WO2020037866A1 - Low molecular weight chitooligosaccharide and preparation method therefor - Google Patents

Abstract

A low molecular weight chitooligosaccharide and a preparation method therefor, comprising the steps of dissolving chitosan in an acetate-sodium acetate buffer solution, preparing a chitosan solution, adding a compound enzyme solution into the chitosan solution for enzymatic digestion, dialyzing a filtrate by using dialysis after the completion of enzymatic digestion, concentrating an obtained dialysate and then spray-drying, and so on. The described preparation method has the advantages of the yield of low molecular weight chitooligosaccharide being high, conditions being simple, time required for preparation being short, the enzymes used being cheap and easy to obtain, and production costs being low, thus being suitable for wide-scale promotion and application.

Description

Low molecular weight chitooligosaccharide and preparation method thereof Technical field

The invention belongs to the field of oligosaccharide preparation methods, and particularly relates to a low molecular weight chitooligosaccharide and a preparation method thereof.

Background technique

Chitosan is the only positively-charged, alkaline, water-soluble polysaccharide found in nature at present. It has superior biological activity and can be prepared from chitin and chitosan. Chito-oligosaccharides with different molecular weights have different functions. Chito-oligosaccharides with a molecular weight of about 5000 Da have the function of lowering blood lipids. Chito-oligosaccharides with a molecular weight of about 3,000-2000 Da can inhibit the growth and metastasis of tumor cells. Chitooligosaccharides below 1000 Da have unique physiological activities and functions, including enhancing humoral immunity and cellular immunity; promoting calcium absorption and bone health; antibacterial, bacteriostatic, and significant moisture-absorbing capacity.

At present, chitosan is mainly produced by enzymatic degradation of chitosan. The enzymatic hydrolysis method has the advantages of specificity, mild reaction conditions, easy control of the process, and less environmental pollution. At present, the commonly used enzymes can be divided into specific enzymes and non-specific enzymes. Specific enzymes have good enzymatic hydrolysis, but they are expensive. Some non-specific enzymes such as papain and bromelain are less expensive. The hydrolysis effect is not good. For example, Chinese Patent Publication No. CN107739418A discloses a method for preparing chitosan oligosaccharides by freeze-drying papain. Although the method has a short enzymolysis time, the molecular weight of the product is about 3000 Da; China with publication number CN107988287A The patent discloses a method for preparing chitooligosaccharides by spray drying of cellulase. The method uses cheap and easy-to-obtain enzymes, but has problems such as long enzymatic hydrolysis time.

Tannase, also known as tanninase, is a tannin hydrolase. The enzyme can be produced by molds such as Aspergillus niger and Aspergillus oryzae. At present, it is mainly used for processing tannin and protein in beer to make it clear and transparent. However, its role in enzymatic hydrolysis of chitosan is not mentioned.

So far, there have been no reports on the use of fig protease and tannase as complex enzymes to hydrolyze chitosan to produce chitooligosaccharides.

Summary of the Invention

In view of the shortcomings of the prior art, the object of the present invention is to provide a chitooligosaccharide and a preparation method thereof. The method for preparing chitosan oligosaccharides provided by the present invention has mild conditions, is simple, requires short preparation time, has a high yield of chitosan oligosaccharides with a molecular weight of about 1000 Da, does not need to add a large number of reaction reagents, has less environmental pollution, and uses figs Proteases and tannins are inexpensive and readily available.

The technical scheme of the present invention is: a method for preparing a low molecular weight chitooligosaccharide, comprising the following steps:

S1. Dissolving chitosan in an acetate-sodium acetate buffer solution to prepare a chitosan solution with a mass concentration of 0.5 to 1.5%;

S2. Add a complex enzyme to the chitosan solution obtained in step S1. The mass ratio of the mass of the complex enzyme to the mass of the chitosan is 2.5 to 25%. The reaction is performed at 65 ° C for 1 to 3 hours to obtain an enzymatic hydrolysis solution. Fig protease and tannin are composed in a weight ratio of 1: (0.1 to 1);

S3. Add 5% NaOH solution with a mass concentration of 5% to the enzymatic hydrolysis solution obtained in step S2 with stirring, adjust the pH to 7.0 to 8.0, filter, and separate the water-insoluble chitosan. The filtrate is placed in a dialysis bag and dialyzed in distilled water. 24h, get dialysate;

S4. The dialysate obtained in step S3 is concentrated, concentrated to a concentrated solution having a solid content concentration of 9 to 15%, and spray-dried.

