JP2845386B2 - Process for producing a macrocyclic cyclodextrin mixture containing an inner branched macrocyclic cyclodextrin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a macrocyclic CD containing an inner-branched macrocyclic CD from a CD product produced by a cyclodextrin (hereinafter sometimes abbreviated as CD) synthase.
Regarding the method of producing the mixture, specifically, a mixture of various CDs is obtained by reacting a starch with CD synthase, and then glucoamylase is reacted to decompose saccharides other than CD into glucose, thereby obtaining α-CD, β. -CD and [gamma] -CD are removed by complexing precipitation followed by a reverse phase column to produce a macrocyclic CD mixture containing internally branched macrocyclic CDs.

[0002]

2. Description of the Related Art In a conventional CD, six glucoses are composed of α-
Α-CD cyclically linked by 1,4 bonds, 7 β-C
D, 8 γ-CDs, Bacillus macerans (Bac
It is well known that starch is produced from starch and other related substrates by the reaction of CD synthase (CGTase) such as illus macerans).

In addition to these CDs, macrocyclic CDs such as δ-, ε-, ζ-, η-CD and the like having nine or more glucose rings belonging to the same family, and branches such as glucose and maltose are attached to the CD rings. Branch CDs are known.

[0004] As for the branched CD, α-
For an inner-branched CD having 1,6 bonds, the existence of inner-branched α- and β-CDs has been predicted by French et al.

A conventional method for producing a macrocyclic CD has been described by Kobayashi et al. (Japanese Society of Agricultural Chemistry, Abstracts of Lectures,
p649 (1986)). In this method, β-amylase, pullulanase and yeast were allowed to act simultaneously on a commercially available CD powdered candy to efficiently prepare a macrocyclic CD.

[0006] However, recent research results have shown that the co-action of glucoamylase or β-amylase with pullulanase significantly reduces the production rate of macrocyclic CD, and it is expected that the cyclic structure is cleaved by pullulanase. Was done.

[0007]

Accordingly, the present inventors have predicted the possibility of the presence of an intrabranched CD in α to γ-CD and the existence of a macrocyclic CD in which the ring is distorted without further existence of pullulanase. And attempted to prepare a macrocyclic CD using only glucoamylase, without using glucoamylase.
It was confirmed that 1,4-glucan was produced, and the present invention was completed by clarifying the existence of an inner-branched macrocyclic CD.

[0008]

That is, the present invention provides a method for producing a cyclodextrin product obtained by reacting a starch with cyclodextrin synthase with glucoamylase.
The present invention relates to a method for producing a cyclodextrin mixture containing an inner branched macrocyclic cyclodextrin.

There are no particular restrictions on the raw materials, reaction conditions, and the like in the production of cyclodextrin products by allowing cyclodextrin synthase to act on starches, and known techniques can be applied. The following is an example of the method of the present invention. 400 g of a commercially available CD powdered candy (trade name: Dexipearl K-50, manufactured by Shimizu Port Refined Sugar) is used as a raw material. 4, 50 ℃ 2
After reacting for one day, dextrins other than CD are hydrolyzed to glucose as completely as possible, the sugar composition is changed to glucose and CD, and after deproteinization and deionization, a complex forming reagent of bromobenzene and tetrachloroethane is prepared. Plus 1
CD from α to γ was precipitated by stirring at low temperature overnight.

After filtration, the filtrate is concentrated (filtrate concentrate),
It was loaded on a column packed with DS silica (manufactured by Nippon Organo) and eluted with water first, followed by elution with an ethanol concentration gradient of 0 to 20% to obtain a water-eluting fraction and an ethanol fraction.

FIG. 1 shows a liquid chromatogram of the filtrate concentrate. As is clear from the figure, at this stage, glucose (G1), maltose (G2), α-, β
-, Γ-CD (A, B, C) are contained in large amounts.

When glucose and maltose produced by the glucoamylase treatment are treated with yeast, 90%
% Or more was removed, and there was no change in the CD component ratio.

When this is passed through an ODS silica column, FIG.
, Glucose, maltose, E
The ethanol fraction contained peaks above the peak, and the ethanol fraction contained peaks A to E, and eluted in the order of A, C, D, E, and B as the ethanol concentration increased. Also, E
The peak was 5 in both the water-eluting fraction and the ethanol fraction.
0% was eluted.

