Application of glucose oxidase gel microspheres in reduction of glucose in natural grape juice

Abstract

The invention relates to a method for improving the stability and efficiency of glucose oxidase (GOD) in a grape juice or acid environment, sodium alginate A is selected to individually encapsulate GOD, the stability and catalytic efficiency of GOD in an environment with the pH value of 3.6-4.0 are remarkably improved, the encapsulation efficiency is the highest, and the GOD can be used for preparing the grape juice or acid environment. When the sodium alginate A gel microspheres are applied to grape juice, the comprehensive effect of reducing glucose is better than that of sodium alginate B and C; sodium alginate A and sodium alginate B are combined according to a specific ratio in an improved manner, and other conditions are cooperated, so that the optimal pH range is expanded from 3.6-4.0 to 3.0-4.0, the application range is increased by 3.0-3.6, the catalytic activity is kept at 10-25 DEG C, the glucose reduction effect of the sodium alginate A + B group is better, and the method has important significance in coping with natural grape juice with different pH values in actual production. The GOD gel microspheres continuously catalyze glucose conversion in the natural grape juice to generate gluconic acid which can regulate the acidity of the juice and improve the taste of the grape wine. The GOD gel microspheres have good mechanical strength and operation stability.

Description

Technical Field

[0001] This invention relates to the field of wine processing technology, specifically to a method for reducing glucose in natural grape juice by encapsulating glucose oxidase (GOD) with sodium alginate based on different M / G ratios and molecular weights, a method for improving the stability and efficacy of glucose oxidase (GOD) in acidic environments, and a method for preparing glucose oxidase gel microspheres. Background Technology

[0002] Climate change is causing an increase in average annual temperatures, leading to a greater and faster accumulation of sugar in ripening grapes. This sugar accumulation translates to higher alcohol content in post-fermentation wines, sometimes exceeding 15% (v / v). High ethanol concentrations produce unpleasant aromas and tastes, and wines with lower alcohol content offer more benefits to both producers and consumers compared to those with higher alcohol content. Therefore, the wine industry must develop new strategies to address the challenges posed by climate change and evolving market demands.

[0003] Glucose oxidase (GOD) treatment is an effective method for reducing alcohol content through enzymatic processes. Pre-fermentation of grape juice before alcoholic fermentation lowers the glucose concentration, resulting in wine with a lower alcohol strength. GOD is a flavoprotein that catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide in the presence of oxygen. However, the low pH (3.0-4.0) of natural grape juice limits the utilization of glucose by glucose oxidase (GOD).

[0004] Encapsulation protects enzymes from pH fluctuations and allows for their reuse. Sodium alginate is recognized as GRAS (Generally Recognized As Safe), non-toxic, non-antigenic, and exhibits satisfactory biocompatibility and sufficient biodegradability. Alginate is a polysaccharide extracted from brown algae, composed of 1-4 randomly linked β-D-mannuronic acid and α-L-guluronic acid units, referred to as M and G units, respectively. The composition of alginate varies with its molecular weight (Mw) and the M / G block ratio, resulting in different properties when alginate crosslinks with calcium ions. There is no universal consensus in the field regarding the microencapsulation properties of alginate. Currently, studies have reported the structural properties of microcapsules made from different types of alginate as intestinal targeted delivery carriers. Several reports have also described the encapsulation of probiotics and fish oil with sodium alginate of different molecular weights and M / G ratios, examining the properties and protective effects of different gels. However, there are no reports on the encapsulation of glucose oxidase (GOD) with sodium alginate of different molecular weights and M / G ratios, or on the encapsulation of glucose oxidase (GOD) with combinations of different types of sodium alginate. Furthermore, there are no reports on the use of the encapsulated glucose oxidase (GOD) to reduce glucose in natural grape juice. Improving the stability and efficacy of glucose oxidase (GOD) in natural grape juice or acidic environments, and making it valuable for industrial transformation applications, is a new challenge facing the winemaking field. Summary of the Invention

[0005] This invention uses different types of sodium alginate (with different M / G and different molecular weights) or combinations thereof to encapsulate glucose oxidase (GOD), and applies the encapsulated GOD gel microspheres to reduce glucose in natural grape juice, thus solving the technical problems of low stability and efficacy of glucose oxidase (GOD) in natural grape juice or lower acidity environments.

[0006] The technical solution adopted in this invention is as follows:

[0007] This application relates to the application of glucose oxidase (GOD) gel microspheres in lowering glucose in natural grape juice at pH 3.6-4.0. The GOD gel microspheres are prepared by encapsulation of sodium alginate A with high G units and low molecular weight (HighG-LowMw). The M / G ratio of sodium alginate A is 1 / 2, and the molecular weight is 30K. The M unit is β-D-mannuronic acid, and the G unit is α-L-guluronic acid.

