JPH02211890A - Production of branched cyclodextrin - Google Patents

Abstract

PURPOSE:To inexpensively and continuously obtain a branched cyclodextrin by reacting starch with a cyclodextrin forming enzyme to give a reaction product, treating the reaction product with a debranching enzyme and beta-amylase simultaneously and ultrafiltering the treated substance. CONSTITUTION:A cyclodextrin-containing dextrin is treated with a debranching enzyme and beta-amylase or a mixture of cyclodextrin and maltooligosaccharide is treated with the debranching enzyme by using reverse reaction of debranching enzyme to form a branched cyclodextrin, which is continuously separated by an ultrafilter to obtain the branched cyclodextrin inexpensively and in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing branched cyclodextrin.

Cyclodextrin contains 6 or more glucose molecules with α-
Oligosaccharides having 1,4 bonds forming a cyclic structure are conventionally known, such as α-1β- and γ-cyclodextrins having 6.7 and 8 glucose units, respectively. Such cyclodextrins incorporate various hydrophobic guests in an aqueous solution to form relatively stable host-guest complexes, so they are used as unstable substances in the pharmaceutical and food industries, for example. It is used for stabilizing substances, solubilizing insoluble substances, etc.

However, cyclodextrin generally has low solubility; for example, α-cyclodextrin has a solubility in water of 14.5 g/d! , β-cyclodextrinka<1.85g/d! , γ-cyclodextrin is 23
．． 2g/d! That's about it. In particular, β-cyclodextrin has extremely low solubility, which severely limits its use in various applications.

On the other hand, research on the structure and properties of branched cyclodextrins has been rapidly progressing in recent years;
Publication No. 2, JP-A-61-236801, JP-A-6
The structure is described in JP-A No. 1-287901, JP-A No. 61-287902, JP-A No. 62-106901, etc., and the structure is known for its solubility in water and organic solvents, acute toxicity, and resistance to various lipophilic substances. The inclusion function and the like are described in "Food Chemical", Vol. 3, No. 7, pp. 20-60. The branched cyclodextrin has malto-oligo I! at -a and the outer ring of α-1β- or γ-cyclodextrin.
(including glucose) is one to three α-1,6-linked. Such branched cyclodextrins are particularly useful because their solubility in water is several to several tens of times higher than that of cyclodextrins.

Various methods for producing branched cyclodextrins are already known. For example, the method of Taylor et al. (Ar
ch, Biochei, Biophys,,
113+500 (1966)), Fre
The method of nch et al. (Biochea+, J, 100
．． 6 (1966)), Kobayashi Rano Method <is Powder Chemistry, 3
0.231 (1983), JP-A No. 61-92592, method of Sakano et al.
83)), the method of Kaida et al. (Starch Chemistry, 33.127
(1986)) are known.

Among these, the method of Kobayashi et al. and the method of Sakano et al. have been implemented industrially, and it is said that branched cyclodextrins can be produced efficiently by these methods. However, since it is a batch process, it is not possible to continuously produce branched cyclodextrin.Furthermore, since it uses a reverse enzyme reaction, it consumes a large amount of expensive debranching enzyme. The debranching enzyme has to be disposable for each preparation, therefore,
Branched cyclodextrins cannot be produced at low cost.

As a result of intensive research in order to solve the problems in the production of conventional branched cyclodextrins as described above, the present inventors found that by utilizing the reverse reaction of debranching enzymes, A branching enzyme and β-amylase are allowed to act on a cyclodextrin-containing dextrin, or a branching enzyme is allowed to act on a mixture of cyclodextrin and malto-oligosaccharide, and the branched cyclodextrin thus produced is continuously separated by an ultrafiltration membrane. The inventors have discovered that branched cyclodextrin can be obtained at low cost and in high yield by this method, leading to the present invention.

The first method for producing branched cyclodextrin according to the present invention for the purpose of After that, the product is treated with a debranching enzyme and β-amylase at the same time, and the branched cyclodextrin produced is continuously separated using an ultrafiltration membrane.

Further, the second method for producing branched cyclodextrin according to the present invention involves allowing a branching enzyme to act on a mixture of cyclodextrin and malto-oligosaccharide, and continuously separating the produced branched cyclodextrin using an ultrafiltration membrane. Features.

First, a first method according to the present invention will be explained.