Further, the complex enzyme in the step S1 is composed of fig protease and tannin in a weight ratio of 1: 0.3.

Further, the pH value of the acetic acid-sodium acetate buffer solution in step S1 is 5.0, and the concentration is 0.2 mol / L.

Further, the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 5-10%.

Further, the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 7.5%.

Further, in step S2, the reaction is performed at 65 ° C for 2h.

Further, the dialysis bag in step S3 is a regenerated cellulose dialysis bag with a molecular weight cut off of 1000 Da.

Further, the solid content mass concentration of the spray-dried concentrated liquid in the step S4 is 10%.

Further, the process conditions for spray drying in step S4 are: an inlet air temperature of 180 ° C., and a feed rate of 700 mL / h.

The invention uses fig protease and tannin to hydrolyze chitosan, which can obtain more small molecular weight chitosan oligosaccharides with a molecular weight of about 1000 Da in a short period of time, and the average yield can reach 39.47%, which significantly shortens the enzyme. Solution time and reduce costs. However, when the fig protease or the tannase is used alone, the same or similar effects as those described above cannot be obtained.

Compared with the prior art, the present invention has the following beneficial effects:

The method provided by the invention has mild conditions, the complex enzyme used is cheap and easy to obtain, the production cost is low, the time required for preparation is short, and more importantly, the yield of low molecular weight chitooligosaccharide is higher, and significant progress has been achieved compared with the prior art. .

detailed description

The following further describes the present invention through the description of specific embodiments, but this is not a limitation on the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but as long as it does not depart from the basics of the present invention The ideas are all within the scope of the present invention.

Chitosan (batch number: 171112A, DD85%, Shandong Aokang Biotechnology Co., Ltd.); fig protease (Tixi Aihuacheng Industrial Development Co., Ltd.); tannase (Henan Xihe Chemical Co., Ltd.); Sheng Ocean Technology Co., Ltd.); regenerated cellulose dialysis bag (cut-off molecular weight 1000 Da, US MYM Biotechnology Company).

Example 1. Preparation of low molecular weight chitooligosaccharides

S1. Dissolve chitosan in a 0.2 mol / L acetic acid-sodium acetate buffer solution with a pH value of 5.0 to prepare a chitosan solution with a mass concentration of 1.0%;

S2. Add a 0.5% complex enzyme solution to the chitosan solution obtained in step S1. The complex enzyme is composed of fig protease and tannin in a weight ratio of 1: 0.3. It was 7.5%, and reacted at 65 ° C for 2h to obtain an enzymatic hydrolysis solution;

S3. Add 5% NaOH solution with a mass concentration of 5% to the enzymatic hydrolysis solution obtained in step S2 under stirring, adjust the pH to 7.0-8.0, filter, separate the water-insoluble chitosan, and fill the filtrate with regeneration with a molecular weight of 1000 Da. In a cellulose dialysis bag, dialysis was performed in distilled water for 24 hours to obtain a dialysate;

S4. The dialysate obtained in step S3 is concentrated, concentrated to a concentrated solution with a solid content concentration of 10%, and spray-dried. The process conditions for spray-drying are: an inlet air temperature of 180 ° C and a feed rate of 700 mL / h.

The complex enzyme solution is prepared from a 0.2 mol / L acetate-sodium acetate buffer solution with a pH value of 5.0.

Example 2. Preparation of low molecular weight chitooligosaccharides

The difference between Example 2 and Example 1 is that in step S1, the mass concentration of the chitosan solution is 1.2%, and the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 5%. The complex enzyme is composed of fig protease and Tannase was composed by a weight ratio of 1: 0.1, and the rest was the same as in Example 1.

Example 3 Preparation of low molecular weight chitooligosaccharides

The difference between Example 3 and Example 1 is that in step S1, the mass concentration of the chitosan solution is 1.2%, and the mass ratio of the compound enzyme to the mass of chitosan in step S2 is 7.5%. The compound enzyme is composed of fig protease and Tannase was composed by a weight ratio of 1: 1, and the rest was the same as in Example 1.

Comparative Example 1. Preparation of low molecular weight chitooligosaccharide

Compared with Example 1, this comparative example is different in that the enzyme used is fig protease, and the rest are the same as in Example 1.

Comparative Example 2: Preparation of low molecular weight chitooligosaccharides

Compared with Example 1, this comparative example is different in that the enzyme used is tannin, and the rest are the same as in Example 1.

Comparative Example 3, Preparation of low molecular weight chitooligosaccharides

Compared with Example 1, this comparative example is different in that Tannase is replaced with cellulase, and the rest are the same as in Example 1.

