JPH1070993A - Production of branched cyclodextrin carboxylic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To industrially and advantageously obtain the subject compound having high purity and useful for a solubilizing agent, especially the solubilizing agent for medicines, etc., by oxidizing a mixture containing plural kinds of specific branched cyclodextrins by microorganisms and separating and purifying the product. SOLUTION: This production of branched cyclodextrincarboxylic acids useful as a solubilizing agent, especially the solubilizing agent for medicines is to oxidize a mixture of branched cyclodextrins or their salts containing at least two of branched dextrins in which 1-8 groups expressed by the formula -Gn -CH2 OH [G is a glucose unit; (n) is 1-6 integer] are bonded at α-1, 6 positions with microorganisms such as Pseudogluconobacter saccharoketogenes K591s (FERM BP-1130), separate and purify by using ion exchangers and/or absorbents.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing a branched cyclodextrin carboxylic acid, particularly to 6-O-cyclomaltoheptaosyl- (6 → 1) -α-D-glucosyl-.
The present invention relates to an improved method for producing (4 → 1) -O-α-D-glucuronic acid or a salt thereof.

[0002]

2. Description of the Related Art Branched cyclodextrin carboxylic acids (hereinafter abbreviated as branched CyD carboxylic acids) or salts thereof, particularly branched β-cyclodextrin carboxylic acids (hereinafter abbreviated as branched β-CyD carboxylic acids) or salts thereof, particularly
6-O-cyclomaltoheptaosyl- (6 → 1) -α-
D-glucosyl- (4 → 1) -O-α-D-glucuronic acid (hereinafter abbreviated as G ₂ -β-CyD carboxylic acid) or a salt thereof is useful as a solubilizing agent in the field of food and medicine. As disclosed in Japanese Patent Application Laid-Open No. 7-76594, maltosyl-β-cyclodextrin (hereinafter abbreviated as G ₂ -β-CyD), which is a kind of branched cyclodextrin, is substituted with a hydroxymethyl group (- CH ₂ OH) and / or hemiacetal OH are produced by microbiologically oxidizing a microorganism belonging to the genus Pseudogluconobacterium having the ability to oxidize a carbon atom having a hemiacetal OH to a carboxyl group. The obtained G ₂ -β-CyD carboxylic acid is obtained, for example, by removing the cells from the microorganism oxidation reaction solution, subjecting the supernatant to activated carbon column chromatography, washing with water, and then adding 10%
The fraction eluted with an aqueous methanol solution and further eluted with a 50% aqueous methanol solution is collected, concentrated, and lyophilized to a powder to be purified.

Although this method is an efficient purification method, G ₂ -β-CyD as a substrate is, for example, maltose and β-β-CyD.
It is produced by allowing pullulanase or isoamylase to act on cyclodextrin (hereinafter abbreviated as β-CyD), and has the formula: —G _n —CH ₂ OH (where G is a glucose unit and n is an integer of 1 to 6) Is a branched β-cyclodextrin having 1 to 7 α-1,6 bonds at the 6-O-position of the glucose unit of the β-cyclodextrin ring (that is, a branched β-cyclodextrin ring).
CyD), an unreacted substance, and a mixture with other cyclodextrin (hereinafter abbreviated as CyD) derivatives. Therefore, it is necessary to isolate and purify a desired branched β-CyD and subject it to the above-mentioned microbial oxidation reaction. There is.

[0004] However, the isolation and purification of branched CyD requires time-consuming and costly and very complicated operations. For example, in Japanese Patent Publication No. 61-197602, maltose and β
-CyD is reacted in the presence of pullulanase or isoamylase, the reaction solution is separated by a gel filtration column, and fractions eluted 15 to 17 hours after sample loading are collected and purified. In JP-A-6-14789, pullulanase or isoamylase is allowed to act on a mixture of β-CyD and maltose, and the reaction solution is purified by high performance liquid chromatography. The branched β-CyD produced by the above method is expensive, and the branched β-CyD carboxylic acid produced by using the same is also expensive, which is not an industrially advantageous method. Therefore, in order to efficiently produce a high-purity branched β-CyD carboxylic acid, further improvement in the production method is required.

The above-mentioned Japanese Patent Application Laid-Open No. 7-76594 does not mention crystallization of a branched cyclodextrin carboxylic acid. On the other hand, regarding the branched cyclodextrin, JP-A-63-150301 discloses that a reaction solution is brought into contact with a chemically modified silica carrier, separated and purified, concentrated, dried and powdered. In JP-A-61-197602, a reaction solution is separated and purified by filtration chromatography using a Toyopearl HW-40S column, and concentrated and dried to obtain a branched β-CyD powder. As described above, conventionally, after purification using a specific separating agent to increase the purity, freeze-drying or concentration drying was performed to form a powder. Further, as described above, both the branched β-CyD carboxylic acid and the branched β-CyD have extremely high solubility in water, and it has been extremely difficult to extract them as crystals. Freeze drying requires expensive drying equipment, is difficult to automate, and has to be batch type.
There is a problem industrially. Spray drying is difficult to aseptically operate.
From the above, it is desired to obtain a branched β-CyD carboxylic acid as an industrial production method in a crystallized form.

[0006]

SUMMARY OF THE INVENTION The main object of the present invention is to:
It is an object of the present invention to provide an improved production method useful for efficiently and industrially obtaining a high-purity branched CyD carboxylic acid useful as a solubilizer, particularly as a solubilizer for pharmaceuticals.

[0007]

In view of such circumstances, the present inventors have studied for the purpose of microbial oxidation reaction of branched CyD and isolation of a reaction product from the reaction solution in a high yield. Was piled up. As a result, the mixture of branched CyDs, such as the reaction solution obtained in the production of branched CyDs, is directly subjected to microbial oxidation and then treated under specific conditions with an ion exchanger and / or adsorbent to achieve its purpose. I found what I could achieve. In addition, they have found that a branched CyD carboxylic acid can be crystallized from an aqueous solution, and have made further studies to complete the present invention. The present invention provides: (1) the above formula: (wherein, G is a glucose unit, n represents 1 to an integer of 6) -G _n -CH ₂ OH groups represented by, the cyclodextrin ring Microbial oxidation of a branched CyD mixture containing at least two kinds of branched CyD in which 1 to 8 α-1,6 bonds are bound to the 6-O position of the glucose unit, and then separating and purifying it. It is intended to provide a method for producing a branched CyD carboxylic acid or a salt thereof. -CH ₂ OH branch portion -G _n -CH ₂ OH indicates that the terminal glucose branch portion has as a 6-position hydroxymethyl group.

Further, the present invention provides (2) that n is 1 to 3
(1) wherein the cyclodextrin is β-cyclodextrin; and (4) the branched cyclodextrin mixture is maltosyl-β-cyclodextrin. (5) The method according to (1), wherein the branched cyclodextrin carboxylic acid is 6-O-cyclomaltoheptaosyl-
(6 → 1) -α-D-glucosyl- (4 → 1) -O-α
The method according to the above (1), which is -D-glucuronic acid,
(6) The production method according to the above (1), wherein separation / purification is performed using an ion exchanger and / or an adsorbent; (7) Separation / purification is performed using an ion exchanger; The production method according to the above (1), wherein the adsorbent is obtained as a crystal,
(9) The production method according to the above (6), which is a synthetic adsorbent and / or an activated carbon, and (9) using an anion exchange resin as an ion exchanger, eluting a branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin with an acid. The method according to (6) above,

(10) The production method according to the above (6), wherein activated carbon is used as an adsorbent and the adsorbed branched cyclodextrin carboxylic acid or a salt thereof is eluted with water or a mixture of water and a hydrophilic organic solvent. (6) The method according to (6), wherein the adsorbed branched cyclodextrin carboxylic acid or a salt thereof is eluted with water or a mixture of water and a hydrophilic organic solvent, using a synthetic adsorbent as the adsorbent. Using a strongly basic anion exchange resin as a product, eluting the branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin with an acid, and desalting the eluate,
(13) The production method according to the above (7), wherein the target product is obtained as crystals by crystallization, (13) a branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin using a strongly basic anion exchange resin as an ion exchanger. After elution with an acid, the resulting eluate is concentrated, then desalted using a cation exchange resin and an anion exchange resin, concentrated, and the concentrated liquid is cooled to precipitate a branched cyclodextrin carboxylic acid, which is then separated. (14) using a strongly basic anion exchange resin as the ion exchanger, eluting the branched cyclodextrin carboxylic acid or its salt loaded on the anion exchange resin with an acid, and further obtaining After adjusting the pH of the eluate to be neutral and concentrating it, the concentrated solution is adsorbed on activated carbon and then eluted with water. The strongly basic anion exchange resin used as the exchanger, loaded onto an anion exchange resin branch cyclodextrin carboxylic acid or eluting a salt thereof with an acid,
The pH of the obtained eluate is adjusted to neutral and concentrated, and then the concentrated solution is adsorbed on a synthetic adsorbent, and then eluted with water.

