Chitosan oligomer fractionation method

Abstract

To provide a chitosan oligomer fractionation method capable of sufficiently fractionating an aqueous solution of low-molecular-weight chitosan oligomers with a molecular weight of 2,000 or less and precipitated high-molecular-weight chitosan oligomers with a molecular weight of 8,000 or more.SOLUTION: Acid is dissolved in heated water, chitosan is added to the solution and dissolved by heating, and then the first enzyme is added to the heated solution to cause a hydrolysis reaction to produce chitosan oligomers. An alkaline substance is added to and mixed with the produced chitosan oligomers, allowing them to be fractionated into an aqueous solution of low-molecular-weight chitosan oligomers with a molecular weight of 2,000 or less and precipitated high-molecular-weight chitosan oligomers with a molecular weight of 8,000 or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for fractionating chitosan oligomers. [Background technology]

[0002] Chitosan is a basic homopolysaccharide consisting of linearly linked 2-amino-2-deoxy-D-glucose (glucosamine) units. In industrial production, it is generally produced by deacetylating natural chitin, which is found in the shells of crustaceans such as crabs and shrimp, and in the skeletons of squid.

[0003] However, chitosan is generally a polymer with a molecular weight of over 100,000, which makes it difficult to handle due to its high viscosity when made into a solution (it is poorly soluble in water and requires the addition of an acid as a solvent), limiting its applications.On the other hand, research on low-molecular-weight chitosan oligomers with a molecular weight of 2,000 or less has been actively conducted, and unique physiological functions not seen in high-molecular-weight chitosan oligomers are being revealed in plants, animals, and microorganisms.

[0004] Under these circumstances, various research and development efforts have been made to improve the utility of chitosan by reducing its molecular weight (see, for example, Patent Document 1).

[0005] However, in order to reduce the molecular weight of such chitosan, it would be desirable to fractionate chitosan into low molecular weight chitosan oligomers with a molecular weight of 2000 or less and high molecular weight chitosan oligomers with a molecular weight of 8000 or more. However, there has been a problem in that such a clear fractionation method has not yet been established.

[0006] In order to solve such problems, the present applicant has proposed a technique as described in Patent Document 2. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 63-63701 [Patent Document 2] Japanese Patent Application Publication No. 2023-135748 Summary of the Invention [Problem to be solved by the invention]

[0008] However, through repeated experiments, the applicant has found that the method described in Patent Document 2 is insufficient for fractionating chitosan into low molecular weight chitosan oligomers having a molecular weight of 2000 or less and high molecular weight chitosan oligomers having a molecular weight of 8000 or more.

[0009] In view of the above problems, an object of the present invention is to provide a method for fractionating chitosan oligomers, which can sufficiently fractionate chitosan oligomers into an aqueous solution of low-molecular-weight chitosan oligomers having a molecular weight of 2000 or less and precipitated high-molecular-weight chitosan oligomers having a molecular weight of 8000 or more. [Means for solving the problem]

[0010] According to the invention of claim 1, an acid is dissolved in heated water, and chitosan is added and dissolved therein. Then, the first enzyme is added to the heated solution to cause a hydrolysis reaction to generate chitosan oligomers. An alkaline substance is added to and mixed with the generated chitosan oligomers, thereby fractionating the solution into an aqueous solution of low molecular weight chitosan oligomers having a molecular weight of 2000 or less and precipitated high molecular weight chitosan oligomers having a molecular weight of 8000 or more.

[0011] According to the invention of claim 2, in the chitosan oligomer fractionation method of claim 1, the first enzyme is added to cause a hydrolysis reaction, and then a second enzyme is added to hydrolyze the first enzyme, thereby producing the chitosan oligomer group. [Effects of the Invention]

[0012] According to the present invention, it is possible to sufficiently fractionate an aqueous solution of low molecular weight chitosan oligomers having a molecular weight of 2000 or less from precipitated high molecular weight chitosan oligomers having a molecular weight of 8000 or more. DETAILED DESCRIPTION OF THE INVENTION

