KR980009287A - Method for preparing high purity crystalline NOCC and method for controlling molecular weight of water soluble chitosan using enzyme - Google Patents

Abstract

purpose

An object of the present invention is to provide a method for preparing high purity crystalline NOCC and a method for controlling molecular weight of NOCC.

Configuration

After dispersing chitosan in isopropanol and warming it to about 30 ° C, NaOH aqueous solution was added dropwise, and after stirring, monochloroacetic acid dissolved in isopropyl alcohol was added, and the white powder in this solution became transparent, and then it was kept at room temperature. After stirring for a certain period of time, the pH was adjusted to 7, the residue was filtered, washed with a mixture of methanol and water, dissolved in methanol, stirred, washed with methanol, filtered, and dried to prepare high-purity crystalline NOCC, and NOCC Warm the aqueous solution to 50 ~ 60 ℃, add cellulase or chitosanase, adjust the pH to 6 ~ 7 after enzymatic digestion, and then concentrate and dry the filtrate to adjust the molecular weight.

Description

Method for preparing high purity crystalline NOCC and method for controlling molecular weight of water soluble chitosan using enzyme

The present invention relates to a method for preparing high purity crystalline NOCC and a method for controlling molecular weight of water-soluble chitosan using an enzyme.

Chitin is widely abundant in nature, especially complexes with proteins in invertebrates and fungi, and similar to the cellulose present in the plant system to form the exoskeleton and shell of the organism. The deacetylate of chitin is called chitosan, and this chitosan is a natural polymer cation that is non-toxic and is involved in the synthesis and degradation of living organisms but does not cause environmental pollution. , Heavy metal adsorbents, seed coatings for protein production, protein recovery agents and wound healing agents.

General chitosan is a prerequisite for the preparation of molecular weight and oligosaccharides. It is an essential prerequisite to dissolve using organic acid. On the other hand, in the case of a derivative in which chitosan is solubilized, it has excellent solubility in water. There is no need for pretreatment. When using such water-soluble chitosan, the use of the water-soluble chitosan has a characteristic that its use can be very differently applied according to the patient's physical property. It is protected in secrecy. Accordingly, the present invention relates to a manufacturing method for easily preparing a water-soluble chitosan and oligosaccharides for each grade controlled patient amount by using a general chitosan as the first raw material to prepare a water-soluble chitosan and applying an enzyme thereto. In general, the prepared water-soluble chitosan is a water-soluble testicular derivative, which has been used extremely limited in the existing cosmetic raw materials, etc., but the molecular weight-controlled water-soluble chitosan obtained by the present invention is considered to be excellent in reinforcing effects such as absorption of the living body. Therefore, the application range of medical, health supplements, and agriculture, which have been used to be limited to existing chitin and chitosan, is considered to be further expanded. In the present invention, the chitosan molecular chain cleavage effect using general chitosanase and cellulase when molecular weight is adjusted. The present invention relates to a process for producing a water-soluble chitosan at a low cost in industrial production by preparing a grade-soluble water-soluble chitosan with controlled patient volume.

