CN110609033A - Method for detecting enzyme value of sucrose invertase in honey - Google Patents
Method for detecting enzyme value of sucrose invertase in honey Download PDFInfo
- Publication number
- CN110609033A CN110609033A CN201911052429.4A CN201911052429A CN110609033A CN 110609033 A CN110609033 A CN 110609033A CN 201911052429 A CN201911052429 A CN 201911052429A CN 110609033 A CN110609033 A CN 110609033A
- Authority
- CN
- China
- Prior art keywords
- solution
- honey
- enzyme
- test tube
- value
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a method for detecting the enzyme value of sucrose invertase in honey, belonging to the technical field of food quality safety detection. The method for detecting the enzyme value of the sucrose invertase comprises the following steps: (1) extracting enzyme liquid in a honey sample; (2) carrying out color development reaction on the enzyme solution; (3) and (4) calculating the enzyme value of the sucrose invertase. The detection method is realized by a sucrose invertase enzyme value in honey established on the basis of the principle that 3, 5-dinitrosalicylic acid can be reduced into brownish red amino compounds after a reducing sugar solution generated by catalyzing sucrose with an enzyme solution and the 3, 5-dinitrosalicylic acid solution are heated together. The method adopts an ultrafiltration method to enrich enzymes in honey, removes interference of reducing sugar in the honey, optimizes the dosage of a color developing agent solution, the pH value of a buffer solution, the reaction temperature and the reaction time in the reaction, further establishes a detection method for evaluating the enzyme value of sucrose invertase in the honey, and has the advantages of simple operation, accuracy, high efficiency and the like.
Description
Technical Field
The invention belongs to the technical field of food quality safety detection, and particularly relates to a method for detecting a sucrose invertase enzyme value in honey.
Background
Honey is one of the most important products in the bee industry. The honey contains a variety of enzymes including amylase, sucrose invertase, glucose oxidase, catalase, acid phospholipase, beta-glucosidase, etc., which are both present in the honey source itself and incorporated by the honey salivary gland secretions during the brewing process. The activity of enzymes present in honey is gradually reduced by the influence of the heat level and storage time during the honey processing, and thus, the freshness and quality of honey can be evaluated by measuring the activity of enzymes in honey. A common evaluation method for the enzyme value of honey is to measure the amylase value in honey, but the comprehensive evaluation of the maturity of honey, the heating condition of the processing process and the storage time is more reasonable by measuring the enzyme value of sucrose invertase. This is mainly because, as an indicator of biological activity, sucrose invertase is more sensitive to environmental stability than amylase. In addition, from the mature process of honey, the biochemical process is mainly the process of converting sucrose into glucose and fructose, and the degree and strength of the conversion of the process depend on the activity of sucrose invertase, not amylase.
At present, more and more countries in the world gradually add the measurement of the enzyme value of sucrose invertase to a system for evaluating the quality and judging the freshness of honey, and the analysis method for measuring the enzyme value in honey mainly adopts a titration method. The titration method usually adopts a potassium iron hydride titration method to determine the enzyme value of the sucrose invertase, but the method has high requirements on the control of titration operating conditions, and whether the operation is skilled or not has great influence on the titration speed, the titration endpoint observation and the determination result. Up to now, no general detection method for the activity of sucrose invertase in honey has been established in China.
Disclosure of Invention
In view of the above, the invention aims to provide a method for detecting the enzyme value of sucrose invertase in honey, which has the advantages of simple operation, high efficiency, accuracy and the like.
The invention provides a method for detecting the enzyme value of sucrose invertase in honey, which comprises 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.
Preferably, the molecular weight cut-off of the ultrafiltration membrane in the step 1) is 3K to 50K.
Preferably, during the ultrafiltration in the step 1), the centrifugal rotating speed is 2500-6000 g, the centrifugal time is 10-60 min, and the centrifugal temperature is 4 ℃.
Preferably, 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.
Preferably, the inactivation solution in the step 2) is a NaOH solution; the molar concentration of the NaOH solution is 1 mol/L.
Preferably, the temperature of the reaction in the step 2) is 40 ℃, and the time of the reaction is 1.0 h.
Preferably, the mass concentration of the sucrose solution in the step 2) is 5.0%.