In the first method, the starches include potato, corn, corn, waxy corn, barley,
Those obtained from arbitrary raw materials such as wheat and tapioca can be used. Examples of the starch composition fraction include amylose, amylopectin, and the like. Furthermore, as starch decomposition reaction products, for example,
White dextrin, yellow dextrin, roasted dextrin such as British gum, oxidized starch, modified starch such as low viscosity modified starch treated with enzymes or acids, or obtained by high-speed mechanical stirring, starch phosphate,
Examples include starch derivatives such as starch ethers and starch esters typified by starch acetate, physically treated starches such as starches that have been irradiated with radiation or neutron beams, or have been subjected to high frequency treatment or heat treatment, and α-starch.

These starches, their composition fractions, and decomposition reaction products are
It can be used as a mixture of two or more types.

When raw starch is used as a substrate, raw starch is
- Pre-solubilized with enzymes such as amylase, cyclodextrin-forming enzyme, pullulanase, acid, alkali, etc.
It is preferable to subject it to an enzymatic reaction.

The cyclodextrin-producing enzyme used in the first method according to the present invention is not limited in any way, but includes, for example, an enzyme produced by Bacillus macerans, an enzyme produced by Bacillus stearothermophilus, and an enzyme produced by Bacillus stearothermophilus.
Enzymes produced by Megatherium are preferably used.

Furthermore, as the debranching enzyme, pullulanase is preferably used, but isoamylase can also be used. As the pullulanase, in addition to normal pullulanase, heat-resistant and acid-resistant pullulanase can also be used. The use of thermostable pullulanase has the advantage of increasing the solubility of the substrate and facilitating the reverse reaction. As such a thermostable pullulanase, for example, pullulanase produced by Bacillus acidoplulyticus (manufactured by Novo Industries Japan) is preferably used.

The β-amylase used together with the debranching enzyme can also be used with various expiration dates.

For all of the enzymes mentioned above, crude products as well as purified products can be used. Further, these enzymes may be used as free enzymes, but they can also be immobilized on a suitable support and used as a so-called bioreactor.

FIG. 1 shows a flow chart of the second method according to the present invention.Hereinafter, one preferred embodiment of the present invention will be described in detail for each step shown in parentheses in FIG. do.

In the following, the units of enzyme activity are as follows. Cyclodextrin-generating enzyme ITHU is 3%
of soluble starch was purified by a 30 minute reaction.
This is the enzyme unit that makes e-brown.

purple brown is 0.0.550 ns
+10. D, refers to the state where 302 ns is 0.7,
IPUN of debranching enzyme activity is 40℃, pH 5.0 (
Hydrolyze pullulan under conditions (0.2% pullulan),
It refers to the amount of enzyme that produces reducing sugars with a reducing power equivalent to 1 micromole of glucose per minute, and IU of β-amylase activity is measured at 60°C, pf 15.0 (1% soluble starch, reaction time 1 per minute under conditions of 30 minutes)
This refers to the amount of enzyme that produces micromoles of reducing sugar.

(1) First, raw starch, which is a substrate, is adjusted to the optimum pH of the enzyme.
The raw starch is liquefied by adjusting the raw starch to 5 to 30 THU, preferably 10 to 20 THU of enzyme units per 1 g of starch, and adding a liquefaction enzyme, cyclodextrin-forming enzyme, to the raw starch. The amount of substrate charged is, for example, about 10°
It is Bx.

(2) The above liquefaction is usually carried out at a temperature of 60 to 70° C. The zero reaction time is usually 1 to 3 hours, depending on the activity of the cyclodextrin-forming enzyme used.

(3) After liquefying this raw starch, the substrate is cooled to about 50° C. for the cyclodextrin production reaction.

(4) For the cyclodextrin production reaction, 2 to 10 THU of cyclodextrin production enzyme was added per 1 g of starch, and then 15 to 7 THU was added at 50°C.
Let react for 2 hours.

(5) When liquefying raw starch, when using an enzyme produced by Bacillus stearothermophilus as a liquefaction enzyme, since this enzyme has heat resistance,
Liquefaction of raw starch and cyclodextrin production reaction can be performed simultaneously. The time required from liquefaction to cyclodextrin production reaction is usually about 24 to 72 hours.

(6) By the steps (4) and (5) above, it is possible to obtain dextrin containing about 40 to 55% cyclodextrin. Next, in the saccharification step, dextrins other than cyclodextrin are combined with pullulanase and β-amylase. is used in combination to degrade maltose, thus providing a substrate for branched cyclodextrin production.

