KR980008299A - Separation and Purification Method of Low Molecular Chitosan Using Multistage Membrane Process - Google Patents

Abstract

The present invention relates to a method for separating and purifying low-molecular chitosan using a multi-stage membrane process, and more particularly, to a method for separating and purifying low-molecular chitosan, by reacting a chitosan mixture of various molecular weights with an acid and an enzyme with ultrasonic treatment. Adjusting the chitosan to a dispersion degree of 1.5 to 2.5 and an average molecular weight of 100,000 or less; Adding pure water to the reaction to provide an aqueous solution having a concentration of 1 to 50% of chitosan; Primary fractionating and separating chitosan by the continuous membrane separation process at a pressure of 0.5 to 5 kg / cm ² in the aqueous solution; And separating and purifying the low molecular weight chitosan with high purity and high yield by repeating the second fractionation and the separation process one or more times using the separation membrane selected according to the desired molecular weight of the primary fraction and the separated permeate. will be.

Description

[Name of invention]

Separation and Purification Method of Low Molecular Chitosan Using Multistage Membrane Process

Detailed description of the invention

[Technical Field to which the Invention belongs and Prior Art in the Field]

The present invention relates to a method for separating and purifying low-molecular chitosan using a multi-stage membrane process. More specifically, the low molecular weight α-chitosan, which is extracted from shellfish such as shrimp and crabs, is easily obtained at high yield using a multi-stage membrane process. A method for separating and purifying with molecular weight chitosan or oligosaccharide chitosan.

The chemical structure of chitin binds to N-acetylglucosamine (β-1,4 bond) in which N-acetylamide (-NHCOCH ₃ ) group is substituted in place of -OH group in cellulose glucopyranose C-2 position. The deacetylated chitin acetyl group as a polymer of the polysaccharide is called chitosan.

Chitosan is a very useful biomaterial derived from shellfish such as shrimp and crab, and its chemical name is called (1 → 4) 2-amino-2-deoxy-β-D-glucosamine. This is not only very valuable in all fields such as environment, chemistry, agriculture, biology, medicine and food, but also the oligosaccharides are widely used in many research papers. The effectiveness of the disease is widely known for its effectiveness, and there are records from Korea, China, and Japan that have been used as foods and pharmaceuticals for treating diseases.

The chitosan has a 1 → 4 β-D-glucosamine bond, which is very stable, and the chemical separation method is very complicated and has environmental problems. The separation method using liquid chromatography and ion exchange resin columns The chitosan low molecular weight or oligosaccharide fractionation and separation has the disadvantage of high cost and low unit productivity. Therefore, there is a need for a method for separating and purifying low molecular weight chitosan or oligosaccharide chitosan in high yield through a simple physical method.

In the prior art, Japanese Patent Application Laid-Open No. 54-148890 discloses chitosan obtained by deacetylating natural chitin in an aqueous solution of 0.007 to 0.35% hydrogen peroxide adjusted to pH 6-12 to have an arbitrary molecular weight. A method for producing low molecular chitosan is disclosed.

In Japanese Patent Laid-Open No. 1-256395, in a method of producing low molecular weight chitosan by applying an enzyme to chitosan obtained by deacetylating natural chitin, an acid is continuously added to the reaction solution while controlling the addition rate. A method for producing a low molecular weight chitosan having a molecular weight of 340 to 50,000 without D-glucosamine by carrying out an enzymatic reaction while maintaining substantially constant value in the range of 3 to 9 is disclosed.

Japanese Patent Laid-Open No. 2-11601 discloses a method for producing low molecular weight chitosan by reacting 0.1 to 20% by weight of hydrogen peroxide with chitosan by adjusting the pH of the reaction solution to 5 or less with hydrochloric acid or acetic acid. Doing.

Japanese Patent Laid-Open No. 2-200196 discloses a method for producing low molecular weight chitosan by treating chitosan with one or more of neutral proteases or lipases having chitosan resolution.

Japan, Monthly Food Chemistry, February 1993, entitled "Chinese and Chitosan Oligosaccharide Separation and Analytical Techniques," discloses a method for fractionating chitosan using a weakly acidic cation ion exchange resin column.

