A method for preparing chitosan microspheres for cell immobilization and drug delivery
By combining microwave induction and a green chelating agent with enzymatic hydrolysis modification, chitosan microspheres with excellent uniformity and mechanical properties were prepared, overcoming the shortcomings of existing preparation methods and realizing efficient industrial production and efficient transformation of enzyme-immobilized cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI SHINJOY IND CO LTD
- Filing Date
- 2021-08-06
- Publication Date
- 2026-07-14
AI Technical Summary
Existing methods for preparing chitosan microspheres suffer from inconsistent sizes, are time-consuming and labor-intensive, making industrial production difficult. Furthermore, traditional chelating agents are environmentally unfriendly, and controlling the pore size of chitosan carriers is challenging, affecting enzyme or cell encapsulation and substrate release efficiency.
Chitosan microspheres were prepared using a microwave-induced process combined with a green chelating agent and a composite enzyme method. Heavy metals were chelated by the chelating agent, proteins and fats were removed by enzymatic hydrolysis, chitosan was modified by enzyme method, and chitosan/polyvinyl alcohol microspheres were formed by spray drying using a polymer gel sphere preparation instrument. The microspheres were then further treated with genipin to improve their mechanical properties.
Chitosan microspheres with smooth shape and uniform size were prepared, with particle size controlled in the range of 1 to 250 μm. The mechanical properties were improved, cell viability retention and reusability were enhanced, and efficient industrial production was achieved. The enzyme immobilization cell transformation capacity was increased by 4.3 times.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of chitosan microsphere preparation, and more specifically to a method for preparing chitosan microspheres for cell immobilization and drug delivery. Background Technology
[0002] Chitosan, a product of chitin deacetylation, is the only basic polysaccharide among natural polysaccharides. It is widely distributed in the exoskeletons of crustaceans, insects, and the cell walls of fungi, with an annual production exceeding tens of billions of tons. As a novel green polymer material, chitosan contains numerous amino and hydroxyl groups, exhibiting high reactivity. It can undergo alkylation, esterification, acylation, carboxylation, hydrolysis, chelation, and other chemical reactions, introducing various functional groups to alter its physical and chemical properties. Chitosan can also be formulated into different application forms such as chitosan microspheres, chitosan nanoparticles, and chitosan membranes, broadening its application scope. Due to its excellent biocompatibility, biodegradability, permeability, ability to form films, filaments, and microspheres, protein affinity, and natural non-toxicity, chitosan has attracted widespread attention from researchers in the fields of medicine, food, chemical engineering, and materials science. Currently, research and development worldwide focuses on the enzyme carrier application of chitosan, with chitosan microspheres being the most widely used in enzyme immobilization.
[0003] However, current methods for preparing chitosan microspheres remain relatively limited, and product quality needs improvement. Researchers both domestically and internationally have prepared chitosan microsphere carriers using methods such as ionogelation, chemical cross-linking precipitation, solvent evaporation, emulsification cross-linking, spray drying, and neutralization precipitation, with emulsification cross-linking and neutralization precipitation being the most commonly used. However, chitosan microspheres prepared by manual hammering and spray drying are currently of inconsistent size, and the preparation process is time-consuming and labor-intensive, making them unsuitable for industrial production. The key is to efficiently prepare chitosan carriers with good mechanical properties, numerous active groups, and a large specific surface area. Secondly, controlling the pore size of the microspheres to effectively encapsulate enzymes or bacterial cells while allowing free entry of substrates and release of products are also critical issues that urgently need to be addressed. Summary of the Invention
[0004] In view of the above-mentioned problems in the prior art, the technical problem to be solved by the present invention is to provide a method for preparing chitosan microspheres for cell immobilization and drug delivery.
[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0006] A method for preparing chitosan microspheres for cell immobilization and drug delivery includes the following steps:
[0007] 1) Weigh chitin and chitin deacetylase at a mass ratio of 1000:1, dissolve in water, place in a microwave device, treat at 40-60℃ and microwave power of 400-600W for 10 minutes, then inactivate the enzyme to obtain chitosan solution.
