JPS60227693A - Method of increasing yield of gamma-cyclodextrin - Google Patents

Abstract

PURPOSE:A specific enzyme forming cyclodextrin is allowed to act on terpenoids and starch whereby gamma-cyclodextrin is selectively obtained in high yield. CONSTITUTION:A starch which can be converted into cyclodextrins such as starch from potatoes or sweet potatoes and its hydrolyzate is converted into 2- 40wt% aqueous suspension, then 1-20units/g starch of cyclodextrin glycosyltransferase and 0.05-2.0wt% (based on the starch) of a tetra- or pentacyclic terpenoid, glycyrrhizin, peryandllin or steviosides are added to the suspension to effect reaction on standing or under stirring at 40-70 deg.C for 5-72hr to effect high-yield production of gamma-cyclodextrin.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for increasing the yield of γ-cyclop-dextrin, and more specifically, when producing cyclodextrin by allowing cyclodextrin lycodyltransferase to act on starch that can be converted to cyclodextrin, The present invention relates to a method for increasing the yield of γ-cyclodextrin, which is characterized by allowing a 4-ring (1) or 5-ring terpenoid to coexist in the reaction solution.

Cyclodextrin is a glucose molecule with α-1,4-
α-9β-2γ-cyclodextrin, which is a non-reducing sugar linked in a cyclic form by a bond, and has 6, 7, and 8 glucose residues, respectively, is generally well known. Among these, r-cyclodextrin in particular has higher solubility in water than α- or β-cyclodextrin, is more easily decomposed by α-amylase in the body, and has a large molecular size. From the standpoint of being able to clathrate molecules, it is thought that it will have a wide range of applications, particularly in the fields of food and pharmaceutical formulations, and the development of an efficient production method has been desired.

However, to date, no cyclodextrin lycosyltransferase (hereinafter referred to as rCD-forming enzyme) that can selectively produce γ-cyclodextrin has been discovered, and the production of γ-cyclodextrin has been limited to starch soluble ) A method is known in which a reaction solution containing α-1β- and γ-cyclodextrin obtained by treating the solution with a CD-producing enzyme known per se is collected as a fraction using a glue filtration agent. (For example, in the manufacturing method of
Since the content of γ-cyclodextrin in all cyclodextrins is small, the above production method has a drawback as a method for selectively producing γ-cyclodextrin.

Therefore, as a result of repeated studies to develop a method for selectively producing γ-cyclo-r-kistrin, the present inventors discovered that the known carbon
This book has succeeded in dramatically increasing the content of γ-cyclodextrin in the total cyclodextrin produced by coexisting tetracyclic or pentacyclic terpenoids when acting on the D-generating enzyme. Completed the invention.

In the present invention, starches that can be converted into cyclodextrins are those that can produce cyclodextrins (which may be either α-2β-1γ-) by the action of a known CD-generating enzyme. If so, starch obtained from any raw material such as starch, sweet potato, corn, waxy corn, barley, wheat, tapioca, etc., regardless of its type or origin, and its decomposition reaction products can be mentioned. can. Here, starch decomposition reaction products include roasted dextrins such as white dextrin, yellow dextrin, and Britelash gum; modified starches such as oxidized starch and low-viscosity modified starch; starch ethers represented by phosphoric acid starch, acetic acid starch, etc. , starch derivatives such as starch esters;
Examples include physically treated starches such as starches that have been irradiated with radiation or neutron beams, or subjected to high frequency treatment or moist heat treatment.

As the CD-producing enzyme, there are known CD-producing enzymes (
E C,2,4,1,19), α-cyclodextrin, β-cyclodextrin and/or γ
- Enzymes that produce any of the cyclodextrins can also be used. For example, Bacillus macerans z Klebsiella pneumonia
eumoni&e), alkaline/kutilis (Alka
riBacillus), Micrococcus variins, Micrococcus e luteus (M, 1uteus), etc., and those produced by their related bacteria (for example, Fragrance Journal A63 (1983), JP-A-55-138390, (See Japanese Patent Publication No. 52-31949). Among these, particularly preferred are CD-generating enzymes of the type that preferentially produce β-cyclodextrin derived from Bacillus opensis, Bacillus circulans, Bacillus megaterium, and the like.

