Method for preparing chitosan with high deacetylation degree based on microbial fermentation method

Abstract

The invention provides a method for preparing chitosan with high deacetylation degree based on a microbial fermentation method. The preparation method comprises the following steps: preparing colloidal chitin turbid liquid; preparing a fermentation culture medium; transferring the prepared culture medium into a fermentation tank, sterilizing, cooling, and adjusting the pH value; inoculating an aspergillus oryzae seed solution and fermenting; inoculating bacillus amyloliquefaciens and fermenting; and carrying out sterilization, centrifugation, precipitate collection, washing, treatment with a NaOH solution, washing and freeze drying so as to obtain the product. The method comprises the following steps: by simulating a microorganism synergistic degradation process in nature and regulating and controlling fermentation conditions of aspergillus oryzae and bacillus amyloliquefaciens in stages, firstly completing wall breaking pretreatment by the aspergillus oryzae, carrying out mild and controllable enzymolysis, destroying crystal structures of the aspergillus oryzae and exposing a large amount of free amino groups and acetyl sites, so that the viscosity of fermentation liquor can be obviously reduced, and the fermentation liquor can be used for fermenting the bacillus amyloliquefaciens; extremely favorable conditions are created for the next-step deacetylation reaction, and deep and thorough deacetylation is realized.

Description

Technical Field

[0001] This invention relates to the technical field of chitosan preparation, and specifically to a method for preparing chitosan with high degree of deacetylation based on microbial fermentation. Background Technology

[0002] Chitin, also known as chitosan, is a natural aminopolysaccharide derived from the exoskeletons of shrimp, crabs, insects, and fungal cell walls. Its annual biosynthesis is enormous, making it the second largest natural polymer material after cellulose. Chitosan is the product of chitin after the removal of some acetyl groups. Due to its rich free amino groups in its molecular chain, it possesses unique properties such as excellent biocompatibility, biodegradability, antibacterial properties, hemostatic properties, and film-forming properties, and is widely used in numerous fields including biomedicine, food and health products, cosmetics, agriculture, textiles, and water treatment. The application value of chitosan largely depends on two key indicators: degree of deacetylation and molecular weight. A high degree of deacetylation (>95%) means more free amino groups, resulting in stronger positive charge, superior biological activity, and better physicochemical properties; while a high molecular weight endows it with better mechanical strength and the ability to form fibers and films. Therefore, how to efficiently and environmentally prepare chitosan with both high degree of deacetylation and high molecular weight has always been a research hotspot and technical challenge in this field.

[0003] Currently, the main method for large-scale industrial production of chitosan is the chemical method. This method typically involves placing chitin raw materials in a 40-50% (w / w) high-concentration sodium hydroxide solution and reacting them at high temperatures (100-120℃) for several hours or even longer to remove acetyl groups. Although this method is technically mature and relatively low-cost, its inherent drawbacks are becoming increasingly prominent: (1) Severe environmental pollution: The reaction process consumes a large amount of concentrated alkali and generates a large amount of organic wastewater with high alkalinity and high color, resulting in high subsequent treatment costs and being extremely unfriendly to the environment. (2) Limited product quality: Under the severe conditions of strong alkali and high temperature, the chitin molecular chain will undergo severe hydrolysis and breakage, leading to a significant reduction in the molecular weight of the product and uneven distribution of the degree of deacetylation, making it difficult to stably obtain products with ultra-high degree of deacetylation (>98%). (3) High energy consumption: The reaction needs to be carried out at high temperatures, resulting in high energy consumption. In order to overcome the disadvantages of the chemical method, the microbial fermentation method has received widespread attention as an environmentally friendly alternative. It utilizes chitin deacetylase produced by microorganisms to specifically catalyze the deacetylation reaction under mild conditions. Therefore, developing a new microbial fermentation process that can efficiently produce chitosan with both ultra-high deacetylation degree and high molecular weight using inexpensive raw materials and under mild conditions is not only of great theoretical significance, but also has huge potential for industrial application and market prospects. Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation. This method simulates the natural microbial synergistic degradation process, controlling the fermentation conditions of *Aspergillus oryzae* and *Bacillus amyloliquefaciens* in stages. Through precise temporal design, *Aspergillus oryzae* first completes the "cell wall disruption" pretreatment. During its growth, *Aspergillus oryzae* secretes chitinase to perform mild and controllable enzymatic hydrolysis of colloidal chitin, destroying its crystal structure and exposing a large number of free amino and acetyl sites. This significantly reduces the viscosity of the fermentation broth, creating extremely favorable conditions for the subsequent deacetylation reaction. Without sterilizing the materials, *Bacillus amyloliquefaciens* is directly inoculated, utilizing its secreted chitin deacetylase to efficiently act on the "pretreated" chitin substrate, achieving deep and thorough deacetylation.

