JP3150266B2 - Glucan having cyclic structure and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a raw material in the starch processing industry, a composition for eating and drinking, a composition for adding food, an infusion solution, a composition for bonding, a clathrate or an adsorbent, an antiaging agent for starch, or a biological material. The present invention relates to a glucan, a derivative thereof, and a method for producing the same, which are useful as a substitute for starch for disintegrable plastics. More specifically, at least 14 α-
The present invention relates to a glucan having one cyclic structure composed of 1,4-glucoside bonds in a molecule, a derivative thereof, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, starch has been used as a raw material for producing maltose, starch syrup, cyclodextrin, etc., a composition for eating and drinking, a composition for adding food, or a material for biodegradable plastic. It is a polymer substance.

[0003] However, existing starches have the problem of having low solubility in water, aging, and high viscosity when used in the above applications. Starch generally has low solubility in water. Therefore, in order to dissolve starch in water, it is necessary to perform heat treatment or treatment with an organic solvent, acid, alkali, or the like.

[0004] The dissolved or gelatinized starch rapidly ages and forms an insoluble precipitate. Aging of starch is
Changes the physical properties such as viscoelasticity of starch solution and adhesiveness of starch, or causes problems such as deterioration of water retention, shape retention, freezing resistance, or digestibility in foods containing starch. I have.

[0005] Furthermore, gelatinized starch has a high viscosity. This is due to the fact that amylopectin in starch is a very long molecule with a large number of tufted structures. Since gelatinized starch has high viscosity, starch is used as a raw material,
When producing maltose or cyclodextrin, there is a problem that handling is difficult. For example, when a gelatinized starch having a certain concentration or more is transported using a pipe, the pipe may be clogged.

[0006] Thus, the above-mentioned properties (low solubility, aging, and high viscosity) of existing starch have limited the use of starch in food and other industries.

[0007] Therefore, studies have been made to improve the solubility and aging resistance by reducing the molecular weight of starch by enzymatic, chemical, and physical treatments, and to some extent, prevent aging. became. However, it is difficult to suppress an excessive decrease in the molecular weight, and there has been a problem that the inherent properties of starch, which is originally a polymer, are lost. Furthermore, these methods increase the reducing power of the starch. Therefore, when proteins and amino acids are mixed and heated, the starch is colored by the reaction with these substances, and its use has been limited.

[0008] As one of the above-mentioned enzyme treatments, a so-called D enzyme (dispatching enzyme (EC 2.
4.1.25)) or a method using amylomaltase (EC 2.4.1.25) is known. This D enzyme was first discovered in potato, but is known to be present in various plants and microorganisms such as Escherichia coli. This enzyme is called D enzyme when it is derived from plants, and is called amylomaltase when it is derived from bacteria.

The D enzyme has been considered to catalyze the sugar transfer reaction (heterogeneization reaction) of maltooligosaccharides. This enzyme transfers a glucosyl group or maltosyl or maltooligosyl unit from the non-reducing end of the donor molecule to the non-reducing end of the acceptor molecule. Accordingly, as a result of the enzymatic reaction, the degree of polymerization of the maltooligosaccharide initially given becomes nonuniform. This heterogeneous reaction is performed, for example, by using a high-molecular-weight starch such as soluble starch or amylose as a donor,
It is known that this occurs when glucose or maltooligosaccharide is used as the receptor.

[0010] However, in studies using this D enzyme, even a clue that solves the above-mentioned problems in starch has not been obtained.

On the other hand, it is conceivable to use a cyclic polysaccharide composed of D-glucose, that is, a cyclic glucan, as an alternative to the starch.

As the existing glucan having a cyclic structure, cyclodextrins, which are glucans having only a cyclic structure constituted by α-1,4-glucoside bonds, are known. These cyclodextrins are synthesized by allowing a cyclodextrin glucanotransferase (CGTase) to act on starch, and the degree of polymerization is usually from 6 to 8. Kobayashi (1993) Denpun K
agaku: vol. 40 103-116 reports that a glucan having only a cyclic structure composed of α-1,4-glucoside bonds having a higher degree of polymerization is synthesized by CGTase. The largest one has a degree of polymerization of 13.

Furthermore, the yield of glucans having a degree of polymerization of 9 to 13 having only a cyclic structure constituted by α-1,4-glucosidic bonds is extremely low and is not practical. Also,
In the reaction with CGTase, α, with a degree of polymerization of tens or hundreds,
It seems that synthesis of glucan having only a cyclic structure constituted by -1,4-glucoside bonds is impossible.

A sugar can be bound to the 6-position of one or more glucose residues in the cyclodextrin, but the size of the sugar to be bound is usually from the viewpoint of yield to maltotetraose. It is size. Such a glucan has a problem that, due to its special structure, it is difficult to digest with digestive enzymes such as saliva and pancreatic juice.

[0015] Other enzymes, such as the D enzyme, are α-1,4-
It is not used for the synthesis of glucan having a cyclic structure constituted by glucosidic bonds.

[0016] In methods other than enzymatic production, 1
There has been no report on the production of glucans having a degree of polymerization of 4 or more, and even a degree of polymerization of several tens or hundreds, and having a cyclic structure composed of at least 14 α-1,4-glucosidic bonds. That is, at least 14 α having a degree of polymerization of 14 or more, and further, tens or hundreds of
The existence of a glucan having a cyclic structure constituted by -1,4-glucoside bonds has not been known.

Therefore, it is not known what properties glucan having a cyclic structure composed of at least 14 α-1,4-glucosidic bonds having a high degree of polymerization has and it is possible to substitute starch. Was not considered.

[0018] Therefore, an alternative to starch, which has excellent properties such as higher solubility in water, lower viscosity of the dissolved size liquid and no aging observed in ordinary starch, as compared with existing starch. The substance is long-awaited.

[0019]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems. Compared with the existing starch, it has higher solubility in water, lower viscosity of the dissolved solution, and has the excellent properties that the aging observed in ordinary starch does not occur. It is an object of the present invention to provide a novel substance useful as a substitute for starch.

[0020]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, an enzyme which catalyzes a sugar transfer reaction, particularly a D enzyme, has been known to have been known in the art. In addition to the disproportionation reaction, a new reaction α-1,
The present inventors have discovered that a 4-glucan cyclization reaction is catalyzed, and utilized this to complete the present invention. Furthermore, the inventors have determined that the D enzyme has at least one α-1,6-
The present inventors have discovered that they catalyze an α-1,4-glucan cyclization reaction containing a glucoside bond, and have completed the present invention using the catalyst. The D enzyme is a glucan having one cyclic structure in its molecule composed of α-1,4-glucosidic bonds having a degree of polymerization of at least 14, and even tens or hundreds of degrees of polymerization (ie, at least 14 Α-1,
It is not known to carry out the synthesis reaction of glucan having one cyclic structure formed by a 4-glucoside bond in the molecule, nor is it easily imaginable, and is a surprising effect. We are new compounds,
The glucan of the present invention has a very high solubility in water,
Confirmed that the viscosity of the solution is low, and that it has the property of not causing aging, it is useful as a substitute for starch for food and drink compositions, food additive compositions, or biodegradable plastics. We have completed the present invention.

The glucan of the present invention has one cyclic structure in the molecule composed of at least 14 α-1,4-glucosidic bonds.

In a preferred embodiment, the glucan of the present invention has only a cyclic structure composed of at least 14 α-1,4-glucosidic bonds.

In a preferred embodiment, the glucan of the present invention has one ring structure in the molecule composed of at least 17 α-1,4-glucosidic bonds.

In a preferred embodiment, the glucan of the present invention has only a cyclic structure composed of at least 17 α-1,4-glucosidic bonds.

In a preferred embodiment, the glucan of the present invention has a molecular structure having a cyclic structure composed of at least 14 α-1,4-glucosidic bonds and at least one α-1,6-glucosidic bond. Have one in.

In a preferred embodiment, the glucan of the present invention has only a cyclic structure composed of at least 14 α-1,4-glucosidic bonds and at least one α-1,6-glucosidic bond. Have.

Further, the present invention provides a derivative of the above glucan. In the glucan derivative of the present invention, at least one of the alcoholic hydroxyl groups of the glucan is derivatized.

[0028] The derivatization is selected from the group consisting of etherification, esterification, crosslinking, and grafting. This achieves the above object.

Further, the present invention provides a method for producing a linear α-1,4-glucan or a saccharide containing the linear α-1,4-glucan and at least 14 α-1,4-glucosidic bonds in a molecule. Reacting with an enzyme capable of producing glucan having at least 14 α-1,
Disclosed is a method for producing a glucan having one cyclic structure formed by a 4-glucoside bond in a molecule or a derivative thereof.

In a preferred embodiment, the production method of the present invention is performed in the presence of phosphorylase and glucose-1-phosphate.

In a preferred embodiment, the production method of the present invention is performed in the presence of an enzyme that cleaves an α-1,6-glucosidic bond.

In a preferred embodiment, the saccharide used in the production method of the present invention is maltooligosaccharide, amylose, amylopectin, glycogen, starch, waxy starch, high amylose starch, soluble starch, dextrin, starch debris, starch At least one linear α-1,4 selected from the group consisting of partial hydrolysates, phosphorylase-enzymatically synthesized starch, and derivatives thereof.
-Glucan or a saccharide containing it.

In a preferred embodiment, the enzyme capable of producing the glucan used in the production method of the present invention is D
It is an enzyme.

