Multicolor detection kit and digital quantification method for total antioxidant capacity of beverages

Abstract

The present invention relates to a multicolor detection kit and digital quantification method for the total antioxidant capacity of beverages, belonging to the field of chemical analysis and testing. The present invention establishes a linear correlation between the hue H value and the total antioxidant capacity of antioxidants. By combining the color development and discoloration process of a color developer, the color development results are collected using digital imaging devices such as mobile phones. The hue H value obtained by digital imaging enables rapid determination of the antioxidant capacity of beverages. The present invention develops a novel multicolor development kit that facilitates digital quantification of total antioxidant capacity, establishing a more efficient and intuitive evaluation method for the quality assessment of the antioxidant capacity of beverages.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis and detection, and relates to a multicolor detection kit for the total antioxidant capacity of beverages and a digital quantification method. Background Art

[0002] In a healthy body, free radicals exist in a dynamic equilibrium at low levels. Excessive free radicals can severely disrupt the normal physiological activities of cells and damage biological macromolecules such as nucleic acids. If this phenomenon persists for a long time, it can lead to cancer and tumors, cardiovascular and cerebrovascular diseases, aging, and neurological diseases. Natural antioxidants, such as tea polyphenols and carotenoids, often contain hydroxyl groups, phenolic groups, benzene rings, and triple bonds in their structures. These can act as hydrogen donors and electron acceptors, combining with free radicals to undergo nucleophilic and electrophilic reactions, thereby reducing their impact on the body. Testing and evaluating the antioxidant properties of natural products can provide a reliable reference for choosing appropriate beverages and promoting good health.

[0003] Existing antioxidant capacity determination mostly uses a monochromatic absorption mode, such as patent application number: 202011221282.X "A high-sensitivity total antioxidant capacity detection kit and its use method". Its principle is to use the iron ion reduction / antioxidant capacity method (FRAP method). At low pH, the antioxidant in the sample converts Fe 3+ TPTZ complex is reduced to Fe 2+ TPTZ appears blue. The absorbance is measured at 593 nm. The absorbance is proportional to the reducing power of the antioxidants in the sample, so the overall antioxidant capacity can be measured. This type of color development method is based on the Lambert-Beer law. The change in absorbance is used to test the antioxidant capacity, and a professional spectrophotometer or microplate reader is required for quantification. However, the absorbance and color depth of the coloring substance are not only related to the concentration of the analyte, but also to the optical path length of the light passing through the solution to be tested. Therefore, the quantification process in the single-color mode is somewhat challenging. There is an urgent need for a simple and rapid kit for evaluating the antioxidant capacity of ingested beverages and related digital quantification methods.

[0004] In color data collection, the most widely used color standard is the RGB color model, which measures the changes in the three color channels of red (R), green (G), and blue (B) and superimposes these three channels to obtain numerical changes, thereby representing different colors and different shades of color. In color recognition, the HSV color space color model has its own unique characteristics. The HSV color space uses three components: hue (H), saturation (S), and brightness (V) to represent color. Affected by the shooting angle, background light, etc., the RGB information of the same object in different shooting environments varies greatly, but the use of the HSV color space can more intuitively express the hue, brightness, saturation, etc. of the color. The hue H value is related to the color presented by the object itself, and a single hue value can more conveniently represent the color of objects in different environments.

[0005] In order to realize the digital quantification and visual quantification of the total antioxidant capacity of beverages through hue H value, it is urgent to develop a multicolor colorimetric reagent that can display the total antioxidant capacity of beverages through hue changes, that is, the detection reagent interacts with beverages with different antioxidant capacities to show different colors, and the hue H value of the color is proportional to the total antioxidant capacity of the beverage. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a multi-color detection kit for the total antioxidant capacity of beverages and a digital quantification method, establishes a linear correlation between the hue H value of the color development result and the total antioxidant activity of the antioxidants, and realizes the rapid determination of the antioxidant capacity of beverages based on the H value obtained from the color development result, thereby establishing a more efficient and intuitive evaluation method for the quality identification of the antioxidant capacity of beverages.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] A multicolor detection kit for the total antioxidant capacity of beverages, comprising a color developing solution A, a detection solution B, and a standard sample. After the color developing solution A and the detection solution B react with the standard sample, multicolor development can be achieved. Specifically:

[0009] The color developing solution A comprises 100-2000 μM 3,3',5,5'-tetramethylbenzidine (TMB), 1-1000 nM horseradish peroxidase (HRP), a buffer solution with a pH of 4-10 and a concentration of 100-1000 mM, and a surfactant with a mass fraction of 0.1%-10.0%.

