KR0136552B1 - Preparation of chitin and chitosan from solenocera prominentis shells - Google Patents

Abstract

The present invention simultaneously obtains high purity and whiteness high molecular weight and low molecular weight chitin from the shell of chitin and chitosan, in particular from the shellfish of solenocera prominentis, from which the high molecular weight having high purity, high deacetylation and high whiteness or A method for producing low molecular weight chitosan for biomedical medicine.

Description

Preparation of Chitin and Chitosan for Biomedical Clinic from Daerong Beard Shrimp Shell

The present invention simultaneously obtains high purity and whiteness of high molecular weight and low molecular weight chitin from the shell of chitin and chitosan, in particular the shellfish of Solenocera prominentis, from which high purity, high deacetylation and high whiteness are obtained. The present invention relates to a method for preparing chitosan for biomedical medicine of molecular weight or low molecular weight.

Chitin is a natural polymer that is the second highest in the world, followed by fiber, and is a high value biopolymer that is commercially extracted from the crustaceans of sensory animals. Such chitin is insoluble in water and thus becomes a major obstacle to its utilization. Therefore, deacetylation of chitin can be used to convert chitosan, which is water soluble in weakly acidic liquids, and this water soluble chitosan is industrially 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 stopped by biotechnology such as food, medical, functional membranes, enzymes and immobilization carriers of enzymes and microorganisms, It is widely used in all fields such as cosmetics, agriculture, chemicals and environment.

Specifically, chitin and chitosan have high crystallinity and high molecular weight of 1000 centipoise (CPS) or more depending on the molecular weight, so that the fiber forming ability or film forming ability is excellent and the strength is excellent. In the case of using a relatively low molecular weight in the range of an average molecular weight of 5000 to 10,000, it can be used as a food preservative, an anticancer agent, a body cholesterol lowering agent, a cosmetic additive, a heavy metal remover in the body, an antibacterial deodorant, and the like.

In the 1990s, the demand for chitin / chitosan is increasing worldwide, and the demand for high quality rather than low quality is increasing rapidly. Prior to 1990, chitin / chitosan, a low-grade product mainly used for water treatment and agriculture, has been mainstream in the application field, but recently, a high-grade product that can be effectively applied in the food field and in medicine and cosmetics It is required.

In the case of high quality, only a few countries have manufacturing technology in the world, and few countries succeed in industrialization and commercialize them. The conditions for high quality chitin / chitosan that are required in the manufacture of bioclinical use are as follows.

First, the most essential requirement should be a low content of impurities. Representative impurities in the produced chitin / chitosan can be listed as lime (CaCO ₃ is the mainstream), proteins, lipids, pigments, heavy metals, etc., about 0.2% or less for lime, 0.1% or less for protein In the case of heavy metals, approximately 5ppm or less must be satisfied before they can be used in medicine and cosmetics. In order to minimize the residual amount of calcareous or protein, theoretically, it can be solved simply by using intensive treatment conditions when removing the calcareous solution with HCl aqueous solution and protein removal with NaOH aqueous solution, but at the same time, abruptly breaking the molecular chain This results in not only a significantly lower molecular weight, but also a wider molecular weight distribution of chitin / chitosan, which is often accompanied by dark brown coloration. The coloration phenomenon in chitin / chitosan production is thought to be a phenomenon that occurs when the molecular chain is broken. Since the coloration phenomenon is removed by a mixed solvent treatment such as ethanol / H ₂ O, the coloration phenomenon contains a kind of impurities inside the manufactured chitin / chitosan. It can be interpreted as a phenomenon.

As a result, in order to prevent the reduction of the molecular weight and the coloring phenomenon caused by the molecular weight decrease, it is determined that the existing chitin / chitosan preparation method should be closely and extensively reviewed. The use of oxidants has been found in patents and papers published in order to remove coloration (P. Broussignac, Chim. Ind. Genie Chim. 99, 1241 (1968); , J. of Korean Ind. Eng. Chemistry Vol. 4, No. 2, June 1933, P325).

Although it is possible to increase the whiteness by using an oxidant from colored chitin or chitosan, the increase in whiteness due to this oxidant treatment is temporary, and as time passes (after about 10 days), the whiteness gradually decreases and after a certain time In many cases, coloring becomes stronger than before oxidizing agent treatment. Therefore, the method of raising the whiteness by the oxidizing agent should be excluded in the production of high quality chitin / chitosan. In the case of chitin, a certain kind of solvent treatment may cause a gradual coloring similar to that of the oxidizer treatment.