In a preferred embodiment, the enzyme capable of producing the glucan used in the production method of the present invention is an immobilized enzyme.

The present invention also relates to at least 14 α-
Disclosed is a composition for food or drink or a composition for adding food, which contains at least one glucan having one cyclic structure composed of 1,4-glucoside bonds in a molecule or a derivative thereof.

Further, the present invention provides that at least 14 α
Provided is an infusion solution containing at least one kind of glucan having one cyclic structure composed of -1,4-glucoside bonds in a molecule or a derivative thereof.

Further, the present invention provides that at least 14 α
Provided is an adhesive composition containing at least one glucan having one cyclic structure composed of -1,4-glucoside bonds in a molecule or a derivative thereof.

Further, the present invention provides a method for producing at least 14 α
A clathrate or an adsorbate comprising at least one kind of glucan or a derivative thereof having one cyclic structure composed of -1,4-glucosidic bond in the molecule, and a compound that is included or adsorbed on them. provide.

In addition, the present invention provides a method for producing at least 14 α
Glucans having one cyclic structure in the molecule constituted by -1,4-glucoside bonds or derivatives thereof,
Provided is a starch containing at least one kind.

The present invention also relates to a method for producing at least 14 α-
Provided is an antioxidant for starch, comprising at least one glucan having one cyclic structure composed of 1,4-glucoside bonds in a molecule or a derivative thereof.

The above object is achieved by the above-described invention.

Hereinafter, the present invention will be described in detail.

The glucan of the present invention is a glucan having one cyclic structure in the molecule composed of at least 14 α-1,4-glucosidic bonds. Specifically, for example, FIG. And various glucans shown in the above. In FIG. 1, horizontal straight lines and curves are represented by α-
The glucan chains connected by 1,4-glucosidic bonds are shown, and the vertical arrows indicate α-1,6-glucosidic bonds (horizontal straight lines and curves in the schematic diagram below, as well as the vertical arrows) ).

As described above, the glucan of the present invention includes a glucan having only a cyclic structure (hereinafter, referred to as a cyclic glucan of the present invention) and a glucan having a non-cyclic structure in addition to the cyclic structure. The cyclic structure may be composed of only α-1,4-glucosidic bond, or α-1,4-glucosidic bond and at least one α-glycoside.
It may be composed of -1,6-glucoside bonds.
A glucan having at least one α-1,6-glucoside bond in a cyclic structure or in an acyclic structure portion is generated when a glucan having a branched structure such as amylopectin is used as a substrate.

The glucan having only a cyclic structure is a glucan having a non-cyclic structure in addition to a cyclic structure.
It is obtained by a glucoamylase treatment for cleaving a glucoside bond and an α-1,6-glucoside bond from a non-reducing end. Alternatively, it can be obtained directly using linear α-1,4-glucan or a glucan having a branched structure as a substrate.

The glucan of the present invention has the following properties.

(1) When glucoamylase (Toyobo Co., Ltd.), which is an exo-type amylase that hydrolyzes α-1,4-glucoside bond and α-1,6-glucoside bond at the non-reducing terminal, Components that are not further decomposed (glucoamylase-resistant components) remain. The component is not decomposed even if glucoamylase is further acted on after the action of dephosphorylation enzyme (Sigma).

(2) The glucoamylase-resistant component is
The starch may be degraded by isoamylase (Hayashibara Biochemical Laboratory Co., Ltd.) that hydrolyzes the α-1,6-glucoside bond in the starch, and may be affected by glucoamylase.

(3) The glucoamylase-resistant component is
It is degraded by α-amylase, which is an endo-type amylase.

Among the cyclic glucans of the present invention, α-1,6
-Having no glucosidic bond and at least 14 α-
A glucan having only a cyclic structure composed only of 1,4-glucoside bonds (hereinafter, referred to as a cyclic glucan having only α-1,4-glucoside bonds of the present invention) has the following properties.

(1) Neither the reducing terminal nor the non-reducing terminal can be detected.

(2) β-, an exo-type amylase that hydrolyzes the α-1,4-glucoside bond at the non-reducing terminal
Amylase (Seikagaku Corporation) and glucoamylase (Toyobo Co., Ltd.) do not decompose.

(3) Isoamylase (Hayashibara Biochemical Laboratory Co., Ltd.) and Pullulanase (Hayashibara Biochemical Laboratory Co., Ltd.) that hydrolyze α-1,6-glucosidic bonds in starch
Or isoamylase, pullulanase and β-
It is not degraded by the combined use of amylase.

(4) α-, which is an endo-type amylase that hydrolyzes α-1,4-glucoside bonds inside starch molecules
It is completely degraded by amylase (Nagase Seikagaku Corporation).

(5) Hydrolysis with bacterial saccharified α-amylase (Nagase Seikagaku Corporation) and analysis by HPLC yield only glucose, maltose, and some maltotriose. That is, there is no bond other than the α-1,4-glucoside bond.

Among the cyclic glucans of the present invention, glucans having only a cyclic structure composed of at least 14 α-1,4-glucosidic bonds and at least one α-1,6-glucosidic bond (hereinafter referred to as the present invention) Α-1,6 of the invention
-A cyclic glucan having a glucosidic bond) has the following properties.

(1) Neither the reducing terminal nor the non-reducing terminal can be detected.

(2) β-, an exo-type amylase that hydrolyzes the α-1,4-glucosidic bond at the non-reducing terminal
Amylase (Seikagaku Corporation) and glucoamylase (Toyobo Co., Ltd.) do not decompose.

(3) Degraded by isoamylase (Hayashibara Biochemical Laboratory Co., Ltd.), which hydrolyzes α-1,6-glucosidic bonds in starch, and is subjected to the action of glucoamylase.

(4) Endo-type α-amylase (manufactured by Nagase Biochemical Co., Ltd.) that hydrolyzes α-1,4-glucosidic bonds in starch is degraded by glucoamylase. It is known that the minimum limit dextrin when the same endo-type α-amylase is caused to act on a cyclic glucan having an α-1,6-glucoside bond is isomaltosyl maltose (IMM) (Yamamoto, T. Handbook of amylase and
related enzymes, Pergamon press, p40-45 (1988)).
IMM is detected by treating the cyclic glucan having an α-1,6-glucoside bond of the present invention with an endo-α-amylase.

The number of glucoses constituting the cyclic structure of the glucan of the present invention is at least 14, and is preferably
14 to about 5000, more preferably 17 to 1000
Individual. When having an α-1,6-glucoside bond,
The number may be at least one, and is usually 1 to 500.
Pieces, preferably 1 to 100 pieces.

The quantification of the reducing end was carried out according to Hizukuri et al. (198
1) By the modified Park Johnson method of Carbohydr.Res: 94: 205-213, quantification of non-reducing ends was determined by Hizukuri et al. (1978) Carboh
ydr.Res: 63: 261-264.

The degradation by β-amylase and glucoamylase, which are exo-type amylase, or α-amylase, which is isoamylase, pullulanase or endo-type amylase, is carried out, for example, by using at least 14 α-1 and α-amylase of the present invention. A glucan having only a cyclic structure constituted by a 4-glucoside bond is dissolved in distilled water so as to have a concentration of 0.1% (w / v), and 100 μl of the solution is taken. And react for several hours. This reaction product was analyzed using a sugar analysis system (DINEX Co., Ltd., DX300, detector: PAD-2, column: CarboPacPA10).
0) can be analyzed. Elution is performed, for example, at a flow rate of 1 m1 / mi.
n, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 m
M, 37 minutes-350 mM, 45 minutes-850 mM, 47 minutes-850 mM, the degree of polymerization and the resulting sugar can be analyzed.

The detection of the glucoamylase-resistant component from the glucan of the present invention or a derivative thereof can be performed as follows. For example, 100 ml of the glucan of the present invention in 5 ml
, And glucoamylase is added to a final concentration of 10 units / ml, and reacted at 40 ° C. overnight. This reaction product is heated at 100 ° C. for 10 minutes, and insoluble substances are removed by centrifugation. Then, 10 times the amount of ethanol is added, and the remaining polysaccharide is recovered as a precipitate by centrifugation. The precipitate was further dissolved in 1 ml of distilled water, and glucoamylase was added to a final concentration of 50 units / ml.
The mixture is reacted at 0 ° C. for 1 hour, heated at 100 ° C. for 10 minutes, and insoluble substances are removed by centrifugation. A 10-fold amount of ethanol is added thereto to obtain a precipitate. When the raw material of the glucan of the present invention is a raw material such as starch partially modified by a phosphate group, the obtained precipitate is subjected to a 10 mM carbonate buffer solution (pH 9.
4, 10 mM MgCl ₂ and 0.3 mM ZnCl ₂
), And 20 units of phosphatase (bovine origin, manufactured by Sigma) is added. The mixture is reacted at 40 ° C. for 24 hours, and 10-fold ethanol is added to collect the precipitate. The glucoamylase was dissolved again in distilled water and the final concentration was 5
It is added so as to have a concentration of 0 units / ml, reacted at 40 ° C. for 1 hour, and a 10-fold amount of ethanol is added to obtain a glucoamylase-resistant component as a precipitate.

