JPS6121102A - 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 adding concentrated hydrochloric acid to chitosan, and carrying out the partial hydrolysis of the chitosan under specific condition. CONSTITUTION:1pt.wt. of chitosan of formula is added with 5-30pts.wt. of concentrated hydrochloric acid having concentration of >=10N, and is partially hydrolyzed at 60-100 deg.C for 1-4hr to obtain the objective chitosan oligosaccharide. The chitosan used as a raw material is produced preferably by using chitin obtained from the crust of crab, and treating the chitin in a 45-50% NaOH solution at 90 deg.C for 5hr, and is used preferably in the form of flakes of >=5 mesh.

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 in which D-glucosamine is condensed 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 within its molecule, making it a chemically very interesting polymer, and research and development of chitosan and its derivatives is being actively conducted. . However, at present, there are very few products that have been put into practical use industrially, and most of them are only used in wastewater treatment in the form of chitosan as a cationic natural flocculant. On the other hand, chitosan oligosaccharides obtained by partially hydrolyzing chitosan are
It is a condensation polymerization of several D-glucosamines with β-1,4-glucosidic bonds. Research and development on these products has lagged behind that of the polymeric chitosan, and their properties have not yet been clarified. However, it is considered to be a very promising substance in the fields of medicine, reagents, cosmetics, food, etc. By the way, the conventional method for preparing chitosan oligosaccharides is to dissolve chitosan, partially hydrolyze it using a large amount of hydrochloric acid for a relatively long time, adsorb the decomposed product on a cation exchange resin column, and then dissolve it in hydrochloric acid. A method of separating D-glucosamine from chitosan oligosaccharides such as bioses, trioses, tetraoses, pentaoses, and hexaoses by performing chromatography using a concentration gradient elution method has been carried out. However, this method requires the preparation of chitosan hydrochloride and a large amount of dilute acid or water to dissolve chitosan in advance during hydrolysis, and furthermore, it requires a large amount of concentrated hydrochloric acid to increase the hydrochloric acid concentration. It required hydrochloric acid.

For this reason, large-scale hydrolytic snowmaking is required relative to the amount of chitosan to be decomposed, and the hydrolysis time also takes a long time.
Industrial production of chitosan oligosaccharides in large amounts is extremely inefficient and unpractical. For example, to date, several reports have been made on methods for preparing chitosan oligosaccharides, and their hydrolysis methods are as follows.

A method of dissolving chitosan in dilute acid, adding 10.8 N hydrochloric acid in an amount 200 times the weight of chitosan, and hydrolyzing it at 53°C for 48 hours (Journal of American C
chemical 5ociety, 79.5046-
5049 (1957)).

Prepare chitosan hydrochloride, dissolve it in water, add 12N hydrochloric acid in an amount 180 times the weight of chitosan, and heat at 50-55°C.
Method of hydrolysis for 48 hours
Carbohydrate Chemistry, I
s 305 (1962)).

A method in which chitosan hydrochloride is prepared from sea bream, dissolved in water, made into a 100-fold volume of 3.6N hydrochloric acid solution, and hydrolyzed at 100°C for 34 hours [Journal of CAemic
alSociety', 221B-2227 (195
B)).

These require a very large amount of hydrochloric acid, 100 to 200 times the weight of chitosan, and a long hydrolysis time of 1 to 2 days. This method is not suitable for manufacturing. Therefore, the present inventors aimed to economically and efficiently produce large quantities of chitosan oligosaccharides, and as a result of various studies, they arrived at the present invention.

That is, the present invention proposes a novel method for producing chitosan oligosaccharides, which is characterized by adding concentrated hydrochloric acid to chitosan and subjecting it to partial hydrolysis at high temperature for a short period of time.

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 addition, for hydrolysis, chitosan does not need to be dissolved in dilute acid or prepared into chitosan hydrochloride and dissolved in water, but considering the ease of hydrolysis, chitosan should be It is appropriate to use flakes. Hydrolysis may be carried out by adding 10N or more hydrochloric acid 5 to 30 times the weight of chitosan at 60 to 100°C for 1 to 4 hours, but particularly preferably, adding 10N or more hydrochloric acid to 10 times the weight of chitosan. Add ~20 times the amount and heat at 70-90°C for 2-3 hours. If the hydrochloric acid concentration is below 10N or the temperature is below 60°C, hydrolysis will take a long time and the yield of chitosan oligosaccharides will be low. Furthermore, if the hydrochloric acid concentration is less than 10N and the temperature is at a high temperature of 100° C. or more or for more than 4 hours, the amount of monosaccharide D-glucosamine produced increases and the yield of chitosan oligosaccharide becomes low.