As a known method for preparing water-soluble chitosan, a method of preparing NOCC (N, O-Carboxymethyl chitosaN) has been published in US Pat. No. 4,619,995 to Hayes et al. (1986). In other words, NOCC was prepared as general chitosan and used as a wound healing agent and a cholesterol lowering agent. In addition, these water-soluble chitosan was proved to be non-toxic as a result of clinical experiments using mice, and it is recognized that up to 5% of the food additive can be added. As a manufacturing method, monochloroacetic acid was treated under alkaline conditions, and this NOCC not only has excellent solubility in water, but also inhibits the growth of certain microorganisms, has a strong film forming ability, and is selected for oxygen and CO ₂ . Since it shows permeability, it was used to extend the shelf life by applying it to fruit surface coatings, etc. In addition, Biagini et al. (Proceedings from 4th international Conference on chitin & chitosan in held in The use of N-Carboxymethyl chitosan reported by Trondheim, Norway, August 22-24, pp 671-677, 1988 has been reported to be used as a porch to replace small vessels as a medical material. At this time, the deacetylation degree of the water-soluble chitosan prepared was 58% of average molecular weight 465,000, which was a polymer property, and there were reports used in shampoos, cosmetics, and mouthwashes, but the above-mentioned derivatives of the water-soluble chitosan were the first raw materials. Although the physical properties of chitosan are important and the physical properties of water-soluble chitosan are changed accordingly, the overall prerequisites are important. However, the previously published data are the results of insoluble matters due to the analysis of intrinsic properties and substitution of NCC and NOCC. Is not mentioned at all, it is limited to manufacturing to retain the characteristics of the polymer as a whole, and insoluble components of the final product also appears to be 1% to about 7%, the use of which has the trouble of removing insoluble matters, the limitation of applicability As shown in the present invention, in order to solve this problem, N, o-Carboxymethy l Chitosan was newly prepared to be insoluble by modifying the method of haze, and the manufacturing method according to the physical properties of the deacetylation degree and molecular weight of chitosan was additionally established. In addition, by using the enzyme, rather than using a chemical method using acid and the like to adjust the patient dose and at the same time to prepare a graded water-soluble chitosan and final soluble chitosan oligosaccharides.

An object of the present invention is to provide a method for preparing high-purity crystalline NOCC and a molecular weight control method of high-purity N, O-Carboxymethyl chitosan (NOCC), which is a water-soluble chitosan, in order to solve the above-mentioned problems.

1 is a photograph showing the process of forming a water-soluble chitosan oligosaccharide by thin layer chromatography formed after the enzyme treatment by time.

2a to 2d are GPC molecular weight distribution charts showing the molecular weights of the polymer water-soluble chitosan decreased over time after the enzyme treatment.

After dispersing chitosan in isopropanol and warming it to about 30 ° C, NaOH aqueous solution was added dropwise, and after stirring, monochloroacetic acid dissolved in isopropyl alcohol was added thereto, and then the white powder in this solution became transparent and then at room temperature. After stirring for a certain time, the pH was adjusted to 7, and then the residue was filtered and washed with a mixture of methanol and water, dissolved in methanol, and then stirred, washed with methanol, filtered and dried to produce a high-purity crystalline NOCC and an aqueous solution of NOCC. After warming up the cellulase or chitosanase, and after enzymatic digestion, the enzyme activity is removed and then adjusted to pH 6-7, and then the filtrate is concentrated and dried.

If it describes in detail the embodiment as follows.

The present invention is illustrated by the following examples, the viscosity of the molecular weight in the entire example was measured at 107rpm using a Brookfield viscometer (USA), and the chitosan solution used in the measurement in a 1% acetic acid solution to 700ml amount 0.5% (W / W) Chitosan was completely dissolved and bubbles were removed, and then measured at 25 ° C., and the measurement of water-soluble chitosan was 0.5% (W / W) in tertiary deionized water to make 600 ml of the powdery aqueous chitosan powder solution. W) Chitosan was completely dissolved, and then the bubble was removed, and the measurement spindle was measured using the 1-4 times. The deacetylation degree for confirming the purity of chitosan used for the first time was measured according to the colloid titration method by applying the Terayama method (J. Polym. Sci., 8. 243, 1952). In order to prepare the water-soluble chitosan and to test the solubility, the mixture was stirred for 1 hour while slowly adding 5 g of the water-soluble chitosan powder to 95 ml of tertiary deionized water, and then filtered using a standard filter paper to measure the amount remaining. The amounts of the chitosans were determined by pH 1, respectively.

For example, it will be described in detail as follows.

