KR100252704B1 - Method of making n,o-carboxy methyl chitosan with high purity crystallization and a method of controlling a molecular weight of water-soluble chitosan - Google Patents

Abstract

PURPOSE: A process for producing high purity crystalline NOCC is provided to solve existing limitations such as removal of insoluble portion and application by controlling molecular weight of NOCC and achieving high purity of the same. CONSTITUTION: The process comprises the following steps of adding NaOH solution to a chitosan dispersion in isopropanol at 30 deg.C; stirring the mixture then adding monochloro acetic acid solution in isopropyl alcohol; agitating the obtained solution at room temperature for desired time after it forms a clear solution; controlling it to pH 7; filtering and washing the residual with a blend of methanol and water; dissolving the washed solution in methanol under stirring; washing again it with methanol; then rinsing, filtering and drying the resultant solution. The NOCC solution is under the molecular weight controlling process by applying cellulase or chitosanase as a specific enzyme after warming the solution at 40-60 deg.C.

Description

Manufacturing method of high purity crystalline NOC and adjusting 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, its use can be applied very differently depending on its molecular weight, which is its physical property. Therefore, various studies on the physical property control method have been conducted at home and abroad. Is protected. Therefore, the present invention relates to a method for preparing a water-soluble chitosan by using general chitosan as an initial raw material and easily preparing a water-soluble chitosan and oligosaccharides of each grade having a molecular weight controlled by applying an enzyme thereto. In general, the prepared water-soluble chitosan is a water-soluble polymer derivative, and these have been used extremely limited in existing cosmetic raw materials, etc., but the molecular weight-controlled water-soluble chitosan obtained by the present invention is determined to be excellent in the reinforcing effect such as absorption of the living body, etc. It is thought that the application range of medical, health supplement food, and agriculture, etc., which have been limited to the existing chitin and chitosan, will be further expanded. In the present invention, the chitosan molecular chain cleavage effect using general chitosanase and cellulase is controlled when molecular weight is adjusted. The present invention relates to a process for preparing a water-soluble chitosan at a low cost in industrial production by preparing a grade-soluble water-soluble chitosan with controlled molecular weight.

As a known method for preparing water-soluble chitosan, a method for producing NO, N, O-Carboxymethyl chitosan (NCC) has been disclosed 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 is excellent in solubility in water, 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. In addition, N-Carboxymethyl reported by Biagini et al. (Proceedings from 4th international Conference on chitin & chitosan in held in Trondheim, Norway, August 22-24, pp671-677, 1988) for the preparation and presentation of other types of water soluble chitosan. It has been published that chitosan is used as a blood vessel to replace small blood 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 by modifying the method of haze so that there was no insoluble content, and additionally, a manufacturing method was established according to the deacetylation degree of chitosan and the properties of molecular weight difference. In addition, the molecular weight was controlled using enzymes, not chemical methods using acid lamps, to prepare graded water-soluble chitosan and final water-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 conventional problems.

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

2A to 2D are GPC molecular weight distribution analysis charts showing molecular weights that decrease with 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 the white powder in this solution became transparent, and then it was kept 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 the warming up, the cellulase or chitosanase was added thereto, after enzymatic digestion, the enzyme activity was removed, and then the pH was adjusted to 6-7, and the filtrate was concentrated and dried.

The embodiment will be described in detail as follows.

The present invention is illustrated by the following examples, the viscosity of the decrease in the molecular weight in the entire example was measured at 100rpm using a Brookfield viscometer (USA), the chitosan solution used in the measurement was 0.5ml in 1% acetic acid solution to 600ml amount % (W / W) Chitosan was completely dissolved and the bubble was removed, and then measured at 25 ℃. The measurement of water-soluble chitosan was 0.5% (W / W) in tertiary deionized water to make 600ml of the powdery water-soluble chitosan powder solution. The 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 aqueous phase chitosan powder to 95 ml of tertiary deionized water, and then filtered using a common filter paper to measure the amount of insoluble content. In addition, the pH value of each chitosan was administered by 1g graded water-soluble chitosan graded in 100ml deionized water, and after stirring, it was measured by pH meta.

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 warmed at 25 ° C., and 50.4 ml of a 30% (w / w) NaOH aqueous solution was added to the slurry over 20 minutes while stirring the resulting slurry six times at four minute intervals. After stirring, 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., and reacted for 3 hours. Cold deionized water was added, then 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, which was 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 for using 46% instead of 30% and 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 the viscosity was 1,320 cps instead of 1,100 cps.

[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. The mixture was sufficiently dispersed for 1 hour, and then heated to 30 ° C., and thus 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 12 hours or more, and then a solution of 12 g of monochloroacetic acid dissolved in 50 ml of isopropyl alcohol was added uniformly in 2 hours, and the white powder particles became completely transparent, and then the temperature was maintained at room temperature. The mixture was stirred for 12 to 24 hours to proceed with stirring and reaction. 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. After 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 having undergone the first reaction, and the whole process was repeated twice, followed by 2 hours at 50 ° C. After 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 having 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 1,140 cps 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 having a 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.

Molecular weight control using enzymes

Example 8

Cellulase (trade name: ACE, Antarctic Amalgamated Resoures Limited. USA.) Was used in this example, 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 from 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 stirred at 150 rpm for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, After reacting for 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, and then the pH was adjusted to 5% (W / W) NaOH aqueous solution. After adjusting to ˜7, and standing for 12 hours, the precipitated enzyme was centrifuged (15,000 rpm, 15 minutes) to completely remove the precipitate, and the filtrate was concentrated and lyophilized to prepare a chitosan having a molecular weight of powdery grade. The results of Example 8 are shown in Table 3.

Example 9

Chitosanase produced in Example 8 by chitosanase instead of cellulase enzyme, produced from Bacillus pumilus BN-262 with a purity of 3,000 to 20,000 units / g and having a molecular weight of about 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

A sample was prepared in Example 9, except that the water-soluble chitosan prepared in Example 3 was used as in Example 9. The results are shown in Table 5.

High purity crystalline NOCCs can be prepared, and low molecular weights and oligomers can be obtained with NOCC as enzymes.

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 thereto, and the white powder in this solution became transparent, and then it was kept at room temperature. After stirring for a certain time to adjust the pH 7, and after filtering the residue was washed with a mixture of methanol and water and dissolved in methanol, 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 enzyme, characterized in that by heating the NOCC aqueous solution, cellulase or chitosanase 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 for 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 decomposed for 1 to 6 hours.