As described above, the conventional method is to decompose chitosan by acid decomposition of hydrochloric acid, phosphoric acid, nitric acid, hydrogen peroxide treatment or enzyme treatment under acid solution, and then soluble in methanol (CH ₃ OH) to separate oligosaccharide. Dividing into oligosaccharides and insoluble components is repeated several times, and each oligosaccharide is separated by changing the concentration of methanol. Subsequently, a separation method using chromatography and ion exchange resin columns is known.

However, since the preparation of such low molecular weight chitosan or oligosaccharide chitosan is almost obtained by the acid decomposition method, it causes a lot of time and high cost, post-treatment problems, toxicity and environmental problems in production. In other words, existing methods have a disadvantage in that industrialization is difficult because of high productivity, low environmental yield, and low yield during long post-treatment.

[Technical problem to be achieved]

In order to solve this problem, the present inventors have conducted extensive research to isolate and purify pure white low molecular weight chitosan for medical, biological and food by physical method through suppressing chemical treatment as much as possible and using a simple multi-stage membrane process. Could.

Accordingly, an object of the present invention is to provide a method for separating and purifying low molecular weight chitosan that can be used for food and medicine using a multi-stage membrane process.

In the method of the present invention for achieving the above object, in the method for separating and purifying low molecular weight chitosan, a mixture of chitosan having various molecular weights is reacted with an acid and an enzyme with sonication to have a dispersion degree of 1.5 to 2.5, and an average molecular weight of 100,000 or less. Preparing a chitosan solution; Adding pure water to the solution to provide an aqueous solution having a concentration of 1 to 50% of chitosan; Primary fractionating and separating chitosan by a continuous membrane separation process using a membrane at a pressure of 0.5 to 5 kg / cm ² in the aqueous solution; And repeating the first fractionation and separation process one or more times using the membrane selected according to the desired molecular weight of the first fraction and the separated permeate.

[Configuration and Function of Invention]

Hereinafter, the method of the present invention will be described in more detail.

First, chitosan obtained from the shells of shrimp and crab is treated with acid to adjust the viscosity to 1-15 cps, and then the chitosan is treated with various acids and enzymes for acid decomposition and solubilization. At this time, when chitosan is treated with an acid and an enzyme, sonication is performed in parallel. The sonication is preferably economically performed at a frequency in the range of 40 to 60 kHz for about 10 to 120 minutes, preferably about 30 to 60 minutes. The ultrasonic treatment has the advantage of shortening the acid and enzyme treatment time by maximizing the acid and enzyme treatment efficiency for chitosan. Acid and enzyme treatment for chitosan is to obtain a water-soluble oligomer suitable for the membrane separation process, which is a subsequent step, it is preferable that the chitosan dispersion degree is carried out to 1.5 to 2.5, the average molecular weight of about 100,000 or less. The reaction is stopped by additionally adding an inorganic acid and an organic acid to the reaction solution or raising the temperature to deactivate the enzyme.

Then, after vacuum drying, the chitosan was dissolved in pure water to adjust the concentration of chitosan to 1-50%, and the pH was adjusted to 6-8 to suit the subsequent membrane separation process, 0.5-5kg / cm Primary fractionation and separation by a continuous membrane separation process at a pressure of ² . Subsequent separations can be made in high yield of low molecular weight chitosan in the desired molecular weight range using a separator having an appropriate fractional molecular weight. The driving pressure at this time is preferably 0.5 to 5 kg / cm ² . In addition, by repeatedly performing the separator process, the desired low molecular weight chitosan can be obtained in high purity and high yield. In addition, the low molecular weight chitosan obtained by the above-described method is further purified by alcohol (C ₂ H ₅ OH) and dried, and then lyophilized to obtain a more pure low molecular weight chitosan.