The acyclic structural moiety in the glucan of the present invention, α
Quantification of a cyclic structure portion having a -1,6-glucoside bond and a cyclic structure portion having only an α-1,4-glucoside bond can be performed as follows. Sample glucan 10
After dissolving the mg in 1 ml DMSO, it is quickly diluted with 8 ml of 100 mM sodium acetate buffer.
Take 900 μl of this diluted solution in four samples, and add 100 μl of distilled water, a glucoamylase solution, a mixed solution of a debranching enzyme and glucoamylase, and a mixed solution of endo-α-amylase and glucoamylase to each sample. In addition, the reaction is performed at 40 ° C. for 4 hours. After completion of the reaction, the resulting glucose is measured using a commercially available glucose quantification kit, whereby the non-cyclic structure portion in the sample glucan, the cyclic structure portion having an α-1,6-glucoside bond, and α-1, A cyclic structure portion having only a 4-glucoside bond can be determined by calculation. Detail is,
An example will be described.

The degree of polymerization of the cyclic structure portion of the glucan of the present invention can be measured by using chromatography. In general, it is known that cyclic polysaccharides behave differently in chromatography than linear polysaccharides having the same degree of polymerization, and this property is used to prove that they are cyclic and to determine the degree of polymerization of cyclic polysaccharides. Can be performed. For example, the cyclic glucan having only the α-1,4-glucoside bond of the present invention obtained by reacting with the enzyme D is separated by the above-mentioned sugar analysis system of DIONEX to obtain a single peak fraction. obtain.
The obtained fraction is treated with, for example, 0.1 N HCl at 100 ° C. for 30 minutes, and after partially hydrolyzing the cyclic structure portion,
Degradation of linear glucans of various degrees of polymerization produced by decomposition
The degree of polymerization can be determined by analysis using EX's sugar analysis system. The detailed analysis method will be described in Examples.

The glucan of the present invention has a linear α-1,4-
It can be obtained by reacting glucan or a saccharide containing these with an enzyme capable of producing a glucan having one cyclic structure in the molecule composed of at least 14 α-1,4-glucosidic bonds. Any enzyme can be used as long as it has such activity. In the present invention,
It is preferred to use the D enzyme.

The D enzyme was first discovered in potato,
It is known to be present in various plants and microorganisms such as Escherichia coli. Therefore, the D enzyme can be used irrespective of its origin, even if a gene encoding a plant-derived enzyme is expressed using a host such as Escherichia coli. Here, a method for purifying the D enzyme from potato and Escherichia coli is disclosed as an example, but the method is not limited thereto.

The method for purifying the D enzyme from potato is described in Ta.
Kaha et al., J. Biol. Chem. vol. 268, 1391-1396 (1993). First, potato tubers were homogenized in an appropriate buffer containing 5 mM mercaptoethanol, centrifuged, passed through a 0.45 μm membrane, and then subjected to Q-Sepharo
Apply to a se column and add, for example, 150 mM NaCl to 20 mM Tris-HCl (pH 7.5) (buffer A) containing 5 mM 2-mercaptoethanol.
Wash with a buffer containing The D enzyme elutes in buffer A containing 450 mM NaCl. This was dialyzed against buffer A, and a solution containing 500 mM ammonium sulfate was added to Phenyl Toyop.
Elution is carried out by loading onto an earl 650M (Toso) column and changing the ammonium sulfate concentration in buffer A from 500 mM to 0 mM, collecting the D enzyme active fraction and dialyzing against buffer A. PL-SAX equilibrated with dialysis buffer with buffer A
Load on a column (Polymer Laboratory UK), elute by changing the NaCl concentration in buffer A to 150 mM-400 mM,
D Collect the enzymatically active fraction. The D enzyme can be purified from potato by the above method. The measurement of the enzyme activity is described in detail in Examples.

Takaha et al., J. Biol. Chem. Vol.
91-1396 (1993) contains the cDNA sequence of potato D enzyme
P.394, Fig.3), Preparation of recombinant plasmid of D enzyme (1392)
P.), Expression of the recombinant plasmid in E. coli, and purification of the D enzyme from the recombinant E. coli,
Naturally, recombinantly produced D enzymes can also be used. A method for purifying D enzyme from E. coli is, for example, first, after culturing E. coli TG-1 strain, which is a D enzyme producing strain, in LB liquid medium at 37 ° C. until the logarithmic growth phase, and then converting maltose to a final concentration of 1% ( w / v), and further culture at 37 ° C. for 2 hours. The cells collected by centrifugation are suspended in the buffer solution A and subjected to sonication and centrifugation to obtain a cell extract. Next, for example, Q-Sephar equilibrated with buffer A
Load onto ose Fast Flow (Pharmacia) column and add buffer A
Elution was performed by changing the NaCl concentration from 0 mM to 500 mM,
D Collect the enzymatically active fraction. Ammonium sulfate was added to the active fraction to a final concentration of 1 M, and the mixture was allowed to stand. An insoluble precipitate was removed by centrifugation, and the supernatant was equilibrated with a buffer A containing 1 M ammonium sulfate to Phenyl Toyopearl 650M (Total).
so) load into the column. Elution is performed by changing the concentration of ammonium sulfate in buffer A from 1 M to 0 mM, and a D enzyme active fraction is collected. This fraction was dialyzed against buffer A, and the dialysate was equilibrated with buffer A to obtain Res.
Load onto a source Q column (Pharmacia) and elute by changing the NaCl concentration in buffer A from 0 mM to 500 mM to purify the D enzyme.

The D enzyme can be purified as described above. If no endo-type amylase acting on the α-1,4-glucoside bond in the starch molecule is detected, the crude enzyme in any of the above-mentioned purification steps is used. Even an enzyme can be used for the synthesis of the glucan of the present invention.

The enzyme used in the present invention is a purified enzyme,
Regardless of the crude enzyme, an immobilized product may be used in the reaction, and the type of the reaction may be a batch system or a continuous system. As a method for immobilization, a carrier bonding method (for example, a covalent bonding method, an ion bonding method, or a physical adsorption method), a cross-linking method, or a comprehensive method (lattice type or microcapsule type) can be used.

When it is desired to obtain only the cyclic glucan of the present invention having only an α-1,4-glucosidic bond, enzymatic synthesis of amylose, starch, cut phosphorylase, amylose, maltooligosaccharide, etc. It can be produced by allowing a linear α-1,4-glucan consisting only of glucosidic bond to act on the above-mentioned enzyme which can be used for the glucan of the present invention, for example, the D enzyme.

Also, amylopectin, glycogen, starch, waxy starch, high amylose starch, soluble starch,
When a saccharide having an α-1,6-branched structure such as dextrin, starch hydrolyzate, or amylopectin is used as a raw material, the α-1,4-amino acid of the present invention is used.
When it is desired to obtain only a cyclic glucan having only a glucoside bond, the enzyme can be produced by directly reacting the above-mentioned enzyme usable for the glucan of the present invention, for example, the D enzyme with a raw material. Alternatively, the α-1,6-glucosidic bond is cleaved,
In the presence or absence of enzymes that do not cleave 1,4-glucosidic bonds, such as isoamylase, pullulanase,
The saccharide can be produced by reacting with the above-mentioned enzyme that can be used for the glucan of the present invention, for example, the D enzyme.

For example, as shown in FIG.
The cyclic glucan (13) having only the α-1,4-glucoside bond is prepared by reacting the amylose (12) having 1) with the D enzyme, and then glucoamylase is added thereto to convert the acyclic glucan. Hydrolysis is performed sequentially from the non-reducing end. Next, ethanol is added to recover the cyclic glucan as a precipitate, followed by freeze-drying to obtain a cyclic glucan (13) having only an α-1,4-glucoside bond.

Alternatively, an amylopectin (14) having a reducing end (11), an isoamylase which cleaves an α-1,6-glucosidic bond and a D enzyme are simultaneously reacted,
After preparing the cyclic glucan (13) having only the α-1,4-glucoside bond, glucoamylase is added and hydrolysis is performed sequentially from the non-reducing end. Next, ethanol is added to recover the cyclic glucan as a precipitate, followed by freeze-drying to obtain a cyclic glucan (13) having only an α-1,4-glucoside bond.

Alternatively, by reacting the amylopectin (14) having a reducing end (11) with the D enzyme,
a cyclic glucan (13) having only an α-1,4-glucosidic bond, a glucan (18) having a cyclic structure composed of only an α-1,4-glucosidic bond and a non-cyclic structural part, and at least 14 And a glucan (15) having one cyclic structure in the molecule composed of α-1,4-glucosidic bond and at least one α-1,6-glucosidic bond in the molecule, and then glucoamylase, isoamylase Is reacted. Further, pullulanase can be reacted. Next, ethanol is added to precipitate a cyclic glucan, and the precipitate is collected and freeze-dried to obtain a cyclic glucan (13) having only an α-1,4-glucoside bond.

Furthermore, at least 14 α-
1,4-glucosidic bond and at least one α-1,6
Glucan having one cyclic structure composed of a glucoside bond in a molecule (hereinafter referred to as glucan having an α-1,6-glucoside bond of the present invention) is amylopectin, glycogen, starch, waxy starch, high amylose starch , Soluble starch, dextrin, starch hydrolysate,
Enzyme synthesis by phosphorylase α such as amylopectin
It can be produced by reacting a raw material having a -1,6-branched structure with the above-mentioned enzyme usable for the glucan of the present invention, for example, the D enzyme.

