WO2020038077A1 - Chitosan oligosaccharide prepared by compound enzyme and preparation method for chitosan oligosaccharide - Google Patents

Abstract

A chitosan oligosaccharide prepared by a compound enzyme and a preparation method for the chitosan oligosaccharide. The preparation method comprises the following steps: dissolving chitosan into an acetic acid-sodium acetate buffer solution; then adding a compound enzyme solution into the chitosan solution for enzymolysis; after the enzymolysis is finished, dialyzing filtrate by adopting a dialysis method after filtering the obtained enzymolysis solution; finally concentrating and drying the obtained dialysis solution to obtain the chitosan oligosaccharide. The chitosan oligosaccharide is small in molecular weight, concentrated in degree of polymerization and high in yield, and is applicable to the fields of medicines, foods, cosmetics and the like; moreover, the preparation method is simple in operation and low in material costs.

Description

Chitooligosaccharide prepared by composite enzyme and preparation method thereof Technical field

The invention belongs to the field of biotechnology, and particularly relates to a chito-oligosaccharide prepared by a composite enzyme and a preparation method thereof.

Background technique

Chitosan is the product of chitosan degradation to a certain degree. Compared with chitosan, chitosan has better water solubility and lower viscosity under physiological conditions. This is due to the sugar chain of chitosan It is shorter and has a large number of free amino groups. Chito-oligosaccharides contain one amino group and two hydroxyl groups in the repeating unit of glycoside residues. It is the only positively charged cationic basic amino oligosaccharide in nature. These characteristics of chitooligosaccharide have attracted more and more researchers' attention. Many literatures have reported the biological activities of chitooligosaccharides, such as anti-oxidation, anti-inflammatory, antagonistic microorganisms, lowering cholesterol and enhancing immune activity, anti-tumor activity and so on.

There are currently three main methods for producing chitooligosaccharides: chemical, physical, and enzymatic. Enzymatic degradation of chitosan is a method that uses specific or non-specific enzymes to degrade chitosan. Although specific enzymes have good degradation efficiency, most of them are derived from fungal cells or artificial synthesis, such as the intensely hot coccus chitinase synthesized by the whole gene synthesis method. If it is used in industrial large-scale production, it will increase its extraction cost and be difficult to obtain. Therefore, non-specific enzymes with high activity and low price are worthy of further study.

For example, Chinese patent application CN101818181A provides an enzymatic method for preparing chitosan oligosaccharides. A mixed enzyme of pectinase, xylanase, cellulase, and amylase is added to the chitosan solution. An oligochitosan solution was prepared in 1 hour. Chinese Patent Publication No. CN107739418A discloses a method for preparing chitosan oligosaccharides by freeze-drying with papain, which shortens the time for enzymolysis of chitosan. Chinese patent application CN1320124C invented a method for fixing a neutral protease to a hydrogel ball on N-succinylchitosan by a cross-linking method, and then degrading chitosan with an immobilized enzyme to prepare a chitooligosaccharide. However, the molecular weight distribution of chitooligosaccharides obtained by these methods is not uniform, and the degree of polymerization is not concentrated. Studies have shown that chitosan molecules with different molecular weights and degrees of polymerization play different roles in biological activities. For example, chitooligosaccharides with a degree of polymerization of 6 to 8 have good antibacterial, immune enhancement, and antitumor activities. Miscellaneous polymerization of oligosaccharide molecules will severely limit the effect on biological activity.

Therefore, there is an urgent need for a chito-oligosaccharide prepared by a complex enzyme. The chito-oligosaccharide has a concentrated molecular weight and a high degree of polymerization, a high yield, a simple preparation method, a low raw material cost, and a good industrial applicability and practicability. Medicine, food, cosmetics and other fields.

Summary of the Invention

In view of the shortcomings of the prior art, the object of the present invention is to provide a chitosan prepared by a complex enzyme. The chitosan has a concentrated molecular weight and a polymerization degree, a high yield, a simple preparation method, a low raw material cost, and a good industry. Popularity and practicality, more suitable for medicine, food, cosmetics and other fields.