In addition, in the production of high-quality chitin / chitosan, the source (crab, shrimp, squid, chrysalis, insects, fungus) that obtain chitin / chitosan in order to manufacture differentiated products having various molecular weights and unique characteristics also differentiates itself. Should be. Recently, as the application range of chitin / chitosan is beginning to expand day by day, the technology of molecular weight control is urgently needed.

In the above, the use of violent reaction conditions in the production of chitin can significantly lower the content of residual impurities, but has been pointed out that it is unsuitable for the production of high molecular weight products due to the decrease in molecular weight, but in the production of low molecular weight products, violent reaction conditions. It is judged that the grant of is not unreasonable.

However, it can be said that induction of coloring with the decrease of molecular weight is inevitable. Although the coloring phenomenon caused by low molecular weight products is not a big problem in the application of food, but it cannot be applied in the field of cosmetic additives and pharmaceuticals, the development of low molecular weight chitin / chitosan product and low degree of coloring is urgently needed. It is becoming. Depending on the source of raw material (crab, shrimp, cuttlefish) from which chitin / chitosan can be obtained, the amount of calcare and protein contained differs in the binding state with chitin. Differentiate accordingly.

In general, crustaceans of crabs are easy to obtain high molecular weight chitin / chitosan because of their tight tissue and extremely high degree of crystallization of chitin. However, in order to obtain low molecular weight, violent reaction conditions must be imparted. The low molecular weight product obtained from the shellfish is extremely unsatisfactory in the manufacture of low molecular weight having excellent whiteness.

On the other hand, shrimp shellfish can be easily prepared in high molecular weight chitin / chitosan because it can be removed even under milder conditions than the crab shell was applied. However, in general, chitin / chitosan derived from shrimp has been found to be impossible to obtain high molecular weight chitin / chitosan as obtained from crab shell because of its low crystallinity and low molecular weight.

Rather, in shrimps, low-molecular-weight chitin / chitosan can be obtained without the same intense Hcl treatment conditions as used in crab shellfish, so it is easy to obtain low-molecular weight chitin / chitosan with much lower coloration than that obtained from crab shellfish.

Conventional methods for preparing chitin have been the Hackman method (Austr. J. Biol, Sci. 7, 168-178 (1954)), Whistler and BeMiller method (JOC 27, 1161-1163 (1962)), Horowitz, Roseeman and Blumenthal (JACS, 179, 5046-5049 (1957)) and Brusignacac (Chim, Ind. Genie Chim. 99, 1241-1247 (1968)), but these methods are generally inconvenient to obtain high molecular weight chitin / chitosan from shrimps due to the cumbersome manufacturing process, too long processing time, or extreme processing conditions. Methods.

In addition, the Brucignac method uses the oxidizing agent H2O2 for decolorization, which causes a decrease in whiteness after a certain time and has a problem as a manufacturing process for chitin / chitosan for biomedical medicine. Chitosan can be obtained by deacetylating insoluble chitin obtained by the various methods described above, which is generally obtained by treating 30 to 50% sodium hydroxide solution using about 20 times the amount of chitin for 5 to 20 hours. Can be.

As described above, it has been known that low molecular weight chitin / chitosan is difficult to obtain from crab shells and high molecular weight chitin / chitosan from shrimp shells. However, the present inventors have conducted extensive research on shrimps solenocera prominentis. The present invention finds that high molecular weight and low molecular weight chitin / chitosan can be easily obtained simultaneously, and in a simpler process, a method for obtaining high purity and high whiteness chitin / chitosan in a short time is completed. Was done.

That is, the solenocera prominentis, which are caught on the south coast of Korea and near the Jeju island, is much simpler than the method applied to crab shellfish because the lime content is much higher than that of other kinds of shrimp and the molecular weight of chitin itself is significantly higher. Not only can high molecular weight chitin / chitosan be easily applied, but also low molecular weight can be easily obtained even under milder conditions than the treatment conditions applied to crab shell during low molecular weight production. In addition, since the lobster has almost no red pigment that ordinary shrimps have, the chitin / chitosan having excellent whiteness can be obtained even if the decolorization process is omitted.

It is an object of the present invention to provide a method for preparing chitin, which can easily obtain high purity, high whiteness and high molecular weight chitin and high purity, high whiteness and low molecular weight chitin simultaneously from the sapling shrimp.

Another object of the present invention is to provide a method for preparing chitosan which simultaneously obtains high purity and high whiteness high molecular weight chitosan and low molecular weight chitosan from the chitin obtained above.

