CN107475050B - Method for reducing sweetness of sugar-containing beverage by using biological enzyme - Google Patents
Method for reducing sweetness of sugar-containing beverage by using biological enzyme Download PDFInfo
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Abstract
The invention relates to a method for reducing sweetness of a sugar-containing beverage by using biological enzyme, which comprises the steps of respectively preparing a glucose oxidase solution and a catalase solution; mixing a glucose oxidase solution and a catalase solution, and then co-curing to obtain co-immobilized glucose oxidase-catalase; adding glucose oxidase solution, catalase solution or co-immobilized glucose oxidase-catalase into glucose solution or sugar-containing beverage, and oscillating at pH of 3.0-10.0 and temperature of 30-60 deg.C for reaction. The invention has wider application range of blood sugar reduction, convenient use, high efficiency and can tolerate alcohol with certain concentration, and the blood sugar reduction rate can be automatically controlled according to the requirement; the method of the immobilized enzyme can reduce the use cost and has good application prospect.
Description
Technical Field
The invention belongs to the field of preparation of low-sugar foods, and particularly relates to a method for reducing sweetness of a sugar-containing beverage by using biological enzyme.
Background
Saccharides are the main source of energy required for various human vital activities, which are composed of elements such as carbon, oxygen, hydrogen, etc., and among them, glucose is an essential nutrient for metabolism in the organism. With the improvement of living standard of people, the excessive intake of sugar caused by poor eating habits causes a plurality of diseases such as decayed teeth, diabetes and obesity. Sugar content in foods is generally reduced by controlling the amount of added sugar or searching for a substitute for sugar, but the sugar content in foods cannot be controlled by such methods for drinks directly fermented from raw materials, particularly alcoholic beverages, and therefore, there is a high necessity for developing new methods for reducing sugar content.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for reducing the sweetness of the sugar-containing beverage by using the biological enzyme, and the method has the advantages of wide application range, safety, no toxic or side effect, controllable degradation rate, capability of tolerating alcohol with certain concentration, low use cost and good application prospect.
The invention relates to a method for reducing sweetness of a sugar-containing beverage by using biological enzyme, which comprises the following steps:
(1) respectively preparing a glucose oxidase solution and a catalase solution;
(2) mixing a glucose oxidase solution and a catalase solution, and then co-curing to obtain co-immobilized glucose oxidase-catalase;
(3) adding glucose oxidase solution and/or catalase solution or co-immobilized glucose oxidase-catalase into glucose solution or sugar-containing beverage, and oscillating at pH of 3.0-10.0 and temperature of 30-60 deg.C for reaction.
And (2) in the step (1), the solvents of the glucose oxidase solution and the catalase solution are buffer solutions.
The pH value of the buffer solution is 3.0-10.0, and the concentration is 50-200 mM.
The buffer solution is prepared from one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid and sodium citrate.
And determining the optimal single enzyme adding amount of the glucose oxidase and the catalase according to the mixing ratio of the glucose oxidase and the catalase and the enzyme activity ratio when the measured glucose degradation rate is highest.
The ratio of the catalase to the glucose oxidase in the step (2) is 0.5:1-150: 1.
And (3) co-curing in the step (2) by adopting bacterial cellulose, wherein the bacterial cellulose is obtained by fermenting acetobacter gluconicum. The method comprises adopting Acetobacter xylinum as strain, inoculating by inoculating loop or membrane inoculation under aseptic condition, and culturing the seed culture solution in shaker at 30 deg.C and 160rpm for 12-24 h. Inoculating into static fermentation culture medium with inoculum size of 5-10% (v/v), and standing in 30 deg.C constant temperature incubator for 5-12 days to obtain bacterial cellulose film.
The co-curing method is one or more of an adsorption method, an embedding method, a crosslinking method and a combination method.
The concentration of the glucose solution in the step (3) is 0-400 g/L; the sugar-containing beverage is a sugar-containing beverage with ethanol concentration of 0-40 v/v%.
