Composite sodium alginate-based aerogel immobilized enzyme carrier and application thereof

Abstract

The invention provides a composite sodium alginate-based aerogel immobilized enzyme carrier and an application thereof. The preparation method comprises the following steps: dispersing sodium alginate and oxidized sodium alginate in deionized water, adding starch and nanocellulose, stirring in a boiling water bath until the starch and nanocellulose are completely dissolved, and cooling to room temperature to obtain a mixed solution; adding a beta-galactosidase solution, and uniformly stirring to obtain an enzyme-containing mixed solution; slowly dripping into a metal ion solution at a constant speed for primary hardening; adding a CaCl2 solution for secondary hardening, and separating to obtain gel microspheres; immersing into a potassium phosphate buffer solution containing polydopamine nanoparticles; freezing and drying; and carrying out heat-moisture treatment. Sodium alginate is used as a substrate material and has good biocompatibility, resistant starch is added in the preparation process, the stability of the aerogel immobilization carrier is improved, and the aerogel immobilization carrier shows a round and full skeleton structure after being freeze-dried to form a supporting effect; and a dynamic dual-network design is adopted, so that the self-repairing capability is provided.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme immobilization, and in particular relates to a composite sodium alginate-based aerogel immobilized enzyme carrier and application thereof. Background Art

[0002] Enzymes are widely used in the development of novel foods and in numerous other fields. However, the inactivation and difficulty of free enzymes in complex industrial environments severely restrict their large-scale application. Immobilization techniques, which physically or chemically bind enzymes to carriers, can significantly improve enzyme reusability and environmental tolerance. Among existing methods, encapsulation is the preferred immobilization method due to its ease of use and ability to maximize enzyme activity. Sodium alginate, with its unique physicochemical properties, has become a core encapsulation material for enzyme immobilization. This natural linear polysaccharide, extracted from the cell walls of brown algae, is composed of alternating 1,4-glycosidic bonds between β-D-mannuronic acid and α-L-guluronic acid. Its molecular chain is rich in active sites such as hydroxyl and carboxyl groups. Its excellent water solubility, biocompatibility, mild gel properties, and low cost have made it widely used as a green and safe immobilization carrier in the food industry, drug delivery, and enzyme engineering. The gelation mechanism of sodium alginate follows the classic “egg-box model”: under neutral conditions, its carboxylic acid groups dissociate into negative charges, and the introduction of Ca 2 Cross-linking with divalent cations such as Zn2+ and Zn2+ can rapidly form a three-dimensional network hydrogel in a mild environment, achieving efficient encapsulation of enzyme molecules. However, single sodium alginate gels have obvious drawbacks: low mechanical strength, excessive porosity, and easy structural disintegration under long-term hydration conditions, making it difficult to meet the stringent requirements of industrial production for the mechanical stability and durability of the carrier. Aerogels are new porous materials that remove the liquid components in hydrogels through freeze-drying or supercritical drying technology to form a solid network structure with ultra-high specific surface area, low density and excellent thermal insulation properties. Compared with ordinary gels, they are easier to store and transport, and the enzyme loading efficiency can be optimized through pore regulation. The development of sodium alginate-based composite aerogels, such as the introduction of resistant starch to construct a hybrid network, can further optimize the performance of aerogels. At the same time, in recent years, self-healing hydrogels have attracted attention due to their ability to recover autonomously after damage. The application of existing self-healing materials in enzyme immobilization still faces challenges. Therefore, it is urgent to break through the performance bottleneck of a single material through dynamic dual-network design and provide an immobilized enzyme carrier with high mass transfer rate and self-healing properties. Summary of the Invention

[0003] Technical problem to be solved: In response to the above problems, the purpose of the present invention is to provide a composite sodium alginate-based aerogel immobilized enzyme carrier and its application, which uses sodium alginate as the base material and has good biocompatibility. Resistant starch is added during the preparation to increase the stability of the aerogel immobilization carrier. After freeze-drying, it exhibits a rounded and full skeleton structure to form a supporting effect, and adopts a dynamic double network design: the aldehyde group of oxidized sodium alginate forms a dynamic Schiff base bond with the hydroxyl group of starch, providing pH-responsive self-repairing ability.