[0008] The method for preparing GOD gel microspheres is as follows: Sodium alginate A is dissolved in distilled water to prepare an aqueous solution with a concentration of 1-2% w / v. GOD is dissolved in the sodium alginate A aqueous solution at a ratio of 0.03 / 10 (g / mL) to form an enzyme-loaded mixture. The enzyme-loaded mixture is added dropwise to a 0.25-0.26 mol / L CaCl2 solution at a rate of 0.5-1 mL / min using a constant flow pump. GOD gel microspheres are formed under magnetic stirring at 150-200 r / min. After immobilization for 30-35 min, the microspheres are washed to obtain the GOD-loaded gel microspheres.

[0009] In one embodiment of the present invention, the GOD gel microspheres maintain catalytic activity at 10-25℃, remain stable in a grape juice environment with pH 3.6-4.0, and can be recycled at least 7 times; the GOD gel microspheres, after being treated at 20℃ and pH 3.6 for 24 hours, still retain an enzyme activity rate of 99.25%.

[0010] This invention also relates to the preparation of another type of glucose oxidase (GOD) gel microspheres, which can be used in natural grape juice at pH 3.0-4.0. The GOD gel microspheres are extended to natural grape juice at pH 3.0-3.6 and in lower acidic environments, thus broadening their application range. The GOD gel microspheres are prepared by encapsulating sodium alginate A (High G-Low Mw) and sodium alginate B (Low G-MEDMw): Sodium alginate A and sodium alginate B are dissolved in distilled water at a mass ratio of 1:1-1.5 to prepare a 2.5-3% w / v sodium alginate aqueous solution. GOD is dissolved in the sodium alginate aqueous solution at a ratio of 0.03 / 10 (g / mL) to form an enzyme-carrying mixture. The enzyme-carrying mixture is then added dropwise at a rate of 0.5-1 mL / min to a concentration of 0.27-0.29 mol / L using a constant flow pump. GOD gel microspheres were formed in CaCl2 solution under magnetic stirring at 150-200 r / min. After immobilization for 30-35 min, the microspheres were washed to obtain loaded GOD gel microspheres. The sodium alginate A had an M / G ratio of 1 / 2 and a molecular weight of 30 K. The sodium alginate B had an M / G ratio of 65 / 35 and a molecular weight of 90-180 K, where the M unit was β-D-mannuronic acid and the G unit was α-L-guluronic acid.

[0011] This application also relates to a method for preparing low-alcohol wine, specifically, adding GOD-loaded gel microspheres prepared by the aforementioned methods to natural grape juice, and reacting with shaking at 20°C for 24 hours. After fermentation, the alcohol content of the treated grape juice is reduced.

[0012] Compared with the prior art, the present invention achieves the following technical effects:

[0013] 1. This invention uses sodium alginate A with an M / G ratio of 1 / 2 and a molecular weight of 30K to encapsulate glucose oxidase (GOD). When applied to natural grape juice, this reduces the glucose content by 67.69 g / L, equivalent to a potential alcohol concentration of 3.98% (v / v). The ordered, dense, rigid gel structure formed by the high G unit content of sodium alginate effectively maintains GOD activity. The gel microspheres bind more calcium ions, resulting in more robust and less porous gel microspheres. The encapsulated enzyme exhibits good pH stability, significantly improving the stability and catalytic efficiency of glucose oxidase in pH 3.6-4.0 solutions, and also enhancing temperature stability. This application further increases the encapsulation efficiency of each microsphere by effectively controlling the volume of the CaCl2 gel solution (by reducing the volume). Compared to LowG-MEDMw (87.17%) and LowG-HighMw (76.64%), HighG-LowMw achieves a better encapsulation efficiency of 97.53%. Under conditions of 20℃ and pH 4.0 for 24 hours, the enzyme activity retention rate of glucose oxidase encapsulated by HighG-LowMw reached 99.25%. HighG-LowMw gel microspheres showed the best overall effect in reducing glucose content in grape juice. Compared to encapsulation with composite gels formed by alginate and other polymers (polysaccharides, proteins, such as sodium alginate, carboxymethyl cellulose, silica gel, gelatin, etc.), the encapsulation process using sodium alginate alone is simple, easy to control, and yields excellent results.