In the step (3), the degree of reaction progress can be controlled by controlling the introduction amount of oxygen.
The oscillation reaction in the step (3) is as follows: the process is carried out under the condition of constant temperature oscillation, and the rotating speed is 100-.
The immobilized glucose oxidase-catalase in the step (3) can be repeatedly used for catalytic degradation of the sugar-containing beverage, and the buffer solution can be used for washing for 1-5 times after each use.
The co-immobilized glucose oxidase-catalase in the step (3) can be repeatedly used for 1-8 times.
Advantageous effects
The invention has wider application range, is safe and has no toxic or side effect when being used in food, and has mild reaction condition and environmental protection; the production cost can be reduced by immobilizing the enzyme during enzyme degradation, and the reaction can be controlled by controlling the introduction amount of oxygen, so that the degradation rate can be controlled automatically; can tolerate alcohol with certain concentration, thus being applicable to blood sugar reduction processing of products such as fermented sweet wine and the like, and having good large-scale application prospect.
Drawings
FIG. 1: degradation rate of free glucose oxidase to 100g/L glucose solution at different temperatures;
FIG. 2: degradation rate of free glucose oxidase-catalase to glucose solution;
FIG. 3: relative enzyme activity of free enzyme and immobilized enzyme at different temperatures;
FIG. 4: degradation rate of free glucose oxidase on glucose solutions with different concentrations;
FIG. 5: degradation rate of free glucose oxidase on glucose solutions with different ethanol contents;
FIG. 6: the degradation rate of the free and immobilized glucose oxidase-catalase to the glucose solution containing ethanol;
FIG. 7: degrading sweet wine samples by using different enzyme adding amounts of immobilized enzymes;
FIG. 8: the number of times of reuse of the immobilized enzyme.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
(1) Dilute glucose oxidase
Preparing 50-200mM buffer solution with pH5.0 from sodium dihydrogen phosphate, disodium hydrogen phosphate or potassium dihydrogen phosphate, dipotassium hydrogen phosphate or acetic acid, sodium acetate or citric acid, and sodium citrate, dissolving and diluting glucose oxidase solid powder, and preparing into glucose oxidase solution.
(2) Degradation of glucose solution by free enzyme liquid
10mL of 50g/L, 100g/L, 200g/L and 400g/L glucose solution with pH5.0 are prepared, 200 mu L of free glucose oxidase solution is added, and the mixture is respectively placed in a constant temperature oscillation water bath kettle at 30-60 ℃ for oscillation reaction under the condition of 200rpm and 100-. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
As a result, it was found that the degradation rate of the glucose solution was the highest at a temperature of 40 ℃ and about 19% at a glucose concentration of 100g/L, as shown in FIG. 1.
Example 2
(1) Diluting glucose oxidase, catalase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., respectively dissolving and diluting glucose oxidase and catalase solid powder, and mixing two enzyme solutions according to enzyme activity ratios of catalase to glucose oxidase of 0.5:1, 2.5:1, 5:1, 25:1, 50:1, 100:1 and 150:1 to obtain mixed enzyme solution.
(2) Degradation of glucose solution by using free glucose oxidase-catalase enzyme liquid
10mL of substrate glucose solutions with the concentrations of 50g/L, 100g/L, 200g/L and 400g/L are respectively prepared, 200 mu L of enzyme solution is added into the solution with the pH value of 5.0, and the solution is placed in a constant-temperature oscillation water bath kettle at 37 ℃ for oscillation reaction under the condition of 200 rpm. Sampling and testing are carried out at intervals, and the amount of generated gluconic acid and the amount of glucose reduction in the solution are detected by High Performance Liquid Chromatography (HPLC), so that the degradation rate is calculated.
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
The results showed that the rate of degradation of free glucose oxidase-catalase was higher than that of free glucose oxidase, as shown in FIG. 2.
