CN110609033A - A method for detecting the enzyme value of sucrose invertase in honey - Google Patents

Abstract

The invention provides a method for detecting the enzyme value of sucrose invertase in honey, which belongs to the technical field of food quality and safety detection. The method for detecting the enzyme value of the sucrose invertase comprises the following steps: (1) extraction of the enzyme liquid in the honey sample; (2) color reaction of the enzyme liquid; (3) calculation of the enzyme value of the sucrose invertase. The detection method is based on the fact that 3,5-dinitrosalicylic acid can be reduced to a brown-red amino compound after co-heating the reducing sugar solution produced by the enzyme solution catalyzing sucrose and the 3,5-dinitrosalicylic acid solution. Schematic established enzymatic value realization of sucrose invertase in honey. The invention adopts the method of ultrafiltration to enrich the enzyme in the honey, and removes the interference of the reducing sugar in the honey, optimizes the amount of the chromogenic agent solution, the pH value of the buffer solution, the reaction temperature and the reaction time in the reaction, and then establishes A detection method for evaluating the value of sucrose invertase in honey has been developed, which has the advantages of simple operation, accuracy and high efficiency.

Description

A method for detecting the enzyme value of sucrose invertase in honey

technical field

The invention belongs to the technical field of food quality and safety detection, and in particular relates to a method for detecting the enzyme value of sucrose invertase in honey.

Background technique

Honey is one of the most important products in the bee industry. Honey contains a variety of enzymes, including amylase, sucrose invertase, glucose oxidase, catalase, acid phospholipase, β-glucosidase, etc. These enzymes exist not only in the honey plant itself, but also in honey. It is mixed with secretions from the salivary glands of bees during the brewing process. The activity of enzymes in honey will gradually decrease due to the degree of heat in honey processing and storage time. Therefore, the freshness and quality of honey can be evaluated by measuring the activity of enzymes in honey. The commonly used evaluation method of honey enzyme value is to measure the amylase enzyme value in honey, but it is more reasonable to comprehensively evaluate honey maturity, processing heat and storage time by measuring the enzyme value of sucrose invertase. This is mainly because, as an indicator of bioactive substances, sucrose invertase is more sensitive to environmental stability than amylase. In addition, from the perspective of the ripening process of honey, its biochemical process is mainly the process of converting sucrose into glucose and fructose. The degree of conversion and the strength of this process depend on the activity of sucrose invertase, not amylase.

At present, more and more countries in the world have gradually added the determination of sucrose invertase enzyme value to the system of evaluating the quality of honey and judging the freshness of honey. There are existing analytical methods for the determination of enzyme value in honey based on titration law. The titration method often uses iron potassium hydride titration method to determine the enzyme value of sucrose invertase, but this method has high requirements for the control of titration operation conditions, and the skilled operation has a great influence on the titration speed, titration end point observation and measurement results. Up to now, my country has not formed a general detection method for the activity of sucrose invertase in honey.

Contents of the invention

In view of this, the object of the present invention is to provide a method for detecting the enzyme value of sucrose invertase in honey, which has the advantages of simple operation, high efficiency and accuracy.

The invention provides a method for detecting the enzyme value of sucrose invertase in honey, comprising the following steps:

1) dissolving the honey and centrifuging to obtain a clarified solution and a precipitate, redissolving the precipitate to obtain a reconstituted solution, performing ultrafiltration on the clarified solution, diluting the obtained concentrated solution and then ultrafiltering again, and then ultrafiltering and concentrating the obtained concentrate solution and the reconstitution solution are mixed to a constant volume to obtain an enzyme solution; the solution for dissolving, redissolving or diluting is a buffer solution with a pH value of 4.0 to 6.8;

2) Measure 2mL of the enzyme solution and place it in two test tubes. Test tube 1 is the experimental group, and test tube 2 is the control group. Add 1 mL of inactivation solution to test tube 2 for inactivation. Put test tube 1, test tube 2 and sucrose Put the solution in a water bath at 30°C to 50°C for 10 minutes, draw 2 mL of sucrose solution into test tube 1 and test tube 2, react at 30°C to 50°C for 0.5 to 3.0 h, add 1 mL of the inactivated solution to test tube 1 Liquid termination reaction;

