KR20030000999A - A continuous oxidation process for the regio-selective oxidation of primary alcohol groups in chitin and chitosan - Google Patents

Abstract

PURPOSE: A method for oxidizing chitin or chitosan is provided, to oxidize only the primary alcohol groups within chitin or chitosan selectively to improve the water-solubility of chitin and chitosan with maintaining the polymer structure of chitin and chitosan. CONSTITUTION: The method comprises the steps of reacting chitin or chitosan with TEMPO (2,2,6,6-tetramethyl-1-piperidine oxoammonium), sodium bromide and sodium hypochlorite at a temperature of 0-50 deg.C and at a pressure of 1 atm to oxidize only the primary alcohol groups within chitin or chitosan into the carboxyl groups; adding chitin or chitosan and sodium hypochlorite by the equal mol ratio and to carry out the second oxidation; and repeating the previous oxidation process for 20-25 times continuously. Preferably the oxidation process is carried out at pH 9-12.

Description

A continuous oxidation process for the regio-selective oxidation of primary alcohol groups in chitin and chitosan}

The present invention relates to a process for the continuous oxidation of chitin or chitosan to selectively oxidize only primary alcohol groups present in chitin or chitosan.

Chitin and chitosan are present in shells of crustaceans such as shrimp and crabs, and in the exoskeleton of arthropods such as the shells of beetles, organs of mollusks, fungal yeasts and mushrooms. Exists in form.

Chitin is a natural polysaccharide with a molecular weight of 1 million or more and a structure similar to that of plant fiber, and is a natural high molecular compound containing 5,000 or more acetylglucosamines, and is an animal dietary fiber that is not digested or absorbed. Therefore, like fiber, there are no calories, and in addition, it has the property of adsorbing fat or harmful metals.

Chitosan (Chitosan) is a deacetylated chitin, soluble in weak acids, less molecular weight than chitin is dissolved in the intestinal digestive juice when drinking it is easy to absorb and use in the body. There is more chitin in nature, and generally speaking chitosan is not pure chitosan, but contains 10-30% chitin, which is called chitin chitosan because it has both the properties of chitin and chitosan.

Chitin and chitosan are natural polymers that have pharmacological effects such as antacid, cholesterol lowering, and tumor suppression. They are used as medical materials for drug delivery systems, blood coagulants, artificial skin, etc. Has been reported to enhance function (Tokura Seiichi, Fragrance Journal, May, 1995). In addition, studies on the inhibitory effect of the hydrolyzate of chitosan and its phosphates on phytopathogenic fungi have been progressing rapidly (J. Biomed. Mat. Res., 19, 413, 1985; J. Appl. Poly. Sci. , 24, 1587, 1979).

However, these chitin and chitosan materials have unique properties depending on their source, and in general, they have disadvantages such as being insoluble in cold water and insoluble in colloidal or water at pH 6.5 or higher. (Carbohydr. Res., 137, 205, 1984; J. Appl. Poly. Sci., 24, 1558, 1985; J. Appl. Poly. Sci., 28, 1909, 1986).

In the past, attempts were made to overcome the reduction of insolubility in water and the solubility in water at low temperatures by modifying natural chitin under extreme conditions such as hydrochloric acid or sulfuric acid, inorganic catalysts, high temperature and high pressure. Under these conditions, grades of the final oxides were degraded with various side reactions, and separation, purification, and recovery processes were required.

Therefore, Kendra et al. Performed a reaction to increase water solubility by hydrolyzing chitin by treating chitin and glucan degrading enzymes (Kendra, DF, D. Christian, and LA Hadwiger: Chitosan oligomers from Fusarium solani / pea interactions, chitinase / b-glucanase digestion of sporelings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance, Physiol.Mol. Plant Path., 35, 215, 1989). However, this method decomposes between the N-acetylglucosamine constituents of chitin, so when applied to polysaccharides, the characteristics of the polymer are lost by exposing the inherent cyclic structure of the basic unit saccharide constituting the polysaccharides. It has a decisive disadvantage.