Fraction and separation process according to the present invention is to adjust the molecular weight distribution of chitosan by using a physical multi-step membrane process conveniently without chemical treatment, the average molecular weight of chitosan can be obtained in the range of less than 10,000 and the degree of dispersion of 1.5 to 2.5 have. In addition, by repeating the separator process, chitosan or oligosaccharide having a lower molecular weight, which is more pure and nontoxic, may be produced at a high yield per unit time. Preferably, the average molecular weight of chitosan after the continuous membrane separation process is 10,000 or less, and the average molecular weight of chitosan after the secondary separation and purification process is about 1,000 or less, depending on the molecular weight fraction of the membrane used.

The various acids used in the present invention include, for example, inorganic acids (sulfuric acid, nitric acid, hydrochloric acid or phosphoric acid), organic acids (citric acid, vinegar or formic acid), or mixed acids thereof, and the concentration of the acid may be combined with ultrasonic treatment. In this case, it can be used at low concentration of 0.1-1.0N.

Examples of the enzyme include chitosanase, chitinase, cellulase, lysozyme, or a mixed enzyme thereof, and are added in an amount of 0.01 to 25 wt% based on chitosan as a substrate.

On the other hand, oligosaccharide is a generic term for sugars from the disaccharide to the 10-saccharide sugar as a single sugar glycosidic bond. Oligosaccharides are produced by hydrolysis of polysaccharides, and the hydrolyzed components mostly belong to low molecular weight oligosaccharides. It is also a polymer produced by polymerizing to a low degree, and is called an oligomer because the molecular weight is low, and an oligomer is also referred to as oligosaccharide.

The oligosaccharides obtained from chitosan by the multistage continuous membrane process according to the present invention are classified according to their type and molecular weight as shown in Table 1 below.

Table 1

The oligosaccharides of chitosan usually have a -Cl group, contain N of free amino groups, and the pH of the aqueous solution that can be used in food is known to be 5.7-7.4. The low molecular weight chitosan used for medical and food use usually has a molecular weight of 10,000 or less.

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

Example 1

The viscosity of chitosan obtained from shrimp and crab was adjusted to about 1-15 cps with hydrochloric acid. Viscosity was measured using a spindle rotational viscometer (Brookfield Co. USA, Model DV-II). Chitosan acid was injected by injecting 0.1-1 N of mixed acid and chitosanase mixed with acetic acid and formic acid at a volume ratio of 1: 1 to 0.1-3 wt% with respect to chitosan, and ultrasonically at about 40 kHz for about 30 to 60 minutes. Was detached. Treatment conditions and the average molecular weight of the obtained chitosan are shown in Table 2 below.

TABLE 2

A solution of chitosan C5 with an average molecular weight of 78,000-80,000 was dried under reduced pressure. The dried chitosan was dissolved in pure water (conductivity: 0.445) to prepare an aqueous solution of about 20% chitosan concentration and adjusted to about pH 7. HFK131 (Koch Co., USA) with a driving pressure of about 0.5-5 kg / cm ² ) Was applied to a continuous membrane separation pilot process. The permeate obtained in this manner had an average molecular weight of 4,500. The permeate was continuously separated with pure white low molecular weight chitosan using ultrafiltration membrane HFK328 (Koch Co., USA) having a driving pressure of about 3 kg / cm ² , and freeze-dried to remove impurities having a deacetylation degree of about 99%. Low molecular weight chitosan with an average molecular weight of 1,000 or less (measured by Gel Permeation Chromatography) was obtained. GPC measurement conditions and deacetylation degree (DA) measurement method is as follows.

※ GPC measurement condition

Model: Waters LC Module with Heating Chamber, I, M410-RI

Ultrahydrogel 250, 1000, Linear Column, M2010 Millennium

Test condition: Flow rate: 1.0 ml / min

Column temperature: 36 ℃

Mobile phase: 0.1M Acetic Acid / 0.1M NaCl

Sample injection volume: 150μl

Sample treatment: dissolved at 0.2 w / v% concentration and filtered through 0.45μl filter

Standard used: Pullulan

※ Deacetylation degree (DA) measuring method

PVSK (Polyvinyl Sulfate Potassium) method was used.

