JP2732107B2 - Method for producing low molecular weight chitosan - Google Patents

Description

[Detailed Description of the Invention] [Industrial application field] The present invention is a method for safely converting low molecular weight chitosan having a molecular weight of 100,000 or less from high molecular weight chitosan obtained by deacetylating chitin in nature by a simple operation. It relates to a method of manufacturing.

In recent years, demand for low molecular weight chitosan has been increasing in various fields such as cosmetics, pharmaceuticals, and antibacterial agents.

[Conventional technology]

Conventionally, methods for producing low molecular weight chitosan include a method using chitosanase derived from a microorganism, a method using heat treatment in a high-concentration aqueous hydrochloric acid solution, a method using chlorine gas, and a method using hydrogen peroxide. I have.

[Problems to be solved by the invention]

These methods have problems in safety and economy, such as unconfirmed safety of chitosanase, complicated post-treatment of the hydrochloric acid method, toxicity of chlorine gas, and problems of residual hydrogen peroxide. The present inventors have conducted various studies and found that the molecular weight is safe and inexpensive.
The present inventors have found a method for obtaining a low molecular weight chitosan of 100,000 or less, and have completed the present invention.

[Means for solving the problem]

The present invention relates to a method for producing low molecular weight chitosan having a molecular weight of 100,000 or less, characterized in that the molecular weight is reduced by treating chitosan having a molecular weight of 1,000,000 or more with one or more neutral proteases and lipases having chitosan decomposability.

Neutral proteases are used in food processing, brewing and fermentation, pharmaceuticals,
Commercially available products used in feeds and the like whose safety has been sufficiently confirmed can be used. Examples of neutral proteases having chitosan resolution include Protin P (manufactured by Daiwa Kasei Co., Ltd.) Punchidase NP-2 (manufactured by Yakult Honsha Co., Ltd.) and Protease A "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.).

Lipase is used for decomposition and reforming of fats and oils, reforming of dairy products,
Commercially available products used in fermentation, pharmaceuticals, etc., whose safety is well confirmed, can be used. Examples of lipases having chitosan resolution include lipase P “Amano”, lipase A
"Amano" (both manufactured by Amano Pharmaceutical Co., Ltd.) and the like.

The chitosan used in the present invention is preferably one obtained by deacetylating natural chitin obtained from shrimp, crab and the like with, for example, caustic soda, and has a molecular weight of 1,000,000 or more.

Chitosan is an inorganic acid such as hydrochloric acid or nitric acid and formic acid, acetic acid,
An aqueous solution can be prepared using an organic acid such as propionic acid or lactic acid.

A low molecular weight chitosan can be obtained by allowing one or more neutral proteases or lipases to act on the chitosan solution.

The conditions for the lowering reaction are not particularly limited in the present invention, but the concentration of the chitosan solution is 1 to 20%, preferably 3 to 10%.
%. The pH of the chitosan solution is preferably from 3 to 6. The amount of the neutral protease and lipase to be used is not particularly limited in order to obtain a low molecular weight chitosan having an arbitrary molecular weight, but is usually used in an amount of about 0.001 to 10% w / w with respect to chitosan. The reaction temperature is from room temperature to about 55 ° C, preferably 40 to 50 ° C, and the reaction time is about 1 to 100 hours.

After the decomposition reaction, the low-molecular-weight chitosan solution is first filtered for impurities, and then adjusted to a pH of about 8 with an alkali metal hydroxide solution.
Alternatively, a low molecular weight chitosan can be obtained by forming a precipitate using a solvent such as ethanol or acetone, and filtering the precipitate. Alternatively, the low molecular weight chitosan solution after the decomposition reaction is filtered, the pH is adjusted to about 8, the resulting precipitate is removed, and the formed salt is then used with an ion exchange resin, electrodialysis, a dialysis membrane or another membrane. ,
And the like, and then concentrated to dryness, freeze-dried or dried with a spray drier to obtain water-soluble low molecular weight chitosan. The molecular weight of the low molecular weight chitosan varies depending on the desired properties, but is about several hundred to several thousand when water solubility is imparted.

 Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 10 g of chitosan was dispersed in 500 ml of water, and 4.2 g of concentrated hydrochloric acid was added.
After stirring and dissolving, 0.5 g of commercially available neutral protease punchase PN-2 (manufactured by Yakult Honsha Co., Ltd.) was dissolved in 10 ml of water and added to the reaction solution. The temperature of the reaction solution was maintained at 45 ° C., and stirring was continued for 12 hours. The pH of the reaction solution at this time was 5.0
Met.

The molecular weight of chitosan is determined by using
The measurement was performed by a GPC method using a TSKgel GMPWxL column manufactured by Co., Ltd. The molecular weight before the reaction was 1.32 million, but the molecular weight of the chitosan after the reaction was reduced to 70,000.

The reaction solution was subjected to a heat treatment at 90 ° C. for 30 minutes, and once filtered, the pH was adjusted to 8 with 5% NaOH to obtain a precipitate. This is filtered, washed with water and dried to obtain low molecular weight chitosan 9.
5 g were obtained.

[Example 2] 10 g of chitosan was dispersed in 500 ml of water, 4.2 g of concentrated hydrochloric acid was added, and the mixture was dissolved by stirring. Then, 0.5 g of commercially available protin PC10 (manufactured by Daiwa Kasei Co., Ltd.) was dissolved in 10 ml of water, and the reaction was carried out. Added to the solution. The temperature of the reaction solution was maintained at 50 ° C., and stirring was continued for 12 hours.

