Glucose oxidase double-layer gel microspheres for eating or feeding and preparation method of glucose oxidase double-layer gel microspheres

Abstract

The invention relates to a preparation method of glucose oxidase double-layer gel microspheres with slow, continuous and remarkable increase of enzyme release amount in an intestinal digestion stage, and provides a product which is high in encapsulation efficiency, good in slow release effect in the whole gastrointestinal stage, resistant to high temperature and excellent in storage stability and swelling degree. According to the invention, sodium alginate, sodium carboxymethyl cellulose, chitosan and calcium ions are effectively utilized to carry out secondary coating on glucose oxidase, so that the mechanical strength, thickness and pore size of the membrane are changed, and the slow release performance is further improved. Compared with a single-layer gel microsphere, the double-layer gel microsphere can still continuously, remarkably and linearly release (Plt; 0.05) at the release amount of 0.15-0.2% per minute within 210-240 minutes at the intestinal digestion stage of people or animals; after the double-layer gel microspheres are placed at 80 DEG C for 10 minutes, the double-layer gel microspheres still have 39.76% of enzyme activity; 80.52% of activity can still be maintained after storage for 30 days; the glucose oxidase gel microspheres have wide application prospects in the field of food and feed processing.

Description

Technical Field

[0001] The invention mainly relates to the field of food and feed processing, and in particular to a double-layer gel microsphere of edible or feed glucose oxidase and a preparation method thereof. Background Art

[0002] Glucose oxidase is an aerobic dehydrogenase that can convert glucose into gluconic acid and hydrogen peroxide in the presence of oxygen. It is widely used in food, feed and medicine because of its functions of removing glucose, deoxygenation and sterilization. However, in actual production applications, operations such as extrusion, granulation, drying, acid-base changes and the presence of metal ions can damage the structural integrity of glucose oxidase and reduce its functional characteristics, which greatly limits the actual development and utilization of glucose oxidase.

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

[0004] Carboxymethyl cellulose is a water-soluble cellulose derivative. Adding carboxymethyl cellulose to gel products will make the gel have excellent water retention. Therefore, the composite carrier formed by alginate and carboxymethyl cellulose is beneficial to improve the encapsulation rate of the enzyme (Xu Shan et al. Ethylene glycol glycidyl ether cross-linked sodium alginate-sodium carboxymethyl cellulose immobilized lipase [J]). However, the study showed that only 28% of the activity of sodium alginate-sodium carboxymethyl cellulose monolayer gel encapsulated lipase remained at 65°C, which has an adverse effect on the thermal stability of the enzyme during production and processing. Cross-linking agents such as ethylene glycol glycidyl ether and glutaraldehyde have certain toxicity and irritation, and there are hidden dangers to food safety. Carboxymethyl cellulose makes the gel have excellent water absorption and water retention, and it is also a key factor affecting the degree of swelling of encapsulated particles in the gastrointestinal tract.

[0005] Chitosan is the deacetylated product of chitin and is the second most abundant polysaccharide in nature. It has the advantages of non-toxicity, biodegradability and biocompatibility. The encapsulation of bioactive substances in alginate / chitosan has been widely studied. Since its positive charge forms an ion complex with the negative charge of alginate, it provides gel strength, barrier properties and controlled release properties in microcapsules. However, due to poor water retention during the encapsulation process, the encapsulation rate is not ideal, and during gastrointestinal digestion, the enzyme activity is still easily lost and has poor stability. The use of emulsifiers such as soybean oil and mono / diglycerides in the alginate / chitosan encapsulation process not only increases the process complexity and production costs, but also affects the sustained release to a certain extent.