[0014] 2. By combining sodium alginate A (M / G = 1 / 2; molecular weight = 30K) and sodium alginate B (M / G = 65 / 35; molecular weight = 90-180K) in a specific ratio, the optimal pH range was expanded from pH 3.6-4.0 to pH 3.0-4.0, and the applicable range of 3.0-3.6 was increased. The enzyme activity retention rate at pH 3.2 increased to 92.6%, and the enzyme activity retention rate at pH 3.4 was 89.5%. This has important guiding significance for dealing with natural grape juice at different pH values ​​in actual production. This is due to the addition of sodium alginate B. The increased molecular weight of sodium alginate, coupled with longer molecular chains, creates more cross-linking sites and facilitates intermolecular entanglement. The loose sodium alginate B molecules entangle within the rigid, relatively porous sodium alginate A network, forming gel microspheres with increased density and hardness. This better prevents the shrinkage that occurs after cross-linking of alginate in the presence of divalent cations, and also prevents hydrophilic enzymes from rapidly diffusing from the alginate beads into the gel solution, thus avoiding enzyme leaching and affecting the encapsulation efficiency. Furthermore, it inhibits, to some extent, the problems caused by the excessive protonation (-COOH) of carboxylic acid groups in sodium alginate due to pH changes (pH < 3.6), which leads to molecular chain shrinkage, acid gel precipitation, and locally low pH within the microspheres. It also avoids excessive pH drops in natural grape juice that could negatively impact enzyme activity. Figure 9 The results showed that, compared to the HighG-LowMw group, the pH value of group A+B could recover to 3.45. The GOD-loaded gel microspheres exhibited high permeability, achieving better glucose consumption. The glucose content in natural grape juice was reduced by 71.87 g / L, equivalent to a potential alcohol concentration of 4.22% (v / v), resulting in more ideal blood sugar reduction. In actual production, the enzyme dosage can be adjusted appropriately based on the blood sugar-reducing effect of the GOD-loaded gel microspheres to reduce enzyme costs and effectively control glucose consumption.

[0015] 3. During the pre-fermentation stage, GOD gel microspheres effectively reduce the sugar content of natural grape juice, resulting in a lower alcohol concentration in the wine after fermentation. This avoids the unpleasant flavors caused by high alcohol concentration. GOD gel microspheres continuously catalyze the conversion of glucose into gluconic acid in natural grape juice, which can regulate the acidity of the juice. By controlling the catalytic process, the wine's taste balance is improved. Compared with exogenous acidulants, it has a better, more mellow and lingering taste, and the overall sensory quality of the wine reaches an ideal state.

[0016] 4. The GOD gel microspheres protected in this application all have good mechanical strength and operational stability (7 cycles), and can be recycled multiple times, reducing production costs. Attached Figure Description

[0017] 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.

[0018] Figure 1 Encapsulation efficiency of three types of gel microspheres.

[0019] Figure 2 Mechanical strength of three types of gel microspheres at different sodium alginate concentrations.

[0020] Figure 3 Electron micrographs of the appearance and cross-sectional structure of three types of enzyme-loaded gel microspheres.

[0021] Figure 4 Enzyme activity retention rates of naked GOD and three types of enzyme-loaded gel microspheres after 24 h of treatment under different pH conditions.

[0022] Figure 5 Enzyme activity retention rates of naked GOD and four types of enzyme-loaded gel microspheres after 24 h of treatment under different pH conditions.

[0023] Figure 6 Effects of different temperatures on the activities of naked GOD and three types of enzyme-loaded gel microspheres.

[0024] Figure 7 Operational stability of three types of enzyme-loaded gel microspheres.

[0025] Figure 8 Glucose concentration and pH of grape juice after 24 h of treatment with untreated and naked GOD and three types of gel microspheres.

[0026] Figure 9 Glucose concentration and pH of grape juice after 24 h of treatment with untreated and naked GOD and four types of gel microspheres. Detailed Implementation

[0027] Main experimental materials and their sources:

[0028] Sodium alginate includes three types: High G unit - Low molecular weight (HighG-LowMw), Low G unit - Medium molecular weight (LowG-MEDMw), and Low G unit - High molecular weight (LowG-HighMw), which correspond to sodium alginate A, sodium alginate B, and sodium alginate C, respectively.

[0029] Sodium alginate A (M / G: 1 / 2, molecular weight: 30K) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0030] Sodium alginate B (M / G: 65 / 35, molecular weight: 90-180K, sodium alginate CR8133) was purchased from Amsty (China) Co., Ltd.

[0031] Sodium alginate C (M / G: 65 / 35, molecular weight: 250-350K, sodium alginate CR8233) was purchased from Amsty (China) Co., Ltd.

[0032] Glucose oxidase was purchased from Weinuoen Biotechnology Co., Ltd.

[0033] Anhydrous calcium chloride was purchased from Tianjin Kemeo Chemical Reagent Co., Ltd.