Preferably, the preparation method of the developer solution in the step 3) is 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.
Preferably, the addition volume of the developer solution in step 3) is 1.5 mL.
Preferably, the honey comprises raw honey or commercial honey.
Compared with the prior art, the invention has the following advantages:
1) the spectrophotometry has the advantages of convenient operation, high sensitivity, accurate measurement result and the like, and is the current international universal method for measuring the amylase value in honey. The spectrophotometric detection method for the enzyme value of the sucrose invertase in the honey is established based on the principle that 3, 5-dinitrosalicylic acid can be reduced into a brownish red amino compound after the 3, 5-dinitrosalicylic acid solution and the reducing sugar solution are heated together, and is simple to operate and good in stability.
2) The invention adopts the ultrafiltration method to enrich the enzyme in the honey, removes the interference of reducing sugar in the honey, strictly limits the dosage of the color developing agent solution, the pH value of the buffer solution, the reaction temperature and the reaction time in the reaction, and has the characteristics of simple sample treatment, high detection accuracy and good stability.
3) So far, China has not formed a general detection method for the enzyme value of the sucrose invertase in honey, and the detection method for the enzyme value of the sucrose invertase in the invention is easy to operate, accurate and efficient, is suitable for detecting all honey samples, and is convenient for large-scale popularization and application.
4) The detection method provided by the invention provides a new idea for measuring the value of sucrose invertase in honey, can be used as one of comprehensive indexes for evaluating the freshness and quality of commercial honey, and has positive significance for perfecting the honey quality evaluation system in China, improving the market competitiveness of honey products in China and promoting the vigorous development of the bee-keeping industry in China.
Drawings
FIG. 1 is a line graph showing the effect of different reaction temperatures on the enzyme value of sucrose invertase.
Detailed Description
The invention provides a method for detecting the enzyme value of sucrose invertase in honey, which comprises 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.
Dissolving honey, centrifuging to obtain clear solution and sediment, redissolving the sediment to obtain composite solution, performing ultrafiltration on the clear solution, diluting the obtained concentrated solution, performing ultrafiltration again, mixing the obtained ultrafiltered concentrated solution and the composite solution to a constant volume to obtain enzyme solution; the solution for dissolving, redissolving or diluting is a buffer solution with the pH value of 4.0-6.8.
In the present invention, the honey preferably comprises honey raw honey or commercial honey. The detection method is suitable for honey from various sources, such as acacia honey, linden honey or osmanthus honey.
In the invention, the solution for dissolving is preferably an acetate buffer solution with the pH value of 4.0-6.8; the concentration of the acetate buffer solution is preferably 0.1 mol/L; the pH of the acetate buffer is preferably 6. The centrifugal temperature is preferably 4 ℃, and the centrifugal rotating speed is preferably 6000-12000 rpm, and more preferably 9000 rpm. The time for centrifugation is preferably 5-20 min, and more preferably 15 min. The purpose of the centrifugation is to remove impurities in the honey, such as pollen.
In the invention, the solution for redissolution or dilution is preferably an acetate buffer solution with the pH value of 4.0-6.8; the concentration of the acetate buffer solution is preferably 0.1 mol/L; the pH of the acetate buffer is preferably 6. The cut-off molecular weight of the ultrafiltration membrane is preferably 3K to 50K, and more preferably 10K. The type of the centrifugal tube used in the ultrafiltration is not particularly limited in the present invention, and an ultrafiltration centrifugal tube known in the art may be used. In the embodiment of the invention, the ultrafiltration centrifugal tube is an Amicon Ultra-15 centrifugal ultrafiltration tube. During ultrafiltration, the centrifugal rotating speed is preferably 2500-6000 g, and more preferably 5000 g; the time for centrifugation is preferably 10-60 min, and more preferably 30 min. The temperature of centrifugation is preferably 4 ℃. The purpose of the two ultrafiltration steps is to effectively remove the sugars contained in the honey sample. The dilution ratio is not particularly limited, and a dilution method well known in the art may be used. The volume of the mixed volume of the re-ultrafiltration concentrated solution and the redissolution is preferably 50 mL.
