Anti-aging beautifying oral liquid containing low-polymerization-degree chitosan oligosaccharide and preparation method of anti-aging beautifying oral liquid

Abstract

The invention discloses an anti-aging beautifying oral liquid containing low-polymerization-degree chitosan oligosaccharide and a preparation method thereof, and belongs to the technical field of marine organisms. Chitosan oligosaccharide mainly comprising chitobiose and chitotriose is prepared by utilizing the synergistic fermentation effect of microorganisms, and the chitosan oligosaccharide is compounded with collagen, sodium hyaluronate, vitamin C and the like to prepare the oral liquid. The low-polymerization-degree chitosan oligosaccharide is an excellent in-vivo antioxidant, can remove excessive free radicals, inhibit lipid peroxidation and slow down damage of metabolites such as peroxided lipid to the body, and then the anti-aging effect is achieved; the anti-aging and beautifying oral liquid prepared by matching natural moisturizing and beautifying components such as collagen and sodium hyaluronate is good in taste and easy to absorb, and animal experiment results show that the anti-aging and beautifying oral liquid can have beautifying and anti-aging effects after being frequently taken.

Description

Technical Field

[0001] This invention relates to an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide and its preparation method, particularly a method for preparing chitosan oligosaccharide using microbial fermentation and using it in an anti-aging and beauty oral liquid, belonging to the field of marine biotechnology. Background Technology

[0002] Aging is a complex physiological process. Under normal physiological conditions, the production and elimination of free radicals in the human body are in dynamic equilibrium. Once this balance is disrupted, the body will produce excessive free radicals, leading to aging characteristics such as wrinkles, dryness, keratinization, dullness, and excessive pigmentation. Furthermore, the increasingly fast pace of modern life, including staying up late, lack of exercise, and prolonged sitting, all accelerate aging. Therefore, eliminating excessive free radicals in the body is a crucial way to slow down aging. Antioxidants can react with common free radicals in the body, such as hydroxyl radicals and superoxide anion radicals, preventing lipid peroxidation and thus playing a role in reducing pigmentation, preventing wrinkles, and combating aging. With the increasing awareness of healthy living, the research and development of anti-aging health products has become a hot topic.

[0003] Currently, chitosan oligosaccharides are increasingly widely used in the health care field. Among existing technologies, patent CN103340340A discloses a health food that enhances immunity, specifically disclosing that chitosan oligosaccharides can reduce oxidative damage caused by various free radicals to the body, improve the body's disease prevention, resistance, and anti-aging abilities, and further enhance physical fitness; patent CN117643555A discloses an anti-aging moisturizing skin care liquid and its preparation method, specifically disclosing the modification of chitosan oligosaccharides for the preparation of the anti-aging moisturizing skin care liquid; patent CN107550753A discloses a composition with skin repair and anti-aging functions and its preparation method, specifically disclosing that the composition containing chitosan oligosaccharides can replenish skin cell nutrients, achieving deep repair, promoting cell metabolism, and activating cell vitality, and has good antibacterial, moisturizing, and anti-aging functions for the skin.

[0004] Chitosan oligosaccharides, degradation products of chitosan, are oligosaccharides with a degree of polymerization of 2-10 composed of 2-aminoglycans linked by β-1,4-glycosidic bonds. Compared with chitosan, they have advantages such as smaller relative molecular mass, better water solubility, higher bioactivity, easier absorption, and wider application range. Chitosan oligosaccharides possess antibacterial, antioxidant, antitumor, and immune-enhancing functions, and are widely used in medicine, food, beverages, cosmetics, and chemical industries. The bioactivity of chitosan oligosaccharides is closely related to their structure. Monomers with well-defined structures are obtained through chromatography and ion chromatography, and the antioxidant properties of each monomer are then tested. Current research results indicate that chitobiose and chitotriose inhibit the hydroxylation of benzoate to salicylate. Furthermore, the inhibitory effects of chitobiose and chitotriose are superior to the other three control groups, with chitobiose showing the best inhibitory effect. Chitobiose and chitotriose also exhibit higher scavenging effects on ·OH free radicals than the three control groups. Animal experiments have shown that the two chitosan oligosaccharide monomers can serve as excellent in vivo antioxidants. Research results indicate that, when chitosan oligosaccharides are orally administered, only the low-molecular-weight chitobiose and chitotriose enter the bloodstream to exert their physiological functions, while the chitosan tetraose and chitosan pentaose cannot enter the bloodstream. Therefore, researching and developing high-content chitobiose and chitotriose has become a key technological approach to improving the bio-health benefits of chitosan oligosaccharides.