[0005] Technical solution: A method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation, comprising the following steps: S1: Slowly add chitin to a 31-33% (w / w) NaOH solution under continuous stirring, treat at 25°C for 3 hours to form alkalized chitin, wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: Inoculate Aspergillus oryzae seed liquid at an inoculation rate of 5% (v / v) and ferment at a temperature of 28°C, a rotation speed of 250-260 rpm, and an aeration rate of 1.0 vvm for 36 h. Aspergillus oryzae grows in large quantities and secretes chitinase, which enzymatically hydrolyzes colloidal chitin, and the viscosity of the fermentation broth is significantly reduced. S4: Inoculate with Bacillus amyloliquefaciens at an inoculum of 5% (v / v), heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at a speed of 350-380 rpm and an aeration rate of 1.0 vvm, maintaining pH at 7.2 throughout the process. During this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetase for a deep and thorough deacetylation reaction. S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash, and centrifuge again. Repeat 3-4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral. S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan.

[0006] Furthermore, the mass-to-volume ratio of the chitin to the NaOH solution is 1 g: 10 mL.

[0007] Furthermore, the method for preparing the Aspergillus oryzae seed liquid is as follows: take a ring of spores from the PDA slant medium, inoculate it into a shake flask containing 100 mL of PDB medium, and culture it at 28°C and 180 rpm for 28-36 h to obtain the Aspergillus oryzae seed liquid.

[0008] Furthermore, the cell dry weight concentration of the Aspergillus oryzae seed liquid is 8-12 g DCW / L.

[0009] Furthermore, the method for preparing the Bacillus amyloliquefaciens seed culture is as follows: take a loop of bacterial growth from LB slant medium, inoculate it into a shake flask containing 100 mL of LB medium, and incubate at 30°C and 200 rpm for 16-18 hours until OD... 600 =1.5-1.6, to obtain Bacillus amyloliquefaciens seed solution.

[0010] The high degree of deacetylation chitosan prepared by the above method.

[0011] Furthermore, the high degree of deacetylation chitosan has a degree of deacetylation ≥98% and a viscosity-average molecular weight ≥500,000 Da.

[0012] Beneficial effects: 1. This invention simulates the natural microbial synergistic degradation process by controlling the fermentation conditions of Aspergillus oryzae and Bacillus amyloliquefaciens in stages. Through precise temporal design, Aspergillus oryzae first completes the "cell wall disruption" pretreatment. During its growth, Aspergillus oryzae secretes chitinase to perform mild and controllable enzymatic hydrolysis of colloidal chitin, destroying its crystal structure and exposing a large number of free amino and acetyl sites. The viscosity of the fermentation broth will be significantly reduced, creating extremely favorable conditions for the next step of deacetylation. Without sterilizing the material, Bacillus amyloliquefaciens is directly inoculated, and its secreted chitin deacetylase acts efficiently on the "pretreated" chitin substrate to achieve deep and thorough deacetylation.

[0013] 2. This invention enables *Bacillus amyloliquefaciens* to undergo a thorough and uniform enzymatic reaction on a pretreated *Aspergillus oryzae* substrate, resulting in chitosan with a stable deacetylation degree exceeding 98%, even approaching 99%, far surpassing the levels achieved by traditional chemical methods and single-strain fermentation. Because the entire deacetylation process is conducted under mild enzymatic reaction conditions, the severe damage to the chitin molecular backbone caused by strong alkalis and high temperatures is avoided, thus maximizing the preservation of the product's molecular weight, resulting in a viscosity-average molecular weight exceeding 500,000 Da. This high molecular weight translates to better film-forming properties, fiber strength, and solution viscosity, significantly expanding its application prospects in high-end fields. Detailed Implementation

[0014] This invention proposes a method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples will be used to further describe the invention in detail. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0015] Example 1 The method for preparing Aspergillus oryzae seed culture is as follows: take a ring of spores from PDA slant medium, inoculate it into a shake flask containing 100 mL of PDB medium, and culture at 28°C and 180 rpm for 28 h to obtain Aspergillus oryzae seed culture.