[0008] 2) The chelating agent and chitosan solution are stirred thoroughly at a molar ratio of 10-20:0.2-1 until they are evenly mixed. The changes in the solution are observed until the solution changes from clear to turbid and then back to clear.
[0009] 3) Add carbonate to the solution from step 2) to allow precipitation, stir thoroughly, and obtain crude chitosan solution;
[0010] 4) Add a complex bio-enzyme to the crude chitosan solution to hydrolyze it and remove proteins and fats. After the enzymatic reaction, inactivate the enzyme, ultrafilter, and dry to obtain refined chitosan.
[0011] 5) Refined chitosan is directly dissolved in water to form a transparent solution. The pH is adjusted, and a modifying enzyme is added. The chitosan is modified using the modifying enzyme. The mass ratio of chitosan to modifying enzyme is 1:0.09-0.15. After treatment at 40-60℃ for 1-24 hours, the modified chitosan is obtained. The modifying enzyme is laccase, glutamine transferase, and tyrosinase.
[0012] 6) Inoculate *Gibberella fuciformis* into potato dextrose liquid medium, shake and culture until the logarithmic phase, centrifuge, remove supernatant, and resuspend in physiological saline to obtain a bacterial suspension. Add the chitosan solution and polyvinyl alcohol solution obtained in step 5) to form a composite solvent system. The mass concentration ratio of bacterial suspension, chitosan solution and polyvinyl alcohol solution is 1.5-2:1:1. Use a polymer gel ball preparation instrument to spray dry inert gas by pumping inert gas through the inlet nozzle in the hot air flow formed by the inert gas to obtain dried chitosan / polyvinyl alcohol microspheres and chitosan / polyvinyl alcohol microsphere immobilized cells.
[0013] 7) The cells immobilized with polysaccharide / polyvinyl alcohol microspheres were treated with genipin and the immobilized cells were stored in physiological saline.
[0014] Furthermore, in step 2), the chelating agent is tetrasodium glutamate diacetate or citric acid, and the concentration of the chelating agent is 0.01-0.02 mol / L.
[0015] Furthermore, in step 3), the amount of carbonate added is 0.5 to 2 g of carbonate per liter of solution.
[0016] Chitosan's molecular chains contain -NH2 and -OH active groups, which can form cage-like molecules with a network-like structure through hydrogen bonds or salt bonds. These molecules can coordinate with heavy metal ions to form complexes, easily chelating many metal ions. This leads to excessive heavy metal content in most chitosan raw materials. Traditional chelating agents have varying degrees of impact on human health and the environment. Although my country's standard GB2760-2011 clearly stipulates that disodium ethylenediaminetetraacetate (EDTA) can be used in jams and various beverages, its usage is generally strictly limited to 0.03-0.25 g / kg. Moreover, EDTA has long been considered environmentally unfriendly due to its non-biodegradable nature. NTA (nitroglycerin triacetic acid) is biodegradable, but the International Agency for Research on Cancer officially listed NTA as "probable human carcinogen" in 1990, classifying it as Group 2B. This invention uses green chelating agents such as tetrasodium glutamate diacetate or citric acid to replace traditional heavy metal chelating agents. Furthermore, the residual metal ions were chelated, extracted, and removed using carbonate-based solubilization technology under critical pH conditions to prepare high-purity chitosan.
[0017] Further, in step 4), the composite bio-enzyme is a specific keratinase and lipase, the mass ratio of chitosan to the composite bio-enzyme is 1:0.01-0.05, the enzymatic hydrolysis reaction temperature is 40-60℃, and the enzymatic hydrolysis reaction time is 3h; the enzymatic hydrolysis reaction solution is rapidly heated to 90℃ for 10min to inactivate the enzyme, and the enzymatic hydrolysis reaction solution is filtered using an ultrafiltration membrane with a molecular weight cutoff of 5000Da, with a membrane operating pressure of 0.05MPa; the ultrafiltrate is further filtered using a 3000Da ultrafiltration membrane, with a membrane operating pressure of 0.20MPa, the filtrate is concentrated, and dried to obtain purified chitosan.