[0005] Currently, in the preparation and application research of chitosan oligosaccharides, the degree of polymerization of chitosan oligosaccharides is difficult to control, and most are mixtures with a wide degree of polymerization range. Their bioactivity is unstable, becoming a bottleneck restricting the application of chitosan oligosaccharides in bio-health products. The chitosan oligosaccharides prepared in this patent are mainly chitobiose and chitotriose, which are excellent in vivo antioxidants. They can scavenge excess free radicals, inhibit lipid peroxidation, and slow down the damage to the body caused by metabolites such as lipid peroxidation, thereby exerting anti-aging effects and significantly enhancing the free radical scavenging and anti-aging effects of oral liquids and other health products. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention aims to provide an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharides and its preparation method. By utilizing the synergistic fermentation of microorganisms, an oligosaccharide mainly composed of chitobiose and chitotriose is prepared and compounded with collagen, sodium hyaluronate, vitamin C, etc., thereby significantly enhancing the anti-aging and beauty effects of the oral liquid.

[0007] The above-mentioned objective of this invention is achieved through the following technical solutions: An anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide comprises the following raw materials in parts by weight: 8-10 parts low-polymerization degree chitosan oligosaccharide, 6-10 parts collagen, 4-8 parts sodium hyaluronate, 5-10 parts vitamin C, 0.5-2 parts DL-malic acid, 1-3 parts peach powder, 0.5-1.5 parts potassium sorbate, and 60-70 parts deionized water.

[0008] Preferably, an anti-aging beauty oral liquid containing low-polymerization degree chitosan oligosaccharide comprises the following raw materials in parts by weight: 9 parts low-polymerization degree chitosan oligosaccharide, 8 parts collagen, 6 parts sodium hyaluronate, 7 parts vitamin C, 1 part DL-malic acid, 2 parts peach powder, 1 part potassium sorbate, and 66 parts deionized water.

[0009] An anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide is prepared by the following process: the raw materials are stirred and mixed evenly, centrifuged at 3000 r / min in a high-speed centrifuge for 10-15 min, and the supernatant is taken to obtain the anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide.

[0010] The preparation method of the above-mentioned low-polymerization degree chitosan oligosaccharide includes the following steps: (1) Dissolution of chitosan: Prepare a 2% solution of chitosan with 0.5% to 1% acetic acid, and heat and stir at 40℃ to 50℃ to dissolve it completely. (2) Enzymatic hydrolysis of chitosan: Mix the prepared chitosan with the chitosan solution at a volume ratio of 1:50 to 1:70, and heat and stir in a water bath at 40℃ to 50℃ for 1 to 3 hours to degrade the chitosan. (3) Inactivation of enzyme hydrolysate: Inactivate the enzyme hydrolysate at 100°C for 15 min; (4) Centrifuge the enzyme hydrolysate at 8000 r / min for 10 min and take the supernatant. (5) The solution obtained in step (4) is freeze-dried under vacuum to obtain low degree of polymerization chitosan oligosaccharide.

[0011] The above-mentioned method for preparing chitosanase includes the following steps: (1) Microbial fermentation: The microbial fermentation strain is first inoculated into the fermentation medium at an inoculation amount of 1% to 3%, and cultured in a shaker at 20℃ to 40℃ and 60 to 100 rpm / min for 30 to 50 hours to obtain crude enzyme solution; (2) Ethanol precipitation and crude separation: Slowly add 2 to 3 times the volume of 95% ethanol to the crude enzyme solution, let it stand for a while, then centrifuge at 8000 to 10000 r / min at 4℃ for 20 to 30 min, discard the supernatant, dissolve the precipitate in 20 mmol / L pH 7.0 phosphate buffer, dialyze overnight, centrifuge at 8000 to 10000 r / min at 4℃ for 20 to 30 min, and take the supernatant; (3) Ion exchange chromatography: The supernatant obtained in the previous step was subjected to ion exchange chromatography on a CM Sepharose Fast Flow column with NaCl as the mobile phase. The active components were collected and concentrated into highly active chitosanase.