[0013] The chitosan oligomer fractionation method according to the present invention involves dissolving an acid in heated water, adding chitosan to the solution and dissolving it by heating, adding a first enzyme to the heated solution to cause a hydrolysis reaction to produce chitosan oligomers, and adding an alkaline substance to the produced chitosan oligomers to fractionate them into an aqueous solution of low-molecular-weight chitosan oligomers with a molecular weight of 2000 or less and precipitated high-molecular-weight chitosan oligomers with a molecular weight of 8000 or more. Furthermore, in producing chitosan oligomers, the first enzyme can be added to cause a hydrolysis reaction, and then a second enzyme can be added to hydrolyze the first enzyme to produce chitosan oligomers.

[0014] Chitosan is a basic homopolysaccharide consisting of linearly linked 2-amino-2-deoxy-D-glucose (glucosamine) units, and is produced by deacetylating natural chitin, which is found in the shells of crustaceans such as crabs and shrimp, and squid skeletons. However, because chitin molecules have a hard crystalline structure, 100% deacetylation does not occur, and approximately 20% of the chitin domain always remains.

[0015] Examples of the acid include organic acids such as citric acid, acetic acid, etc. Chitosan is known to dissolve in an aqueous solution of an organic acid such as citric acid or acetic acid, and therefore, when an aqueous solution of an organic acid is added to chitosan, the chitosan will dissolve.

[0016] The first enzyme can be lysozyme (muramitase), and can be prepared by dissolving an acid in water, adding chitosan to the solution, and then adding the first enzyme to the heated solution to hydrolyze the acid, thereby dissolving the chitin region present in the solution. The solution can be heated by first heating the water, or by dispersing or dissolving the acid, chitosan, and first enzyme in water, and then heating the solution.

[0017] The second enzyme, such as pepsin, inactivates the first enzyme. Specifically, when fractionated chitosan oligomers are used, for example, in the agricultural field, long-term storage must be considered. If the first enzyme remains in the chitosan oligomer during long-term storage, the first enzyme may act to destroy the factors that form the chitosan oligomer clusters. Therefore, by inactivating the first enzyme using the second enzyme, the factors that form the chitosan oligomer clusters can be maintained consistently for a long period of time, thereby enabling long-term storage. To illustrate this point using a specific example, when lysozyme is used as the first enzyme and pepsin as the second enzyme, lysozyme is contained in egg white and its activity spans a wide pH range. Lysozyme is a polysaccharide hydrolase, but its main component is a protein. Pepsin, on the other hand, is a protease found in large amounts in gastric juices and exhibits its activity at a relatively low pH. Therefore, by combining these two enzymes and inactivating lysozyme at low pH, the factors that form chitosan oligomers can be maintained for a long period of time. Note that storage methods other than dry storage can also be used.

[0018] Thus, chitosan oligomers are produced in this manner, and by adding an alkaline substance such as an aqueous solution of sodium hydroxide to the produced chitosan oligomers and cooling the mixture, the chitosan oligomers can be fractionated into an aqueous solution of low molecular weight chitosan oligomers having a molecular weight of 2000 or less and precipitated high molecular weight chitosan oligomers having a molecular weight of 8000 or more. [Example]

[0019] Here, the present inventors conducted the following experiment to prove the above content. Note that the following experiment is merely an example and is not intended to be limiting.

[0020] The inventors prepared a commercially available bottle, placed 10 g of citric acid in the bottle, dissolved the citric acid in 200 to 300 ml of deionized water (ion-exchanged water) heated to approximately 40 to 50°C, and added and dissolved 10 g of chitosan powder to the bottle.

[0021] Next, when the chitosan powder had dissolved, the liquid volume was adjusted to 900 ml, and a solution of 0.01 g of lysozyme dissolved in 10 ml of deionized water (ion-exchanged water) was added, followed by hydrolysis reaction at 42°C for 9 hours.

[0022] After the hydrolysis reaction, 0.02 g of pepsin dissolved in 10 ml of deionized water (ion-exchanged water) was added and reacted at 30°C for 2 hours to hydrolyze and inactivate the lysozyme, thereby obtaining an aqueous solution of chitosan oligomers at pH 4.