After obtaining the enzyme solution, respectively weighing 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, placing the test tube 1, the test tube 2 and the sucrose solution in a water bath at 30-50 ℃ for 10min, respectively sucking 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.
In the present invention, the temperature of the water bath is preferably 40 ℃. The temperature of the reaction is preferably 40 ℃ and the time of the reaction is preferably 1.0 h. The reaction is that sucrose invertase contained in the enzyme solution catalyzes sucrose to produce reducing sugar under appropriate conditions.
In the present invention, the inactivation solution is preferably a 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 finished, 1-3 mL of reaction solution with the same volume is sucked from a test tube 1 and a test tube 2 and respectively displaced from two colorimetric tubes with plugs, 0.25-3.5 mL of color development reagent solution is respectively added, the reaction is carried out for 5min in a boiling water bath, the cooling is carried out, the solution of each colorimetric tube with plugs is subjected to constant volume to scale with water, the OD value of absorbance of the obtained solution in the two colorimetric tubes with plugs is measured at the wavelength of 540nm, and blank zeroing is carried out by the color development reagent solution which is not contained in the reaction solution and is subjected to constant volume to the scale.
In the present invention, the volume of the reaction solution is preferably 2 mL. The addition volume of the developer solution is preferably 1.5 mL.
In the present invention, the developer solution is preferably 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.
In the invention, the reaction is carried out for 5min in a boiling water bath by utilizing the action of the generated reducing sugar and the 3, 5-dinitrosalicylic acid solution, the 3, 5-dinitrosalicylic acid can be reduced into a brownish red amino compound, and the enzyme value of the sucrose invertase is quantitatively detected by adopting a spectrophotometry. The cooling is preferably carried out to room temperature (23-28 ℃).
After obtaining the OD values of the test tube 1 and the test tube 2, the invention calculates the enzyme value of the sucrose invertase according to the formula (2) according to the drawn standard reducing sugar curve.
The method for drawing the standard curve of reducing sugar is not particularly limited in the invention, and a scheme for drawing the standard curve of reducing sugar, which is well known in the field, can be adopted. The obtained OD values of the test tube 1 and the test tube 2 are substituted into the formula (1), the amount of reducing sugar in the sample is calculated according to the amount of corresponding reducing sugar in the standard curve equation of reducing sugar, and the calculated amount of reducing sugar in the sample is substituted into the expression (2) to obtain the enzyme value of the sucrose invertase.
The method for detecting the value of sucrose invertase in honey according to the present invention is described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
2g of acacia honey was weighed out and placed in a beaker, and 10ml of acetate buffer (0.1mol/L) with pH 6.0 was added to dissolve sufficiently, followed by centrifugation at 4 ℃. After centrifugation, the precipitate was redissolved with acetate buffer, the supernatant was transferred to a 10KAmicon ultra-15 centrifugal ultrafiltration tube and centrifuged at 5000 Xg for 30min at 4 ℃ to recover the concentrate. Acetate buffer was added to the concentrate and the centrifugation step was repeated. And (4) diluting the concentrated solution obtained again and the precipitate redissolved solution I in the step to a 50mL volumetric flask by using acetate buffer solution, namely enzyme solution.
2.0mL of the obtained enzyme solution was taken and placed in two test tubes, wherein test tube 1 was the experimental group and test tube 2 was the control group. To tube 2 was added 1mL of NaOH solution (1 mol/L). Tube 1, tube 2 and 5% sucrose solution were placed together in a water bath and preheated at 40 ℃ for 10 min. 2mL of 5% sucrose solution was added to test tube 1 and test tube 2, and the reaction was started at 40 ℃ for 1.0h in a water bath, and 1mL of NaOH solution (1mol/L) was added to test tube 1 immediately after the reaction time was reached to terminate the reaction. Sucking 2mL of reaction liquid with the same volume from the test tube 1 and the test tube 2 respectively, displacing the reaction liquid into a 25mL colorimetric tube with a plug, adding 1.5mL of color developing agent solution into the colorimetric tube with the plug, placing the colorimetric tube with the plug into a boiling water bath, immediately taking out the colorimetric tube after 5min, cooling the colorimetric tube with the plug in cold water to room temperature, and adding water to the solution of each colorimetric tube with the plug to a constant volume to be calibrated. Taking the solution in each cuvette with a stopper, carrying out blank zeroing on a color developing agent solution (with a constant volume of 25mL to the scale of the cuvette with the stopper) under the wavelength condition of 540nm, measuring the absorbance value, and measuring the OD value of the test tube 1 to be 0.259 and the OD value of the test tube 2 to be 0.095.
