KR20010105888A - Maunfacturing method of high purity chitosan oligosaccarid - Google Patents

Abstract

A method of microwave heating of a substrate in a plasma processing chamber wherein a heatup gas is supplied into the processing chamber, the heatup process gas is energized with microwave power to heat an exposed surface of the substrate, a reactant gas is supplied into the processing chamber and the reactant gas is energized into a plasma gas state to process the substrate.

Description

Manufacturing method of high purity chitosan oligosaccharides {MAUNFACTURING METHOD OF HIGH PURITY CHITOSAN OLIGOSACCARID}

The present invention relates to a method for producing a high purity chitosan oligosaccharide, and more particularly, to obtain a chitosan oligosaccharide reaction mixture by an enzymatic hydrolysis method, and to remove the acid groups and unreacted polymer chitosan used to dissolve chitosan by adding calcium carbonate to the reaction mixture. It relates to a method for producing only pure chitosan oligosaccharides.

In recent years, as the bioactive functions of chitosan and its derivatives have been discovered, studies and a lot of interests have been heightened. However, chitosan is insoluble in water and has many limitations in its application. In particular, in order to be able to apply chitosan in the food field, the problem of the taste of chitosan must be solved. To use the chitosan in the food field, it must be dissolved and added to an acid capable of dissolving chitosan.

However, when chitosan is dissolved in acid, bitter and astringent taste becomes very strong, so that even at low concentration, bitter taste is detected. Therefore, it is almost impossible to add chitosan to acid in terms of taste. In addition, when added to the food that taste is important in this state, the utility value is inevitably lowered due to the bitter taste of the chitosan and the acidity of the acid.

For this reason, many studies have been conducted to increase the water solubility of chitosan. For example, a method of preparing a new chitosan derivative by introducing chemical substituents such as carboxymethyl and hydrocypropyl to chitosan is known (Japanese Patent Laid-Open No. 61-21664). The problem is that there is insufficient validation for the system.

In addition, when viewed in terms of taste, it is common that the molecular weight should be at least 10,000 or less in order for the taste to be satisfied so that the low molecular weight chitosan can be used in food and the like. Japanese Laid-Open Patent Publication No. 5-49684 also points out the relationship between chitosan and rice bran, which point out that when the molecular weight of low molecular weight chitosan is in the range of several hundred to 10,000, the bitter and astringent taste is weakened compared to high molecular weight chitosan. .

Therefore, attention has been focused on solving the above problems with low molecular weight chitosan oligosaccharides that can be easily dissolved in water while maintaining the physiological activity of chitosan.

Japanese Patent Application Laid-Open No. 60-186504 discloses a method for producing a low molecular weight water-soluble chitosan by contacting chitosan and chlorine gas, and Japanese Patent Laid-Open No. 62-184002 uses a low molecular weight using sodium nitrite and acetic acid. A method for producing a water-soluble chitosan is known, and Japanese Patent Laid-Open No. 63-120701 discloses a method for producing a low molecular weight water-soluble chitosan by adding sodium chlorite, hydrogen peroxide, and hydrochloric acid. No. 2-41301 is known to produce low molecular water soluble chitosan by adding hydrogen peroxide and sulfuric acid.

In addition, Korean Patent Publication No. 97-015604 discloses a method for producing a low molecular weight water-soluble chitosan by adding hydrogen peroxide, acetic acid and sulfuric acid, and Korean Patent Publication No. 99-0049775 adds a bezenelenic acid mixed solution and hydrogen peroxide. It is known to produce low molecular weight water soluble chitosan.

However, when producing low molecular weight chitosan oligosaccharides by chemical treatment as described above, it is pointed out that the problem is extremely dangerous process, or the safety of the various chemicals remaining after the reaction and irregular cutting of the chitosan molecular chain, and the environmental pollution, etc. It is becoming.

Therefore, a method of preparing chitosan oligosaccharides by treating enzymes, which are biological methods, has been studied instead of chemical treatment methods that have problems such as biosafety and environmental pollution. In Korean Patent Publication No. 98-034824, chitosan was dissolved in lactic acid. It is known to produce low molecular weight chitosan oligosaccharides using ultrafiltration membranes (molecular weight cut off 3,000 to 5,000) after the bioreaction by adding chitosan hydrolase.

However, since the lactic acid cannot be removed by the above method, there is still a problem that it is difficult to produce high purity chitosan oligosaccharides due to the remaining acid groups with the problem of taste. In addition, since the ultrafiltration membrane itself is very expensive and has no durability, there is a problem that an increase in manufacturing cost is inevitable during industrialization.