Tetracyclic or pentacyclic terpenoids are compounds widely distributed in the plant world as free radicals, sugar-bonded glycosides, and acid-bonded esters, or compounds obtained from these by chemical treatment. It also includes derivatives such as Among these terpenoids, γ-CD1 from the viewpoint of application in the field of food or pharmaceutical formulations,
It is preferable that it can be used as a food additive itself.
Specific examples include sweet components collectively called glycyrrhizin, periandrin, and steviocyto, aglycone (free radical) moieties derived therefrom, and various glycosyl derivatives of the glycosides (for example,
7-18779), derivatives obtained by dulling these with an appropriate bifunctional crosslinking agent, or derivatives obtained by bonding them to an appropriate polymeric substrate.

Furthermore, the reaction between starch and CD-generating enzyme according to the present invention is as follows:
Refers to a method known per se of adding a CD-generating enzyme to an aqueous suspension or solution of starches, for example, at a concentration of an aqueous solution of starches of 2 to 40 percent by weight, preferably 5 to 2 percent by weight.
1 to 0 weight percent aqueous solution or suspension
20 units/(6) g (starch), preferably 5-10 units/g (
A method in which a CD-generating enzyme is added to starch and allowed to react while standing or stirring, and the reaction temperature is determined depending on the CD used.
Depending on the enzyme produced, the optimum temperature should be selected and is not critical, but generally 40-70°C, preferably 50°C.
Conducted at 0-60°C.

As for the method of causing the tetracyclic or pentacyclic terpenoids to coexist in the reaction solution, it is possible to make them coexist from the beginning of the reaction, or from the middle of the reaction, but it is usually preferable to make them coexist from the beginning of the reaction. good. The amount of the terpenoids to be allowed to coexist varies depending on the type, and is difficult to specify especially when using derivatives thereof, but it is generally 0.05 to 2.0 parts by weight based on the starch, preferably,
It is preferable to use 0.1 to 1 part by weight. Furthermore, the effects of the present invention can be achieved regardless of whether the reaction solution containing the terpenoids is in the form of a suspension (heterogeneous system) or a solution (homogeneous system).

The reaction after the chilloids are allowed to coexist can be carried out at the above temperature for 5 to 72 hours, preferably for 16 to 48 hours.

In this way, γ-cyclodextrin, which includes the terpenoids or is added to the terpenoids through some kind of interaction, is selectively obtained in a high yield in the reaction solution. Furthermore, the effect of the present invention is simply α- or β
- Compared to cyclodextrin, it is significantly superior to the conventional method in that it not only increases the production ratio of γ-cyclodextrin but also improves the conversion rate of sugar to r-cyclodextrin. I can say that.

To isolate and collect γ-cyclodextrin from this reaction solution, a method known per se (for example, the method described above) is used to separate and collect γ-cyclodextrin from a sugar solution in which α-2β-9γ-cyclodextrin is usually mixed. Japanese Unexamined Patent Publication 1987-
146600).

For example, the reaction solution may be treated with a hydrophobic synthetic adsorption resin made of a porous polymer, an ion exchange resin, and/or a glue-applied resin, or by using an appropriate solvent, γ-cyclodextrin This can be carried out extremely easily due to the dramatic increase in the content of .

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited thereto.

Example 1 Potato starch (50.9 g) was well suspended in 800 ml of water, 0.03 g of starch liquefying enzyme Neosvitase (manufactured by Nagashio Sangyo Co., Ltd.) was added, heated with stirring, and held at 80°C for 10 minutes. This resulted in a liquefied starch solution, which was then treated at 120°C for 10 minutes and then cooled to 50°C. To this liquefied starch solution, 25 g of commercially available glycyrrhizin (glytinone A most pure, manufactured by Tokiwa Phytochemical Research Institute Co., Ltd.) and Bacillus oh were added.
CGTags derived from bensig (JP-A-49-124
285 Publication)) was added to make the total amount 1.0
It was set to 0Qml. At this time, -1 of the reaction solution was adjusted to 7.0 with a diluted sodium bicarbonate solution.

(9) When the enzymatic reaction was carried out at 50°C for 65 hours, the amount of each cyclodextrin in the reaction solution was analyzed by high performance liquid chromatography.
g, β-cyclodextrin was produced in an amount of 1.4 g. The amount of r-cyclodextrin produced was a high conversion rate of 4 oq6 per weight of starch charged, and almost no α-cyclodextrin was produced at this time.