[0010] The detection solution B comprises 100-2000 μM of an oxidant, a buffer solution with a concentration of 100-1000 mM and a pH of 4-10.

[0011] The standard samples include ascorbic acid (Vc) with concentrations of 0 μM, 25 μM, 50 μM and 75 μM.

[0012] Furthermore, the buffer solution in the color developing solution A includes acetic acid-sodium acetate buffer solution, Tris-HCl buffer solution, PBS buffer solution or sodium carbonate-sodium bicarbonate buffer solution.

[0013] Furthermore, the surfactant in the color developing solution A includes Tween 20, Tween 60, Tween 80 or Triton X100.

[0014] Furthermore, the oxidant in the detection solution B includes urea peroxide or hydrogen peroxide.

[0015] Furthermore, the buffer solution in the detection solution B includes acetic acid-sodium acetate buffer solution, Tris-HCl buffer solution, PBS buffer solution or sodium carbonate-sodium bicarbonate buffer solution.

[0016] A digital quantification method for a multicolor detection kit for the total antioxidant capacity of beverages is disclosed. Based on the characteristic of TMB that it can display multiple colors under different oxidation levels, a detection solution capable of multicolor development is formulated. The detection solution comprises a color development solution A and a detection solution B. Color development solution A contains a certain concentration of TMB and HRP to accelerate the reaction rate, and detection solution B contains a certain amount of an oxidant. Equal volumes of solution A and solution B are first mixed to obtain a detection solution C with a certain oxidizing capacity. The reducing sample to be tested is then diluted 10-100 times to obtain the test sample. Using vitamin C as a standard sample, the total antioxidant capacity of the beverage is detected through multicolor display, where the total antioxidant capacity is measured using vitamin C equivalents. The method comprises the following steps:

[0017] Step 1: Take equal volumes of colorimetric solution A and detection solution B and add them to a centrifuge tube or a larger container. React at room temperature for 1-10 minutes to obtain detection solution C. Detection solution B uses carbamide peroxide or hydrogen peroxide as an oxidant and utilizes the catalytic activity of horseradish peroxidase to accelerate the reaction rate. Before detection, TMB in colorimetric solution A is oxidized to TMB. 2+ , yellow.

[0018] Step 2: Mix equal volumes of the standard sample and the detection solution C, perform color development, and obtain a linear relationship between the antioxidant capacity (Vc equivalent) and the color development H value.

[0019] 2.1) Add equal volumes of four standard samples of different concentrations to four containers, and add detection solution C to each container. The volume of detection solution C added is the same as that of each standard sample. Incubate at room temperature for 1-10 minutes to develop color. During the color development process: TMB in detection solution C 2+Reduced by different concentrations of Vc, it appears yellow-green, green, blue-green or blue, and the blue is TMB + Other colors are TMB 2+ With TMB + Mixed colors, TMB in this color change process 2+ The degree of reduction is proportional to the concentration of Vc.

[0020] 2.2) Take a mobile phone photo of the four color development results from the test kit and use imaging software to obtain the four H values ​​of the color development results. Using the concentration of the standard sample Vc as the X-axis and the H value as the Y-axis, a linear relationship between the H value and the Vc concentration is obtained, where the Vc concentration corresponds to the total antioxidant capacity. Studies have shown that the H value and Vc concentration show a good linear relationship only when the Vc concentration is between 0 and 75 μM.

[0021] Step 3: Obtain the total antioxidant capacity of the sample to be tested

[0022] 3.1) Dilute the sample to be tested N-fold, add the diluted sample solution to the same container as step 2.1), then add detection solution C, where the volumes of detection solution C and the sample solution to be tested are the same as step 2.1), react at room temperature for 1-10 minutes, and develop color. TMB in detection solution C 2+ It can also be reduced by reducing substances in the sample solution to be tested, showing color.

[0023] 3.2) Determine an appropriate dilution factor N by visual comparison. Compare the color of the diluted sample solution in step 3.1) with the four colors in step 2.1) to roughly determine whether the H value of the diluted sample solution in step 3.1) is within the color development range of 0 to 75 μM Vc. If the color is pure blue or light blue, the H value can be directly determined to be greater than the color development H value of 75 μM Vc. Increase the dilution factor N and repeat steps 3.1) and 3.2). If the color is between yellow-green and blue, the H value can be determined to be within the color development range of 0 to 75 μM Vc and proceed to step 3.3).

