Edible or feed glucose oxidase double gel microspheres and a method for preparing the same

Abstract

The application relates to a preparation method of glucose oxidase double-layer gel microspheres with a slow and continuous significant increase in enzyme release amount in the intestinal digestion stage, and simultaneously provides a product with high encapsulation efficiency, good sustained-release effect in the whole gastrointestinal period, good high-temperature resistance, excellent storage stability and swelling degree. The application effectively utilizes sodium alginate, sodium carboxymethyl cellulose, chitosan and calcium ions to perform secondary coating on glucose oxidase, changes the mechanical strength, thickness and pore size of the film, and further improves the sustained-release performance. Compared with single-layer gel microspheres, the double-layer gel microspheres can still be continuously and significantly linearly released at a release amount of 0.15-0.2% per minute (P<0.05) within 210-240 minutes in the intestinal digestion stage of human or animals; after being placed at 80 DEG C for 10 minutes, the double-layer gel microspheres still have 39.76% of enzyme activity; after being stored for 30 days, the double-layer gel microspheres still have 80.52% of activity; and the glucose oxidase gel microspheres have wide application prospects in the field of food and feed processing.

Description

Technical Field

[0001] This invention mainly relates to the fields of food and feed processing, specifically to a bilayer gel microsphere of glucose oxidase for food or feed and its preparation method. Background Technology

[0002] Glucose oxidase is an aerobic dehydrogenase that converts glucose into gluconic acid and hydrogen peroxide in the presence of oxygen. Due to its functions of glucose removal, deoxygenation, and sterilization, it is widely used in the food, feed, and pharmaceutical industries. However, in actual production applications, operations such as extrusion, granulation, and drying, as well as acid-base changes and the presence of metal ions, can damage the structural integrity of glucose oxidase and reduce its functional properties, greatly limiting its practical development and utilization.

[0003] Sodium alginate is a natural polysaccharide derived from brown algae. It can crosslink with multivalent ions such as calcium and barium ions to form gels. Due to its excellent immobilization ability, hydrophilicity, porosity and thermal stability, it is often used as a wall material for enzyme encapsulation. Although sodium alginate encapsulation alone has the advantages of simple synthesis and increased enzyme stability, problems such as low encapsulation efficiency, enzyme leakage and poor enzyme recycling often occur in industrial production.

[0004] Carboxymethyl cellulose (CMC) is a water-soluble cellulose derivative. Adding CMC to gel products gives the gel excellent water retention. Therefore, composite carriers formed by alginate and CMC are beneficial for improving enzyme encapsulation efficiency (Xu et al. Ethylene glycol glycidyl ether crosslinked sodium alginate-sodium carboxymethyl cellulose immobilized lipase [J]). However, this study showed that only 28% of the activity of lipase encapsulated in sodium alginate-sodium carboxymethyl cellulose monolayer gel remained at 65℃, which has an adverse effect on the thermal stability of the enzyme during production and processing. Crosslinking agents such as ethylene glycol glycidyl ether and glutaraldehyde have certain toxicity and irritation, posing potential food safety hazards. While CMC gives the gel excellent water absorption and retention, it is also a key factor affecting the degree of swelling of encapsulated particles in the gastrointestinal tract.

[0005] Chitosan, the deacetylated product of chitin, is the second most abundant polysaccharide in nature, possessing advantages such as non-toxicity, biodegradability, and biocompatibility. Encapsulation of bioactive substances with alginate / chitosan has been extensively studied. Due to the formation of ionic complexes between the positive charge of chitosan and the negative charge of alginate, it provides gel strength, barrier properties, and controlled-release characteristics in microcapsules. However, poor water retention during encapsulation leads to unsatisfactory encapsulation rates. Furthermore, enzyme activity is easily lost and stability is poor during gastrointestinal digestion. The use of emulsifiers such as soybean oil and mono / diglycerides in the alginate / chitosan encapsulation process not only increases process complexity and production costs but also affects sustained-release properties to some extent.