[0006] Since sodium alginate, carboxymethyl cellulose and chitosan are all macromolecular materials, their combined use or multi-layer coating may lead to many bottleneck problems such as complex polymer structure, poor sustained release (especially poor sustained release in the small intestine), and complex processes. Currently, few researchers have explored and tried. How to effectively utilize sodium alginate, carboxymethyl cellulose and chitosan in a coordinated manner, seek the best balance point among the three, and then improve the technical effects of glucose oxidase encapsulation products in terms of sustained release performance, catalytic efficiency, stability, recovery rate, etc., is still a technical barrier in current research. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a glucose oxidase double-layer gel microsphere with a slow, continuous and significant increase in enzyme release during the small intestinal stage or the intestinal digestion stage, and to provide a corresponding glucose oxidase gel microsphere product with a continuous upward trend in the enzyme release during the entire gastrointestinal stage, high temperature resistance, storage stability and excellent swelling index, thereby solving technical problems such as low thermal stability, poor storage stability, inaccurate regulation of swelling index, inability to slowly and continuously increase enzyme release linearly during the small intestinal stage (or intestinal digestion stage), and weak activity in the gastrointestinal tract. The method can achieve a slow, continuous and significant increase in enzyme release in the late stage of small intestinal digestion.

[0008] In order to achieve the above objectives and solve the corresponding technical problems, the present invention adopts the following solutions:

[0009] A method for preparing glucose oxidase double-layer gel microspheres with a slow, continuous and significant increase in enzyme release during the intestinal digestion period, comprising the following steps:

[0010] (1) First coating: 27-36 mL of a sodium alginate solution with a mass concentration of 0.05%-4% and 3-4 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.05%-0.4% are mixed, and 1-2 mL of a glucose oxidase solution with a mass concentration of 0.1%-0.5% is added and mixed to prepare a mixed solution of glucose oxidase and a dual carrier; the mixed solution is dripped into 100-120 mL of a calcium chloride solution with a mass concentration of 0.5%-4% using a peristaltic pump, and cured at 3-4° C. for 15-120 min, and filtered to obtain immobilized glucose oxidase (GOD) gel particles for later use;

[0011] (2) Second coating layer: 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) into 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 the step (1), 27 mL of a sodium alginate solution with a mass concentration of 1.5% and 3 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.1% are mixed, 1-2 mL of a glucose oxidase solution with a mass concentration of 0.3% is added, and mixed to prepare a mixed solution of glucose oxidase and a dual carrier; a peristaltic pump is used to drop the mixed solution into 100-120 mL of a calcium chloride solution with a mass concentration of 1%, and the mixture is cured at 3-4°C for 30 minutes.

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

[0014] Preferably, in the step (2), the chitosan solution is prepared by: preparing a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0; transferring the immobilized glucose oxidase gel particles prepared in step (1) into 100 mL of the chitosan solution, stirring with a magnetic stirrer at 150 rpm, curing at 4°C for 30 min, and filtering to obtain glucose oxidase gel microspheres.

[0015] Preferably, the peristaltic pump is used with a rotation speed of 12 rpm, and a silicone double manifold with an inner diameter of 3 mm is used, and a stainless steel needle with an inner diameter of 0.45 mm is connected to the end, 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) The present application is the first to use a mixture of sodium alginate and sodium carboxymethyl cellulose to perform the first layer encapsulation of glucose oxidase, which is beneficial to reduce the decrease in the encapsulation rate of hydrophilic enzymes caused by the dehydration shrinkage of gel particles during the ion gelation process, and obtain an immobilized glucose oxidase activity yield greater than 78.58%, up to 92.56%. The core-shell structure of glucose oxidase and the carrier is beneficial to the enzyme maintaining its structural stability in harsh environments, thereby maintaining the activity of the enzyme in harsh environments. The use of sodium carboxymethyl cellulose adjusts the swelling degree and thus regulates the sustained release rate. Further synergistic 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 above three, changes the mechanical strength, thickness, pore size, etc. of the membrane, and the secondary coating further improves the various properties of the microspheres. The double-layer gel microspheres obtained in the present application improve the thermal stability, storage stability and activity of glucose oxidase in the gastrointestinal tract of animals. After being placed at 80°C for 10 minutes, the enzyme activity of the double-layer gel microspheres still retained 39.76%; the storage stability was good, and the double-layer gel microspheres still retained 80.52% of their activity after 30 days.