A group represented by the formula: -G _n -COOH (wherein G is a glucose unit and n is an integer of 1 to 6) is a group represented by the 6-O position of the glucose unit of the cyclodextrin ring. Characterized by separating and purifying a mixture of a branched cyclodextrin carboxylic acid or a salt thereof containing at least two kinds of a branched cyclodextrin carboxylic acid or a salt thereof, wherein 1 to 8 α-1,6 bonds are bonded to each other. carboxylic acid or separation and purification of a salt thereof, (17): (wherein, G is a glucose unit, n represents shows the integer of 1 to 6) -G _n -COOH groups represented by, cyclodextrin ring Containing at least two kinds of branched cyclodextrin carboxylic acids or salts thereof having 1 to 8 α-1,6 bonds at the 6-O position of the glucose unit Trinh carboxylic acid or mixtures of their salts, (18): (wherein, G is a glucose unit, n represents shows the integer of 1 to 6) -G _n -CH ₂ OH groups represented by the cyclodextrin ring Microbial oxidation of a mixture of branched β-cyclodextrin or a salt thereof containing at least two kinds of branched cyclodextrins having 1 to 7 α-1,6 bonds at the 6-O position of the glucose unit of And / or a method for producing a branched cyclodextrin carboxylic acid or a salt thereof, wherein the method is subjected to a treatment with activated carbon.

The -COOH of the branched moiety -Gn-COOH of the branched cyclodextrin carboxylic acid indicates that the hydroxymethyl group at the 6-position of the terminal glucose of the branched moiety is oxidized to form a carboxyl group. In particular, the present invention is, as a branching beta-CyD mixture, G ₂ using a reaction mixture obtained by the action of pullulanase or isoamylase to a solution containing a beta-CyD and maltose -β
It is suitable for producing -CyD carboxylic acid, and the mixture is subjected to a microbial oxidation reaction, which is then separated and purified to obtain the desired product. Separation and purification methods include, inter alia, loading the reaction solution obtained by the microbial oxidation reaction onto an ion exchanger, eluting with an acid, loading the eluate on activated carbon, and then eluting with a mixture of water and a hydrophilic organic solvent. Alternatively, a preferable method is to adjust the pH of the eluate to neutral, then load the solution on activated carbon, and then elute with water. Alternatively, adjust the pH of the eluate to neutral,
After concentration, the concentrate is further adsorbed on a synthetic adsorbent and eluted with water. Preferably, activated carbon or a synthetic adsorbent is used in combination with a strongly basic anion exchange resin among ion exchangers.

A more preferable separation / purification method is a method of subjecting the target substance to crystallization by crystallization after subjecting it to an ion exchanger. Crystallizing the branched cyclodextrin carboxylic acid (free form) is often preferred. More specifically, the reaction solution obtained by the microbial oxidation reaction is loaded on an ion exchanger, eluted with an acid, and the obtained eluate is concentrated if necessary, and then desalted (eg, a cation exchange resin and an anion exchange resin are used). Then, depending on the method used)
Concentration and cooling of the concentrated solution to precipitate and separate the branched cyclodextrin carboxylic acid. Preferably, among the ion exchangers, it is preferable to purify in combination with a strongly basic anion exchange resin and subject it to crystallization. After ion-exchanger treatment, high-purity branched β-CyD carboxylic acid can be isolated and purified by crystallization, but it is further purified by crystallization after the combined use of ion-exchanger treatment and adsorbent treatment. You can also increase the degree.

[0013] as a mixture of branched CyD in the production process of the present invention is not particularly limited as long as the object can be achieved, for example, the formula: -G _n -CH ₂ OH (wherein, G is a glucose unit, n represents A group represented by an integer of 1 to 6) containing at least two kinds of branched CyDs in which 1 to 8 α-1,6 bonds are bonded to the glucose unit of the cyclodextrin ring at the 6-O-position at the 6-O-position. Cy
As D, α-CyD, β-CyD and γ-CyD are usually used. The present invention is particularly applicable to a mixture of branched β-CyD. In particular, those containing G ₂ -β-CyD and at least one other type of branched CyD are preferably used. The content and ratio are not particularly limited. Reaction solution, heat treatment reaction in branched CyD production,
It is preferable to use the concentrate or the like directly. For example, in the case of branched β-CyD, such a reaction solution or the like may be, for example, a branched β-CyD such as maltosyl-β-cyclodextrin, maltotriosyl-β-cyclodextrin, maltotetraosyl-β-cyclodextrin. , Other reaction products and unreacted raw materials. As such a reaction solution, for example, a reaction solution of a branched cyclodextrin obtained by the method described in JP-A-6-14789 can be preferably used. In particular, branched β obtained by allowing pullulanase or isoamylase to act on a solution containing β-cyclodextrin and maltose.
Mixtures of -CyD are preferred. At this time, G ₂ -β-CyD
Having a content of 50% or less (for example, containing only about 40%) can be used. A more preferred mixture is G ₂ -β-CyD about 30 to 50.
%, -G ₂ -CH ₂ OH branched CyD branched at two places
(Hereinafter abbreviated as (G ₂ ) ₂ -β-CyD) about 30 to 50%,
-G ₂ -CH ₂ OH is bifurcated branches CyD in 3 places and the like (hereinafter, (G _{2) 3} abbreviated as-beta-CyD) mixture containing about 20-50%. In particular, those having a branched β-CyD content of 90% or more, preferably 95% or more, are preferred.

The branched α-CyD and the branched γ-CyD can also be used in the form of low purity or mixtures which are commercially available or can be obtained according to known methods. In the present invention,
Such a mixture of branched CyDs is subjected to a microbial oxidation reaction to obtain an oxidation reaction solution containing a mixture of the corresponding branched CyD carboxylic acids. Microbial oxidation of the branched CyD mixture
It can be performed by a known method, for example, a method similar to the method disclosed in JP-A-7-76594. That is,
A microorganism belonging to the genus Pseudogluconobacter, branch C
Using a microorganism capable of specifically oxidizing the hydroxymethyl group (—CH ₂ OH) group of yD, the branched C
Oxidizes to yD carboxylic acid. Examples of microorganisms belonging to the genus Pseudogluconobacter that can be used include, for example, bacteria of Pseudogluconobacter saccharoketogenes, typically the following strains described in European Patent Publication No. 221707. .