The above reaction solution was heat-treated at 100°C for 5 minutes, cooled to 45°C, filled with 21 Diamond Ion HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.) and passed through a nine column.
When the mixture was eluted with warm water at 5°C, unreacted starch and dextrin were eluted first, and then cyclodextrins were eluted. As a result of high performance liquid chromatography analysis of the elution fraction of cyclodextrins, γ-
Cyclodextrin content was 93.5q6. Furthermore, this cyclodextrin fraction was reduced to a solid content of 30%.
When concentrated to 0.5% at room temperature. jir β-cyclodextrin was crystallized. β-cyclodextrin (10) When the liquid was cooled to 5°C and heated overnight, needle-shaped γ-cyclodextrin was obtained.
Crystalline γ-cyclodextrin is reduced to 102 by drying under reduced pressure.
g was obtained. The purity of this crystal was tested by high performance liquid chromatography and was found to be 99.5%. The crystal mother liquor was again left at room temperature to crystallize β-cyclodextrin, and then cooled to 5°C to obtain a purity of 99.
4.6 g of 2% γ-cyclodextrin crystals were obtained, giving a total of 14.8.9 γ-cyclodextrin. The yield based on starch was 29.6%.

The added dalicyrrhizin was completely recovered by passing the reaction solution through an HP-20 column and eluting it with an 80% ethanol solution. The solvent of the recovered glycyrrhizic acid was removed by concentration under reduced pressure, and it could be used again for the reaction.

Example 2 In the same manner as in Example 1, 50 g of potato starch was poured into an 800-g liquefied starch solution, treated at 120°C for 10 minutes, and then cooled to 50°C. This liquefied starch solution was added with commercially available Stehi, t-site (5T
EVIX-80, manufactured by Tama Biochemical Co., Ltd.) 10g and B,
CG Tame derived from 6hbens1g (Example 1
500 units of (same as ) were added to make the total volume 1,000 ml. At this time, - of the reaction solution was 6.5.

When the enzymatic reaction was carried out at 50° C. for 68 hours, analysis of the reaction solution by high performance liquid chromatography revealed that 111.5.9 cyclodextrins were produced. Moreover, almost no α-cyclodextrin was produced. The amount of γ-cyclodextrin produced was a high conversion rate of 24 g per weight of starch charged.

After heat treatment at 100°C for 5 minutes, the above reaction solution had a solid content of 4.
After concentrating to 0% and leaving it overnight at 5℃, β-cyclodextrin crystallized, so I went door to door and found that 10
．． Crystalline β-cyclodextrin (29) IJ was obtained. When the crystallization mother liquor was purified in the same manner as in Example 1, 8.4 g of γ-cyclodextrin with a purity of 99.5% was obtained as crystals.

The yield based on starch was 16.8%.

Moreover, the stevioside used in the reaction was recovered in the same manner as in Example 1 and could be used again in the reaction.

Example 3 50 g of potato starch was made into 800 ml of liquefied starch liquid in the same manner as in Example 1, treated at 120°C for 10 minutes, and then cooled to 50°C. To this liquefied starch solution, 5 g of commercially available glycyrrhetinic acid (Agritinone, manufactured by Tokiwa Phytochemical Research Institute) and 500 units of CGTai+e derived from B. ohbensia (same as Example 1) were added, and the total amount was 1.000 m
/ Closed. At this time, the pI of the reaction solution (7.0 with diluted sodium bicarbonate solution)
Adjusted to.

When the enzyme reaction was carried out at 50°C for 65 hours with constant stirring using a stirrer, analysis by high performance liquid chromatography revealed that γ-cyclodextrin was 8.2
1 and β-cyclodextrin are 12. '25g was produced.

When the above reaction solution was purified by the same operation as in Example 2, it was found that γ-cyclodext with a purity of 99.3% and phosphorus were
8 g of β-cyclodextrin with a purity of 99.0% was obtained.

(13) Glycyrrhetinic acid used in the reaction was recovered in the same manner as in Example 1 and could be used again in the reaction.

Patent applicant Sanraku Ocean Co., Ltd. (14)

Claims (1)

[Scope of Claims] 1. When producing cyclodextrin by allowing cyclodextrin lycosyltransferase to act on starch that can be converted into cyclodextrin, it is possible to coexist tetracyclic or pentacyclic terpenoids in the reaction solution. A method for increasing the yield of γ-cyclodextrin. 2. The method according to claim 1, wherein the four- or pentacyclic terpenoids are natural products collectively referred to as glycyrrhizin, periandrin, or steviocyto, or derivatives thereof.