The amount of enzyme to be used is 0.0% debranching enzyme per 1g of starch.
Preferably, the range is 2 to 1.0 PUN, and the β-amylase is in the range of 10 to 30 U per gram of starch. The reaction time for the above saccharification depends on the amount of enzyme used, but is usually 15
~72 hours.

(7) The glycated product obtained by this saccharification may be directly subjected to the branched cyclodextrin production reaction, but in consideration of the efficiency of the reverse reaction of the debranching enzyme, it is preferable to concentrate the glycated product. This method of concentrating saccharides is particularly
Advantageously, but not exclusively, by reverse osmosis, the concentration is preferably from 20 to 80"Bx, particularly preferably from 40 to 70"Bx. Since β-cyclodextrin is precipitated when concentrating the glycated product using such a membrane, the concentration is preferably carried out at 50° C. or higher.

(8) Next, a debranching enzyme is added to the obtained substrate, and while reacting, the reaction mixture is treated with an ultrafiltration membrane, and the membrane permeate containing the branched cyclodextrin, which is the reaction product, is continuously filtered out. obtain. The amount of debranching enzyme used is 50 to 150 PUN, preferably 80 to 100 PUN per gram of substrate.
The range is N.

The reaction temperature is 50-65°C, preferably about 60°C.

The operating conditions for ultrafiltration, such as pressure and membrane surface velocity, may be appropriately set depending on the concentration of branched cyclodextrin in the membrane permeate. When the concentration of branched cyclodextrin in the membrane permeate is low, the membrane permeate may be returned to the circulating liquid side, if necessary. Alternatively, add debranching enzyme to the obtained membrane permeate and proceed in the same manner as above.
Branched cyclodextrin can also be obtained via an ultrafiltration membrane, and by such a method the concentration of branched cyclodextrin can be increased. Furthermore, in order to increase the concentration of branched cyclodextrin in the membrane permeate, unreacted maltooligosaccharides contained in the membrane permeate may be separated by chromatography.

(9) The substrate for branched cyclodextrin production is
In the above branched cyclodextrin production reaction, the concentration and amount of liquid obtained as the membrane permeate are continuously replenished, and at this time, the debranching enzyme is deactivated during the branched cyclodextrin production reaction. Therefore, in order to supplement this inactivated bone, add 20 to 60% of debranching enzyme to the replenishment solution.
It is desirable to add 0PUN.

A second method according to the present invention is that in FIG.
Instead of the substrate for branched cyclodextrin production obtained in step ), a commercially available mixture of cyclodextrin and maltooligosaccharide is used as the substrate for the branched cyclodextrin production reaction, so the subsequent steps are as follows: This is the same as explained earlier.

In a mixture of cyclodextrin and maltooligosaccharide, maltooligo I! /cyclodextrin weight ratio ranges from 1 to 5, preferably from 3 to 4, and the substrate concentration ranges from 20 to 80°Bx, preferably from 40 to 70°B
It is x.

The ultrafiltration membrane used in the method of the present invention preferably consists of a dense layer with a molecular weight cut-off in the range of 1,000 to 1,000,000 and a porous layer having micropores with a pore size of several μm to 100 μm. The shape is not limited in any way and may be arbitrary, for example, flat, tubular, hollow fiber, etc. In order to increase the effective membrane area, it is preferable to use a hollow fiber membrane as the ultrafiltration membrane.

The polymer constituting the ultrafiltration membrane is preferably, for example, polysulfone, polyethersulfone, polyamide, polyimide, cellulose acetate, polyacrylonitrile, etc., but is not particularly limited.

However, among the above-mentioned polymers, it is preferable to use polysulfone, polyamide, or polyimide as those that satisfy the strict molecular weight cutoff required for the production of foods and pharmaceuticals, and polysulfone is particularly suitable.

According to the method of the present invention, a colorless and transparent aqueous solution containing 15 to 55% of branched cyclodextrin can be obtained as a membrane permeate. This branched cyclodextrin aqueous solution can be concentrated by an appropriate concentration means, if necessary. As the concentration means, for example, general means such as a heating evaporation method may be employed, but concentration can be easily and efficiently carried out by a reverse osmosis method.

Therefore, for example, when obtaining a maltooligosaccharide powder containing branched cyclodextrin, the membrane permeate is appropriately concentrated as described above, and then powdered by means such as spray drying. Furthermore, in order to obtain individual branched cyclodextrins, the membrane permeate may be concentrated, and then a low-temperature crystallization method, a precipitation method using ethanol, a purification method using column chromatography, etc. may be used.

