JP4588205B2 - Chitin oligosaccharide production method - Google Patents

Description

[0001]
[Technical field to which industry belongs]
The present invention relates to a method for producing chitin oligosaccharides and a chitin oligosaccharide having a high composition ratio of high-polymerization degree oligomers obtained by the method.
[0002]
[Prior art]
Chitin is a natural polysaccharide widely distributed mainly in invertebrates, and its resource is said to be next to cellulose and is expected to be used effectively. However, chitin is insoluble in water and common organic solvents, so its utility is low, and only a part of existing resources is used as chitosan raw material. Chitin is a polymer whose molecular weight is estimated to be several millions of N-acetylglucosamine linearly linked by glycosidic bonds, but oligosaccharides (oligomers) that partially decomposed the glycosidic bonds, especially about 6 sugars or less. Are known to dissolve in water. Further, these chitin oligomers are known to exhibit physiological activities such as antitumor activity and immunostimulatory activity, and are used in health foods and the like.
[0003]
These physiological activities show strong activity for components having a relatively high degree of polymerization among oligomers exhibiting water solubility such as pentasaccharide and hexasaccharide or more, but it is extremely difficult to efficiently produce them. This is because the acid decomposition method using concentrated hydrochloric acid, which is usually used in industrial production methods, can only obtain monosaccharides when the reaction is completely advanced, and the oligomer is obtained by interrupting the reaction at a relatively large amount of oligomer components. In this case, an oligomer with a lower polymerization degree is produced more and chitin that is not sufficiently decomposed is removed as an insoluble part, so the yield is limited to 40 to 50%, and 30 to 30 monosaccharides that cannot be originally called oligomers. 40 percent is also included. Furthermore, 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]
In addition, production methods using enzymes have been studied, but since chitin is a polymer that is insoluble in water, the efficiency of the enzymatic reaction is poor, and most of the currently known chitin degrading enzymes are chitin in disaccharide units. Since it is cut out, it is difficult to obtain an oligomer with a high degree of polymerization.
Each component of the chitin oligomer can be purified and concentrated by using a chromatographic method, but it is inefficient to concentrate a low content component originally, and the cost is extremely increased.
[0005]
[Problems to be solved by the invention]
An object of this invention is to provide the manufacturing method which improved the yield of the product of the oligomer composition equivalent to the former significantly.
An object of the present invention is to provide a chitin oligomer that can be expected to have high physiological activity at low cost.
[0006]
[Means for Solving the Problems]
The gist of the present invention is a method for producing a chitin oligosaccharide characterized in that, in the acid hydrolysis of chitin as a raw material , limited hydrolysis is performed simultaneously using a polyvalent acid and hydrochloric acid .
[0008]
The polyvalent acid is sulfuric acid, and in this case, the present invention provides a chitin oligosaccharide characterized in that limited hydrolysis is performed using sulfuric acid and hydrochloric acid simultaneously in the acid hydrolysis of chitin as a raw material. The method is summarized.
[0009]
The polyvalent acid is phosphoric acid, and in this case, the present invention provides a chitin oligosaccharide characterized in that in the acid hydrolysis of chitin as a raw material, phosphoric acid and hydrochloric acid are simultaneously used for limited hydrolysis. The manufacturing method is the gist.
[0010]
The gist of the present invention is a chitin oligomer mixture having a high composition ratio of the high degree of polymerization oligomer produced by any of the above methods.
[0011]
Furthermore, this invention makes the summary the composition containing said chitin oligomer mixture.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The origin and shape of the chitin to be used are not particularly limited, but finely pulverized chitin is preferable when rapid dispersion is expected.
[0013]
Two or more acids, preferably two or more acids composed of a combination of a polyvalent acid and hydrochloric acid are used. The reaction solution only needs to contain hydrochloric acid and a polyvalent acid, and the kind of acid and the number of acids to be mixed are not particularly limited, but preferably the polyvalent acid is sulfuric acid or phosphoric acid. Since the concentration is not particularly limited, it can be used by diluting with a small amount of water. For example, concentrated hydrochloric acid: sulfuric acid: water = 100: 1 to 100: 1 to 100, preferably 100: 10 to 20: 1 to 10. For example, if the amount of water is too much, the acid concentration becomes thin, and if the amount of polyvalent acid is too large, neutralization requires a large amount of alkali. 75: 6: 4 is one of the preferred formulations.