Example 3
(1) Diluting glucose oxidase, catalase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., dissolving and diluting solid glucose oxidase and catalase powder, respectively, and mixing the two enzyme solutions according to a certain proportion to obtain a mixed enzyme solution.
(2) Preparation of immobilized glucose oxidase-catalase
Fixing the addition amount of glucose oxidase, changing the addition amount of catalase to enable the enzyme activity ratio of the glucose oxidase to the catalase to be 0:1, 0.5:1, 2.5:1, 5:1, 25:1, 50:1, 100:1 and 150:1 respectively, testing the product generation amount, and when the enzyme activity ratio is 25:1, the glucose degradation rate is maximum, and the enzyme activity ratio of the catalase to the glucose oxidase is the optimal enzyme activity addition ratio.
Preparing immobilized enzyme by using one of an adsorption method, an embedding method, a crosslinking method or a covalent bond combination method according to the enzyme activity ratio of 25:1 of catalase to glucose oxidase, taking out the immobilized enzyme, washing the immobilized enzyme for 1-5 times by using PBS (phosphate buffer solution) with the pH value of 6.0, absorbing surface moisture by using filter paper, and storing the immobilized enzyme in a refrigerator at the temperature of 4 ℃ for later use;
(3) degradation of glucose solution by immobilized enzyme
10mL of 100g/L glucose solution with pH of 5.0 was prepared, 200. mu.L of free glucose oxidase solution was added, and the mixture was placed in a constant temperature shaking water bath at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ and subjected to shaking reaction at 100 rpm. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
As a result, as shown in FIG 3, the immobilized enzyme had the highest enzyme activity at a temperature of 40 ℃ and the enzyme activity ratio of catalase to glucose oxidase was 25: 1.
Example 4
(1) Dilute glucose oxidase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., dissolving and diluting glucose oxidase solid powder, and preparing into glucose oxidase solution.
(2) Degradation of glucose solution by free enzyme liquid
10mL, 50g/L, 100g/L, 200g/L and 400g/L glucose solutions with the pH value of 3.0-7.0 are respectively prepared, 200 mu L of free glucose oxidase solution is added, and the mixture is placed in a constant-temperature oscillation water bath kettle at 37 ℃ for oscillation reaction under the condition of 100 rpm. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
The results show that free glucose oxidase has the greatest degradation rate at a pH of 5.0.
Example 5
(1) Dilute glucose oxidase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., dissolving and diluting glucose oxidase solid powder, and preparing into glucose oxidase solution.
(2) Degradation of glucose solution by free enzyme liquid
10mL of substrate glucose solutions with concentrations of 50g/L, 100g/L, 200g/L and 400g/L were prepared, 200. mu.L of the enzyme solution was added at pH5.0, and the mixture was placed in a 37 ℃ constant temperature shaking water bath at 100rpm for shaking reaction. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
The results showed that the degradation rate of free glucose oxidase was about 36% at a substrate concentration of 50g/L at the maximum, as shown in FIG. 4.
Example 6
(1) Dilute glucose oxidase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., dissolving and diluting glucose oxidase solid powder, and preparing into glucose oxidase solution.
(2) Degradation of glucose solution by free glucose oxidase liquid
Preparing 200g/L glucose solution with ethanol concentration (v/v%) of 0-40%, adding 200 μ L glucose oxidase solution, and reacting at 37 deg.C and pH of 5.0 under stirring at 100 r/min. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
The results show that the degradation rates of 0-40% (v/v%) alcohol concentration were all about 4.5% when the free glucose oxidase degrades the glucose solution containing ethanol, as shown in FIG. 5.
Example 7
(1) Diluting glucose oxidase, catalase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., respectively dissolving and diluting glucose oxidase and catalase solid powder, and mixing two enzyme solutions according to enzyme activity ratios of catalase to glucose oxidase of 0.5:1, 2.5:1, 5:1, 25:1, 50:1, 100:1 and 150:1 to obtain mixed enzyme solution.