Test example one, product performance measurement

The moisture content, reducing sugar content, 5% aqueous solution viscosity, and 1000 Da chitooligosaccharide yield of the chitosan samples prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested, and the corresponding chitosan raw materials were used. As a control group, the test results are shown in Table 1 below.

Table 1 test results

Group	Moisture (%)	Reducing sugar content (%)	5% aqueous solution viscosity	Yield (%)
Example 1	5.02	62.71	1.38	39.47%
Example 2	5.15	61.03	1.41	37.36%
Example 3	5.29	64.69	1.35	38.54%
Comparative Example 1	5.18	43.74	1.42	20.06%
Comparative Example 2	5.00	30.65	1.43	11.57%
Comparative Example 3	5.23	56.53	1.40	25.36%
1000Da Chitosan API	4.96	57.66	1.42	-

It can be known from Table 1 that the difference between Comparative Example 1 and Comparative Example 2 and Example 1 is only that the composite degrading enzyme is replaced by a single enzyme of the two, but the yield of 1000 Da chitooligosaccharide is much lower than that of Example 1. The yield of complex degrading enzymes, especially the tannin digestion, was only 11.57%. In addition, compared with Example 3, although both use the complex enzymatic hydrolysis of chitosan, The enzymatic hydrolysis performance of the fig enzyme and the cellulase complex enzyme used in the ratio 3 is not as good as the fig protease and tannin complex enzymes described in Examples 1 to 3. In summary, the present invention has achieved unexpected effects.

What has been disclosed above are only preferred embodiments of the present invention, and of course, the scope of rights of the present invention cannot be limited by this. Therefore, equivalent changes made in accordance with the scope of patent application of the present invention still fall within the scope of the present invention.

Claims (10)

1. A method for preparing low molecular weight chitooligosaccharides, which comprises the following steps:

   S1. Dissolving chitosan in an acetate-sodium acetate buffer solution to prepare a chitosan solution with a mass concentration of 0.5 to 1.5%;

   S2. Add a complex enzyme to the chitosan solution obtained in step S1. The mass ratio of the mass of the complex enzyme to the mass of the chitosan is 2.5 to 25%. The reaction is performed at 65 ° C for 1 to 3 hours to obtain an enzymatic hydrolysis solution. Fig protease and tannin are composed in a weight ratio of 1: (0.1 to 1);

   S3. Add 5% NaOH solution with a mass concentration of 5% to the enzymolysis solution obtained in step S2 under stirring conditions, adjust the pH to 7.0-8.0, filter, separate the water-insoluble chitosan, and place the filtrate in a dialysis bag. Dialyzed for 24 hours in distilled water to obtain dialysate;

   S4. The dialysate obtained in step S3 is concentrated, concentrated to a concentrated solution having a solid content concentration of 9 to 15%, and spray-dried.
2. The method for preparing a low-molecular-weight chitooligosaccharide according to claim 1, wherein in step S1, the complex enzyme is composed of fig protease and tannin in a weight ratio of 1: 0.3.
3. The method for preparing a low-molecular-weight chitooligosaccharide according to claim 1, wherein in the step S1, the pH value of the acetate-sodium acetate buffer solution is 5.0 and the concentration is 0.2 mol / L.
4. The method for preparing a low molecular weight chitooligosaccharide according to claim 1, wherein the mass ratio of the mass of the complex enzyme to the mass of chitosan in the step S2 is 5-10%.
5. The method for preparing a low-molecular-weight chitooligosaccharide according to claim 4, wherein the mass ratio of the complex enzyme to the mass of chitosan is 7.5%.
6. The method for preparing a low-molecular-weight chitooligosaccharide according to claim 1, wherein in step S2, the reaction is carried out at 65 ° C for 2 hours.
7. The method for preparing a low molecular weight chitooligosaccharide according to claim 1, wherein the dialysis bag in the step S3 is a regenerated cellulose dialysis bag with a molecular weight cut-off of 1000 Da.
8. The method for preparing a low-molecular-weight chitooligosaccharide according to claim 1, wherein the step S4 is concentrated to a concentrated solution having a solid content concentration of 10%.
9. The method for preparing a low-molecular-weight chitooligosaccharide according to claim 1, wherein the spray drying process conditions in the step S4 are: an inlet air temperature of 180 ° C, and a feed rate of 700 mL / h.
10. A chitooligosaccharide prepared by the method according to any one of claims 1 to 9.