[0006] Since sodium alginate, carboxymethyl cellulose, and chitosan are all macromolecular materials, their combined use or multi-layer coating may lead to numerous bottlenecks, such as complex polymer structures, poor sustained-release properties (especially in the small intestine), and complex processes. Currently, few researchers have explored this approach. Finding the optimal balance between sodium alginate, carboxymethyl cellulose, and chitosan to synergistically and effectively improve the sustained-release performance, catalytic efficiency, stability, and recovery rate of glucose oxidase-encapsulated products remains a significant technical challenge in current research. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing glucose oxidase bilayer gel microspheres in which the enzyme release rate increases slowly, continuously, and significantly during the small intestinal phase or intestinal digestion. Simultaneously, it provides a glucose oxidase gel microsphere product in which the enzyme release rate shows a continuous upward trend throughout the entire gastrointestinal process, and exhibits excellent high-temperature resistance, storage stability, and swelling index. This addresses technical problems such as low thermal stability, poor storage stability, inaccurate control of the swelling index, the inability to achieve a slow, continuous, and linear increase in enzyme release during the small intestinal phase (or intestinal digestion), and weak activity in the gastrointestinal tract. The method enables a slow, continuous, and significant increase in enzyme release even in the later stages of small intestinal digestion.

[0008] To achieve the above objectives and solve the corresponding technical problems, the present invention adopts the following solution:

[0009] A method for preparing glucose oxidase bilayer gel microspheres with a slow, sustained, and significant increase in enzyme release during intestinal digestion includes the following steps:

[0010] (1) First coating: Mix 27-36 mL of sodium alginate solution with a mass concentration of 0.05%-4% with 3-4 mL of sodium carboxymethyl cellulose solution with a mass concentration of 0.05%-0.4%, add 1-2 mL of glucose oxidase solution with a mass concentration of 0.1%-0.5%, mix well, and prepare a mixture of glucose oxidase and dual carrier; use a peristaltic pump to drop the mixture into 100-120 mL of calcium chloride solution with a mass concentration of 0.5%-4%, solidify at 3-4℃ for 15-120 min, filter to obtain immobilized glucose oxidase (GOD) gel particles, and set aside;

[0011] (2) Second coating: preparation of chitosan solution: prepare a chitosan solution with a mass concentration of 0.2%-1% and a pH value of 3.0-5.0; transfer the immobilized glucose oxidase gel particles prepared in step (1) to 100 mL of the chitosan solution, stir magnetically at 100-300 rpm for 10-50 min, and filter to obtain glucose oxidase gel microspheres.

[0012] Preferably, in step (1), 27 mL of sodium alginate solution with a mass concentration of 1.5% is mixed with 3 mL of sodium carboxymethyl cellulose solution with a mass concentration of 0.1%, and 1-2 mL of glucose oxidase solution with a mass concentration of 0.3% is added and mixed to prepare a mixture of glucose oxidase and dual carrier; the mixture is then dripped into 100-120 mL of calcium chloride solution with a mass concentration of 1% using a peristaltic pump and solidified at 3-4°C for 30 min.

[0013] Preferably, in step (1), 27 mL of sodium alginate solution with a mass concentration of 1.5% is mixed with 3 mL of sodium carboxymethyl cellulose solution with a mass concentration of 0.05%, and 1-2 mL of glucose oxidase solution with a mass concentration of 0.3% is added and mixed to prepare a mixture of glucose oxidase and dual carrier; the mixture is then dripped into 100-120 mL of calcium chloride solution with a mass concentration of 0.5% using a peristaltic pump and solidified at 3-4°C for 15 min.

[0014] Preferably, in step (2), the preparation of the chitosan solution is as follows: a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0 is prepared; the immobilized glucose oxidase gel particles prepared in step (1) are transferred to 100 mL of the chitosan solution, magnetically stirred at 150 rpm, cured at 4°C for 30 min, and filtered to obtain glucose oxidase gel microspheres.