[0004] Technical solution: A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier, comprising the following steps: Step 1: Sodium alginate (SA) and oxidized sodium alginate (ADA) are dispersed in deionized water, starch and nanocellulose (CNF) are added, and the mixture is stirred in a boiling water bath until completely dissolved, and cooled to room temperature to obtain a mixed solution; Step 2: Add β-galactosidase solution and stir evenly to obtain an enzyme-containing mixture; Step 3: Prepare the metal ion solution, draw the enzyme mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface for the initial hardening for 60-240 minutes; Step 4: Remove the supernatant and continue to add 75-200 mM / L CaCl2 solution for secondary hardening for 60 minutes, and separate with a nylon membrane to obtain gel microspheres; Step 5: Immerse the gel microspheres in potassium phosphate buffer containing polydopamine nanoparticles for 2 h; Step 6: Wash the gel 3-4 times with pure water, pre-freeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, treat in an environment of 50° C. and 80% relative humidity for 10-13 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. Furthermore, in step 1, the concentration of sodium alginate is 1-4%, the concentration of oxidized sodium alginate is 0.25%, the concentration of starch is 0.1-2.0%, and the concentration of nanocellulose is 0.05%. Furthermore, the starch includes potato starch (MLS), tapioca starch (MS), corn starch (YM), sweet potato starch (GS) and resistant starch (RS). Furthermore, the concentration of the β-galactosidase solution in step 2 is 0.2-2.0 mg / mL. Furthermore, the metal ion solution in step 3 includes calcium chloride, manganese chloride and cobalt chloride solutions. Furthermore, the CaCl2 solution in step 4 contains 5 mM / L histidine. Furthermore, in step 4, the pore size of the nylon membrane is 0.45 μm. Furthermore, in step 5, the potassium phosphate buffer solution has a pH of 7.0, 10 mM, and the content of polydopamine nanoparticles is 0.1%. The composite sodium alginate-based aerogel immobilized enzyme carrier prepared by the above preparation method. Application of the composite sodium alginate-based aerogel immobilized enzyme carrier in the preparation of low-lactose food. Beneficial effects: 1. The composite sodium alginate-based aerogel immobilized enzyme carrier of the present invention uses sodium alginate as a base material and has good biocompatibility. 2. Resistant starch is added during the preparation of the composite sodium alginate-based aerogel immobilized enzyme carrier of the present invention, which increases the stability of the aerogel immobilized carrier. After freeze-drying, it exhibits a rounded and full skeleton structure, forming a supporting effect. 3. This invention utilizes a dynamic dual-network design: the aldehyde groups of oxidized sodium alginate form dynamic Schiff-base bonds with the hydroxyl groups of starch, providing pH-responsive self-healing capabilities. Moisture-heat conditions trigger the recombination of dynamic bonds (Schiff-base bonds and coordination bonds), repairing microcracks. After damage, the material can be self-healed (with a repair efficiency >90%) by gentle heating (50°C) or pH adjustment (pH 5-8). 4. Ca in the present invention 2+ -Histidine coordination bond: Dynamic regulation of Ca 2 + Cross-linking density can enhance cross-linking reversibility and avoid the rigidity defects of traditional ionic cross-linking. 5. In the present invention, polydopamine is synthesized by catechol-Ca 2+ Coordinate bonds stabilize the network while providing antioxidant protection. 6. Nanocellulose in the present invention serves as a reinforcing phase, which can improve the mechanical strength of the aerogel and prevent collapse during freeze drying. 7. The composite sodium alginate-based aerogel immobilized enzyme carrier of the present invention was successfully applied to the immobilization of β-galactosidase from Aspergillus oryzae. When containing a single lactose system, a lactose conversion rate of 85% could be achieved within 1 hour. 8. The preparation method of the present invention does not require the use of complex instruments and equipment, has low material costs, and is easy to produce in batches. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The SEM images of the composite sodium alginate-based aerogel-immobilized enzyme carriers prepared in Examples 1-3 under their respective optimal conditions, wherein (a) shows the optimal CaCl2 concentration (200 mM) in Example 1; (b) shows the optimal SA concentration (3%) in Example 2; and (c) shows the optimal hardening time (60 min) in Example 3. Figure 2These are SEM morphologies of the composite sodium alginate-based aerogel-immobilized enzyme carrier prepared in Example 5, wherein Figure (a) shows an RD addition amount of 0% without secondary hardening; Figure (b) shows an optimal RS addition amount (0.5%) without secondary hardening; and Figure (c) shows an optimal RS addition amount (0.5%) with secondary hardening. Figure 3 These are SEM morphologies of the composite sodium