[0018] (2) Through specific processes and raw material dosage ratios, an appropriate swelling index and sustained-release rate are obtained, so that the double-layer gel microspheres show a continuous upward trend throughout the gastrointestinal stage. In particular, in the intestinal stage within the range of 210-240 minutes, the double-layer gel microspheres can still maintain a significant linear release at a release rate of 0.15-0.2% per minute (P<0.05), while the release amount of single-layer gel particles has tended to be flat. The double-layer gel microspheres have a good continuous and uniform sustained-release effect. It may be that when the double-layer gel microspheres are exposed to neutral or alkaline media, a large number of carboxyl groups in sodium carboxymethyl cellulose form hydrogen bonds with water molecules in the first coating stage to form a dense three-dimensional network gel structure, which restricts the movement of water molecules. Combined with the support of the highly viscous chitosan complex membrane, the dissociation rate of the carboxylic acid group slows down, and a relatively small amount of positively charged chitosan in an alkaline environment can adsorb part of the negatively charged carboxylic acid groups of sodium carboxymethyl cellulose, which to a certain extent delays the process of carboxylic acid groups converting into negatively charged carboxylate ions. However, with the increasing electrostatic repulsion between different polymer chains, the polymer network is forced to expand over time to reach an ideal microscopic equilibrium state. There is a mutual balance and restraint relationship in the system, which is the result of the synergistic enhancement of sodium alginate, sodium carboxymethyl cellulose and chitosan.

[0019] (3) The process of the present application is simple, easy to operate, and the product is highly safe and widely used. In addition to being used in the food field, it can also be extended to the feed and medicine fields to provide corresponding products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0022] Figure 2 : Swelling degree of double-layer gel particles at different pH values;

[0023] Figure 3 : Thermal stability of free enzyme, single-layer gel microspheres (particles), and double-layer gel microspheres at 60°C;

[0024] Figure 4 : Thermal stability of free enzyme, single-layer gel microspheres (particles), and double-layer gel microspheres at 70°C;

[0025] Figure 5 : Thermal stability of free enzyme, single-layer gel microspheres (particles), and double-layer gel microspheres at 80°C;

[0026] Figure 6 :Effects of free enzymes, single-layer gel particles, and double-layer gel microspheres in simulated digestive fluid of the gastrointestinal tract;

[0027] Figure 7 : Storage stability of free enzyme, single-layer gel particles, and double-layer gel particles;

[0028] Figure 8 : Appearance and morphology of single-layer gel particles;

[0029] Fig. 9 :Appearance and morphology of double-layer gel particles;

[0030] Fig.10 :Scanning electron microscopy images of single-layer and double-layer gel particles;

[0031] Fig.11 :Surface roughness of monolayer and bilayer gel particles. DETAILED DESCRIPTION

[0032] The material of the present application is food grade and / or reagent grade, preferably food grade. The deacetylation degree of chitosan is ≥ 90.0%, preferably 92%.

[0033] Embodiment 1:

[0034] The invention discloses a preparation method for glucose oxidase double-layer gel microspheres whose enzyme release amount increases slowly and continuously during the intestinal digestion period, comprising the following steps: (1) first layer 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%, and 1 mL of glucose oxidase solution with a mass concentration of 0.3% are mixed and mixed to prepare a mixed solution of glucose oxidase and a dual carrier; the mixed solution is dripped into 100 mL of calcium chloride solution with a mass concentration of 1% by using a peristaltic pump, and solidified at 4°C for 30 minutes, and filtered and washed to obtain single-layer sodium alginate-sodium carboxymethyl cellulose gel particles; (2) second layer coating: preparation of chitosan solution: preparation of 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) into 100 mL of the chitosan solution, stir with a magnetic force of 150 rpm, solidify at 4°C for 30 minutes, and wash and filter to obtain glucose oxidase double-layer gel microspheres.

[0035] Embodiment 2:

[0036] A preparation method for glucose oxidase double-layer gel microspheres with a slow, continuous and significant increase in enzyme release during the intestinal digestion period, comprising the following steps: (1) first layer coating: 27 mL of a sodium alginate solution with a mass concentration of 1.5% and 3 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.1%, and 1 mL of a glucose oxidase solution with a mass concentration of 0.3% are added and mixed to prepare a mixed solution of glucose oxidase and a double carrier; a peristaltic pump is used to drop the mixed solution into 100 mL of a calcium chloride solution with a mass concentration of 1%, and the mixture is cured at 4°C for 30 minutes; (2) a first layer coating: 27 mL of a sodium alginate solution with a mass concentration of 1.5% and 3 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.1% are mixed and mixed to prepare a mixed solution of glucose oxidase and a double carrier; a peristaltic pump is used to drop the mixed solution into 100 mL of a calcium chloride solution with a mass concentration of 1%, and the mixture is cured at 4°C for 30 minutes; ) Second layer coating: Preparation of chitosan solution: Prepare 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) into 100 mL of the chitosan solution, stir with a magnetic force of 150 rpm, solidify at 4°C for 30 min, wash and filter to obtain glucose oxidase double-layer gel microspheres, wherein the peristaltic pump is used to assist, running at a speed of 12 rpm, and using a silicone double 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. If there are bubbles before injection, they can be removed by ultrasound (40 kHz, 5 minutes).