[0015] Preferably, in the use of the peristaltic pump, it operates at a speed of 12 rpm and uses a silicone manifold with an inner diameter of 3 mm, the end of which is connected to a stainless steel needle with an inner diameter of 0.45 mm, and the injection height is 10 cm. A 0.45 mm 0.45×16RWLB stainless steel needle is further preferred.

[0016] Beneficial effects:

[0017] (1) This application is the first to use a mixture of sodium alginate and sodium carboxymethyl cellulose for the first layer encapsulation of glucose oxidase. This helps to reduce the decrease in encapsulation rate of hydrophilic enzymes caused by the dehydration and shrinkage of gel particles during ion gelation. The resulting immobilized glucose oxidase has an enzyme activity recovery rate greater than 78.58%, reaching a maximum of 92.56%. The core-shell structure of glucose oxidase and the carrier helps the enzyme maintain its structural stability under harsh environments, thereby maintaining its activity. The use of sodium carboxymethyl cellulose adjusts the swelling degree and thus regulates the sustained-release rate. Furthermore, by synergizing with chitosan, the ionic complexation relationship between the positive charge of chitosan and the negative charge of alginate, as well as the cross-linking effect of the aforementioned three components, is utilized to change the mechanical strength, thickness, and pore size of the membrane. The secondary coating further improves the various properties of the microspheres. The bilayer gel microspheres obtained in this application improve the thermal stability, storage stability, and activity of glucose oxidase in the animal gastrointestinal tract. After being placed at 80℃ for 10 minutes, the enzyme activity of the bilayer gel microspheres was still 39.76%; the storage stability was good, and after 30 days, the bilayer gel microspheres still retained 80.52% of the activity.

[0018] (2) By using specific processes and raw material ratios, suitable swelling index and sustained release rate are obtained, so that the bilayer gel microspheres show a continuous upward trend throughout the gastrointestinal phase. In particular, the bilayer gel microspheres can still release significantly linearly at a rate of 0.15-0.2% per minute in the intestinal phase (P<0.05) within the range of 210-240 min, while the enzyme release of the monolayer gel particles has become relatively slow. The bilayer gel microspheres have a good continuous and uniform sustained release effect. It is possible that when the bilayer gel microspheres are exposed to neutral or alkaline media, the large number of carboxyl groups in sodium carboxymethyl cellulose form hydrogen bonds with water molecules during the first coating stage, forming a dense three-dimensional network gel structure. This restricts the movement of water molecules. Combined with the support of the highly viscous chitosan complex film, the dissociation rate of carboxylic acid groups slows down. Furthermore, in an alkaline environment, a relatively small amount of positively charged chitosan can adsorb some of the negatively charged carboxylic acid groups of sodium carboxymethyl cellulose, all of which to some extent delay the process of carboxylic acid groups converting into negatively charged carboxylate ions. However, as the electrostatic repulsion between different polymer chains increases, the polymer network is forced to expand over time, reaching an ideal microscopic equilibrium state. There is a mutual balance and constraint relationship in the system, which is the result of the synergistic effect of sodium alginate, sodium carboxymethyl cellulose, and chitosan.

[0019] (3) The process of this application is simple, easy to operate, and the product is highly safe and widely used. In addition to its application in the food industry, it can also be extended to the feed and pharmaceutical industries to provide corresponding products. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Swelling degree of monolayer gel particles at different pH values;

[0022] Figure 2 Swelling degree of bilayer gel particles at different pH values;

[0023] Figure 3 Thermal stability of free enzymes, monolayer gel microspheres (particles), and bilayer gel microspheres at 60℃;

[0024] Figure 4 Thermal stability of free enzymes, monolayer gel microspheres (particles), and bilayer gel microspheres at 70℃;

[0025] Figure 5 Thermal stability of free enzymes, monolayer gel microspheres (particles), and bilayer gel microspheres at 80℃;

[0026] Figure 6 The effects of free enzymes, monolayer gel particles, and bilayer gel microspheres on simulated digestive fluids in the gastrointestinal tract;

