JPH06239902A - Production of high-purity chitin and chitosan - Google Patents

Abstract

PURPOSE: To obtain high-purity, high-whiteness, high molecular weight chitin and chitosan by drying crusts of the crustaceans, then pulverizing them, treating the formed fine powder with hydrochloric acid plural times under specific conditions, washing and drying the substance thus treated, then treating with NaOH, washing with water, separating the substance thus obtained by filtration, washing with an organic solvent and drying the substance thus obtained.

CONSTITUTION: Crusts of the crustaceans are dried and then pulverized to a fine powder. This fine powder is subjected to a first hydrochloric acid treatment by immersing the powder in a 0.4-3M hydrochloric acid solution at -10°C-+10°C for 10-25 hours, successively to a second hydrochloric acid treatment by immersing the substance thus treated at 10-20°C for 2-8 hours. Subsequently, the hydrochloric acid treated substance is washed with water, separated by filtration, washed with an organic solvent and dried to obtain crude chitin. Then, this crude chitin is immersed in a 2-10 wt.% sodium hydroxide solution and heated, and this sodium hydroxide treated chitin is washed with water, separated by filtration, washed with an organic solvent and then dried to obtain high-purity, high-whiteness, high molecular weight chitin and high molecular weight chitosan with a high degree of deacetylation with good efficiency.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing chitin and chitosan, and more particularly to a method for producing high-purity and high-whiteness chitin having a high molecular weight from crab shells for biomedical use, and high deacetylation. The present invention relates to a method for producing a high molecular weight chitosan having a certain degree for biomedical treatment.

[0002]

Chitin is a high-value-added biopolymer substance industrially extracted from the shells of crustaceans as a polysaccharide which is the most abundant available on earth after fibrin. Since such chitin is insoluble in water, it is a great obstacle to use it. Therefore, deacetylating chitin,
It is converted to chitosan which is water-soluble, and such water-soluble chitosan is industrially more useful than chitin.

[0003] Recently, as interest in disposing of shellfish, which is a marine waste, has increased due to the large-scale catch of crabs and shrimp, much research has been conducted on various uses of chitin extracted from shellfish. Initially, these chitins and chitosans were found to be active substances (eg proteins) in food waste.
Was mainly used as a flocculant for recovering
Recently, it has been widely used in various fields such as food field, medical field, biotechnology field such as functional membrane, immobilization carrier for enzymes and microorganisms, cosmetic field, agricultural field, chemical engineering field, environmental protection field. There is. Chitin is usually produced from the shell by a method comprising pretreatment, deproteinization and demineralization steps and is represented by the following structural formula.

[0004]

[Chemical 1]

Known methods for producing chitin include:
Wash the shell and dry it in a vacuum oven at 50 ° C; crush the dried shell and soak it in 10% NaOH solution at room temperature for 3 days to deproteinize; wash the deproteinized material with water until the alkaline component is gone After that, the product was washed several times with an organic solvent; the obtained white product was dried under reduced pressure and then kept in a 37% aqueous hydrochloric acid solution at -20 ° C for 4 hours to obtain crude chitin; Wash with cold hydrochloric acid solution and repeat washing process Whistler & Bemirror
miller) There is a method.

As another method, the shell is washed and the temperature is 100 ° C.
Dried in an oven; treated dry shell with a 2N hydrochloric acid solution at room temperature for 5 hours; washed and dried the treated shell and then pulverized into a fine powder, and the fine powder was treated with a 2N hydrochloric acid solution at 0 ° C. for 2 hours.
There is a Hackman method in which the extract is washed with water, washed with water and then extracted with a 1N NaOH aqueous solution at 100 ° C. for 12 hours with stirring; this NaOH aqueous solution treatment step is repeated four times or more. On the other hand, chitosan is obtained by deacetylating insoluble chitin and is represented by the following structural formula.

[0007]

[Chemical 2]

Chitosan has an advantage in chemical reactivity and chemical reformability because one of the hydroxyl groups present in the fibrin is substituted with an organic amino group. Chitosan is easily obtained by deacetylating chitin generally with 30-50% NaOH solution for 5-20 hours.

However, the above-mentioned conventional method for preparing chitin is carried out under relatively vigorous reaction conditions in order to reduce the contents of protein and CaCO ₃ . That is, in the case of the Whistler-Bemirer method, since the CaCO ₃ removal step is performed after the deproteinization step, it is difficult to obtain a high molecular weight chitin due to an increase in the cleavage of the molecular chain. When it is left in the air, it discolors over time, has a high content of impurities, and even if the CaCO ₃ removal step is carried out at a low temperature of −20 ° C., since a high-concentration hydrochloric acid aqueous solution is used, the molecular chain is broken. I will end up. Also, in the case of Hackman method, CaC removal
Since the O ₃ process is carried out at room temperature or at a temperature of 10 ° C. or higher, there is a problem that the molecular chain is broken.