That is, the present invention comprises the steps of keeping the shrimp crust intact, crushing the dried shrimp crust, processing the pulverized shrimp crust in an aqueous hydrochloric acid solution to remove the calcite, the calcareous shrimp crust in NaOH aqueous solution The present invention relates to a method for preparing chiropractic chitin, comprising the steps of removing the protein and filtering, washing, neutralizing and drying the prepared jochitin.

The present invention also relates to a method for producing chitosan comprising the step of deacetylating chitin prepared by the above method in aqueous NOaH solution, washing with water and filtering.

The present invention will be described in more detail as follows.

First, wash the shrimp crust with running water to effectively remove any succulents or suspended matter remaining on the shrimp crust.

Residual broth or suspended solids must be removed completely, as the shrimp shell may rot in a short time, possibly degrading the quality of chitin / chitosan and acting as a residual impurity.

Dehydrated shrimp shells are dehydrated with a dehydrator and then immersed in organic solvents such as acetone, ethanol, methanol, methyl ether ketone and dioxane for 24-48 hours, dehydrated with a dehydrator and dried in the shade to promote the decay of shrimp shells. Moisture can be removed and the anti-corrosion effect by the organic solvent itself can be obtained.

Shrimp shells dried from the solvent treatment were decomposed to a size of 0.5-3 mm in diameter, 50 g were placed in a 2-liter three-necked flask, and slowly added to a solution of 1-6 N hydrochloric acid at -40 ° C. to + 30 ° C. Once all the carapaces have subsided, they are decalcified by stirring at 50-200 rpm for 1.5 to 24 hours. In this case, it is advantageous to mix an organic solvent with an aqueous hydrochloric acid solution at a temperature below −10 ° C. to prevent freezing of the aqueous hydrochloric acid solution, and preferred organic solvents are methanol, ethanol, isopropanol, butanol, acetone, and methyl ethyl ketone. In addition, the molecular weight of the chitin / chitosan finally obtained according to the concentration of the hydrochloric acid aqueous solution when the hydrochloric acid aqueous solution is treated, and at a low temperature (below +10 ℃) high molecular weight chitin / chitosan, high temperature (+20 ℃ or more) ), Relatively low molecular weight chitin / chitosan can be obtained. Beef crayfish has much higher lime content than crab shellfish or other kinds of shrimps, so the bubbles are intense, so special care should be taken to prevent the contents from overflowing the container.

When calcification is completed by hydrochloric acid aqueous solution, all processes such as filtration, washing and neutralization, solvent treatment for removing pigments required for crab shells and other kinds of shrimp can be omitted, and the deproteinization process can be entered immediately. .

In the deproteinization process, 5-10% (w / w) NaOH aqueous solution is used to heat the first two times at 90 ° C. to 100 ° C. for 0.5 hours to 6 hours and the second 0.5 hours to 5 hours to completely remove residual protein. can do.

The chitin obtained by the above method was deacetylated by treating with NaOH aqueous solution of 40-50% concentration for 2 to 8 hours at 70-120 ° C. for 2-8 hours while passing through a nitrogen stream to obtain high purity, high whiteness, high viscosity ( Or low viscosity) to obtain chitosan.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Comparative Examples, but the present invention is not limited thereto.

In the examples, the residual CaCO3 content in the chitin was measured as the loss of ignition after burning the chitin at 800 ° C. for 1 hour, and the protein content was determined using Bradford (Bio-Rad) protein analysis kit. According to the Bradford method, it was measured by a UV / VIS spectrometer of Hewlett-packard. In addition, the metal content was measured by using ICP-AES manufactured by Seiko, Japan and AA spectrometer, manufactured by Perkin-Elmer, USA. Deacetylation degree is described in 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 # 4 spins on a Brookfield viscometer.

Example 1

Step 1: Preparation of Chitin

The meat shells were washed with running water, dehydrated and immersed in ethanol for 24 hours and dried.

60g of solvent-treated and dried shrimp shells (about 10% water content) are crushed evenly to a particle diameter of 2-3mm,

Into a 3 volume flask equipped with 2ι volume, slowly add 1.0ι of aqueous 2N hydrochloric acid solution cooled to -10 ℃ and keep it at -10 ℃ until all the shrimp shells floating on the surface of hydrochloric acid solution subside. It was.