[0037] Embodiment 3:

[0038] A preparation method for glucose oxidase double-layer gel microspheres with a slow, continuous and significant increase in enzyme release during the intestinal digestion period, comprising the following steps: (1) first layer coating: 27 mL of a sodium alginate solution with a mass concentration of 1.5% and 3 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.05%, and 1 mL of a glucose oxidase solution with a mass concentration of 0.3% are added and mixed to prepare a mixed solution of glucose oxidase and a double carrier; a peristaltic pump is used to drop the mixed solution into 100 mL of a calcium chloride solution with a mass concentration of 0.5%, and the mixture is cured at 4°C for 15 minutes. ; (2) Second coating: Preparation of chitosan solution: Prepare 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) into 100 mL of the chitosan solution, stir with a magnetic force of 150 rpm, solidify at 4°C for 30 min, wash and filter to obtain glucose oxidase gel microspheres, wherein a peristaltic pump is used to assist, running at a speed of 12 rpm, and using a silica gel double 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. If there are bubbles before injection, they can be removed by ultrasound (40 kHz, 5 minutes). The immobilized glucose oxidase enzyme activity yield (embedding rate) obtained in this embodiment can reach 92.56%.

[0039] Controlled trials:

[0040] Test 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] Test Example 1, Control Example 1 and Control Example 2 were subjected to a comparative test.

[0044] Determination method: (not limited to the following method, other conventional methods in the art may also be used)

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

[0046] The samples to be tested were placed in a 37°C oven to dry for 3 hours. Subsequently, the dried samples were placed in coffee filter paper bags and placed in PBS buffers with pH values ​​of 2.5 and 7.4, respectively. They were incubated in a shaker at 37°C and 180 rpm. The samples were taken out every 30 minutes, wiped dry with filter paper, and then weighed. After weighing, the samples were placed back in the original phosphate buffer solution and continued to be incubated. The swelling degree can be calculated according to the following formula.

[0047] Calculation formula:

[0048]

[0049] Among them, m s is the weight of the sample after swelling in the buffer, m d is the weight of the sample in a dry state.

[0050] The swelling degree (swelling index) of the sample in Example 1 is measured as follows: Figure 1 , Figure 2 :

[0051] Depend on Figure 2 It can be seen that at pH 2.5, the double-layer gel microspheres reached swelling equilibrium at 30 minutes, with a swelling index of 13.18%, while at pH 7.4, the gel microspheres continued to swell with the increase of time, reaching the highest at 60 minutes, with a swelling index of about 93.22%. It can be seen that the double-layer gel microspheres basically do not swell under acidic conditions, and have a higher swelling degree under neutral or alkaline conditions, which helps to release the enzymes embedded in them in the intestine. The stomach is a digestive organ, and the small intestine is the main organ for absorbing nutrients. In the intestinal fluid, due to the higher pH value, deacetylated chitosan is deprotonated and weakens the integrity of sodium alginate-sodium carboxymethyl cellulose-chitosan, and glucose oxidase is released.

[0052] (2) Thermal stability

[0053] The samples to be tested (naked enzyme, single-layer sodium alginate-sodium carboxymethyl cellulose gel particles, and double-layer gel microspheres) were placed in a water bath at 60°C, 70°C (taken out every 10 minutes to measure enzyme activity), and 80°C (taken out every 2 minutes to measure enzyme activity) to investigate their thermal stability.