In order to achieve the above objective, the technical solution of the present invention is:

A chito-oligosaccharide prepared by a complex enzyme. The preparation method of the chito-oligosaccharide includes the following steps:

S1, dissolving chitosan in an acetate-sodium acetate buffer solution to prepare a chitosan solution;

S2. Add the complex enzyme to the chitosan solution obtained in step S1, and hydrolyze in a water bath at 45 ° C to 55 ° C for 1 to 3 hours. After the completion of the digestion, place in a boiling water bath, inactivate it for 10 minutes, and cool to room temperature. NaOH solution with a concentration of 5% was adjusted to pH = 7-9, filtered, the filter residue was discarded, and the filtrate was taken;

S3. The filtrate obtained from S2 is dialyzed for 24 hours in a regenerated cellulose dialysis bag, and the dialysate is changed every 8 hours to collect the dialysate. The collected dialysate is heated and concentrated and then spray-dried.

Further, in the step S1, the pH value of the acetic acid-sodium acetate buffer solution is 4.5, and the concentration is 0.2 mol / L.

Furthermore, the mass concentration of the chitosan solution prepared in the step S1 is 0.5 to 1.5%.

Further, the mass ratio of the mass of the complex enzyme to the mass of chitosan in the step S2 is 8-15%, wherein the complex enzyme is composed of pectinase and pepsin in a weight ratio of 1: (0.1 to 1).

Further, the mass ratio of the complex enzyme to the mass of chitosan in step S2 is 10%, wherein the complex enzyme is composed of pectinase and pepsin in a weight ratio of 1: 0.6.

Further, the step S2 is hydrolyzed in a water bath environment at 50 ° C. for 2 h.

Further, the cut-off molecular weight of the regenerated cellulose dialysis bag in step S3 is 3000 Da.

Further, the process conditions for spray drying in the step S3 are: an inlet air temperature of 180 ° C., and a feed rate of 700 mL / h.

In addition, the present invention also provides a method for preparing the chitosan, including the following steps:

I. Dissolve chitosan in an acetate-sodium acetate buffer solution with a pH of 4.5 and a concentration of 0.2 mol / L, and prepare a chitosan solution with a mass concentration of 0.5 to 1.5%;

II. Add the pectinase and pepsin to the chitosan solution obtained in step I at a weight ratio of 1: (0.1 to 1), and the mass ratio of the complex enzyme to the mass of chitosan is 8 to 15 %, Enzymolysis in a water bath at 45 ° C to 55 ° C for 1 to 3 hours. After the completion of the enzymolysis, place in a boiling water bath for 10 minutes to inactivate. After cooling to room temperature, adjust the pH = 7 ~ 9 with a 5% NaOH solution. Filter, discard the filter residue, and take the filtrate;

III. The filtrate obtained in step II is dialyzed in a regenerated cellulose dialysis bag with a cut-off molecular weight of 3000 Da for 24 hours, and the dialysate is changed every 8 hours. The dialysate is collected, and the collected dialysate is heated and concentrated. ℃, spray drying at a feed rate of 700mL / h, that is.

The inventive combination of pectinase and pepsin can degrade chitosan in a short period of time, and can obtain more chitosan oligosaccharides with a molecular weight of 3000 Da and an average degree of polymerization of 6 to 8 in a short period of time. The rate can reach 56.43%, and the method is easy to operate. When pectinase, pepsin, or other complex enzymes are used alone, the same or similar effects as described above cannot be obtained.

Therefore, compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The chito-oligosaccharide prepared by a complex enzyme of the present invention creatively combines pectinase and pepsin to degrade chitosan, improve the enzymolysis efficiency, shorten the enzymolysis time, and the prepared product has a small molecular weight and polymerizes. Degree of concentration, high output.

(2) A method for preparing chitosan oligosaccharides by a complex enzyme of the present invention has simple process, convenient operation, simple source of raw materials, and is beneficial to industrialization.

