KR20010054390A - The processing method of chito oligosaccharides using enzyme - Google Patents

Abstract

PURPOSE: A method for producing chito oligosaccharides using enzymes is provided, thereby high purity chito oligosaccharides can be produced and their preservability and stability are improved by the inhibition of brown reaction. CONSTITUTION: The method for producing chito oligosaccharides comprises the steps of: mixing chitosan powder with water in a vessel; adding acid solution into the mixture and adding enzyme into the mixture to decompose chitosan; transferring the mixture through microfilter into a cooler, cooling the mixture, and ultrafiltering it to separate oligosaccharides having low molecular weight; and passing the oligosaccharides of low molecular weight through anionic exchange resin in the flow rate of 10 to 20 m/h to remove acid components.

Description

The processing method of chito oligosaccharides using enzyme}

The present invention relates to a method for preparing chito oligosaccharides, and more particularly, to prevent the browning phenomenon of the finally prepared oligosaccharide product, an enzyme capable of producing chitosan oligosaccharides having excellent long-term integrity and stability as well as high purity It relates to a method for producing chito oligosaccharides used.

In general, chitin is N-acetylglucopyranose, in which N-acetyl group (-N-acetyl) is bonded instead of hydroxyl group (-OH) of glucopyranose No. 2 carbon, a basic monosaccharide constituting cellulose. (N-acetylglucopyranose) has a β-1,4 bonded structure continuously and is contained in large quantities in shellfish such as crabs and shrimps.

Deacetylation of chitin of the above structure with strong alkali changes the acetyl group to an amine group to obtain chitosan. The chitosan and chitin are industrial wastewater treatment agents or heavy metal adsorbents. Carriers, resins for chromatography and functional membranes, pesticides as agricultural materials, a variety of uses, such as artificial skin as a medical material has been of great interest.

Recently, it has been found that chitin and chitosan and its derivatives have various physiological activities such as anticancer activity, antimicrobial activity, body cholesterol improvement and hypertension suppression by immunopotentiation and reactivation. It's going on.

However, chitin and chitosan itself is a high molecular material, as in the case of cellulose, which is not digested and absorbed in the human body, and is discharged as it is. At present, chito oligosaccharides prepared in the form of low molecular weight oligosaccharides by decomposing the chitin and chitosan are present. By using it, the digestive absorption is possible in the human body.

The method for preparing chito oligosaccharides can be broadly divided into a method by acid hydrolysis and a method by enzymatic decomposition.

First, a general method for preparing chito oligosaccharides by acid hydrolysis is hydrolyzing at high temperature by adding concentrated hydrochloric acid or sulfuric acid to chitosan, and then separating low molecular weight oligosaccharides.

However, the above-described method has a problem that the oligosaccharide content of 5 or more saccharides having excellent physiological activity is very low, and the low molecular oligosaccharides of less than 5 saccharides having low physiological activity are produced in a large amount, and the oligosaccharides prepared contain a large amount of harmful substances (acid components) There is a problem that can be.

Therefore, a method for preparing chito oligosaccharides by enzymatic digestion has been proposed to increase oligosaccharide content of at least 5 saccharides. The general method for enzymatic digestion is to dissolve chitosan in an acid solution to maintain an appropriate pH, and then to prepare an enzyme in a solution. After the addition of the reaction for a certain time to decompose chitosan is a method of producing an oligosaccharide through a separation process.

Referring to the accompanying drawings in more detail with reference to the accompanying drawings, Figure 2 is a reaction vessel 11 equipped with a stirring fan 12 as shown in Figure 2 as a manufacturing process of chito oligosaccharides using a conventional enzyme. Into the chitosan powder raw material with water and stirred for 30 minutes while maintaining the temperature at 30 ℃ to 40 ℃ using a thermostat (13), after adjusting the pH to pH 4.2 to pH 5.3 by adding an acid Agitate for 20 to 24 hours to decompose chitosan, pass the decomposition mixture through a microfilter 14 to the cooler 15, cool to 15 ° C. to 20 ° C., and then back to the ultrafilter 16 and the nanofilter. The low molecular weight oligosaccharide is separated through (17).