[0012] The aforementioned microbial fermentation strains are one or a mixture of two of Aspergillus and Candida tropicalis.

[0013] Preferably, the microbial fermentation strain is a mixed strain of Aspergillus and Candida tropicalis, with a ratio of 5:2.

[0014] The above-mentioned Aspergillus has the preservation number ATCC 16888, and the Tropical Pseudomonas has the preservation number ATCC 60556. Both were obtained through market channels.

[0015] The above fermentation medium formula is: 0.05% MgSO4·7H2O, 0.06% KCl, 0.002% FeSO4, 0.03% KH2PO4, 1.5% chitosan, 0.5% peptone, and the balance is purified water.

[0016] This invention provides an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharides and its preparation method, which has the following significant advantages compared with the prior art: (1) The low degree of polymerization chitosan oligosaccharide in this invention is an excellent in vivo antioxidant that can remove excess free radicals and inhibit lipid peroxidation, thus slowing down the damage of lipid peroxidation and other metabolites to the body and thus exerting anti-aging effects. The anti-aging oral liquid prepared in combination with natural moisturizing and beauty ingredients such as collagen and sodium hyaluronate has a good taste and is easy to absorb. Animal experiments show that regular use can have beauty and anti-aging effects. (2) Collagen has a structure similar to human skin collagen. It is a non-water-soluble fibrous glycoprotein with a large number of amino acids and hydrophilic groups in its molecules. It has certain surface activity and good compatibility. At the same time, because its molecules contain a large number of hydroxyl groups, it has a very good moisturizing effect. It has the effects of pure natural moisturizing, whitening, anti-wrinkle and spot removal. Sodium hyaluronate is a transparent natural moisturizing ingredient in the human body. It can moisturize for a long time, promote the absorption of other active ingredients, provide a good environment for the synthesis of dermal collagen and elastic fibers, and reduce the signs of aging wrinkles. Vitamin C is a water-soluble substance that can rebuild the dermal-epidermal junction and promote the production of collagen fibers. In addition, it has a strong ability to scavenge free radicals and is also an indispensable component for enhancing the body's immunity. This invention significantly improves the anti-aging and beauty effects of oral liquid through the scientific compatibility of raw materials. (3) The present invention utilizes microbial fermentation to prepare highly active chitosan degrading enzyme, which significantly increases the content of chitobiose and chitotriose in low-polymerization degree chitosan oligosaccharides, and plays a key role in enhancing the anti-aging and health care effects of oral liquid. Attached Figure Description

[0017] Figure 1 This is an HPLC chromatogram of low-polymerization degree chitosan oligosaccharides. Detailed Implementation

[0018] The fermentation medium formulations in the following examples are: 0.05% MgSO4·7H2O, 0.06% KCl, 0.002% FeSO4, 0.03% KH2PO4, 1.5% chitosan, 0.5% peptone, and the remainder is purified water.

[0019] The preparation process of an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide specifically includes the following steps: (1) Microbial fermentation: Aspergillus and Candida tropicalis were inoculated into the fermentation medium at a ratio of 5:2, with an inoculation amount of 2%. The medium was cultured at 30℃ and 100 rpm / min for 36 h to obtain crude enzyme solution. (2) Ethanol precipitation and crude separation: Slowly add 2.5 times the volume of 95% ethanol to the crude enzyme solution, let it stand for a while, then centrifuge at 8000 r / min at 4℃ for 20 min, discard the supernatant, dissolve the precipitate in 20 mmol / L pH7.0 phosphate buffer, dialyze overnight, centrifuge at 10000 r / min at 4℃ for 30 min, and take the supernatant. (3) Ion exchange chromatography: The supernatant obtained in the previous step was subjected to ion exchange chromatography on a CM Sepharose Fast Flow column with NaCl as the mobile phase. The active components were collected and concentrated into highly active chitosanase. (4) Dissolution of chitosan: Prepare a 2% solution of chitosan with 1% acetic acid, and heat and stir at 40℃~50℃ to dissolve it completely; (5) Enzymatic hydrolysis of chitosan: The prepared chitosanase is mixed with the chitosan hydrolysate from step (4) at a volume ratio of 1:60, and the mixture is heated and stirred in a water bath at 40℃~50℃ for 2 hours to degrade the chitosan. (6) Inactivation of enzyme hydrolysate: Inactivate the enzyme hydrolysate at 100°C for 15 min; (7) Centrifuge the enzyme hydrolysate at 8000 r / min for 10 min and take the supernatant. (8) The solution obtained in step (7) is freeze-dried under vacuum to obtain low degree of polymerization chitosan oligosaccharide.

