JP2002177000A - Method for manufacturing chitin olygosaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for manufacturing a product having an oligomer composition similar to that of a conventional product in widely improved yield, and a method for manufacturing a chitin oligomer expectably having high biological activity at a low cost. SOLUTION: The method for manufacturing a chitin oligomer comprises limited hydrolysis using two or more kinds of acids simultaneously in the acid hydrolysis of a raw material. The two or more kinds of acids consist of a combination of a polyvalent acid and hydrochloric acid. The polyvalent acid is sulfuric acid or malic acid. A chitin oligomer mixture has a high composition ratio of a high polymeric oligomer which has been manufactured by the above method. A composition contains the chitin oligomer mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a chitin oligosaccharide and a chitin oligosaccharide having a high composition ratio of a high-polymerization degree oligomer obtained by the method.

[0002]

2. Description of the Related Art Chitin is a natural polysaccharide widely distributed mainly in invertebrates, and its resource is said to be second only to cellulose, and its effective utilization is expected. However, chitin is insoluble in water and common organic solvents, and therefore has low utility. Only a part of the existing resources is used for chitosan raw materials and the like. Chitin is a macromolecule in which N-acetylglucosamine is linearly linked by glycosidic bonds and the molecular weight is estimated to be several million. Oligosaccharides (oligomers) partially decomposing the glycosidic bonds,
Particularly, it is known that those having about 6 sugars or less dissolve in water. In addition, these chitin oligomers have antitumor activity,
It is known to exhibit physiological activities such as immunostimulatory activity and is used in health foods and the like.

[0003] Although these physiological activities have a strong activity on components having a relatively high degree of polymerization among water-soluble oligomers such as pentasaccharide and hexasaccharide, it is extremely difficult to produce them efficiently. It is. This is because the acid decomposition method using concentrated hydrochloric acid, which is usually used in industrial production methods, only allows monosaccharides to be obtained when the reaction proceeds completely, and the oligomer is obtained by interrupting the reaction where the oligomer component is relatively large. In this case, the lower the degree of polymerization, the more oligomers are produced, and chitin that is not sufficiently decomposed is removed as an insoluble portion, so that the yield is limited to 40 to 50%. Includes 40 percent. Further, if the treatment is interrupted at the beginning of the reaction, the proportion of the oligomer component increases somewhat, but in this case, the yield is inevitably reduced.

[0004] Further, a production method using an enzyme has been studied, but the efficiency of the enzymatic reaction is low because chitin is a polymer that is insoluble in water, and most of the currently known chitinolytic enzymes use chitin as a derivative of chitin. It is difficult to obtain an oligomer having a high degree of polymerization because it is cut out in sugar units. Each component of the chitin oligomer can be purified or concentrated by using a chromatographic technique. However, it is inefficient to concentrate components having a low content from the beginning, and the cost is extremely increased.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a process for producing a product having an oligomer composition equivalent to that of the prior art, with a greatly improved yield. An object of the present invention is to provide a low-cost chitin oligomer that can be expected to have high physiological activity.

[0006]

SUMMARY OF THE INVENTION The gist of the present invention is to provide a method for producing chitin oligosaccharides, wherein the acid hydrolysis of a raw material is carried out with limited hydrolysis using two or more acids simultaneously. .

[0007] The two or more kinds of acids described above are composed of a combination of a polyacid and hydrochloric acid. In this case, in the present invention, in the acid hydrolysis of the raw material, limited hydrolysis is performed by simultaneously using the polyacid and hydrochloric acid. A gist of the present invention is a method for producing a chitin oligosaccharide.

The polyacid is sulfuric acid. In this case, the present invention provides a method for producing chitin oligosaccharides, wherein sulfuric acid and hydrochloric acid are simultaneously used in the acid hydrolysis of a raw material. The method is summarized.

[0009] The polyacid is phosphoric acid, in which case
The gist of the present invention is to provide a method for producing a chitin oligosaccharide, wherein a limited hydrolysis is carried out by simultaneously using phosphoric acid and hydrochloric acid in the acid hydrolysis of a raw material.

The present invention also provides a chitin oligomer mixture having a high composition ratio of a high-polymerization degree oligomer produced by any of the above methods.

Further, the present invention provides a composition containing the above-mentioned chitin oligomer mixture.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The origin and shape of chitin to be used are not particularly limited, but if rapid dispersion is expected, finely ground chitin is preferred.

Two or more acids are used, preferably two or more acids comprising a combination of a polyacid and hydrochloric acid. The type of acid and the number of acids to be mixed are not particularly limited as long as the reaction solution contains hydrochloric acid and a polyacid, but sulfuric acid or phosphoric acid is preferably used as the polyacid. Since the concentration is not particularly limited, it can be used after being diluted with a small amount of water. For example, concentrated hydrochloric acid: sulfuric acid: water = 100: 1-100: 1-100, preferably 100: 10-20: 1-10. For example, if the amount of water is too large, the acid concentration becomes low, and if the amount of the polyvalent acid is large, it is not preferable because a large amount of alkali is required for neutralization. 75: 6: 4 is one of the preferred formulations.

