separating chemically modified cyclodextrins. He discovered that a silica carrier selectively adsorbs only cyclodextrins, and successfully used this to separate oligosaccharides and cyclodextrins, and filed a patent application for a ``method for purifying cyclodextrins.'' did.

However, since this method usually uses hot water of 80°C or higher to elute cyclodextrins, various cyclodextrins are eluted almost simultaneously, so it is not possible to separate cyclodextrins from pond sugars. However, it has the disadvantage that it is virtually impossible to separate cyclodextrins from each other, especially to separate cyclodextrins from branched cyclodextrins.

As a result of various studies on methods for obtaining purified products of cyclodextrin, the present inventors have found that when cyclodextrins adsorbed on a chemically modified silica carrier are eluted with hot water of about 20°C or higher, depending on the type of cyclodextrin, We found that there were significant differences in elution rates.

[Measures to Solve the Problemsg'i1] The present invention was completed based on the above-mentioned new findings. Therefore, a sugar solution containing maltosyl-cyclodextrins, cyclodextrins, and oligosaccharides was prepared in a chemical manner. The cyclodextrins are adsorbed onto the silica carrier, and then the adsorbed cyclodextrins are fractionally eluted using hot water at about 20-80'C, and maltosyl-cyclodextrins and cyclodextrins are separated from each other by fractional elution using warm water at about 20-80'C. This is a method for separating maltosyl-cyclodextrins, which consists of separating dextrins.

The chemically modified silica carrier used in the method of the present invention has a structure in which the silica 7-l group of the silica sol is substituted with a linear alkylsilyl group of C, -C, etc. Especially preferred is The silica carrier is substituted with a linear alkylsilyl group of C1 so that the carbon content is 7-20%.Also, the residual silanol group of the silica sol (silica sol substituted with an octadecylsilyl group) Furthermore, it is possible to use those having a structure in which the above-mentioned octadecylsilyl group is substituted with a trimethylsilyl group (endocamping), or an octylsilyl group in place of the octadecylsilyl group.

These chemically modified silica carriers can be produced by reacting silica sol with furkylchlorosilane and, if desired, reacting the reaction product with trimethylchlorosilane. However, such chemically modified silica carriers are already available in Prevaratiboo 18 (product of Waters), YMC-ODS-ALL, and YMC-G.
Since they are commercially available under trade names such as EL-C8 (all products manufactured by Yamamura Kagaku Kenkyusho), it is convenient to purchase and use them as appropriate.

According to the present invention, the contact between the sugar solution and the chemically modified silica carrier can be carried out in various ways. The most preferred method is to allow the sugar solution to flow down a column filled with these silica carriers, but it is also possible to use a method in which the silica carrier is added to the sugar solution and mixed.

In addition to maltosyl-cyclodextrins and unbranched cyclodextrins, other saccharides or two or more types of saccharides may be present in the sugar solution that is brought into contact with the chemically modified silica carrier, and as the cyclodextrins, Maltosyl-cyclodextrins in which several maltosyl groups are bonded in a branched manner and unbranched cyclodextrins may be contained in any proportion. maltosyl-cyclodextrin in these sugar solutions)
The content of the IJ neck may be as low as 1% or less, and the sugar solution may contain enzymes, etc., as long as they do not inhibit the adsorption of cyclodextrins. The concentration of the sugar solution brought into contact with the silica carrier can be used in an extremely 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 present invention, cyclodextrins adsorbed on a chemically modified silica carrier are eluted as follows.

First, if necessary, the chemically modified silica support is washed with a predetermined amount of water (2-3 times the volume of the support) to remove oligosaccharides.Next, the temperature of the eluate is continuously maintained between 20-80℃. cyclodextrins are differentially eluted.

According to the present invention, by using hot water among the cyclodextrins adsorbed on the chemically modified silica carrier, those having maltosyl branch molecules are eluted quickly, and the cyclodextrins having no branch molecules are eluted later. In this way, in the fractional elution of the present invention, most of the cyclodextrin (IJ) nons are eluted with hot water at 20-80°C, but
Furthermore, if necessary, the cyclodextrin remaining in the column can be eluted with hot water at 80° C. or higher.

In addition, in the present invention, as described above, the eluent (warm water)
In addition to the so-called gradient elution method in which elution is performed by continuously increasing the temperature of the eluate between 20 and 80'C, there is also a method in which elution is performed by increasing the temperature of the eluate stepwise between 20 and 80'C. This is possible by using In addition, in the elution method in which the temperature of the eluate is increased stepwise, the elution speed of cyclodextrins from the chemically modified silica carrier varies somewhat depending on the type of chemically modified silica carrier used, so it depends on the carrier used. Depending on the temperature of the hot water 10-2
It is necessary to raise or lower the temperature by about 0°C.

Therefore, in this elution method, the temperature of the eluate (hot water) is adjusted appropriately according to the cyclodextrin composition in the raw material solution, taking the above points into account, so as to obtain a preferable elution pattern. It is necessary to.