Substituting the measured OD value of test tube 1 and OD value of test tube 2 into the standard curve equation y of 0.5398x-0.0275, and substituting the result into formula (1)
Substituting the result of the amount of reducing sugar in the sample obtained by the calculation into formula (2)
The sucrose invertase enzyme value of the acacia honey of the brand was calculated to be 9.49 mg/(g.h).
Example 2
The bee research institute of Chinese academy of agricultural sciences detects the value of sucrose invertase in raw linden tree honey collected from Heilongjiang.
The operation method comprises the following steps:
weighing 3g of linden raw honey, placing in a beaker, adding 10ml of acetate buffer solution (0.1mol/L) with the pH value of 6.0, fully dissolving, and centrifuging at 4 ℃. After centrifugation, the precipitate was redissolved with acetate buffer, the supernatant was transferred to a 10KAmicon ultra-15 centrifugal ultrafiltration tube and centrifuged at 5000 Xg for 30min at 4 ℃ to recover the concentrate. Acetate buffer was added to the concentrate and the centrifugation step was repeated. And (4) diluting the concentrated solution obtained again and the precipitate redissolved solution I in the step to a 50mL volumetric flask by using acetate buffer solution, namely enzyme solution.
2.0mL of the obtained enzyme solution was taken and placed in two test tubes, wherein test tube 1 was the experimental group and test tube 2 was the control group. To tube 2 was added 1mL of NaOH solution (1 mol/L). Tube 1, tube 2 and 5% sucrose solution were placed together in a water bath and preheated at 40 ℃ for 10 min. 2mL of 5% sucrose solution was added to test tube 1 and test tube 2, and the reaction was started at 40 ℃ for 1.0h in a water bath, and 1mL of NaOH solution (1mol/L) was added to test tube 1 immediately after the reaction time was reached to terminate the reaction. Sucking 1mL of reaction solution with the same volume from the test tube 1 and the test tube 2 respectively, displacing the reaction solution with the same volume to a 25mL colorimetric tube with a plug, adding 1.5mL of color developing agent solution into the colorimetric tube with the plug, placing the colorimetric tube with the plug in a boiling water bath, immediately taking out the colorimetric tube after 5min, cooling the colorimetric tube with the plug in cold water to room temperature, and adding water to the solution of each colorimetric tube with the plug to a constant volume to be calibrated. Taking the solution in each colorimetric tube with a plug, carrying out blank zeroing on a developer solution (with a constant volume of 25mL to the scale of the colorimetric tube with a plug) under the wavelength condition of 540nm, measuring the absorbance value, measuring the OD value of the test tube 1 to be 0.474 and the OD value of the test tube 2 to be 0.078, substituting the measured OD values of the test tube 1 and the test tube 2 into a standard curve equation
y is 0.5398x-0.0275, and the results are substituted in formula (1) as follows:
the results of substituting the amount of reducing sugar in the sample into formula (2) are as follows:
the sucrose invertase enzyme value of the acacia honey of the brand is calculated to be 30.58 mg/(g.h).
Example 3
The sucrose invertase enzyme values of 20 acacia honey and 20 linden honey on the market were measured according to the test method of example 1, and the results are shown in Table 1.
Method for detecting enzyme value of sucrose invertase in table 140 brands of honey
The results of 20 brands of commercially available acacia honey samples and 20 brands of commercially available basswood honey samples detected by the method for measuring the enzyme value of the sucrose invertase in honey provided by the invention. Wherein, the sucrose invertase enzyme value of 20 brands of commercially available acacia honey samples is between 4.53 and 42.19mg/(g.h), the average value is 13.50mg/(g.h), the sucrose invertase enzyme value of 4 brands of samples is more than 20mg/(g.h), and the sucrose invertase enzyme value of 7 brands of samples is less than 8 mg/(g.h); the sucrose invertase enzyme value of 20 brand commercially available basswood honey samples is 7.42-31.78 mg/(g.h), the average value is 21.29mg/(g.h), the sucrose invertase enzyme value of 11 brand samples is more than 20mg/(g.h), and the sucrose invertase enzyme value of only 4 brand samples is less than 18 mg/(g.h).