3) Draw 1-3mL of the same volume of reaction solution from test tube 1 and test tube 2 and transfer to two stoppered colorimetric tubes respectively, then add 0.25-3.5mL of color developer solution, react in boiling water bath for 5min, cool down, Each stoppered colorimetric tube solution is fixed to the scale with water, and the absorbance OD value is measured at a wavelength of 540nm by the solutions in the two obtained stoppered colorimetric tubes, and the solution is adjusted to the scale without the reaction solution. The chromogen solution is blanked and zeroed;

4) draw reducing sugar standard curve, calculate sucrose invertase enzyme value according to formula (2);

In the formula (2): 5 represents the enzyme reaction system, and the unit is mL;

2 means take 2mL enzyme solution to participate in the reaction;

50 represents the total volume of the enzyme solution, in mL;

X represents the mass of the honey sample, in g;

1 means the reaction time is 1h;

The calculation method of the amount of reducing sugar in the sample is calculated according to formula (1);

In formula (1): Y represents the volume of reaction solution, and the unit is mL;

The sucrose invertase enzyme value is the number of milligrams of sucrose invertase contained in 1 g of honey that can hydrolyze sucrose to release reducing sugar for 1 hour under the experimental conditions of 40° C. and pH 6.0 buffer solution.

Preferably, the molecular weight cut-off of the filter membrane for ultrafiltration in step 1) is 3K-50K.

Preferably, during the ultrafiltration described in step 1), the centrifugation speed is 2500-6000g, the centrifugation time is 10-60min, and the centrifugation temperature is 4°C.

Preferably, the buffer solution with a pH value of 4.0 to 6.8 in step 1) includes a 0.1mol/L acetate buffer solution;

The pH value of the acetate buffer is 6.

Preferably, the inactivation solution in step 2) is NaOH solution; the molar concentration of the NaOH solution is 1mol/L.

Preferably, the reaction temperature in step 2) is 40° C., and the reaction time is 1.0 h.

Preferably, the mass concentration of the sucrose solution in step 2) is 5.0%.

Preferably, the preparation method of the developer solution described in step 3) is as follows:

Dissolve 6.9g of crystalline phenol with 15.2mL of NaOH solution with a concentration of 100g/L, dilute to 69mL with water, and then add 6.9g of sodium bisulfite to dissolve to obtain solution A;

Dissolve 25g of potassium sodium tartrate in 300mL of NaOH solution with a concentration of 100g/L, and then add 880mL of 3,5-dinitrosalicylic acid solution with a concentration of 10g/L to obtain solution B;

Mixing the solution A and the solution B to obtain a developer solution;

The order of preparation of the solution A and the solution B is not limited.

Preferably, the added volume of the developer solution in step 3) is 1.5 mL.

Preferably, the honey includes raw honey or commercial honey.

Compared with the prior art, the present invention has the following advantages:

1) Spectrophotometry has the advantages of convenient operation, high sensitivity, and accurate measurement results. It is the current international general method for the determination of amylase in honey. The present invention is based on the principle that 3,5-dinitrosalicylic acid can be reduced to brown-red amino compound after co-heating 3,5-dinitrosalicylic acid solution and reducing sugar solution, and establishes a kind of sucrose in honey The spectrophotometric detection method of invertase enzyme value has simple operation and good stability.

2) The present invention enriches the enzyme in the honey by ultrafiltration, removes the interference of reducing sugar in the honey, and strictly limits the amount of developer solution, buffer pH value, reaction temperature and reaction time in the reaction , which has the characteristics of simple sample processing, high detection accuracy and good stability.

3) So far, my country has not yet formed a general detection method for sucrose invertase enzyme value in honey. The detection method for sucrose invertase enzyme value in the present invention is easy to operate, accurate and efficient, suitable for the detection of all honey samples, and convenient for large-scale Scale application.

4) The detection method provided by the present invention provides a new train of thought for the determination of the sucrose invertase enzyme value in honey, and can be used as one of the comprehensive indicators for evaluating the freshness and quality of commercially available commercial honey, which can improve my country's honey quality evaluation system and improve It is of positive significance to improve the market competitiveness of my country's honey products and promote the vigorous development of my country's beekeeping industry.