[0034] Example 1

[0035] Preparation of GOD gel microspheres: Sodium alginate A, sodium alginate B, and sodium alginate C were dissolved in distilled water to prepare aqueous solutions with concentrations (w / v) of 1%, 1.5%, and 2%, respectively. GOD was dissolved in the aqueous solutions of sodium alginate A, B, and C at a ratio of 0.03 g / 10 mL to form enzyme-loaded mixtures. The enzyme-loaded mixtures were then added dropwise to 0.25 mol / L CaCl2 solution at a rate of 0.5 mL / min using a 0.45 × 16 RWLB needle and a constant flow pump. GOD gel microspheres were formed under magnetic stirring at 150 r / min. The microspheres were collected at a distance of 10 cm, immobilized for 30 min, and then washed three times with distilled water to obtain different types and concentrations of GOD-loaded gel microspheres.

[0036] Example 2

[0037] GOD gel microspheres were prepared by encapsulating sodium alginate A (high G-low molecular weight) and sodium alginate B (low G-medium molecular weight) with high G-units. Sodium alginate A and sodium alginate B were dissolved in distilled water at a mass ratio of 3:1 to prepare a 2.5% w / v sodium alginate aqueous solution. GOD (g / mL) was dissolved in the sodium alginate aqueous solution at a ratio of 0.03 / 10 to form an enzyme-carrying mixture. The enzyme-carrying mixture was then added dropwise at a rate of 0.5 mL / min to a 0.27 mol / L sodium alginate solution. GOD gel microspheres were formed in CaCl2 solution under magnetic stirring at 150 r / min. After immobilization for 30 min, the microspheres were washed to obtain loaded GOD gel microspheres, referred to as A+B gel microspheres. The sodium alginate A had an M / G ratio of 1 / 2 and a molecular weight of 30 K. The sodium alginate B had an M / G ratio of 65 / 35 and a molecular weight of 90-180 K.

[0038] Example 3

[0039] GOD gel microspheres were prepared by encapsulating sodium alginate A (High G-Low Mw) and sodium alginate B (Low G-MEDMw). Sodium alginate A and sodium alginate B were dissolved in distilled water at a mass ratio of 2:1 to prepare a 3% w / v sodium alginate aqueous solution. GOD (g / mL) was dissolved in the sodium alginate aqueous solution at a ratio of 0.03 / 10 to form an enzyme-carrying mixture. The enzyme-carrying mixture was then added dropwise at a rate of 0.5 mL / min to a 0.29 mol / L solution using a constant flow pump. GOD gel microspheres were formed in CaCl2 solution under magnetic stirring at 150 r / min. After immobilization for 30 min, the microspheres were washed to obtain loaded GOD gel microspheres. The sodium alginate A had an M / G ratio of 1 / 2 and a molecular weight of 30 K. The sodium alginate B had an M / G ratio of 65 / 35 and a molecular weight of 90-180 K, where the M unit was β-D-mannuronic acid and the G unit was α-L-guluronic acid.

[0040] Example 4: Treatment of natural grape juice with glucose oxidase-loaded gel microspheres

[0041] Fresh grapes were crushed using a cell wall blender, placed in centrifuge tubes, and centrifuged at 5000 rpm for 15 min. The supernatant was collected to obtain grape juice (the initial pH of the grape juice was 3.7). 600 U of naked enzyme with enzyme activity and 1% concentration of enzyme-loaded gel microspheres prepared in Example 1 were added to an Erlenmeyer flask containing 20 mL of grape juice. The flask opening was covered with perforated aluminum foil, and the flask was kept at 20°C with oscillation at 53 rpm for 24 h. The control grape juice without microspheres was kept under the same conditions.

[0042] Example 5: Treatment of natural grape juice with glucose oxidase-loaded gel microspheres

[0043] Fresh grapes were crushed using a cell wall blender and placed into centrifuge tubes. The mixture was centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected to obtain grape juice (the initial pH of the grape juice was 3.7). 600 U of naked enzyme with enzyme activity and the enzyme-loaded gel microspheres prepared in Example 2 were added to an Erlenmeyer flask containing 20 mL of grape juice. The flask opening was covered with perforated aluminum foil, and the mixture was kept at 20°C with shaking at 53 rpm for 24 hours. The control grape juice (without capsules) was kept under the same conditions. After treatment, glucose concentration and pH were measured, and the process was repeated three times.

[0044] Experimental methods and index determination:

[0045] 1. Determination of GOD enzyme activity

[0046] Standard curve plotting

[0047] The activity of glucose oxidase (GOD) was assessed by measuring the amount of H2O2 generated. 0 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.8 mL, 0.9 mL, and 1.1 mL of hydrogen peroxide standard solution were pipetted and diluted to 25 mL with phosphate buffer to prepare a series of hydrogen peroxide standard solutions with final concentrations of 0 μg / mL, 28.8 μg / mL, 48 μg / mL, 67.2 μg / mL, 76.8 μg / mL, 86.4 μg / mL, and 105.6 μg / mL.