Put the chitosan powder raw material together with water in the reaction vessel (11) equipped with a stirring fan 12 and stirred for 30 minutes while maintaining the temperature at 30 ℃ to 40 ℃ using a thermostat 13, such as hydrochloric acid Acids such as inorganic acids and organic acids such as acetic acid are added to adjust the pH to 4.2.

At this time, the reaction temperature and pH are general conditions that allow the enzyme to be added to decompose chitosan under optimum conditions, and acid is usually added to dissolve insoluble chitosan.

As described above, chitosan and acid are mixed, followed by an enzyme such as chitosanase and the like, followed by stirring for 20 to 24 hours to obtain a chitosan decomposition mixture, wherein high molecular weight chitosan is decomposed into low molecular weight chito oligosaccharides by an enzyme. Will be.

The decomposition mixture is passed through the micro filter 14 to the cooler 15 to be cooled to 15 ° C. to 20 ° C., in particular, when the decomposition mixture passes through the micro filter 14, impurities having a relatively large size of 1 μm or more are removed. In addition, if the transfer to the cooler 15 is cooled to 15 ℃ to 20 ℃ will reduce the activity of the enzyme is not greatly degraded.

The cooled mixture is passed through the ultra filter 16 and the nano filter 17 to separate the low molecular weight oligosaccharides, wherein the ultra filter 16 separates the chito oligosaccharides having a molecular weight of less than 20,000, When the nano filter 17 is passed again, a low molecular weight chito oligosaccharide, which is a final product, is obtained.

However, the chito oligosaccharide prepared by the above-mentioned conventional manufacturing method does not completely stop or remove the activity of the enzyme, so that the enzyme is contained in the product, and the acid component added to dissolve the raw material is not completely removed. Therefore, the final product will show a pH of about 4 to pH 6.

Since the pH of the final product satisfies the conditions for the activity of the enzyme, the time lapse occurs because the chito oligosaccharide is not only continuously degraded but acidified by the enzyme that has not been removed in the final manufactured product, and thus the decomposition of the chito oligosaccharide is caused. As a result, a large amount of oligosaccharides less than 5 saccharides with low bioactivity are generated, and browning of the final product occurs, as well as a problem of lowering the purity of the final product.

Accordingly, the present invention is to solve the above-mentioned problems, and to prevent the browning phenomenon of the finally produced oligosaccharide not only excellent long-term integrity and stability, but also using a chito using an enzyme to prepare a high-purity chito oligosaccharides Its purpose is to provide a method for preparing oligosaccharides.

1 is a manufacturing process diagram of the chito oligosaccharides using the enzyme of the present invention.

Figure 2 is a production process diagram of chito oligosaccharides using a conventional enzyme.

<Explanation of symbols for the main parts of the drawings>

1: reaction vessel 2: stirring pan 3: thermostat

4 micro filter 5 cooler 6 ultra filter

7: anion exchange resin

The present invention for achieving the above object is to put chitosan powder raw material with water in the reaction vessel and stirred, add acid solution, and then add the enzyme to decompose the chitosan by stirring, to the cooler through the decomposition mixture microfilter In the method for producing a chito oligosaccharide using an enzyme that separates the low-molecular-weight oligosaccharides by passing through the filtration device such as ultra-filter after cooling to transfer, the low-molecular-weight oligosaccharides passed through the filtration device such as filter 10 It can achieve by providing the manufacturing method of the chito oligosaccharide using the enzyme by removing an acid component by passing an anion exchange resin at the flow rate of -20 m / h.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a manufacturing process diagram of a chito oligosaccharide using the enzyme of the present invention.