Pseudogluconobacter saccharoketogenes strain K591s (FERM BP-1130, IF
O14464), Pseudogluconobacter saccharoketogenes 12-5 strain (FERM BP-1129, I
FO14465), Pseudogluconobacter saccharoketogenes TH14-86 (FERMBP-112)
8, IFO14466), Pseudogluconobacter
Saccharoketogenes strain 12-15 (FERM BP-1
132, IFO14482), Pseudogluconobacter saccharoketogenes strain 12-4 (FERM BP-
1131, IFO14483 strain), Pseudogluconobacter saccharoketogenes 22-3 strain (FERM B)
P-1133, IFO14484)

In the oxidation reaction, the cells of the Pseudogluconobacter microorganism may be allowed to act, or a processed product thereof may be used. As the processed product, for example, a culture solution of these microorganisms can be used. Further, enzymes produced by these microorganisms may be used. In the case of Pseudogluconobacter microorganisms, enzymes are usually accumulated in the cells. Usually, it is convenient to use the cells themselves and contact and act on the branched CyD mixture to produce a carboxylic acid. The cells or a culture solution thereof are described in, for example,
It can be produced according to the method described in JP-A-85088. That is, seed culture is performed from the slant, and then main culture is performed to obtain a fermentation broth. If necessary, the fermentation broth is centrifuged, the precipitate is collected, and then washed several times with a saline solution. The resulting precipitate can be subjected to a bacterial reaction. The culture is performed at pH 4-9, preferably at pH
6 to 8. The preferred culture temperature is 10-4
0 ° C., preferably 25-35 ° C.

In the present invention, the crude mixture of the branched CyD may be dissolved or suspended in water or a water-miscible solvent to contact the microorganism. The amount of the solvent to be used may be selected within a range that does not delay the reaction, and the effective substrate concentration is usually 0.1 to 20% (w / v), preferably 1 to 5% (w / v). It is. The preferred temperature range for performing the oxidation reaction by microorganisms is 10 to 40 ° C,
Preferably it is 25-35 degreeC. The reaction is preferably carried out under aerobic conditions.
While ventilating at liters / minute, if necessary,
It can also be stirred at 00 rpm. In this reaction, the pH is preferably adjusted. Usually, pH 4-9, preferably pH
It is effective to carry out in the range of 6 to 8. The base used for pH adjustment may be any as long as it does not inhibit the reaction. For example, inorganic salts such as sodium hydroxide, potassium hydroxide, calcium carbonate, magnesium hydroxide, and ferrous hydroxide, and organic salts such as sodium morpholinoethanesulfonate and calcium morpholinoethanesulfonate can be used.

The microbial oxidation reaction solution thus obtained depends on the composition of the branched CyD mixture used. For example, when oxidizing a branched β-CyD mixture, in addition to G ₂ -β-CyD carboxylic acid, 6-O -Cyclomaltoheptaosyl- (6
→ 1) -α-D-glucosyl- (4 → 1) -O-α-D
-Glucosyl- (4 → 1) -O-α-D-glucuronic acid (hereinafter abbreviated as G ₃ -β-CyD carboxylic acid), -GG
A branched β-CyD carboxylic acid in which -COOH is branched at two places (hereinafter abbreviated as (G ₂ ) ₂ -β-CyD carboxylic acid) and a branched β-Cy in which -GG-COOH is branched at three places
Other branched β-CyD carboxylic acids such as D carboxylic acid (hereinafter abbreviated as (G ₂ ) ₃ -β-CyD carboxylic acid) or salts thereof, for example, salts with alkali metals such as sodium and potassium, magnesium, Includes salts with alkaline earth metals such as calcium. When the microorganism is oxidized while being neutralized with the above inorganic salt or organic salt (particularly hydroxide) for pH adjustment, it is usually obtained with the above salt. When obtained as a salt, it can be converted to a free carboxylic acid by a conventional method (eg, contact with a cation exchange resin). When obtained with a carboxylic acid, it can be converted into a salt thereof by a conventional method.

In the present invention, if necessary, the cells are removed from the reaction solution by filtration, centrifugation, etc., and then separated and purified by means such as treatment with an ion exchanger and / or adsorbent. preferable. In this specification, the classification and distinction of the separation / purification means are in accordance with "Isolation and Purification of Substances" (Tokyo Publishing Co., Noboru Ohtake et al.). When separating and purifying using an adsorbent,
As the adsorbent to be used, it is particularly preferable to use one or more selected from active substances and synthetic adsorbents. As the ion exchanger, any can be used which can isolate the desired branched CyD carboxylic acid by ion exchange or adsorption. These ion exchangers include ion exchange resin, ion exchange cellulose, ion exchange dextran and the like, but usually anion exchange resin is conveniently used. Among them, strong basic anion exchange resins are preferred. More preferably, a strongly basic anion exchange resin having a degree of crosslinking of 8% or less, for example, PA-406, PA-408,
PA-412, PA-416, PA-306, PA-3
08, PA-312, PA-316 (all manufactured by Mitsubishi Chemical Corporation), IRA-401, IRA-402, IRA-41
1, IRA-900, IRA-904 (all manufactured by Organo Corporation) and the like. If the degree of cross-linking is too high, the amount of adsorption per unit amount of the branched CyD carboxylic acid per unit resin decreases, and a large amount of resin is required, which is economically problematic.

The treatment with an ion exchanger can be carried out by contacting with an ion exchanger such as an ion exchange resin to bind a branched CyD carboxylic acid or a salt thereof, and then eluting with an appropriate acid. By collecting the fraction containing the branched CyD carboxylic acid or a salt thereof and removing the solvent by a method known per se, a desired branched CyD carboxylic acid or a salt thereof can be isolated and purified. Examples of the acid include inorganic acids (eg, hydrochloric acid, sulfuric acid, etc.), organic acids (eg, acetic acid,
Formic acid or the like is used, and hydrochloric acid or acetic acid is particularly preferred. When an inorganic acid is used as the acid, its concentration is 0.0
005 to 0.5N, preferably 0.001 to 0.1N. When an organic acid is used as the acid, its concentration is 0.1 to 0.1.
3N, preferably 0.5-2N. For example, G ₂ -β
When isolating -CyD carboxylic acid, 0.001 to 0.00.
It is good to elute at 01N, preferably at 0.003 to 0.008N.

As the activated carbon, those having a large surface area, for example, having a pore size of about 10 to 100 °, preferably about 20 to 40 °, for example, commercially available specially made Shirasagi and K-1 coal (above,
(Takeda Pharmaceutical Co., Ltd.) is preferably used. Activated carbon treatment is performed by contacting activated carbon to adsorb a branched CyD carboxylic acid or a salt thereof, and then water or water and methanol,
It can be carried out by eluting with a mixed solvent with an organic solvent such as isopropanol.
The desired branched CyD carboxylic acid or a salt thereof can be isolated and purified by collecting the fraction containing D carboxylic acid or a salt thereof and removing the solvent. Examples of the synthetic adsorbent include aromatic modified resins in which halogen such as bromine is chemically bonded to a styrene-divinylbenzene-based base to enhance hydrophobic adsorption power. The synthetic adsorbent has a large surface area, for example, a pore diameter of 1 to 1000 ° (usually 10 to 10 °).
500 °), preferably about 50 to 250 °, and a particle size of 1 to 1000 μm (normally 5 to 500 μm), preferably 50 to
Those having a thickness of about 180 μm are preferable. For example, Sepabeads SP207 (manufactured by Mitsubishi Chemical), Sepabeads SP207SS
(Mitsubishi Chemical) etc. (for example, "Adsorption Technology Handbook"
(Refer to Hiroshi Shimizu, published by NTS on February 2, 1993). The synthetic adsorbent treatment can be carried out by adsorbing the branched CyD carboxylic acid or a salt thereof by contact with the synthetic adsorbent, and then eluting with water. Further, elution with a mixed solution of a hydrophilic organic solvent and water can also be performed. Similarly, by collecting a fraction containing the desired branched CyD carboxylic acid or a salt thereof and removing water, the desired branched CyD carboxylic acid and a salt thereof can be isolated and purified.