In order to improve the water solubility of chitin and chitosan, Iva also prepared chitosan, which is a deacetylating substance of chitin, or made various derivatives of chitosan (Aiba, S .: Preparation of N-acetylchitooligosaccharides from lysozymic hydrolysates of partially). N-acetylated chitosans, Carbohydr.Res., 261, 297, 1994). However, this method also has a disadvantage in that the water solubility is increased because the decomposition of the monomer or oligomer must be preceded, but the intrinsic functionality of chitin is rapidly reduced.

Therefore, there is an urgent need to establish a method of preventing water loss while increasing the water solubility while maintaining the polymer structure of chitin or chitosan.

In order to solve the above problems, the present invention selectively reacts chito or chitosan under conditions of room temperature and atmospheric pressure using tempo, sodium bromide and sodium hypochlorite, thereby selectively selecting only primary alcohol groups among functional groups of chitin or chitosan constituents. It is an object to provide a continuous oxidation method of chitin or chitosan for oxidation.

1 is a ¹³ C-NMR diagram of chitin oxide prepared according to Example 1 of the present invention.

2 is a ¹³ C-NMR diagram of chitosan oxide prepared by Example 2 of the present invention.

The present invention relates to a process for the continuous oxidation of chitin or chitosan to selectively oxidize only primary alcohol groups in chitin or chitosan.

The continuous oxidation method of the present invention uses a tempo, sodium bromide and sodium hypochlorite to oxidatively convert only the primary alcohol group among the functional groups of chitin or chitosan constituents at 0 to 50 ° C. and 1 atm to convert to a carboxyl group. After the completion of once, chitin or chitosan and sodium hypochlorite are added simultaneously at the same molar ratio to perform the second oxidation reaction, and the oxidation reaction is characterized in that it is carried out continuously 20 to 25 times.

Hereinafter, the continuous oxidation method of the present invention will be described in more detail.

Chitin or chitosan commonly used at room temperature is sufficiently dispersed in water at 0 ~ 50 ℃, then tempo and sodium bromide are added to disperse and disperse uniformly. After adding a small amount of sodium hypochlorite, the pH of the reaction solution is 9 ~ 12. By selectively oxidizing chitin or chitosan. As the oxidation reaction proceeds, the pH of the reaction solution is lowered by the carboxylic acid formed.Only the primary alcohol group in chitin or chitosan is corrected by adding an appropriate amount of 0.5N-NaOH solution to maintain the pH at 10.8. Is selectively oxidized.

After the first oxidation reaction is completed, only the chitin or chitosan and sodium hypochlorite are added simultaneously in the same molar ratio to react the second oxidation reaction in the same manner as in the first time. From the subsequent oxidation method, tempo and sodium bromide are not added, but only chitin or chitosan and sodium hypochlorite are added in the same molar ratio.

As in the above method, the selective oxidation reaction is continuously performed 20 to 25 times so that the concentration of the final oxidation reactant reaches 5.5 to 21% (w / v). The reaction is terminated by addition of 4N-HCl solution to obtain an aqueous chitin or chitosan solution in which only the primary alcohol group is selectively oxidized.

2 to 3 times (v / v) isopropanol or a nonpolar organic solvent having a polarity index of about 3.5 to 5.1 is added to the chitin oxide or chitosan aqueous solution obtained above, and the tempo, sodium bromide, sodium hypochlorite, etc. The mixture is dissolved in the supernatant, and chitin oxide or chitosan oxide is precipitated. By centrifuging the aqueous solution to which isopropanol was added and taking out only the precipitated part, only the primary alcohol group selectively isolates and purifies oxidized chitin or chitosan.

Separated and purified chitin oxide or chitosan liquid was dried in a vacuum drying oven at 50 ° C. for 1 day, and then powdered to prepare a final product.

Hereinafter, examples of the present invention will be described in more detail, but the present invention is not limited to these examples.

Example 1 Continuous Oxidation of Chitin

510 mg of chitin was sufficiently dispersed in a 50 ml of water at 25 ° C. using a homogenizer for 1-2 minutes to prepare a suspension, and then dissolved by adding 3.9 mg of tempo and 125 mg of sodium bromide.