[0018] Chitosan is a deacetylated product of chitin, containing high levels of crude protein and crude fat, requiring further removal of these components. Traditional methods for removing protein and lipids primarily employ chemical methods. Percot A et al. analyzed the kinetics of acid-base deproteinization and optimized the extraction method, using 0.25 mol / L HCl solution for 15 min followed by 1 mol / L NaOH solution at 70°C for 24 h, resulting in a product with high purity. Nguyen Van Toan et al. pretreated chitosan with 0.016 mol / L benzoic acid, then removed protein and lipids with 0.62 mol / L NaOH solution; however, this method suffers from four highs and one low: high acid-base concentration, high acid-base usage, high water consumption, high pollutant emissions, and low product purity. This invention addresses the trace amounts of protein and lipids present in chitosan by employing a green production process to hydrolyze and remove these components. Specifically, it utilizes specific keratinase and lipase to hydrolyze and purify crude chitosan, thereby improving its purity.
[0019] Furthermore, in step 5), the pH is adjusted to 6.0-8.0, and the mass ratio of laccase, glutamine transferase, and tyrosinase is 1:1:1.
[0020] Chitosan's low antioxidant properties limit its widespread application. Molecular modification can effectively improve its performance. Traditional modification methods mainly employ chemical methods, which are costly and generate toxic and harmful substances that pollute the environment. This application utilizes enzymatic modification of chitosan to endow chitosan and its derivatives with higher bioactivity and other application properties, exhibiting specificity, particularity, and environmental friendliness that chemical methods cannot match.
[0021] Furthermore, in step 6), the particle size of the chitosan / polyvinyl alcohol microspheres is controlled within the range of 1 to 250 μm.
[0022] Further, in step 7), dried chitosan / polyvinyl alcohol microspheres immobilized cells are added to PBS buffer containing 1 wt.%-5 wt.% genipin and treated at a water bath temperature of 20-40℃ for 5-18 hours. After treatment, the cells are rinsed several times with PBS buffer to remove residual genipin. Finally, the immobilized cells are stored in physiological saline at 0-10℃.
[0023] The application of genipin to treat chitosan / polyvinyl alcohol microspheres immobilized cells improved the mechanical properties of the immobilized cells, as well as enhanced cell viability and reusability.
[0024] The application of the chitosan microspheres in the immobilization of live cells of Gibberella fuciformis.
[0025] The application of the chitosan microspheres in the continuous conversion production of nicotinic acid.
[0026] Furthermore, the optimal reaction conditions for continuous conversion were: temperature 35℃, pH 7.0, and a substrate concentration of 350 mmol·L⁻¹. -1 The volume ratio of immobilized cells to substrate 3-cyanopyridine was 1:1, and the retention time was 35.2 min.
[0027] Beneficial effects: Compared with the prior art, the advantages of this invention are:
[0028] 1) The chitosan / polyvinyl alcohol microspheres prepared by this invention have smooth shapes, uniform sizes, and particle sizes controlled within the range of 1 to 250 μm, and have a core-shell structure.
[0029] 2) The chemical method (using more than 40% sodium hydroxide) currently used to produce chitosan has many problems, such as high energy consumption, unstable product quality (mainly referring to average molecular weight and degree of deacetylation), and, more seriously, the emissions cause huge environmental pollution, severely damage the surrounding ecology, and have high costs for subsequent environmental treatment. This invention adopts a composite enzyme combined with microwave induction process to deacetylate chitin: improving the deacetylation rate of chitin and the degree of deacetylation of the obtained chitosan.