In the present invention, by treating with any of the above-mentioned ion exchangers and / or adsorbents, it is possible to efficiently and industrially advantageously isolate high-purity branched CyD carboxylic acids or salts thereof. It can be purified, but in particular, after the treatment with an ion exchanger, the treatment with activated carbon or the treatment with a synthetic adsorbent is carried out very efficiently to obtain highly purified branched Cy.
D carboxylic acid or its salt can be isolated. The degree of purification can be further increased by using the activated carbon treatment and the synthetic adsorbent treatment together. Preferred processing methods include, for example,
As an example of isolation of a branched β-CyD carboxylic acid, a branched CyD carboxylic acid bonded to a strongly basic anion exchange resin having a degree of crosslinking of 8% or less (eg, G ₂ -β-CyD carboxylic acid, G ₃
-Β-CyD carboxylic acid) and hydrochloric acid (0.003 to 0.003).
(About 008N, usually about 0.005N). At this time, the other branched β-CyD carboxylic acid remains in the resin without being eluted with about 0.003 to 0.008 N hydrochloric acid. Eluted G ₂ -β-CyD
After adjusting the eluate of carboxylic acid and G ₃ -β-CyD carboxylic acid to neutral (pH 6 to 8), activated carbon having a pore diameter of 10 to 100 °, preferably 20 to 40 ° or a pore diameter of 1 to 100
0 ° (usually 10-500 °), preferably 50-250
Å, When contacted with a synthetic adsorbent having a particle size of 1 to 1000 μm (normally 5 to 500 μm), preferably 50 to 180 μm,
G ₂ -β-CyD-carboxylic acid and G _3-beta-CyD acids are adsorbed.

G ₂ from activated carbon and / or synthetic adsorbent
The elution of -β-CyD carboxylic acid generally requires only passing water or a mixture of water and an organic solvent (preferably water) through an activated carbon column or a synthetic adsorbent column. G ₂ -β
-CyD carboxylic acid is selectively eluted because of its weak adsorbing power to the adsorbent. Fractions containing G ₂ -β-CyD carboxylic acid are collected, for example, after concentration,
When freeze-dried, G ₂ − has a high purity, for example, 99% or more.
A powder of β-CyD carboxylic acid is obtained. When it is desired to obtain a free branched CyD carboxylic acid, the branched CyD carboxylic acid or a salt thereof is decationized, then concentrated, and an organic solvent such as acetone is added to the concentrated solution to precipitate the branched CyD carboxylic acid. can get. In the present invention, the eluate from the ion exchanger may be brought into contact with activated carbon without adjusting the pH. In that case, to elute the branched CyD carboxylic acid from the activated carbon, a mixed solution of water and a hydrophilic organic solvent is used. As the hydrophilic organic solvent, for example, methanol, ethanol, n-butanol, isopropanol, acetone or the like is used, and methanol or isopropanol is preferable. The concentration of the hydrophilic organic solvent in the mixture of water and the hydrophilic organic solvent is 1 to 50% (v / v), preferably
5 to 40% (v / v). As described above, when an ion exchanger and activated carbon or a synthetic adsorbent are used in combination, a high-purity branched CyD carboxylic acid can be obtained. For example, purity 9
9% or more of branched CyD carboxylic acid can be obtained in high yield, which is an industrially advantageous method.

On the other hand, in order to obtain the branched CyD carboxylic acid in a crystalline form, it is preferably applied to an ion exchanger such as a strongly basic anion exchange resin and eluted with an acid such as hydrochloric acid or acetic acid.
Desalting. Desalting can be performed at room temperature. In particular, desalting is preferably performed using a cation exchange resin and an anion exchange resin. The cation exchange resin is not particularly limited as long as the ionic form is H-form. For example, for example, Diaion series (SK1B, SK102, SK104,
SK106, SK110, SK112, SK116, P
K208, PK212, PK216, PK220, PK
228, WK10, WK11, WK20) (above, manufactured by Mitsubishi Chemical Corporation), Amberlite series (IR-120B,
IR-121, 200C, 252, IR-122, IR
-124, IRC-50) (all manufactured by Organo Corporation). The ionic form is OH as an anion exchange resin
The shape is not particularly limited as long as the shape is, for example, Diaion series (SA20A, SA21A, SA10A, SA1
1A, SA12A, PA306, PA308, PA31
2, PA316, PA318, PA40b, PA40
8, PA412, PA416, PA418, WA10,
WA20, WA30) (above, manufactured by Mitsubishi Chemical Corporation), Amberlite series (IRA-400, IRA-401, I
RA-402, IRA-900, IRA-911, IR
A-94S, IRA-93, IRA-68, IR-4
5,2R-4B) (all manufactured by Organo Corporation).

After desalting, the branched CyD carboxylic acid can be crystallized by a conventional method. Most commonly, it is crystallized by cooling after concentration. As a preferable crystallization method, after desalting, for example, in the case of a branched β-CyD carboxylic acid, a concentrated solution having a concentration of 40 to 80 w / v% of the carboxylic acid is stirred at 5 ° C. with a temperature gradient of 1 ° C./1 hr. Then, G ₂ -β-CyD carboxylic acid crystals are precipitated.
Seed crystals may be used to grow the crystals. Separation of the precipitated crystals is performed by a known method such as centrifugation or filter press filtration. As a crystallization method, an organic solvent may be added to a concentrated solution of the branched β-CyD carboxylic acid for precipitation. As the organic solvent, water-soluble organic solvents such as alcohols such as methanol and ethanol, and ketones such as acetone can be used. As described above, in particular, in the method of crystallizing the target substance, a high-purity branched CyD carboxylic acid can be precipitated from a relatively low-purity aqueous solution, and simple drying can be performed without requiring expensive freeze-drying equipment. There is an advantage that equipment is sufficient. The branched CyD carboxylic acid thus obtained is used as a solubilizing agent in the field of pharmaceutical veterinary medicine, food, etc.
It can be used as a stabilizer, a flavoring agent, and the like. For example, the solubilizing agent can be used according to the method described in JP-A-7-215895.

[0026]

Next, the present invention will be described in more detail with reference to Reference Examples and Examples. Reference Example 1 Pseudogluconobacter Saccharoketogenes K59
Preparation of 1s culture solution The following medium composition (“%” in the medium composition is w /
The pre-culture was performed using a pre-culture medium having the following formula: Ingredient % Lactose 1 Yeast extract 1 Ammonium sulfate 0.3 Corn steep liquor 3 Calcium carbonate 2 Prior to feeding calcium carbonate, the above preculture medium 3 adjusted to pH 7.0 with a 30% (w / w) aqueous sodium hydroxide solution
One platinum loop of Pseudogluconobacter saccharoketogenes K591s was inoculated into a 1-liter Sakaguchi flask containing 00 ml, and shake-cultured at 28 ° C. for 48 hours to obtain a preculture solution.

The obtained preculture was cultured in a seed medium having the following composition. Ingredient % Lactose 1 Yeast extract 1 Ammonium sulfate 0.3 Corn steep liquor 3 Calcium carbonate 2 Actol 0.05 Silicone 0.05 Before adding calcium carbonate, pH is adjusted to 7.0 with 30% (w / w) aqueous sodium hydroxide solution. Pre-culture solution 30 in a 200-liter tank containing 150 liters of the above-described seed medium adjusted to
0 ml was inoculated and cultured at 32 ° C. for 46 hours under aeration conditions of 120 liters / min while stirring at 150 rpm to obtain a seed culture solution.

The whole amount of the obtained seed culture was main-cultured in a main culture medium having the following composition. Ingredient % Corn steep liquor 2 Yeast extract 0.5 Ammonium sulfate 0.3 Ferrous sulfate heptahydrate 0.1 Vitamin B ₂ 1 mg / L Actol 0.075 Silicone 0.006 30% (w / w) hydroxylated Glufinal 2 (separate sterilization) Lanthanum chloride heptahydrate 0.01 (separate sterilization) The whole amount of the seed culture solution was inoculated into a 2000-liter tank containing 1200 liters of the main culture medium, and adjusted to pH 7.0 with a sodium aqueous solution. liter/
The cells were cultured at 32 ° C. for 24 hours while stirring at 100 rpm under aeration conditions for 1 minute to obtain a main culture solution. The obtained main culture solution was washed and concentrated, and the cells were suspended in water to obtain a washed and concentrated bacterial solution (concentration: about 4 times).