As shown in FIG. 1, the present invention puts a chitosan powder raw material together with water in a reaction vessel 1 equipped with a stirring fan 2, and maintains the temperature at 30 ° C. to 40 ° C. using a thermostat 3 for 30 minutes. After stirring, the acid was added to adjust pH to pH4.2 to pH5.3, the enzyme was added thereto, and the mixture was stirred for 20 to 24 hours to decompose the chitosan, and the decomposition mixture was passed through a microfilter 4 to cooler (5 After cooling to 15 ℃ to 20 ℃, and then through the ultra filter 6 and the anion exchange resin (7) to remove the low molecular weight oligosaccharides.

Put the chitosan powder raw material together with water in the reaction vessel (1) equipped with a stirring fan (2) and stirred for 30 minutes while maintaining the temperature at 30 ℃ to 40 ℃ using a thermostat (3), such as hydrochloric acid Acids such as inorganic acids or organic acids such as acetic acid are added to adjust the pH to 4.2 to pH 5.3 so that the added enzyme can decompose chitosan under optimal conditions.

As described above, the chitosan and the acid are mixed, followed by addition of an enzyme such as a chitosanase, and the mixture is stirred for 20 to 24 hours to decompose the high molecular weight chitosan into low molecular weight chito oligosaccharides.

The decomposition mixture decomposed to low molecular weight is subjected to a micro filter 4 to remove impurities having a relatively large size of 1 μm or more, and then transferred to the cooler 5 and cooled to 15 ° C. to 20 ° C. to decompose chitosan by enzymes. To lower the

The cooled mixture is further separated through a filtration device such as an ultra filter (6) to separate the chito oligosaccharide having a molecular weight of less than 20,000 to remove the enzyme.

The low molecular weight chito oligosaccharides passed through a filtration device such as an ultra filter 6 are passed through the anion exchange resin 7 at a flow rate of 10 m / h to 20 m / h, wherein the low molecular weight chito oligosaccharides are anion exchange resin ( When passing through 7) the anionic impurities contained in the oligosaccharides are attached to the anion exchange resin (7), and only pure chito oligosaccharides are passed to increase the purity of the final product.

In addition, the anionic material is removed by the anion exchange resin (7) in the case of the conventional manufacturing method, while the final product exhibits an acidity of pH 4 to pH 6, in the case of the present invention, the final product is close to neutral at pH 6.8 to pH 7.5 do.

Therefore, the pH of the final product according to the present invention is close to neutral, and since the enzyme is removed, the decomposition of the chito oligosaccharides is prevented so that browning of the product no longer occurs, so that long-term integrity and stability is possible.

As described above, in order to separate a product having high purity and close to neutrality of pH 7, it is preferable that the flow rate of the anion exchange resin 7 is 10 m / h to 20 m / h, in which case the purity is less than 10 m / h. It has the advantage of high pH and close to neutral, but it is inefficient because it takes a long time to separate, and when the flow rate exceeds 20m / h, the anionic impurities are not attached to the anion exchange resin (7) and a large amount of impurities Since this ion exchange resin passes through, there is a problem that the purity decreases and that the separated product exhibits an acidity of less than pH 7.

Hereinafter, the present invention will be described in detail with reference to the following examples and comparative examples, but the present invention is not limited to the following examples.

<Example 1>

6 parts by weight of chitosan powder raw materials were added together with 100 parts by weight of water to a reaction vessel equipped with a stirring fan, and stirred at 35 ° C. for 30 minutes. The mixture was adjusted to pH 4.5 by adding 2 parts by weight of acetic acid and acetic acid, respectively, and 0.01 weight of chitosanase enzyme. After addition, the mixture was stirred for 24 hours, and then the mixture was passed through a micro filter to a cooler, cooled to 15 ° C., passed through an ultra filter, and passed through an anion exchange resin at a flow rate shown in Table 1 below to obtain a low molecular weight oligosaccharide. After separation, the browning phenomenon and pH were measured by the following method using the separated oligosaccharides, and are shown in Table 1 below.

-pH Measurement Method-

PH was measured with a pH meter from Denver instrument.