[0048] Take 10 mL test tubes (two replicates for each concentration), and add 2.5 mL of o-benzylamine buffer, 0.3 mL of glucose solution, and 0.1 mL of peroxidase solution sequentially. Vortex mix, then add 0.1 mL of the above peroxide standard series solutions. Mix well, and add 2 mL of 2 mol / L sulfuric acid solution. Use a test tube containing 0 μg / mL hydrogen peroxide as a standard blank. Measure the absorbance at 540 nm. Plot a hydrogen peroxide standard curve with absorbance on the x-axis and hydrogen peroxide concentration on the y-axis.

[0049] Test Procedure

[0050] Add 2.5 mL of o-benzylamine buffer, 0.3 mL of glucose solution, and 0.1 mL of peroxidase solution sequentially to a test tube. Vortex the mixture until homogeneous and preheat in a 37°C water bath for 5 min. Add 0.1 mL of the glucose oxidase solution to be tested to the test tube and react precisely at 37°C for 3 min. Add 2 mL of 2 mol / L sulfuric acid solution, shake well, terminate the reaction, and cool to room temperature. Measure the absorbance at 540 nm, using a standard blank solution as a blank control. Record the net absorbance as A.

[0051] Experimental data processing

[0052] The glucose oxidase activity in the sample is represented by X, and the value is expressed in enzyme activity units per gram or enzyme activity units per milliliter (U / g or U / mL). The calculation formula is as follows:

[0053]

[0054] X—Glucose oxidase activity in the sample, U / g (or U / mL); A—Net absorbance of the enzyme reaction group; K—Slope of the standard curve; C0—Intercept of the standard curve; N—Total dilution factor of the sample; m—Sample weight, g or mL; T—Reaction time, 3 min; 34.01—Molecular weight of hydrogen peroxide

[0055]

[0056] 2. Determination of Encapsulation Efficiency (EE)

[0057] The gel microspheres were added to PBS buffer at pH 6.0. After homogenization using a homogenizer (5000 rpm, 1 min), the mixture was centrifuged to assess enzyme activity. Encapsulation efficiency (EE) was calculated using the formula:

[0058]

[0059] Wherein, U2 represents the total GOD activity used to prepare the gel microspheres, and U1 represents the GOD activity released from the gel microspheres.

[0060] Encapsulation efficiency is an important indicator in the enzyme encapsulation process. This application increases the encapsulation efficiency by effectively controlling the volume of the CaCl2 gel solution (by reducing the volume). The encapsulation efficiencies of the three types of gel microspheres (1% concentration prepared in Example 1) are as follows: Figure 1 As shown:

[0061] Depend on Figure 1 The encapsulation efficiency of GOD encapsulated by HighG-LowMw gel microspheres was 97.53%, that of LowG-MEDMw gel microspheres was 87.17%, and that of LowG-HighMw gel microspheres was 76.64%. These results indicate that by reducing the gel solution volume, all three types of gel microspheres achieved good encapsulation efficiency for GOD. HighG-LowMw gel microspheres exhibited the highest encapsulation efficiency.

[0062] 3. Mechanical performance testing

[0063] The mechanical properties of the GOD-loaded gel microspheres (1% concentration prepared in Example 1) were evaluated using a texture analyzer, with the trigger measured at 2 mm / s. -1 The gel sample was compressed twice at the specified rate to a depth of 20 mm using 0.3 N. The delay time between the two compressions was 15 s. In each case, at least five replicate analyses were performed on each formulation using fresh samples at room temperature. Results are as follows: Figure 2 As shown:

[0064] Depend on Figure 2It can be seen that the hardness of the three types of gel microspheres generally increases with increasing sodium alginate concentration. At a sodium alginate concentration of 1%, the hardnesses of HighG-LowMw, LowG-MEDMw, and LowG-HighMw gel microspheres are 0.92N, 1.02N, and 1.12N, respectively. At a sodium alginate concentration of 1.5%, the hardnesses are 2.16N, 2.7N, and 2.9N, respectively. Although studies have shown that G-rich alginate can form more ordered and harder hydrogels, the LowG-HighMw gel microspheres exhibit the highest hardness at these two sodium alginate concentrations. At a sodium alginate concentration of 2%, the hardnesses of HighG-LowMw, LowG-MEDMw, and LowG-HighMw gel microspheres are 3.19N, 2.85N, and 2.57N, respectively. This indicates that at higher concentrations, G-rich alginate can form gel microspheres with greater rigidity. Furthermore, at a concentration of 2%, the rigidity of the higher molecular weight LowG-HighMw gel microspheres was lower than that of the LowG-MEDMw gel microspheres. This is because at a 2% concentration, the LowG-HighMw gel solution has a higher viscosity, and unremovable air bubbles are generated during stirring and dissolving, which affects the rigidity of the gel microspheres.