[0030] 3) This invention refines chitosan through enzymatic hydrolysis and performs enzymatic molecular modification to endow chitosan with more characteristics and functions.
[0031] 4) Applying genipin to treat chitosan / polyvinyl alcohol microspheres to immobilize cells improves the mechanical properties of the immobilized cells, as well as enhances cell viability retention and reusability. The cells can be reused multiple times, have high substrate tolerance, and improve the production capacity per unit cell.
[0032] 5) The prepared chitosan microspheres were used to immobilize live Gibberella fusarium cells producing nitrile hydrolase, and nicotinic acid was continuously produced by conversion using a packed-bed bioreactor. The conversion capacity was increased by 4.3 times compared to free cells. This achieved the efficient industrial production of high-quality chitosan microspheres. Attached Figure Description
[0033] Figure 1 This is a technical roadmap for the present invention. Detailed Implementation
[0034] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of the invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.
[0035] Example 1
[0036] A method for preparing chitosan microspheres for cell immobilization and drug delivery includes the following steps:
[0037] 1) Weigh chitin and chitin deacetylase at a mass ratio of 1000:1, dissolve in water, place in a microwave device, treat at 50°C and microwave power of 600W for 10 minutes to inactivate the enzyme and obtain chitosan solution.
[0038] 2) Prepare a chelating agent with a concentration of 0.02 mol / L using tetrasodium diacetate of glutamic acid. Mix the chelating agent and chitosan solution at a molar ratio of 10:0.2 thoroughly and observe the changes in the solution until the solution changes from clear to turbid and then back to clear.
[0039] 3) Add 0.5 g / L of carbonate to the solution in step 2) for sedimentation, stir thoroughly to improve the extraction and removal ability of the chelating agent for residual metal ions, and obtain crude chitosan solution.
[0040] 4) Add a complex bio-enzyme (specific keratinase and lipase) to the crude chitosan solution to hydrolyze it and remove proteins and fats. The mass ratio of chitosan to the complex bio-enzyme is 1:0.01. React in a water bath at 50℃ for 3 hours. The enzymatic reaction solution is then rapidly heated to 90℃ to inactivate the enzyme for 10 minutes. The enzymatic reaction solution is filtered using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.05 MPa. The ultrafiltrate is then further filtered using a 3000 Da ultrafiltration membrane at an operating pressure of 0.20 MPa. Simultaneously, the filtrate concentration is concentrated to 20 ± 2 wt.%.
[0041] 5) Refined chitosan is directly dissolved in water to form a transparent solution. The pH is adjusted to 7.0, and modifying enzymes are added: laccase, glutamine transferase, and tyrosinase. The mass ratio of laccase, glutamine transferase, and tyrosinase is 1:1:1, and the mass ratio of chitosan to modifying enzymes is 1:0.09. After treatment at 50℃ for 5 hours, modified chitosan is obtained. Modifying chitosan with modifying enzymes can effectively improve its application functions, endowing chitosan and its derivatives with higher biological activity or other application properties.
[0042] 6) A novel polymer gel sphere preparation instrument designed and assembled by the author (see patent application "A method for preparing gel spheres", application number 2018111818341) was used to prepare and obtain chitosan microspheres with a shell-core structure that are smooth in shape and uniform in size by selecting the pore size of the dropper, controlling the speed of the peristaltic pump, controlling the concentration and temperature of the crosslinking agent, and using screening filters with different pore sizes. A double-layer dropper was innovatively used to prepare gel spheres with a shell-core structure through direct embedding. Gibberellinia spp. (purchased from the National Standards Network) was inoculated into potato dextrose liquid culture medium, shaken and cultured to the logarithmic phase, centrifuged, and the supernatant was removed. The culture was then resuspended in physiological saline to obtain a bacterial suspension. The chitosan solution and polyvinyl alcohol solution obtained in step 5) were added to form a composite solvent system. The mass concentration ratio of the bacterial suspension, chitosan solution and polyvinyl alcohol solution was 1.5:1:1. The green and environmentally friendly spray drying method is adopted. Inert gases such as CO2 are pumped in through the liquid inlet nozzle, and spray drying is carried out in the hot air flow formed by the inert gases. The composite solvent is rapidly evaporated to obtain dried chitosan / polyvinyl alcohol (PVA-CS) microspheres and chitosan / polyvinyl alcohol microsphere immobilized cells. The microsphere particle size is controlled in the range of 1-250μm and the porosity is high.