Description of drawings

Fig. 1 is a line graph of the influence of different reaction temperatures on the enzyme value of sucrose invertase.

Detailed ways

The invention provides a method for detecting the enzyme value of sucrose invertase in honey, comprising the following steps:

1) dissolving the honey and centrifuging to obtain a clarified solution and a precipitate, redissolving the precipitate to obtain a reconstituted solution, performing ultrafiltration on the clarified solution, diluting the obtained concentrated solution and then ultrafiltering again, and then ultrafiltering and concentrating the obtained concentrate solution and the reconstitution solution are mixed to a constant volume to obtain an enzyme solution; the solution for dissolving, redissolving or diluting is a buffer solution with a pH value of 4.0 to 6.8;

2) Measure 2mL of the enzyme solution and place it in two test tubes. Test tube 1 is the experimental group, and test tube 2 is the control group. Add 1 mL of inactivation solution to test tube 2 for inactivation. Put test tube 1, test tube 2 and sucrose Put the solution in a water bath at 30°C to 50°C for 10 minutes, draw 2 mL of sucrose solution into test tube 1 and test tube 2, react at 30°C to 50°C for 0.5 to 3.0 h, add 1 mL of the inactivated solution to test tube 1 Liquid termination reaction;

3) Draw 1-3mL of the same volume of reaction solution from test tube 1 and test tube 2 and transfer to two stoppered colorimetric tubes respectively, then add 0.25-3.5mL of color developer solution, react in boiling water bath for 5min, cool down, Each stoppered colorimetric tube solution is fixed to the scale with water, and the absorbance OD value is measured at a wavelength of 540nm by the solutions in the two obtained stoppered colorimetric tubes, and the solution is adjusted to the scale without the reaction solution. The chromogen solution is blanked and zeroed;

4) draw reducing sugar standard curve, calculate sucrose invertase enzyme value according to formula (2);

In the formula (2): 5 represents the enzyme reaction system, and the unit is mL;

2 means take 2mL enzyme solution to participate in the reaction;

50 represents the total volume of the enzyme solution, in mL;

X represents the mass of the honey sample, in g;

1 means the reaction time is 1h;

The calculation method of the amount of reducing sugar in the sample is calculated according to formula (1);

In formula (1): Y represents the volume of reaction solution, and the unit is mL;

The sucrose invertase enzyme value is the number of milligrams of sucrose invertase contained in 1 g of honey that can hydrolyze sucrose to release reducing sugar for 1 hour under the experimental conditions of 40° C. and pH 6.0 buffer solution.

In the present invention, honey is dissolved and then centrifuged to obtain clarified liquid and precipitate, and the precipitate is redissolved to obtain a reconstituted solution, the clarified liquid is subjected to ultrafiltration, the obtained concentrated liquid is diluted and then ultrafiltered again, and the obtained concentrated liquid is again ultrafiltered and concentrated The solution is mixed with the reconstituted solution to obtain an enzyme solution; the solution for dissolving, redissolving or diluting is a buffer solution with a pH value of 4.0-6.8.

In the present invention, the honey preferably includes raw honey or commercial honey. The detection method is suitable for honeys from various sources, such as acacia honey, linden honey or sweet-scented osmanthus honey and the like.

In the present invention, the solution for dissolving is preferably an acetate buffer solution with a pH value of 4.0 to 6.8; the concentration of the acetate buffer solution is preferably 0.1mol/L; the pH value of the acetate buffer solution is 6 is preferred. The temperature of the centrifugation is preferably 4° C., and the rotation speed of the centrifugation is preferably 6000-12000 rpm, more preferably 9000 rpm. The centrifugation time is preferably 5-20 min, more preferably 15 min. The purpose of the centrifugation is to remove impurities in the honey, such as pollen.