[0024] 3.3) Take a photo of the color development result of the sample solution using a mobile phone, and use imaging software to obtain the H value of the color development result:

[0025] 3.3.1) If the H value is within the color-developing H value range of 0-75 μM Vc, substitute this H value into the linear relationship obtained in step 2.2) to obtain the corresponding Vc equivalent concentration. Multiply this by the dilution factor N to obtain the final Vc equivalent concentration of the test sample, which also provides the total antioxidant capacity of the test sample.

[0026] 3.3.2) If the H value is greater than the color-developed H value of 75 μM Vc, return to step 3.1) and further dilute the diluted sample solution to ensure that the color-developed H value of the sample solution is within the color-developed H value range of 0 to 75 μM Vc; then repeat step 3.3.1) to determine the total antioxidant capacity of the sample.

[0027] This method is suitable for detecting one sample to be tested or for high-throughput simultaneous detection of multiple samples to be tested.

[0028] The beneficial effects of the present invention are:

[0029] TMB 2+ Digital quantification of the degree of reduction is usually done using a microplate reader or spectrophotometer, TMB 2+ It is yellow and its absorption wavelength is 450nm, TMB + It is blue and its absorption wavelength is 650nm. 2+ Reduced to TMB by different concentrations of Vc or reducing substances + During the process, the absorption of 450nm wavelength is weakened, while the absorption of 650nm wavelength is enhanced. However, the equipment required for this method is expensive and requires professional technicians to operate. The present invention combines the color development and discoloration process of the color developer, and collects the color development results through digital imaging equipment such as mobile phones. The hue H value obtained by digital imaging is proportional to the amount of 0-75μM Vc or the equivalent of Vc in reducing substances. That is, the present invention has developed a novel multi-color color development kit, which for the first time associates the H value of the color development result with the total antioxidant capacity of the beverage, realizing a method that is convenient for digital quantification and visual quantification of the total antioxidant capacity of the beverage, and can accurately and efficiently obtain the total antioxidant capacity of the sample solution to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the principle of TMB color development in the present invention.

[0031] Figure 2 This is the working curve for detecting standard sample concentrations of 0, 25, 50, and 75 μmol / LVc in Example 1 of the present invention.

[0032] Figure 3 This is the H value and total antioxidant capacity (Vc equivalent) of the color development results of beverage samples (black tea, Pu'er, and certain tea) used in Example 1 of the present invention.

[0033] Figure 4 This is the working curve for detecting standard sample concentrations of 0, 25, 50, and 75 μmol / LVc in Example 2 of the present invention.

[0034] Figure 5This is the H value and total antioxidant capacity (Vc equivalent) of the color development results of beverage samples (black tea, Pu'er, and certain tea) used in Example 2 of the present invention.

[0035] Figure 6 This is the working curve for detecting standard sample concentrations of 0, 25, 50, and 75 μmol / LVc in Example 3 of the present invention.

[0036] Figure 7 This is the H value and total antioxidant capacity (Vc equivalent) of the color development results of beverage samples (black tea, Pu'er, and certain tea) used in Example 3 of the present invention.

[0037] Figure 8 This is the working curve for detecting standard sample concentrations of 0, 25, 50, and 75 μmol / LVc in Example 4 of the present invention.

[0038] Figure 9 This is the H value and total antioxidant capacity (Vc equivalent) of the color development results of beverage samples (black tea, Pu'er, and certain tea) used in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention clearer, the above scheme is further described below with reference to the accompanying drawings and specific examples. It should be understood that the specific examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. The implementation conditions not specified are generally those in routine experiments.

[0040] Example 1

[0041] A multicolor detection kit for the total antioxidant capacity of beverages, comprising the following solutions:

[0042] Color development solution A: includes 200 μM TMB, 10 nM HRP, PBS buffer solution with a pH of 5.4 and a concentration of 100 mM, and a surfactant Tween 20 with a mass fraction of 0.2%.

[0043] Detection solution B: Contains 200 μM carbamide peroxide, PBS buffer solution with a pH of 5.4 and a concentration of 500 mM.

[0044] Standard samples: including ascorbic acid (Vc) at concentrations of 0 μM, 25 μM, 50 μM, and 75 μM.

[0045] A digital quantification method for a multicolor detection kit for the total antioxidant capacity of beverages comprises the following steps:

[0046] Step 1: Take equal volumes of colorimetric solution A and detection solution B and add them to a centrifuge tube. Incubate at room temperature for 1-10 minutes to obtain detection solution C.

[0047] Step 2: Mix equal volumes of the standard sample and detection solution C, perform color development, and obtain a linear relationship between the antioxidant capacity and the H value.

