Chitosan degrading enzyme composition, chitosan oligosaccharide composition, preparation method of chitosan degrading enzyme composition and chitosan oligosaccharide composition, and application of chitosan degrading enzyme composition and chitosan oligosaccharide composition in preparation of hangover-alleviating and liver-protecting products

Abstract

The invention belongs to the technical field of preparation of bioactive substances, and discloses a chitosan degrading enzyme composition, a chitosan oligosaccharide composition, a preparation method of the chitosan degrading enzyme composition and the chitosan oligosaccharide composition and application of the chitosan degrading enzyme composition and the chitosan oligosaccharide composition in preparation of products for dispelling effects of alcohol and protecting liver. When the chitosan degrading enzyme composition provided by the invention is used for performing enzymolysis on chitosan with relatively low deacetylation degree, the chitosan oligosaccharide composition with a specific polymerization degree can be obtained, and the chitosan oligosaccharide composition can reduce adverse effects of alcohol on intestinal flora, increase metabolism of the intestinal tract on the alcohol, improve the intestinal tract metabolism and improve the intestinal tract metabolism. The amount of alcohol entering blood is reduced, the concentration of blood alcohol is reduced, and finally good effects of dispelling the effects of alcohol and protecting the liver are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of bioactive substances, and particularly relates to a chitosan degrading enzyme composition, a chitosan oligosaccharide composition, a preparation method thereof, and an application thereof in the preparation of products for relieving hangover and protecting the liver. Background Art

[0002] Chitosan is a natural polysaccharide with the second largest reserve in nature after cellulose, and is widely present in the cell walls of plants, insects, fungi, and microorganisms. It has the advantages of high biocompatibility, biodegradability, non-toxicity, and high biological activity, and is widely used in the fields of food, medicine, agriculture, etc. However, chitosan is insoluble in water and common organic solvents, which limits its further application. Chitosan oligosaccharide is a degradation product of chitosan. Compared with chitosan, it has a lower molecular weight and better water solubility, and also has biological activities such as antifungal, antibacterial, antitumor, anti-inflammatory, antioxidant, bone strengthening, blood cholesterol lowering, anti-allergy, immune enhancement, and promotion of plant protection, and has a broader application prospect.

[0003] The Chinese patent application with the publication number CN101461822A discloses that chitosan oligosaccharide can reduce the activity of glutamate transaminase in serum, reduce the production of lipid peroxides, and increase the activities of superoxide dismutase and alcohol dehydrogenase, thereby achieving the hangover effect. The Chinese patent application with the publication number CN117223858A discloses a composition for relieving hangover and protecting the liver. This composition realizes a better hangover and liver protection effect through the mutual cooperation of chitosan oligosaccharide, fucoidan, and trehalose. However, when the chitosan oligosaccharide used in the above-mentioned prior art is applied alone, the hangover and liver protection effect is not ideal, and there are great limitations. Summary of the Invention

[0004] The first object of the present invention is to provide a chitosan degrading enzyme composition, which specifically includes chitinase ChiA-Sa62 and chitosanase, and the chitosanase has the activity of specifically catalyzing the cleavage of the glycosidic bond between D-glucosamine and D-aminoglucose. Through the synergistic effect between chitinase ChiA-Sa62 and chitosanase, chitosan with a low degree of deacetylation is enzymatically hydrolyzed. In the enzymatic hydrolysis product, in addition to mainly including chitosan oligosaccharides with a polymerization degree of 2-4 (content above 50%), there also includes a certain amount of chitosan oligosaccharides with a relatively high polymerization degree (10-12) (content above 10%), thereby obtaining a chitosan oligosaccharide composition. The inventors further studied and found that this chitosan oligosaccharide composition can cooperate synergistically between low-degree and high-degree glucans, reduce the adverse effects of alcohol on the intestinal flora, and promote the metabolism of alcohol by the intestine to reduce the amount of alcohol entering the blood and lower the blood alcohol concentration, and finally achieve a good hangover and liver protection effect.

[0005] The second object of the present invention is to provide a method for preparing a chitosan oligosaccharide composition.

[0006] The third object of the present invention is to provide a chitosan oligosaccharide composition.