[0065] 4. Scanning electron microscope

[0066] The gel microspheres (1% concentration prepared in Example 1) were lyophilized and observed using a scanning electron microscope. To obtain cross-sectional images, the samples were additionally treated with liquid nitrogen and immediately cut in half with a blade. The prepared samples were mounted on double-sided gold-plated metal fixtures and observed under an accelerating voltage of 15 kV. Results are shown in […]. Figure 3 :

[0067] Depend on Figure 3 It is evident that HighG-LowMw can form a more uniform, porous, and organized structure. High G content results in larger microspheres, with more and more uniform pores. The other two types of microspheres have more porous pores. The lower molecular weight allows for better organization of the bead-like structure, resulting in fewer porous bead-like structures.

[0068] 5. pH stability

[0069] The pH stability of free and encapsulated GOD was investigated using potassium hydrogen phthalate buffer (pH range 3.0 to 4.0) to simulate the pH environment of natural grape juice. 600 U (or more) of naked enzyme and enzyme-loaded gel microspheres (1% concentration prepared in Example 1) were added to 30 mL of potassium hydrogen phthalate buffer at different pH values ​​(3.0, 3.2, 3.4, 3.6, 3.8, 4.0) and incubated at 20 °C for 24 h. Enzyme activity was then measured. Untreated naked enzyme served as a control.

[0070] Encapsulation is crucial for improving enzyme stability at low pH levels. This application investigated the protective effects of three types of sodium alginate-encapsulated GOD at simulated grape juice pH (3.0-4.0), using naked GOD as a control. Results are shown below. Figure 4 :

[0071] Depend on Figure 4 It can be seen that the enzyme activity retention rates of naked GOD, HighG-LowMw, LowG-MEDMw, and LowG-HighMw gel microspheres generally increase with increasing pH. At pH 3.6, the enzyme activity retention rate of HighG-LowMw gel microspheres is 76.58%, while that of naked GOD is 36.36%. At pH 3.8, the enzyme activity retention rate of HighG-LowMw gel microspheres is 92.48%, while that of naked GOD is 40.47%. At pH 4.0, the enzyme activity retention rate of HighG-LowMw gel microspheres is 99.25%, essentially retaining all enzyme activity.

[0072] This application further determined the protective effects of four types (i.e., with the addition of A+B, prepared according to Example 2) of sodium alginate-encapsulated GOD at simulated grape juice pH (3.0-4.0), using naked GOD as a control. The results are shown in [Figure 1]. Figure 5 :

[0073] Depend on Figure 5 It is evident that the combined use of sodium alginate A and B (prepared according to Example 2) expands the applicable pH range of GOD enzyme-loaded gel microspheres, exhibiting excellent performance within the pH range of 3-3.6. The enzyme activity retention rate increases to 92.6% at pH 3.2, 89.5% at pH 3.4, and 70.2% at pH 3.6. This is significant for handling natural grape juice at different pH values ​​in actual production. The sodium alginate A and B combination maintains a high and stable overall enzyme activity retention rate within the pH range of 3.0-4.0.

[0074] 6. Temperature stability

[0075] The effect of temperature was investigated using four different temperatures within the range of 10 to 25°C in a winery. 600 U of naked enzyme and enzyme-loaded gel microspheres (1% concentration prepared in Example 1) were added to 30 mL of potassium hydrogen phthalate buffer at pH 3.6 and incubated for 24 h at different temperatures (10, 15, 20, 25°C). Enzyme activity was then measured. Untreated naked enzyme was used as a control. This application investigated the effect of temperature on the activities of naked GOD and three enzyme-loaded gel microspheres at pH 3.6 using four different temperatures within the range of 10 to 25°C in a winery. The results are shown in [Figure number missing]. Figure 6 :

