Method for accurately controlling molecular weight of chitosan oligosaccharide by using enzyme digestion-ultra-nanofiltration coupling technique

Abstract

The invention discloses a method for accurately controlling the molecular weight of chitosan oligosaccharide by using an enzyme digestion-ultra-nanofiltration combined technology, and belongs to the field of chitosan oligosaccharide preparation. The method comprises the following steps: (1) enzyme digestion: adding papain into chitosan according to the ratio of enzyme to a substrate; and (2) ultrafiltration: enabling the chitosan oligosaccharide obtained in the step (1) to sequentially pass through ultrafiltration membranes and nanofiltration membranes with different molecular cut-off weights so as to recover the chitosan oligosaccharide with different molecular weights. The chitosan oligosaccharide obtained after enzymolysis is filtered by using the ultrafiltration membrane and the nanofiltration membrane with different molecular weight cutoff, so that the chitosan oligosaccharide with different polymerization degrees can be more accurately obtained, the use requirements of a user on different polymerization degrees of the chitosan oligosaccharide are met, and the method has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of chitosan oligosaccharide preparation, and in particular to a method for accurately controlling the molecular weight of chitosan oligosaccharide by utilizing an enzyme cutting-ultra-nanofiltration combined technology. Background Art

[0002] Chitosan is a natural polysaccharide composed of β-(1-4)-linked 2-amino-2-deoxy-D-glucopyranose and 2-acetylamino-2-deoxy-D-glucopyranose. It is nontoxic, biodegradable, and biocompatible. Chitosan is commercially produced by deacetylation of chitin, which can be extracted from the exoskeletons of crustaceans (such as shrimp, lobster, crab, and fish) and the cell walls of fungi. Chitosan is considered a functional biopolymer and is widely used in various industrial applications, such as the food and nutrition, medical and pharmaceutical, and cosmetics industries, as well as the environmental and agricultural sectors. However, its low solubility in common solvents and high viscosity due to its high molecular weight and fibrous structure have hindered its wider use. According to literature reports, low molecular weight chitosan oligosaccharides (typically ≤3200) exhibit better bioactivity than high molecular weight chitosan. Furthermore, the lower the molecular weight of chitosan oligosaccharides, the higher their water solubility and bioactivity. Therefore, reducing the molecular weight of chitosan to produce more chitosan oligosaccharides is increasingly important for expanding the application of chitosan.

[0003] Currently, several methods have been proposed to reduce the molecular weight of chitosan. Generally speaking, these methods include (1) chemical depolymerization using O3, NaNO2 or H2O2 through acid hydrolysis or redox reaction, (2) physical depolymerization using acoustic radiation or hydrodynamic shearing, and (3) enzymatic depolymerization. Chemical depolymerization has several disadvantages, including the difficulty in obtaining a high degree of depolymerization, harsh hydrolysis conditions (e.g., high concentration and temperature), low product yield, especially side reactions caused by glucose ring modification, while physical depolymerization requires special equipment and the resulting molecular weight cannot be well controlled. Enzymatic hydrolysis has some advantages, including mild and specific reaction conditions, high product yield, no glucose ring modification, and easier control. In addition, enzymatic hydrolysis can retain the original biological properties of chitosan oligosaccharides. Due to these conditions, enzymatic hydrolysis is more important from a practical point of view.

[0004] The Chinese patent application document with publication number CN111718972A discloses a method for preparing chitosan oligosaccharides with a specific degree of polymerization, belonging to the technical field of chitosan oligosaccharide preparation. The preparation method comprises the following steps: S1, chitosan purification; S2, preparation of a chitosan solution; S3, stage one hydrolysis: adding a non-specific enzyme to the chitosan solution, ultrasonic reaction for 0.5-1.5 hours, then heating and inactivating, neutralizing, and centrifuging to obtain a low molecular weight chitosan solution with a degree of polymerization of 65-115; S4, stage two hydrolysis: adding glucanase to the low molecular weight chitosan solution, reacting for 1-2 hours, then heating and inactivating, neutralizing, and centrifuging to obtain a chitosan oligosaccharide mixture solution with a degree of polymerization of 5-7, and separating, purifying, and drying to obtain chitosan oligosaccharide powder with a degree of polymerization of 5-7. This patent uses a nonspecific enzyme with high affinity for large molecules and a specific enzyme with high affinity for small molecules, glucanase, to act on chitosan, greatly increasing the reaction speed. The enzymatic hydrolysis reaction can be completed within 3.5 hours, greatly improving production efficiency. However, the preparation steps of this patent are relatively cumbersome, and the molecular weight control of chitosan oligosaccharides is limited, so further improvement is needed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to accurately control the molecular weight of chitosan oligosaccharides by combining enzyme cleavage and ultrafiltration.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The present invention provides a method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme cleavage-ultra-nanofiltration combined technology, comprising the following steps:

[0008] (1) Enzymatic digestion: Chitosan is mixed with papain and fully reacted to obtain chitosan oligosaccharides;

[0009] (2) Ultrafiltration: The chitosan oligosaccharide obtained in (1) is subjected to ultrafiltration and nanofiltration in sequence to recover chitosan oligosaccharides of different molecular weights; the temperature of the ultrafiltration and nanofiltration is 20-30° C., the pH value is 6-8, and the pressure is 0.6-1 MPa.