-Browning phenomenon-

20 ml of the separated oligosaccharide solution was placed in a beaker, sealed, and placed in a thermostat maintained at 35 ° C. to determine the time required for browning to occur.

Comparative Example 1

6 parts by weight of chitosan powder raw materials were added together with 100 parts by weight of water to a reaction vessel equipped with a stirring fan, and stirred at 35 ° C. for 30 minutes. The mixture was adjusted to pH 4.5 by adding 2 parts by weight of acetic acid and acetic acid, respectively, and 0.01 weight of chitosanase enzyme. After addition, the mixture was stirred for 24 hours, and then the mixture was passed through a micro filter to a cooler, cooled to 15 ° C., and passed through an ultra filter and a nano filter to separate low molecular weight oligosaccharides, followed by the same method as in Example 1. The browning phenomenon and pH were measured and shown in Table 1.

division pH Browning phenomenon (time) Example 1 Flow rate of ion exchange resin (m / h) - - 25 6.0 24 20 6.7 96 15 7.2 168 10 7.3 172 Comparative Example 1 4.5 14

As shown in Table 1, when the flow rate of the ion exchange resin was 25 m / h in Example 1, while changing the flow rate of the ion exchange resin, pH was 6.0 and browning occurred after 24 hours. When the flow rate of resin was 20m / h, pH was 6.7 and browning phenomenon occurred after 96 hours. When the flow rate of ion exchange resin was 15m / h, pH was 7.2 and browning phenomenon occurred after 168 hours. When the flow rate of the ion exchange resin was 10 m / h, the pH was shown to be 7.3, and browning phenomenon occurred after 172 hours through Table 1 above.

In particular, when the flow rate is 20m / h or less, the pH shows a value close to neutral, and when the flow rate exceeds 20m / h, it can be seen that the pH shows an acidity of less than 7, which is when the flow rate exceeds 20m / h. Since anionic impurities do not adhere to the anion exchange resin and a large amount of impurities pass through the ion exchange resin as it is, it shows an acidity of less than pH 7 and when the flow rate is less than 20 m / h, the ion exchange resin is in contact with the impurities. The longer the time is, the more anionic impurities are removed, which results in a neutral pH close to 7.

In addition, the time taken for browning phenomenon to appear according to pH shows that the time taken for browning phenomenon to appear shorter when the pH indicates a strong acidic side. As the pH moves toward the neutral side, it takes longer for browning to appear. Can be.

This is because as the pH moves toward the neutral side, the activity of the enzyme contained in the final product is suppressed, thereby preventing the decomposition of chito oligosaccharides by the enzyme, and the browning phenomenon progresses slowly.

However, in Comparative Example 1, which did not use an ion exchange resin, the pH was 4.5, and the time required for browning phenomenon appeared after 14 hours, indicating that chito oligosaccharides were rapidly decomposed.

At this time, comparing the case of Comparative Example 1 and the flow rate of Example 1 is 15m / h it can be seen that the pH difference is very large, which means that in Comparative Example 1 contains a lot of impurities including an anionic material In the case of Example 1, it means that the impurity containing an anionic material is hardly contained, and thus, the purity of the present invention may be high.

As described above, the presently prepared oligosaccharide prevents browning due to complete removal of the enzyme and ion exchange, thereby providing excellent long-term integrity and stability as well as an enzyme capable of producing high purity chito oligosaccharides. It is a useful invention to provide a method for preparing chito oligosaccharides.

Claims (1)

Put chitosan powder raw material together with water and stir in the reaction vessel, add acid solution, add enzyme and stir to decompose chitosan, transfer it to the cooler through the decomposition mixture microfilter, cool, and then again In the method for producing a chito oligosaccharide using an enzyme that separates the low molecular weight oligosaccharides by passing through a filtration device, the low molecular weight oligosaccharides passed through the filtration device such as the filter is again an anion exchange resin at a flow rate of 10 to 20 m / h Method for producing a chito oligosaccharide using an enzyme, characterized in that to remove the acid component by passing through.