[0014]
Further, the procedure for mixing the reaction solution and chitin is not particularly limited, but it is preferably mixed by a method in which cold chitin is added to the reaction solution mixed in advance. The amount of the reaction solution with respect to chitin is sufficient if the chitin can be sufficiently dispersed, and a small amount is desirable in order to reduce the amount of alkali used for neutralization when the reaction is stopped, and preferably a ratio of 15 ml to 20 ml with respect to 1 g of chitin. Used.
[0015]
It is desirable to treat at a low temperature until the chitin can be sufficiently dispersed or dissolved by stirring at the initial stage of the reaction, preferably 0 to 20 ° C., more preferably 5 to 15 ° C. Not. After 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 time treatment is continued from 30 minutes to 10 hours.
[0016]
The reaction temperature and time are important to be combined and are adjusted according to the target chitin oligomer composition or yield. The treatment time is adjusted to be long at a low temperature and short at a high temperature, but in particular when the side reaction of deacetylation is to be avoided, the treatment is performed for a time of 4 hours or less, and the temperature at this time is preferably 25 ° C. or more. For example, a combination of 2.5 hours at 30 ° C. is a preferable combination.
[0017]
The reaction is stopped by neutralization with an arbitrary alkali, but is usually neutralized with an inexpensive alkali such as a sodium hydroxide solution. In order to avoid heating due to neutralization, the reaction solution and the neutralizing alkali are generally cooled before neutralization to avoid the 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 or ice.
[0018]
The salt produced by neutralization varies depending on the acid and alkali used, and includes sodium sulfate and sodium phosphate in addition to sodium chloride. Dialysis is widely used.
[0019]
The obtained chitin oligomer solution can be used as it is depending on the purpose, and can be concentrated or completely dried and used as a powder. In addition, it is possible to adjust the composition and refine the composition using membrane fractions such as RO membranes and ultrafiltration membranes, and to concentrate and purify each component using general chromatographic techniques. It is. Even when performing such purification, the ratio of oligomers with a higher degree of polymerization than conventional products is high, so yield and efficiency are high, and high-quality products with a high concentration of oligomers with a high degree of polymerization can be obtained. it can.
[0020]
[Action]
The oligomer produced by the method of the present invention contains a high content of oligomer components having a high degree of polymerization, although the production of monosaccharides is unavoidable. The method of the present invention can produce an oligomer containing 3 sugars more than 2 sugars and 4 sugars more than 3 sugars by controlling the production conditions, and 5 sugars can be obtained by selecting more suitable conditions. Oligomers containing up to 4,6 sugars at the same level can be produced. Also in this case, the yield is not inferior to the conventional production method. In other currently known methods, it is not possible to obtain a chitin oligomer product containing a high degree of polymerization oligomer component without any operation for adjusting the composition.
The method of the present invention also contains a component of 7 or more sugars that was hardly obtained by conventional decomposition of hydrochloric acid alone. Therefore, by using the method of the present invention, a chitin oligomer that can be expected to have high physiological activity at low cost can be provided.
[0021]
The hydrolysis reaction used in the present invention can exert a very strong action on the cleavage of glycosidic bonds compared to 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. It is possible to suppress the deacetylation of the side reaction as well as to increase the pH. Thus, 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 an advantageous method in terms of cost. In addition, by controlling the conditions, it is possible to produce a product having an oligomer composition equivalent to the conventional one, and in that case, the yield can be greatly improved.
[0022]
【Example】
The details of the present invention will be described in Examples. The present invention is not limited to these examples.
[0023]
Example 1
150 ml of concentrated hydrochloric acid, 13 ml of concentrated sulfuric acid and 7 ml of water were mixed, 10 g of chitin powder (80 mesh pass) was gradually added with 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 put into 100 g of ice, 100 ml of water was added, and while stirring, 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. The mixture was left overnight at room temperature until undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalting apparatus (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized and weighed. The obtained oligomer composition was determined by HPLC (Asahipack NH2P-50 4E) as follows: monosaccharide 28.8%, disaccharide 10.3%, trisaccharide 11.7%, tetrasaccharide 13.8%, saccharide 14.0%, 6 sugars 12.1%, 7 sugars 6.7%, 8 sugars 2.3%, 9 sugars 0.4%. The yield was 55.4%.