(2) Preparation of immobilized glucose oxidase-catalase
Preparing immobilized enzyme by taking the enzyme activity ratio of catalase to glucose oxidase as the optimal enzyme activity adding proportion when the glucose degradation rate is maximum and adopting an adsorption method, an embedding method, a crosslinking method and a combination method according to the enzyme activity ratio of 25:1, taking out, washing for 1-5 times by using PBS (phosphate buffer solution) with the pH value of 6.0, absorbing surface moisture by using filter paper, and storing in a refrigerator at 4 ℃ for later use;
(3) degradation of glucose solution by free double enzyme and immobilized double enzyme
10mL of 0-400g/L of pH5.0 is prepared, 0-40 (v/v%) of glucose solution containing ethanol is added, 200 μ L of free glucose oxidase solution is added, and the mixture is respectively placed in a constant temperature oscillation water bath kettle at 40 ℃ under the condition of 100-200rpm for oscillation reaction. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
As a result, as shown in FIG. 6, the degradation rate of the immobilized double enzyme was higher than that of the free double enzyme, and the degradation rate of the immobilized double enzyme was not ethanol-containing crystals.
Example 8
(1) Diluting glucose oxidase, catalase
Preparing 50-200mM buffer solution with pH5.0 with sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, etc., respectively dissolving and diluting glucose oxidase and catalase solid powder, and mixing two enzyme solutions according to enzyme activity ratios of catalase to glucose oxidase of 0.5:1, 2.5:1, 5:1, 25:1, 50:1, 100:1 and 150:1 to obtain mixed enzyme solution.
(2) Preparation of immobilized glucose oxidase-catalase
Adopting the optimal enzyme activity ratio of 25:1 of catalase to glucose oxidase, standing and adsorbing for 24 hours to prepare immobilized enzyme, then removing the immobilized enzyme, washing the immobilized enzyme with PBS buffer solution with the pH value of 6.0, sucking surface moisture with filter paper, and storing the immobilized enzyme in a refrigerator at 4 ℃ for later use;
(3) degradation of sugar-containing beverage by immobilized enzyme
Adding the immobilized enzyme into sugar-containing beverage, reacting in a shaker at 37 deg.C and 200r/min for 1 hr, repeatedly using for several times, and detecting the amount of gluconic acid generated in the solution and the reduction of glucose by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
As a result, as shown in FIG. 8, the enzyme activity was less decreased when the immobilized enzyme was repeatedly used less than 4 times. After the application of the enzyme inhibitor for 7 to 8 times, the relative enzyme activity is sharply reduced, and finally, the relative enzyme activity is only remained 22.5 percent.
Example 9
(1) Dilute glucose oxidase
And (3) preparing a PBS buffer solution with the pH value of 5.0 by using sodium dihydrogen phosphate and disodium hydrogen phosphate, and dissolving and diluting the solid glucose oxidase powder to obtain a glucose oxidase solution.
(2) Degradation of glucose solution by free enzyme liquid
10mL of 100g/L glucose solution with pH of 5.0 is prepared, 200 mu L of free glucose oxidase liquid is added, oxygen is continuously pumped, and the solution is respectively placed in a constant-temperature oscillation water bath kettle at 30-60 ℃ under the condition of 200rpm for oscillation reaction. Samples were taken at intervals and the amount of gluconic acid formed in the solution and the amount of glucose reduction was measured by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
The result shows that the conversion rate of converting glucose into saccharic acid can reach 100 percent, and the sweetness is reduced to 0. And the glucose reduction rate can be controlled by adjusting the oxygen input.
Example 10
(1) Diluting glucose oxidase, catalase
Preparing 50-200mM buffer solution with pH5.0 with acetic acid and sodium acetate, or citric acid and sodium citrate, respectively dissolving and diluting solid powders of glucose oxidase and catalase, and mixing the two enzyme solutions according to enzyme activity ratio of catalase to glucose oxidase of 0.5:1, 2.5:1, 5:1, 25:1, 50:1, 100:1 and 150:1 to obtain mixed enzyme solution.