[0048] 2.1) Add equal volumes of four standard samples of different concentrations to four containers. Add detection solution C to each container, where the volume of detection solution C added is the same as that of each standard sample. Incubate at room temperature for 5 minutes to develop color.

[0049] 2.2) Take photos of the four color development results from the test kit using a mobile phone. Use imaging software to obtain the four H values ​​of the color development results. Use the Vc concentration of the standard sample as the X-axis and the H value as the Y-axis to fit the linear relationship between the H value and the Vc concentration, where the Vc concentration corresponds to the total antioxidant capacity.

[0050] Step 3: Obtain the total antioxidant capacity of the sample to be tested

[0051] 3.1) Dilute the test samples (black tea, Pu'er tea, and a certain tea) 50-fold and add the diluted test sample solution to the same container as in step 2.1). Then, add detection solution C, where the volumes of detection solution C and the test sample solution are the same as in step 2.1). Incubate at room temperature for 5 minutes to develop color.

[0052] 3.2) Visually compare the color of the diluted sample solution in step 3.1) with the four colors in step 2.1) to roughly determine that the H value in step 3.1) is within the color development range of 0 to 75 μM Vc, and the appropriate dilution factor is 50.

[0053] 3.3) Use a mobile phone to photograph the color development results of each of the three test sample solutions. Use imaging software to obtain the H value of the color development results. Substitute the obtained H value into the linear relationship obtained in step 2.2) to obtain the corresponding Vc equivalent concentration. Multiply this by the dilution factor of 50 to obtain the final Vc equivalent concentration of the test sample, which also determines the total antioxidant capacity of the test sample.

[0054] The color development principle of this method is as follows Figure 1 As shown, colorless TMB is added to urea peroxide in an equal molar ratio, and the catalytic activity of horseradish peroxidase is used to accelerate the reaction rate, and TMB is oxidized to TMB before detection. 2+ , yellow, TMB 2+ Reduction by different concentrations of Vc or reducing substances shows yellow-green, green, blue-green, and blue respectively, and the blue is TMB + Other colors are TMB 2+ With TMB + Mixed colors, TMB in this color change process 2+The degree of reduction is proportional to the concentration of Vc or reducing substances.

[0055] The working curve of hue H value and Vc concentration obtained by the four-point method is:

[0056] H=A*C Vc +B (1)

[0057] In the formula, H represents the hue value, C Vc Represents the concentration equivalent of Vc, A represents the slope coefficient of the method, and B represents the hue value when the Vc concentration is 0 μM. The A and B values ​​are related to the formula of the detection solution C. The linear relationship obtained in step 2.2) is as follows Figure 2 shown.

[0058] The method of the present invention was used to detect the test samples (black tea, Pu'er tea and a certain tea), and the H values ​​and Vc equivalents of the total antioxidant capacity obtained were shown in Table 1 below:

[0059] Table 1 H value and Vc equivalent of total antioxidant capacity of standard samples and test samples in Example 1

[0060]

[0061] like Figure 3 As shown, since the actual sample dilution factor is 50, the total antioxidant capacity of each sample to be tested can be calculated according to the working curve: black tea is 2.33mmol / L, Pu'er is 1.37mmol / L, and a certain tea is 2.65mmol / L (Vc equivalent).

[0062] Example 2

[0063] A multicolor detection kit for the total antioxidant capacity of beverages, comprising the following solutions:

[0064] Color development solution A: includes 100 μM TMB, 1 nM HRP, 500 mM acetic acid-sodium acetate buffer solution with a pH of 4.0, and 0.1% by mass of surfactant Tween 60.

[0065] Detection solution B: contains 100 μM carbamide peroxide, pH 4.0, and 100 mM acetic acid-sodium acetate buffer solution.

[0066] Standard samples: including ascorbic acid (Vc) at concentrations of 0 μM, 25 μM, 50 μM, and 75 μM.

[0067] A digital quantification method for a multicolor detection kit for the total antioxidant capacity of beverages comprises the following steps:

[0068] Step 1: Take equal volumes of colorimetric solution A and detection solution B and add them to a centrifuge tube. Incubate at room temperature for 1 minute to obtain detection solution C.

[0069] Step 2: Mix equal volumes of the standard sample and detection solution C, perform color development, and obtain a linear relationship between the antioxidant capacity and the H value.

[0070] 2.1) Add equal volumes of four standard samples of different concentrations to four containers. Add detection solution C to each container, where the volume of detection solution C added is the same as that of each standard sample. Incubate at room temperature for 1 minute to develop color.

