JPS62106901A - Diglucosyl-beta-cyclodextrin and its production - Google Patents

Abstract

PURPOSE:To obtain diglucosyl-beta-cyclodextrin nearly comparable in an inclusion power to well-known beta-cyclodextrin and markedly excellent in solubility, by allowing glucoamylase to act upon dimaltosyl-beta-cyclodextrin. CONSTITUTION:The titled compound is obtained by allowing glucoamylase to act upon dimaltosyl-beta-cyclodextrin. This reaction is preferably carried out at 30-60 deg.C and a pH in the range of 3.5-6.5. The obtained diglucosyl-beta- cyclodextrin is a white powder having a molecular formula of C54H90O45, a MW of 1,459 (mass spectrometry), a m.p. of 277.5 deg.C and easy solubility in water.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel branched cyclodextrin and a method for producing the same, and more particularly to noglucosyl-β-cyclodextrin and a method for producing the same.

Imitate me! Lunar branch - Cyclodextrin has glucose residues in β-1,4-
Oligosaccharides bonded in a cyclic manner through bonds, such as α-cyclodextrin consisting of 6 glucose residues, β-cyclodextrin consisting of 7 glucose residues, and γ-cyclodextrin consisting of 8 residues, are generally known.

Cyclodextrin has voids inside due to its structure, and since the inside of these voids is a lipophilic region, it can take in various oily substances.

Therefore, various applications are being considered by utilizing these properties, such as: - Stabilization of unstable substances - Retention of volatile substances - Masking of off-flavors - Solubilization of difficult or insoluble substances.
These cyclodextrins are generally expensive, and this is a major factor hindering the expansion of their use. However, among these, β-cyclodextrin is the most powerful of the other cyclodextrins and is considered promising for use, but its solubility in water at low temperatures (below room temperature) is extremely low (i, ss%; 20° C.), and improvements in this respect have been awaited.

In order to improve the solubility of β-cyclodextrin, the present inventors first separated maltosyl residues by reacting β-cyclodextrin with maltose in the presence of pullulanase. However, we succeeded in obtaining a branched β-cyclodextrin having
No. 694, No. 60-77598), and after further investigation, it was found that by allowing glucoamylase to act on these branched β-cyclodextrins, especially dimaltosyl-β-cyclodextrin, nozzulcosyl-β-cyclodextrin was produced. They discovered this and completed the present invention.

-kflRRIl:j 11-1-S/
Il--7? -→Toy '7 m F
The present invention relates to a method for producing cosyl-β-cyclodextrin for jigs, which comprises reacting glucoamylase to triglyceride to produce diglucosyl-β-cyclodextrin, and to the thus obtained cosyl-β-cyclodextrin for jigs.

The cosyl-β-cyclodextrin for jigs obtained by the present invention is a new compound having the following physical and chemical properties.

1) Molecular formula CS 4 H! . 0452) Molecular weight
1459 Secondary Ion Mass Spectrometry; S1M
S method) (measured from this. (See Figure 1) 3) Melting point 277.5°C (amorphous; decomposed) 4) Specific optical rotation [a12D'+169.3 (C=0,2; 11□
0) 5>Paperk Roma F-Graphy 1-butanol:1-propanol:water=3:5:
One spot is shown in each case, after development on paper using 7ilrgi solvent of 4, color development using iodine solution and color development using silver nitrate after pretreatment with crude glucoamylase.

6) Thin layer chromatography ■ Improbanol: ethanol: water = 5:5:2 ■
1-butanol:pyrinone:water = 6:4:3 or ■
A thin layer plate (DC-Fertigpl) was prepared using each developing solvent of 1-butanol:ethanol:water=5:5:2.
atten Kiese1ge160 (manufactured by Merck & Co.)
After developing on top, when coloring is carried out by color development using iodine solution and color development using phosphomolybdic acid/sulfuric acid,
1 slot each) (Rf value is 0.41 for each)
0.54■0.25).