[0043] 7) Take the dried chitosan / polyvinyl alcohol microspheres immobilized cells and add them to PBS buffer containing 2 wt.% genipin. Treat at 30°C for 5 hours. After treatment, rinse repeatedly with PBS buffer to remove residual genipin. Finally, store the immobilized cells in physiological saline at 10°C.
[0044] The chitosan / polyvinyl alcohol microspheres prepared in this embodiment have the following purity: degree of deacetylation ≥90%, viscosity ≤2000 mpa.s, loss on drying ≤10%, residue on ignition ≤2.0%, acid-insoluble matter ≤1.0%; and the amount of alkali used is reduced by 80%.
[0045] According to the enterprise standard Q / 321084 RXSW37-2020, the hardness of chitosan / polyvinyl alcohol microspheres is required to be ≥289g. After testing, the hardness of the product prepared in this example reached 345g.
[0046] According to the enterprise standard Q / 321084 RXSW37-2020, the particle diameter of chitosan / polyvinyl alcohol microspheres is required to be ≤2.5mm. After testing, the particle diameter of the product prepared in this embodiment reaches 2.0mm.
[0047] The microstructure of the chitosan / polyvinyl alcohol microspheres conforms to the claimed requirements according to the enterprise standard Q / 321084 RXSW37-2020. After testing, the microstructure of the product prepared in this embodiment conforms to the claimed requirements.
[0048] According to the enterprise standard Q / 321084 RXSW37-2020, the porosity of chitosan / polyvinyl alcohol microspheres is required to be ≥85.4%. After testing, the porosity of the product prepared in this embodiment reaches 88.9%.
[0049] Example 2
[0050] A method for preparing chitosan microspheres for cell immobilization and drug delivery includes the following steps:
[0051] 1) Weigh chitin and chitin deacetylase at a mass ratio of 1000:1, dissolve in water, place in a microwave device, treat at 50°C and microwave power of 400W for 10 minutes to inactivate the enzyme and obtain chitosan solution.
[0052] 2) Prepare a chelating agent with a concentration of 0.01 mol / L using citric acid. Mix the chelating agent and chitosan solution at a molar ratio of 20:1 thoroughly and observe the changes in the solution until the solution changes from clear to turbid and then back to clear.
[0053] 3) Add 2 g / L of carbonate to the solution in step 2) for precipitation, stir thoroughly to improve the extraction and removal ability of the chelating agent for residual metal ions, and obtain crude chitosan solution.
[0054] 4) Add a complex bio-enzyme (specific keratinase and lipase) to the crude chitosan solution to hydrolyze it and remove proteins and fats. The mass ratio of chitosan to the complex bio-enzyme is 1:0.05. React in a water bath at 50℃ for 3 hours. The enzymatic reaction solution is then rapidly heated to 90℃ to inactivate the enzyme for 10 minutes. The enzymatic reaction solution is filtered using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.05 MPa. The ultrafiltrate is then further filtered using a 3000 Da ultrafiltration membrane at an operating pressure of 0.20 MPa. Simultaneously, the filtrate concentration is concentrated to 20 ± 2 wt.%.