[0016] The cell dry weight concentration of Aspergillus oryzae seed culture was measured to be 9.21 g DCW / L.

[0017] Example 2 The method for preparing Aspergillus oryzae seed culture is as follows: take a ring of spores from PDA slant medium, inoculate it into a shake flask containing 100 mL of PDB medium, and culture at 28°C and 180 rpm for 30 h to obtain Aspergillus oryzae seed culture.

[0018] The cell dry weight concentration of Aspergillus oryzae seed culture was measured to be 10.54 g DCW / L. Example 3

[0019] The method for preparing Aspergillus oryzae seed culture is as follows: take a ring of spores from PDA slant medium, inoculate it into a shake flask containing 100 mL of PDB medium, and culture at 28°C and 180 rpm for 36 h to obtain Aspergillus oryzae seed culture.

[0020] The cell dry weight concentration of Aspergillus oryzae seed culture was measured to be 11.97 g DCW / L. Example 4

[0021] The method for preparing Bacillus amyloliquefaciens seed culture is as follows: take a loop of bacterial growth from LB slant medium, inoculate it into a shake flask containing 100 mL of LB medium, and culture at 30°C and 200 rpm for 16 h to obtain Bacillus amyloliquefaciens seed culture.

[0022] OD of Bacillus amyloliquefaciens seed liquid was measured 600 =1.55. Example 5

[0023] The method for preparing Bacillus amyloliquefaciens seed culture is as follows: take a loop of bacterial growth from LB slant medium, inoculate it into a shake flask containing 100 mL of LB medium, and culture at 30°C and 200 rpm for 18 h to obtain Bacillus amyloliquefaciens seed culture.

[0024] OD of Bacillus amyloliquefaciens seed liquid was measured 600 =1.59. Example 6

[0025] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 1 was inoculated at a rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with Bacillus amyloliquefaciens from Example 4 at an inoculum of 5% (v / v), heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at a speed of 370 rpm and an aeration rate of 1.0 vvm, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral. S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 7

[0026] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 1 was inoculated at a rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with 5% (v / v) of Bacillus amyloliquefaciens from Example 5, heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at 370 rpm and 1.0 vvm aeration, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0027] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 8

[0028] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 2 was inoculated at an inoculation rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with Bacillus amyloliquefaciens from Example 4 at an inoculum of 5% (v / v), heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at a speed of 370 rpm and an aeration rate of 1.0 vvm, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0029] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 9

[0030] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 2 was inoculated at an inoculation rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with 5% (v / v) of Bacillus amyloliquefaciens from Example 5, heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at 370 rpm and 1.0 vvm aeration, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0031] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 10

[0032] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 3 was inoculated at a rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm, and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with Bacillus amyloliquefaciens from Example 4 at an inoculum of 5% (v / v), heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at a speed of 370 rpm and an aeration rate of 1.0 vvm, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0033] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 11

[0034] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 3 was inoculated at a rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm, and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with 5% (v / v) of Bacillus amyloliquefaciens from Example 5, heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at 370 rpm and 1.0 vvm aeration, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0035] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 12

[0036] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 2 was inoculated at an inoculation rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with 5% (v / v) of Bacillus amyloliquefaciens from Example 5, heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at 370 rpm and 1.0 vvm aeration, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0037] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Example 13