[0024]
Example 2
150 ml of concentrated hydrochloric acid, 12 ml of concentrated sulfuric acid, and 8 ml of water were mixed and stirred while maintaining at 10 ° C. in a water bath, 10 g of chitin powder (80 mesh pass) was gradually added, and stirring was continued for 30 minutes. The reaction vessel was moved into a water bath maintained at 34 ° C. and stirring was continued for 3 hours. The reaction solution was put into 100 g of ice, 100 ml of water was added, and while stirring, 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. The mixture was left overnight at room temperature until undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalting apparatus (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized and weighed. The obtained oligomer composition was determined by HPLC (Asahipack NH2P-50 4E) 26.7% monosaccharide, 13.7% disaccharide, 14.0% trisaccharide, 13.9% tetrasaccharide, 13.4% 5 sugar, 6 sugars 10.4%, 7 sugars 5.5%, 8 sugars 2.0%, 9 sugars 0.3%. The yield was 73.0%.
[0025]
Example 3
1 g of chitin powder (80 mesh pass) was gradually added to 15 ml of concentrated hydrochloric acid with stirring in a water bath at 10 ° C., and stirring was continued for 15 minutes. The reaction vessel was moved into a water bath maintained at 40 ° C. and stirred for 10 minutes, and 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 20% sodium hydroxide solution was gradually added while stirring until the solution became neutral, and the reaction was stopped by neutralization. The mixture was left overnight at room temperature until undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalting apparatus (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized and weighed. The resulting oligomer composition was determined by HPLC (Asahipack NH2P-50 4E) 28.1% monosaccharide, 9.4% disaccharide, 11.4% trisaccharide, 13.4% tetrasaccharide, 13.0% 5 sugar, 6 sugars 11.0%, 7 sugars 7.4%, 8 sugars 4.0%, 9 sugars 1.7%, 10 sugars 0.5%. The yield was 61.1%.
[0026]
Example 4
Concentrated hydrochloric acid (30 ml), phosphoric acid (2.4 ml), and water (1.6 ml) were mixed, and while stirring under ice cooling, 2 g of chitin powder (80 mesh pass) was gradually added and stirring was continued for 15 minutes. The reaction vessel 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, 20% sodium hydroxide solution was gradually added until the solution became neutral, and the reaction was stopped by neutralization. It was left overnight until undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalting apparatus (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized and weighed. The obtained oligomer composition was determined by HPLC (Asahipack NH2P-50 4E) 19.0% monosaccharide, 9.3% disaccharide, 11.8% trisaccharide, 14.3% tetrasaccharide, 14.7% five sugar, 6 sugars 13.8%, 7 sugars 9.3%, 8 sugars 5.5%, 9 sugars 2.3%. The yield was 52.0%.
[0027]
Comparative Example 1 g of chitin powder (80 mesh pass) was gradually added while stirring 10 ml of concentrated hydrochloric acid at 40 ° C., and stirring was continued for 2 hours. The reaction solution was poured into 10 g of ice, 10 ml of water was added and 20% sodium hydroxide solution was gradually added while stirring until the solution became neutral, and the reaction was stopped by neutralization. It was left overnight until undegraded chitin was sufficiently precipitated, and the precipitate was removed by filtration. The filtrate was desalted with a desalting apparatus (Asahi Kasei Micro Acilyzer G3), concentrated under reduced pressure, lyophilized and weighed. The resulting oligomer composition was determined by HPLC (Asahipack NH2P-50 4E) 33.1% monosaccharide, 18.2% disaccharide, 15.3% trisaccharide, 11.6% tetrasaccharide, 8.6% 5-saccharide, 6 sugars 6.3%, 7 sugars 4.0%, 8 sugars 2.2%, 9 sugars 0.7%. The yield was 44.8%.
[0028]
【The invention's effect】
A chitin oligomer product containing a high degree of polymerization oligomer component can be provided.
The yield of products having the same oligomer composition as the conventional one can be greatly improved.
A chitin oligomer that can be expected to have high physiological activity at low cost can be provided.

Claims (5)

A method for producing a chitin oligosaccharide characterized in that, in the acid hydrolysis of chitin as a raw material , limited hydrolysis is performed simultaneously using a polyvalent acid and hydrochloric acid . Polyvalent acid process according to claim 1 of the chitin oligosaccharide sulfuric above. Polyvalent acid phosphate method for producing chitin oligosaccharide of claim 1 is described above. A chitin oligomer mixture having a high composition ratio of the high polymerization degree oligomer produced by the method according to claim 1, 2 or 3 . A composition comprising the chitin oligomer mixture of claim 4 .