alginate-based aerogel-immobilized enzyme carriers prepared in Example 6, where Figure (a) is the aerogel without RS added as a control; Figure (b) is RS; Figure (c) is GS; Figure (d) is MS; Figure (e) is YM; and Figure (f) is MLS. Figure 4 The graph shows the change in lactose concentration corresponding to the application of the composite sodium alginate-based aerogel-immobilized enzyme carrier prepared in the best embodiment in the hydrolysis of lactose. DETAILED DESCRIPTION The present invention provides a composite sodium alginate-based aerogel-immobilized enzyme carrier and its application. To clarify the objectives, technical solutions, and effects of the present invention, the present invention will be further described in detail with reference to the following examples. It should be understood that the specific examples described herein are intended only to illustrate the present invention and are not intended to limit the present invention. Example 1 (Screening of CaCl2 solution concentration) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Disperse sodium alginate and oxidized sodium alginate in deionized water, add resistant starch and nanocellulose, stir in a boiling water bath until completely dissolved, and cool to room temperature to obtain a mixed solution with a sodium alginate concentration of 2%, a sodium alginate concentration of 0.25%, a resistant starch concentration of 1.0%, and a nanocellulose concentration of 0.05%; Step 2: Add β-galactosidase solution and stir evenly to obtain a mixture containing 0.5 mg / mL β-galactosidase; Step 3: Prepare 200 mM CaCl2 solution, draw the enzyme mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface at a uniform speed for 30 minutes of primary hardening; Step 4: Remove the supernatant and continue to add 75mM / L, 100mM / L, 200mM / L, 400mM / L and 600mM / L CaCl2 solution containing 5mM / L histidine, respectively, for secondary hardening for 60min, and separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution with a pH of 7.0 containing 0.1% polydopamine nanoparticles (PDA) for 2 h; Step 6: Wash the gel 4 times with pure water, prefreeze at -80°C for 6 h, and freeze-dry for 36 h; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. Performance testing: Protein precipitation rate: 0.5 g of the composite sodium alginate-based aerogel-immobilized enzyme carrier prepared above was placed in 5 mL of 10 mM potassium phosphate buffer (pH 7.0) and placed at 4°C for 24 h. The amount of protein precipitation in the supernatant was then measured. The results showed that when the CaCl2 concentration was 200mM, SA was 2%, and the hardening time was 30min, the protein precipitation rate after 24h was 4.16%. The CaCl2 solution concentration of 75-200mM was the best, with 200mM being the most effective. Example 2 (Screening of sodium alginate concentration) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Sodium alginate and oxidized sodium alginate are dispersed in deionized water, resistant starch and nanocellulose are added, and the mixtures are stirred in a boiling water bath until completely dissolved. The mixtures are cooled to room temperature to obtain mixed solutions with sodium alginate concentrations of 1%, 2%, 3%, and 4%, oxidized sodium alginate concentrations of 0.25%, resistant starch concentrations of 1.0%, and nanocellulose concentrations of 0.05%. Step 2: Add β-galactosidase solution and stir evenly to obtain a mixture containing 0.5 mg / mL β-galactosidase; Step 3: Prepare 200 mM CaCl2 solution, draw the enzyme mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface at a uniform speed for 30 minutes of primary hardening; Step 4: Remove the supernatant and continue to add 200mM / L CaCl2 solution containing 5mM / L histidine for secondary hardening for 60 minutes. Separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. Performance testing: Protein precipitation rate: 0.5 g of the composite sodium alginate-based aerogel-immobilized enzyme carrier prepared above was placed in 5 mL of 10 mM potassium phosphate buffer (pH 7.0) and placed at 4°C for 24 h. The amount of protein precipitation in the supernatant was then measured. The results showed that when the CaCl2 concentration was 200mM, the SA content was 3%, and the hardening time was 30 minutes, the protein precipitation rate after 24 hours was 3.91%. The sodium alginate concentration of 1-4% (w / v) was the best, with 3% (w / v) being the most effective. Example 3 (Screening of initial hardening time) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Sodium alginate and oxidized sodium alginate are dispersed in deionized water, resistant starch and nanocellulose are added, and the mixture is stirred in a boiling water bath until completely dissolved. The mixture is cooled to room temperature to obtain a mixture having a sodium alginate concentration of 3%, a sodium alginate oxidized concentration of 0.25%, a resistant starch concentration of 1.0%, and a nanocellulose concentration of 0.05%; Step 2: Add β-galactosidase solution and stir evenly to obtain a mixture containing 0.5 mg / mL β-galactosidase; Step 3: Prepare a 200 mM CaCl2 solution, draw the enzyme-containing mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface. Perform the initial hardening for 30 minutes, 60 minutes, 120 minutes, and 240 minutes respectively; Step 4: Remove the supernatant and continue to add 200mM / L CaCl2 solution containing 5mM / L histidine for secondary hardening for 60 minutes. Separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. Performance testing: The SEM morphology images are all images of aerogels at a scale of 200 μm, with an accelerating voltage of 3-5 kV; Protein precipitation rate: 0.5 g of the composite sodium alginate-based aerogel-immobilized enzyme carrier prepared above was placed in 5 mL of 10 mM potassium phosphate buffer (pH 7.0) and placed at 4°C for 24 h. The amount of protein precipitation in the supernatant was then measured. The results showed that when the CaCl2 concentration was 200mM, SA was 3%, and the hardening time was 60min, the protein precipitation rate after 24h was 3.63%. The initial hardening time was 60-240min, which had the best effect. Figure 1 shown. Example 4 (Screening of β-galactosidase solution concentration) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Disperse sodium alginate and oxidized sodium alginate in deionized water, add resistant starch and nanocellulose, stir in a boiling water bath until completely dissolved, and cool to room temperature to obtain a mixed solution with a sodium alginate concentration of 3%, a sodium alginate concentration of 0.25%, a starch concentration of 1.0%, and a nanocellulose concentration of 0.05%; Step 2: Add β-galactosidase solution and stir evenly to obtain mixed solutions containing 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, and 5.0 mg / mL β-galactosidase, respectively; Step 3: Prepare a 200 mM CaCl2 solution, draw the enzyme-containing mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface for initial hardening for 60 minutes; Step 4: Remove the supernatant and continue to add 200mM / L CaCl2 solution containing 5mM / L histidine for secondary hardening for 60 minutes. Separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. Performance testing: Protein precipitation rate: 0.5 g of the composite sodium alginate-based aerogel-immobilized enzyme carrier prepared above was placed in 5 mL of 10 mM potassium phosphate buffer (pH 7.0) and placed at 4°C for 24 h. The amount of protein precipitation in the supernatant was then measured. The results showed that when the CaCl2 concentration was 200mM, SA was 3%, the hardening time was 60min, and the β-galactosidase solution concentration was 0.2mg / mL, the protein precipitation rate after 24h was 3.56%. The concentration of 0.2mg / mL had the best effect. Example 5 (Screening of starch addition amount) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Sodium alginate and oxidized sodium alginate are dispersed in deionized water, resistant starch and nanocellulose are added, and the mixture is stirred in a boiling water bath until completely dissolved. The mixture is cooled to room temperature to obtain a mixture having a sodium alginate concentration of 3%, a sodium alginate concentration of 0.25%, resistant starch concentrations of 0%, 0.1%, 0.5%, 1.0%, and 2.0%, respectively, and a nanocellulose concentration of 0.05%. Step 2: β-galactosidase solution is added and stirred uniformly to obtain a mixture containing 0.2 mg / mL β-galactosidase. Step 3: 200 mM CaCl2 solution is prepared, the enzyme-containing mixture is aspirated, and the mixture is slowly dripped into the metal ion solution at a height of 20 cm from the liquid surface for initial hardening for 60 minutes. Step 4: Remove the supernatant and continue to add 200mM / L CaCl2 solution containing 5mM / L histidine for secondary hardening for 60 minutes. Separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. The amount of supernatant protein precipitation was determined, and the enzyme protein embedding rate and the SEM of the composite sodium alginate-based aerogel immobilized enzyme carrier were also determined. The results showed that when the CaCl2 concentration was 200mM, SA was 3%, the hardening time was 60 minutes, the β-galactosidase solution concentration was 0.2mg / mL, and the RS addition was 0.5%, the protein precipitation rate after 24 hours was 2.25%. The best effect was achieved when the starch addition was 0.1-2.0%, with the optimal effect being 0.5%. The SEM morphologies of the composite sodium alginate-based aerogel immobilized enzyme carrier prepared above after primary hardening and secondary hardening under the conditions of 0% and 0.5% starch addition are shown in Figure 2. Figure 2 shown. Example 6 (Screening of starch types) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Sodium alginate and oxidized sodium alginate are dispersed in deionized water, and different types of starch (including potato starch, tapioca starch, corn starch, sweet potato starch, and resistant starch) and nanocellulose are added separately. The mixture is stirred in a boiling water bath until completely dissolved, and then cooled to room temperature