[0007] The fourth object of the present invention is to provide the application of the above chitosan oligosaccharide composition in the preparation of products for relieving alcohol and protecting the liver.

[0008] Specifically, the chitosan degrading enzyme composition provided by the present invention includes: chitinase ChiA-Sa62 and chitosanase, the chitosanase specifically acts on the glycosidic bond between D-glucosamine and D-aminoglucose, and the enzyme activity ratio of the chitinase ChiA-Sa62 to the chitosanase is greater than 1:1.

[0009] Further, the chitosanase is chitosanase GH46.

[0010] Further, the enzyme activity ratio of the chitinase ChiA-Sa62 to the chitosanase is (3-8):1. In some specific embodiments, the enzyme activity ratio of the chitinase ChiA-Sa62 to the chitosanase can be 3:1, 3.2:1, 4:1, 5:1, 6:1, 7:1, 8:1 or any value between them.

[0011] The method for preparing the chitosan oligosaccharide composition provided by the present invention includes: taking the above chitosan degrading enzyme composition and mixing it with a chitosan raw material, performing enzymatic hydrolysis treatment to obtain an enzymatic hydrolysis product; taking the enzymatic hydrolysis product and performing purification treatment to obtain the chitosan oligosaccharide composition.

[0012] Further, in step S1, the deacetylation degree of the chitosan raw material is 25-50%. In some specific embodiments, the deacetylation degree of the chitosan raw material can be 25%, 28%, 30%, 36%, 40%, 45%, 50% or any value between them.

[0013] Further, in step S1, based on the weight of the chitosan raw material, the addition amount of the chitosan degrading enzyme composition is 10-15U / g. In some specific embodiments, the addition amount of the chitosan degrading enzyme composition can be 10U / g, 10.5U / g, 12U / g, 15U / g or any value between them.

[0014] Further, in step S1, the solvent for the enzymatic hydrolysis treatment is Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 5-20 mM, and the pH is 4-5. In some specific embodiments, the concentration of the Tris-HCl buffer solution can be 5 mM, 7.5 mM, 10 mM, 15 mM, 20 mM or any value between them; the pH can be 4, 4.1, 4.3, 4.5, 4.8, 5 or any value between them.

[0015] Further, in step S1, the temperature of the enzymatic hydrolysis treatment is 30-40 °C, and the time is 3-6 h. In some specific embodiments, the temperature of the enzymatic hydrolysis treatment can be 30 °C, 31 °C, 34 °C, 38 °C, 40 °C or any value between them, and the time can be 3 h, 3.5 h, 4 h, 5 h, 6 h or any value between them.

[0016] Further, in step S1, the enzymatic hydrolysis treatment further includes ultraviolet irradiation, and the irradiation intensity of the ultraviolet irradiation is 5-25 Gy. In some specific embodiments, the irradiation intensity of the ultraviolet irradiation can be 5 Gy, 5.8 Gy, 6 Gy, 8 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy or any value between them.

[0017] Further, in step S2, the purification treatment includes centrifugation treatment and freeze-drying treatment.

[0018] In the present invention, the centrifugation treatment is a method commonly used in the prior art, limited to being able to separate chitosan oligosaccharide from other substances such as chitosan and proteins, and the present invention does not particularly limit its specific conditions.

[0019] In the present invention, the freeze-drying treatment is a method commonly used in the prior art, limited to being able to remove moisture and prepare chitosan oligosaccharide composition powder, and the present invention does not particularly limit its specific conditions.

[0020] The chitosan oligosaccharide composition provided by the present invention is prepared by the above-mentioned preparation method of the oligosaccharide composition.

[0021] The present invention also provides the application of the above chitosan oligosaccharide composition in the preparation of products for relieving hangover and protecting the liver.

[0022] Further, the products for relieving hangover and protecting the liver are health products and / or drugs. Description of the Drawings

[0023] Figure 1 One of the experimental result graphs of the colonic content flora abundance test provided in Example 7 of the present invention (feeding day 0);

[0024] Figure 2 One of the experimental result graphs for testing the abundance of colonic flora provided in Example 7 of the present invention (fed for 56 days). Detailed implementation mode

[0025] The embodiments of the present invention will be described in detail below. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those without specific technical or conditions noted in the examples, the techniques or conditions described in the literature in the art or according to the product specifications are followed. Reagents or instruments without the manufacturer noted are all conventional products that can be obtained through commercial purchase.