<Production of Water-soluble Chitosan>

Comparative Example 1

In order to prepare a water-soluble chitosan to be used in the present invention, the deacetylation degree of chitosan was 30%, and the water-soluble chitosan was prepared by the above-described haze method with chitosan having a molecular weight (viscosity) of 1,100 cps. That is, 200 ml of isopropanol was added to 20 g of chitosan and heated to 25 ° C., while stirring the resultant slurry, 50.4 ml of 30% (w / w) NaOH aqueous solution was added to the slurry over 4 minutes at 6 minute intervals. After the addition, the resulting alkaline slurry was stirred for 45 minutes, and then 24 g of monochloroacetic acid was added five times at intervals of 5 minutes over 25 minutes, heated to 60 ° C., reacted for 3 hours, and then 17 ml. Of cold deionized water was then cooled and the pH was adjusted to 7 with glacial acetic acid. The reaction product was filtered and washed with 300 ml of alcohol mixed with methanol and water at 70:30, then washed with 300 ml of anhydrous methanol and dried at 60 ° C. to obtain NOCC.

Comparative Example 2

In Comparative Example 1, the deacetylation degree of chitosan was carried out in the same manner as in Comparative Example 1 except that 46% instead of 30% and the viscosity was 1,210 cps instead of 1,100 cps.

Comparative Example 3

In Comparative Example 1, the deacetylation degree of chitosan was carried out in the same manner as in Comparative Example 1 except that 54% instead of 30% and the viscosity was 1.140 cps instead of 1,100 cps.

[Comparative Example 4]

In Comparative Example 1, the deacetylation degree of chitosan was carried out in the same manner as in Comparative Example 1 except that 65% instead of 30% and 1,320 cps instead of 1,100 cps were used.

[Comparative Example 5]

In Comparative Example 1, the deacetylation degree of chitosan was carried out in the same manner as in Comparative Example 1 except that 74% instead of 30% and the viscosity was 1.450 cps instead of 1,100 cps.

Comparative Example 6

In Comparative Example 1, the deacetylation degree of chitosan was performed in the same manner as in Comparative Example 1 except that 86% instead of 30% and a viscosity of 1,230 cps instead of 1,100 cps were used.

Comparative Example 7

In Comparative Example 1, the deacetylation degree of chitosan was performed in the same manner as in Comparative Example 1 except that 99% instead of 30% and a viscosity of 1,050 cps instead of 1,100 cps were used. In the present invention, the results of Comparative Examples 1 to 7 are shown in Table 1.

Example 1

150 ml of isopropanol was added to 10 g of chitosan having a deacetylation degree of 30% and a molecular weight (viscosity) of 1,100 cps, followed by sufficient dispersion for 1 hour, followed by heating to 30 ° C., followed by a 30% (w / v) NaOH solution. 25.2 ml was slowly added dropwise over 2 hours. After the completion of the NaOH solution dropping, the mixture was sufficiently stirred for at least 12 hours, and then a solution of 12 g of monochloroacetic acid dissolved in 50 ml of isopropyl alcohol was added uniformly in 2 hours. The mixture was dropped to room temperature and stirred and reacted for 12 to 24 hours. Subsequently, glacial acetic acid was added to adjust the pH to 7, and the reaction product was filtered, which was then sufficiently washed with a mixture of methanol and water at 80:20 upon filtration, and the intermediate was added to the reactor again in an 80% (v / v) methanol solution. While stirring for 1 to 3 hours, the pH was additionally adjusted to 7, and the resultant was filtered and washed with 300 ml of anhydrous methanol, followed by filtration to obtain the NOCC in which the first reaction proceeded. After time drying, high purity crystalline NOCC without insoluble content was prepared.

Example 2

In Example 1, it carried out similarly to Example 1 except having used the deacetylation degree of chitosan 46% instead of 30%, and a viscosity of 1,210 cps.

Example 3

In Example 1, it carried out similarly to Example 1 except having used the deacetylation degree of chitosan 54% and viscosity of 1,140cps instead of 30%.