7) High performance liquid chromatography (conditions 1) Column size: 6φ x 50mm Support: Nucleosil-5NH2 (manufactured by Nadel) Solvent Niacetonitrile:Water = 70:30 flow rate: 2.
Oat/+in Detector: Differential refractometer ERC7520 type (manufactured by Elma Kou Co., Ltd.) (Condition 2) Column size: 6φ x 150 mm Support: 5pherisorb 0DS-11 (7x-
(Manufactured by Zsep) Solvent: Methanol: Water = 8:92 Flow rate and detector were the same as Condition 1.

This product shows one peak under each of the above conditions.

8) Solubility Easily soluble in water, slightly soluble in ethanol.

9) Sexual condition The powder is white and the aqueous solution is colorless.

10) Infrared absorption spectrum (see Figure 2) ν” 3y
400cm-1, 2,930cm-1, 1,150c
Absorption is observed at m-'1.030 cm-'.

11) 1″C nuclear magnetic resonance spectrum (see Figure 3) δ
([)20) QQQ/IQ*YI') 100.6 (C of 1-6 bond,) 12) Methylation analysis After methylation according to the Hakomori method, the methylated product was hydrolyzed to produce The hydrolyzate was reduced and acetylated to induce fludidol-acetate, which was identified by chromatography as 2,3,4,6-tetra-0.
The molar ratio of -methylglucose, 2,3.6-) to 0-methylglucose and 2,3-di-O-methylglucose is 1.9:5.0:1°9.

In addition, cosyl-β-cyclodextrin for jigs having the above-mentioned physicochemical properties has two glucosyl groups in the cyclic glucose residue (6th position) that constitutes β-cyclodextrin, and α-1, 6 glucosyl groups in separate positions. - It was shown by methylation analysis and molecular weight (mass spectrometry) to consist of a linked structure.

According to the present invention, such cosyl-β-cyclodextrin for jigs is produced as follows.

That is, after preparing a solution of dimaltosyl-β-cyclodextrin with a solid concentration (substrate concentration) of 5 to 35%, a predetermined amount of glucoamylase is added, and the temperature and PL of the solution are maintained within the enzyme's preferred action range. , 1 to 24 hours to produce diglucosyl-β-cyclodextrin, and then, if desired, the reaction solution is separated and collected by a method such as chromatography.

Glucoamylase used in the uninvention is as follows:
Rhizopus n1veus
), Rhizopus delemer (R1+1zopus del
commercially available products derived from microorganisms, such as Dimaltosyl-β-Cyclodextrin, and the amount of these enzymes used varies depending on the quality of the substrate and the method of reaction, but in general, it is 5 units per gram of dimaltosyl-β-cyclodextrin. Used above.

In addition, dimaltosyl-β used as a raw material of the present invention
- Cyclodextrin is a new substance, and can be obtained, for example, by reacting β-cyclodextrin and maltose in the presence of pullulanase, and separating and collecting it from the reaction solution.

The reaction of the present invention is basically carried out by using normaltosyl-β-cyclodextrin (with glucoamylase), but in some cases, β-cyclodextrin and maltose are reacted in the presence of pullulanase, It is also possible to produce nozzle fusyl-β-cyclodextrin by subsequently allowing glucoamylase to directly act on this reaction solution without isolating the produced dimaltosyl-β-cyclodextrin.

According to the method of the present invention, the reaction is carried out in accordance with the action conditions of glucoamylase, and the reaction temperature, reaction pH, etc. vary somewhat depending on the type (origin) of the enzyme used, but are generally 30 to 60%. It is desirable that the temperature is within the range of 13.5 to 6.5 °C, p).