For example, as shown in FIG.
The D-enzyme is reacted with amylopectin (14) having 1) to form a cyclic structure composed of at least 14 α-1,4-glucosidic bonds and at least one α-1,6-glucosidic bond. A glucan (15) having one in the molecule, a cyclic glucan (13) having only an α-1,4-glucosidic bond, and a cyclic structure and a non-cyclic structural part constituted solely by an α-1,4-glucosidic bond And then producing a glucan (18) having at least 14 α-amino acids by gel filtration chromatography.
1,4-glucosidic bond and at least one α-1,6
-Glucan (15) having one cyclic structure in the molecule constituted by a glucoside bond can be separated. Glucamylase is added to the glucan (15) to hydrolyze the non-cyclic structure portion sequentially from the non-reducing end. After the hydrolysis, the cyclic glucan is recovered as a precipitate by adding ethanol, and then lyophilized to obtain a glucan (16) formed of only a cyclic structure having at least one α-1,6-glucoside bond.

The linear α-1,4-series which can be used in the present invention
Glucans or sugars having the same include maltotriose, maltotetraose, maltooligosaccharides such as maltopentaose, amylose, amylopectin, glycogen, starch, waxy starch, high amylose starch,
Examples include soluble starch, dextrin, debranched starch, partially hydrolyzed starch, enzymatically synthesized starch by phosphorylase, and derivatives thereof. These may be used alone or in combination. Here, the term "branched starch" refers to a product obtained by enzymatically cleaving all or a part of a bond other than α-1,4-glucoside bond in starch. The starch partial hydrolyzate is a part of α-1,4-glucosidic bond in starch enzymatically,
Alternatively, it refers to one obtained by chemical cleavage, for example, amylopectin having a degree of polymerization of about 100 or more, amylose having a degree of polymerization of about 20 or more, and the like can be used as a raw material.

Further, a linear α-
The reaction between 1,4-glucan or a saccharide having the same and an enzyme capable of producing a glucan having one cyclic structure in a molecule composed of at least 14 α-1,4-glucosidic bonds in a molecule includes phosphorylase and Glucose-1
-Can be performed in the presence of phosphoric acid. Phosphorylase, when glucose-1-phosphate is present in excess, has
It catalyzes the extension reaction of 1,4-glucan chains. For this reason,
When the α-1,4-glucan chain of the raw material does not have a sufficient length to undergo the cyclization reaction of the enzyme, phosphorylase and glucose-1-phosphate are allowed to coexist. It is possible to increase the yield of the glucan of the present invention.

Further, derivatives of the above-mentioned starch or partially decomposed products of starch may be used as raw materials. For example, a derivative in which at least one of the alcoholic hydroxyl groups of the starch is etherified (carboxymethylated, hydroxyalkylated, etc.), esterified (phosphorylated, acetylated, sulfated, etc.), crosslinked or grafted. Etc. can also be used. Further, a mixture of two or more of these may be used as a raw material.

The reaction between the above-mentioned raw material and the enzyme which can be used in the present invention may be carried out under any conditions such as pH and temperature at which the glucan of the present invention is produced.

Taking the enzyme D as an example, the pH of the reaction is usually 3 to 10, preferably 4 to 9, and more preferably 6 to 8, from the viewpoints of reaction rate, efficiency, enzyme stability and the like. is there. The temperature is preferably from about 20 ° C to 60 ° C, more preferably from 30 ° C to 40 ° C, from the viewpoint of reaction rate, efficiency, enzyme stability and the like. When using enzymes obtained from heat-resistant microorganisms, etc.
Can be used at high temperatures of 90 ° C. Concentration of the above raw materials (substrate concentration)
Can be determined in consideration of the degree of polymerization of the substrate used and the reaction conditions. Usually, about 0.1% to 30%, preferably 0.1% from the viewpoint of the reaction rate, efficiency, and ease of handling the substrate solution.
From 10%, more preferably 0.5 considering the solubility and the like.
% To 5%. The amount of the enzyme to be used is determined depending on the reaction time and the concentration of the substrate, and it is usually preferable to select the amount of the enzyme so that the reaction is completed in about 1 to 48 hours. 50 to 10,000 units,
Preferably 70 to 2,500 units, more preferably 40
0 to 2,000 units.

The glucan of the present invention can be separated and purified by applying a method known per se. For example, the glucan of the present invention can be separated and purified after the completion of the above reaction, by precipitation with a solvent, separation by a membrane, separation by chromatography, and the like. Preferably, the reaction solution is heated or purified as it is. Α-1,4- of the present invention
Cyclic glucans having only glucosidic bonds can be obtained by adding exo-type β-amylase or glucoamylase and decomposing the remaining linear α-1,4-glucan. When a branched polysaccharide such as starch is used as a raw material, an exo-type β-amylase or glucoamylase is used in combination with an enzyme that cleaves an α-1,6-glucosidic bond to form a cyclic α-1,4. -React so that only glucan remains. Thereafter, separation and purification means such as precipitation using a solvent, membrane separation, and chromatographic separation can be applied.

The glucan of the present invention can be easily etherified, esterified, cross-linked, and grafted, can be used as a derivative, and can be used for the above purposes and applications. As a method of derivatization, a method usually used for modifying starch can be used (Biochemical Experimental Method 19, “Starch and Related Carbohydrate Experimental Method”: Nakamura et al., Gakkai Shuppan Center, 19).
86, 273-303). As an example of phosphorylation, the glucan of the present invention is reacted with phosphorus oxychloride in dimethylformamide to obtain a phosphorylated derivative of the glucan of the present invention.

The glucan of the present invention, which is a novel compound obtained as described above, has a significantly improved solubility in water as compared with existing starch, amylose and amylopectin. It has the excellent property that the aging observed in aqueous solutions of starch, amylose and amylopectin does not occur. Further, the glucan of the present invention has an effect of suppressing or preventing aging of existing starch. Furthermore, the aqueous solution has an excellent property that the viscosity is lower than that of an existing aqueous solution such as starch. Furthermore, the glucan of the present invention has low reactivity, and when mixed and heated with proteins and amino acids,
Compared with existing syrup and dextrin, it has an excellent property of being hardly colored. Further, the glucan of the present invention has an excellent property of including or adsorbing various substances. Further, since the glucan of the present invention has the same basic structure as ordinary starch, in which glucose is only linked by α-1,4- and α-1,6-glucosidic bonds, glucose can be easily produced by in vivo enzymes. It is excellent in digestibility and energy conversion efficiency.

Due to these properties, the glucan of the present invention can be used for all foods in which starch, dextrin and the like can be used conventionally, and can be used for almost all food and drink compositions or food additive compositions. can do. The composition for eating and drinking, human food, animal feed,
Pet food is a generic term. That is, liquid and powdered beverages such as coffee, black tea, Japanese tea, oolong tea, juice and sports drinks, bakery products such as bread, cookies, crackers, biscuits, cakes, pizzas, pies, etc., and pasta products such as spaghetti and macaroni. , Noodles such as udon, buckwheat, ramen, confectionery such as caramel, gum, chocolate, snacks such as oysters, potato chips, snacks, cold desserts such as ice cream, sherbet, cream, margarine, cheese, milk powder, condensed milk, Dairy products such as dairy drinks, Western confectionery such as jelly, pudding, mousse, yogurt, Japanese confectionery such as buns, uiro, mochi, rice balls, soy sauce, sauce, noodle soup, sauce, dash sauce, stew sauce, soup No ingredients, complex seasonings, curry ingredients, mayonnaise, dressings, Seasonings such as chaps, curry, stew, soup, retort or canned foods such as bowls, ham, hamburger, meatballs, croquettes, pilaf, rice balls and other refrigerated and frozen foods, and fish processing such as chikuwa and kamaboko It can be effectively used for food, rice balls, rice for lunch, rice for sushi, etc., as well as gyoza skin and Shumai skin. Further, it can be used for infant milk, baby food, baby food, pet food, animal feed, sports drinks, sports foods, dietary supplements, and the like by utilizing its high digestibility.

The glucan of the present invention can be used for infusion.

The glucan of the present invention can be used for an adhesive composition. The glucan of the present invention has adhesiveness and tackiness. Therefore, it can be conventionally used in the adhesive field where starch or dextrin or a derivative thereof is used. The adhesive composition includes, for example, papermaking, surface sizing agents in the paper processing industry, coating agents, interlayer adhesives, etc., binders in the food industry, fibers, sizing agents in the building materials industry, binders, and the like. .

The glucan of the present invention includes or adsorbs various substances. Although it has the ability to include and adsorb cychirodextrin and existing amylose, the glucan of the present invention has a remarkably higher solubility in water than amylose and cyclodextrin, so that a wider range of application can be expected. Furthermore, since the degree of polymerization is significantly higher than that of cyclodextrin, it is considered that the cyclodextrin has guest specificity different from that of cyclodextrin. The substance can change its property or acquire a new property by being included or adsorbed to amylose or cyclodextrin. For example, improvements in solubility, non-volatization of volatile substances, stabilization of unstable substances, and masking of unpleasant odor are well known. The substance to be adsorbed or included on the glucan of the present invention is not particularly limited, for example, wasabi, soy sauce, tea, sansho, citron, flavor, seasoning, foodstuffs such as pigments,
There are medicines such as menthol and linoleic acid. The clathrate or adsorbate thus obtained can be used as foods and pharmaceuticals. For example, it can be used for foods such as those described above and pharmaceuticals such as bath salts, swallows, and powdered medicines.

The glucan of the present invention is used as a raw material in the starch processing industry, such as a raw material for biodegradable plastics and an intermediate material for producing, for example, cyclodextrin from starch, by focusing on the low viscosity of the size liquid. can do.