[0010] Preferably, in (1), the deacetylation degree of the chitosan is in the range of 50 to 90%.

[0011] Preferably, in (1), the ratio of papain to chitosan is 1000-1400 u / mg.

[0012] Preferably, in (1), the reaction temperature ranges from 40 to 50° C., the reaction pH ranges from 4.5 to 5, and the reaction time ranges from 22 to 25 hours.

[0013] Preferably, the molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration is 1000-3000 Da, more preferably 3000 Da, 2000 Da, or 1000 Da.

[0014] Preferably, the molecular weight cut-off of the nanofiltration membrane used in the nanofiltration is 300 to 1000 Da, more preferably 800 Da, 500 Da, or 300 Da.

[0015] Preferably, the temperature of the ultrafiltration and nanofiltration is 25° C., the pH value is 7, and the pressure is 0.8 and 1 MPa.

[0016] Preferably, the number of cycles of ultrafiltration and nanofiltration is 5 to 9 times.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention proposes a method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme cleavage-ultrafiltration combined technology. By adopting the enzyme cleavage-ultrafiltration combined technology, the chitosan oligosaccharides obtained after enzymatic hydrolysis are filtered by using ultrafiltration membranes and nanofiltration membranes with different molecular weight cut-offs. Chitosan oligosaccharides with different degrees of polymerization can be obtained more accurately, solving the user's demand for the use of chitosan oligosaccharides with different degrees of polymerization, and having good application prospects.

[0019] 2. The present invention uses this method to prepare chitosan oligosaccharides, which is more environmentally friendly and can separate chitosan oligosaccharides of different molecular weights. The papain used is a food-grade enzyme that is easily available in large quantities and has been widely recognized for its safety. It does not produce toxic or harmful byproducts during the enzymatic hydrolysis process. Moreover, due to its large quantity and reusability, the production cost of chitosan oligosaccharides can be reduced. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0021] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0022] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0023] Example 1:

[0024] A method for accurately controlling the molecular weight of chitosan oligosaccharides using enzyme digestion-ultra-nanofiltration combined technology comprises the following steps:

[0025] (1) Enzymatic digestion: Papain was added to chitosan with a deacetylation degree of 50% according to the ratio of enzyme to substrate of 1000 u / mg, mixed, and the reaction temperature was set at 40°C and pH 4.5. The reaction was carried out for 24 hours to obtain chitosan oligosaccharides.

[0026] (2) Ultrafiltration: The chitosan oligosaccharide obtained in step (1) is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 3000Da, 2000Da, and 1000Da and nanofiltration membranes with molecular weight cutoffs of 800Da, 500Da, and 300Da to recover chitosan oligosaccharides of different molecular weights; during ultrafiltration, the filtration temperature is set to 25°C, the pH value is 7, the filtration pressure is 0.8MPa, and the cycle is repeated 5 times; during nanofiltration, the temperature is set to 25°C, the pH value is 7, the filtration pressure is 1MPa, and the cycle is repeated 7 times.

[0027] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 3000 was 95.02%; the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 2000 was 74.29%; and the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 1000 was 58.39%.

[0028] Example 2:

[0029] The difference between this embodiment and embodiment 1 is that:

[0030] The reaction temperature in step (1) is 45°C;

[0031] In step (2), the ultrafiltration was circulated 6 times, and the nanofiltration was circulated 8 times; the rest was the same as in Example 1.

[0032] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight less than 3000 was 95.84%; the acquisition rate of chitosan oligosaccharides with a molecular weight less than 2000 was 77.29%; and the acquisition rate of chitosan oligosaccharides with a molecular weight less than 1000 was 60.43%.

[0033] Example 3:

[0034] The difference between this embodiment and embodiment 1 is that:

[0035] In step (1), the ratio of enzyme to substrate was 1200 u / mg, and the rest was the same as in Example 1.

[0036] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 3000 was 96.97%; the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 2000 was 80.2%; and the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 1000 was 61.53%.

[0037] Example 4:

[0038] The difference between this embodiment and embodiment 1 is that:

[0039] In step (1), the enzyme to substrate ratio is 1200 u / mg and the reaction temperature is 45°C;

[0040] In step (2), the ultrafiltration was circulated 6 times, and the nanofiltration was circulated 8 times; the rest was the same as in Example 1.

[0041] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight less than 3000 was 97.93%; the acquisition rate of chitosan oligosaccharides with a molecular weight less than 2000 was 81.73%; and the acquisition rate of chitosan oligosaccharides with a molecular weight less than 1000 was 65.77%.