[0029]

【Example】

Example 1 200 g of a G ₂ -β-CyD mixture (manufactured by Saltwater Port Refining Co., Ltd.
Branched β-CyD content 98.2%, loss on drying 5.4%: G ₂ −
β-CyD 30%, G ₃ -β-CyD 1%, (G ₂ ) ₂ −
1500 ml of an aqueous solution containing 44% β-CyD, 24% (G ₂ ) ₃ -β-CyD, and 1% other β-CyD (hereinafter referred to as β-CyD mixture A) and the washing obtained in Reference Example 1. While stirring 500 ml of the concentrated bacterial solution in a 5 liter jar fermenter at 32 ° C. and 800 rpm,
Air was bubbled in at 1.6 liters / minute, 1N sodium hydroxide solution was added dropwise to maintain the pH at 6.3, and the reaction was carried out for 24 hours to microbial oxidation to convert to the corresponding carboxylic acid. 2 L of the obtained oxidation reaction solution was filtered using a hollow fiber membrane to remove cells, and the hollow fiber membrane was washed with water to obtain 3.4 L of a clear filtrate. 1.1 liters of this filtrate was used as a strongly basic anion exchange resin DIAION PA-406.
The solution was passed through a 1000 ml column (Cl-type, manufactured by Mitsubishi Chemical Corporation). After washing with 3.0 liters of water, elution was performed with 12 liters of 0.005N hydrochloric acid heated to 30 ± 5 ° C., and 16.6 g (purity: 97.5%) of the desired fraction was collected. Activated carbon (Special Shirasagi PLP charcoal Takeda Pharmaceutical Co., Ltd.)
The mixture was passed through an 80 g column, washed with 3.2 liters of water, and eluted with 2.4 liters of 30% (v / v) isopropanol-water to obtain a fraction (16.2 g, purity) of the target compound. 97.6%).

The collected fractions were concentrated under reduced pressure to completely remove isopropanol. 400 ml of the resulting aqueous solution of the desired product was treated with 1N sodium hydroxide solution to a pH of 7.2 ± 0.2.
Adjusted to 5. This was passed through a 20 g column of activated carbon (special Shirasagi PLP charcoal manufactured by Takeda Pharmaceutical Co., Ltd.), and then water 50
Elution was performed at 0 ml, and the desired product fraction (1) was 730 ml.
(12.6 g, 100% purity) was obtained. The activated carbon column was then washed with 30% (v / v) isopropanol-water 1.6.
The product was eluted in 1 liter, and a fraction (3.2 g, purity: 96.3%) of the desired product was collected. The collected fractions were concentrated under reduced pressure to completely remove isopropanol, and 200 ml of the obtained aqueous solution of the target product was adjusted to pH with 1N sodium hydroxide solution.
Adjusted to 7.2 ± 0.5. Activated carbon (special Shirasagi)
After passing through a column of 11 g of PLP charcoal (manufactured by Takeda Pharmaceutical Co., Ltd.), the column was eluted with 300 ml of water, and 330 ml (2.1 g, purity 98.8%) of the target fraction (2) was collected.
The desired fractions (1) and (2) were mixed, adjusted to pH 7.3 ± 0.1 with 1N sodium hydroxide solution, concentrated under reduced pressure, lyophilized, and treated with G ₂ -β-CyD.
13.8 g of a white powder of carboxylic acid Na salt was obtained. The yield as G ₂ -β-CyD carboxylic acid from the reaction solution was 81%.
The Na salt powder had an HPLC area percentage purity of 99.6.
%Met. The HPLC analysis conditions were as follows: Column: Asahip
ak NH ₂ P-50 (4.6 ID × 250 mmL), mobile phase:
CH ₃ CN / H ₂ O = 48/52 at a concentration of PIC reagent of 0.
005 M / liter. Flow rate: 0.
8 ml / min, detection: RI.

Example 2 200 g of a G ₂ -β-CyD mixture (manufactured by Shiosui Minato Seika Co., Ltd., 98.5% branched β-CD content, 3.3% loss on drying, G ₂ -β
-CyD 41%, G ₃ -β-CyD 2%, (G ₂ ) ₂ -β
-CyD 46%, (G ₂ ) ₃ -β-CyD 10%, and other β-CyD 1%, hereinafter referred to as β-CyD mixture B). 1.0 liter of the filtrate was passed through 500 ml of a strong basic anion exchange resin DIAION PA-406 (AcO type, manufactured by Mitsubishi Chemical Corporation). After washing with water (1.5 liters), 1N acetic acid 3.
Elution was performed at 0 liter, and the fraction of the target substance (19.2) was eluted.
g, purity 94.3%). This fraction was passed through an 80 g column of activated carbon (special Shirasagi PLP charcoal manufactured by Takeda Pharmaceutical Co., Ltd.), washed with 2.4 liters of water, and then 30%
(V / v) Eluted with 2.4 liters of isopropanol-water, fractions of the desired product (17.5 g, purity 94.7)
%) Were collected. The collected fractions were concentrated under reduced pressure to completely remove isopropanol, and 450 ml of the obtained aqueous solution of the target product was adjusted to pH 7.2 ± with a 1N sodium hydroxide solution.
Adjusted to 0.2. This solution was added to two activated carbon columns (Special Shirasagi PLP charcoal, 10 g, manufactured by Takeda Pharmaceutical Co., Ltd.) and Special Shirasagi P
LP charcoal (40 g, manufactured by Takeda Pharmaceutical Co., Ltd.) was passed in series, and eluted with 1.2 liters of water to collect the desired fraction (1) (9.6 g, purity 99.8%). Further, a column of 40 g of PLP charcoal was eluted with 10% (v / v) methanol-water (2.0 liter) to collect the desired fraction (2) (2.8 g, purity 98.7%). Then, a column of 40 g of PLP charcoal was eluted with 1.2 liters of 30% (v / v) isopropanol-water, and a fraction (3.8 g, purity 82.6%) of the desired product was collected.

The collected fractions were concentrated under reduced pressure to completely remove isopropanol, and to obtain an aqueous solution 2 of the desired product.
00 ml with 1N sodium hydroxide solution pH 7.2 ± 0.2.
Was adjusted. This solution was passed through a 16 g column of activated carbon (special Shirasagi PLP charcoal, manufactured by Takeda Pharmaceutical Co., Ltd.), and then water 40
Elution was performed at 0 ml, and the desired fraction (3) was 420 ml.
(1.8 g, 98.9% purity). The desired fraction (1), fraction (2) and fraction (3) were mixed and the pH was adjusted to 7.3 with 1N sodium hydroxide solution.
± adjusted to 0.1, was concentrated under reduced pressure, and lyophilized, G ₂ -
13.6 g of a white powder of β-CyD carboxylic acid Na salt was obtained. The yield as G ₂ -β-CyD carboxylic acid from the reaction solution was 67%, and the Na area powder had an HPLC area percentage purity of 99.3%.