The amount of 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 (although it can be carried out at any speed, the range of 5V = 3-30 is usually selected in consideration of work efficiency, etc.). It will be done.

The eluate from the column is separated into eluates by a 7-lux collector. As mentioned above, in elution with warm water, maltosyl-cyclodextrins are eluted slowly, and unbranched cyclodextrins are eluted later, so the cyclodextrins in the eluate collected in each 7-lacticin collector are The composition can be confirmed by HPLC, etc., and the same components can be collected, concentrated, and, if necessary, dried to form a powder. In addition, in the case of large-scale processing, similar fractionation and fractionation can be performed by peak fractionation using an R1 detector or fractionation based on the amount of eluate using a fractionation device.

Also, cyclodextrin isolated in this way) +
If the above-mentioned adsorption/separation/elution operations are carried out with respect to 7 compounds, even higher purity maltosyl-cyclodextrins can be obtained.

[Effect of the invention 1] According to the present invention, not only can maltosyl-cyclodextrins and unbranched cyclodextrins be efficiently separated without using an organic solvent, but also maltosyl-cyclodextrins of extremely high purity can be obtained. It has the characteristic that it can be done. Therefore, the present invention is extremely useful for the industrial production of malton-leux cyclodextrin.

[Example 1] Next, an example will be shown to explain the present invention in more detail and concretely.

Example 1 A mixed solution of cyclodextrins and oligosaccharides containing maltosyl-a-cyclodextrin (Bx, 21.
5%, composition: branched α-cyclodextrin 19.9%
, α-cyclodextrin 21.7%, oligosaccharides such as maltose 58.4%) was added to an octadecylsilyl (ODS) carrier column (diameter 2.Oca+Xff1).
Sa6. 0 then loaded onto the carrier fi8g)
Water 481 is passed through the column at 5v=io to elute and remove coexisting oligosaccharides.

Next, while passing the liquid at the same speed, the column elution temperature was adjusted to 60°C.
Gradually rises to around (average temperature increase rate 0.4°C/-1n
) to elute the cyclodextrins adsorbed on the carrier (the eluate is fractionated every 2.7+al with a 7-lacsilane collector).

The sugar composition of each 7 laxicone fractionated as above was analyzed by HPLC.
The two fractions (Pl) containing branched α-cyclodextrin as the main component are
, a mixed fraction of branched a-cyclodextrin and α-cyclodextrin (R2), and a-cyclodextrin fraction (R3), each fraction was collected, concentrated, and dried to obtain three types of Powders were obtained. Table 1 shows the yield and cyclodextrin composition of each powder.

Table 1 (blank below) Example 2 Mixed solution of cyclodextrins and oligosaccharides containing 71retosyl-β-cyclodextrin (Bx, 5.
0%, composition: branched β-cyclodexcillin 45.0%
, β-cyclodextrin 14°9%, oligo W such as maltose 40.1%) was added to a column of octadecylsilyl (○DS) carrier (diameter 2, Oc theory x length 6).
, Ocm, carrier amount 8 g).

Next, water 89 value 1 was passed through the column at 5V=20,
Coexisting oligosaccharides are eluted and removed.

Next, the column elution temperature was gradually increased to around 80°C while passing the liquid at the same rate (average temperature increase rate 0.85'C/m
i n ), and the cyclodextrins adsorbed on the carrier are eluted (the eluate is fractionated every 4.05 μl using a 7-laxis collector).

HP L
Check with C to obtain the elution pattern shown in Figure 1 (in the figure,
The curve (■) represents maltosyl-β-cyclodextrin, the curve (■) represents fumartosyl-β-cyclodextrin, the curve (■) represents β-cyclodextrin, and the curve (■) represents the temperature at which the bathing solution passes through the column. . ).

Next, each of these 7-lactones is divided into a fraction (Pl) containing branched β-cyclodextrin as a main component, a fraction containing branched β-cyclodextrin and β-cyclodextrin, depending on the cyclodextrin composition.
- After dividing the mixture into cyclodextrin mixed fractions (P2), each fraction was concentrated and dried to obtain two types of powder. Table 2 shows the yield and cyclodextrin composition of each powder.

Table 2 (Note: CD is an abbreviation for cyclodextrin) (The following is a blank space)

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a curve diagram showing the elution pattern of the cyclodextrins obtained in Example 2.

Claims (1)

[Claims] 1. A sugar solution containing maltosyl-cyclodextrins, cyclodextrins, and oligosaccharides is brought into contact with a chemically modified silica carrier, and the cyclodextrins in the solution are adsorbed onto the silica carrier, and then the adsorbed cyclodextrins are adsorbed onto the silica carrier. 1. A method for separating maltosyl-cyclodextrins, which comprises separating maltosyl-cyclodextrins and cyclodextrins by fractionating and eluting the cyclodextrins with warm water. 2. The method for separating maltosyl-cyclodextrins according to claim 1, wherein the fractional elution with hot water is carried out by increasing the temperature of the eluate stepwise or continuously.