The present inventor has studied the method for obtaining chitin and chitosan having high purity and high molecular weight, which are suitable for pharmaceutical and food applications, by solving the above-mentioned problems, and as a result, the treatment temperature during the hydrochloric acid treatment was relatively low. By doing so, chitin with a high molecular weight and a low CaCO ₃ content is obtained by preventing the molecular chain from being cleaved, and even when treated with a NaOH solution, it is injected twice for 3 hours while injecting an inert gas to prevent the molecular chain from being cleaved. The treatment has led to the completion of the method of the invention, which simplifies the process and reduces the time.

[0011]

The object of the present invention is to provide a method for obtaining chitin with high purity, high molecular weight and high whiteness. Another object of the present invention is to provide a method for producing chitosan having a high molecular weight and a high degree of deacetylation from the obtained chitin.

[0012]

That is, the present invention provides (a)
Drying the crustacean crust and then crushing it to obtain a fine powder,
(B) The fine powder was -10 in a 0.4 to 3 M HCl solution.
A step of applying a primary hydrochloric acid treatment for 10 to 25 hours at a temperature of -10 ° C and then a secondary hydrochloric acid treatment for immersing at a temperature of 10 to 20 ° C for 2 to 8 hours, (c) washing the hydrochloric acid-treated substance with water Filtering, washing with an organic solvent and then drying to obtain crude chitin, (d) 2-10 wt% N of the crude chitin.
a step of heating by immersing in an aOH solution, and (e) the N
A method for producing high-purity chitin, which comprises a step of washing aOH-treated chitin with water, filtering, washing with an organic solvent and then drying, and (a) the chitin obtained by the above method
a step of immersing in an aOH solution at a temperature of 80 to 100 ° C. for 2 to 12 hours to obtain crude chitosan, (b) filtering the crude chitosan, rinsing with water, immersing in deionized water, filtering and miscibility with water It is a method for producing high-purity chitosan in which the steps of immersing in a mixed solution of an organic solvent, filtering and drying are repeated.

The present invention will be described in more detail below.
The method for producing high-purity chitin according to the present invention comprises the following three steps.

Pretreatment Step First, the shell is dipped in water at 40 to 50 ° C. for 30 minutes to 1 hour and subjected to hot water treatment to effectively separate the fleshy portion from the shell. Then, the shell is washed with water and then dried with a dehydrator, and is kept in an organic solvent such as ethanol, methanol, acetone, tetrahydrofuran (THF), dioxane or methyl ethyl ketone (MEK) at room temperature for a certain time, for example, 3
After soaking for 0 minutes to 1 hour, in the shade, the water content is 8 to 12
Dry until%. If the dried shell is not used immediately, it is important to store the dried shell in a cool place. If the storage condition is poor, it will rot during storage and the molecular weight will decrease, making it impossible to obtain high-quality chitin. The crust from which meat is removed is crushed for effective treatment with hydrochloric acid. Generally, it is preferable to grind to a roughness of 0.5 to 3 mm or 200 to 300 mesh by a common grinder.

Demineralization Step Subsequently, the crushed shell (water content 8 to 12%) is subjected to hydrochloric acid treatment to remove mineral components such as CaCO ₃ . In the hydrochloric acid treatment, after adjusting the hydrochloric acid aqueous solution to a temperature of −10 to 10 ° C., preferably −5 to 5 ° C., shell powder (usually water content 8 to 12%) stirred with a mechanical stirrer is added, 10-25 hours at that temperature, preferably 15-20
After treating for a period of time, the temperature of the aqueous hydrochloric acid solution is rapidly increased by 10 to 20
C., preferably 12-18.degree. C. for 2-8 hours,
It is preferably carried out by holding for 3 to 5 hours. At this time, the concentration of the hydrochloric acid aqueous solution is preferably 0.4 to 3M, and the mechanical stirrer is stirred at a speed of 50 to 200 rpm, preferably 100 rpm, and a new hydrochloric acid aqueous solution is added once or twice to a suitable time during the treatment process. It is preferable to replace it. It should be noted that the effect of hydrochloric acid treatment decreases when the stirring speed is 200 rpm or more.