Example 3 A filtrate 1.0 obtained in the same manner as in Example 1 except that 200 g of β-CyD mixture B (manufactured by Shimizu Port Refining Co., Ltd.) was used.
Liter of strong basic anion exchange resin Diaion PA
The solution was passed through a 600 ml column of -406 (manufactured by Mitsubishi Chemical Corporation, AcO type). After washing with 1.8 liters of water, the residue was eluted with 3.0 liters of 1N acetic acid, and the desired fraction (19.3 g, purity 94.1%) was collected. The collected fraction was passed through a column of 80 g of activated carbon (K-1 charcoal manufactured by Takeda Pharmaceutical Co., Ltd.), washed with 2.0 liters of water, and then washed with 2.4 liters of 30% (v / v) isopropanol-water. The eluted fraction (18.5 g, purity 94.2%) was collected. The collected fractions were concentrated under reduced pressure to completely remove isopropanol, and 450 ml of the obtained aqueous solution of the target product was adjusted to pH 7.2 ± 0.2 with a 1N sodium hydroxide solution. This liquid is used for two activated carbon columns (K-1 coal Takeda Pharmaceutical Co., Ltd.)
After passing through a series of 10 g manufactured by K.K. and 40 g of specially made Shirasagi PLP charcoal manufactured by Takeda Pharmaceutical Co., Ltd., the mixture was eluted with 1.3 liters of water, and the target fraction (1) (10.9 g, purity 99) .6%).

Further, the column of 40 g of PLP charcoal was
(V / v) Eluted with 1.2 liters of isopropanol-water, fractions of the desired product (6.3 g, purity 88.1%)
Collected. This fraction was concentrated under reduced pressure to completely remove isopropanol, and to obtain an aqueous solution 360 of the obtained target substance.
The ml was adjusted to pH 7.2 ± 0.2 with 1N sodium hydroxide solution. Activated carbon (K-1 coal Takeda Pharmaceutical Co., Ltd.)
After passing through a 24 g column, elution was carried out with 600 ml of water, and 700 ml (2.4 g, purity 99.4%) of the desired fraction (2) was collected. Fraction of target (1)
And fraction (2) were mixed, adjusted to pH 7.3 ± 0.1 with 1N sodium hydroxide solution, and concentrated under reduced pressure.
After freeze-drying, 12.7 g of white powder of Na salt of G ₂ -β-CyD carboxylic acid was obtained. The yield as G ₂ -β-CyD carboxylic acid from the reaction solution was 62%, and the Na area powder had an HPLC area percentage purity of 99.3%.

Example 4 A filtrate obtained in the same manner as in Example 1 except that 200 g of β-CyD mixture B (manufactured by Shiosui Minato Sugar Co., Ltd.) was used.
Liter of strong basic anion exchange resin Diaion PA
The solution was passed through a 300 ml column of -406 (Mitsubishi Chemical, AcO type). After washing with 0.9 liter of water, 1N acetic acid 1.5
The fraction was eluted in 1 liter, and a fraction (9.1 g, purity 94.5%) of the desired product was collected. The collected fraction was passed through a 40 g column of activated carbon (K-1 charcoal manufactured by Takeda Pharmaceutical Co., Ltd.), washed with 0.6 liter of water, and then washed with 2.0 liter of 30% (v / v) isopropanol-water. The eluted fraction (8.9 g, purity 95.1%) was collected. This fraction was concentrated under reduced pressure to completely remove isopropanol. 500 ml of the obtained aqueous solution of the target product was passed through a 27 g column of activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.), washed with 0.7 liter of water, and then 10% (v / v) methanol-water. The fraction was eluted with 1.4 liters, and the desired fraction (1) (2.7 g, purity 99.6%) was collected.

Then, the column of 27 g of K-1 coal was reduced to 50%
(V / v) The mixture was eluted with 2.7 liters of methanol-water, and the desired fraction (4.4 g, purity 96.3%) was collected. This fraction was concentrated under reduced pressure to completely remove methanol, and 500 ml of the obtained aqueous solution of the desired product was passed through a 13 g column of activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.), and 0.3 liter of water was added. After elution, elution was carried out with 0.7 liter of 15% (v / v) methanol-water, and the desired fraction (2) (1.6 g, purity 100%) was collected. Then, a column of 13 g of K-1 charcoal was eluted with 1.0 liter of 30% (v / v) isopropanol-water, and a fraction (2.6 g, purity 94.5%) of the desired product was collected. 350 liters of an aqueous solution of the target product from which isopropanol had been removed was passed through a column of activated carbon (K-1 charcoal, manufactured by Takeda Pharmaceutical Co., Ltd.) 7 g, washed with 0.2 liters of water, and then 20% (v / v)
The fraction was eluted with 0.4 liter of methanol-water, and the desired fraction (3) (1.1 g, purity 99.6%) was collected.
The desired fraction (1), fraction (2), and fraction (3) were mixed, adjusted to pH 7.3 ± 0.1 with 1N sodium hydroxide solution, concentrated under reduced pressure, lyophilized, and subjected to G _2- β-CyD carboxylic acid Na salt white powder 5.
4 g were obtained. The yield as G ₂ -β-CyD carboxylic acid from the reaction solution was 53%, and the Na area powder had an HPLC area percentage purity of 99.1%.

Example 5 785 ml of the filtrate obtained in the same manner as in Example 2 was passed through a strong basic anion exchange resin Diaion PA-406 (manufactured by Mitsubishi Chemical Corporation, Cl type, 1.0 liter). After washing with water (3.0 liters), it was heated to 30 ± 5 ° C. and 0.005.
Elution was performed with 13.0 liters of N-HCl, and 11.5 liters (17.6 g, HPLC area percentage purity 95.5%) of the desired fraction was collected. Activated carbon column (Special Shirasagi PLP charcoal Takeda Pharmaceutical Company Limited 72
g), washed with water (3.6 liters), and then 30%
Eluted with (v / v) isopropanol-water (2.2 liter), the desired fraction 2.2 (16.7 g, HPLC area percentage purity 95.8%) was collected. The target fraction was concentrated under reduced pressure to completely remove isopropanol. 500 ml of an aqueous solution of the target substance from which isopropanol has been removed is 1N-N
The pH was adjusted to 7.2 ± 0.2 with aOH solution. pH 7.2 ±
The solution adjusted to 0.2 was mixed with Sepabeads SP-207SS.
(1.4 liters, manufactured by Mitsubishi Chemical Corporation), and eluted with water (8.0 liters).
5.1 g, 99.7% HPLC area percent purity). 6.0 liters of the desired fraction was treated with a 1N NaOH solution.
The pH was adjusted to 7.3 ± 0.1, and the mixture was concentrated under reduced pressure, lyophilized, and dried to obtain a dry powder of G ₂ -β-CyD carboxylic acid Na salt.
g was obtained. The yield as G ₂ -β-CyD carboxylic acid from the reaction solution was 83%, and the Na area powder had an HPLC area percentage purity of 99.7%.

Example 6 790 ml of the filtrate obtained in the same manner as in Example 2 was passed through a strong basic anion exchange resin Diaion PA-406 (manufactured by Mitsubishi Chemical Corporation, Cl type, 1.0 liter). After washing with water (3.0), the mixture was heated to 30 ± 5 ° C. 0.005N-H
Elution was carried out with 12.0 liters of Cl, and 10.5 liters (17.4 g, HPLC area percentage purity of 95.9%) of the desired fraction was collected. Fraction of this target is 1
After adjusting the pH to 7.2 ± 0.5 with N sodium hydroxide solution, the mixture was concentrated under reduced pressure. 150 ml of the concentrate obtained was passed through a Sepabeads SP207SS (manufactured by Mitsubishi Chemical Corporation, 1.4 liter) column. Then, elution was carried out with 7.0 liters of water, and 5.1 liters (15.9 g,
(99.9% purity). 1 fraction of target
PH was adjusted to 7.3 ± 0.1 with N sodium hydroxide solution,
After concentration under reduced pressure, the residue was freeze-dried to obtain 15.8 g of white powder of G ₂ -β-CyD carboxylic acid Na salt. G ₂ from reaction solution
The yield as -β-CyD carboxylic acid was 87%, and the Na area powder had an HPLC area percentage purity of 99.9%.