In the present invention, the solution for reconstitution or dilution is preferably an acetate buffer solution with a pH value of 4.0 to 6.8; the concentration of the acetate buffer solution is preferably 0.1mol/L; the acetate buffer solution The pH value is preferably 6. The molecular weight cut-off of the filter membrane for ultrafiltration is preferably 3K-50K, more preferably 10K. The present invention has no special limitation on the type of centrifuge tube used in ultrafiltration, and the centrifuge tube for ultrafiltration well known in the art can be used. In the embodiment of the present invention, the ultrafiltration centrifuge tube is an Amicon Ultra-15 centrifugal ultrafiltration tube. During the ultrafiltration, the centrifugation speed is preferably 2500-6000 g, more preferably 5000 g; the centrifugation time is preferably 10-60 min, more preferably 30 min. The temperature of centrifugation is preferably 4°C. The purpose of the two ultrafiltrations is to effectively remove the sugar contained in the honey sample. The dilution factor is not particularly limited, and any dilution method known in the art can be used. The volume of the re-ultrafiltration concentrated solution mixed with the complex solution to a constant volume is preferably 50 mL.

After obtaining the enzyme liquid, the present invention measures 2 mL of the enzyme liquid respectively and places them in two test tubes. Test tube 1 is the experimental group, and test tube 2 is the control group. Add 1 mL of inactivation solution to test tube 2 for inactivation. , test tube 2 and sucrose solution in a water bath at 30°C to 50°C for 10 minutes, pipette 2 mL of sucrose solution into test tube 1 and test tube 2, react at 30°C to 50°C for 0.5 to 3.0 hours, and add to test tube 1 1 mL of the inactivation solution was used to terminate the reaction.

In the present invention, the temperature of the water bath is preferably 40°C. The temperature of the reaction is preferably 40° C., and the reaction time is preferably 1.0 h. The reaction is that the sucrose invertase contained in the enzyme liquid catalyzes the sucrose to produce reducing sugar under suitable conditions.

In the present invention, the inactivation solution is preferably NaOH solution; the molar concentration of the NaOH solution is preferably 1 mol/L. The mass concentration of the sucrose solution is preferably 5.0%.

After the reaction is over, the present invention draws 1-3 mL of the same volume of reaction solution from test tube 1 and test tube 2, and respectively transfers two stoppered colorimetric tubes, then adds 0.25-3.5 mL of chromogenic agent solution, and reacts in a boiling water bath. 5min, cool down, dilute the solution of each stoppered colorimetric tube with water to the scale, measure the absorbance OD value of the solution in the two obtained two stoppered colorimetric tubes at a wavelength of 540nm, use the solution that is dilute to the scale without the The developer solution of the above reaction solution was used as a blank to adjust to zero.

In the present invention, the volume of the reaction solution is preferably 2 mL. The added volume of the developer solution is preferably 1.5 mL.

In the present invention, the preparation method of the developer solution is preferably as follows:

Dissolve 6.9g of crystalline phenol with 15.2mL of NaOH solution with a concentration of 100g/L, dilute to 69mL with water, and then add 6.9g of sodium bisulfite to dissolve to obtain solution A;

Dissolve 25g of potassium sodium tartrate in 300mL of NaOH solution with a concentration of 100g/L, and then add 880mL of 3,5-dinitrosalicylic acid solution with a concentration of 10g/L to obtain solution B;

The solution A and the solution B are mixed to obtain a developer solution; the preparation order of the solution A and the solution B is not limited.

In the present invention, reacting in a boiling water bath for 5 minutes utilizes the reducing sugar produced to react with the 3,5-dinitrosalicylic acid solution, and the 3,5-dinitrosalicylic acid can be reduced to a brown-red amino compound, The enzyme value of sucrose invertase was quantitatively detected by spectrophotometry. The cooling is preferably cooling to room temperature (23-28° C.).

After obtaining the OD values of test tube 1 and test tube 2, the present invention calculates the sucrose invertase enzyme value according to the formula (2) according to the drawn reducing sugar standard curve.

In the present invention, there is no special limitation on the method for drawing the standard curve of reducing sugar, and the scheme for drawing the standard curve of reducing sugar well known in the art can be used. Bring the OD values of test tube 1 and test tube 2 into the formula (1), calculate the amount of reducing sugar in the sample according to the amount of the corresponding reducing sugar in the reducing sugar standard curve equation, and use the calculated amount of reducing sugar in the sample Bring it into Publication (2) to get the sucrose invertase enzyme value.