On the other hand, during the above treatment, the step of treating the hydrochloric acid aqueous solution as a one-step method is -10 to 8 ° C, preferably -5 to 5 ° C.
The temperature may be 20 to 60 hours, preferably 30 to 50 hours. The shellfish from which CaCO ₃ has been removed by the hydrochloric acid treatment is washed with water to remove the remaining hydrochloric acid component. Instead of washing with water, an aqueous solution of NaOH may be added dropwise to adjust the pH to 7-9, and then immersed in deionized water for a certain time, for example, 1-6 hours. Then, after filtration, for example, ethanol, methanol, acetone, THF, dioxane, ME
It is washed with an organic solvent such as K once or twice and then dried. The solvent washing is for removing the red color of the crustacean crustacea and increasing the whiteness of chitin, and this solvent acts as a decolorizing agent, and in the present invention, another oxidizing agent or a decolorizing agent is used. It has the advantage that no reducing agent need be used.

Deproteinization step The crude chitin obtained in the above step is immersed in an aqueous solution of NaOH for heat treatment. The concentration of the NaOH aqueous solution is 2 to 10
% By weight is preferred, the reaction temperature is in the range of 80 to 100 ° C. and the heating time is generally 2 to 4 hours. This step is preferably carried out while continuously injecting an inert gas such as nitrogen, argon, helium or neon gas. In this way, breaking of the chitin molecular chain can be prevented. Then the crude chitin is washed with deionized water to
After removing the aOH aqueous solution or dropping a hydrochloric acid solution to adjust the pH of the crude chitin immersion aqueous solution to 7 to 8, it is immersed in deionized water for a certain time, for example, 1 to 5 hours. Then, after filtering the chitin, as in the subsequent treatment after the hydrochloric acid treatment, for example, ethanol, methanol, acetone, T
It is washed once or twice with an organic solvent such as HF, dioxane or MEK and then dried. In order to enhance the purity of the NaOH-treated chitin and accelerate decolorization, it is preferable to repeat the above-mentioned NaOH treatment step and washing step twice or more. The obtained chitin was added to deionized water to increase the whiteness, and 2
After soaking for ~ 5 days, it is filtered and vacuum dried to obtain high-purity chitin.

From the high-purity chitin obtained by the present invention, chitosan having a high molecular weight and a high degree of deacetylation can be obtained as follows. First, for example, 30 to 50% NaOH aqueous solution is added to the chitin prepared by the above method and heated at a temperature of 80 to 100 ° C. for 2 to 12 hours. At this time, it is preferable to continuously inject an inert gas such as nitrogen, argon, helium or neon gas. Further, when the temperature and the time are out of the above ranges during the treatment of the NaOH aqueous solution, the molecular chain may be broken, which is not preferable. Then, the chitosan treated with the NaOH aqueous solution is washed with deionized water, and then washed with a mixed aqueous solution of an organic solvent such as acetone, ethanol, methanol, isopropanol, dioxane, THF or MEK (concentration of usually 5 to 30% by weight). After soaking, filtration,
Dry to increase the whiteness of chitosan and remove impurities.

Then, the dried chitosan is further subjected to the above-mentioned aqueous NaOH treatment and organic solvent treatment to obtain high-purity chitosan having a high degree of deacetylation. Preferably,
In order to further increase the deacetylation degree of chitosan, the above-mentioned NaOH aqueous solution treatment and organic solvent treatment steps can be repeated twice or more.

[0020]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto. In the examples, the residual CaCO ₃ content in chitin was measured as the loss on ignition after burning for 1 hour at 800 ° C. The protein content is
The measurement was carried out with a UV-visible spectrophotometer manufactured by Hewlett-Packard, USA according to the Bradford method using a protein analysis kit manufactured by Bio-Rad. The metal content was measured using an inductively coupled high frequency plasma optical emission spectrophotometer manufactured by Seiko Instruments Inc. and an atomic absorption spectrophotometer manufactured by Perkin-Elmer, USA. The degree of deacetylation was determined using an infrared spectrophotometer [J. Appl. Polym. Sc.
i. 28, 1909 (1983)]. As for the whiteness over time, the appearance state of chitin after 30 days or more was visually judged. Viscosity was also measured with a Brookfield viscometer using a # 4 spindle at 60 rpm with a 0.5% chitosan solution in 1% acetic acid.