[0076] Depend on Figure 6 It was found that, under pH 3.6 and after 24 hours of treatment at different temperatures, the enzyme activity retention rates of naked GOD, LowG-MEDMw, and LowG-HighMw gel microspheres gradually decreased with increasing temperature, while the enzyme activity retention rate of HighG-LowMw gel microspheres showed a trend of first increasing and then decreasing with increasing temperature. Specifically, the enzyme activity retention rates of LowG-MEDMw and LowG-HighMw gel microspheres were lower than those of naked GOD, indicating a negative impact on enzyme protection. The enzyme activity retention rate of HighG-LowMw gel microspheres was higher than that of naked GOD, reaching its highest at 20℃, retaining 76.58% of the enzyme activity, compared to only 36.36% for naked GOD. This indicates that HighG-LowMw gel microspheres have a good protective effect on GOD at different temperatures, and these conditions are more conducive to the utilization of HighG-LowMw gel microspheres at grape juice fermentation temperatures, thereby effectively reducing glucose content. This application further tested sodium alginate A+B (prepared according to Example 2), which retained 70.2% of enzyme activity at 20°C. The enzyme activity retention rate was high and stable in the temperature range of 10-25°C. This is because after combination, the continuous positions of G blocks on alginate form cavities for calcium ion connection. HighG can bind more calcium ions, thereby forming a more uniform, porous, and organized structure. The structure of the gel beads is more stable, which is beneficial to maintaining the spatial conformation of the enzyme.

[0077] 7. Operational stability

[0078] 200 g of glucose was added to 1 L of water, and the final pH was adjusted to 3.7 using a pH meter to prepare simulated grape juice. The operational stability (600 U) of the enzyme-loaded gel microspheres (1% concentration prepared in Example 1) was determined by quantifying the reduction in reducing sugar content in 20 mL of simulated grape juice at 20 °C (stirred at 53 rpm) for 7 consecutive enzyme cycles. After each cycle of 24 h, the microspheres were filtered out, washed with PBS buffer (0.1 M, pH 6), and then added to fresh simulated grape juice. Reducing sugars were determined by the 3,5-dinitrosalicylic acid (DNS) method, using D-glucose as a standard. DNS is an aromatic compound that reacts with reducing sugars to produce 3-amino-5-nitrosalicylic acid, which absorbs light at 540 nm. Results are shown in […]. Figure 7 :

[0079] Depend on Figure 7 It was found that all three types of gel microspheres could be reused. Specifically, the LowG-MEDMw gel microspheres consumed more glucose during initial use, but after three cycles, their glucose consumption capacity significantly decreased, and the microspheres broke down after three cycles. The LowG-HighMw gel microspheres could be reused for 5 cycles; the high molecular weight played a positive role in the structure of the gel microspheres, ensuring that the microsphere structure remained intact for a longer period. The HighG-LowMw gel microspheres could be reused for 7 cycles; the high G content formed a more robust gel, ensuring that the gel structure remained intact under harsh environments and providing better protection for the enzyme. Further testing with sodium alginate A+B (prepared according to Example 2) also showed that they could be reused for 7 cycles.

[0080] 8. Glucose concentration determination using the DNS method

[0081] Three enzyme-carrying microspheres, HighG-LowMw, LowG-MEDMw, and LowG-HighMw (prepared at a 1% concentration according to Example 1), were applied to grape juice, with naked GOD as a control, to investigate their effectiveness in consuming glucose in practical applications. The results are as follows: Figure 8 As shown:

[0082] Depend on Figure 8The glucose content of untreated grape juice was 212.77 g / L, and the initial pH was 3.7. After treatment with various gel microspheres, the glucose content of the grape juice decreased significantly. The glucose consumption after treatment with naked GOD, enzyme-loaded HighG-LowMw, LowG-MEDMw, and LowG-HighMw gel microspheres was 24.62 g / L, 67.69 g / L, 80 g / L, and 49.99 g / L, respectively. A 1.0% (v / v) alcohol concentration is approximately equivalent to about 17 g / L of glucose produced by yeast during alcoholic fermentation. Therefore, after treatment with naked GOD and enzyme-loaded gel microspheres, the potential alcohol concentration of the grape juice decreased by approximately 1.45% (v / v), 3.98% (v / v), 4.7% (v / v), and 2.94% (v / v), respectively. The initial pH of the grape juice was 3.7. After treatment with naked GOD and enzyme-loaded HighG-LowMw, LowG-MEDMw, and LowG-HighMw gel microspheres, the pH decreased to 3.48, 3.38, 3.45, and 3.46, respectively, due to the conversion of glucose to gluconic acid. Although the LowG-MEDMw group showed a high glucose consumption of 80 g / L, its glucose consumption capacity decreased significantly after three cycles; furthermore, the microspheres had low mechanical strength, leading to microsphere breakage after three cycles, resulting in poor overall performance.