A method for detecting the enzyme value of sucrose invertase in honey provided by the present invention will be described in detail below in conjunction with the examples, but they cannot be interpreted as limiting the protection scope of the present invention.

Example 1

Weigh 2g of acacia honey into a beaker, add 10mL of acetate buffer (0.1mol/L) with a pH value of 6.0 to fully dissolve, and centrifuge at 4°C. After centrifugation, the precipitate was redissolved with acetate buffer, and the clarified solution was transferred to a 10KAmiconUltra-15 centrifugal ultrafiltration tube and centrifuged at 5000×g for 30 min at 4°C to recover the concentrated solution. Add acetate buffer to the concentrate and repeat the centrifugation step. The concentrated solution obtained again, together with the precipitate reconstituted solution in the above step, was fixed to a 50 mL volumetric flask with acetate buffer solution, that is, the enzyme solution.

Take 2.0 mL of the obtained enzyme solution and place them in two test tubes, of which test tube 1 is the experimental group and test tube 2 is the control group. Add 1 mL of NaOH solution (1 mol/L) to test tube 2. Put test tube 1, test tube 2 and 5% sucrose solution together in a water bath and preheat at 40°C for 10 min. Pipette 2 mL of 5% sucrose solution into test tube 1 and test tube 2 and start timing, react in a water bath at 40°C for 1.0 h, and immediately add 1 mL of NaOH solution (1mol/L) to test tube 1 to terminate the reaction time reaction. Draw 2mL of the same volume of reaction solution from test tube 1 and test tube 2 and transfer it to a 25mL stoppered colorimetric tube, add 1.5mL of chromogenic reagent solution to it, place the stoppered colorimetric tube in a boiling water bath, and immediately after 5min Take it out and cool it to room temperature in cold water, and dilute the solution of each stoppered colorimetric tube to the mark with water. Take the solution in each stoppered colorimetric tube under the condition of 540nm wavelength, use the chromogen solution (fixed volume to 25mL stoppered colorimetric tube scale) as blank zero adjustment, measure the absorbance value, and measure the OD value of test tube 1 as 0.259, test tube The 2OD value was 0.095.

Substitute the measured test tube 1OD value and test tube 2OD value into the standard curve equation y=0.5398x-0.0275, and substitute the result into formula (1)

Substitute the result of the calculated amount of reducing sugar in the sample into the formula (2)

The calculated sucrose invertase enzyme value of this brand of acacia honey is 9.49mg/(g.h).

Example 2

The Bee Research Institute of the Chinese Academy of Agricultural Sciences detected the sucrose invertase enzyme value of the linden raw honey collected from the Heilongjiang area.

Operation method:

Weigh 3g of linden raw honey and place it in a beaker, add 10mL of acetate buffer (0.1mol/L) with a pH value of 6.0 to fully dissolve, and centrifuge at 4°C. After centrifugation, the precipitate was redissolved with acetate buffer, and the clarified solution was transferred to a 10KAmiconUltra-15 centrifugal ultrafiltration tube and centrifuged at 5000×g for 30 min at 4°C to recover the concentrated solution. Add acetate buffer to the concentrate and repeat the centrifugation step. The concentrated solution obtained again, together with the precipitate reconstituted solution in the above step, was fixed to a 50 mL volumetric flask with acetate buffer solution, that is, the enzyme solution.

Take 2.0 mL of the obtained enzyme solution and place them in two test tubes, of which test tube 1 is the experimental group and test tube 2 is the control group. Add 1 mL of NaOH solution (1 mol/L) to test tube 2. Put test tube 1, test tube 2 and 5% sucrose solution together in a water bath and preheat at 40°C for 10 min. Pipette 2 mL of 5% sucrose solution into test tube 1 and test tube 2 and start timing, react in a water bath at 40°C for 1.0 h, and immediately add 1 mL of NaOH solution (1mol/L) to test tube 1 to terminate the reaction time reaction. Draw 1mL of the same volume of reaction solution from test tube 1 and test tube 2 and transfer it to a 25mL stoppered colorimetric tube, add 1.5mL of chromogenic reagent solution to it, place the stoppered colorimetric tube in a boiling water bath, and immediately after 5min Take it out and cool it to room temperature in cold water, and dilute the solution of each stoppered colorimetric tube to the mark with water. Take the solution in each stoppered colorimetric tube under the condition of 540nm wavelength, use the chromogenic agent solution (fixed volume to 25mL stoppered colorimetric tube scale) as blank zero adjustment, measure the absorbance value, and measure the OD value of test tube 1 as 0.474, test tube The 2OD value is 0.078, and the measured test tube 1OD value and test tube 2OD value are substituted into the standard curve equation