EXAMPLE 1 Shell 300 of snow crab (scientific name: Chionoecetes opilio)
After being left in a water bath at 40 to 50 ° C. for 30 minutes, g was washed with water until the meat quality of the crab was completely removed. After removing water from the shell with a dehydrator and immersing it in an ethanol bath for 1 hour, the water was further removed with a dehydrator to obtain 200 g of dried shell (water content 10%) dried in the shade for 10 days. Then, 200 g of the dried shell was used for the (Dietz-Mottren)
-Motoren GmbH & Co. KG) crusher Mot. WRB
After pulverizing with 90 LB / 4P to a particle size of 0.5 to 3 mm, the mixture was put into a reaction vessel equipped with a mechanical stirrer, and the mixture was added at 0 ° C. to 1
After adding 5 L of N hydrochloric acid aqueous solution and maintaining at 0 ° C. for 20 hours while stirring at a speed of 50 rpm, the temperature was rapidly raised to 15 ° C. and maintained at 15 ° C. for 4 hours. The shell treated with hydrochloric acid was filtered, then immersed in water, the pH of the immersion liquid was adjusted to 7 to 8 with a 5 wt% NaOH aqueous solution, and then left for 6 hours. The filtered crude chitin was washed twice with 3 L of acetone, filtered, and dried in a vacuum oven at 30 ° C. for 36 hours.

The dried crude chitin was immersed in 6 L of a 5% by weight NaOH aqueous solution and then at 90 ° C. while injecting nitrogen gas.
Heated for 3 hours. Then, a 2N aqueous hydrochloric acid solution was added to adjust the pH of the crude chitin-containing aqueous solution to 7 to 8, and then the crude chitin was filtered, immersed in 6 L of deionized water for 6 hours, and then filtered. After washing the filtered material twice with 3 L of acetone,
It was dried in a vacuum oven at 30 ° C. for 12 hours. The dried chitin thus obtained was further subjected to the above-mentioned NaOH aqueous solution treatment step. The chitin treated with the secondary NaOH aqueous solution was washed with deionized water 5 to 10 times, immersed in 6 L of deionized water for 3 days, filtered, and dried in a vacuum oven to finally obtain 50 g of high-purity chitin. Obtained.

Example 2 Chitin was produced in the same manner as in Example 1 except that the treatment with the aqueous hydrochloric acid solution was carried out by maintaining the temperature at 0 ° C. for 48 hours.

Example 3 Chitin was produced in the same manner as in Example 1 except that the treatment with the hydrochloric acid aqueous solution was carried out by keeping the temperature at 5 ° C. for 24 hours.

Example 4 Chitin was produced in the same manner as in Example 1 except that the treatment with the aqueous hydrochloric acid solution was carried out by maintaining the temperature at -8 ° C for 40 hours.

Comparative Example 1 Chitin was produced in the same manner as in Example 1 except that the treatment with the hydrochloric acid aqueous solution was carried out by keeping the temperature at 15 ° C. for 8 hours.

Comparative Example 2 Chitin was produced in the same manner as in Example 1 except that the treatment with the hydrochloric acid aqueous solution was carried out by keeping the temperature at 10 ° C. for 24 hours.

Comparative Example 3 200 g of dried snow crab shell was crushed to 0.5 to 3 mm and charged into a reaction vessel equipped with a mechanical stirrer.
5 L of a 2N hydrochloric acid aqueous solution was added, and the mixture was reacted at room temperature for 5 hours with stirring. The hydrochloric acid solution was cooled to 0 ° C. and reacted for 48 hours. After filtration, the filtrated product is washed with water and kept at 100 ° C. for 3.85.
It was immersed in a wt% NaOH aqueous solution for 12 hours. After washing with water and drying, 80 g of chitin was obtained. Examples 1-4 and Comparative Examples 1-3
Table 1 shows the reaction conditions and the characteristics of the obtained chitin.

[0029]

[Table 1]

Example 5 45 g of chitin obtained in Example 1 was charged into a 4-liter three-necked flask, and 3 L of 40 wt% NaOH aqueous solution was added.
After adding, while continuously injecting nitrogen gas,
Was heated for 7 hours. The obtained crude chitosan was washed with deionized water until the pH of the washing water became neutral, filtered, immersed in 3 L of deionized water for 2 days, filtered, and immediately added to 3 L of 10% aqueous ethanol solution. It was immersed for 3 days. The crude chitosan was filtered and dried in a vacuum oven at 40 ° C. for 2 days (treatment with primary NaOH aqueous solution). After adding 3 L of 40 wt% NaOH aqueous solution to the dried chitosan, it was heated at a temperature of 90 ° C. for 3 hours while continuously injecting nitrogen gas. The resulting chitosan was washed with deionized water until the pH of the washing water became neutral, then filtered and washed with 2 L of 3 L of deionized water.
After soaking for a day, it was filtered, immediately immersed in a 10% aqueous ethanol solution for 3 hours, then filtered and dried (secondary N
aOH aqueous solution treatment). Thus, chitosan having a deacetylation degree of 90% or more was obtained. The above-mentioned primary and secondary Na
The OH aqueous solution treatment was further repeated once to obtain chitosan having a degree of deacetylation of 92% or more.