Thus, the ODS column allows A (α
-CD), B (β-CD) and C (γ-CD) are not eluted with water, and macrocyclic CDs larger than δ-CD (D peak) are eluted with water, so that separation is extremely high. Easy and clear.

The E, F, G, and H peaks in the obtained water-eluted fraction were analyzed by high performance liquid chromatography (HPLC) using a column: Merck's analytical column, Liclosphere NH ₂ , 5 μm, 2 Separation was performed at a solvent flow rate of 0.9 mL / min and a column temperature of 20 ° C. The D peak in the ethanol fraction of the ODS column was compared with the preparative column YMC-Pack.
It was fractionated by C18 (YMC). The conditions for liquid chromatography are as follows: solvent: 3% ethanol, flow rate: 3 mL / min, column temperature: 4
0 ° C.

The peaks obtained in this manner correspond to HPL
It was C-single and showed a single spot in the paper chromatogram, indicating that each component of the macrocyclic CD was purified.

The sample of each peak was adjusted to 1% W / W, and 0.8 IU of commercially available crystal pullulanase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was added to 400 μL, and reacted at 30 ° C. for 20 hours. The resulting sugar was analyzed by HPLC. H
The PLC conditions were as follows: solvent: 55% acetonitrile; column: analytical column Licrosphere NH ₂ manufactured by Merck;
m, flow rate: 0.8 mL / min, column temperature: 20 ° C.

In the HPLC chart when the E peak fraction was reacted with pullulanase, an E peak and a new E ′ peak having a longer retention time appeared after 20 hours.
E 'retention time of the present HPLC conditions, fully consistent with malto deca ose maltooligosaccharides series standards (G _10), further, when the action of β- amylase, were all converted to maltose. Thus, E ′ is G ₁₀ and E is a cyclic sugar (ε-C
D) and an inner-branched macrocyclic CD having one α-1,6 bond in the ring (IBr-ε-CD).

Regarding the structure of ε-CD, when α-amylase is allowed to act, G10 is formed in the early stage of the reaction, and this is completely converted to maltose by β-amylase. It is clear that this is a macrocyclic CD.

A macrocyclic intrabranched CD having two or more α-1,6 bonds in the ring has not been discovered yet, but its existence can be expected.

The other peak fractions were similarly examined to determine the content of inner-branched CD. As a result, A, B, C, and D did not contain inner-branched CD, E was 13.14%, and G was 33.42
% And H were 27.25%.

The F peak fraction has a content in the raw material of 0.0
6% and the retention time of HPLC is adjacent to E
Close to the peak and G peak, but it is difficult to obtain highly pure fractions, the crude F peak fractions to act on pullulanase was similarly generated by F 'corresponding to G _11.

The E peak, F peak, G peak, and H peak are ε-, ζ-, η-, and θ-CD, respectively, and each CD contains IBr-ε-, ζ-, η-, and θ-CD. Revealed that you are.

From the above results, there is an inner branch CD having an α-1,6 bond in a macrocyclic CD in which a large strain is applied to the ring, and no distortion is applied to the ring, or α〜
It was found that δ-CD did not have an inner branch CD.

[0025]

Next, the present invention will be described with reference to examples. Example 1 Commercially available CD powder candy Dexipearl K-50 (manufactured by Shiosui Minato Sugar)
Using 400 g as a raw material, an excess of 6000 IU of a commercially available glucoamylase enzyme (manufactured by Seikagaku Corporation) was added, and a substrate concentration of 30 g was added.
%, PH 5.4, 50 ° C. for 24 hours, hydrolyze dextrins other than CD to glucose as completely as possible, and make the sugar solution composition glucose and CD. The reaction was continued at 24 ° C. for 24 hours to obtain an inner branched macrocyclic CD and a CD mixture containing 10% or more of the macrocyclic CD.

After deproteinization and deionization, a complex forming reagent of bromobenzene and tetrachloroethane was added, and CD was precipitated from α to γ by low-temperature stirring overnight. After filtration, the filtrate was concentrated (filtrate concentrate). Macrocyclic CD of δ or more 7
A CD mixture containing 0% or more was obtained as Macrocyclic CD Standard I.

Example 2 The macrocyclic CD product obtained in Example 1 was loaded on a column packed with ODS silica (manufactured by Nippon Organo), the water-eluted portion was collected and concentrated to reduce 90% of the macrocyclic CD of δ or more. CD included above
The mixture was obtained as macrocyclic CD preparation II.