[0027] Figure 7 Storage stability of free enzymes, monolayer gel particles, and bilayer gel particles;

[0028] Figure 8 Appearance and morphology of monolayer gel particles;

[0029] Figure 9 Appearance and morphology of bilayer gel particles;

[0030] Figure 10 Scanning electron microscope images of monolayer and bilayer gel particles;

[0031] Figure 11 Surface roughness of single-layer and double-layer gel particles. Detailed Implementation

[0032] The materials used in this application are food-grade and / or reagent-grade, preferably food-grade. The degree of deacetylation of chitosan is ≥90.0%, preferably 92%.

[0033] Example 1:

[0034] The preparation of a glucose oxidase bilayer gel microsphere with a slow and sustained significant increase in enzyme release during intestinal digestion includes the following steps: (1) First coating: 27 mL of sodium alginate solution with a mass concentration of 1.5% and 3 mL of sodium carboxymethyl cellulose solution with a mass concentration of 0.1% are mixed, and 1 mL of glucose oxidase solution with a mass concentration of 0.3% is added and mixed to prepare a mixture of glucose oxidase and dual carriers; the mixture is dripped into 100 mL of calcium chloride solution with a mass concentration of 1% using a peristaltic pump, solidified at 4°C for 30 min, filtered and washed to obtain monolayer sodium alginate-sodium carboxymethyl cellulose gel particles; (2) Second coating: preparation of chitosan solution: prepare a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0; transfer the immobilized glucose oxidase gel particles prepared in step (1) to 100 mL of the chitosan solution, stir magnetically at 150 rpm, solidify at 4°C for 30 min, wash and filter to obtain glucose oxidase bilayer gel microspheres.

[0035] Example 2:

[0036] The preparation of a glucose oxidase bilayer gel microsphere with a slow and sustained significant increase in enzyme release during intestinal digestion includes the following steps: (1) First coating: 27 mL of 1.5% sodium alginate solution and 3 mL of 0.1% sodium carboxymethyl cellulose solution are mixed, and 1 mL of 0.3% glucose oxidase solution is added and mixed to prepare a mixture of glucose oxidase and dual carriers; the mixture is then dripped into 100 mL of 1% calcium chloride solution using a peristaltic pump and cured at 4°C for 30 min; (2) Second coating: Preparation of chitosan solution: Prepare a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0; transfer the immobilized glucose oxidase gel particles prepared in step (1) to 100 mL of the chitosan solution, stir magnetically at 150 rpm, solidify at 4℃ for 30 min, wash and filter to obtain glucose oxidase bilayer gel microspheres. The peristaltic pump is used in conjunction with the microspheres, running at a speed of 12 rpm, and a silicone bimanifold with an inner diameter of 3 mm is used, with a stainless steel needle with an inner diameter of 0.45 mm connected to the end. The injection height is 10 cm. If there are air bubbles before injection, they can be removed by ultrasound (40 kHz, 5 minutes).

[0037] Example 3:

[0038] The preparation of a glucose oxidase bilayer gel microsphere with a slow and sustained significant increase in enzyme release during intestinal digestion includes the following steps: (1) First coating: 27 mL of 1.5% sodium alginate solution and 3 mL of 0.05% sodium carboxymethyl cellulose solution are mixed, and 1 mL of 0.3% glucose oxidase solution is added and mixed to prepare a mixture of glucose oxidase and dual carriers; the mixture is then dripped into 100 mL of 0.5% calcium chloride solution using a peristaltic pump and cured at 4°C for 15 min. (2) Second coating: Preparation of chitosan solution: Prepare a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0; transfer the immobilized glucose oxidase gel particles prepared in step (1) to 100 mL of the chitosan solution, stir magnetically at 150 rpm, solidify at 4°C for 30 min, wash and filter to obtain glucose oxidase gel microspheres. The peristaltic pump is used in conjunction with the microspheres, running at 12 rpm, and a silicone bimanifold with an inner diameter of 3 mm is used, with a stainless steel needle with an inner diameter of 0.45 mm connected to the end. The injection height is 10 cm. If there are air bubbles before injection, they can be removed by ultrasound (40 kHz, 5 minutes). The enzyme activity recovery rate (encapsulation rate) of the immobilized glucose oxidase obtained in this embodiment can reach 92.56%.