[0042] Example 5:

[0043] The difference between this embodiment and embodiment 4 is that:

[0044] In step (1), the deacetylation degree of chitosan is 70%, and the rest is the same as in Example 4.

[0045] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight less than 3000 was 93.57%; the acquisition rate of chitosan oligosaccharides with a molecular weight less than 2000 was 71.93%; and the acquisition rate of chitosan oligosaccharides with a molecular weight less than 1000 was 54.68%.

[0046] Example 6:

[0047] A method for accurately controlling the molecular weight of chitosan oligosaccharides using enzyme digestion-ultra-nanofiltration combined technology comprises the following steps:

[0048] (1) Enzymatic digestion: Papain was added to chitosan with a deacetylation degree of 70% according to the ratio of enzyme to substrate of 1400 u / mg, mixed, and the reaction temperature was set at 50°C and pH 5. The reaction was carried out for 25 hours to obtain chitosan oligosaccharides.

[0049] (2) Ultrafiltration: The chitosan oligosaccharide obtained in step (1) is sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 3000Da, 2000Da, and 1000Da and nanofiltration membranes with molecular weight cutoffs of 800Da, 500Da, and 300Da to recover chitosan oligosaccharides of different molecular weights; during ultrafiltration, the filtration temperature is set to 25°C, the pH value is 7, the filtration pressure is 0.8MPa, and the cycle is repeated 8 times; during nanofiltration, the temperature is set to 25°C, the pH value is 7, the filtration pressure is 1MPa, and the cycle is repeated 9 times.

[0050] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight less than 3000 was 94.28%; the acquisition rate of chitosan oligosaccharides with a molecular weight less than 2000 was 73.57%; and the acquisition rate of chitosan oligosaccharides with a molecular weight less than 1000 was 54.97%.

[0051] Example 7:

[0052] The difference between this embodiment and embodiment 6 is that:

[0053] The deacetylation degree of chitosan in step (1) is 90%,

[0054] The filtration pressure during ultrafiltration in step (2) was 1 MPa, and the process was repeated 9 times; the rest was the same as in Example 6.

[0055] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight less than 3000 was 93.38%; the acquisition rate of chitosan oligosaccharides with a molecular weight less than 2000 was 69.48%; and the acquisition rate of chitosan oligosaccharides with a molecular weight less than 1000 was 49.34%.

[0056] Comparative Example 1:

[0057] The difference between this comparative example and Example 1 is that in step (1), the chitosan deacetylation degree is 40%, the enzyme to substrate ratio is 700u / mg, the number of ultrafiltration cycles in step (2) is 3, and the number of nanofiltration cycles is 4. The rest is the same as in Example 1.

[0058] In this embodiment, HPLC detection showed that the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 3000 was 72.62%; the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 2000 was 48.25%; and the acquisition rate of chitosan oligosaccharides with a molecular weight of less than 1000 was 28.85%.

[0059] Table 1 The acquisition rate of chitosan oligosaccharides with different molecular weights under different reaction conditions

[0060]

[0061] As shown in Table 1, the chitosan oligosaccharide yield in Example 4 is the highest.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for accurately controlling the molecular weight of chitosan oligosaccharides using enzyme digestion-ultra-nanofiltration technology, characterized in that: The following steps are involved: (1) Enzymatic digestion: Chitosan is mixed with papain and fully reacted to obtain chitosan oligosaccharides; (2) Ultrafiltration: The chitosan oligosaccharide obtained in (1) is subjected to ultrafiltration and nanofiltration in sequence to recover chitosan oligosaccharides of different molecular weights; the temperature of the ultrafiltration and nanofiltration is 20-30° C., the pH value is 6-8, and the pressure is 0.6-1 MPa.

2. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: In (1), the deacetylation degree of the chitosan is in the range of 50 to 90%.

3. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: (1), the dosage ratio of papain to chitosan is 1000-1400 u / mg.

4. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: In (1), the reaction temperature range is 40-50°C, the reaction pH range is 4.5-5, and the reaction time range is 22-25 hours.

5. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: The ultrafiltration membrane used in the ultrafiltration has a molecular weight cut-off range of 1000 to 3000 Da.

6. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 5, characterized in that: The ultrafiltration membrane used in the ultrafiltration has a molecular weight cut-off of 3000Da, 2000Da or 1000Da.

7. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: The nanofiltration membrane used in the nanofiltration has a molecular weight cut-off of 300 to 1000 Da.

8. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 7, characterized in that: The nanofiltration membrane used in the nanofiltration has a molecular weight cut-off of 800Da, 500Da or 300Da.

9. The method for accurately controlling the molecular weight of chitosan oligosaccharides by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: The temperature of the ultrafiltration and nanofiltration was 25° C., the pH value was 7, and the pressures were 0.8 and 1 MPa.

10. The method for accurately controlling the molecular weight of chitosan oligosaccharide by using enzyme digestion-ultra-nanofiltration combined technology according to claim 1, characterized in that: The number of cycles of ultrafiltration and nanofiltration is 5 to 9 times.

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