[0026] Among them, the chitinase ChiA-Sa62 used in the following examples was prepared according to the method provided in the reference [1] ; the GH46 chitosanase was prepared according to the method provided in the reference [2] .

[0027] [1] Zhao Jiechang, Wang Qiyuan, Song Kexin, et al. Enzymatic properties of antifungal endochitinase and functional analysis of preparing chito-oligosaccharides [J]. Food Science, 2024, 45(05): 49-58

[0028] [2] Heggset E B, Dybvik A I, Hoell I A, et al. Degradation of chitosans with a family 46 chitosanase from Streptomyces coelicolor A3(2) [J]. Biomacromolecules, 2010, 11(9): 2487

[0029] Example 1

[0030] This example is used to illustrate the preparation of a chito-oligosaccharide composition, which specifically includes:

[0031] S1. Take the chitosan degrading enzyme composition at an addition amount of 10 U / g and mix it with 25 g of chitosan (degree of deacetylation 50%, weight average molecular weight 100000 Da), and add Tris-HCl buffer solution (20 mM, pH = 4) to make up the volume to 1000 mL to obtain an enzymatic hydrolysis reaction solution, and carry out enzymatic hydrolysis treatment in a shaker at 35 °C and 150 rpm for 6 h to obtain an enzymatic hydrolysis product;

[0032] S2. Centrifuge the enzymatic hydrolysis product at 5000 rpm for 5 min, take the supernatant, and freeze-dry the supernatant to obtain a chito-oligosaccharide composition.

[0033] Among them, in the chitosan-degrading enzyme composition used in this embodiment, the enzyme activity ratio of chitinase ChiA-Sa62 to chitosanase GH46 is 5:1.

[0034] Example 2

[0035] This example uses the method provided in Example 1 to prepare a chitosan oligosaccharide composition. The difference is that ultraviolet irradiation is also set in the enzymatic hydrolysis treatment in step S1, the irradiation intensity is 15 Gy, and other conditions are the same, obtaining a chitosan oligosaccharide composition.

[0036] Example 3

[0037] This example uses the method provided in Example 2 to prepare a chitosan oligosaccharide composition. The difference is that the enzyme activity ratio of chitinase ChiA-Sa62 to chitosanase GH46 in the chitosan-degrading enzyme composition is 3:1, and other conditions are the same, obtaining a chitosan oligosaccharide composition.

[0038] Example 4

[0039] This example uses the method provided in Example 2 to prepare a chitosan oligosaccharide composition. The difference is that the enzyme activity ratio of chitinase ChiA-Sa62 to chitosanase GH46 in the chitosan-degrading enzyme composition is 8:1, and other conditions are the same, obtaining a chitosan oligosaccharide composition.

[0040] Example 5

[0041] This example uses the method provided in Example 2 to prepare a chitosan oligosaccharide composition. The difference is that in step S1, an equal mass of chitosan raw material (deacetylation degree 75%, weight average molecular weight 100,000 Da) is used to replace the chitosan raw material (deacetylation degree 50%, weight average molecular weight 100,000 Da), and other conditions are the same, obtaining a chitosan oligosaccharide composition.

[0042] Comparative Example 1

[0043] This comparative example uses the method provided in Example 1 to prepare a chitosan oligosaccharide composition. The difference is that the enzyme activity ratio of chitinase ChiA-Sa62 to chitosanase GH46 in the chitosan-degrading enzyme composition is 0:1 (that is, the chitosan-degrading enzyme composition only includes chitosanase GH46), and other conditions are the same, obtaining a chitosan oligosaccharide composition.

[0044] Comparative Example 2

[0045] This comparative example uses the method provided in Example 1 to prepare a chitosan oligosaccharide composition. The difference is that the enzyme activity ratio of chitinase ChiA-Sa62 to chitosanase GH46 in the chitosan-degrading enzyme composition is 1:0 (that is, the chitosan-degrading enzyme composition only includes chitinase ChiA-Sa62), and other conditions are the same, obtaining a chitosan oligosaccharide composition.