Accordingly, the present inventors have completed the present invention as a result of studying a method for preparing high purity chitosan oligosaccharides which can solve the above problems.

It is an object of the present invention to provide a method for efficiently and economically preparing pure chitosan oligosaccharides by removing acid groups and unreacted polymer chitosan used for chitosan dissolution in the process of producing chitosan oligosaccharides from chitosan by enzymatic hydrolysis.

1 is a high-performance liquid chromatogram of chitosan oligosaccharide used in the present invention.

The present invention relates to a method for producing a high purity chitosan oligosaccharide, comprising the steps of: (1) dissolving chitosan in a tartaric acid solution to prepare a chitosan salt solution; (2) decomposing chitosan by adding chitosan degrading enzyme to the sample solution obtained in (1) to obtain chitosan oligosaccharides; (3) preparing a high purity low molecular chitosan oligosaccharide by adding calcium carbonate to the chitosan oligosaccharide-containing reaction mixture obtained in (2) to remove unreacted chitosan and tartaric acid.

The present invention will be described in detail for each step as follows.

(1) Chitosan Salt Solution Preparation Step

Chitosan is dissolved in tartaric acid solution to prepare a chitosan salt solution. At this time, the reaction temperature is preferably 40 to 80 ° C.

Chitosan used as a substrate has a suitable viscosity of 5 to 20 cps, particularly preferably 8 to 12 cps, and a concentration of 2 to 7%, of which 3 to 4% is preferable.

The concentration of tartaric acid for dissolving chitosan is suitably 0.05 to 0.2 mole, of which 0.075 to 1.5 mole is preferable.

(2) chitosan oligosaccharide manufacturing step

Chitosan oligosaccharides are prepared by adding chitosan degrading enzyme to the chitosan salt solution prepared in step (1). At this time, the ratio of the chitosan 1% solution to the enzyme is 0.05 to 0.2 unit / ml, and particularly preferably 0.1 to 0.2 unit / ml.

Bioreaction temperature is 30-55 degreeC, 45-55 degreeC is especially preferable, bioreaction time is 12-48 hours, and 18-30 hours are preferable.

(3) adding calcium carbonate

Calcium carbonate is added to the chitosan oligosaccharide-containing reaction mixture to remove unreacted chitosan and tartaric acid.

Calcium carbonate is insoluble in water, but has a characteristic of increasing pH and precipitating by dissolving in small amounts under acidic conditions to combine with acid and form a salt. In addition, calcium carbonate generates carbon dioxide in the presence of acid at a high temperature, and calcium ions are generated when combined with acid to give water. Because of its reactivity, the calcium ions easily bind to the anions and form salts.

In this reaction, even if calcium carbonate is not used, the solubility is lowered due to the characteristic of tartaric acid, and the precipitated or colloidal state is combined with chitosan ions. Therefore, the acid can be removed by lowering the temperature. However, by lowering the temperature it is not possible to completely remove the acid, so if a small amount of calcium carbonate is treated at high temperature to remove the remaining dissolved tartaric acid, it will bind with the acid and precipitate together.

In the present invention, the calcium carbonate added to the reaction mixture is 0.05 to 0.5 mol, particularly preferably 0.1 to 0.3 mol.

The reaction time after addition of calcium carbonate is 0.5 to 2 hours, particularly preferably 0.5 to 1 hour, and the reaction temperature after addition of calcium carbonate is 40 to 80 ° C, particularly preferably 60 to 70 ° C.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely examples of the present invention and the present invention is not limited to the following examples.

Example 1

50 kg of dried crab shells were cut to a size of about 3 to 5 cm, and 800 L of 7% sodium hydroxide solution was added thereto, followed by treatment at 100 ° C. for at least 5 hours to remove proteins, and washed with water to neutrality. 800L of 7% hydrochloric acid was added to the crab shell, followed by immersion for 2 to 3 hours at 40 to 50 ° C, followed by washing until neutral with water, followed by immersion in 600L of 50% sodium hydroxide solution and reaction at 100 ° C for 5 hours. Then washed with water until neutral, dried and prepared 10.2 kg of chitosan.

Example 2

3 g of distilled water was added to 105 g of chitosan of Example 1, the mixture was stirred for 30 minutes, the temperature was raised to 60 ° C., and the amount of tartaric acid was added to 0.12 mol, followed by stirring for 1 hour to prepare a chitosan tartarate solution.