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.); pectinase (Shanghai McLean Biochemical Technology Co., Ltd.); pepsin (Henan Xihe Chemical Co., Ltd.); chitosan (Qingdao Xiangsheng) Ocean Technology Co., Ltd.); regenerated cellulose dialysis bag (cut-off molecular weight 3000 Da, Shanghai Anpu Experimental Technology Co., Ltd.).

Example 1. Chito-oligosaccharide prepared by a complex enzyme

The preparation method includes the following steps:

S1. Dissolve chitosan in an acetate-sodium acetate buffer solution with a pH of 4.5 and a concentration of 0.2mol / L to prepare a 1% chitosan solution;

S2. To the chitosan solution obtained in step S1 is added a complex enzyme composed of pectinase and pepsin in a weight ratio of 1: 0.6. The mass ratio of the complex enzyme to the mass of chitosan is 10%. After 2 hours of enzymolysis, put in a boiling water bath after enzymatic hydrolysis, inactivate for 10 minutes, cool to room temperature, adjust pH = 8 with 5% NaOH solution, filter, discard the filter residue, and take the filtrate;

S3. The filtrate obtained from S2 is dialyzed for 24 hours in a regenerated cellulose dialysis bag with a cut-off molecular weight of 3000 Da, and the dialysate is changed every 8 hours. The dialysate is collected, and the collected dialysate is heated and concentrated. , Spray drying at a feed rate of 700mL / h, that is.

Example 2. Chito-oligosaccharide prepared by a complex enzyme

The difference between Example 2 and Example 1 is that in step S1, the mass concentration of the chitosan solution is 0.5%, and the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 8%. It is composed of pepsin in a weight ratio of 1: 0.1, and the remaining parameters and operations refer to Example 1.

Example 3, a chitooligosaccharide prepared by a complex enzyme

The difference between Example 3 and Example 1 is that in step S1, the mass concentration of the chitosan solution is 1.5%, and the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 15%. It is made up with pepsin in a weight ratio of 1: 1. For the remaining parameters and operations, refer to Example 1.

Comparative Example 1, a chitooligosaccharide

Compared with Example 1, this comparative example is different in that the complex enzyme is replaced with pectinase, and the remaining parameters and operations refer to Example 1.

Comparative Example 2, a chitooligosaccharide

Compared with Example 1, this comparative example is different in that the complex enzyme is replaced with pepsin, and the remaining parameters and operations refer to Example 1.

Comparative Example 3, a chitooligosaccharide

Compared with Example 1, this comparative example is different in that pepsin is replaced with lignin, and the remaining parameters and operations refer to Example 1.

Test example 1, performance measurement

1. Test materials: Chitosan prepared in Examples 1 to 3 and Comparative Examples 1 to 3.

2. Test method:

The chitosan samples prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested for reducing sugar content, 5% aqueous solution viscosity, and a yield of 3000 Da chitosan oligosaccharides. As a control group, specifically:

DNS reagent (3,5-dinitrosalicylic acid reagent): Weigh 6.3g of 3,5-dinitrosalicylic acid, and dissolve 21.0g of NaOH in 500mL of distilled water, add 182.0g of potassium sodium tartrate, phenol 5.0g (melted in water at 50 ° C), 5.0g of sodium sulfite, stir until fully dissolved, make up to 1000mL. After being fully dissolved, it should be stored in a brown bottle and allowed to stand before use. (This reagent is valid for 1 month)

(1) DNS method for reducing sugar content

Preparation of glucosamine hydrochloride standard curve

Add 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL of 0.1% glucosamine hydrochloride solution in a 10mL colorimetric tube, and then add 1.00mL, 0.80mL, 0.60mL, 0.20 mL, 0.00mL of distilled water, followed by the addition of 1.0mL of DNS reagent, boiling water bath for 5min to develop color, take out and cool to room temperature, make up to 10mL, first use a UV spectrometer to perform a spectral scan to obtain the largest absorption peak at 480nm, and then The absorbance was measured at a wavelength of 480 nm. A standard curve was drawn with the absorbance value on the ordinate and the volume of glucosamine hydrochloride as the abscissa.

