KR970008132B1 - Method for manufacturing chitin and chitosan for biomedical medicine - Google Patents

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Description

Method for manufacturing chitin and chitosan for biomedical medicine

The present invention relates to a method for preparing chitin and chitosan, in particular, a method for preparing chitosan for biomedical medicine having high purity, high molecular weight, high deacetylation degree and high whiteness from the chitin by separating the chitin with high purity from the crab shell.

Chitin is the next most abundant natural polymer in the world after fibrin, a high value-added biopolymer that is commercially extracted from the crustaceans of crustaceans. Such chitin is insoluble in water and thus becomes a major obstacle to its utilization. Accordingly, by deacetylating chitosan to obtain chitosan, it is possible to convert chitosan to which water solubility is expressed in weakly acidic liquid, and such water-soluble chitosan is industrially used more useful than chitin.

Recently, the interest in the disposal of shellfish, aquatic wastes, has led to the development of various uses of chitin that can be extracted from large-scale fishing of crabs and shrimp. These chitin and chitosan were initially used as coagulants for recovering effective substances (proteins, etc.) in food plant wastewater, but recently, in the fields of biotechnology and cosmetics, such as food, medical, functional membranes, enzymes and immobilization carriers of enzymes and microorganisms. It is widely developed in all fields such as field, agriculture, chemical field and environment.

The chitin manufacturing method widely used up to now is pretreatment of chitin or crab shrimp, which is the main ingredient of chitin, and then finely chopped or broken to remove protein as a connective tissue, and it is immersed in aqueous hydrochloric acid to remove calcium carbonate to obtain a chitin of the following structural formula. It is.

The chitosan of the following structural formula can be obtained by removing the acetyl group by biological or chemical treatment in the chitin of the structural formula to generate free amino (-NH ₂ ).

As can be seen from the structural formula, chitosan is advantageous in terms of its chemical reactivity and chemical modification since one of the hydroxyl groups present in the fiber is substituted with a free amino group.

The general manufacturing process for chitin is as follows: crab or shrimp are ground and then ground to a size of 0.5 to 3 cm to increase production efficiency. Subsequently, decalcium carbonate reaction is performed at room temperature in about 5% hydrochloric acid solution and then immersed in 5% aqueous sodium hydroxide solution to remove proteins by heating at room temperature or high temperature for several hours. Finally reflux for several hours in 95% ethanol to obtain chitin.

As another known method, the shell is washed, dried in a vacuum oven at 50 ° C. and then ground to immerse in 10% NaOH solution. The deproteinized chitin is washed with water and washed several times with a solvent. The product obtained is dried under reduced pressure and then kept in an aqueous hydrochloric acid solution at -20 ° C for 4 hours and then washed. Dipping and washing in hydrochloric acid are repeated (Whistler & BeMiller method).

Another method is to wash the shell and dry it at 100 ° C., then treat at room temperature for 5 hours with 2N hydrochloric acid solution, wash and dry to grind to fine powder. The powder is extracted with 2N hydrochloric acid solution under stirring at 0 ° C., the collected material is washed and then extracted with 1N NaOH aqueous solution at 100 ° C. The NaOH aqueous solution treatment is repeated several times (Hackman method).

Chitosan can be obtained by deacetylating insoluble chitin, which is typically treated for 5 to 20 hours using a solution of 30% to 50% sodium hydroxide about 20 times the amount of chitin.

In the conventional chitin separation methods described above, relatively excessive reaction conditions are introduced to reduce the content of protein and CaCO _{3. In} the case of the decarbonated reaction, the aqueous solution of hydrochloric acid is performed at room temperature or at a temperature of about 10 ° C. or more, thereby It is easy to remove but it is difficult to obtain a high molecular weight chitin due to the increased molecular chain cleavage, and also has a disadvantage that it takes a long time more than 12 hours in several steps in the NaOH solution treatment for protein removal. The chitin thus obtained is left in the air to discolor over time and has a high content of impurities.

Therefore, the present inventors have continued to study how to solve the above-mentioned problems and obtain high purity and high molecular weight chitin and chitosan suitable for the pharmaceutical and food applications that are required, and the discoloration of the obtained chitin is determined by the molecular chain. This is due to the destruction of, so that the treatment temperature is lowered during hydrochloric acid treatment to prevent the cleavage of the molecular chain, thereby obtaining chitin with high molecular weight and low calcium carbonate content, and inert gas is also injected to prevent the cleavage of the molecular chain during NaOH solution treatment. The present invention was completed by simplifying the steps and shortening the time by processing twice every three hours.