[0039] Controlled experiment:

[0040] Experimental Example 1: Example 1

[0041] Comparative Example 1: Free glucose oxidase

[0042] Comparative Example 2: Monolayer sodium alginate-sodium carboxymethyl cellulose gel particles (prepared in step (1) of Example 1)

[0043] Experimental Example 1, Control Example 1, and Control Example 2 were used as control experiments.

[0044] Measurement methods: (Not limited to the methods described below, other conventional methods in this field may also be used)

[0045] (1) Determination of swelling degree (swelling index)

[0046] The samples to be tested were dried in a 37°C oven for 3 hours. Then, the dried samples were placed in coffee filter paper bags and incubated in PBS buffer solutions with pH values ​​of 2.5 and 7.4, respectively, at 37°C and 180 rpm in a shaker. Every 30 minutes, the samples were removed, wiped dry with filter paper, and weighed. After weighing, the samples were returned to the original phosphate buffer solution for further incubation. The degree of swelling can be calculated using the following formula.

[0047] Calculation formula:

[0048]

[0049] Where, m s It is the weight of the sample after swelling in the buffer solution, m d This is the weight of the sample in its dry state.

[0050] The swelling degree (swelling index) measurement results of the sample in Example 1 are shown below. Figure 1 , Figure 2 :

[0051] Depend on Figure 2 It was found that at pH 2.5, the bilayer gel microspheres reached swelling equilibrium within 30 minutes, with a swelling index of 13.18%. However, at pH 7.4, the microspheres continued to swell over time, reaching a maximum swelling index of approximately 93.22% at 60 minutes. This indicates that the bilayer gel microspheres do not swell significantly under acidic conditions, but exhibit higher swelling under neutral or alkaline conditions, which facilitates the release of encapsulated enzymes in the intestine. The stomach is a digestive organ, while the small intestine is the primary organ for nutrient absorption. In intestinal fluid, due to the higher pH, deacetylated chitosan is deprotonated, weakening the integrity of the sodium alginate-carboxymethyl cellulose-chitosan complex, thus releasing glucose oxidase.

[0052] (2) Thermal stability

[0053] The samples to be tested (naked enzyme, monolayer sodium alginate-sodium carboxymethyl cellulose gel particles, and bilayer gel microspheres) were placed in water baths at 60℃, 70℃ (enzyme activity was measured every 10 minutes), and 80℃ (enzyme activity was measured every 2 minutes) to investigate their thermal stability.

[0054] Calculation formula:

[0055]

[0056] See results Figure 3 , Figure 4 , Figure 5 :

[0057] The results show that, under water bath heating at different temperatures, the enzyme activity retention rate of enzyme-loaded bilayer microspheres generally trended higher than that of monolayer gel microspheres and free enzymes. After incubation at 60℃ for 30 min, the free enzyme retained 75.23% of its activity, while the enzyme-loaded bilayer gel microspheres retained 93.27% of their activity, an increase of approximately 18% compared to the free enzyme. After incubation at 70℃ for 60 min, the free enzyme retained 64.42% of its activity, while the enzyme-loaded bilayer gel microspheres retained 85.28%. After incubation at 80℃ for 10 min, the free enzyme retained 7.5% of its activity, while the enzyme-loaded bilayer gel microspheres retained 39.76%, an increase of 32.26% compared to the free enzyme. The improved thermal stability of enzyme-carrying bilayer gel microspheres is due to the egg-shaped structure of the bilayer gel microspheres having a certain degree of "rigidity." The GOD is bound within the mesh, which is beneficial to the stability of the enzyme's spatial structure. At the same time, the chitosan membrane reduces the dissociation of enzyme cofactors, thereby improving the enzyme's heat resistance.