[0054] Calculation formula:

[0055]

[0056] Results Figure 3 , Figure 4 , Figure 5 :

[0057] The results show that when heated in a water bath at different temperatures, the enzyme activity retention rate of the enzyme-loaded double-layer microspheres is generally higher than that of the single-layer gel microspheres and free enzymes. The free enzyme was kept at 60°C for 30 minutes, and the enzyme activity retention rate was 75.23%, while the enzyme-loaded double-layer gel microspheres were kept at 60°C for 30 minutes, and there was still 93.27% enzyme activity, which was about 18% higher than the free enzyme activity. After being placed at 70°C for 60 minutes, the enzyme activity of the free enzyme remained 64.42%, and the enzyme activity of the enzyme-loaded double-layer gel microspheres was still 85.28%. After being placed at 80°C for 10 minutes, the enzyme activity of the free enzyme remained 7.5%, and the enzyme activity of the enzyme-loaded double-layer gel microspheres was still 39.76%. The enzyme activity was increased by 32.26% compared with the free enzyme. The reason for the improved thermal stability of enzyme-loaded double-layer gel microspheres is that the egg-shaped structure of the double-layer gel microspheres has a certain "rigidity". 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 the enzyme cofactor and improves the enzyme's heat resistance.

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

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

[0060] Preparation of simulated intestinal fluid: Weigh 0.68g of potassium dihydrogen phosphate, place it in a beaker, and dissolve it in 25mL of distilled water. Subsequently, add 7.7mL of 0.2mol / L sodium hydroxide solution, 50mL of distilled water and 1g of trypsin to the beaker, and then use a pH meter to accurately adjust the pH value to 7.7. The prepared gastrointestinal simulation fluid is stored in a refrigerator at 4°C.

[0061] Take 1g of the sample to be tested and add it to 10mL of simulated gastric fluid. Incubate it in a shaker at 37°C and 180rpm. Take out 0.1mL of the reaction solution every 30min for enzyme activity determination. Add 0.1mL of simulated gastric fluid after each sampling to maintain the balance of the reaction solution. The entire gastric stage cycle is 2h. After the gastric stage, adjust the pH value to 7.7. The incubation method of the small intestine stage is similar to that of the gastric stage. After each sample is collected, an equal volume of simulated intestinal fluid is added. The results are expressed as the cumulative release data in the solution. Free glucose oxidase is used as the control group. The average value of three parallel measurements is calculated for each sample. Results are shown in Figure 6 .

[0062] Figure 6The results showed that the release rate of free glucose oxidase increased rapidly in the first 30 minutes of the culture medium, reached the maximum after 90 minutes, and gradually decreased in the small intestine stage, and dropped to 26.10% after 240 minutes. The cumulative enzyme release of the single-layer gel particles continued to rise in the gastric stage, but when it reached the small intestine stage, the release of the enzyme released by the single-layer gel particles increased from 41.09% to 84.32%, and the enzyme release in the late intestinal stage tended to be flat. The double-layer gel microspheres showed a continuous upward trend throughout the gastrointestinal stage, and the enzyme release in the intestinal digestion stage increased from 41.06% to 74.98%. In particular, the double-layer gel microspheres were able to continue to release significantly linearly at a release rate of 0.15-0.2% (preferably 0.175%) per minute within the time range of 210-240 minutes in the intestinal stage (the cumulative release at 210 minutes was 69.70%) (P<0.05), which shows that the double-layer gel microspheres have a good sustained release effect.

[0063] In addition, in the experiment of simulating the gastrointestinal digestive fluid of aquatic fish (the pH of the intestinal fluid was selected to be 6.8), the release rate of free glucose oxidase also increased rapidly in the first 30 minutes of the culture medium, reaching a maximum of 63.55% after 90 minutes, and gradually decreased in the small intestine stage, falling to 28.11% after 240 minutes. The cumulative enzyme release of the monolayer gel particles continued to rise in the gastric stage, but when reaching the small intestine stage, the release of the enzyme released by the monolayer gel particles increased from 40.07% to 80.33%, and the enzyme release in the late intestinal stage tended to be flat. The double-layer gel microspheres showed a continuous upward trend throughout the gastrointestinal period, and the enzyme release amount increased from 41.04% to 70.32% during the intestinal digestion stage. In particular, the double-layer gel microspheres were within the time range of 210-240 minutes during the intestinal digestion stage (the cumulative release amount was 65.74% at 210 minutes), and they were also able to continuously and significantly release linearly at a release amount of 0.15-0.2% (preferably 0.153%) per minute (P<0.05). The double-layer gel microspheres had a good sustained release effect.