[0020] Low degree of polymerization chitosan oligosaccharide detection The above-mentioned low-polymerization degree chitosan oligosaccharides were detected using high-performance liquid chromatography (HPLC), and the results are shown in Table 1. Figure 1 As shown; Detection instrument: Shimadzu liquid chromatograph (Japan); Chromatographic conditions: Shodex NH2 P 50 4E column (4.6×250mm); Mobile phase: A (water), B (acetonitrile); Detector: evaporative light scattering detector.

[0021] Table 1. HPLC Detection Results of Low Degree of Polymerization Chitosan Oligosaccharides Pk # Area Area % Monosaccharides 629.22351 2.9526 Chitobiose 1.21276e4 56.9080 Chitosan 7132.26221 33.4677 Chitosan 669.56909 3.1419 Chitosan 235.64682 1.1058 Chitosan 311.04349 1.4596 Chitosan 205.55489 0.9646 Total 2.13109e4 100 From Table 1, Figure 1 It can be seen that the low degree of polymerization chitosan oligosaccharides are mainly chitobiose and chitotriose, with a content of 90.38% for chitobiose and chitotriose.

[0022] The preparation process of an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide specifically includes the following steps: (1) Microbial fermentation: Aspergillus, a microbial fermentation strain, was inoculated into the fermentation medium at an inoculation amount of 2%, and cultured in a shaker at 30℃ and 100 rpm / min for 36 h to obtain crude enzyme solution; (2) Ethanol precipitation and crude separation: Slowly add 2.5 times the volume of 95% ethanol to the crude enzyme solution, let it stand for a while, then centrifuge at 8000 r / min at 4℃ for 20 min, discard the supernatant, dissolve the precipitate in 20 mmol / L pH7.0 phosphate buffer, dialyze overnight, centrifuge at 10000 r / min at 4℃ for 30 min, and take the supernatant. (3) Ion exchange chromatography: The supernatant obtained in the previous step was subjected to ion exchange chromatography on a CM Sepharose Fast Flow column with NaCl as the mobile phase. The active components were collected and concentrated into highly active chitosanase. (4) Dissolution of chitosan: Prepare a 2% solution of chitosan with 1% acetic acid, and heat and stir at 40℃~50℃ to dissolve it completely; (5) Enzymatic hydrolysis of chitosan: The prepared chitosanase is mixed with the chitosan hydrolysate from step (4) at a volume ratio of 1:60, and the mixture is heated and stirred in a water bath at 40℃~50℃ for 2 hours to degrade the chitosan. (6) Inactivation of enzyme hydrolysate: Inactivate the enzyme hydrolysate at 100°C for 15 min; (7) Centrifuge the enzyme hydrolysate at 8000 r / min for 10 min and take the supernatant. (8) The solution obtained in step (7) is freeze-dried under vacuum to obtain low degree of polymerization chitosan oligosaccharide.

[0023] The low-polymerization degree chitosan oligosaccharide was tested according to the method described in Example 1. The contents of chitobiose and chitotriose were 60.8214% and 34.1471%, respectively, and the total content of chitobiose and chitotriose was 94.97%, which is higher than the content in Example 1.