Example 4
The research on the influence of the reaction temperature on the enzyme value of the sucrose invertase is carried out by the bee research institute of Chinese agricultural academy of sciences on the raw acacia honey collected from the region of Shaanxi.
The operation method comprises the following steps:
2g of acacia raw honey was weighed out and placed in a beaker, and 10ml of acetate buffer (0.1mol/L) with pH 6.0 was added to dissolve sufficiently, followed by centrifugation at 4 ℃. After centrifugation, the precipitate was redissolved with acetate buffer, the supernatant was transferred to a 10KAmicon ultra-15 centrifugal ultrafiltration tube and centrifuged at 5000 Xg for 30min at 4 ℃ to recover the concentrate. Acetate buffer was added to the concentrate and the centrifugation step was repeated. And (4) diluting the concentrated solution obtained again and the precipitate redissolved solution I in the step to a 50mL volumetric flask by using acetate buffer solution, namely enzyme solution.
Placing the test tube 1, the test tube 2 and the 5% sucrose solution together in a water bath kettle for preheating for 10min, respectively sucking 2mL of the 5% sucrose solution, adding the 5% sucrose solution into the test tube 1 and the test tube 2, reacting for 1h, respectively controlling the reaction temperature at 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, keeping other experimental conditions unchanged (the pH value of the buffer solution is 6.0, the color developing agent solution is 1.5mL, and the reaction time is 1h), and measuring the sucrose invertase enzyme value of the acacia raw honey at different reaction temperatures. FIG. 1 is obtained by plotting the enzyme value of sucrose invertase/reaction temperature.
As can be seen from FIG. 1, the reaction temperature was gradually increased with the increase of the reaction temperature at 30 to 40 ℃ and reached the maximum value at 40 ℃. When the temperature exceeds 40 ℃, the enzyme value of the sucrose invertase starts to decrease, and when the temperature exceeds 50 ℃, the decrease of the enzyme value exceeds 50%, which means that an excessively high temperature will result in the decrease or even the loss of the sucrose invertase activity. Therefore, in the detection method provided by the invention, the reaction temperature is preferably controlled to be 30-45 ℃.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911052429.4A CN110609033B (en) | 2019-10-31 | 2019-10-31 | Method for detecting enzyme value of sucrose invertase in honey |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911052429.4A CN110609033B (en) | 2019-10-31 | 2019-10-31 | Method for detecting enzyme value of sucrose invertase in honey |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110609033A true CN110609033A (en) | 2019-12-24 |
| CN110609033B CN110609033B (en) | 2022-04-08 |
Family
ID=68895618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911052429.4A Active CN110609033B (en) | 2019-10-31 | 2019-10-31 | Method for detecting enzyme value of sucrose invertase in honey |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110609033B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101676719A (en) * | 2008-09-19 | 2010-03-24 | 河南瑞驰生物科技有限公司 | Method for measuring diastatic enzyme activity using a spectrophotometer |
| US20140260562A1 (en) * | 2013-03-14 | 2014-09-18 | Safa MOTLAGH | Method of measuring the digestibility of a food protein |
| CN104132939A (en) * | 2014-07-12 | 2014-11-05 | 福建农林大学 | Method for rapidly detecting amylase value in honey |
| CN106248664A (en) * | 2016-07-18 | 2016-12-21 | 中国科学院东北地理与农业生态研究所 | The assay method of soil saccharase |
| CN109182314A (en) * | 2018-07-11 | 2019-01-11 | 郑州力酷生物科技有限公司 | A kind of extracting method of honey enzyme activity substance |
-
2019
- 2019-10-31 CN CN201911052429.4A patent/CN110609033B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101676719A (en) * | 2008-09-19 | 2010-03-24 | 河南瑞驰生物科技有限公司 | Method for measuring diastatic enzyme activity using a spectrophotometer |