[0064] The research results of the subject have also repeatedly shown that the double-layer gel microspheres prepared in this application show the same trend whether in simulated intestinal fluid pH 7.7 or pH 6.8, and also show excellent sustained-release effect, and can be applied to food and aquatic feed processing.

[0065] (4) Storage stability analysis

[0066] The prepared gel particles and free enzyme were placed in a centrifuge tube filled with phosphate buffer (pH 6.0) and stored at 4°C. The 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 relative activity was used to express the enzyme activity. The results are shown in Figure 7 .

[0067] Figure 7 The results showed that as the storage time increased, the activity of the two gel microspheres and free enzymes gradually decreased. The activity of the double-layer gel microspheres decreased more slowly than that of the single-layer gel microspheres, and the activity of the single-layer gel microspheres decreased more slowly than that of the free enzymes. The enzyme activity retention rate reached 96.32% in the initial 5 days. After 30 days, the activity of the free enzyme decreased to 66.35%, while the activity of the double-layer gel microspheres was still 80.52%. Therefore, the use of chitosan solution to coat the sodium alginate-sodium carboxymethyl cellulose gel microspheres twice improved the storage stability of the enzyme to a certain extent.

Claims

1. A method for preparing glucose oxidase double-layer gel microspheres with a slowly and continuously increasing enzyme release during the intestinal digestion period, characterized in that: The following steps are involved: (1) First layer coating: Mix 27-36 mL of a sodium alginate solution with a mass concentration of 0.05%-4% and 3-4 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.05%-0.4%, and then add 1-2 mL of a glucose oxidase solution with a mass concentration of 0.1%-0.5%, mix well, and prepare a mixed solution of glucose oxidase and a dual carrier; use a peristaltic pump to drop the mixed solution into 100-120 mL of a calcium chloride solution with a mass concentration of 0.5%-4%, solidify at 3-4°C for 15-120 minutes, filter to obtain immobilized glucose oxidase (GOD) gel particles, and set aside; (2) Second coating layer: 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) into 100 mL of the chitosan solution, stir magnetically at 100-300 rpm for 10-50 min, and filter to obtain glucose oxidase double-layer gel microspheres.

2. The method according to claim 1, characterized in that In the step (1), 27 mL of a sodium alginate solution with a mass concentration of 1.5% and 3 mL of a sodium carboxymethyl cellulose solution with a mass concentration of 0.1% are mixed, 1-2 mL of a glucose oxidase solution with a mass concentration of 0.3% are added, and mixed to prepare a mixed solution of glucose oxidase and a dual carrier; a peristaltic pump is used to drop the mixed solution into 100-120 mL of a calcium chloride solution with a mass concentration of 1%, and the mixture is cured at 3-4°C for 30 minutes.

3. The method according to any one of claims 2, characterized in that In the step (2), the chitosan solution is prepared by preparing a chitosan solution with a mass concentration of 0.6% and a pH value of 4.0; the immobilized glucose oxidase gel particles prepared in the step (1) are transferred into 100 mL of the chitosan solution, stirred with a magnetic stirrer at 150 rpm, cured at 4° C. for 30 min, and filtered to obtain glucose oxidase gel microspheres.

4. The method according to any one of claims 3, characterized in that The peristaltic pump was used with the aid of a rotation speed of 12 rpm and a silicone double manifold with an inner diameter of 3 mm was used, with a stainless steel needle with an inner diameter of 0.45 mm connected at the end, and the injection height was 10 cm.

5. Use of the method according to any one of claims 1 to 4 for increasing the amount of enzyme released in the small intestine slowly, continuously and significantly, characterized in that: The microspheres can still be continuously released linearly at a release rate of 0.15-0.2% per minute within the time range of 210-240 minutes in the small intestine.

6. Glucose oxidase gel microspheres prepared by the method according to any one of claims 1 to 4, characterized in that: The microspheres have excellent thermal stability, storage stability and swelling index, and can also improve the activity of glucose oxidase in the gastrointestinal tract of animals.

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