JPS6121103A - Preparation of chitosan oligosaccharide - Google Patents

Abstract

PURPOSE:To produce a large amount of chitosan oligosaccharide useful in the field of pharmaceuticals, cosmetics, foods, etc., economically and efficiently, by partially hydrolyzing chitosan with hydrochloric acid, adding a methanol-contaning solvent to the hydrolyzed product, and dissolving and separating the objective material. CONSTITUTION:Chitosan of formula is partially hydrolyzed with hydrochloric acid. 1pt. of the hydrolyzed product is added with usually 5-20pts.wt. of a solvent containing >=60% (V/V) methanol (preferably ethanol, acetone, ether, etc. containing >=90% methanol), and the mixture is stirred at <=20 deg.C for 2-3hr. The objective chitosan oligosaccharide can be produced by the dissolution and separation process taking advantage of the solubility difference.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing chitosan oligosaccharide, which is a partially hydrolyzed product of chitosan.

Chitosan is a basic polysaccharide formed by condensation of D-glucosamine with β-1,4-glucosidic bonds. It is obtained by heating the saccharide chitin in a concentrated alkaline solution to deacetylate it, and its structure is as shown below. As described above, chitosan has a highly reactive free amino group in its molecule, and is a chemically very interesting polymer, and research and development of chitosan and its derivatives is being actively conducted. But now,
Very few of them have been put to practical use industrially, and most of them are in the form of chitosan, which is used as a cationic natural flocculant for wastewater treatment. On the other hand, chitosan oligosaccharide obtained by partial hydrolysis of chitosan is a condensation polymerization of several D-glucosamines with β-1,4-glucosidic bonds. Although it is lagging behind in comparison and its properties have not been clarified, it is considered to be a very promising substance supply in the fields of medicine, reagents, cosmetics, food, etc. By the way, the conventional method for preparing chitosan oligosaccharides is to partially hydrolyze chitosan with hydrochloric acid, adsorb the decomposed product on a cation exchange column, and then perform chromatography using a concentration gradient elution method using hydrochloric acid to obtain D-glucosamine. Methods have been carried out to separate chitosan oligosaccharides such as bios, triose, tetraose, pentaose, and hexaose. In addition, at this time, the proportion of D-glucosamine in the decomposition product varies depending on the hydrolysis conditions, but
As long as hydrolysis with acid is carried out, the amount will be considerable. For example, as a method for preparing chitosan oligosaccharide, chitosan 5f is
Using 0 times volume 0 weight 0.8N hydrochloric acid, at 53'℃
It is reported that when partially hydrolyzed for several hours, D-glucosamine in the decomposition product is 1.3'lf, and the yield is 27.5% based on chitosan. [Journal of America
ican Chemical 5ociety, 79
, 5046-5049 (1957)]. Furthermore, the present inventors hydrolyzed chitosan under various conditions and investigated the amount of D-glucosamine and chitosan oligosaccharide produced in the decomposition product. As the amount of chitosan oligosaccharide produced increased, It was confirmed that the amount of D-glucosamine produced also increased significantly. In this way, in the conventional method, the decomposition product of chitosan with a high content of D-glucosamine is adsorbed on a cation exchange resin column, so a large amount of ion exchange resin is required to separate a large amount of oligosaccharides. The drawback is that the equipment is large-scale and inefficient. In order to overcome the drawbacks of this conventional method and efficiently produce a large amount of chitosan oligosaccharide using equipment on a small scale, the present inventors conducted various studies and found that D-glucosamine hydrochloride is soluble in methanol. Although it was difficult, the hydrochloride of chitosan oligosaccharide was.

The present invention was completed based on this knowledge,
This method for producing chitosan oligosaccharides is characterized by adding a methanol-containing solvent to a hydrochloric acid partial hydrolyzate of chitosan, and dissolving and separating chitosan oligosaccharides using the difference in solubility.