Take 0.5mL of the test solution in a 10mL colorimetric tube, add distilled water to 1mL, then add 1.0mL DNS reagent, develop color in a boiling water bath for 5min, cool the water to room temperature after taking out, and make up to 10mL. Using a blank tube as a reference, the absorbance was measured at a wavelength of 480 nm. Substitute the absorbance into the standard curve of glucosamine hydrochloride to calculate the value of reducing sugar content.

(2) Determination of dynamic viscosity

Under the condition of 25 ℃, the dynamic viscosity of the solution to be measured was read directly by using a rotational viscometer during the process of enzymatic degradation of chitosan. In the present invention, an NDJ-8S digital viscometer is used, and a L ₀ rotor uses a digital viscometer to measure the viscosity value of chitosan before and after enzymolysis, and the solution temperature is kept constant during the measurement. As the enzymolysis reaction progresses, the viscosity of the solution gradually decreases. In order to ensure the accuracy of the reading, the rotor should be replaced to keep the reading on the scale between 30 and 80.

(3) Determination of chitosan water

Collect the spray-dried powder in a dish, bake in an oven at a temperature of 50 ° C for 2h, cool in a desiccator for 30min, re-bake for 30min, weigh after cooling, and repeat the baking process until the difference between the two weighings is less than 0.003g. The formula for calculating the moisture of chitosan powder is:

Among them: ω ₁ is the calculated chitosan water content; g is the mass of chitosan for measurement; g ′ is the mass of chitosan after drying.

(4) Calculation of chitosan polymerization degree

Operation method: Take 0.5mL of chitosan test solution and measure absorbance A ₀ according to the above reducing sugar measurement method. Another 0.5 mL of the same test solution was added, 1.5 mL of a 6 mol / L hydrochloric acid solution was added, and the solution was placed in a boiling water bath for 2 h. After neutralizing the hydrolysis solution with an appropriate amount of NaOH solution, it was diluted to 10 mL with distilled water. Take 1 mL of the diluted hydrolysate, measure it according to the method for measuring reducing sugars, and calculate the absorbance to be A _1. The average degree of polymerization of chitooligosaccharide is: n = 10A ₁ / A ₀ .

(5) Calculation of spray-dried chitooligosaccharide yield

The formula for the chitosan yield is:

Among them: m ₁ is the mass of chitosan powder obtained by spray drying; m ₂ is the mass of raw material sample; ω ₁ is the moisture of chitosan powder obtained by spray drying; ω ₂ is the moisture of raw material sample.

3. Test results: See Table 1 for test results.

Table 1 Performance measurement results

It can be known from Table 1 that the chitosan oligosaccharides prepared in Examples 1 to 3 have a low water content, a reducing sugar content of more than 87.95%, a 5% aqueous solution viscosity of less than 1.35, and a polymerization degree distribution between 6 to 8, and the standard The small difference indicates that the polymerization degree distribution is concentrated. From the yield of 3000 Da chitosan oligosaccharides, the chitosan oligosaccharides produced in the examples are greater than 55.54%. Among them, each index of Example 1 is better and is the best embodiment. Compared with the examples, the comparative examples 1 to 2 are different only in that the composite enzyme is replaced with a single enzyme of the two, but the detection effect is far inferior to that of the example in terms of water content, reducing sugar content, and viscosity of a 5% aqueous solution. The larger the degree of polymerization and the larger the standard deviation, it means that the prepared chitosan has a broader degree of polymerization distribution and is relatively heterogeneous. The yield of the 3000 Da chitooligosaccharide is also far inferior to the examples. In addition, compared with Comparative Example 3 and the Examples, although both use a complex enzyme to hydrolyze chitosan, the enzymatic hydrolysis performance of the pectinase and papain complex enzyme used in Comparative Example 3 is not as good as that described in Examples 1 to 3. Pectinase and pepsin complex enzyme. In summary, the present invention has achieved unexpected effects.