(2) Preparation of immobilized glucose oxidase-catalase
Adopting the optimal enzyme activity ratio of 25:1 of catalase to glucose oxidase, standing and adsorbing for 24 hours to prepare immobilized enzyme, then flushing with PBS buffer solution with the pH value of 6.0, sucking dry surface moisture with filter paper, and storing in a refrigerator at 4 ℃ for later use;
(3) degradation of sugar-containing alcohol beverage by different enzyme adding amount of immobilized enzyme
The immobilized enzyme is prepared by the enzyme activity ratio of catalase to glucose oxidase of 25:1, wherein the GOD content is 701.25U/L, 1402.5U/L, 2805U/L, 5610U/L and 11220U/L.
Adding the immobilized enzyme into sugar-containing alcoholic beverage (ethanol concentration of 10-40 v/v%), reacting in shaker at 37 deg.C and 200r/min for 1 hr, repeating for several times, and detecting the amount of generated gluconic acid and glucose reduction in the solution by High Performance Liquid Chromatography (HPLC).
Detection method of HPLC (AminexHPX-87H Ion Exclusion Column, evaporative light scattering detector and ultraviolet detector used together), mobile phase: 0.0015mol/L dilute sulfuric acid; flow rate of mobile phase: 0.6 mL/min; column temperature: 50 ℃; detection wavelength: 235 nm; sample introduction amount: 20 μ L.
The result shows that the larger the enzyme adding amount is in the preparation of the immobilized enzyme, the larger the degradation rate of the immobilized enzyme on the sugar content in the wine sample is.
Claims (9)
1. A method for reducing sweetness of a sugar-containing beverage using a biological enzyme, comprising:
(1) respectively preparing a glucose oxidase solution and a catalase solution;
(2) mixing a glucose oxidase solution and a catalase solution, and then co-immobilizing to obtain co-immobilized glucose oxidase-catalase; wherein the enzyme activity ratio of catalase to glucose oxidase is 0.5:1-150: 1;
(3) adding the co-immobilized glucose oxidase-catalase into glucose solution or sugar-containing beverage, and oscillating to react at pH of 3.0-7.0 and temperature of 40-60 deg.C.
2. The method for reducing the sweetness of a sugar-containing beverage using a biological enzyme according to claim 1, wherein: and (2) in the step (1), the solvents of the glucose oxidase solution and the catalase solution are buffer solutions.
3. The method for reducing the sweetness of a sugar-containing beverage using a bio-enzyme according to claim 2, wherein: the pH value of the buffer solution is 3.0-7.0, and the concentration is 50-200 mM.
4. The method for reducing the sweetness of a sugar-containing beverage using a bio-enzyme according to claim 2, wherein: the buffer solution is prepared from one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, citric acid and sodium citrate.
5. The method for reducing the sweetness of a sugar-containing beverage using a biological enzyme according to claim 1, wherein: and (3) co-immobilizing in the step (2) by adopting bacterial cellulose.
6. The method for reducing the sweetness of a sugar-containing beverage using a biological enzyme according to claim 5, wherein: the co-immobilization method is one or more of an adsorption method, an embedding method, a crosslinking method and a covalent bond combination method.
7. The method for reducing the sweetness of a sugar-containing beverage using a biological enzyme according to claim 1, wherein: the concentration of the glucose solution in the step (3) is 50-400 g/L; the sugar-containing beverage is a sugar-containing beverage with ethanol concentration of 0-40 v/v%.
8. The method for reducing the sweetness of a sugar-containing beverage using a biological enzyme according to claim 1, wherein: the oscillation reaction in the step (3) is as follows: the process is carried out under the condition of constant temperature oscillation, and the rotating speed is 100-.
9. The method for reducing the sweetness of a sugar-containing beverage using a biological enzyme according to claim 1, wherein: and (4) repeatedly using the co-immobilized glucose oxidase-catalase in the step (3) for 1-8 times.
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