Example 7 5.0 kg of a G ₂ -β-CyD mixture (G ₂
1.94 kg of -β-CyD, branched β-CD content 98.
_{5%, (G 2 -β-} CD 41%, G 3 -β-CD 2
%, (G ₂ ) ₂ -β-CD 46%, (G ₂ ) ₃ -β-CD 1
0%, other branching β-CD 1%) loss on drying 3.3
% Of an aqueous solution containing 50% and 50 L of the washed concentrated bacterial solution obtained in Reference Example 1 were added to a culture tank to make 100 L. While stirring at 32 ° C. and 230 rpm, air was bubbled in at 20 liters / minute, 1N sodium hydroxide solution was added dropwise, the pH was maintained at 6.3, and reaction was carried out for 24 hours to oxidize microorganisms. To the corresponding carboxylic acid. 89 liters of the obtained oxidation reaction solution was filtered using a ceramic membrane to remove cells, and 160 liters of a clear filtrate was obtained. 123 liters of this filtrate was used as a strongly basic anion exchange resin Diaion PA-406 (manufactured by Mitsubishi Chemical Corporation, C
(l-type, 90 liters). After washing with water (220
Liter), 0.005N hydrochloric acid 7 heated to 30 ± 5 ° C.
Elute with 30 liters and extract the desired fraction (1,2
57 g, purity 95.9%). A 10N sodium hydroxide solution was added to the desired fraction to adjust the pH to 7.0.
It was adjusted to ± 0.5. This solution was concentrated under reduced pressure, and concentrated solution 10
Liters were obtained. 5 L of the concentrated solution was passed through a column of a synthetic adsorbent (Sepabeads SP207SS, manufactured by Mitsubishi Chemical Corporation, 50 L), and eluted with 250 L of water to obtain a fraction (463 g, purity 99.8%) of the desired product.
Fractions of the desired product were p-p with 1N sodium hydroxide solution.
After adjusting to H 7.3 ± 0.1, the mixture was concentrated under reduced pressure and freeze-dried to give a white powder of G ₂ -β-CyD carboxylic acid Na salt.
0 g was obtained. The yield as G ₂ -β-CyD carboxylic acid from the reaction solution was 22%, and the Na area powder had an HPLC area percentage purity of 99.8%.

Example 8 14.6 liters of the filtrate obtained in the same manner as in Example 7 was passed through a strongly basic anion exchange resin Diaion PA-406 (manufactured by Mitsubishi Chemical Corporation, type C1, 8.0 liters). . After washing with water (24.0 liters), it was heated to 30 ± 5 ° C.
Elution was carried out with 90.0 liters of 0.005N-HCl, and the desired fraction was obtained in an amount of 78.0 liters (142.1 g,
C area percent purity 94.3%). The fraction of this target substance was adjusted to pH 7.2 ± with 1N sodium hydroxide solution.
After adjusting to 0.5, the mixture was concentrated and 0.5 liter of concentrated solution (1
(40.0 g, purity 94.3%). 50 ml of the obtained concentrated solution of the target product was separated by Sepabeads SP207 (manufactured by Mitsubishi Chemical Corporation,
180 ml). Subsequently, elution was carried out with 500 ml of water, and the desired fraction (200 ml, 43 g, purity 9) was obtained.
9.0%). The target fraction was adjusted to pH 7.3 ± 0.1 with a 1N sodium hydroxide solution, concentrated under reduced pressure, freeze-dried, and G ₂ -β-CyD carboxylic acid Na
42 g of a white powder of salt were obtained. G ₂ -β-Cy from reaction solution
The yield as D carboxylic acid is 28%, and the Na salt powder H
The PLC area percentage purity was 99.0%.

Example 9 Sepabead SP-207S obtained in the same manner as in Example 5
S treatment solution (15.1 g, HPLC area percentage purity 99.7)
%) Was passed through a strongly acidic cation resin Diaion PK-228 column (H-type, 1.0 liter, manufactured by Mitsubishi Chemical Corporation). Wash with water (1.5 liters) and decationize solution 7.5
Liters (15.1 g, HPLC area percent purity 99.
7%). 7.5 liters of the decationized solution was concentrated under reduced pressure to obtain 130 ml of a concentrated solution. 130 ml of the concentrated solution is treated with Ca (O
H) After adjusting the pH to 7.3 ± 0.1 with the ₂ -slurry, 35
Heated to ± 5 ° C. While stirring the aqueous solution maintained at a temperature of 35 ± 5 ° C., 110 ml of 99% (w / w) acetone was added thereto, and the mixture was cooled to 5 ° C. with stirring. The deposited precipitate was separated from the mother liquor using a glass filter (PGF17G3, manufactured by Iwaki Glass KK). 99% (w /
w) Washed with 100 ml of acetone. The washed precipitate was placed in a desiccator containing diphosphorus pentoxide as a desiccant and dried under reduced pressure to obtain 12.6 g of Ca powder of G ₂ -β-CyD carboxylic acid Ca salt. Sepabeads SP-2
The yield as G ₂ -β-CyD carboxylic acid from the 07SS-treated solution was 80%, and the purity of the Ca salt powder by HPLC area percentage was 99.7%.

Example 10 7.5 liters of the decationized solution obtained in the same manner as in Example 5 was concentrated under reduced pressure to obtain 115 ml of a concentrated solution. Concentrate 115
ml was heated to 35 ± 5 ° C. While stirring the aqueous solution maintained at a temperature of 35 ± 5 ° C., 99% (w / w) acetone 1
75 ml was added and cooled to 5 ° C. The deposited precipitate was separated from the mother liquor using a glass filter (PGF17G3, manufactured by Iwaki Glass KK). The separated precipitate was washed with 100 ml of 99% (w / w) acetone. The washed precipitate was placed in a desiccator containing diphosphorus pentoxide as a desiccant and dried under reduced pressure. G ₂ -β-CyD carboxylic acid white powder 1
0.1 g was obtained. The yield of G ₂ -β-CyD carboxylic acid from the Sepabeads SP-207SS treatment solution was 65%, and the HPLC area percentage purity of the powder was 99.7%.

Example 11 6.74 liters of a filtrate obtained in the same manner as in Example 7 (7
1.8 g, HPLC area percentage purity 46.3%) was passed through a strong basic anion exchange resin DIAION PA-406 (Mitsubishi Chemical, Cl type, 4 liters). Wash with water (1
2.0 liters) and then heated to 30 ± 5 ° C.
Elution was carried out with 45 liters of HCl, and 40.0 liters (69.5 g, purity 93.6%) of the desired fraction was collected. The fraction of this target substance was concentrated, and the concentrated liquid 0.5
1 (68.9 g, purity 93.6%) was obtained. 0.5 liter of the obtained concentrated solution of the target product was added to a strongly acidic cation exchange resin DIAION PK-228 (manufactured by Mitsubishi Chemical Corporation, H type,
(0.4 liter) and then a column packed with a weakly basic anion exchange resin Amberlite IRA-94 (manufactured by Organo, OH type, 0.3 liter). Then, the mixture was extruded with 2.0 liters of water, and 2.0 liters (94.6 g,
A purity of 93.1) was collected. The desired fraction was concentrated under reduced pressure to obtain a concentrated liquid (90 ml, Bri × value: 60.7%).
The concentrated solution is immersed in a constant temperature water bath at 35 ° C., and stirred at 1 ° C.
Crystallized to 5 ° C. with a temperature gradient of / 1 hr. The precipitated crystals are filtered using a glass filter (PGF 17G, manufactured by Iwaki Glass).
It was separated from the mother liquor using 3). The separated wet crystals were washed with 5 ml of water at 5 ° C. The washed wet crystals were dried at 5 ° C. under reduced pressure to obtain 45.3 g (40.3 g, purity 97.9%) of the first crystals.
I got The mother liquor (45 ml) was concentrated to give a concentrated liquid (35 ml, Bri × 59.7%). The concentrate was immersed in a constant temperature water bath at 35 ° C., and cooled and crystallized to 5 ° C. with a temperature gradient of 1 ° C./1 hr while stirring. The precipitated crystals were separated from the mother liquor using a glass filter (PGF 17G3, manufactured by Iwaki Glass). The separated wet crystals were washed with 5 ml of water at 5 ° C. The washed wet crystals were dried at 5 ° C. under reduced pressure to obtain 17.5 g (15.4 g) of the second crystals.
g, purity 95.5%). 45.3 g of the first crystal and the second
17.5 g of the crystals were dissolved in water to obtain 200 ml of a solution.
The lysate is concentrated and the concentrate is 85 ml (Bri x value 58.1%)
I got The concentrated solution was immersed in a constant temperature water bath at 35 ° C., and cooled and crystallized to 5 ° C. with a temperature gradient of 1 ° C./1 hr while stirring.
The precipitated crystals are filtered using a glass filter (GF, manufactured by Iwaki Glass).
17G3) to separate from the mother liquor. The separated wet crystals were washed with 5 ml of water at 5 ° C. The washed wet crystals were dried at 5 ° C. under reduced pressure to obtain 50.3 g (45.5 g, purity: 99.5%) of purified crystals of G ₂ -β-CyD carboxylic acid (free form). A diffraction diagram obtained by subjecting the obtained crystal to powder X-ray diffraction under the following conditions is shown in FIG.