To separate the produced diglucosyl-β-cyclodextrin from the reaction solution, for example, Toyopearl HW-4O
Although this can be easily carried out by using column chromatography using 3 or paper chromatography using Whatman 17 chromium, industrially, for reasons of cost, ion exchange resin chromatography, large-scale del filtration, etc. It is advantageous to use

The novel branched cyclodextrin obtained by the present invention has a strong adhesion force comparable to that of the known β-cyclodextrin, and is much superior in solubility, so it can be used in pharmaceuticals and foods. It will greatly contribute to the development of applications for cyclodextrin in cosmetics and other general chemical industry fields.

EXAMPLE 1L Next, the present invention will be explained in more detail and concretely by referring to Reference Examples and Examples.

Reference example (manufacture of dimaltosyl-β-cyclodextrin) Maltose (Nippon Starp Industry KK5!!, purity 99%) 2
．． 00g and β-cyclodextrin (Nihon Shokuhin Kako K
K? ! , purity 98%) to 0.40 g, p) (540,5
Add 0.63 ml of 0 InM sodium acetate buffer and dissolve by heating in a boiling bath. After cooling, add Bacillus sp (
Bacillus sp) thermostable pullulanase (/manufactured by Bo Industry Japan, 200 units/[1)
Add 400 mg and react at 60°C for 72 hours.

After the completion of the reaction, the reaction solution was subjected to Delfiltration chromatography using columns (4,5X 100c+n: 2 columns) packed with Toyovar HW-4OS for separation and purification. The 7 lactyone eluted 13 to 14 hours after loading the sample was collected and concentrated and dried using a rotary evaporator to obtain a white powder of dimaltosyl-β-cyclodextrin.
Get 4B.

Example 1 Dimaltosyl-β-cyclodextrin powder 5.0g
, pH 540, 50mM sodium acetate 11 buffer solution 10
0+nl and add Rhizopus niveas (Rbi
zopus n1veus) glucoamylase (manufactured by Seikagaku Kogyo KK, 33.6 units/TI1g) was added,
After reacting at 40°C for 24 hours, heat is applied to stop the enzyme reaction.

After cooling the reaction solution, use a membrane filter (manufactured by Raiyo Filter Paper KK, Cellulose Nidrei) T M-2,0,45.
The concentration was further adjusted to 10%, and 10 ml of this solution was subjected to gel filtration chromatography using a column (4,5X 100c+++: 2 columns) packed with Toyovar HW-40S for separation and purification. 740m of Kosilu β-cyclodextrin for jigs
g get.

The elemental analysis values and mass spectrometry values of this product were as follows.

Elemental analysis value (C54H, ○1.) Calculated value C=, i4, 45% l+=6.22% 0=4
9.34% Actual value C = 44.51% H = 6.24% Quality Main analysis value (SIMS method) Molecular weight 1459.2924 Mass 1458.4748
A (M 1 H) ion was detected at m/z 1459.

Moreover, this product decomposed at 277.5°C.

Example 2 20g of dimaltosyl-β-cyclodextrin powder was added to 1100mM of 50mM sodium acetate buffer at pHs, o.
Rhizopus niveas (Rbizop
200 units of glucoamylase (manufactured by Seikagaku Kogyo KK, 33.6 units/h) were added, and the mixture was allowed to react at 40°C for 10 hours, followed by heating to stop the enzymatic reaction.

After cooling the reaction solution, filter it using a membrane filter (Toyo Roshi KKlil, Cellulose Nidorate TM-2,0,45).
3 μm mesh) to further reduce the concentration to 10%.
1! ! 'L, 10ml of this solution was added to Toyobar HW-4
Column packed with O3 (4,5X 100cm: 2 columns)
Separation and purification was performed by gel filtration chromatography using
Obtain 0mH.

Example 3 10g of dimaltosyl-β-cyclodextrin powder was added to pH 540, 50mM sodium acetate Ifc solution 110
Dissolve in 0ml and add Rhizopus niveas (Rhiz
opus n1veus) glucoamylase (Seikagaku KK 33.6 units/...8), add 200 units, and add 50
After reacting at ℃ for 5 hours, heating is performed to stop the enzyme reaction.