[0058] (3) In vitro simulated enzyme release test

[0059] Preparation of simulated gastric juice: Weigh 0.2g sodium chloride and 0.32g pepsin, place them in a 500mL beaker, dissolve them with distilled water and adjust the pH to 2.5, and finally accurately fill a 100mL volumetric flask.

[0060] Preparation of simulated intestinal fluid: Weigh 0.68g of dipotassium hydrogen phosphate and place it in a beaker. Dissolve it in 25mL of distilled water. Then, add 7.7mL of 0.2mol / L sodium hydroxide solution, 50mL of distilled water, and 1g of trypsin to the beaker. Then, use a pH meter to precisely adjust the pH value to 7.7. Store the prepared gastrointestinal simulated fluid in a refrigerator at 4°C.

[0061] 1g of the sample to be tested was added to 10mL of simulated gastric fluid and incubated in a shaker at 37℃ and 180rpm. Every 30 minutes, 0.1mL of the reaction solution was taken for enzyme activity assay. After each sampling, 0.1mL of simulated gastric fluid was added to maintain the equilibrium of the reaction solution. The entire gastric phase lasted 2 hours. After the gastric phase, the pH was adjusted to 7.7. The incubation method for the small intestine phase was similar to that for the gastric phase; after each sample collection, an equal volume of simulated intestinal fluid was added. Results are expressed as cumulative release data in solution. Free glucose oxidase was used as a control group. The average of three parallel measurements was calculated for each sample. See [Results are listed below]. Figure 6 .

[0062] Figure 6The results showed that the release rate of free glucose oxidase increased rapidly in the first 30 minutes of culture, reached its maximum after 90 minutes, and gradually decreased in the small intestine, dropping to 26.10% after 240 minutes. The cumulative enzyme release of monolayer gel particles continued to increase in the gastric phase, but by the time they reached the small intestine, the enzyme release from monolayer gel particles increased from 41.09% to 84.32%, and then leveled off in the later intestinal phase. Bilayer gel microspheres showed a continuous upward trend throughout the gastrointestinal tract, with the enzyme release increasing from 41.06% to 74.98% during intestinal digestion. In particular, bilayer gel microspheres maintained a significant linear release rate of 0.15-0.2% (preferably 0.175%) per minute within the 210-240 minute time range in the intestinal phase (cumulative release at 210 minutes was 69.70%) (P<0.05), indicating that bilayer gel microspheres have a good sustained-release effect.

[0063] Furthermore, in experiments simulating the gastrointestinal digestive fluids of aquatic animals (with intestinal fluid pH set at 6.8), the release rate of free glucose oxidase also increased rapidly within the first 30 minutes of culture, reaching a maximum of 63.55% after 90 minutes, while gradually decreasing in the small intestine stage, dropping to 28.11% after 240 minutes. The cumulative enzyme release from monolayer gel particles continued to increase during the gastric stage, but by the time they reached the small intestine stage, the enzyme release from monolayer gel particles increased from 40.07% to 80.33%, and the enzyme release tended to level off in the later stages of the intestinal stage. The bilayer gel microspheres showed a continuous upward trend throughout the gastrointestinal phase, with the enzyme release increasing from 41.04% to 70.32% during the intestinal digestion stage. In particular, the bilayer gel microspheres maintained a significant linear release at a rate of 0.15-0.2% (preferably 0.153%) per minute within the 210-240 min time range of the intestinal digestion stage (cumulative release at 210 min was 65.74%) (P<0.05). The bilayer gel microspheres exhibited a good sustained-release effect.

[0064] The research results have also repeatedly shown that the bilayer gel microspheres prepared in this application exhibit the same trend in both simulated intestinal fluid pH 7.7 and pH 6.8, and at the same time show excellent sustained-release effects, making them applicable to food and aquatic feed processing.