[0046] Comparative Example 3

[0047] In this example, the method provided in Example 2 was used to prepare the chitosan oligosaccharide composition. The difference was that the enzyme activity ratio of chitinase ChiA-Sa62 and chitosanase GH46 in the chitosan degrading enzyme composition was 1:1, and other conditions were the same, and the chitosan oligosaccharide composition was obtained.

[0048] Example 6

[0049] This example was used to illustrate the main components of the chitosan oligosaccharide compositions provided in the above examples and comparative examples. The specific tests included:

[0050] 1. Drawing of the standard curve: Glucosamine and chitosan oligosaccharide standards (degree of polymerization 2-24) were mixed with deionized water to prepare a series of standard solutions with different concentrations. After sterilization through a 0.22 μm membrane, ion chromatography analysis was performed to obtain the peak areas of each standard solution, and the standard curve was drawn.

[0051] 2. Preparation of the solution to be tested: 0.5 g of the chitosan oligosaccharide composition was mixed with deionized water and made up to 50 mL to obtain the solution to be tested.

[0052] 3. Liquid chromatography analysis: Using a Shodex Asahipak NH2P 50-4E (4.6 mm × 250 mm) amino column as the chromatographic column, liquid chromatography analysis was performed on the solution to be tested to obtain the peak areas of each chitosan oligosaccharide. According to the standard curve, the specific concentration was obtained, and the content of each chitosan oligosaccharide in the chitosan oligosaccharide composition was calculated. The conditions of liquid chromatography included: mobile phase acetonitrile: water = 70:30, flow rate 0.9 mL / min, column temperature 30 °C, and injection volume 10 μL.

[0053] The results are shown in Table 1.

[0054] Table 1.

[0055]

[0056] Note: "-" indicates that no peak appears in the ion chromatogram or the peak area cannot be calculated for the peak that appears.

[0057] As can be seen from the results shown in Table 1, compared with the methods provided in Comparative Examples 1-3, the yield of chitosan oligosaccharide prepared by the methods provided in Examples 1-5 of the present invention is high, and in the chitosan oligosaccharide composition prepared, the proportion of DP2-6 is more than 50%, and the proportion of DP10-12 is more than 10%.

[0058] Example 7

[0059] This example is used to illustrate the biological activity of the chitosan oligosaccharide composition provided in the above examples and its effect on the abundance of the intestinal flora of mice. The specific tests include:

[0060] 1. Construction of an alcohol-induced liver injury mouse model: SPF-grade male C57BL / 6J mice aged 6 - 8 weeks were used as experimental animals. After 7 days of adaptive feeding with a standard diet, each group was administered and raised according to the following protocol (4 mice per group, N = 32):

[0061] (1) Blank control group: Intragastric administration of normal saline, with a dosage of 10 mL / (kg×d) from day 1 to day 14, and a dosage of 20 mL / (kg×d) starting from day 14;

[0062] (2) Model group: Intragastric administration of an alcohol solution (95% alcohol: water = 0.33:0.67), with a dosage of 10 mL / (kg×d) from day 1 to day 14, and a dosage of 20 mL / (kg×d) starting from day 14;

[0063] (3) Experimental group: Intragastric administration of an alcohol solution according to the dosage of the model group, and starting from day 14, intragastric administration of a chitosan oligosaccharide solution (concentration of chitosan oligosaccharide composition 0.8 g / mL) every day, with a dosage of 5 mL / (kg×d);

[0064] The total feeding time was 56 days. During the feeding period, the mice were kept in a quiet, clean, ventilated, and warm environment, with the environmental temperature being (22 ± 1) °C and the humidity being 45% - 65%. Ensure that the mice have free access to water and standard feed.

[0065] 2. Blood alcohol concentration of mice:

[0066] 1 hour after intragastric administration of alcohol on day 56, 15 mL of blood was collected from the mouse orbital cavity. The blood sample was processed and the blood alcohol concentration was measured by headspace gas chromatography, and the average value was taken. The results are shown in Table 2.