The procedure for mixing the reaction solution and chitin is not particularly limited. Preferably, the reaction solution and the chitin are mixed by a method in which chitin is added to the previously mixed reaction solution at a cold time. The amount of the reaction solution with respect to chitin may be any amount as long as chitin can be sufficiently dispersed, and is preferably as small as possible in order to reduce the amount of alkali used for neutralization at the time of stopping the reaction. A ratio of 15 ml to 20 ml per 1 g of chitin is preferable. Used.

At the beginning of the reaction, it is desirable to treat at low temperature until the chitin can be sufficiently dispersed or dissolved by stirring, preferably at 0 to 20 ° C, more preferably at 5 to 1 ° C.
Although 5 ° C. is desirable, there is no particular limitation as long as it is 100 ° C. or less. After the chitin is sufficiently dispersed in the reaction solution, the reaction solution is heated to a target temperature in the range of 0 to 100 ° C. while maintaining stirring, and the target treatment is continued for 30 minutes to 10 hours.

The reaction temperature and time are important in combination, and are adjusted according to the target composition or yield of chitin oligomer. The treatment time is adjusted to be long at a low temperature and short at a high temperature. In particular, when it is desired to avoid a side reaction of deacetylation, the treatment is performed for a time of 4 hours or less, and the temperature at this time is desirably 25 ° C. or more. For example, a combination at 30 ° C. for 2.5 hours is one of the preferable combinations.

The termination of the reaction is carried out by neutralization with an arbitrary alkali, but is usually carried out with an inexpensive alkali such as sodium hydroxide solution. Also, in order to avoid heating due to neutralization, the reaction solution and the alkali for neutralization are generally cooled before neutralization to avoid generation of heat of neutralization, but the same treatment is effective in this method. For the same reason, it is desirable to neutralize the reaction solution after diluting it with water and ice.

The salt formed by the neutralization differs depending on the acid and alkali used, and includes sodium sulfate and sodium phosphate in addition to salt, which can be removed by a general desalting apparatus. Electrodialysis is widely used for the purpose.

The obtained chitin oligomer solution can be used as it is according to the purpose, and can be concentrated or completely dried and used as a powder. In addition, it is possible to adjust the composition and use a membrane fraction such as an RO membrane or an ultrafiltration membrane, and to perform more advanced purification, and it is also possible to concentrate and purify each component by a general chromatographic technique. It is. Even in the case of such purification and the like, since the ratio of oligomers having a higher degree of polymerization is higher than that of conventional products, the yield and efficiency are high, and it is also possible to obtain high-quality products in which the oligomers having a high degree of polymerization are highly concentrated. it can.

[0020]

The oligomer produced by the method of the present invention contains a high content of an oligomer component having a high degree of polymerization, although formation of a monosaccharide cannot be avoided. The method of the present invention can produce oligomers containing more trisaccharides than disaccharides and more tetrasaccharides than trisaccharides by controlling the production conditions, and pentasaccharides by selecting more suitable conditions. Up to 4,
Oligomers containing hexasaccharides at the same level can be produced. Also in this case, the yield is not inferior to the conventional production method. According to other methods known at present, it is not possible to obtain a chitin oligomer product containing a high degree of polymerization oligomer component at a high level without performing any operation for adjusting the composition. In addition, the method of the present invention also contains components of seven or more sugars, which were hardly obtained by conventional decomposition of only hydrochloric acid. Therefore, by using the method of the present invention, a chitin oligomer which can be expected to have high biological activity at low cost can be provided.

The hydrolysis reaction used in the present invention can exert an extremely strong effect on the cleavage of glycosidic bonds as compared with the conventional reaction consisting only of hydrochloric acid. In this case, the treatment can be completed at a lower temperature and in a shorter time. Therefore, not only can the production efficiency be increased, but also the deacetylation of side reactions can be suppressed. Accordingly, the treatment with a cation exchange resin, which is a step of removing a by-product generated by a side reaction called deacetylation, is not always necessary and is a method advantageous in cost. In addition, by controlling the conditions, it is possible to produce a product having an oligomer composition equivalent to that of the conventional product, in which case the yield can be significantly improved.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to embodiments. The present invention is not limited by these examples.

Example 1 150 ml of concentrated hydrochloric acid, 13 ml of concentrated sulfuric acid and 7 ml of water were mixed, and 10 g of chitin powder (80 mesh pass) was gradually added while stirring under ice cooling, and stirring was continued for 20 minutes. The reaction vessel was moved into a water bath maintained at 30 ° C., and stirring was continued for 2.5 hours. The reaction solution was poured into 100 g of ice, and 100
Then, while stirring, a 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. It was left overnight at room temperature until the undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalter (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized, and weighed. The obtained oligomer composition was analyzed by HPLC (Asahipack NH2P-50 4E) for 28.8% monosaccharide, 10.3% disaccharide, 11.7% trisaccharide, 11.7% tetrasaccharide,
3.8%, pentasaccharide 14.0%, hexasaccharide 12.1%, heptasaccharide6.
7%, 2.3% octasaccharide, 0.4% nin sugar. Yield 55.4%
Met.