Highlights: Fruit As described above, according to the method of the present invention, cyclodextrin-containing dextrin is treated with a debranching enzyme and β-amylase by utilizing the reverse reaction of a debranching enzyme, or it is treated with a cyclodextrin and a β-amylase. A branching enzyme is applied to the mixture of malto-oligosaccharides to produce branched cyclodextrin, which is simultaneously filtered through an ultrafiltration membrane.
Since the separation is continuous, branched cyclodextrin can be produced with high productivity.

Further, according to the method of the present invention, the enzyme is not consumed in each reaction and can be used repeatedly, so that branched cyclodextrin can be produced at a low level.

EXAMPLES The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 After adding reverse osmosis-treated water 22, 51 to potato starch 2.5 ktr to form a slurry, cyclodextrin-producing enzyme 37 derived from Bacillus macerans was added to the slurry.
500 THU was added, heated to 66-70°C, and stirred for about 2 hours to liquefy the starch.

Next, the mixture was cooled to 50°C, 7500 THU of cyclodextrin-forming enzyme was added, and the mixture was reacted at 50°C for 24 hours to obtain a cyclodextrin-containing dextrin. After the reaction, pullulanase 12500PUN and β
- Add 50,000 U of amylase and add 30
Allowed time to react.

Before the glycated product obtained in this way is subjected to the branched cyclodextrin production reaction, the 60'
It was concentrated to Bx. Next, using this concentrate as a substrate,
After adding pullulanase 250,000PUN to this, 6
While reacting at 0°C, the branched cyclodextrin produced was filtered using an ultrafiltration membrane (NTU-3250CIR manufactured by Nitto Denko ■).
) and separated as a membrane permeate.

As a result of analyzing the branched cyclodextrin in the membrane permeate by HPLC (column: Fine 5il-NO3, solvent niacetonitrile/water (65/35)), the result was 17.
It was 8%.

Furthermore, when the maltooligosaccharide in the membrane permeate was separated by chromatography and the obtained maltooligosaccharide was returned to the circulating fluid side, the content of branched cyclodextrin in the membrane permeate increased to 48.2%.

Example 2 Pullulanase was added to the substrate for cyclodextrin production obtained in Example 1 at a rate of 10% per gram of starch.
After adding 0PUN, the reaction was continued at 60° C. for 10 days, and at the same time, the reaction product was separated using an ultrafiltration membrane. The substrate concentration in the replenishment fluid was the same as that in the circulating fluid. In addition, the pullulanase activity of the replenishment solution is 3 per gram of starch.
Added so that it would be 0PUN. As a result, during continuous operation, the content of branched cyclodextrin in the membrane permeate is 1
The value was around 7%.

Example 3 2.5 kg of potato starch was treated with reverse osmosis water 2
After adding 2.51 to make a slurry, add Bacillus to this.
40,000 THU of a cyclodextrin-producing enzyme derived from Stearothermophilus was added, heated to 66 to 70°C, and reacted for 24 hours to obtain a cyclodextrin-containing dextrin.

Thereafter, the same operation as in Example 1 was performed to obtain a branched cyclodextrin.

The content of branched cyclodextrin in the membrane permeate is 16.
It was 3%.

Example 4 0.75 kg of β-cyclodextrin and 2.75 kg of maltose were dissolved in reverse osmosis-treated water 51, and 350,000 PUN of pullulanase was added thereto. While reacting at 60°C, maltose in the membrane permeate was dissolved. was separated by chromatography, and while returning the obtained maltose to the circulating fluid side, using the same ultrafiltration membrane as in Example 1,
The reaction products were separated. I responded. As a result, the content of branched cyclodextrin in the membrane permeate was 51.1%.
Met.

Example 5 Example 3 except that α-cyclodextrin was used instead of β-cyclodextrin.
In the same manner as above, a membrane permeate containing 50.9% of branched cyclodextrin was obtained.

[Brief explanation of the drawing]

FIG. 1 is a flow chart illustrating the method of the present invention.

Claims (2)

[Claims] (1) After reacting a cyclodextrin-forming enzyme with at least one substrate selected from starch, starch composition fractions, and starch decomposition reaction products, debranching enzyme and β-amylase are simultaneously applied to the product. 1. A method for producing a branched cyclodextrin, which comprises continuously separating the produced branched cyclodextrin using an ultrafiltration membrane. (2) A method for producing branched cyclodextrin, which comprises applying a branching enzyme to a mixture of cyclodextrin and malto-oligosaccharide, and continuously separating the produced branched cyclodextrin using an ultrafiltration membrane.