[0067] Table 2. Blood alcohol concentration of mice

[0068]

[0069]

[0070] It can be seen from the test results shown in Table 2 that compared with Comparative Examples 1 - 3, treating mice with the chitosan oligosaccharide composition provided in Examples 1 - 5 of the present invention can effectively reduce the blood alcohol concentration of mice. It should be noted that when the concentration of DP2 - 4 in chitosan oligosaccharide is 60 - 70% and the concentration of DP10 - 12 is 10 - 20, it has a better effect on reducing blood alcohol.

[0071] 3. Changes in the abundance of the intestinal flora of mice: After the feeding ended, the total DNA of the mouse colon content samples was extracted, and the V3-V4 region of bacterial 16S rDNA was amplified using primers. The library was quantified using Qubit and Q-PCR to construct a qualified sequencing library. The qualified sequencing library was subjected to paired-end sequencing using the NovaSeq 6000 sequencing system. The data of each sample set were filtered, and after checking and detecting with the species annotation database, the effective data were finally obtained. The effective data were analyzed by OTUs clustering. Using the Silva 132 database, R v3.4.1, GraphPad Prism, and SPSS software, and using the RDP classifier to classify the representative sequences of OTUs, the richness, diversity, and evenness indexes of the microbial community were calculated, and the obtained data were all expressed as mean ± standard deviation (SD). The test results are as Figure 1 and 2 shown.

[0072] From Figure 1 and 2 the test results shown, it can be seen that treating mice with the chitosan oligosaccharide composition provided in Example 1 of the present invention can effectively alleviate the damage of alcohol to the intestinal flora of mice, increase the intestinal metabolic capacity of alcohol, so as to reduce the amount of ethanol entering the blood, and finally achieve a good effect of relieving alcohol and protecting the liver.

[0073] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. A chitosan-degrading enzyme composition, characterized in that, The chitosan degrading enzyme composition includes chitinase ChiA-Sa62 and chitosanase, the chitosanase specifically acts on the glycosidic bond between D-glucosamine and D-aminoglucose, and the enzyme activity ratio of the chitinase ChiA-Sa62 to the chitosanase is greater than 1:

1.

2. The chitosan-degrading enzyme composition according to claim 1, characterized in that, The chitosanase is chitosanase GH46; Optionally, the enzyme activity ratio of the chitinase ChiA-Sa62 to the chitosanase is (3-8):

1.

3. A method for preparing a chitosan oligosaccharide composition, characterized in that, The preparation method includes: mixing the chitosan degrading enzyme composition according to claim 1 or 2 with a chitosan raw material, performing enzymatic hydrolysis treatment to obtain an enzymatic hydrolysis product; and subjecting the enzymatic hydrolysis product to purification treatment to obtain the chitosan oligosaccharide composition.

4. The preparation method of the chitosan oligosaccharide composition according to claim 3, wherein, In step S1, the degree of deacetylation of the chitosan raw material is 25-75%; Optionally, based on the weight of the chitosan raw material, the addition amount of the chitosan degrading enzyme composition is 10-15 U / g; Optionally, the solvent for the enzymatic hydrolysis treatment is Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 5-20 mM, and the pH is 4-5.

5. The preparation method of the chitosan oligosaccharide composition according to claim 3, characterized in that, In step S1, the temperature of the enzymatic hydrolysis treatment is 30-40°C, and the time is 3-6 h.

6. The preparation method of the chitosan oligosaccharide composition according to claim 3, wherein In step S1, ultraviolet irradiation is further included in the enzymatic hydrolysis treatment, and the irradiation intensity of the ultraviolet irradiation is 5-25 Gy.

7. The preparation method of the chitosan oligosaccharide composition according to claim 3, characterized in that, In step S2, the purification treatment includes centrifugation treatment and freeze-drying treatment.

8. A chitosan oligosaccharide composition, characterized in that, Prepared by the preparation method of the chitosan oligosaccharide composition according to any one of claims 3-7.

9. Use of the chitosan oligosaccharide composition according to claim 8 in the preparation of a product for relieving hangover and protecting the liver.

10. The application according to claim 9, wherein The product for relieving hangover and protecting the liver is a health product and / or a drug.

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