to obtain a mixed solution with a sodium alginate concentration of 3%, a sodium alginate concentration of 0.25%, a starch concentration of 0.5%, and a nanocellulose concentration of 0.05%; Step 2: Add β-galactosidase solution and stir evenly to obtain a mixture containing 0.2 mg / mL β-galactosidase; Step 3: Prepare 200 mM CaCl2 solution, draw the enzyme mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface at a uniform speed for 60 minutes of primary hardening; Step 4: Remove the supernatant and continue to add 200mM / L CaCl2 solution containing 5mM / L histidine for secondary hardening for 60 minutes. Separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. Measurement of supernatant protein precipitation revealed that RS had the lowest protein precipitation rate of 2.07% when the CaCl2 concentration was 200 mM, SA was 3%, the hardening time was 60 minutes, the β-galactosidase solution concentration was 0.2 mg / mL, and the starch addition was 0.5%. After secondary hardening, the protein precipitation rate of all samples decreased, with RS alone experiencing a rate below 2%, reaching 1.56%. Its SEM morphology is as follows Figure 3 As shown, the addition of resistant starch has the best morphology and the best effect. Example 7 (Screening of Metal Ion Solutions) A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Sodium alginate and oxidized sodium alginate are dispersed in deionized water, resistant starch and nanocellulose are added, and the mixture is stirred in a boiling water bath until completely dissolved. The mixture is cooled to room temperature to obtain a mixture having a sodium alginate concentration of 3%, a sodium alginate concentration of 0.25%, a resistant starch concentration of 0.5%, and a nanocellulose concentration of 0.05%; Step 2: Add β-galactosidase solution and stir evenly to obtain a mixture containing 0.2 mg / mL β-galactosidase; Step 3: Prepare 200 mM MnCl2 / CoCl2 / CaCl2 solutions respectively, draw the enzyme mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface at a uniform speed for initial hardening for 60 minutes; Step 4: Remove the supernatant and continue to add 200mM / L CaCl2 solution containing 5mM / L histidine for secondary hardening for 60 minutes. Separate with a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier. The protein precipitation rate of the supernatant was measured. The protein precipitation rates of SA-Ca, SA-Mn and SA-Co were 0.86%, 1.24% and 4.33% respectively. The protein precipitation rates of SA-Ca+RS, SA-Mn+RS and SA-Co+RS were 0.61%, 1.56% and 1.45% respectively. Taking all factors into consideration, the gel material prepared with the participation of Ca is still more solid. Best Mode From the data and SEM morphology observations, it can be seen that when the SA, RS and CaCl2 concentrations are 3%, 0.5% and 200mM respectively, the enzyme addition amount is 0.2mg / mL, the primary hardening time is 60min, and the secondary hardening time is 60min, the composite sodium alginate-based aerogel immobilized enzyme carrier shows a relatively complete and full aerogel spherical morphology. At this time, the 24h protein precipitation rate can be kept below 2%. Specifically: A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier comprises the following steps: Step 1: Sodium alginate and oxidized sodium alginate are dispersed in deionized water, resistant starch and nanocellulose are added, and the mixture is stirred in a boiling water bath until completely dissolved. The mixture is cooled to room temperature to obtain a mixture having a sodium alginate concentration of 3%, a sodium alginate concentration of 0.25%, a resistant starch concentration of 0.5%, and a nanocellulose concentration of 0.05%; Step 2: Add β-galactosidase solution and stir evenly to obtain a mixture containing 0.2 mg / mL β-galactosidase; Step 3: Prepare 200 mM CaCl2 solution, draw the enzyme mixture, and slowly drip it into the metal ion solution at a height of 20 cm from the liquid surface at a uniform speed for 60 minutes of primary hardening; Step 4: Remove the supernatant and continue to add 200mM / L and 5mM / L histidine-containing CaCl2 solutions, respectively, for secondary hardening for 60 minutes, and separate using a nylon membrane with a pore size of 0.45μm to obtain gel microspheres; Step 5: Immerse the gel microspheres in a potassium phosphate buffer solution (pH 7.0) containing 0.1% polydopamine nanoparticles for 2 hours; Step 6: Wash the gel four times with pure water, prefreeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, the mixture was treated in an environment of 50° C. and 80% relative humidity for 12 minutes to obtain a composite sodium alginate-based aerogel immobilized enzyme carrier, which was designated as SA-Ca+RS. Comparative Example 1 The difference between this embodiment and the best embodiment lies in the absence of oxidized sodium alginate and histidine. Comparative Example 2 The difference between this embodiment and the optimal embodiment is that there is no nanocellulose and PDA modification. Performance testing: Determination of self-repair efficiency: By