Example 3 Pullulanase was allowed to act on the macrocyclic CD preparation II obtained in Example 2 to obtain a preparation containing linear saccharides of G _{10 to} G ₁₃ and macrocyclic CD. Further, pullulanase was allowed to act on the ε-, η-, and θ-CD fractions separated by HPLC, and G ₁₀ + ε-CD, G ₁₂ +
Standards of η-CD and G ₁₃ + θ-CD were obtained.

[0029]

According to the method of the present invention, a CD mixture containing an inner branched macrocyclic CD can be produced by using glucoamylase from a CD product produced by a CD synthase. Furthermore, since glucose and maltose are assimilated by the action of yeast, α-, β-,
A macrocyclic CD mixture containing γ-CD is obtained. Depending on the application, the product can be commercialized in this state, but an ODS column is used to produce a product with an increased macrocyclic CD content.
That is, since a CD having a ring length of ε or more is obtained by elution with water, the CD may be concentrated and commercialized. At this time, straight-chain sugars such as glucose and maltose are also eluted at the same time. Therefore, in order to increase the content of the macrocyclic CD, it is desirable to remove them in advance by treatment with yeast or the like.

Examples of the use of the macrocyclic CD include inclusion of chlorophyll, steroids, saturated fatty acids, unsaturated fatty acids, and other hydrophobic substances having a large molecular weight, and inclusion of an unpleasant odor which is difficult to include with ordinary ring CD. It is also excellent in solubility and can be used for solubilization. Other, ordinary ring C
Like D and branched CD, it can also be used for medicine, cosmetics, chemical industry, and the like.

[0031] When caused to act pullulanase macrocyclic CD mixture was separated by reverse-phase column can be a G ₁₀ or more linear sugar macrocyclic CD. Further, if the macrocyclic CD and the inner-branched macrocyclic CD can be separated, each can be used as a single product.
It can be easily obtained by separation with a column or the like.

Although it is extremely difficult to prepare a linear sugar having a G of ₁₀ or more using HPLC, IBr-ε-, η-, θ-C
Separation of D is relatively easy. Therefore, G ₁₀ ,
It can be used for preparing G ₁₂ , G _{13, and the} like. Furthermore, the inner branch C
Does not contain D .epsilon., .eta., maltose added in excess to the theta-CD, if caused to act on the CD synthase, such as _{G 12 ~G 15, G 22 ~G} 28 by its coupling effect, separately heretofore It is also possible to prepare a megalosaccharide that has not been obtained as a product.

[Brief description of the drawings]

FIG. 1 is a liquid chromatogram showing the saccharide composition of macrocyclic preparation I before yeast treatment. G ₁ : glucose G ₂ : maltose A: α-CD B: β-CD C: γ-CD D: δ-CD E: ε-CD F: ζ-CD G: η-CD H: θ-CD I: ι-CD J: κ-CD HPLC conditions are as follows: Column: Licrosphere NH ₂ 5 μm
, Column temperature: 20 ° C, elution solvent system: 55% acetonitrile, flow rate: 0.8 mL / min

FIG. 2 is a liquid chromatogram showing the sugar composition of Macrocyclic Standard II. A: α-CD B: β-CD C: γ-CD D: δ-CD E: ε-CD F: ζ-CD G: η-CD H: θ-CD I: ι-CD J: κ-CD

FIG. 3 is a liquid chromatogram showing the sugar composition of a sugar solution in which macrocyclic sample I was loaded on an ODS column, eluted with water, and the portions eluted with 20% ethanol were collected and concentrated. A: α-CD B: β-CD C: γ-CD D: δ-CD E: ε-CD

Claims (3)

(57) [Claims] 1. A cyclodextrin product obtained by allowing a cyclodextrin synthase to act on starches, and glucoa.
Cyclodextrin mixture containing an inner-branched macrocyclic cyclodextrin characterized by acting on a mirase
Manufacturing method . 2. The method according to claim 1 , wherein after the glucoamylase is allowed to act on the cyclodextrin product, the inner branched macrocyclic cyclodextrin is separated by a reversed-phase column. 3. The method according to claim 1, wherein the macrocyclic cyclodextrin mixture separated by the reversed-phase column is subjected to pullulanase to separate linear saccharides to obtain the macrocyclic cyclodextrin.
The method according to 2.