Example 2 150 ml of concentrated hydrochloric acid, 12 ml of concentrated sulfuric acid, and 8 ml of water were mixed, and the mixture was stirred while being kept at 10 ° C. in a water bath to obtain chitin powder (8%).
(0 mesh pass), 10 g was gradually added, and stirring was continued for 30 minutes. The reaction tank was moved into a water bath maintained at 34 ° C., and stirring was continued for 3 hours. Pour the reaction solution into 100 g of ice,
100 ml of water was added, and while stirring, a 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. It was left overnight at room temperature until the undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate is desalted by a desalting device (Asahi Kasei Micro Acilyzer G3),
After concentration under reduced pressure, freeze-drying was performed and the weight was measured. The obtained oligomer composition was analyzed by HPLC (Asahipack NH2P-50 4E) for 26.7% of monosaccharide, 13.7% of disaccharide, and 14.0% of trisaccharide.
%, 4 sugars 13.9%, pentasaccharide 13.4%, hexasaccharide 10.4
%, 7 sugars 5.5%, 8 sugars 2.0%, 9 sugars 0.3%. The yield was 73.0%.

Example 3 1 g of chitin powder (80 mesh pass) was gradually added to 15 ml of concentrated hydrochloric acid while stirring at 10 ° C. in a water bath, and stirring was continued for 1 hour.
Continued for 5 minutes. The reaction vessel was moved into a water bath maintained at 40 ° C., and stirred for 10 minutes. Then, 1.6 ml of concentrated sulfuric acid and 0.4 ml of water were added, and stirring was continued for 1.4 hours. The reaction solution was poured into 10 g of ice, 10 ml of water was added, and while stirring, a 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. It was left overnight at room temperature until the undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalter (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized, and weighed.
The obtained oligomer composition was analyzed by HPLC (Asahipack NH2P-5
According to 4E), monosaccharide 28.1%, disaccharide 9.4%, trisaccharide 1
1.4%, tetrasaccharide 13.4%, pentasaccharide 13.0%, hexasaccharide 1
1.0%, 7.4% of 7 sugars, 4.0% of 8 sugars, 1.7 of 9 sugars
%, 10 sugar 0.5%. The yield was 61.1%.

Example 4 30 ml of concentrated hydrochloric acid, 2.4 ml of phosphoric acid and 1.6 ml of water were mixed, and 2 g of chitin powder (80 mesh pass) was gradually added while stirring under ice cooling, and stirring was continued for 15 minutes. The reaction tank was moved into a water bath maintained at 35 ° C., and stirring was continued for 2.5 hours. The reaction solution was poured into 30 g of ice, 30 ml of water was added, and while stirring, a 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization.
It was left overnight until the undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate is desalted (Asahi Kasei Micro A
The solution was desalted with cilyzer G3), concentrated under reduced pressure, lyophilized, and weighed. The resulting oligomer composition is HP
19.0% of monosaccharide by LC (Asahipack NH2P-504E),
9.3% disaccharide, 11.8% trisaccharide, 14.3% tetrasaccharide, 14.7% pentasaccharide, 13.8% hexasaccharide, 9.3% heptasaccharide, 9.3% octasaccharide.
5%, 2.3% 9 sugars. The yield was 52.0%.

Comparative Example While stirring 10 ml of concentrated hydrochloric acid at 40 ° C., chitin powder (8
(0 mesh pass), 1 g was gradually added, and stirring was continued for 2 hours. The reaction solution was poured into 10 g of ice, 10 ml of water was added, and while stirring, a 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. It was left overnight until the undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate is desalted (Asahi Kasei Micro Acily
The solution was desalted by zer G3), concentrated under reduced pressure, lyophilized, and weighed. The obtained oligomer composition is HPLC
(Asahipack NH2P-50 4E), 33.1% monosaccharide, 18.2% disaccharide, 15.3% trisaccharide, 11.6% tetrasaccharide, 8.6% pentasaccharide, 6.3% hexasaccharide, 7% sugar 4.0%, 8 sugars 2.2
%, 9% sugar 0.7%. The yield was 44.8%.

[0028]

According to the present invention, a chitin oligomer product containing a high degree of polymerization oligomer component at a high level can be provided. The yield of a product having an oligomer composition equivalent to that of a conventional product can be greatly improved. A chitin oligomer which can be expected to have high physiological activity at low cost can be provided.

Claims (6)

[Claims] 1. A method for producing a chitin oligosaccharide, comprising simultaneously performing limited hydrolysis using two or more acids at the time of acid hydrolysis of a raw material. 2. The method for producing a chitin oligosaccharide according to claim 1, wherein the two or more kinds of acids are a combination of a polyacid and hydrochloric acid. 3. The method for producing a chitin oligosaccharide according to claim 2, wherein said polyacid is sulfuric acid. 4. The method for producing chitin oligosaccharide according to claim 2, wherein said polyacid is phosphoric acid. 5. A chitin oligomer mixture having a high composition ratio of a high polymerization degree oligomer produced by the method of claim 1, 2, 3, or 4. 6. A composition comprising the chitin oligomer mixture of claim 5.