The result of measuring the molecular weight of the reaction solution by the same GPC method as in Example 1 was 46,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

Example 3 10 g of chitosan was dispersed in 500 ml of water, 4.2 g of concentrated hydrochloric acid was added, and the mixture was dissolved by stirring. Then, 0.5 g of protease A "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.) was dissolved in 10 ml of water, and the reaction was performed. Added to the solution. The temperature of the reaction solution was maintained at 45 ° C., and stirring was continued for 12 hours.

The molecular weight of the reaction solution measured by the same GPC method as in Example 1 was 30,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

[Example 4] 10 g of chitosan was dispersed in 500 ml of water, and acetic acid, a reagent-grade acetic acid 3.
After adding 2 g and stirring and dissolving, 0.5 g of Protease A “Amano” was dissolved in 10 ml of water and added to the reaction solution. The temperature of the reaction solution was maintained at 45 ° C., and stirring was continued for 12 hours.

The result of measurement of the molecular weight of the reaction solution by the same GPC method as in Example 1 was 25,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

Example 5 10 g of chitosan was dispersed in 500 ml of water, and 4.2 g of concentrated hydrochloric acid was added.
After stirring and dissolving, 0.5 g of protease A “Amano” is added to 10 ml.
In water and added to the reaction mixture. The temperature of the reaction solution is 45 ° C
And stirring was continued for 48 hours.

The result of measurement of the molecular weight of the reaction solution by the same GPC method as in Example 1 was 1,500.

The reaction solution was heated at 90 ° C. for 30 minutes, the pH was adjusted to 8 with 5% NaOH, and the resulting precipitate was removed. Then, 1500 ml of absolute ethanol was added to obtain a precipitate. This was filtered and dried to obtain 6 g of low molecular weight chitosan.

 The resulting low molecular weight chitosan was dissolved in neutral water.

Example 6 A low molecular weight chitosan solution was obtained in the same manner as in Example 5. This solution is subjected to a heat treatment at 90 ° C. for 30 minutes, and 5% Na
After adjusting the pH to 8 using OH and removing the resulting precipitate,
Desalting was performed by electrodialysis. After concentrating the solution to double the chitosan concentration, the solution was dried with a spray drier to obtain 5 g of fine powdered low molecular weight chitosan.

 The resulting low molecular weight chitosan was dissolved in neutral water.

Example 7 10 g of chitosan was dispersed in 500 ml of water, and 4.2 g of concentrated hydrochloric acid was added.
After stirring and dissolving, 0.5 g of commercially available lipase P “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.) was dissolved in 10 ml of water and added to the reaction solution. The concentration of the reaction solution was maintained at 50 ° C., and stirring was continued for 12 hours.

The result of measuring the molecular weight of the reaction solution by the same GPC method as in Example 1 was 62,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

Example 8 10 g of chitosan was dispersed in 500 ml of water, and 4.2 g of concentrated hydrochloric acid was added.
After stirring and dissolving, 0.5 g of commercially available lipase A “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.) was dissolved in 10 ml of water and added to the reaction solution. The temperature of the reaction solution was maintained at 45 ° C., and stirring was continued for 12 hours.

The result of measuring the molecular weight of the reaction solution by the same GPC method as in Example 1 was 64,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

Example 9 10 g of chitosan was dispersed in 500 ml of water, 4.2 g of concentrated hydrochloric acid was added, and the mixture was dissolved by stirring. Then, 0.25 g of protease A “Amano” was added.
Dissolve 0.25 g of lipase A "Amano" in 10 ml of water,
Added to the reaction. The temperature of the reaction solution was maintained at 45 ° C., and stirring was continued for 12 hours.

The result of measurement of the molecular weight of the reaction solution by the same GPC method as in Example 1 was 38,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

Example 10 10 g of chitosan was dispersed in 500 ml of water, 4.2 g of concentrated hydrochloric acid was added, and the mixture was stirred and dissolved.Protin PC10, 0.25 g and lipase P "Amano", 0.25 g were dissolved in 10 ml of water, and reacted. Added to the solution. The temperature of the reaction solution was maintained at 45 ° C., and stirring was continued for 12 hours.

The result of measuring the molecular weight of the reaction solution by the same GPC method as in Example 1 was 46,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

Example 11 10 g of chitosan was dispersed in 500 ml of water, 4.2 g of concentrated hydrochloric acid was added, and the mixture was dissolved by stirring. Then, Lipase P “Amano”, 0.1 g, Protease A “Amano”, 0.2 g, Protin PC10, 0.2 g To
Dissolved in 10 ml of water and added to the reaction.

 The temperature of the reaction solution was maintained at 50 ° C., and stirring was continued for 12 hours.

The result of measurement of the molecular weight of the reaction solution by the same GPC method as in Example 1 was 35,000.

Thereafter, the same treatment as in Example 1 was performed to obtain low molecular weight chitosan.

〔effect〕

As is clear from the results of the examples, the method for producing low molecular weight chitosan of the present invention is an efficient and safe method for producing low molecular weight chitosan by dissolving chitosan and treating it with one or more neutral proteases and lipases. Is obtained.

Claims (1)

(57) [Claims] 1. Production of low molecular weight chitosan having a molecular weight of 100,000 or less, characterized in that the molecular weight is reduced by treating chitosan having a molecular weight of 1,000,000 or more with one or more neutral proteases or lipases having chitosan decomposability. Method.