After cooling the reaction solution, it was filtered using a membrane filter (manufactured by Toyo Roshi KK, Cellulose Nidrei TM-2, 0, 45μ mesh), and 10 ml of the filtrate was filtered using Toyovar H.
Column packed with W-4O8 (4゜5 x 100cm
: 2 bottles) for separation and purification by gel filtration chromatography to obtain 72 On+g of cosyl-β-cyclodextrin for jigs.

[Brief explanation of drawings]

Fig. 1 shows the mass spectrum of cosyl-β-cyclodextrin for jig, and Fig. 2 shows the mass spectrum of cosyl-β-cyclodextrin for jig.
Showing the infrared absorption spectrum of cyclodextrin,
Figure fjS3 shows the I'C nuclear magnetic resonance spectrum of diglucosyl-β-cyclodextrin.

Claims (1)

[Scope of Claims] (1) Diglucosyl-β- having the following physical and chemical properties
cyclodextrin. 1) Molecular formula C_5_4H_9_0O_4_52) Molecular weight 1459 Secondary Ion Mass Spectrometry (SIM)
Measured by S method). (See Figure 1) 3) Melting point 277.5°C (amorphous; decomposed) 4) Specific rotation [α]^2^0_D+169.3 (C=0
．． 2; H_2O) 5) Paper chromatography After developing on paper using a developing solvent of 1-butanol: 1-propanol: water = 3:5:4, color development using iodine solution and pretreatment with crude glucoamylase When the color is developed using silver nitrate, one spot is shown for each spot. 6) Thin layer chromatography (1) Isopropanol:ethanol:water=5:5:2
(2) 1-butanol:pyridine:water = 6:4:3 or (3) 1-butanol:ethanol:water = 5:5:
A thin layer plate (DC-Fertig
platten Kieselgel 60 (manufactured by Merck & Co., Ltd.), and then color development using iodine solution and phosphomolybdic acid/sulfuric acid, respectively.
Spot (Rf value is (1) 0.41 (2) 0, respectively)
．． 54(3)0.25). 7) High performance liquid chromatography (conditions 1) Column size: 6φ x 50mm Support: Nucleosil-5NH_2 (manufactured by Nagel) Solvent: acetonitrile:water = 70:30 Flow rate: 2.0ml/min Detector: Differential refractometer ERC7520 Type (manufactured by Elma Optical Co., Ltd.) (Condition 2) Column size: 6φ x 150 mm Support: Spherisorb ODS-II (manufactured by Phase Sep Co., Ltd.) Solvent: Methanol: Water = 8:92 Flow rate and detector are the same as Condition 1. This product shows one peak under each of the above conditions. 8) Solubility: Easily soluble in water, poorly soluble in ethanol. 9) Properties The powder is white and the aqueous solution is colorless. 10) Infrared absorption spectrum (see Figure 2) ν=3,4
00cm^-^1, 2,930cm^-^1), 1,1
Absorption is observed at 50cm^-^11,030cm^-^1. 11)^1^3C nuclear magnetic resonance spectrum (see Figure 3)
δ(D_2O) 68.2 (C_6 of 1-6 bond) 100.5 (C_1 of 1-8 bond) 12) Methylation analysis After methylation according to the box-mori method, the methylated product was hydrolyzed to produce When the hydrolyzate was reduced and acetylated to induce alditol-acetate and identified by gas chromatography, it was found that 2,3,4,6-tetra-O
The molar ratio of -methylglucose, 2,3,6-tri-O-methylglucose, and 2,3-di-O-methylglucose is 1.9:5.0:1.9. (2) A method for producing diglucosyl-β-cyclodextrin, which comprises reacting dimaltosyl-β-cyclodextrin with glucoamylase to produce diglucosyl-β-cyclodextrin.