First, 100 ml of 0.5% acetic acid is taken. Melt 0.5 g of the measured chitosan, take 1 g, mix 30 ml of distilled water, add toluidine blue as an indicator, and read the point of discoloration from blue to purple by titration with PVSK. For reference, the formula is as follows.

x = 1/400 × 1/1000 × F × 161 × V

y = 0.5 × 1/100-x

DA = (x / 161) ÷ ((x / 161) + (y / 203)]

Example 2

Chitosan C7 having an average molecular weight of 100,000 to 105,000 treated in Example 1 was separated by passing through a microfiltration membrane (MFK603, Koch Co., USA), followed by an ultrafiltration membrane (HFK328, Koch Co., USA). The chitosan obtained at this time had an average molecular weight of 1,000 and a dispersion degree of 1.5-2.0 as measured by GPC.

Example 3

The chitosan C6 having an average molecular weight of 75,000-79,000 treated in Example 1 was separated by passing only through ultrafiltration membrane (UF) HFK328, or passed through microfiltration membrane (MF) MFK603, and then through ultrafiltration membrane (UF) HFK328. The effect of the multistage membrane separation process on the separation of low molecular weight chitosan is shown in Table 3 below.

TABLE 3

Example 4

Example 1 was performed in the same manner as in Example 1 except that the first filtration membrane MFK603 (Koch Co., USA) having a driving pressure of about 0.5-5 kg / cm ^{2 was} used first. The chitosan permeate of the separation membrane process had an average molecular weight of about 9,000. The average molecular weight of the permeate obtained at this time using the ultrafiltration membrane HFK328 (Koch Co., USA) was about 1,000.

The heavy metals (Pb, As) of the final product thus obtained were quantitatively analyzed. As for the determination of Pb and arsenic, lead was measured at 217 nm and arsenic at 193.7 nm using an atomic absorption spectrometer (Varian Co., SpectrAA-300, USA). It was found that 0.3 ppm or less, and arsenic was 0.1 ppm or less, which is a very good component for food and medicine. For reference, the regulation of heavy metals for food is regulated to less than 5 ppm of lead and 1 ppm of arsenic.

In addition, the antioxidant effect of the final product of Example 1 and Example 2 was compared with vitamin C, a representative antioxidant in the food and cosmetics industry. The final product of Example 1 and Example 2 was prepared by 0.1%, 0.5%, 1.0% and 2.0% solution and tested. The vitamin C was 21.23%, 28.51%, 47.83% and 58.28%, and the final product was 13.84%, 25.50%, 30.98% and 53.47% showed a very stable state and excellent antioxidant capacity. As a device used, a UV absorber (Cary I. UV-Visible Spectrophotometer, Varian Co., USA) was immediately measured at 520-532 nm at room temperature.

[Effects of the Invention]

As described above, the method of the present invention is a method for separating and preparing low molecular weight chitosan using a multi-stage membrane process having an industrial production value that can easily and shortly increase the yield while solving the disadvantages of conventional low molecular weight chitosan production. .

Claims (5)

A method for separating and purifying low molecular weight chitosan, comprising: reacting chitosan mixtures of various molecular weights with an acid and an enzyme with sonication to adjust the chitosan to a dispersity of 1.5 to 2.5 and an average molecular weight of 100,000 or less; Adding pure water to the reaction to provide an aqueous solution having a concentration of 1 to 50% of chitosan; Primary fractionating and separating chitosan by a continuous membrane separation process using a membrane at a pressure of 0.5 to 5 kg / cm ² in the aqueous solution; Separation and purification method of low molecular weight chitosan using a multi-stage membrane process comprising the step of repeating the secondary fraction and separation process one or more times using the separator selected according to the desired fractional molecular weight of the primary fraction and the separated permeate. . The method of claim 1, wherein the ultrasonic treatment is performed for 10 to 120 minutes at 40 to 60 kHz. The method of claim 2, wherein the ultrasonic treatment is performed for 30 to 60 minutes at 40 to 60 kHz. 2. The method of claim 1, wherein the average molecular weight of permeated chitosan after the continuous membrane separation process is 10,000 or less. The method of claim 1, wherein the average molecular weight of permeated chitosan after the secondary fractionation and separation process is 1,000 or less, and the dispersion degree is in the range of 1.5 to 2.5. ※ Note: The disclosure is based on the initial application.