After adding 7.88 ml of a 25 ° C. aqueous solution in which sodium hypochlorite was dissolved at a concentration of 10% (w / v) to the prepared suspension, 5.0 ml of 0.5N-NaOH solution was automatically added to maintain the pH of the suspension at 10.8. The addition was carried out regularly.

A selective oxidation reaction was started a second time by newly adding 510 mg of chitin and 1.56 ml of 10% (w / v) hypochlorous acid solution in the selectively oxidized chitin aqueous solution, and in order to maintain the pH of the reaction solution at 10.8. 5.0 ml of N-NaOH solution was added to terminate the second selective oxidation reaction.

The above oxidation reaction was performed up to the 20th time, and the continuous selective oxidation of chitin was terminated by adding 4N-HCl solution to change the pH of the final reaction solution to 7.0.

Two times isopropanol was added to the optionally oxidized chitin solution, followed by centrifugation at 6,000 xg for 5 minutes to discard the supernatant in which tempo, sodium bromide and sodium hypochlorite were dissolved, and containing only optionally oxidized chitin. Only precipitate was taken.

The oxidized chitin liquid was selectively dried in a 50 ° C. vacuum dryer for 24 hours, and then powdered to give 9.9 g of a powder product. The final product yield was 90.8% (w / w).

¹³ C-NMR confirmed that the peak at which the primary alcohol group of the chitin oxide of the present invention was converted to the carboxyl group was found at 175 ppm, and the peak at which the secondary alcohol and other reactors were converted to the ketone group was not found at 215 ppm. (Fig. 1).

Therefore, it can be seen that the continuous oxidation method of the present invention selectively converts only the primary alcohol group in the chitin to the carboxyl group.

Example 2 Continuous Oxidation of Chitosan

810 mg of chitosan was sufficiently dispersed in a 100 ml of 5 ° C. water for 1 to 2 minutes using a homogenizer to prepare a suspension, and then dissolved by adding 7.8 mg of tempo and 250 mg of sodium bromide.

After adding 15.76 ml of a 5 ° C. aqueous solution in which sodium hypochlorite was dissolved at a concentration of 10% (w / v) to the prepared suspension, 5.0 ml of 0.5N-NaOH solution was automatically added to maintain the pH of the suspension at 10.8. The addition was carried out regularly.

When 10.0 ml of 0.5N-NaOH solution was added, all primary alcohol groups in chitosan were converted into carboxyl groups.

The selective oxidation reaction was started a second time by newly adding 810 mg of chitosan and 3.12 ml of 10% (w / v) hypochlorous acid solution in the selectively oxidized aqueous chitosan solution, and in order to maintain the pH of the reaction solution at 10.8, 0.5 10.0 ml of N-NaOH solution was added to terminate the second selective oxidation reaction.

The above oxidation reaction was carried out up to the 25th time, and the continuous selective oxidation of chitosan was terminated by adding 4N-HCl solution to change the pH of the final reaction solution to 7.0.

Two times isopropanol was added to the selectively oxidized chitosan solution, followed by centrifugation at 6,000 xg for 5 minutes to discard the supernatant in which tempo, sodium bromide, and sodium hypochlorite were dissolved, and only optionally oxidized chitosan was contained. Only precipitate was taken.

The oxidized chitosan liquid was selectively dried in a 50 ° C. vacuum dryer for 24 hours, and then powdered to give 20.0 g of a powder product. The final product yield was 90.9% (w / w).

¹³ C-NMR confirmed that the peak at which the primary alcohol group of the oxidized chitosan of the present invention was converted to the carboxyl group was found at 175 ppm, and the peak at which the secondary alcohol and other reactors were converted to the ketone group was not found at 215 ppm. (Fig. 2).

Example 3 Test of Solubility in Water

In order to confirm the degree of increase in water solubility of the final product obtained in Examples 1 and 2, the solubility of water, optionally oxidized chitin or chitosan and non-oxidized chitin or chitosan, was measured at room temperature (25 ° C).

After preparing 5 ml of chitin oxide or chitosan dispersion liquid (600 mg / ml) obtained in Examples 1 and 2, the mixture was stirred at 25 ° C for 24 hours. The supersaturated chitin oxide or chitosan dispersion was centrifuged at 6,000 xg for 15 minutes, then 2 ml of the supernatant was added and 6 ml of ethanol was added. The precipitate was recovered by centrifugation at 10,000 xg for 15 minutes and dried in a vacuum oven at 60 ℃.