Example 3

After adjusting the temperature of the chitosan tartrate solution of Example 2 to 45 to 55 ℃ and 200 hours of chitosan hydrolase was added to the reaction for 24 hours, the temperature of the reaction solution was adjusted to 60 ℃, so that the final concentration is 0.1 mol After adding calcium carbonate and stirring for 30 minutes, the reaction solution was filtered and then lyophilized to prepare a low molecular weight chitosan oligosaccharide powder.

Viscosity measurement experiments were carried out on the chitosan prepared according to Example 1 as follows.

Experimental Example 1

2.5 g of chitosan of Example 1 was added to 497.5 ml of distilled water, followed by stirring for 20 minutes to disperse. Then, 2.5 ml of glacial acetic acid (reagent 1) was added and stirred for 60 minutes at a rotational speed of 500 rpm, followed by standing at room temperature for 18 hours. The solution was stirred at 500 rpm for 30 minutes and the sample solution was placed in a constant temperature dryer controlled at 20 ° C. to a temperature of 20 ° C., and the solution viscosity was measured using a digital viscometer (LVDV-II +, Brookfield). cps.

Experimental Example 2

The composition analysis of the low molecular weight chitosan oligosaccharide of Example 3 was analyzed by high performance liquid chromatography using NH2-60 reversed phase column (4.6X250 mm). 10 mg of the chitosan oligosaccharide powder of Example 3 was dissolved in 10 ml of 60% acetonitrile solution, and 10 µl of which was injected into the column, and eluted with 60% acetonitrile by adjusting the outflow rate to 1.0 ml per minute. A refractive index detector was used for the detection of chitosan oligosaccharides. The results are shown in FIG. 1, and the results of FIG. 1 are shown in Table 1 in detail.

TABLE 1

Minutes Peak name Content according to area ratio (%) 3.952 Dimer 1.201 4.258 Trimer 12.724 4.608 Tetramer 19.193 5.045 Pentamer 32.447 5.605 Hexamer 15.192 6.282 Heptamer 8.814 7.125 Octamer 6.949 7.742 Nonamer 1.163 8.553 Decamer 2.317

Experimental Example 3

0.4 g of the low molecular weight chitosan oligosaccharide sample of Example 3 was precisely taken, degreased if necessary, dissolved in water and neutralized if the liquid acid was acidic. 100 ml of water was added to the solution, and centrifuged at 3000 rpm for 10 minutes, and the supernatant was used as the sample solution. 1.0 ml was taken in a test tube with a stopper, 2.0 ml of acetyl acetone solution was added, mixed, heated at 96 ° C. for 1 hour, cooled in running water, 20.0 ml of 96% (v / v) ethanol was added thereto, and ρ-dimethylaminobenzaldehyde solution was added. 2.0 ml was added and mixed, and the absorbance was measured at a wavelength of 530 nm after 2 hours at room temperature.

Separately, 1.0 ml of a solution containing 100-500 µg / ml of D-glucoseamine or N-acetyl D-glucoseamine was taken and the same procedure as described above was performed to measure absorbance at 530 nm. As a result, the low molecular weight chitosan oligosaccharide content of Example 3 was 94%.

As described above, since the present invention uses a biological method by treating enzymes, it is safe for living organisms and has no problem of environmental pollution, and efficiently and economically solves conventional taste problems by efficiently removing residual acid groups. It is effective to provide a method for producing phosphorus chitosan oligosaccharides.

Claims (3)

Preparing a chitosan salt solution by dissolving chitosan in 0.05-0.5 mol of tartaric acid at 40-80 ° C. to dissolve the chitosan at a concentration of 2-7%; Decomposing chitosan by adding chitosan degrading enzyme to the chitosan salt solution; Method for producing a high-purity chitosan oligosaccharides comprising the step of preparing low molecular weight chitosan oligosaccharides by adding calcium carbonate to the decomposed chitosan-containing mixture by heating and stirring, followed by filtration and drying to remove unreacted chitosan and tartaric acid. According to claim 1, wherein the temperature of the chitosan degrading enzyme addition step is 30 to 55 ℃, the ratio of chitosan to enzyme is in the range of 5 to 20ml of chitosan 1% solution to 1 unit of enzyme, the reaction time is 12 to 48 hours High purity chitosan oligosaccharide manufacturing method characterized in that the range. The method of claim 1, wherein the calcium carbonate addition step is added with 0.05 to 0.5 mol of calcium carbonate, heated and stirred at 40 to 80 ° C. for 0.5 to 2 hours, and then the filtrate filtered to 0.2 to 2 μm is dried and unreacted chitosan. And chitosan oligosaccharide manufacturing method characterized in that the step of removing tartaric acid.