[0024] The preparation process of an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide specifically includes the following steps: (1) Microbial fermentation: The microbial fermentation strain Candida tropicalis was inoculated into the fermentation medium at an inoculation amount of 2%, and cultured in a shaker at 30℃ and 100 rpm / min for 36 h to obtain crude enzyme solution; (2) Ethanol precipitation and crude separation: Slowly add 2.5 times the volume of 95% ethanol to the crude enzyme solution, let it stand for a while, then centrifuge at 8000 r / min at 4℃ for 20 min, discard the supernatant, dissolve the precipitate in 20 mmol / L pH7.0 phosphate buffer, dialyze overnight, centrifuge at 10000 r / min at 4℃ for 30 min, and take the supernatant. (3) Ion exchange chromatography: The supernatant obtained in the previous step was subjected to ion exchange chromatography on a CM Sepharose Fast Flow column with NaCl as the mobile phase. The active components were collected and concentrated into highly active chitosanase. (4) Dissolution of chitosan: Prepare a 2% solution of chitosan with 1% acetic acid, and heat and stir at 40℃~50℃ to dissolve it completely; (5) Enzymatic hydrolysis of chitosan: The prepared chitosanase is mixed with the chitosan hydrolysate from step (4) at a volume ratio of 1:60, and the mixture is heated and stirred in a water bath at 40℃~50℃ for 2 hours to degrade the chitosan. (6) Inactivation of enzyme hydrolysate: Inactivate the enzyme hydrolysate at 100°C for 15 min; (7) Centrifuge the enzyme hydrolysate at 8000 r / min for 10 min and take the supernatant. (8) The solution obtained in step (7) is freeze-dried under vacuum to obtain low degree of polymerization chitosan oligosaccharide.

[0025] The low-polymerization degree chitosan oligosaccharide was tested according to the method described in Example 1. The contents of chitobiose and chitotriose were 28.6872% and 11.2417%, respectively, and the total content of chitobiose and chitotriose was 39.92%, which was significantly lower than the contents of Example 1 and Example 2.

[0026] An anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide is prepared by the following process: the raw materials are mixed in the following proportions: 9 parts low-polymerization degree chitosan oligosaccharide, 8 parts collagen, 6 parts sodium hyaluronate, 7 parts vitamin C, 1 part DL-malic acid, 2 parts peach powder, 1 part potassium sorbate, and 66 parts deionized water. The mixture is stirred and mixed evenly, centrifuged at 3000 r / min for 10-15 min, and the supernatant is collected to obtain the anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide.

[0027] Animal grouping: Eighty mice were randomly divided into four groups of 20 each. These were: Experimental group 1 (oral solution prepared from low-polymerization degree chitosan oligosaccharide as described in Example 1), Experimental group 2 (oral solution prepared from low-polymerization degree chitosan oligosaccharide as described in Example 2), Experimental group 3 (oral solution prepared from low-polymerization degree chitosan oligosaccharide as described in Example 3), and a negative control group (administered physiological saline). Administration method: Mice in the experimental group were given oral solution by gavage at a dose of 0.2 ml / 10 g body weight daily; mice in the negative control group were given physiological saline by gavage at a dose of 0.2 ml / 10 g body weight daily for 30 consecutive days. Antioxidant index determination: On day 30 of drug administration, mice were sacrificed and liver tissue was collected. A 10% liver homogenate was prepared with physiological saline, centrifuged at 3000 r / min for 10 min, and the supernatant was collected. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the content of malondialdehyde (MDA) in the liver homogenate, were measured using an enzyme-linked immunosorbent assay (ELISA) reader. Higher SOD and GSH-Px activities and lower MDA content indicated stronger antioxidant capacity and better anti-aging effects.

[0028] The experimental results are shown in the table below: Table 2 Results of the oral liquid anti-aging test Experimental Groups SOD activity (U / mg) GSH-Px (U / mg) MDA content (nmol / mg) Experimental group 1 139.69±8.85 86.97±5.72 3.01±1.09 Experimental group 2 112.57±9.42 71.05±6.32 4.22±0.86 Experimental group 3 101.74±10.35 64.69±7.24 5.09±1.17 negative control group 91.08±7.67 61.95±6.15 5.35±0.74 As shown in the table above, comparing the anti-aging indicators of the oral liquid, the SOD activity in the liver homogenate of mice in experimental groups 1-3 was significantly higher than that in the negative control group (P<0.05), indicating that the oral liquid of this invention can effectively enhance the body's ability to scavenge superoxide anion free radicals. Regarding GSH-Px activity, experimental groups 1-2 were significantly higher than the negative control group (P<0.05), and experimental group 3 was also higher than the negative control group but not significantly, demonstrating the positive effect of the oral liquid in promoting glutathione participation in the antioxidant defense system. Regarding MDA content, experimental groups 1-2 were significantly lower than the negative control group (P<0.05), and experimental group 3 was also lower than the negative control group but not significantly, indicating that the oral liquid can effectively reduce lipid peroxidation and reduce oxidative damage in the body. In summary, the experimental groups performed excellently in various antioxidant indicators, fully demonstrating that the oral liquid has significant antioxidant and anti-aging effects, with experimental group 1 showing the best anti-aging effect.