[0023] Next, the bottle containing the chitosan oligomer aqueous solution at pH 4 was immersed in ice water for about 1 hour to cool it down.

[0024] Next, a 10 mol / L aqueous solution of sodium hydroxide was added to the pH 4 chitosan oligomer solution, mixed, and placed in a refrigerator for approximately 2-3 hours. During the neutralization process (including after neutralization), precipitation occurred, resulting in separation into two layers: the lower layer containing the precipitate and the upper layer containing the aqueous solution. These two layers were then dehydrated using a cloth and filtered to obtain a pH 7 sodium salt aqueous solution containing chitosan oligomers. The precipitate was then further dehydrated using a cloth and squeezed to separate it. After washing with water and air-drying, a pH 7 chitosan oligomer precipitate was obtained. Analysis of the pH 7 chitosan oligomer precipitate revealed that it was primarily composed of high molecular weight chitosan oligomers with molecular weights of 8000 or greater. Analysis of the pH 7 sodium salt aqueous solution containing chitosan oligomers revealed that it was primarily composed of low molecular weight chitosan oligomers with molecular weights of 2000 or less.

[0025] Next, for easier storage, the inventors dried the chitosan oligomer precipitate at pH 7, removing moisture and storing the chitosan oligomer as a citrate salt. Furthermore, the inventors concentrated a pH 7 aqueous solution of the chitosan oligomer-containing sodium salt, added acetone to crystallize it, and then stored the resulting low-molecular-weight chitosan oligomer, mainly consisting of a molecular weight of 2000 or less, as a sodium salt salt.

[0026] The above experimental results prove that by dissolving an acid in heated water, adding chitosan to the solution and dissolving it, and then adding the first enzyme to the heated solution to cause a hydrolysis reaction to produce chitosan oligomers, and then adding an alkaline substance to the produced chitosan oligomers and mixing them, it is possible to sufficiently fractionate them into an aqueous solution of low molecular weight chitosan oligomers with a molecular weight of 2000 or less and precipitated high molecular weight chitosan oligomers with a molecular weight of 8000 or more.

[0027] In this Example, the first enzyme was added to cause a hydrolysis reaction, and then the second enzyme was added to hydrolyze the first enzyme to produce chitosan oligomers, but if the second enzyme is not needed, it does not have to be added. However, adding the second enzyme is preferable because, as explained above, by inactivating the first enzyme, the factors that form the chitosan oligomers can be maintained consistently for a long period of time, thereby enabling long-term storage.

[0028] Furthermore, in this embodiment, ice water and a refrigerator are used as examples of cooling methods, but the present invention is not limited to these and any cooling method may be used as long as it can provide cooling.

[0029] Furthermore, in this embodiment, a cloth is used as an example of a filtering method, but the filtering method is not limited to this, and any filtering method may be used as long as it can perform filtering, such as a centrifugal separator.

[0030] Furthermore, in this embodiment, wind drying is exemplified as the drying method, but this is not limiting, and an evaporator may also be used, and any drying method may be used as long as it can dry.

[0031] Furthermore, in this embodiment, an example was shown in which an aqueous sodium hydroxide solution was used as the alkaline substance, but the invention is not limited to this, and any alkaline substance such as an aqueous potassium hydroxide solution may be used.

Claims

1. The chitosan oligomer fractionation method comprises dissolving an acid in heated water, adding chitosan to the solution and dissolving it therein, adding a first enzyme to the heated solution to cause a hydrolysis reaction to produce chitosan oligomers, and then adding an alkaline substance to the produced chitosan oligomers and mixing them to fractionate them into an aqueous solution of low molecular weight chitosan oligomers having a molecular weight of 2,000 or less and precipitated high molecular weight chitosan oligomers having a molecular weight of 8,000 or more.

2. 2. The method for fractionating chitosan oligomers according to claim 1, wherein after the first enzyme is added to cause a hydrolysis reaction, a second enzyme is added to hydrolyze the first enzyme, thereby producing the chitosan oligomer group.