[0093]

EXAMPLES Hereinafter, the glucan of the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited to the following examples.

Example 1 Preparation of D Enzyme Takaha et al.
The enzyme D was purified by the method described in Biol. Chem. Vol. 268, 1391-1396 (1993). First, potato tubers were washed with 20 mM Tris-HCl containing 5 mM 2-mercaptoethanol (pH 7.
5) Homogenized in a buffer (buffer A), centrifuged, passed through a 0.45 μm membrane, applied to a Q-Sepharose column (16 × 100 mm Pharmacia), and washed with buffer A containing 150 mM NaCl. The D enzyme eluted in buffer A containing 450 mM NaCl. After the elution, the mixture was dialyzed against buffer A, and ammonium sulfate was added to a final concentration of 500 mM. This solution is applied to a Phenyl Toyopearl 650M (Toso) column (1
0 × 100 mm), and elution was performed by changing the concentration of ammonium sulfate in the buffer A from 500 mM to 0 mM. The D-enzyme-active fraction was collected, dialyzed against buffer A, and the dialysate was concentrated using an Amicon Centricon 30 microconcentrator, and then purified using a PL-SAX HPLC column (Polymer).
Laboratory UK), 150-400 mM NaC in buffer A
Elution was performed by applying a linear concentration gradient.
It was concentrated on a miconCentricon 30 microconcentrator.

The enzyme activity was measured using 100 mM Tris-HCl (pH 7.
0), 5 mM 2-mercaptoethanol, 1% (W / V) maltotriose, and 100 μl reaction mixture containing enzyme
At 10 ° C. for 10 minutes, the reaction solution was heated in boiling water for 3 minutes to stop the reaction, and glucose released by the reaction was analyzed using glucose oxidase (Barham et al. (1972)
Analyst 97: 142). The amount of the enzyme that produces 1 μmol of glucose per minute was defined as 1 unit.

Example 2 Preparation of Cyclic Glucan Having Only α-1,4-Glucoside Bond of the Invention 500 mg of commercially available amylose (average molecular weight 30,000) was completely dissolved in 10 ml of 1N-NaOH, and then dissolved in 1 ml of 1M NaOH. 10 ml of Tris-HCl buffer (pH 7.5), 20 ml of distilled water, and 10 ml of 1N-hydrochloric acid were sequentially added to prepare an amylose solution. To this amylose solution, 200 units of potato-derived D enzyme was added and reacted at 30 ° C. for 24 hours.

After the reaction solution was centrifuged, the supernatant was treated at 100 ° C. for 5 minutes and centrifuged again to remove the denatured enzyme protein. After adding about 100 units of β-amylase and 100 units of glucoamylase to the supernatant and reacting at 50 ° C. for 3 hours,
A 10-fold amount of ethanol was added for precipitation. This precipitate was freeze-dried to obtain about 400 mg of powder.

Example 3 Preparation of Cyclic Glucan Having Only α-1,4-Glucosidic Bond of the Present Invention 500 mg of a commercially available soluble starch was completely dissolved in 10 ml of 1N-NaOH, and then 1M was added.
Tris-HCl buffer (pH7.5) 10ml, distilled water 20ml, 1N-HCl 1
0 ml was added in order to make a starch solution. To this starch solution, 10 units of commercially available isoamylase and 200 units of potato-derived D enzyme were added and reacted at 30 ° C. for 24 hours.

After the reaction solution was centrifuged, the supernatant was treated at 100 ° C. for 5 minutes and centrifuged again to remove denatured enzyme protein. After adding about 100 units of β-amylase and 100 units of glucoamylase to the supernatant and reacting at 50 ° C. for 3 hours,
Cyclic amylose was precipitated by adding 10 volumes of ethanol. The precipitate was freeze-dried to obtain about 150 mg of a cyclic glucan powder having only an α-1,4-glucoside bond.

Example 4 Preparation of Cyclic Glucan Having Only α-1,4-Glucoside Bond of the Present Invention 20 mg of commercially available amylose (average molecular weight: 320,000) was added to DMSO.
After completely dissolving in 1 ml, a 20 mM citrate buffer containing 68 units of the D enzyme purified in Example 1 (pH 0.7.
0) 9 ml was added and the mixture was added at 30 ° C. for 10 minutes, 20 minutes, and 30 minutes.
Minutes, 2 hours, 6 hours, and 18 hours. After heating the reaction solution at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation. Five units of glucoamylase were added to 1 ml of the supernatant and reacted at 40 ° C. for 4 hours to remove non-cyclic amylose. Again, the reaction solution was
After heating for 1 minute, the denatured enzyme protein was removed by centrifugation, and a cyclic glucan was precipitated by adding 10 volumes of ethanol to 250 μl of the supernatant.

(Example 5: Confirmation of cyclic glucan having only α-1,4-glucosidic bond) (1) Determination of reducing end and non-reducing end Reduction of powder obtained in Example 2 Hizuku
(1981) Carbohydr. Res: 94: 205-213. For quantification of non-reducing ends, see Hizukuri
(1978) Carbohydr. Res: 63: 261-264. As a result, neither reducing terminal nor non-reducing terminal could be detected.

(2) Digestion with exo-type enzyme and α-1,6-glucoside bond-degrading enzyme The powder obtained in Example 2 was dissolved in distilled water so as to have a concentration of 0.1% (w / v). One unit of each of the following starch-degrading enzymes was added to 100 μl of the aqueous substance solution, and reacted at 40 ° C. for 2 hours.
The reaction product was analyzed using a sugar analysis system (DINEX Co., Ltd .: DX300, detector: PAD-2, column: CarboPacPA100). Elution was performed at a flow rate of 1 m1 / min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 mM, 37 min-350 mM (Gradient
curve No.3), 45min-850mM (Gradient curve No.7), 4
The test was performed for 7 minutes at -850 mM. FIG. 4 shows the results.

As shown in FIG. 4, the powder obtained in Example 2 is a β-exo-amylase that is an exo-type amylase that hydrolyzes the α-1,4-glucoside bond at the non-reducing end of the starch molecule.
Amylase (Seikagaku Corporation) and glucoamylase (Toyobo Co., Ltd.) did not decompose. Also, a combination of isoamylase (Hayashibara Biochemical Laboratories Co., Ltd.) and pullulanase (Hayashibara Biochemical Laboratories Co., Ltd.), which hydrolyze α-1,6-glucosidic bond in starch, or isoamylase, pullulanase and β- It was not degraded even in combination with amylase. However, this powder was completely degraded by α-amylase (Nagase Seikagaku Corporation), which is an endo-type amylase that hydrolyzes α-1,4-glucoside bonds inside starch molecules.

(3) Digestion with endo-type enzyme The powder obtained in Example 2 was dissolved in distilled water so as to have a concentration of 0.1% (W / V), and 100 μl of this aqueous solution was mixed with bacterial saccharified α-amylase (Nagase Seikagaku Corporation) Add 1 unit and add 40
The reaction was carried out at 2 ° C. for 2 hours. Analysis of the reaction by HPLC showed only glucose, maltose, and some maltotriose. From this, it was proved that the powder obtained in Example 2 had no bond other than the α-1,4-glucoside bond, and the powder obtained had only α-1,4-glucoside bond. It was found to be a cyclic glucan having

As a result of the same confirmation, it was found that the powders obtained in Examples 3 and 4 were also cyclic glucans having only α-1,4-glucosidic bonds.

Example 6 Measurement of Degree of Polymerization of Cyclic Glucan Having Only α-1,4-Glucoside Bond
It is known that cyclic polysaccharides have different behaviors in various chromatography from linear polysaccharides having the same degree of polymerization. Using this property, it was proved to be cyclic and the degree of polymerization of the cyclic polysaccharide was determined.

(1) Purification of cyclic glucan having only α-1,4-glucoside bonds having a specific degree of polymerization The substance obtained in Example 2 was obtained only from α-1,4-glucoside bonds having various degrees of polymerization. Thus, the cyclic glucan having the above specific degree of polymerization was purified.

FIG. 5-1 shows the substance 10 obtained in Example 2.
The elution pattern when 0 μg was analyzed by the sugar analysis system of DIONEX (the equipment and elution conditions were the same as described above) is shown. FIG. 5-2 shows that linear α-
1 shows an elution pattern of 1,4-glucan.

The earliest detectable peak eluted as shown in FIG. 5A is denoted by A, and subsequent peaks are denoted by B, C, D... L, and the peaks from G to L are fractionated. And purified.

(2) Analysis of acid hydrolyzate The separated G to L peaks were each 100
Hydrolyzed at 30 ° C for 30 minutes. These conditions are those for partial hydrolysis. The degraded product was analyzed using a sugar analysis system manufactured by DIONEX (the equipment and elution conditions were the same as in Example 5). FIG.
The results are shown in FIG.

The fraction of the peak G was partially hydrolyzed by an acid and decomposed into glucose and linear α-1,4-glucan having a polymerization degree of 2 to 23.

The peak G before acid decomposition has a linear α-1,4
Glucan was eluted at a degree of polymerization of about 20 and a phenomenon was found in which the degraded product was eluted at a higher degree of polymerization than the undegraded product. This phenomenon was also found in the other peaks from H to L, which seems to indicate that the G to L peaks are cyclic glucans having only α-1,4-glucoside bonds.