One object of the present invention is to provide a method for obtaining chitin with high purity, high molecular weight and high whiteness.

It is another object of the present invention to provide a method for producing high molecular weight and high deacetylation chitosan from chitin thus obtained.

That is, the present invention, the step of drying and storing the crab crust, the step of crushing the dried crab crust, holding the ground crab crust for 10 to 25 hours at a temperature of less than -10 to 10 ℃ in hydrochloric acid aqueous solution Is rapidly increased to maintain at a temperature of 10 to 20 ℃ for 2 to 8 hours, or in another step for 20 to 60 hours at a temperature of -10 to 8 ℃, hydrochloric acid treated crab shell continuously washing, Filtration, organic solvent treatment and drying to obtain crude chitin, immersing the obtained crude chitin in an aqueous NaOH solution for heat treatment, and washing the NaOH treated chitin with water, filtration, organic solvent treatment and drying. The present invention relates to a method for preparing chitin for clinical medicine.

The present invention is also added to the chitin obtained by the method as described above, NaOH aqueous solution and heat treatment at a temperature of 80 to 100 ℃ for 2 to 12 hours, followed by washing and filtration, the crude chitosan obtained by deionized water The present invention relates to a chitosan preparation comprising immersing in an aqueous solution of an organic solvent, followed by filtration and drying, and repeating the NaOH aqueous solution treatment and an organic solvent treatment process.

The present invention is described in more detail as follows.

In the present invention, hot water treatment is usually performed to effectively separate the meat portion from the shell before drying the shell, and is generally carried out by immersing the shell in hot water at a temperature of 40 to 50 ° C. for 30 minutes and 1 hour.

The collected shellfish were washed with water, dried with a dehydrator, and dried at room temperature in an organic solvent such as ethanol, methanol, acetone, tetrahydrofuran (THF), dioxane or methyl ethyl ketone (MEK) for a period of time, for example, 30 minutes to 1 minute. After soaking for a period of time, dried in the shade (usually water content of 8 to 12%), stored in a cool state, and used for the next step. Poor storage results in decay during storage, which leads to molecular weight degradation and alteration, making it difficult to obtain high quality chitin.

It is preferable that the shelled shell is ground for more effective hydrochloric acid treatment. In general, it is preferable to grind and react to a size of 0.5 to 3mm or a size of 200 to 300 mesh (mesh) by a conventional mill.

Subsequently, in order to remove calcite (CaCO ₃ ) from the shell, the aqueous hydrochloric acid solution was adjusted to a temperature of less than -10 to 10 ° C, preferably -5 to 5 ° C, followed by stirring with a mechanical stirrer (usually water content of 8 to 12%) and treated at that temperature for 10 to 25 hours, preferably 15 to 20 hours, and then the temperature of the aqueous hydrochloric acid solution is rapidly raised to 10 to 20 ° C, preferably 12 to 18 ° C, at that temperature. By holding for 2 to 8 hours, preferably 3 to 5 hours, the hydrochloric acid treatment step of the red crab shell is carried out. At this time, the aqueous hydrochloric acid solution is suitable for the range of 0.8N to 3N, the mechanical stirrer is stirred at a speed of 50 to 200rpm, usually 100rpm, it is preferable to exchange once or twice with fresh aqueous hydrochloric acid solution at a suitable time during the treatment process. Also, if the stirring speed is more than 200rpm hydrochloric acid treatment efficiency is lowered, so be careful.

Treatment of aqueous hydrochloric acid in the above method can be carried out by maintaining in one step, 20 to 60 hours, preferably 30 to 50 hours at a temperature of -10 to 8 ℃, preferably -5 to 5 ℃. .

The carapace decalcified by hydrochloric acid treatment then removes the remaining hydrochloric acid component by washing with water. Instead of washing with water, NaOH aqueous solution may be added dropwise to adjust the pH of the crab shell immersion aqueous solution to pH 7-9, and then left in deionized water for a certain time, for example, 1-6 hours. After filtration, it is washed once or twice with an organic solvent such as ethanol, methanol, acetone, THF, dioxane, MEK and the like and then dried. The solvent washing is intended to remove the red color of the crab shell and to increase the whiteness of the chitin. Therefore, the solvent acts as a decolorizing agent, and thus, in the present invention, there is an advantage in that a separate oxidizing agent or reducing agent is not used as the decolorizing agent.