The above-mentioned embodiments merely illustrate the principle of the present invention and its effects, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims (10)

1. A chitooligosaccharide prepared by a complex enzyme, characterized in that the preparation method of the chitooligosaccharide includes the following steps:

   S1, dissolving chitosan in an acetate-sodium acetate buffer solution to prepare a chitosan solution;

   S2. Add the complex enzyme to the chitosan solution obtained in step S1, and hydrolyze in a water bath at 45 ° C to 55 ° C for 1 to 3 hours. After the completion of the digestion, place in a boiling water bath, inactivate it for 10 minutes, and cool to room temperature. NaOH solution with a concentration of 5% was adjusted to pH = 7-9, filtered, the filter residue was discarded, and the filtrate was taken;

   S3. The filtrate obtained from S2 is dialyzed for 24 hours in a regenerated cellulose dialysis bag, and the dialysate is changed every 8 hours to collect the dialysate. The collected dialysate is heated and concentrated and then spray-dried.
2. The chito-oligosaccharide prepared by a complex enzyme according to claim 1, wherein in the step S1, the pH value of the acetate-sodium acetate buffer solution is 4.5 and the concentration is 0.2 mol / L.
3. The chito-oligosaccharide prepared by a complex enzyme according to claim 1, wherein the mass concentration of the chitosan solution prepared in the step S1 is 0.5 to 1.5%.
4. The chitooligosaccharide prepared by a complex enzyme according to claim 3, wherein the mass concentration of the chitosan solution prepared in step S1 is 1%.
5. The chito-oligosaccharide prepared by a complex enzyme according to claim 1, wherein the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 8-15%, wherein the complex enzyme is made of pectinase It is composed of pepsin in a weight ratio of 1: (0.1 to 1).
6. The chito-oligosaccharide prepared by a complex enzyme according to claim 5, wherein the mass ratio of the mass of the complex enzyme to the mass of chitosan in step S2 is 10%, wherein the complex enzyme comprises pectinase and stomach The protease is composed in a weight ratio of 1: 0.6.
7. The chitooligosaccharide prepared by a complex enzyme according to claim 1, wherein in step S2, the enzyme is hydrolyzed in a water bath environment at 50 ° C for 2h.
8. The chitooligosaccharide prepared by a complex enzyme according to claim 1, wherein the molecular weight cut-off of the regenerated cellulose dialysis bag in step S3 is 3000 Da.
9. The chito-oligosaccharide prepared by a complex enzyme according to claim 1, wherein the process conditions for spray drying in step S3 are: an air inlet temperature of 180 ° C and a feed speed of 700 mL / h.
10. The method for preparing chitooligosaccharides according to any one of claims 1 to 9, further comprising the following steps:

    I. Dissolve chitosan in an acetate-sodium acetate buffer solution with a pH of 4.5 and a concentration of 0.2 mol / L, and prepare a chitosan solution with a mass concentration of 0.5 to 1.5%;

    II. Add the pectinase and pepsin to the chitosan solution obtained in step I at a weight ratio of 1: (0.1 to 1), and the mass ratio of the complex enzyme to the mass of chitosan is 8 to 15 %, Enzymolysis in a water bath at 45 ° C to 55 ° C for 1 to 3 hours. After the completion of the enzymolysis, place in a boiling water bath for 10 minutes to inactivate. After cooling to room temperature, adjust the pH = 7 ~ 9 with a 5% NaOH solution. Filter, discard the filter residue, and take the filtrate;

    III. The filtrate obtained in step II is dialyzed in a regenerated cellulose dialysis bag with a cut-off molecular weight of 3000 Da for 24 hours, and the dialysate is changed every 8 hours. The dialysate is collected, and the collected dialysate is heated and concentrated. ℃, spray drying at a feed rate of 700mL / h, that is.