y=0.5398x-0.0275, and substitute the result into formula (1), the result is as follows:

Substituting the result of the amount of reducing sugar in the sample into formula (2), the result is as follows:

The calculated sucrose invertase enzyme value of this brand of acacia honey is 30.58mg/(g.h).

Example 3

According to the detection method of Example 1, the sucrose invertase enzyme values of 20 acacia honeys and 20 linden honeys on the market were measured, and the results are shown in Table 1.

Table 1 Determination of sucrose invertase enzyme value in 40 brands of honey

The results of 20 brand commercially available acacia honey samples and 20 brand commercially available linden honey samples detected by the sucrose invertase enzyme value assay method in honey provided by the invention. Among them, the sucrose invertase enzyme values of 20 brands of commercially available acacia honey samples were between 4.53 and 42.19 mg/(g.h), with an average value of 13.50 mg/(g.h), and the sucrose invertase enzyme values of 4 brand samples were greater than 20mg/(g.h), the sucrose invertase enzyme value of 7 brand samples is below 8mg/(g.h); the sucrose invertase enzyme value of 20 brands of commercially available linden tree honey samples is between 7.42-31.78mg/(g.h) During the period, the average value was 21.29mg/(g.h), the sucrose invertase enzyme value of 11 brand samples was greater than 20mg/(g.h), and only 4 brand samples had a sucrose invertase enzyme value below 18mg/(g.h).

Example 4

The Bee Research Institute of the Chinese Academy of Agricultural Sciences conducted a study on the effect of reaction temperature on the enzyme value of sucrose invertase in acacia raw honey collected from Shaanxi.

Operation method:

Weigh 2g of raw acacia honey into a beaker, add 10mL of acetate buffer (0.1mol/L) with a pH value of 6.0 to fully dissolve, and centrifuge at 4°C. After centrifugation, the precipitate was redissolved with acetate buffer, and the clarified solution was transferred to a 10KAmiconUltra-15 centrifugal ultrafiltration tube and centrifuged at 5000×g for 30 min at 4°C to recover the concentrated solution. Add acetate buffer to the concentrate and repeat the centrifugation step. The concentrated solution obtained again, together with the precipitate reconstituted solution in the above step, was fixed to a 50 mL volumetric flask with acetate buffer solution, that is, the enzyme solution.

Put test tube 1, test tube 2 and 5% sucrose solution together in a water bath to preheat for 10 minutes, draw 2 mL of 5% sucrose solution and add them to test tube 1 and test tube 2 for 1 hour. The reaction temperature is controlled at 30°C, 35°C, 40°C, 45°C and 50°C, keeping other experimental conditions unchanged (buffer pH value 6.0, chromogen solution 1.5mL, reaction time 1h), the sucrose invertase enzyme value of raw acacia honey at different reaction temperatures was tested. Determination. Figure 1 was obtained by plotting the enzyme value of sucrose invertase/reaction temperature.

It can be seen from Figure 1 that when the reaction temperature is 30-40°C, it increases gradually with the increase of the reaction temperature, and reaches the maximum at 40°C. When the temperature exceeds 40°C, the enzyme value of sucrose invertase begins to decrease, and when it exceeds 50°C, the enzyme value decreases by more than 50%, which means that too high temperature will cause the activity of sucrose invertase to be weakened or even lost. Therefore, in the detection method provided by the present invention, the reaction temperature is preferably controlled at 30-45°C.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. A method for detecting the enzyme value of sucrose invertase in honey is characterized by comprising the following steps:

1) dissolving honey, centrifuging to obtain clear liquid and precipitate, redissolving the precipitate to obtain a redissolution, performing ultrafiltration on the clear liquid, diluting the obtained concentrated solution, performing ultrafiltration again, mixing the obtained concentrate subjected to ultrafiltration again with the redissolution to a constant volume to obtain an enzyme solution; the solution for dissolving, redissolving or diluting is a buffer solution with the pH value of 4.0-6.8;

2) respectively measuring 2mL of the enzyme solution, placing the enzyme solution into two test tubes, taking the test tube 1 as an experimental group and the test tube 2 as a control group, adding 1mL of inactivation solution into the test tube 2 for inactivation, carrying out water bath on the test tube 1, the test tube 2 and the sucrose solution at 30-50 ℃ for 10min, respectively absorbing 2mL of the sucrose solution, adding the sucrose solution into the test tube 1 and the test tube 2, reacting at 30-50 ℃ for 0.5-3.0 h, and adding 1mL of the inactivation solution into the test tube 1 to terminate the reaction;

3) sucking 1-3 mL of reaction liquid with the same volume from a test tube 1 and a test tube 2, respectively displacing the reaction liquid with the same volume from the test tube 1 to the test tube 2, respectively adding 0.25-3.5 mL of color development reagent solution, reacting in a boiling water bath for 5min, cooling, fixing the volume of each color comparison tube solution with a plug to a scale with water, measuring the absorbance OD value of the obtained solutions in the two color comparison tubes with the plug at a wavelength of 540nm, and carrying out blank zeroing by using the color development reagent solution which is fixed to the scale and does not contain the reaction liquid;

4) drawing a reducing sugar standard curve, and calculating the enzyme value of the sucrose invertase according to a formula (2);

in formula (2): 5 represents an enzyme reaction system with the unit of mL;

2, taking 2mL of enzyme solution to participate in the reaction;

50 represents the total volume of the enzyme solution, and the unit is mL;

x represents the mass of the honey sample and the unit is g;

1 represents a reaction time of 1 h;

the method for calculating the amount of reducing sugar in the sample is calculated according to the formula (1);

in formula (1): y represents the volume of the reaction solution in mL;

the sucrose invertase enzyme value is the number of milligrams of sucrose invertase contained in 1g of honey which can hydrolyze sucrose to release reducing sugar in 1 hour under the experimental conditions of 40 ℃ and pH 6.0 buffer solution.

2. The detection method according to claim 1, wherein the ultrafiltration membrane of step 1) has a molecular weight cut-off of 3 to 50K.

3. The detection method according to claim 1, wherein in the step 1), during the ultrafiltration, the centrifugal rotation speed is 2500-6000 g, the centrifugal time is 10-60 min, and the centrifugal temperature is 4 ℃.

4. The detection method according to claim 1, wherein the buffer solution with the pH value of 4.0-6.8 in the step 1) comprises 0.1mol/L acetate buffer solution;

the pH value of the acetate buffer solution is 6.

5. The detection method according to claim 1, wherein the inactivation solution in step 2) is a NaOH solution; the molar concentration of the NaOH solution is 1 mol/L.

6. The detection method according to claim 1, wherein the reaction temperature in step 2) is 40 ℃ and the reaction time is 1.0 h.

7. The detection method according to claim 1, wherein the sucrose solution in step 2) has a mass concentration of 5.0%.

8. The detection method according to claim 1, wherein the developer solution in step 3) is prepared as follows:

dissolving 6.9g of crystallized phenol by using 15.2mL of NaOH solution with the concentration of 100g/L, diluting the solution to 69mL by using water, and then adding 6.9g of sodium bisulfite for dissolving to obtain solution A;

dissolving 25g of potassium sodium tartrate by 300mL of NaOH solution with the concentration of 100g/L, and then adding 880mL of 3, 5-dinitrosalicylic acid solution with the concentration of 10g/L to obtain solution B;

mixing the solution A and the solution B to obtain a color developing agent solution;

the order of preparation of the solution A and the solution B is not limited.

9. The detection method according to claim 1, wherein the addition volume of the developer solution in step 3) is 1.5 mL.

10. The assay method of any one of claims 1 to 9, wherein the honey comprises raw honey or commercial honey.