Example 6 and Comparative Examples 4 to 7 Chitosan was produced in the same manner as in Example 5 except that the conditions of the treatment with the aqueous NaOH solution were as shown in Table 2.

Comparative Example 8 40 g of the chitin obtained in Comparative Example 1 was placed in a flask,
After adding further 2.4 L of 40 wt% NaOH aqueous solution,
Heated at a temperature of 115 ° C. for 6 hours. Washed, filtered and dried in a vacuum oven. Table 2 shows the reaction conditions used in Examples 5 to 6 and Comparative Examples 4 to 8 and the properties of the obtained chitosan.

[0033]

[Table 2]

[0034]

EFFECTS OF THE INVENTION According to the present invention, it is possible to prevent the cleavage of molecular chains under mild hydrochloric acid treatment conditions, and to use an organic solvent after hydrochloric acid treatment and NaOH treatment, thereby making it possible to remove chitin without using a decolorizing agent. The effect of increasing the whiteness is obtained. Further, during the NaOH treatment, a high molecular weight chitin can be obtained by injecting an inert gas to prevent the molecular chain from breaking. Further, by repeating treatment of such chitin with an aqueous NaOH solution, chitosan having a high degree of deacetylation, high purity and high molecular weight can be obtained.

Claims (13)

[Claims] 1. A step of (a) drying a crustacean shell and then pulverizing it to obtain a fine powder, (b) -10 in a 0.4 to 3 M hydrochloric acid solution of the fine powder.
A step of performing primary hydrochloric acid treatment for 10 to 25 hours at a temperature of 10 ° C., and then performing secondary hydrochloric acid treatment for dipping at a temperature of 10 to 20 ° C. for 2 to 8 hours, (c) washing the hydrochloric acid treated material with water, Filtering, washing with an organic solvent and then drying to obtain crude chitin, (d) a step of immersing the crude chitin in a 2 to 10 wt% sodium hydroxide solution and heating, and (e) the sodium hydroxide Wash the treated chitin with water,
A method for producing high-purity chitin, which comprises the steps of filtering, washing with an organic solvent, and then drying. 2. Drying the shell in the step (a),
The method according to claim 1, wherein the method is performed in the shade until the water content is 12% or less. 3. The primary hydrochloric acid treatment in the step (b) is performed at a temperature of −5 to 5 ° C. for 15 to 20 hours, and the secondary hydrochloric acid treatment is performed at a temperature of 12 to 18 ° C. for 3 to 5 hours. The method described. 4. The method according to claim 1, wherein the treatment with hydrochloric acid in the step (b) is carried out while stirring with a mechanical stirrer at a speed of 50 to 200 rpm. 5. The method according to claim 1, wherein the solvent used in steps (c) and (f) is selected from ethanol, methanol, acetone, tetrahydrofuran, dioxane and methyl ethyl ketone. 6. The method according to claim 1, wherein the sodium hydroxide treatment in the step (d) is performed in an inert gas. 7. The method according to claim 1, wherein the steps (d) and (e) are repeated twice or more. 8. A method for producing high-purity chitin, which comprises performing the hydrochloric acid treatment step (b) of claim 1 in a one-step method at a temperature of −10 ° C. to 8 ° C. for 20 to 60 hours. 9. The method according to claim 8, wherein the hydrochloric acid treatment step is performed at a temperature of −5 to 5 ° C. for 30 to 50 hours. 10. (a) The chitin produced by the method according to claim 1 or 8 is heat-treated with a sodium hydroxide solution at a temperature of 80 to 100 ° C. for 2 to 12 hours to obtain crude chitosan. And (b) filtering the crude chitosan, rinsing with water, immersing in deionized water, filtering, immersing in a mixed aqueous solution of a water-miscible organic solvent, and then filtering and drying. A method for producing high-purity chitosan. 11. The method according to claim 10, wherein the step (a) is performed in an inert gas. 12. The method according to claim 10, wherein the mixed aqueous solution in the step (b) contains 5 to 30% by weight of an organic solvent. 13. The method of claim 12, wherein the organic solvent is selected from acetone, ethanol, methanol, isopropanol, dioxane, tetrahydrofuran and methyl ethyl ketone.