The linear α-1,4-glucan having the highest degree of polymerization detected in the above-mentioned partial hydrolysis experiment was obtained by polymerization of a cyclic glucan having only α-1,4-glucosidic bonds at the respective peaks. It is considered to represent the degree. Therefore, it was found that the polymerization degrees of the peaks G, H, I, J, K, and L were 23, 24, 25, 26, 27, and 28, respectively. Further, from the results, the degree of polymerization of peak A, which is a cyclic glucan having only the α-1,4-glucoside bond having the minimum degree of polymerization, is 17%.
It is considered to be.

On the other hand, the degree of polymerization of a cyclic glucan having only α-1,4-glucoside bond having a higher degree of polymerization can be analyzed by gel filtration chromatography. In Example 4, 250 g of the cyclic glucan powder having only an α-1,4-glucosidic bond obtained at each reaction time was added to 250 g of the powder.
ul of distilled water, and the whole amount is superose 6
(Φ1cm × 30cm, manufactured by Pharmacia) and Super
ose30 (φ1 cm × 30 cm, manufactured by Pharmacia) was loaded on a connected column, and eluted with a 150 mM aqueous sodium acetate solution.

As a result, as shown in FIG. 7, in the sample in which the reaction time of the D enzyme was 10 minutes, the generated α-1,
The average molecular weight of the cyclic glucan having only 4-glucoside bonds was about 70,000. As the reaction time of the enzyme D increases, the molecular weight of the generated cyclic glucan having only α-1,4-glucosidic bonds decreases. However, after the reaction time of 6 hours, the average molecular weight was about 1
The molecular weight did not change from 5000, and no further lowering of the molecular weight occurred. The molecular weight of cyclic glucan is a value calculated using enzyme-synthesized amylose as a standard.

As described above, α-produced by the D enzyme
It was found that the degree of polymerization of the cyclic glucan having only a 1,4-glucoside bond can be arbitrarily controlled in the range of 17 to several hundreds, depending on the degree of polymerization of amylose used as a substrate, the amount of D enzyme, and the reaction time. .

Example 7 Preparation of Glucan of the Present Invention 40 mg of commercially available waxy corn starch was added to 2 ml of DMS.
Example 1 after suspension in O (dimethyl sulfoxide)
18 ml of a 20 mM citrate buffer (pH 7.0) containing 680 units of the D enzyme purified in the above was added and reacted at 30 ° C. for 40 hours.

After heating the reaction solution at 100 ° C. for 10 minutes,
The denatured enzyme protein was removed by centrifugation. Glucan was precipitated by adding 10 volumes of ethanol to the supernatant. Next, the obtained precipitate was freeze-dried to obtain about 40 mg of the glucan powder of the present invention. This powder has the α of the present invention.
A glucan having only a -1,4-glucoside bond, a glucan having an α-1,6-glucoside bond of the present invention, a cyclic structure comprising only an α-1,4-glucoside bond of the present invention, and an acyclic ring And a glucan having a structural part.

Example 8: At least one α of the present invention
Preparation of a glucan having a -1,6-glucoside bond in a cyclic structure portion or an acyclic structure portion) The precipitate obtained in Example 7 was fractionated by gel filtration chromatography. 25 precipitates
Dissolve in 0 μl of distilled water and add Superose 6 (φ1c
mx 30cm, manufactured by Pharmacia) and Superdex3
0 (φ1 cm × 30 cm, manufactured by Pharmacia) was loaded in its entirety on a connected column. Next, elution was performed using a 150 mM aqueous sodium acetate solution. As shown in FIG. 8, the amylopectin eluted in the void volume was reduced in molecular weight by the reaction of the enzyme D, and the peak I having an average molecular weight of about 30,000 and the average molecular weight of about 30 were obtained.
Two components of peak II, which was 00, were produced. Among them, the peak II having a molecular weight of about 3000 is mainly composed of α-
It was a cyclic glucan having only a 1,4-glucoside bond. On the other hand, peak I having an average molecular weight of about 30,000
Was a glucan having an α-1,6-glucosidic bond. The fraction of peak I was collected, and a 10-fold amount of ethanol was added thereto to precipitate glucan having an α-1,6-glucoside bond. The precipitate was collected by centrifugation and freeze-dried. Thus, 20 mg of a glucan having an α-1,6-glucoside bond was obtained. The molecular weight of the cyclic glucan is a value calculated using enzyme-synthesized amylose as a standard.

(Example 9: Preparation of cyclic glucan having only α-1,4-glucoside bond of the present invention) Maltohexan was added to 10 ml of 100 mM citrate buffer (pH 7.0) containing 5 mM adenosine monophosphate. 20 mg of aose and 200 mg of glucose monophosphate were dissolved, and 20 units of the D enzyme prepared in Example 1 and 1 mg of phosphorylase a (Sigma) were added and reacted at 30 ° C. for 2 hours.

After the reaction solution was centrifuged, the supernatant was
The mixture was centrifuged again to remove the denatured enzyme protein. Add 50 units of glucoamylase to the supernatant,
After decomposing linear amylose to glucose, 10
Cyclic amylose was precipitated by adding twice the volume of ethanol. The precipitate contained about 30 mg of cyclic glucan having only α-1,4-glucosidic bonds. Furthermore, glucose monophosphate present in the precipitate was removed by performing gel filtration chromatography.

(Example 10: Confirmation that the substance of Example 8 contains at least one cyclic structure portion having an α-1,6-glucosidic bond) Glucoamylase is synthesized from the non-reducing end of glucan such as starch. It is an enzyme that sequentially hydrolyzes α-1,4-glucosidic bonds. It is known that, although at a slower rate, it can hydrolyze α-1,6-glucosidic bonds from the non-reducing end. As shown in FIG. 9, amylose and amylopectin having no cyclic structure are completely degraded to glucose (17) by glucoamylase. However, glucans (15) and (13) having a cyclic structure in the molecule have only a non-cyclic structure portion decomposed by glucoamylase, only a cyclic structure is decomposed by glucoamylase, and a cyclic structure portion is not glucoamylase. It remains as a substance that does not undergo decomposition (hereinafter, referred to as a glucoamylase-resistant component). Furthermore, the glucoamylase-resistant component has a cyclic glucan (16) having an α-1,6-glucosidic bond and a cyclic glucan (13) having only an α-1,4-glucosidic bond, depending on the sensitivity to the starch debranching enzyme. Can be classified. That is, the glucoamylase-resistant component that is not decomposed by the combined use of the debranching enzyme and glucoamylase is α-
Cyclic glucan having only 1,4-glucoside bond (1
3) is considered. On the other hand, the glucoamylase-resistant component decomposed by the combined use of the branching enzyme and glucoamylase is considered to be a cyclic glucan (16) having an α-1,6-glucoside bond. However, the cyclic glucan (13) having only α-1,4-glucoside bond, which is not decomposed by the combined use of the debranching enzyme and glucoamylase, is completely decomposed to glucose by using the endo-type α-amylase and glucoamylase together. Can be done. By utilizing these properties, a non-cyclic structural portion, a cyclic structural portion having α-1,6-glucosidic bond, and a cyclic structural portion having only α-1,4-glucosidic bond in the sample glucan can be determined. It is possible to do.

By using this method, the cyclic structures of the glucan and amylopectin obtained in Example 8 were quantified. Table 1 shows the results of quantification of the cyclic structures of glucan and amylopectin obtained in Example 8 using this method.

[0124]

[Table 1]

10 mg of the substance obtained in Example 8 or 10 mg of amylopectin were dissolved in 1 ml of DMSO, and then rapidly diluted with 8 ml of 100 mM sodium acetate buffer. Add 900 µl of this diluted solution to 4
Dispensed into tubes. Next, 100 μl of (1) distilled water, (2) glucoamylase solution, (3) mixed solution of debranching enzyme and glucoamylase, and (4) mixed solution of endo-α-amylase and glucoamylase were added to each tube. In addition, the reaction was carried out at 40 ° C. for 4 hours. After completion of the reaction, the produced glucose was measured using a commercially available glucose quantification kit. Then, the non-cyclic structure portion, the cyclic structure portion having α-1,6-glucosidic bond, and the cyclic structure portion having only α-1,4-glucosidic bond in the sample glucan were determined by the following calculation formulas.

[0126]

(Equation 1)

Here, c, x, y, and z are the amounts of glucose produced from the reaction solutions (1), (2), (3), and (4), respectively. From this result, Example 8
It was found that the substance obtained in (1) contained glucan having a cyclic structure containing at least one α-1,6-glucoside bond.

(Example 11: Measurement of degree of polymerization of cyclic structure part of substance of Example 8) 10 mg of the substance obtained in Example 8 was
After dissolving in 1 ml of DMSO, 1 ml of 1 M sodium acetate buffer (pH 5.5), 8 ml of distilled water, and 20 ml
0 units of glucoamylase were added and reacted at 40 ° C. for 1 hour. After heating at 100 ° C. for 10 minutes, the denatured enzyme was removed by centrifugation. After adding 10 times ethanol to the supernatant to precipitate the polysaccharide, the precipitate was dried.
The obtained polysaccharide was dissolved in 1 ml of distilled water. 50 units of glucoamylase were added to this solution, and reacted at 40 ° C for 1 hour. After heating the reaction solution at 100 ° C. for 10 minutes, the denatured enzyme was removed by centrifugation. 1 in the supernatant
A 0-fold amount of ethanol was added, and the resulting precipitate was dried to obtain 1.1 mg of a powder of a glucoamylase-resistant component (cyclic structure portion).