[0029] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. An anti-aging and beautifying oral liquid containing low-polymerization-degree chitosan oligosaccharide, characterized in that... The ingredients include the following parts by weight: 8-10 parts of low-polymerization chitosan oligosaccharide, 6-10 parts of collagen, 4-8 parts of sodium hyaluronate, 5-10 parts of vitamin C, 0.5-2 parts of DL-malic acid, 1-3 parts of peach powder, 0.5-1.5 parts of potassium sorbate, and 60-70 parts of deionized water.

2. The anti-aging and beautifying oral liquid containing low-polymerization degree chitosan oligosaccharide according to claim 1, characterized in that... The oral liquid comprises the following ingredients by weight: 9 parts low-polymerization chitosan oligosaccharide, 8 parts collagen, 6 parts sodium hyaluronate, 7 parts vitamin C, 1 part DL-malic acid, 2 parts peach powder, 1 part potassium sorbate, and 66 parts deionized water.

3. The anti-aging and beautifying oral liquid containing low-polymerization degree chitosan oligosaccharide according to claim 1, characterized in that... The oral liquid is prepared by the following process: the raw materials are stirred and mixed evenly, centrifuged at 3000 r / min in a high-speed centrifuge for 10 to 15 minutes, and the supernatant is taken to obtain an anti-aging and beauty oral liquid containing low-polymerization degree chitosan oligosaccharide.

4. The anti-aging and beautifying oral liquid containing low-polymerization degree chitosan oligosaccharide according to claim 1, characterized in that... The preparation method of the low-polymerization degree chitosan oligosaccharide includes the following steps: (1) Dissolution of chitosan: Prepare a 2% solution of chitosan with 0.5% to 1% acetic acid, and heat and stir at 40℃ to 50℃ to dissolve it completely. (2) Enzymatic hydrolysis of chitosan: Mix the prepared chitosan with the chitosan solution at a volume ratio of 1:50 to 1:70, and heat and stir in a water bath at 40℃ to 50℃ for 1 to 3 hours to degrade the chitosan. (3) Inactivation of enzyme hydrolysate: Inactivate the enzyme hydrolysate at 100°C for 15 min; (4) Centrifuge the enzyme hydrolysate at 8000 r / min for 10 min and take the supernatant. (5) The solution obtained in step (4) is freeze-dried under vacuum to obtain low degree of polymerization chitosan oligosaccharide.

5. The anti-aging and beautifying oral liquid containing low-polymerization degree chitosan oligosaccharide according to claim 4, characterized in that... The method for preparing the chitosanase includes the following steps: (1) Microbial fermentation: The microbial fermentation strain is first inoculated into the fermentation medium at an inoculation amount of 1% to 3%, and cultured in a shaker at 20℃ to 40℃ and 60 to 100 rpm / min for 30 to 50 hours to obtain crude enzyme solution; (2) Ethanol precipitation and crude separation: Slowly add 2 to 3 times the volume of 95% ethanol to the crude enzyme solution, let it stand for a while, then centrifuge at 8000 to 10000 r / min at 4℃ for 20 to 30 min, discard the supernatant, dissolve the precipitate in 20 mmol / L pH 7.0 phosphate buffer, dialyze overnight, centrifuge at 8000 to 10000 r / min at 4℃ for 20 to 30 min, and take the supernatant; (3) Ion exchange chromatography: The supernatant obtained in the previous step was subjected to ion exchange chromatography on a CM Sepharose Fast Flow column with NaCl as the mobile phase. The active components were collected and concentrated into highly active chitosanase.

6. The anti-aging and beautifying oral liquid containing low-polymerization degree chitosan oligosaccharide according to claim 5, characterized in that... The microbial fermentation strain is one or a mixture of two of Aspergillus and Candida tropicalis.

7. The anti-aging and beautifying oral liquid containing low-polymerization degree chitosan oligosaccharide according to claim 6, characterized in that... The microbial fermentation strain is a mixture of Aspergillus and Candida tropicalis, with a ratio of 5:2.

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