| US20140260562A1 (en) * | 2013-03-14 | 2014-09-18 | Safa MOTLAGH | Method of measuring the digestibility of a food protein |
| CN104132939A (en) * | 2014-07-12 | 2014-11-05 | 福建农林大学 | Method for rapidly detecting amylase value in honey |
| CN106248664A (en) * | 2016-07-18 | 2016-12-21 | 中国科学院东北地理与农业生态研究所 | The assay method of soil saccharase |
| CN109182314A (en) * | 2018-07-11 | 2019-01-11 | 郑州力酷生物科技有限公司 | A kind of extracting method of honey enzyme activity substance |
Non-Patent Citations (1)
| Title |
|---|
| 张忠义 等: "蜂蜜中蔗糖转化酶测定方法探讨", 《食品科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110609033B (en) | 2022-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Improved colorimetric determination of cell wall chitin in wood decay fungi | |
| McFeeters | A manual method for reducing sugar determinations with 2, 2′-bicinchoninate reagent | |
| CN101676719A (en) | Method for measuring diastatic enzyme activity using a spectrophotometer | |
| CN106868096B (en) | High-stability and low-cost glucose detection reagent by hexokinase method | |
| CN106248664B (en) | The measuring method of soil saccharase | |
| CN110609033B (en) | Method for detecting enzyme value of sucrose invertase in honey | |
| CN102980856B (en) | Carboxymethyl cellulase activity determination method | |
| Wiseman et al. | Silage analysis, determination of sugar in silages and forages | |
| CN103789400B (en) | A kind of detection method of beta-amylase | |
| CN102818779A (en) | Method for detecting content of water-insoluble glucan in yeast glucan | |
| CN108277290A (en) | The dry powdered LAMP quick detection kits of staphylococcus aureus | |
| Johnson | Determination of 2, 3-butylene glycol in fermentations | |
| CN100554939C (en) | Serum sodium ion by enzymatic method is measured reagent and method | |
| CN116413421A (en) | PYF factor-based barley or malt PYF value prediction method | |
| Fossati | Phosphate determination by enzymatic colorimetric assay | |
| CN109001189B (en) | Method for detecting amylase content range in sugar product prepared from sugarcane and application of method | |
| CN110358869B (en) | Preparation method of low-molecular-weight hyaluronic acid based on near infrared spectrum technology | |
| CN106290606A (en) | A kind of method of Quantitative detection trehalose synthase enzyme activity in antibacterial TreS approach trehalose producing strains screens | |
| CN104931443A (en) | Method for measuring yellow rice wine yeast growth curve by separation and purification | |
| CN107576651B (en) | The prefabricated reagent and its preservation, detection method that sucrose quickly detects in a kind of honey | |
| CN109266719B (en) | Method for evaluating hydrolysis capacity of malt starch | |
| CN111257447A (en) | Method for analyzing stable hydrogen isotope ratio of non-exchangeable hydrogen in total sugar of food | |
| CN114486870B (en) | Nanometer enzyme functionalized paper-based sensor for rapid blood glucose detection and detection method thereof | |
| CN105352952B (en) | A kind of quick determination method of LBP-X | |
| CN106556595B (en) | A kind of Picric kinetic method detection kit of strong antijamming capability |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230206 Address after: 831111 No. 612 Tuanjie Road, Junhu Farm Town, Changji City, Changji Hui Autonomous Prefecture, Xinjiang Uygur Autonomous Region Patentee after: Xinjiang xinxingyuan Agriculture Co.,Ltd. Address before: 100093, Xiangshan North Road, Beijing, Haidian District Patentee before: INSTITUTE OF APICULTURAL RESEARCH CHINESE ACADEMY OF AGRICULTURAL SCIENCES |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231007 Address after: No. 20 Malan Lane, Junzhureke Village, Jilande Pasture, Yumin County, Tacheng Prefecture, Xinjiang Uygur Autonomous Region, 834804 Patentee after: Xinjiang Silk Road Herbal Medicine Co.,Ltd. Address before: 831111 No. 612 Tuanjie Road, Junhu Farm Town, Changji City, Changji Hui Autonomous Prefecture, Xinjiang Uygur Autonomous Region Patentee before: Xinjiang xinxingyuan Agriculture Co.,Ltd. |