The crude chitin thus obtained enters a protein removal process, which is carried out by immersing the crude chitin obtained in an aqueous NaOH solution and heating. The concentration of the NaOH aqueous solution is suitable 2 to 10% by weight, the reaction temperature is in the range of 80 to 100 ℃, the heating time is generally 2 to 4 hours. This process is preferably carried out while continuously injecting an inert gas such as nitrogen, argon, helium or neon gas. This prevents the cleavage of the molecular chain of chitin.

Subsequently, the crude chitin is washed with deionized water until the pH of the washing solution is neutral, or the hydrochloric acid solution is added dropwise to adjust the pH of the crude chitin immersion aqueous solution to 7 to 8, followed by a predetermined time in deionized water, for example, 1 to 5 Immerse in time. Then, the crude chitin is filtered and then organic solvents such as ethanol, methanol, acetone, THF, dioxane, MEK and the like are washed once or twice with a solvent as in the case of hydrochloric acid treatment and then dried.

In order to make the primary NaOH treated crude chitin more high purity and promote decolorization, it is preferable to repeat the above-described NaOH treatment process and washing process two or more times. The final NaOH treated and washed chitin can be left in deionized water for 2-5 days in order to increase the whiteness of the chitin, filtered and dried in vacuo to obtain a biomedical chitin.

From the chitin for bioclinical medicine obtained according to the present invention, chitosan having high molecular weight and high deacetylation degree can be obtained as follows.

First, for example, 30-50% NaOH aqueous solution is added to the obtained chitin, heated at a temperature of 80-100 ° C. for 2-12 hours, washed with deionized water and filtered until the pH of the washings is neutral. At this time, it is preferable to carry out while continuously injecting an inert gas, for example nitrogen, argon, helium or neon gas. In addition, there is a fear that the molecular chain is broken when the temperature and time when the NaOH aqueous solution treatment is out of the above range.

Subsequently, the crude chitosan treated with aqueous NaOH solution was immersed in deionized water, followed by filtration to increase the whiteness of the chitosan and to remove and remove impurities, such as acetone, ethanol, methanol, isopropanol, dioxane, THF, MEK, and the like. It was immersed in an aqueous solution of a solvent of (usually 5 to 30% by weight), then filtered and dried.

Subsequently, the dried crude chitosan is treated with an aqueous NaOH solution and an aqueous organic solvent solution to obtain a final chitosan having a high degree of deacetylation.

In order to further increase the degree of deacetylation of chitosan, the above-described primary and secondary aqueous NaOH solutions and washing processes may be repeated two or more times.

As described above, in the present invention, the cleavage of the molecular chain is largely prevented by mild HCl treatment conditions, and the organic solvent after HCl treatment and NaOH treatment is used to increase the whiteness of the chitin without using a separate bleaching agent. An effect can be obtained, and high molecular weight chitin can be obtained by injecting an inert gas during NaOH treatment to prevent molecular chain breakage, and can also obtain chitosan with high acetylation degree by repeating NaOH aqueous solution treatment from such chitin.

The invention is illustrated by the following examples, which are not intended to limit the invention.

In the examples, the residual CaCO ₃ content in the chitin was measured as ignition loss after burning the chitin at 800 ° C. for 1 hour, and the protein content was determined by using a protein analysis kit from Bio-Rad. It was measured by UV / VIS spectrometer of Hewlett-Packard, USA according to the Bradford method. In addition, the metal content was measured by using ICP-AES of Seiko, Japan, and AA spectrometer of Perkin-Elmer, USA. Deacetylation degree is described in the paper [J. Appl. Polym. Sci. 28, 1909 (1983)] was measured according to the IR method (Mima method) published in. The whiteness over time was visually determined by visual observation after 30 days or more and divided into excellent (white) and poor (yellow). Viscosity was also measured as a 0.5% chitosan solution in 1% acetic acid solution at 60 rpm using a # 4 spindle on a Brookfield viscometer.

Example 1

300 g of crustacean (Chinoecetes opilio) of red crab was left for 30 minutes in a water bath at 40 to 50 ° C. and washed until the meat of the crab was not suspended. Water was removed using a dehydrator, immersed in an ethanol bath for 1 hour, and then water was removed again using a dehydrator, followed by drying in a shade for 10 days to obtain 200 g of dried red crab shells.

Subsequently, 200 g of dried red crab shell (water content 10%) was ground to a particle size of 0.5 to 3 mm of a grinder (Mot. WRB 90LB / 4P of Dietz-Motoren Gmbh & Co. KG), followed by mechanical It was placed in a reaction vessel equipped with a stirrer, and 5 liters of 1N aqueous hydrochloric acid solution at 0 ° C. was added thereto and reacted at that temperature for 20 hours while stirring at 50 rpm, and then the temperature was rapidly increased to 15 ° C. for 4 hours at that temperature. Reacted for a while.