[0038] A method for preparing chitosan with high degree of deacetylation based on microbial fermentation includes the following steps: S1: Slowly add 100g of chitin to 1L of 32% (w / w) NaOH solution under continuous stirring, and treat at 25°C for 3h to form alkalized chitin. Wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. 2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: make up to 1 L; transfer the prepared culture medium to a fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: The Aspergillus oryzae seed liquid prepared in Example 2 was inoculated at an inoculation rate of 5% (v / v) and fermented for 36 hours at a temperature of 28°C, a rotation speed of 260 rpm and an aeration rate of 1.0 vvm. Aspergillus oryzae grew in large quantities and secreted chitinase, which enzymatically hydrolyzed the colloidal chitin, and the viscosity of the fermentation broth was significantly reduced. S4: Inoculate with 5% (v / v) of Bacillus amyloliquefaciens from Example 5, heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at 370 rpm and 1.0 vvm aeration, maintaining pH at 7.2 throughout the process; at this stage, Bacillus amyloliquefaciens becomes the dominant microbial community, utilizing the more readily reacting chitin substrate pretreated with Aspergillus oryzae to efficiently secrete chitin deacetylase for a deep and thorough deacetylation reaction; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash it, and centrifuge it again. Repeat this process 4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour to remove residual bacterial protein and pigment. It was then centrifuged again and washed with water until neutral.

[0039] S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan. Single-strain fermentation - Aspergillus oryzae only) Step S3: Inoculate the Aspergillus oryzae seed culture prepared in Example 2 at an inoculum rate of 5% (v / v) and ferment for 84 h at a temperature of 28°C, a rotation speed of 260 rpm, and an aeration rate of 1.0 vvm. Step S4 is omitted, and the remaining steps are the same as in Example 6. Comparative Example 2 (Single-strain fermentation - Bacillus amyloliquefaciens only)

[0040] Step S4: Inoculate the Bacillus amyloliquefaciens seed culture prepared in Example 5 at an inoculum rate of 5% (v / v), heat to 30°C, adjust the pH to 7.2, and ferment for 48 hours at a rotation speed of 370 rpm and an aeration rate of 1.0 vvm. S3 is omitted, and the remaining steps are the same as in Example 6. Comparative Example 3 (Chemical Method)

[0041] A method for preparing chitosan includes the following steps: Take 100g of the pretreated alkalized chitin from Example 1 and add it to 1L of 50% (w / w) NaOH solution. Stir the mixture in an oil bath at 100°C for 3 hours. After the reaction is complete, cool the mixture and wash it with water until neutral. The subsequent drying and grinding steps are the same as in Example 6. Simultaneous fermentation with two bacteria

[0042] S3: Simultaneously inoculate the Aspergillus oryzae seed culture from Example 2 at a 2.5% (v / v) inoculation rate and the Bacillus amyloliquefaciens seed culture from Example 5 at a 2.5% (v / v) inoculation rate (total inoculation rate remains 5%). Ferment at 30°C, pH 7.2, 370 rpm, and 1.0 vvm aeration for 84 h. S4 is omitted; the remaining steps are the same as in Example 6.

[0043] The degree of deacetylation and viscosity-average molecular weight of chitosan from the above examples and comparative examples were determined, and the results are shown in Table 1 below: Table 1 Degree of deacetylation (%) Viscosity-average molecular weight (10,000 Da) Product yield (%)* Example 6 98.2 55.3 90.1 Example 7 98.5 56.8 91.5 Example 8 98.7 58.1 92.0 Example 9 98.9 59.5 92.8 Example 10 98.0 57.2 89.5 Example 11 98.4 58.9 90.9 Example 12 97.8 52.1 88.3 Example 13 98.1 54.7 89.0 Comparative Example 1 36.5 62.0 93.5 Comparative Example 2 76.8 32.5 82.0 Comparative Example 3 91.5 15.8 85.0 Comparative Example 4 68.4 41.2 79.6 *Note: Product yield (%) = (dry weight of chitosan obtained / dry weight of chitin added) × 100% In all embodiments of this invention, the degree of deacetylation of chitosan remained consistently high, ranging from 97.8% to 98.9%, significantly superior to all comparative examples. This indicates that the staged synergistic strategy of "fungal cell disruption-bacterial deacetylation" is highly effective. Their molecular weights were all above 500,000 Da, with the highest approaching 600,000 Da, possibly because microbial fermentation maximizes the preservation of the chitin molecular chain integrity. Comparative Example 1 had an extremely low degree of deacetylation, and the product was essentially low-deacetylated chitosan or chitosan oligosaccharide, failing to meet the high degree of deacetylation requirement; it had the highest molecular weight (620,000 Da), but since it was not deacetylated, this data has no practical application value. Comparative Example 2 had a relatively low degree of deacetylation, far below the high degree of deacetylation standard, indicating that untreated chitosan is difficult for bacteria to effectively deacetylate; its low molecular weight (325,000 Da) may be due to degradation of the substrate by other hydrolytic enzymes secreted by bacteria (such as chitinase). Comparative Example 3 showed acceptable deacetylation, but its molecular weight decreased sharply. This is an inherent and unavoidable drawback of the traditional strong alkali method, resulting in poor mechanical and viscosity properties of the product. Comparative Example 4 performed the worst, mainly because the optimal growth conditions (pH, temperature) of the two bacteria were different. Simultaneous fermentation caused them to inhibit each other, preventing either from functioning efficiently.