When all the floating shrimp shells sank underneath, it was stirred for 6 hours at a speed of 50-200 rpm to remove calcification, filtration, neutralization and drying to obtain 16.8 g of lime shells. 16.8 g of decalcified shrimp shells were immersed in a 1% aqueous NaOH solution at a concentration of 5% (w / w), followed by a first NaOH aqueous solution treatment which was heated at 95 ° C. for 2 hours while injecting nitrogen gas. After heating for 2 hours, filtered and washed 2-3 times with deionized water, and then immediately treated with a second NaOH aqueous solution, and heated for 1.5 hours while maintaining 95 ℃, the contents were filtered, washed, neutralized and dried to have excellent whiteness and transparency. 12.8 g chitin was obtained.

Step 2: Preparation of Chitosan

15 g of the chitin prepared by the above-described method was placed in a 1 L three-necked flask, and 600 ml of 40% NaOH aqueous solution was added thereto, followed by deacetylation at 100 ° C. for 5 hours while continuously injecting nitrogen gas. After completion of the first deacetylation reaction, the aqueous NaOH solution was filtered, washed 2-3 times with deionized water, and immediately proceeded to the second deacetylation reaction in the same manner as the first deacetylation reaction. In the deacetylation reaction, the reaction time was changed to 3 hours. After completion of the second deacetylation reaction, the mixture was filtered and washed with deionized water until neutral, and left in 80/20 (v / v) ethanol / water solution for 20 hours, followed by filtration and drying to obtain 11.69 g of chitosan. . The viscosity of the obtained chitosan was 2400 cpc (1% acetic acid solution, 0.5% chitosan solution, Brookfield Viscometer, Spindle # 4, 60 rpm).

Example 2

Except for changing the removal time of the calcareous solution by the hydrochloric acid aqueous solution in step 1 of Example 1 was carried out in the same manner as in Example 1 to obtain 11.58g of chitosan and the viscosity of the chitosan was 1500cps.

Example 3

In the same manner as in Example 1, except that the aqueous hydrochloric acid concentration was changed to 6N in step 1 of Example 1, the treatment temperature of the aqueous hydrochloric acid solution was changed to -20 ° C, and the treatment time was changed to 6 hours, and 11.87 g of chitosan was used. And the viscosity was 1740 cps.

Example 4

In the same manner as in Example 1 except that the concentration of the aqueous hydrochloric acid solution was changed to 6N in step 1 of Example 1, the treatment temperature of the aqueous hydrochloric acid solution was changed to -20 ° C, and the treatment time was changed to 1.5 hours. Chitosan was obtained and the viscosity was 2440 cps.

Example 5

Example 1 was carried out in the same manner as in Example 1 except that the concentration of the aqueous hydrochloric acid solution was changed to 6N, the treatment temperature of the aqueous hydrochloric acid solution was changed to -30 ° C, and the treatment time was changed to 3 hours. Chitosan was obtained and the viscosity was 2390 cps.

Example 6

Example 1 was carried out in the same manner as in Example 1 except that the concentration of the aqueous hydrochloric acid solution was changed to 6N, the treatment temperature of the aqueous hydrochloric acid solution was changed to -30 ° C, and the treatment time was changed to 9 hours. Chitosan was obtained and the viscosity was 2100 cps.

Example 7

Example 1 was carried out in the same manner as in Example 1 except that the concentration of the aqueous hydrochloric acid solution was changed to 6N, the treatment temperature of the aqueous hydrochloric acid solution was changed to -40 ° C, and the treatment time was changed to 6 hours. Chitosan was obtained and the viscosity was 1560 cps.

Example 8

12.03 g of Example 1 was changed in the same manner as in Example 1 except that the concentration of the aqueous hydrochloric acid solution was changed to 6N, the treatment temperature of the aqueous hydrochloric acid solution was changed to -40 ° C, and the treatment time was changed to 9 hours. Chitosan was obtained and the viscosity was 2040 cps.

Example 9

In the same manner as in Example 1, except that the treatment temperature of the aqueous hydrochloric acid solution was changed to 0 ° C. and the treatment time was changed to 3 hours in Step 1 of Example 1, 11.42 g of chitosan was obtained and the viscosity was 2690 cps.

Example 10

In Example 1, except that the treatment temperature of the aqueous hydrochloric acid solution was changed to 0 ° C. and the treatment time was changed to 6 hours, 11.28 g of chitosan was obtained and the viscosity was 2200 cps.

Example 11

In the same manner as in Example 1, except that the treatment temperature of the aqueous hydrochloric acid solution was changed to 10 ° C. and the treatment time was changed to 3 hours in Step 1 of Example 1, 11.23 g of chitosan was obtained and the viscosity was 2260 cps.