Example 4

In Example 1, it carried out similarly to Example 1 except having used the deacetylation degree of chitosan 65% instead of 30%, and the viscosity of 1,320 cps.

Example 5

In Example 1, it carried out similarly to Example 1 except having used the deacetylation degree of chitosan 74% instead of 30%, and having a viscosity of 1,450 cps.

Example 6

In Example 1, it carried out similarly to Example 1 except having used the deacetylation degree of chitosan 86% instead of 30%, and having a viscosity of 1,230 cps.

Example 7

In Example 1, it carried out similarly to Example 1 except having used the deacetylation degree of chitosan 99% instead of 30%, and having a viscosity of 1,050 cps. In the present invention, the results of Examples 1 to 7 are shown in Table 2.

Example 8

In this example, cellulase (trade name: ACE, Antarctic Amalgamated Resoures Limited.U.S.A.) Was used, and the enzyme had a titer of 24,733 units / g. 2 L of tertiary deionized water was added to a 3 L beaker and stirred at 150 rpm. Then, 20 g of NOCC prepared in Example 7 was slowly dissolved to complete dissolution. Then, 0.25 L of each of 5 0.5 L three-necked round flasks was added. Then, the temperature was warmed differently in the range of 40 to 60 ° C., and then 1.5 mL or 2.5 mL or 5 mL or 10 mL or 20 mL of cellulase was added thereto, followed by stirring at 150 rpm for 1 hour, 2 hours, 3 hours, 4 hours, After 5 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, and 24 hours, respectively, the mixture was stirred at 80 ° C. for 30 minutes to remove enzyme activity, followed by 5% aqueous solution of NaOH (W / W). The pH was adjusted to 6-7, and after standing for 12 hours, the precipitated enzyme was centrifuged (15,000 rpm, 15 minutes) to completely remove the precipitate, and then the filtrate was concentrated and lyophilized to prepare a powdered molecular weight of chitosan. It was. The results of Example 8 are shown in Table 3.

Example 9

A chitosan degrading enzyme produced in Example 8 by chitosanase instead of cellulase enzyme, produced by Bacillus pumilus BN-262 with a purity of 3.000 to 20,000 units / g. The enzyme of 31,000 (SDS-PAGE method) was used, and the amount of chitosanase was added in the same manner as in Example 8 except that 0.011 g or 0.022 g or 0.044 g or 0.066 g of chitosanase was added to 1 g of water-soluble chitosan. Is shown in Table 4.

Example 10

Example 9 was carried out in the same manner as in Example 9, except that the water-soluble chitosan prepared in Example 3, the results are shown in Table 5.

High purity crystalline NOCC can be produced, and low molecular weight and oligomer can be obtained with NOCC as an enzyme.

Claims (5)

After dispersing chitosan in isopropanol and warming it to about 30 ° C, NaOH aqueous solution was added dropwise, and after stirring, monochloroacetic acid dissolved in isopropyl alcohol was added, and the white powder in this solution became transparent and then room temperature. After stirring for a certain period of time to adjust the pH, and after filtering the residue was washed with a mixture of methanol and water and then reacted with methanol, and stirred and washed with methanol, filtered and dried to produce a high purity crystalline NOCC. Method of controlling the molecular weight of water-soluble chitosan using an enzyme, characterized in that by heating the NOCC aqueous solution, cellulase or chitosanase is added thereto to remove enzymatic activity after enzymatic digestion, and then adjusting the pH to 6-7. The method for controlling molecular weight of water-soluble chitosan using an enzyme according to claim 2, wherein the aqueous NOCC solution is 40 to 60 ° C. The method of controlling molecular weight of water-soluble chitosan using an enzyme according to claim 2, wherein the degree of deacetylation of the water-soluble chitosan is 30% or more. The method for controlling molecular weight of water-soluble chitosan using an enzyme according to claim 2, wherein the water-soluble chitosan is enzymatically decomposed for 1 to 6 hours. ※ Note: The disclosure is based on the initial application.