The glucoamylase-resistant component obtained as described above is dissolved in distilled water so as to have a concentration of 0.4% (w / v), and then a sugar analysis system (Dionex DX300, Dionex) is used. Detector: PAD-2, Column: CarboP
acPA100). Elution was performed at a flow rate of 1 ml /
min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 mM, 37 min-350 m
M (Gradient curve No. 3), 45 minutes-850 mM (Graent curve No. 3)
dient curve No. 7) for 47 minutes at 850 mM.

As a marker for comparing the degree of polymerization,
Linear α-1,4-glucan and glucan having only α-1,4-glucoside bond obtained in Example 2 were also analyzed under the same conditions.

As shown in FIG. 10, the obtained glucoamylase-resistant component was as clean as that obtained with linear α-1,4-glucan and glucan having only α-1,4-glucoside bond. No separation pattern was obtained. This suggests that the glucoamylase-resistant component contains various numbers of α-1,6-glucosidic bonds. However, the peak (arrow) considered to be the smallest corresponds to the position of the degree of polymerization 15 of linear α-1,4-glucan, and as shown in Example 6, glucan having a cyclic structure is Considering that it elutes faster than a linear glucan having the same degree of polymerization, the degree of polymerization of the cyclic structure portion having at least one α-1,6 bond, which is considered to be the smallest, is considered to be at least 15. Was done.

(Example 12: Solubility of glucan of the present invention) The glucans obtained in Examples 2 and 8 and the comparative products amylose, waxy corn starch, and soluble starch (Wako Pure Chemical Industries) were 10% each
(W / v), suspended in distilled water, stirred vigorously with a vortex mixer, and then centrifuged.
At 60 ° C. and 100 ° C., the concentration of sugar dissolved in the supernatant was measured.
Table 2 shows the results.

[0133]

[Table 2]

The glucans of the examples were all shown to be significantly more soluble than the conventional starch.

(Example 13: Aging test of glucan of the present invention) The glucans obtained in Examples 2 and 8 and the comparative products amylose, waxy corn starch and soluble starch (Wako Pure Chemical Industries) were used. , 20 each
0 mg was placed in a screw vial, 10 ml of water was added, and the mixture was boiled to dissolve. Four samples were each left at 4 ° C., and after 0 hour, 3 hours, 6 hours, and 20 hours, 1 ml of each sample was transferred to a centrifuge tube, centrifuged, and sugar dissolved in the supernatant was removed. The concentration was measured. Table 3 shows the results.

[0136]

[Table 3]

[0137] Compared with the conventional starch, it was shown that all of the glucans of the examples had remarkably low aging properties.

Example 14: Glucan of the present invention
(Aging inhibitory effect of starch) When a 4% (W / V) aqueous solution of soluble starch which has been completely heated and laked is stored at 4 ° C, the soluble starch rapidly ages and the lake liquor becomes cloudy. Using this system, the effect of the glucan of the present invention on starch aging inhibition was examined. Each of the glucans obtained in Example 8 was 0% in final concentration.
(W / V), 2% (W / V), and 4% (W / V) were added to a 4% (W / V) aqueous solution of soluble starch and stored at 4 ° C. Sampling is performed at regular intervals,
The cloudiness was measured at an absorbance of 660 nm. The results are shown in FIG.

It was shown that the glucan of Example 6 had an effect of inhibiting starch aging.

(Example 15: Viscosity of glucan of the present invention)
Glucan obtained in Example 8, conventional waxy corn starch, soluble starch (manufactured by Wako Pure Chemical Industries), and Pinedex # 1 (manufactured by Matsutani Chemical Co., Ltd.)
1.6 g each, and put into a vial, and add distilled water 8
After adding and dispersing, dimethyl sulfoxide 7 was added.
2 ml was added, and each glucan was completely dissolved by sufficiently stirring at room temperature. Digital Viscosity
scometer DVL-B (manufactured by TOKYO KEIKI, rotor: No.
1, rotation speed: 60 rpm, measured for 10 seconds).

As a control, a solution (90% DMSO solution) obtained by adding 72 ml of dimethyl sulfoxide to 8 ml of distilled water was also measured. In addition, Paindex # 1 used here is starch which was more strongly hydrolyzed with α-amylase than the soluble starch. Table 4 shows the results.

[0142]

[Table 4]

It was shown that the viscosity of the size liquid of the example was significantly lower than that of the conventional starch.

(Example 16: Reactivity of the glucan of the present invention) The glucans obtained in Examples 2 and 8, soluble starch (manufactured by Wako Pure Chemical Industries), and Pinedex # 1 (Matsutani for comparison) Chemical Co., Ltd.) 20 each
mg was dissolved in 1 ml of distilled water, and the reducing power was examined by the dinitrosalicylic acid method (Sakuzo Fukui, Biological Chemistry Experimental Method 1 Quantitative method for reduction, etc., Gakkai Shuppan Center) using glucose as a standard. Table 5 shows the results.

[0145]

[Table 5]

It was shown that the reactivity was significantly lower than that of conventional starch.

(Example 17: Phosphorylation of glucan of the present invention) The glucans obtained in Examples 2 and 8 were treated with 8 m
g was suspended in 1 ml of dimethylformamide and reacted with 46 mg of phosphorus oxychloride. 9 ml of acetone was added to the reaction solution to obtain 8 mg of phosphorylated glucan.

(Example 18: Inclusion property 1 of glucan of the present invention) 8-anilino-1-naphthalenesulfonic acid (AN
It is known that S) shows very weak fluorescence in an aqueous solution, but shows strong fluorescence when included in the cavity of cyclodextrin. In order to confirm whether the glucan of the present invention includes ANS and increases its fluorescence, the cyclic glucan having only α-1,4-glucoside bond obtained in Example 2, α-cyclodextrin , Pullulan, and dextran were each dissolved in 1 ml of 100 mM phosphate buffer. Next, a 400 μM ANS aqueous solution was added, and the mixture was stirred well, and the fluorescence spectrum was measured using a fluorescence spectrophotometer. As shown in FIG. 12, the fluorescence intensity of ANS did not increase in the presence of non-inclusion pullulan and dextran. In the presence of α-cyclodextrin having inclusion properties, the fluorescence intensity of ANS increased. In the presence of the glucan obtained in Example 2, the fluorescence intensity of ANS increased to more than α-cyclodextrin, and the glucan of the present invention
It was shown to have the property of including S.

(Example 19: Inclusion property of glucan of the present invention 2) The cyclic glucan having only α-1,4-glucoside bond obtained in Example 2, commercially available amylose and soluble starch were each 2%. Was dissolved in distilled water so that Next, a 1/10 amount of a higher alcohol (such as 1-octanol) and a higher fatty acid (such as oleic acid) were added, and the mixture was vigorously stirred. The state at that time was visually evaluated as follows. Table 6 shows the results. In Table 6, ○ indicates that a large amount of precipitate was formed, Δ indicates that a small amount of precipitate was formed, and x indicates that no precipitate was formed.

[0150]

[Table 6]

As shown in Table 6, the cyclic glucan having only α-1,4-glucosidic bond obtained in Example 2 produced a large amount of white precipitate. From this, it was found that the glucan of the present invention has an inclusion ability.

(Example 20: Sports drink containing glucan of the present invention) A sports drink using the glucan of the present invention was blended in the ratio shown in Table 7.

[0153]

[Table 7]

The obtained sports drink had excellent digestibility and a high energy conversion efficiency.

(Example 21: Inclusion complex of glucan of the present invention and linoleic acid) The α-1,4- obtained in Example 2
900 parts of water was added to 100 parts of a cyclic glucan having only a glucoside bond, and the mixture was stirred and dissolved. 100 parts of linoleic acid was added to this solution and stirred well. After stirring, the resulting white precipitate fraction was freeze-dried to obtain an inclusion complex powder containing the glucan of the present invention and linoleic acid.

(Example 22: Inclusion complex of the glucan of the present invention and menthol) The α-1,4- obtained in Example 2
900 parts of water was added to 100 parts of a cyclic glucan having only a glucoside bond, and the mixture was stirred and dissolved. This solution was heated, 100 parts of menthol dissolved in advance was added, and the mixture was stirred well. After stirring, the resulting white precipitate fraction was freeze-dried to obtain an inclusion complex powder containing glucan and menthol.

(Example 23: Adhesive composition containing glucan of the present invention) At least one α-1,6-
60 parts of water was added to 40 parts of glucan having a glucoside bond, and the mixture was heated and dissolved. This solution showed good adhesion.

(Example 24: Preparation of cyclic glucan of the present invention having only α-1,4-glucoside bond) Chitopearl BCW-3503 for enzyme immobilization washed with distilled water
(Fuji Spinning Co., Ltd.) 130 units (5 ml) of purified D enzyme solution containing 1 g and 20 mM Tris-HCl buffer (pH 7.0)
Was incubated for 2 hours at room temperature with gentle agitation to allow the D enzyme to adsorb to the carrier. The reaction solution was filtered, and the D enzyme activity of the filtrate was measured. As a result, almost no D enzyme activity was detected. Therefore, most of the D enzyme was considered to be bound to the carrier. In this D enzyme-bound carrier,
10 ml of a 0.5% (W / V) enzyme-synthesized amylose (AS-30) solution was added, and the mixture was reacted at 30 ° C. for 24 hours under the condition of pH7. The reaction solution was centrifuged, and the supernatant was centrifuged at 100 ° C., 5
For a minute and centrifuged again. After adding 10 units of glucoamylase to the supernatant and reacting at 50 ° C. for 3 hours,
The cyclic glucan was precipitated by adding 0 volumes of ethanol. This precipitate was freeze-dried to obtain 30 mg of powdery glucan. In the same manner as in Example 5, it was confirmed that a cyclic glucan having only an α-1,4-glucoside bond was obtained.