Example 12

In the same manner as in Example 1 except that the treatment temperature of the aqueous hydrochloric acid solution was changed to 10 ° C. and the treatment time to 9 hours in Step 1 of Example 1, 11.17 g of chitosan was obtained and the viscosity was 1290 cps.

Example 13

In the same manner as in Example 1, except that the treatment temperature of the aqueous hydrochloric acid solution was changed to 20 ° C. and the treatment time was changed to 6 hours in Step 1 of Example 1, 11.21 g of chitosan was obtained and the viscosity was 782 cps.

Example 14

In the same manner as in Example 1, except that the treatment temperature of the aqueous hydrochloric acid solution was changed to 20 ° C. and the treatment time to 9 hours in Step 1 of Example 1, 11.18 g of chitosan was obtained and the viscosity was 671 cps.

Comparative Example (1954. Based on Heckman Act)

5 L of 2N hydrochloric acid aqueous solution was added to 220 g of shrimp shells, and the mixture was left for 5 hours while maintaining at 25 ° C., filtered, washed and neutralized. 500 ml of 2N hydrochloric acid aqueous solution was added to the shrimp shell obtained by the above method, and decalcification was carried out for 48 hours while maintaining 0 ° C., and then filtered, washed, neutralized and dried to obtain 48.4 g of decalcified shrimp shell. . The decalcification shrimp shell obtained by the above method was added to 500 ml of 1N NaOH aqueous solution, and heated at 12 ° C. for 12 hours to remove protein. The protein removal process of the same method as described above was repeated four times, filtered and neutralized, washed with ethanol and ether, and dried to obtain 33 g of chitin. 40 g of chitin obtained by the above security was deacetylated twice over 5 hours for the first time and 3 hours for the second time while maintaining 100 ° C. in 40% NaOH aqueous solution to obtain 28.38 g of chitosan, and its viscosity was 432 cps. The whiteness of the obtained chitosan is yellowish and opaque.

The reaction conditions used in Examples 1 to 14 and the above and the properties of chitin and chitosan obtained therefrom are shown in Tables 1 and 2, respectively.

Table 1

TABLE 2

1) Preparation of 6N Hcl aqueous solution used at -40 ° C, -30 ° C and -20 ° C: 1600 ml of concentrated HCl, 1400 ml of tally hot water, and 200 ml of ethanol were prepared.

2) Preparation of 2N Hcl used at -10 ° C: 6N Hcl aqueous solution prepared by the above method was diluted three times with H ₂ O: ethanol = 1/1 (v / v) mixture to prepare a 2N Hcl aqueous solution.

As can be seen in Table 1 and Table 2, according to the method of the present invention can be recorded simultaneously high purity and high molecular weight chitin / chitosan of high purity, chitin / chitosan prepared by the method of the present invention It is particularly useful in the field of biomedical medicine where purity is important.

Claims (7)

(A) drying and grinding the shrimp shells. (B) treating the ground shrimp shells with a solution of hydrochloric acid at a concentration of 1 to 6N at a temperature of -40 ° C to + 30 ° C for 1.5 to 24 hours to remove calcification, and (C) Crushing dehydrated shrimp shells in a 5 to 10% (w / w) NaOH solution at 90 ° C to 100 ° C for 0.5 to 6 hours for the first time and 0.5 to 5 hours for the second time to remove protein Comprising a, method for producing a chitin for biomedical medicine from solenocesa prominentus. The method according to claim 1, wherein the shrimp shell is washed in the step (a), immersed in an organic solvent for 24 to 48 hours, then dehydrated and dried in the shade. The method of claim 2, wherein the organic solvent is selected from the group consisting of methanol, ethanol, butanol, isoprotanol, acetone, methyl ethyl ketone and dioxane. The method of claim 1, wherein the size of the shrimp crust ground in step (a) is 0.5 to 3 mm in diameter. 40-50% by weight of aqueous NaOH solution was added to the chitin obtained by the method of claim 1 in an amount of 400 to 700 ml based on 10% of the chitin, followed by degassing at a temperature of 70 to 120 ° C. for 2 to 8 hours while injecting nitrogen gas. Method for producing chitosan comprising the step of acetylation and filtration and washing step 2 to 4 times. The hydrochloric acid aqueous solution of step (b) is prepared by mixing concentrated hydrochloric acid, deionized water and an organic solvent in order to prevent freezing of the aqueous hydrochloric acid solution when the hydrochloric acid aqueous solution is treated at −10 ° C. or lower. Way. 9. The method of claim 8, wherein the organic solvent is selected from the group consisting of methanol, ethanol, butanol, isopropanol, acetone and methyl ethyl ketone.