Filtration of the treated red crab shells with hydrochloric acid was immersed in water, and the pH of the immersion liquid was adjusted to 7 to 8 with a 5 wt% NaOH aqueous solution, followed by filtration for 6 hours. The filtered crude chitin was washed twice with 3 L of acetone and then filtered and dried in a vacuum oven at 30 ° C. for 36 hours.

The dried crude chitin was immersed in 6 L of 5 wt% NaOH aqueous solution, heated to 90 ° C. for 3 hours while injecting nitrogen gas, and then the pH of the crude chitin immersed aqueous solution was adjusted to 7-8 by adding 2N hydrochloric acid aqueous solution and 6 L. It was immersed in deionized water for 6 hours and then filtered. The filtered material was washed twice with 3 L of acetone, filtered and dried in a vacuum oven at 30 ° C. for 12 hours.

For the chitin treated with the dried primary NaOH aqueous solution, the above-described NaOH aqueous solution treatment process was repeated. Chitin treated with a secondary NaOH aqueous solution was washed 5 to 10 times with deionized water, and then left in 6 L of deionized water for 3 days, followed by filtration and drying in a vacuum oven to finally obtain 50 g of biomedical chitin.

Example 2

In Example 1, the hydrochloric acid aqueous solution treatment process was carried out in the same manner as in Example 1 except that it was performed to maintain 48 hours at a temperature of 0 ℃.

Examples 3 and 4

The same procedure as in Example 1 was carried out except that the aqueous hydrochloric acid treatment conditions in Example 1 were 5 ° C., 24 hours, and −8 ° C. and 40 hours (2N hydrochloric acid) in one step.

Comparative Examples 1 and 2

This comparative example was tested for changes in temperature and time conditions during the hydrochloric acid aqueous solution treatment according to the present invention, the hydrochloric acid aqueous solution treatment conditions in Example 1 to 1 step, 15 ℃, 8 so as to deviate from the scope of the present invention, respectively It carried out similarly to Example 1 except having set to time, 10 degreeC, and 24 hours.

Comparative Example 3

200 g of dried red crab shells were ground to a particle size of 0.5 to 3 mm, placed in a reaction vessel equipped with a mechanical stirrer, and then 5 L of 2N hydrochloric acid aqueous solution was added at room temperature and reacted for 5 hours while stirring. The temperature of the hydrochloric acid solution was lowered to 0 ° C. and reacted at that temperature for 48 hours. After washing and filtration the filtration material was washed with water and treated with 1N aqueous NaOH solution at 100 ° C. for 12 hours. Washing and drying gave 80 g of chitin.

Important experimental conditions and the properties of the obtained chitin used in Examples 1 to 4 and Comparative Examples 1 to 3 are shown in Table 1 below.

TABLE 1

* In the present invention, only K, Ca, Mg, Fe, Ba, Zn, but in the comparison method also included other metals and heavy metals.

As can be seen from Table 1, according to the method of the present invention, the obtained chitin has high purity, high molecular weight and excellent whiteness.

Example 5

45 g of chitin obtained in Example 1 was placed in a 4 L three-necked flask, and 3 L of 40 wt% NaOH aqueous solution was added thereto, followed by heating at 90 ° C. for 7 hours while continuously injecting nitrogen gas. The crude chitosan was washed with deionized water until the pH of the immersion aqueous solution was neutral, filtered and left for 2 days in 3 L of deionized water, and then filtered and immediately immersed in 3 L of 10% ethanol aqueous solution for 3 days. The soaked chitosan was filtered and dried in a vacuum oven at 40 ° C. for 2 days (primary NaOH aqueous solution treatment).

3 L of 40 wt% NaOH aqueous solution was added to the dried chitosan, followed by heating at 90 ° C. for 3 hours while continuously injecting nitrogen gas. The pH of the immersion aqueous solution was washed with deionized water until the pH was neutral, and then immersed in a deionized water bath for 2 days, filtered, and then immersed in a 10% aqueous ethanol solution for 3 days, followed by filtration and drying (treated with a secondary NaOH aqueous solution). In this way, chitosan with a deacetylation degree of 90% or more was obtained.

The first and second aqueous NaOH aqueous solution treatment was repeated once more to obtain chitosan having a deacetylation degree of 92% or more.