[0055] 5) Refined chitosan is directly dissolved in water to form a transparent solution. The pH is adjusted to 7.0, and modifying enzymes are added: laccase, glutamine transferase, and tyrosinase. The mass ratio of laccase, glutamine transferase, and tyrosinase is 1:1:1, and the mass ratio of chitosan to modifying enzymes is 1:0.15. After treatment at 50℃ for 20 hours, modified chitosan is obtained. Modifying chitosan with modifying enzymes can effectively improve its application functions, endowing chitosan and its derivatives with higher biological activity or other application properties.
[0056] 6) A novel polymer gel sphere preparation instrument designed and assembled by the author (see patent application "A method for preparing gel spheres", application number 2018111818341) was used to prepare and obtain chitosan microspheres with a shell-core structure that are smooth in shape and uniform in size by selecting the pore size of the dropper, controlling the speed of the peristaltic pump, controlling the concentration and temperature of the crosslinking agent, and using screening filters with different pore sizes. A double-layer dropper was innovatively used to prepare gel spheres with a shell-core structure through direct embedding. Gibberellinia spp. (purchased from the National Standards Network) was inoculated into potato dextrose liquid culture medium, shaken and cultured to the logarithmic phase, centrifuged, and the supernatant was removed. The culture was then resuspended in physiological saline to obtain a bacterial suspension. The chitosan solution and polyvinyl alcohol solution obtained in step 5) were added to form a composite solvent system. The mass concentration ratio of the bacterial suspension, chitosan solution and polyvinyl alcohol solution was 2:1:1. The green and environmentally friendly spray drying method is adopted. Inert gases such as CO2 are pumped in through the liquid inlet nozzle, and spray drying is carried out in the hot air flow formed by the inert gases. The composite solvent is rapidly evaporated to obtain dried chitosan / polyvinyl alcohol (PVA-CS) microspheres and chitosan / polyvinyl alcohol microsphere immobilized cells. The microsphere particle size is controlled in the range of 1-250μm and the porosity is high.
[0057] 7) Take the dried chitosan / polyvinyl alcohol microspheres immobilized cells and add them to PBS buffer containing 5 wt.% genipin. Treat them at 30°C for 18 hours. After treatment, rinse them several times with PBS buffer to remove residual genipin. Finally, store the immobilized cells in physiological saline at 10°C.
[0058] Example 3
[0059] Immobilized cells prepared in Example 1 were used for the continuous production of nicotinic acid via a packed-bed bioreactor. Nicotinic acid, also known as nicotinic acid or anti-pellagra factor, is converted into nicotinamide in the human body and participates in lipid metabolism, tissue respiration oxidation, and anaerobic carbohydrate decomposition. A packed-bed bioreactor was constructed using chromatography columns, peristaltic pumps, and a super water bath, among other equipment, for the continuous production of nicotinic acid. The optimal reaction conditions for continuous production were: temperature 35°C, pH 7.0, and a substrate concentration of 350 mmol·L⁻¹. -1 The volume ratio of immobilized cells to substrate 3-cyanopyridine was 1:1, the retention time was 35.2 min, and the transformation capacity of cross-linked PVA-CS immobilized cells was 348.4 g (3-cyanopyridine)·g. -1 (dcw). In fed-batch continuous conversion, up to 6 feeds can be performed within a 600 mL conversion system, with a product concentration of approximately 250.7 g·L⁻¹ in the conversion solution. -1 The transformation capacity of cross-linked PVA-CS immobilized cells was 409.9 g (3-cyanopyridine)·g. -1(dcw). The conversion capacity was 4.3 times higher than that of free cells. High-quality chitosan microspheres were successfully produced industrially, providing a carrier for enzyme / whole-cell immobilization and drug sustained release.
[0060] The preferred embodiments of the present invention have been described in detail above. In addition, drugs, probiotics, nutritional chemicals, or microbial enzyme catalysts can be dispersed in a chitosan solution to form a composite solvent system. Spray drying is then performed in a hot gas stream containing inert gas to rapidly evaporate the composite solvent, obtaining dried microspheres and broadening the engineering applications of chitosan microspheres. Simple modifications and combinations should also be considered as part of the content disclosed in this invention and are all within the scope of protection of this invention.