simulating the material to be repaired at 50°C after compression damage, the degree of mechanical property recovery or morphological integrity before and after repair is compared. Determination of enzyme activity retention rate: By measuring the changes in the catalytic activity of the immobilized enzyme before and after repair, β-galactosidase catalyzes the hydrolysis of ONPG (o-nitrophenyl-β-D-pyranogalactoside) to generate a yellow product (o-nitrophenol, ONP), and the absorbance is measured at 420 nm. Self-repair efficiency Enzyme activity retention Best Mode 94.2% (50℃ damp heat) 85% (5 cycles) Comparative Example 1 none 55% (3 cycles) Comparative Example 2 60％ 65% (5 cycles) It can be seen from Comparative Example 1 that oxidized sodium alginate + histidine is the key to the self-repair function; and it can be seen from Comparative Example 2 that CNF + PDA synergistically improves mechanical strength and enzyme stability. The best embodiment is applied to the preparation of low-lactose food, specifically: First, dissolve 10 g of lactose in 10 mM citric acid-sodium citrate buffer at pH 4.5, then use a 25 mL volumetric flask to dilute to the mark to obtain a 200 g / L lactose solution for later use. Place all the SA-Ca+RS in a 100 mL conical flask with a lid, add 20 mL of the prepared lactose solution, and react at 55°C and 130 r / min for 24 h. Sampling is performed at time points of 0, 1, 2, 4, 8, 12, and 24 h. Take out 0.4 mL of sample at each point and heat in boiling water for 2 min to inactivate the enzyme. The changes in lactose concentration of the samples are analyzed by high performance liquid chromatography (HPLC). The results are as follows Figure 4 As shown, a lactose conversion experiment was carried out using 200 g / L lactose solution. The results showed that after 1 hour of reaction, the lactose consumption rate was as high as 83.89%, and 168.8 g / L of glucose and galactose were produced. In summary, the novel composite food-grade sodium alginate-based aerogel immobilization carrier of the present invention achieves efficient enzyme immobilization by leveraging the good biocompatibility of sodium alginate and the supporting effect of resistant starch. At the same time, its excellent self-repair effect is of great significance for the development of low-lactose products. The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a composite sodium alginate-based aerogel immobilized enzyme carrier, characterized in that: The following steps are involved: Step 1: Disperse sodium alginate and oxidized sodium alginate in deionized water, add starch and nanocellulose, stir in a boiling water bath until completely dissolved, and cool to room temperature to obtain a mixed solution; Step 2: Add β-galactosidase solution and stir evenly to obtain an enzyme-containing mixture; Step 3: Prepare the metal ion solution, draw the enzyme mixture, and slowly drip it into the metal ion solution at a uniform speed for the initial hardening for 60-240 minutes; Step 4: Remove the supernatant and continue to add 75-200 mM / L CaCl2 solution for secondary hardening for 60 minutes, and separate with a nylon membrane to obtain gel microspheres; Step 5: Immerse the gel microspheres in potassium phosphate buffer containing polydopamine nanoparticles for 2 h; Step 6: Wash the gel 3-4 times with pure water, pre-freeze at -80°C for 6 hours, and freeze-dry for 36 hours; Step 7: After taking out, treat in an environment of 50° C. and 80% relative humidity for 10-13 minutes to obtain a composite sodium alginate-based aerogel-immobilized enzyme carrier.

2. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: In the step 1, the concentration of sodium alginate is 1-4%, the concentration of oxidized sodium alginate is 0.25%, the concentration of starch is 0.1-2.0%, and the concentration of nanocellulose is 0.05%.

3. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: The starches include potato starch, tapioca starch, corn starch, sweet potato starch and resistant starch.

4. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: The concentration of the β-galactosidase solution in step 2 is 0.2-2.0 mg / mL.

5. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: The metal ion solution in step 3 includes calcium chloride, manganese chloride and cobalt chloride solutions.

6. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: The CaCl2 solution in step 4 contains 5 mM / L histidine.

7. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: The pore size of the nylon membrane in step 4 is 0.45 μm.

8. The method for preparing a composite sodium alginate-based aerogel-immobilized enzyme carrier according to claim 1, characterized in that: In step 5, the potassium phosphate buffer has a pH of 7.0, 10 mM, and the content of polydopamine nanoparticles is 0.1%.

9. A composite sodium alginate-based aerogel-immobilized enzyme carrier prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the composite sodium alginate-based aerogel immobilized enzyme carrier according to claim 9 in the preparation of low-lactose food.

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