[0065] (4) Storage stability analysis

[0066] The prepared gel particles and free enzyme were placed in centrifuge tubes containing phosphate buffer (pH 6.0) and stored at 4°C. Enzyme activity was measured every 5 days for a total of 30 days. The enzyme activity on the first day was recorded as the maximum activity, and then the enzyme activity was expressed as relative activity. Results are shown below. Figure 7 .

[0067] Figure 7 The results showed that the activities of both types of gel microspheres and the free enzyme gradually decreased with prolonged storage time. The bilayer gel microspheres showed a slower decrease than the monolayer gel microspheres, and the monolayer gel microspheres showed a slower decrease than the free enzyme. The enzyme activity retention rate reached a maximum of 96.32% in the initial 5 days. After 30 days, the free enzyme activity decreased to 66.35%, while the bilayer gel microspheres still retained 80.52% activity. Therefore, secondary coating of sodium alginate-sodium carboxymethyl cellulose gel microspheres with chitosan solution improved the storage stability of the enzyme to some extent.

Claims

1. An application of glucose oxidase bilayer gel microspheres, characterized in that, Glucose oxidase bilayer gel microspheres were prepared using only the following two steps: (1) First coating: Mix 27-36 mL of sodium alginate solution with a mass concentration of 0.05%-4% with 3-4 mL of sodium carboxymethyl cellulose solution with a mass concentration of 0.05%-0.4%, then add 1-2 mL of glucose oxidase solution with a mass concentration of 0.1%-0.5%, mix well, and prepare a mixture of glucose oxidase and dual carrier; use a peristaltic pump to drop the mixture into 100-120 mL of calcium chloride solution with a mass concentration of 0.5%-4%, solidify at 3-4℃ for 15-120 min, filter to obtain immobilized glucose oxidase (GOD) gel particles, and set aside; (2) Second coating: preparation of chitosan solution: prepare a chitosan solution with a mass concentration of 0.2%-1% and a pH value of 3.0-5.0; transfer the immobilized glucose oxidase gel particles prepared in step (1) to 100 mL of the chitosan solution, stir magnetically at 100-300 rpm for 10-50 min, and filter to obtain glucose oxidase bilayer gel microspheres; The glucose oxidase bilayer gel microspheres exhibit a slow but significant increase in enzyme release during the small intestinal phase, maintaining a linear release rate of 0.15-0.2% per minute within the small intestinal phase (210-240 min). The glucose oxidase bilayer gel microspheres are edible or suitable for feeding aquatic animals such as fish.

2. The application of the glucose oxidase bilayer gel microspheres according to claim 1, characterized in that, In step (1), 27 mL of sodium alginate solution with a mass concentration of 1.5% and 3 mL of sodium carboxymethyl cellulose solution with a mass concentration of 0.1% are mixed, and 1-2 mL of glucose oxidase solution with a mass concentration of 0.3% is added and mixed to prepare a mixture of glucose oxidase and dual carriers; the mixture is then dripped into 100-120 mL of calcium chloride solution with a mass concentration of 1% using a peristaltic pump and solidified at 3-4℃ for 30 min.

3. The application of the glucose oxidase bilayer gel microspheres according to claim 2, characterized in that, In step (2), the chitosan solution is prepared as follows: a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0 is prepared; the immobilized glucose oxidase gel particles prepared in step (1) are transferred to 100 mL of the chitosan solution, magnetically stirred at 150 rpm, solidified at 4 °C for 30 min, and filtered to obtain glucose oxidase bilayer gel microspheres.

4. The application of the glucose oxidase bilayer gel microspheres according to claim 3, characterized in that, When used with the peristaltic pump, it runs at a speed of 12 rpm and uses a silicone bimanifold with an inner diameter of 3 mm. The end is connected to a stainless steel needle with an inner diameter of 0.45 mm, and the injection height is 10 cm.

5. The application of the glucose oxidase bilayer gel microspheres according to claim 4, characterized in that, The glucose oxidase bilayer gel microspheres have excellent thermal stability, storage stability and swelling index, and can also improve the activity of glucose oxidase in the animal gastrointestinal tract.

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