Comparative Examples 4 to 8

In the present embodiment, the effect of the change in the treatment conditions in the case of the present invention was tested, as shown in Table 2 to remove the NaOH aqueous solution treatment conditions from the scope of the present invention in Example 5, and the organic solvent treatment after NaOH treatment is omitted. Except that it was carried out in the same manner as in Example 5.

Comparative Example 9

The chitin obtained in Comparative Example 3 was placed in a flask, and 40 wt% NaOH aqueous solution was added thereto, followed by heating at 115 ° C. for 6 hours. Washed, filtered and dried in vacuum oven.

The experimental conditions and the properties of the obtained chitosan used in Example 5 and Comparative Examples 4 to 9 are shown in Table 2 below.

TABLE 2

* In the case of the present invention, only K, Ca, Mg, Fe, Ba, Zn, but in the comparison method also included other metals and heavy metals.

As can be seen in Table 2, chitosan having high deacetylation degree, purity and molecular weight can be obtained by repeating the treatment of NaOH aqueous solution and the solvent washing process two or more times according to the present invention. Processing at causes breakage resulting in poor whiteness. Chitosan prepared by the method of the present invention is particularly useful in the medical field where purity is important. Chitosan prepared by the present invention can be used to prepare N, O-carboxymethylchitosan by substitution with a carboxy methyl group.

Claims (14)

(1) drying and storing Korean red crab shells, (2) crushing the dried crab shell, (3) treating the ground crab shell with an aqueous hydrochloric acid solution of less than 0.8N to 3N for 10 to 25 hours at a temperature of -10 ° C to less than 10 ° C and for 2 to 8 hours at a temperature of 10 to 20 ° C, (4) washing and filtering the hydrochloric acid-treated crab shell, (5) washing and filtering the filtered crab shell, (6) drying the organic solvent-treated crab shell to obtain crude chitin, (7) heating the dried crude chitin by dipping in an aqueous NaOH solution, (8) washing and filtering NaOH treated chitin, (9) treating the filtered crude chitin with an organic solvent, and (10) A method for producing chitin from crab shells, which comprises drying the organic solvent treated crude chitin. The process according to claim 1, wherein the treatment conditions in the hydrochloric acid treatment step (3) are 15 to 20 hours at a temperature of -5 to 5 ° C and 3 to 5 hours at a temperature of 12 to 18 ° C. (1) drying and storing Korean red crab shells, (2) crushing the dried crab shell, (3) treating the ground crab shell with an aqueous hydrochloric acid solution of less than 0.8N to 3N for 20 to 60 hours at a temperature of -10 ° C to 8 ° C, (4) washing and filtering the hydrochloric acid-treated crab shell, (5) treating the filtered crab shell with an organic solvent, (6) drying the organic solvent-treated crab shell to obtain crude chitin, (7) heating the dried crude chitin by dipping in an aqueous NaOH solution, (8) washing and filtering NaOH treated chitin, (9) treating the filtered crude chitin with an organic solvent, and (10) A method for producing chitin from crab shells, which comprises drying the organic solvent treated crude chitin. The process according to claim 3, wherein the treatment conditions in the hydrochloric acid treatment step (3) are from 30 to 50 hours at -5 to 5 ° C. The method according to claim 1, wherein in the drying step (6), the crab shell is dried in a shade with a moisture content of 12% or less and stored in a cool state. The process according to claim 1, wherein in the hydrochloric acid treatment step (3), hydrochloric acid treatment is carried out while stirring at a speed of 50 to 200 rpm with a mechanical stirrer. The process according to claim 1, wherein the solvent used in the solvent treatment steps (5) and (9) is at least one solvent selected from the group consisting of ethanol, methanol, acetone, tetrahydrofuran, dioxane and methylethylketone. The method of claim 1, wherein the steps (7) to (10) are repeated two or more times. The process according to claim 1, wherein the NaOH treatment step (7) is carried out under an inert atmosphere. The method according to claim 8, wherein after the final NaOH treated chitin is washed and filtered (8), it is immersed in deionized water for 3 to 4 days and then filtered and dried. The method of claim 1, wherein the Korean red crab is Chionoecetes opillio. The method of claim 1, wherein the hydrochloric acid aqueous solution concentration in the hydrochloric acid treatment step (3) is 1N to 2N. The method according to claim 3, wherein the hydrochloric acid aqueous solution concentration in the hydrochloric acid treatment step (3) is 1N to 2N. The process according to claim 3, wherein the NaOH treatment step (7) is carried out under an inert atmosphere.