[0159]

The glucans and derivatives thereof of the present invention, which are novel compounds obtained as described above, have greatly improved solubility in water as compared with existing starch, amylose and amylopectin. The aqueous solution
It has the excellent property that the aging observed in aqueous solutions of existing starch, amylose and amylopectin does not occur. Furthermore, the glucans of the present invention also act as starch aging inhibitors. Further, the aqueous solution has an excellent property that the viscosity is lower than that of an existing aqueous solution such as starch. Furthermore, since the glucans and derivatives thereof of the present invention have low reactivity, when mixed and heated with proteins and amino acids, they have excellent properties that they are hardly colored as compared with existing starch syrup and dextrin. . Further, the glucan and its derivative of the present invention have an excellent property of including or adsorbing various substances.
In addition, the glucans and derivatives thereof of the present invention have the same basic structure as ordinary starch, in which glucose is linked only by α-1,4-glucoside bonds and α-1,6-glucoside bonds, and thus can be used in vivo. It can be easily decomposed into glucose by the above enzyme, so that it has excellent digestibility and high energy conversion efficiency.

The process for producing glucan and its derivatives according to the present invention comprises a starch, a partially decomposed product of starch, a derivative thereof, or a mixture thereof, and at least 14 α-1,4-glucosidic bonds. And the step of reacting with an enzyme capable of producing a glucan having one cyclic structure in the molecule. The glucan of the present invention and its derivative can be produced very easily.

[Brief description of the drawings]

FIG. 1 is a schematic view of a glucan of the present invention.

FIG. 2 is a schematic diagram showing a series of processes for obtaining a cyclic glucan having only α-1,4-glucoside bond by reacting D enzyme with amylose or amylopectin.

FIG. 3 is a schematic diagram showing a series of processes for reacting amylopectin with a D enzyme to obtain a glucan formed only of a cyclic structure having at least one α-1,6-glucoside bond.

FIG. 4 is an elution pattern when a cyclic glucan having only an α-1,4-glucoside bond of the present invention is digested with various glycolytic enzymes.

5 is an elution pattern of a cyclic glucan having only an α-1,4-glucoside bond obtained in Example 2, and 5-2 is a linear α-1,4-glucan obtained in Example 2. FIG. FIG. Each peak-like number indicates the degree of polymerization.

FIG. 6 is an elution pattern of partially hydrolyzed and non-decomposed products of each peak GL. Each peak-like number indicates the degree of polymerization.

FIG. 7 is an elution pattern of a cyclic glucan having only α-1,4-glucoside bond obtained in Example 4 by gel filtration.

FIG. 8 shows the elution patterns of waxy corn starch by gel filtration before and after the action of enzyme D.

FIG. 9 shows a non-cyclic structure portion, a cyclic structure portion having at least one α-1,6-glucoside bond, and a cyclic structure having only α-1,4-glucoside bond in the glucan obtained in Example 8. It is a schematic diagram which shows the process of quantifying a part.

10 shows at least one α- obtained in Example 8. FIG.
The elution pattern of the cyclic structure portion of glucan having a 1,6-glucoside bond, the linear α-1,4-glucan, and the cyclic glucan having only the α-1,4-glucoside bond obtained in Example 2 were obtained. is there. Each peak-like number indicates the degree of polymerization.

FIG. 11 is a graph showing the effects of glucan having an α-1,6-glucoside bond obtained in Example 8 on starch aging.

FIG. 12 shows cyclic glucans having only α-1,4-glucosidic bond (solid line), α-cyclodextrin (dashed line), pullulan (one dotted line), and dextrin (two dotted line) obtained in Example 2. It is a fluorescence spectrum of ANS under existing conditions and control (dotted line) conditions. The horizontal axis is the wavelength (nm), and the vertical axis is the spectrum intensity.

[Explanation of symbols]

Reference Signs List 11 reducing terminal 12 amylose 13 cyclic glucan having only α-1,4-glucosidic bond 14 amylopectin 15 composed of at least 14 α-1,4-glucosidic bonds and at least one α-1,6-glucosidic bond Glucan having one cyclic structure in the molecule 16 Glucan formed only by a cyclic structure composed of at least one α-1,6-glucosidic bond 17 Glucose 18 Consisting only of α-1,4-glucosidic bond Glucans having a cyclic structure and a non-cyclic structure portion

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C08L 5/00 C08L 5/00 C09J 105/00 C09J 105/00 // C12N 9/10 C12N 9/10 (72) Inventor Nakamura Hiroyasu 1-30-33, Mizucho-cho, Amagasaki-shi, Hyogo Pref. Ewa Ryo (72) Inventor Kazutoshi Fujii 1-30-33, Mizu-cho, Amagasaki-shi, Hyogo Pref. Ewa-ryo (56) References Patent 3107358 (JP, B2) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 19/14 C08B 37/00 BIOSIS (DIALOG)

Claims (21)

(57) [Claims] 1. A one having glucan 14-5 000 intramolecular cyclic structure composed of alpha-1,4-glucosidic bonds, the glucan, (i) α-1,4- Ring composed only of glucosidic bonds
A glucan having an acyclic structure in addition to a cyclic structure; (ii) at least one α-1,4-glucosidic bond
Cyclic structure composed of α-1,6-glucosidic bond
And a glucan having an acyclic structure, in addition to (iii) consisting of only α-1,4-glucosidic bond
A glycan having only a cyclic structure, and (iv) at least one α-1,4-glucosidic bond
Cyclic structure composed of α-1,6-glucosidic bond
A glucan selected from the group consisting of: 2. A 14-5 by 000 alpha-1,4 glucosidic bonds having only a cyclic structure composed of glucan of claim 1. 3. The glucan according to claim 1, wherein the glucan has one cyclic structure in the molecule composed of 17 to 1,000 α-1,4-glucosidic bonds. 4. The glucan according to claim 1, which has only a cyclic structure composed of 17 to 1,000 α-1,4-glucosidic bonds. Having one to 5. 14-5 000 alpha-1,4-glucosidic bonds and at least one alpha-1,6-glucosidic bonds and intramolecular cyclic structure formed by,
The glucan according to claim 1. 6. The compound according to claim 1, which has only a cyclic structure composed of 14 to 5000 α-1,4-glucosidic bonds and at least one α-1,6-glucosidic bond.
The glucan described in the above. 7. The glucan derivative according to claim 1, wherein at least one of the alcoholic hydroxyl groups of the glucan is derivatized. 8. The glucan derivative according to claim 7, wherein the derivatization is a derivatization selected from the group consisting of etherification, esterification, cross-linking, and grafting. 9. Producing a glucan having a linear α-1,4-glucan or a saccharide containing the same, and a cyclic structure having at least 14 α-1,4-glucosidic bonds in the molecule. A method for producing a glucan having at least one α-1,4-glucosidic bond in a molecule and having a single cyclic structure in the molecule, or a derivative thereof, comprising a step of reacting the enzyme with a D enzyme, which is a possible enzyme. 10. The method according to claim 9, wherein the step is performed in the presence of phosphorylase and glucose-1-phosphate. 11. The method according to claim 9, wherein the step is performed in the presence of an enzyme that cleaves an α-1,6-glucosidic bond. 12. The linear α-1,4-glucan or a saccharide containing the same, comprising maltooligosaccharide, amylose,
Amylopectin, glycogen, starch, waxy starch,
At least one linear α-1,4- selected from the group consisting of high amylose starch, soluble starch, dextrin, starch debranched product, partially hydrolyzed starch, enzymatically synthesized starch by phosphorylase, and derivatives thereof. The glucan or a saccharide containing the glucan.
2. The production method according to any one of 1. 13. The production method according to claim 9, wherein an immobilized enzyme is used. 14. It contains at least one glucan or a glucan derivative selected from the group consisting of the glucan according to claim 1 and the glucan derivative according to claim 7 and claim 8. , Composition for eating and drinking
object. 15. It contains at least one glucan or a glucan derivative selected from the group consisting of the glucan according to claim 1 and the glucan derivative according to claim 7 and claim 8. , Infusion. 16. It contains at least one glucan or glucan derivative selected from the group consisting of the glucan according to claim 1 and the glucan derivative according to claim 7 and claim 8. , An adhesive composition. 17. At least one glucan or glucan derivative selected from the group consisting of the glucan according to claim 1 and the glucan derivative according to claim 7 and claim 8, and them to the inclusion
Clathrate containing the compound to be prepared. 18. A glucan or a glucan derivative selected from the group consisting of the glucan according to claim 1 and the glucan derivative according to claim 7 and claim 8. ,starch. 19. It comprises at least one glucan or glucan derivative selected from the group consisting of the glucan according to claim 1 and the glucan derivative according to claim 7 and claim 8. , Starch aging inhibitor. 20. The gluka according to claim 1 to claim 6.
And the glucan of claim 7 and claim 8.
At least one group selected from the group consisting of derivatives;
For food additives containing lucan or glucan derivatives
Composition. 21. Gurkha according to claim 1
And the glucan of claim 7 and claim 8.
At least one group selected from the group consisting of derivatives;
Lucan or glucan derivatives and their adsorption
Adsorbate containing the compound to be obtained.