Obtained by the method of the present invention. A partially hydrolyzed solution of chitosan is prepared by removing insoluble matter by centrifugation, decolorizing the supernatant by adding activated carbon, and then removing hydrochloric acid by concentration under reduced pressure. From this partial hydrolyzate of chitosan, D-glucosamine and a series of chitosan oligosaccharide hydrochlorides can be separated by conventional cation exchange resin column chromatography.

The method of the present invention is a method for preparing chitosan oligosaccharides in which the partial hydrolysis step is significantly improved, and a large amount of chitosan oligosaccharides can be produced economically and efficiently.

Next, the present invention will be explained in detail based on examples, but it goes without saying that the present invention is not limited by such explanation.

Example (1) 11.5N hydrochloric acid (200ml) was added to 20fI of chitosan ground into 10 meshes, and the mixture was hydrolyzed in a water bath at 80°C for 1 hour.

After adding 200+/ml of water to this hydrolyzate, insoluble materials were removed by centrifugation. Activated carbon was added to the obtained supernatant to decolorize it, and the mixture was concentrated to dryness under reduced pressure to obtain a decomposed product. This decomposition product is 10
0g/ion exchange resin column (Dowex 50w
) and eluted using a linear concentration gradient from O to 4N hydrochloric acid.

D-glucosamine and chitosan oligosaccharide were detected by measuring absorbance at 570 nm using ninhydrin coloring.

In addition, each fraction was concentrated and dried to obtain chitosan oligosaccharide.

Example (2) Hydrolysis was carried out for 2 hours under the same conditions as in Example (1).

The subsequent operations were also performed in the same manner as in Example (1).

Example (3) Hydrolysis was carried out for 4 hours under the same conditions as in Example (1). The subsequent operations were performed in the same manner as in Example (1).

Comparative Example Comparative Examples (11 and 21) were hydrolyzed for 6 hours and 7 hours under the same conditions as in Example (1).

Chitosan 2 crushed into 10 meshes as comparative example (3)
6N hydrochloric acid 680111 was added to 0F and hydrolyzed at 70°C for 38 hours.

As Comparative Example (4), 11.5N hydrochloric acid 21 was added to chitosan 2Of and hydrolyzed at 53° C. for 38 hours.

The decomposition rate of chitosan, the yield of chitosan oligosaccharide hydrochloride and D-glucosamine hydrochloride in Examples (1) to (3) and Comparative Examples (1) to (4) are shown in Table 1 below.

In addition, chitosan decomposition rate, chitosan oligosaccharide yield, and D
- The yield of glucosamine was calculated using chitosan as a factor of 100.

Further, the amount of hydrochloric acid was expressed as a value obtained by dividing the weight of chitosan used by 1.

Example 4 11.5N hydrochloric acid (400°C) was added to 40f of chitosan ground into 10 meshes, and the mixture was hydrolyzed in a water bath at 80°C for 2 hours. After adding 400w1 of water to this hydrolysis solution, insoluble matter was removed by centrifugation.

Activated carbon was added to the obtained supernatant to decolorize it, and it was concentrated to dryness under reduced pressure to obtain a decomposition product of 31. Of got it. This decomposition product is 1000
g/ion exchange resin column (DOW-EX 50W
), and the same operation as in Example 1 was performed to obtain D-glucosamine and a series of chitosan oligosaccharides. The yield and yield of chitosan oligosaccharide hydrochloride and D-glucosamine hydrochloride in the method of the present invention are shown in Table 2 below. The yield was calculated based on the weight of chitosan subjected to decomposition as 100 (@).

Table 2 Procedural amendments dated August 73, 1980 Manabu Shiga, Commissioner of the Patent Office1, Indication of the case Patent No. 143783 of 19822, Name of the invention Process for producing chitosan oligosaccharide3, Person making the amendment Representative: Kei Matsumoto - Department 4, Agent Address: 4-22-12 Nishi-Shinjuku, Shinjuku-ku, Tokyo (1
) ゛”=,,,”’ 7. Contents of the amendment (1) The statement “There are almost no things” in the 11th line from the bottom of the second page of the specification has been amended to “There are almost no things” do.

(2) Lines 10 and 9 from the bottom of page 2: In the form of chitosan, it is used as a cationic natural flocculant for wastewater treatment.
・" has been corrected to read, "In the form of chitosan, it is used as a cationic natural flocculant for wastewater treatment..."

(3) On page 6, line 3, the statement ``below the regulations'' is amended to read ``beyond the regulations.''

(4) “Chitosan-Latraose” on page 11, line 6
The certain description has been corrected to "chitosan-tetraose."

Claims (1)

[Claims] (1) Add 5 to 30 times the weight of chitosan with concentrated hydrochloric acid of 10N or more to chitosan, and add 1 to 4 times the weight of chitosan at a temperature of 60 to 100℃
A method for producing chitosan oligosaccharides, which comprises partially hydrolyzing chitosan for a period of time.