<Measurement conditions> Tube: Cu Tube voltage: 40 kV Tube current: 50 mA Monochromator used Light receiving slit: 0.60 mm and 0.3 mm Goniometer: Vertical goniometer 1 axis Sampling width: 0.020 ゜ Scanning speed : 6.00 ゜ / min. Divergent slit: 1 ゜ Scattering slit: 1 ゜

Example 12 Separation of Separated β-CyD Carboxylic Acids Using Diaion PA-406 3.27 L of a filtrate obtained by the same method as in Example 7 (G ₂
-B-CyD carboxylic acid (34.8 g, HPLC area percentage purity: 46.3%) was passed through a strong basic anion exchange resin DIAION PA-406 (manufactured by Mitsubishi Chemical Corporation, Cl type, 2.2 liter). After washing with water (7.0 liters), 30
Elution was carried out with 45.0 liters of 0.008 N-HCl heated to ± 5 ° C., and 8.9 liters (32.6 g, purity 93.6%) of G ₂ -β-CyD carboxylic acid fraction was collected. Further, elution with 0.008N-HCl was continued, and 4.0 liters of a fraction after the eluate amount reached 36 liters was collected. This fraction contains G ₂ -β-Cy
2.5 g of (G ₂ ) ₂ -β-CyD carboxylic acid, an analog of D carboxylic acid, was obtained, and its purity was 98.1%. Add 1N sodium hydroxide to this fraction
The pH was adjusted to 7.0 ± 0.5. This solution was concentrated under reduced pressure,
10 ml of a concentrate were obtained. After passing 10 ml of the concentrated solution through a column of synthetic adsorbent (Sepabeads SP207SS, 1 liter, manufactured by Mitsubishi Chemical Corporation), the column was eluted with 5 liters of water to give (G ₂ ) ₂ -β
-Fraction of CyD carboxylic acid Na salt (2.0 g,
HPLC area percentage purity 99.5%).

[0046]

According to the present invention, the corresponding branched CyD carboxylic acid can be industrially advantageously and efficiently produced from the mixture of branched CyD, and then isolated and purified.

[Brief description of the drawings]

FIG. 1 shows a powder X-ray diffraction diagram of G ₂ -β-CyD carboxylic acid (free form) crystal obtained in Example 11.

Claims (18)

[Claims] 1. A formula: (wherein, G is a glucose unit, n represents 1 shows a to integer 6) -G _n -CH ₂ OH groups represented by, 6-O of glucose units of the cyclodextrin rings Characterized in that a mixture of a branched cyclodextrin or a salt thereof containing at least two kinds of branched cyclodextrins having 1 to 8 α-1,6 bonds at the 1-position is microbial oxidized, and then separated and purified, A method for producing an acid or a salt thereof. 2. The method according to claim 1, wherein n is an integer of 1 to 3. 3. The method according to claim 1, wherein the cyclodextrin is β-cyclodextrin. 4. The process according to claim 1, wherein the branched cyclodextrin mixture contains maltosyl-β-cyclodextrin. 5. The method according to claim 1, wherein the branched cyclodextrin carboxylic acid is 6-
O-cyclomaltoheptaosyl- (6 → 1) -α-D-
The production method according to claim 1, wherein the production method is glucosyl- (4 → 1) -O-α-D-glucuronic acid. 6. The method according to claim 1, wherein the separation and purification are performed using an ion exchanger and / or an adsorbent. 7. The method according to claim 7, wherein the separation and purification are performed using an ion exchanger.
2. The process according to claim 1, wherein the target product is obtained as crystals by crystallization. 8. The method according to claim 6, wherein the adsorbent is a synthetic adsorbent and / or activated carbon. 9. The method according to claim 6, wherein an anion exchange resin is used as the ion exchanger, and the branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin is eluted with an acid. 10. The process according to claim 6, wherein activated carbon is used as the adsorbent and the adsorbed branched cyclodextrin carboxylic acid or a salt thereof is eluted with water or a mixture of water and a hydrophilic organic solvent. 11. The process according to claim 6, wherein a synthetic adsorbent is used as the adsorbent, and the adsorbed branched cyclodextrin carboxylic acid or a salt thereof is eluted with water or a mixture of water and a hydrophilic organic solvent. 12. A strongly basic anion exchange resin is used as an ion exchanger, and a branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin is eluted with an acid.
8. The production method according to claim 7, wherein the target substance is obtained as crystals by crystallization after desalting the eluate. 13. Use of a strongly basic anion exchange resin as an ion exchanger, eluting a branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin with an acid,
8. The obtained eluate is concentrated, desalted using a cation exchange resin and an anion exchange resin, then concentrated, and the concentrated liquid is cooled to precipitate and separate a branched cyclodextrin carboxylic acid. Manufacturing method. 14. A strongly basic anion exchange resin is used as an ion exchanger, and a branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin is eluted with an acid.
7. The process according to claim 6, further comprising adjusting the pH of the obtained eluate to neutral, concentrating the solution, adsorbing the concentrated solution on activated carbon, and eluted with water. 15. Use of a strongly basic anion exchange resin as an ion exchanger, eluting a branched cyclodextrin carboxylic acid or a salt thereof loaded on the anion exchange resin with an acid,
7. The process according to claim 6, wherein the pH of the obtained eluate is adjusted to neutral, concentrated, and then the concentrated solution is adsorbed on a synthetic adsorbent, followed by elution with water. 16. A group represented by the formula: -G _n -COOH ( _where G is a glucose unit and n is an integer of 1 to 6) is attached at the 6-O position of the glucose unit of the cyclodextrin ring. A branched cyclodextrin carboxylic acid or a salt thereof containing at least two kinds of branched cyclodextrin carboxylic acids or salts thereof having 1 to 8 α-1,6 bonds, wherein the mixture is separated and purified. A method for separating and purifying an acid or a salt thereof. 17. A group represented by the formula: -G _n -COOH (wherein G is a glucose unit and n is an integer of 1 to 6) is attached to the 6-O position of the glucose unit of the cyclodextrin ring. A mixture of a branched cyclodextrin carboxylic acid or a salt thereof containing at least two kinds of 1 to 8 α-1,6 linked branched cyclodextrin carboxylic acids or salts thereof. 18. A group represented by the formula: -G _n -CH ₂ OH (wherein G is a glucose unit and n is an integer of 1 to 6) is a group represented by 6-O of a glucose unit of a cyclodextrin ring. Microbial oxidation of a mixture of branched β-cyclodextrins or salts thereof containing at least two kinds of branched cyclodextrins having 1 to 7 α-1,6 bonds at the 1-position, followed by treatment with an ion exchange resin and / or activated carbon A process for producing a branched cyclodextrin carboxylic acid or a salt thereof, characterized by comprising the following.