Claims

1. A method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation, characterized in that, Includes the following steps: S1: Slowly add chitin to a 31-33% (w / w) NaOH solution under continuous stirring, treat at 25°C for 3 hours to form alkalized chitin, wash with deionized water until neutral, add deionized water to homogenize, and obtain a stable colloidal chitin suspension. S2: Prepare the fermentation medium with the following components per 1 L: colloidal chitin suspension: 200-250 mL, yeast extract: 10 g, wheat bran: 5 g, K2HPO4: 1 g, MgSO4·7H2O: 0.5 g, deionized water: bring to 1 L; transfer the prepared medium to the fermenter, sterilize at 121°C for 20 min, cool to 28°C, and adjust the pH to 5.5; S3: Inoculate with Aspergillus oryzae seed culture at an inoculum rate of 5% (v / v) and ferment for 36 h at a temperature of 28°C, a rotation speed of 250-260 rpm, and an aeration rate of 1.0 vvm; S4: Inoculate with Bacillus amyloliquefaciens at an inoculum of 5% (v / v), heat to 30°C, adjust pH to 7.2, and continue fermentation for 48 hours at a speed of 350-380 rpm and an aeration rate of 1.0 vvm, maintaining pH at 7.2 during this process; S5: Heat the fermentation broth to 95°C and maintain it for 15 min. Centrifuge at 4°C and 8000 rpm for 20 min. Discard the supernatant, collect the precipitate, resuspend the precipitate with deionized water, wash, and centrifuge again. Repeat 3-4 times until the supernatant is neutral. S6: The washed precipitate was treated with 1% (w / v) NaOH solution in a 60°C water bath with gentle stirring for 1 hour, centrifuged again, and washed with water until neutral; S7: Freeze-dry, grind, and pass through a 100-mesh sieve to obtain high degree of deacetylation chitosan.

2. The method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation according to claim 1, characterized in that, The mass-to-volume ratio of chitin to NaOH solution is 1g:10mL.

3. The method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation according to claim 1, characterized in that, The method for preparing the Aspergillus oryzae seed liquid is as follows: take a ring of spores from the PDA slant medium, inoculate it into a shake flask containing 100 mL of PDB medium, and culture it at 28°C and 180 rpm for 28-36 h to obtain the Aspergillus oryzae seed liquid.

4. The method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation according to claim 3, characterized in that, The cell dry weight concentration of the Aspergillus oryzae seed liquid is 8-12 g DCW / L.

5. The method for preparing high-degree-of-deacetylation chitosan based on microbial fermentation according to claim 1, characterized in that, The method for preparing the Bacillus amyloliquefaciens seed culture is as follows: Take a loop of bacterial growth from LB slant medium and inoculate it into a shake flask containing 100 mL of LB medium. Incubate at 30°C and 200 rpm for 16-18 hours until OD reaches the target value. 600 = 1.5-1.6, to obtain Bacillus amyloliquefaciens seed solution.

6. The high degree of deacetylation chitosan prepared by the method according to any one of claims 1-5.

7. The high degree of deacetylation chitosan according to claim 6, characterized in that, The high degree of deacetylation chitosan has a degree of deacetylation ≥98% and a viscosity-average molecular weight ≥500,000 Da.