The chitosan used in the method of the present invention may be prepared by a conventional method, and examples of its preparation are as follows. Chitin obtained from crab shells is treated in a 45-50% sodium hydroxide solution at 90°C for 5 hours, then washed with water and dried. In the method of the present invention, the decomposition product is prepared, for example, as follows. Partially hydrolyze chitosan with hydrochloric acid, remove undegraded materials by centrifugation,
Furthermore, it is prepared by adding activated carbon to the semen to decolorize it, and then removing hydrochloric acid by vacuum concentration. Further, the partial hydrolysis may be carried out at high temperature for a short time or at low temperature for a long time, and the ratio of D-glucosamine and chitosan oligosaccharide in the decomposition product is not limited. In the method of the present invention, the solvent used for dissolving and separating chitosan oligosaccharide from the decomposition product includes water or ethanol containing 601 (V/V) or more of methanol, acetone, ether, propatool, isopropanol, n-butanol, pyridine. The mixed organic solvent is particularly preferably one containing 90% or more of methanol. This methanol-containing solvent is added in an amount of 1 to 50 times, preferably 5 to 20 times, based on the weight of the decomposed product, and stirred. Stirring may be carried out at 40°C or lower for 1 to 5 hours, but preferably at 20°C or lower for 2 to 3 hours. Most of what is not dissolved in this operation is D-glucosamine hydrochloride, which is removed by suction filtration, and the filtrate is concentrated under reduced pressure to obtain the oligosaccharide of chitosan. The chitosan oligosaccharide obtained by the method of the present invention has a significantly reduced content of D-glucosamine, and a series of chitosan oligosaccharides are produced in large quantities by column chromatography using a conventional cation exchange resin. and can be separated efficiently.

Next, embodiments of the present invention will be explained in more detail.
It goes without saying that the present invention is not limited in any way by this explanation.

Example 1 Chitosan 90F was dissolved in 1.51 dilute hydrochloric acid, and
．． 51 concentrated hydrochloric acid was added and hydrolyzed at 70°C for 38 hours. Insoluble matter in this decomposition solution was removed by centrifugation, and activated carbon was added to the supernatant to decolorize it, followed by concentration to dryness under reduced pressure to obtain decomposition product 20f. Add 200g/methanol to this decomposition product and heat to 15°C.
After stirring for 3 hours, insoluble matter was removed by suction filtration, and the filtrate was concentrated to dryness under reduced pressure. Here, by dissolving and separating methanol, 1
A partial decomposition product of chitosan of 6.41 was obtained.

5. of the obtained partial decomposition product. Of was adsorbed on a 200g/ion exchange resin column (DoX50W), and
It was eluted with a linear concentration gradient using defined acidic acid. Chitosan oligosaccharides were detected by measuring the absorbance of 57(1)1 using ninhydrin coloring, and a series of chitosan oligosaccharides were fractionated. Yield and composition ratio of D-glucosamine hydrochloride and chitosan oligosaccharide hydrochloride in the method of the present invention, in which both parts were concentrated and dried to obtain hydrochloride of chitosan biose, triose, tetraose, pentaose, and hexaose. Table 1 shows a comparison with the conventional method. The decomposition products of chitosan subjected to ion-exchange chromatography were evaluated as 5. for both the method of the present invention and the conventional method. Of.

Table 1 As shown in Table 1, the composition ratio of D-glucosamine hydrochloride is reduced and the yield of chitosan oligosaccharide is increased by the method of the present invention.

Example 2 1.5 Il of concentrated hydrochloric acid was added to 150 f of chitosan, and 80
Hydrolyzed at ℃ for 5 hours. ``This decomposition solution was subjected to the same operation as in Example 1 to obtain 125f of a decomposition product of chitosan.

1000 g of methanol was added to this decomposition product, and after stirring at 20°C for 2 hours, insoluble matter was removed by suction filtration, and the filtrate was concentrated to dryness under reduced pressure. Here, by dissolution and separation of methanol, 61
．． A partially decomposed product of chitosan of 9f was obtained. 5. of the obtained partial decomposition product. Of was adsorbed onto a 200 g/ion exchange resin column (DOX 50W), and the same operation as in Example 1 was performed to obtain a series of chitosan oligosaccharides.

The yield and composition ratio of D-glucosamine hydrochloride and chitosan oligosaccharide hydrochloride in the method of the present invention are shown in Table 2 in comparison with the conventional method. In addition, the decomposition product of chitosan subjected to ion exchange chromatography was 5.0 g in both the method of the present invention and the conventional method.

As shown in Table 2, the composition ratio of D-glucosamine hydrochloride is reduced and the yield of chitosan oligosaccharide is increased by the method of the present invention.

Claims (1)

[Claims] (1) Add 6 methanol to the hydrochloric acid partial hydrolyzate of chitosan.
A method for producing chitosan oligosaccharides, which comprises adding a solvent containing 0% (V/V) or more and dissolving and separating chitosan oligosaccharides by utilizing the difference in solubility.