Table 1 shows the results of the solubility of the unoxidized chitin or chitosan and the water of chitin or chitosan selectively oxidized by the continuous oxidation method of the present invention.

Solubility in water of the original product without oxidation (%, w / v) Solubility in water (%, w / v) of products selectively oxidized by the continuous oxidation method of the present invention Chitin 0.0 29.7 Chitosan 7.9 33.4

As can be seen from Table 1, the water-soluble chitin or chitosan produced by the continuous oxidation method of the present invention had an increase in water solubility of 29.7% (w / v) and 33.4% (w / v), respectively, compared to the unoxidized chitin or chitosan. .

Example 4 Stability Experiment

In order to test the stability of the final product obtained in Examples 1 and 2, Salmonella typimurium TA 98 and TA 100 bacteria obtained from the Korean spawn association (KCTC) to the Ames test (Ames test) Mutagenicity test was performed.

The Ames test was performed by administering 25, 50, 75, or 100 mg of the oxidized chitin or chitosan concentrations obtained in Examples 1 to 2 in a petri-dish having a diameter of 10 cm.

The mutagenicity test results are shown in Table 2.

Strains Used in the Ames Test Selectively oxidized chitin Optionally oxidized chitosan Concentration (mg / plate) Mutagenicity (positive, negative) Concentration (mg / plate) Mutagenicity (positive, negative) Salmonella typhimurium TA 98 25 voice 25 voice Salmonella typhimurium TA 98 50 voice 50 voice Salmonella typhimurium TA 98 75 voice 75 voice Salmonella typhimurium TA 98 100 voice 100 voice Salmonella typhimurium TA 100 25 voice 25 voice Salmonella typhimurium TA 100 50 voice 50 voice Salmonella typhimurium TA 100 75 voice 75 voice Salmonella typhimurium TA 100 100 voice 100 voice

As can be seen in Table 2, both mutagenicity and chitosan oxidation showed negative mutagenicity. Therefore, it can be seen that the chitin oxide or chitosan powder product prepared by the continuous oxidation method of the present invention is a safe product which does not cause mutation.

Example 5 Antimicrobial Activity Test

The final product obtained in Examples 1 to 2 was tested for antimicrobial activity by 37 ° C. constant temperature shaking culture. Table 3 shows the results after contacting the test bacteria and various samples for 3 hours.

Antimicrobial Test Strains Optionally Oxidized Chitin of the Invention Optionally Oxidized Chitosan of the Invention Addition amount (ppm) Antimicrobial Activity (%) Addition amount (ppm) Antimicrobial Activity (%) Staphylococcus aureus (KCCM 11335) 50 97.8 ± 2.2 50 96.3 ± 1.2 Eschrichia coli (KCCM 12119, Gram-positive bacteria) 50 94.2 ± 4.7 50 90.3 ± 3.4

As can be seen from Table 3, chitin or chitosan selectively oxidized by the continuous oxidation method of the present invention showed a high antimicrobial activity of more than 90% in all test groups.

The chitin oxide or chitosan oxide prepared by the continuous oxidation method of the present invention is not only obtained in high yield, but also has high solubility in water, does not cause mutation, and exhibits high antibacterial activity, so that it is functional food, pharmaceutical use, It can be effectively used in cosmetics and agricultural antibacterial agents.

Claims (3)

In the oxidation method of chitin or chitosan which selectively oxidizes only the primary alcohol group in chitin or chitosan, using tempo, sodium bromide and sodium hypochlorite under normal temperature (0 to 50 ° C) and normal pressure (1 atm) Method of continuous oxidation of chitin or chitosan after reacting with chitin or chitosan and then oxidizing continuously by adding only chitin or chitosan and sodium hypochlorite in the same molar ratio. The method for continuous oxidation of chitin or chitosan according to claim 1, wherein the step is carried out continuously 20 to 25 times. The method of continuous oxidation of chitin or chitosan according to claim 1, wherein the pH is 9-12.