Claims
1. A method for preparing chitosan microspheres for cell immobilization and drug delivery, characterized in that, Includes the following steps: 1) Weigh chitin and chitin deacetylase at a mass ratio of 1000:1, dissolve in water, place in a microwave device, treat at 40-60℃ and microwave power of 400-600W for 10 minutes, then inactivate the enzyme to obtain chitosan solution. 2) The chelating agent and chitosan solution are stirred thoroughly at a molar ratio of 10-20:0.2-1 until they are evenly mixed. The solution changes until it changes from clear to turbid and then back to clear. The chelating agent is tetrasodium glutamate diacetate or citric acid, and the concentration of the chelating agent is 0.01-0.02 mol / L. 3) Add carbonate to the solution from step 2) to allow precipitation, stir thoroughly to obtain crude chitosan solution; the amount of carbonate added is 0.5~2g per liter of solution; 4) Add a complex bio-enzyme to the crude chitosan solution to hydrolyze it and remove proteins and fats. After the enzymatic hydrolysis reaction, inactivate the enzyme, ultrafilter, and dry to obtain refined chitosan. The complex bio-enzyme is a specific keratinase and lipase. The mass ratio of chitosan to complex bio-enzyme is 1:0.01-0.
05. The enzymatic hydrolysis reaction temperature is 40-60℃, and the enzymatic hydrolysis reaction time is 3h. After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysis reaction solution is rapidly heated to 90℃ for 10min to inactivate the enzyme. The enzymatic hydrolysis reaction solution is filtered using an ultrafiltration membrane with a molecular weight cutoff of 5000Da and a membrane operating pressure of 0.05MPa. The ultrafiltrate is further filtered using a 3000Da ultrafiltration membrane with a membrane operating pressure of 0.20MPa. The filtrate is concentrated and dried to obtain refined chitosan. 5) Refined chitosan is directly dissolved in water to form a transparent solution. The pH is adjusted, and a modifying enzyme is added. The chitosan is modified using the modifying enzyme. The mass ratio of chitosan to modifying enzyme is 1:0.09-0.
15. After treatment at 40-60℃ for 1-24 hours, the modified chitosan is obtained. The modifying enzyme is laccase, glutamine transferase and tyrosinase. 6) Inoculate *Gibberellic Aureobasidium* into potato dextrose liquid medium, culture with shaking until the logarithmic growth phase, centrifuge, remove supernatant, and resuspend in physiological saline to obtain a bacterial suspension. Add the chitosan solution and polyvinyl alcohol solution obtained in step 5) to form a composite solvent system. The mass concentration ratio of bacterial suspension, chitosan solution, and polyvinyl alcohol solution is 1.5-2:1:
1. Use a polymer gel sphere preparation instrument to spray dry dried chitosan / polyvinyl alcohol microspheres and chitosan / polyvinyl alcohol microsphere-immobilized cells by pumping inert gas through the inlet nozzle and the hot gas flow formed by the inert gas. The particle size of the chitosan / polyvinyl alcohol microspheres is controlled within the range of 1~250 μm. 7) Treat chitosan / polyvinyl alcohol microsphere immobilized cells with genipin and store the immobilized cells in physiological saline; take dried chitosan / polyvinyl alcohol microsphere immobilized cells and add them to PBS buffer containing 1wt.%-5wt.% genipin, treat at a water bath temperature of 20-40℃ for 5-18h, after which rinse repeatedly with PBS buffer to remove residual genipin, and finally store the immobilized cells in physiological saline at 0-10℃.
2. The method for preparing chitosan microspheres for cell immobilization and drug delivery according to claim 1, characterized in that, In step 5), adjust the pH to 6.0-8.0, and the mass ratio of laccase, glutamine transferase, and tyrosinase is 1:1:1.