[0083] Further application of sodium alginate A+B enzyme-carrying gel microspheres (prepared according to Example 2) to grape juice yielded the following results: Figure 9 As shown:

[0084] Depend on Figure 9 It can be seen that, compared with the HighG-LowMw group which retained 76.58% enzyme activity at 20℃ (pH 3.6), the sodium alginate A+B group (prepared according to Example 2) retained a slightly lower enzyme activity of 70.2% at 20℃ (pH 3.6), but showed a more superior blood sugar lowering ability in natural grape juice (pH 3.7). The glucose content in natural grape juice was reduced by 71.87 g / L, which is equivalent to a potential alcohol concentration of 4.22% (v / v), and the blood sugar lowering was more ideal. Compared with the pH 3.38 of the HighG-LowMw group, the pH value of the A+B group also rose to 3.45. This may be related to the high permeability of LowG-MEDMw, where the A+B substrate can come into more complete contact with the enzyme, and the enzyme inactivation rate is slow. The reaction system is dynamic and complex; the enzyme-loaded gel microspheres begin to react as soon as they are placed in the grape juice system, catalyzing while inactivating. The reaction rate and extent of action are affected by various factors such as changes in the microsphere's spatial structure, mechanical strength, temperature, pH, encapsulation efficiency, substrate dosage, and time. The study also found that the enzyme activity retention rate of the microspheres is not entirely positively correlated with the glucose reduction.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of glucose oxidase (GOD) gel microspheres in lowering glucose in natural grape juice at pH 3.6-4.0, characterized in that... GOD gel microspheres are prepared by encapsulating sodium alginate A with high G units and low molecular weight (HighG-LowMw). The M / G ratio of sodium alginate A is 1 / 2, and the molecular weight is 30K. The M unit is β-D-mannuronic acid, and the G unit is α-L-guluronic acid.

2. The application according to claim 1, wherein the method for preparing the GOD gel microspheres is characterized in that, Sodium alginate A was dissolved in distilled water to prepare an aqueous solution with a concentration of 1-2% w / v. GOD was dissolved in the sodium alginate A aqueous solution at a ratio of 0.03 / 10 (g / mL) to form an enzyme-loaded mixture. The enzyme-loaded mixture was added dropwise to a 0.25-0.26 mol / L CaCl2 solution using a constant flow pump at a rate of 0.5-1 mL / min. GOD gel microspheres were formed under magnetic stirring at 150-200 r / min. After immobilization for 30-35 min, the microspheres were washed to obtain the GOD-loaded gel microspheres.

3. The application according to claim 2, characterized in that, The GOD gel microspheres maintain catalytic activity at 10-25℃, remain stable in natural grape juice environment at pH 3.6-4.0, and can be recycled at least 7 times; when the GOD gel microspheres are treated at 20℃ and pH 3.6 for 24 hours, the enzyme activity retention rate can reach 99.25%.

4. A method for preparing low-alcohol wine, characterized in that, The method includes the step of using the GOD gel microspheres described in any one of claims 1-3, specifically adding the GOD gel microspheres to natural grape juice and reacting with shaking at 20°C for 24 hours to reduce the glucose content. After fermentation, the alcohol content of the treated grape juice is reduced.

5. The application of glucose oxidase (GOD) gel microspheres in lowering glucose in natural grape juice at pH 3.0-4.0, characterized in that... GOD gel microspheres were prepared by encapsulating sodium alginate A with sodium alginate B of low G-unit-medium molecular weight (LowG-MEDMw): Sodium alginate A and sodium alginate B were dissolved in distilled water at a mass ratio of (2-3):1 to prepare a sodium alginate aqueous solution with a concentration of 2.5-3% w / v. GOD was dissolved in the sodium alginate aqueous solution at a ratio of 0.03 / 10 (g / mL) to form an enzyme-loaded mixture. The enzyme-loaded mixture was added dropwise to a 0.27-0.29 mol / L CaCl2 solution using a constant flow pump at a rate of 0.5-1 mL / min. GOD gel microspheres were formed under magnetic stirring at 150-200 r / min. After immobilization for 30-35 min, the microspheres were washed to obtain the GOD-loaded gel microspheres. The sodium alginate A had an M / G ratio of 1 / 2 and a molecular weight of 30 K; the sodium alginate B had an M / G ratio of 65 / 35 and a molecular weight of 90-180 K.

6. The application according to claim 5, characterized in that, The GOD gel microspheres can be applied to natural grape juice at pH 3.0-3.

6.

7. A method for preparing low-alcohol wine, characterized in that, The method includes the step of using the GOD gel microspheres described in any one of claims 5-6, specifically adding the GOD gel microspheres to natural grape juice and reacting with shaking at 20°C for 24 hours to reduce the glucose content. After fermentation, the alcohol content of the treated grape juice is reduced.

8. A method for improving the enzyme activity retention rate of glucose oxidase (GOD) in an acidic or alkaline environment with a pH value of 3.0-3.6, characterized in that, GOD-loaded gel microspheres are prepared according to the method for preparing gel microspheres as described in claim 5.

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