[0071] 2.2) Take photos of the four color development results from the test kit using a mobile phone. Use imaging software to obtain the four H values ​​of the color development results. Use the Vc concentration of the standard sample as the X-axis and the H value as the Y-axis to fit the linear relationship between the H value and the Vc concentration, where the Vc concentration corresponds to the total antioxidant capacity.

[0072] Step 3: Obtain the total antioxidant capacity of the sample to be tested

[0073] 3.1) Dilute the test sample solutions (black tea, Pu'er tea, and a certain tea, respectively) 25-fold and add the diluted test sample solutions to the same container as in step 2.1). Then, add detection solution C, where the volumes of detection solution C and the test sample solutions are the same as in step 2.1). Incubate at room temperature for 1 minute to develop color.

[0074] 3.2) Visually compare the color of the diluted sample in step 3.1) with the four colors in step 2.1) to roughly determine that the H value in step 3.1) is within the color development range of 0 to 75 μM Vc, and the appropriate dilution factor is 50.

[0075] 3.3) Use a mobile phone to photograph the color development results of each of the three test sample solutions. Use imaging software to obtain the H value of the color development results. Substitute the obtained H value into the linear relationship obtained in step 2.2) to obtain the corresponding Vc equivalent concentration. Multiply this by the dilution factor of 50 to obtain the final Vc equivalent concentration of the test sample, which also determines the total antioxidant capacity of the test sample.

[0076] The linear relationship obtained in step 2.2) is as follows Figure 4 shown.

[0077] The method of the present invention was used to detect the test samples (black tea, Pu'er tea and a certain tea), and the H values ​​and Vc equivalents of the total antioxidant capacity obtained were shown in Table 2 below:

[0078] Table 2 H value and Vc equivalent of total antioxidant capacity of standard samples and test samples in Example 2

[0079]

[0080] like Figure 5 As shown, since the actual sample dilution factor is 50, the total antioxidant capacity of each sample to be tested can be calculated according to the working curve: black tea is 2.25mmol / L, Pu'er is 1.36mmol / L, and a certain tea is 2.52mmol / L (Vc equivalent).

[0081] Example 3

[0082] A multicolor detection kit for the total antioxidant capacity of beverages, comprising the following solutions:

[0083] Color development solution A: includes 500 μM TMB, 500 nM HRP, 100 mM Tris-HCl buffer solution with a pH of 7.0, and 5% by mass of a surfactant Tween 80.

[0084] Detection solution B: Contains 500 μM carbamide peroxide, pH 7.0, and 100 mM Tris-HCl buffer solution.

[0085] Standard samples: including ascorbic acid (Vc) at concentrations of 0 μM, 25 μM, 50 μM, and 75 μM.

[0086] A digital quantification method for a multicolor detection kit for the total antioxidant capacity of beverages comprises the following steps:

[0087] Step 1: Take equal volumes of colorimetric solution A and detection solution B and add them to a centrifuge tube or a larger container. React at room temperature for 10 minutes to obtain detection solution C.

[0088] Step 2: Mix equal volumes of the standard sample and detection solution C, perform color development, and obtain a linear relationship between the antioxidant capacity and the H value.

[0089] 2.1) Add equal volumes of four standard samples of different concentrations to four containers. Add detection solution C to each container, where the volume of detection solution C added is the same as that of each standard sample. Incubate at room temperature for 10 minutes to develop color.

[0090] 2.2) Take photos of the four color development results from the test kit using a mobile phone. Use imaging software to obtain the four H values ​​of the color development results. Use the Vc concentration of the standard sample as the X-axis and the H value as the Y-axis to fit the linear relationship between the H value and the Vc concentration, where the Vc concentration corresponds to the total antioxidant capacity.

[0091] Step 3: Obtain the total antioxidant capacity of the sample to be tested

[0092] 3.1) Dilute the sample solutions (black tea, Pu'er tea, and a certain tea) 50-fold and add the diluted sample solutions to the same container as in step 2.1). Then, add detection solution C, where the volumes of detection solution C and the sample solutions are the same as in step 2.1). Incubate at room temperature for 10 minutes to develop color.

[0093] 3.2) Visually compare the color of the diluted sample in step 3.1) with the four colors in step 2.1) to roughly determine that the H value in step 3.1) is within the color development range of 0 to 75 μM Vc, and the appropriate dilution factor is 50.

[0094] 3.3) Use a mobile phone to photograph the color development results of each of the three test sample solutions. Use imaging software to obtain the H value of the color development results. Substitute the obtained H value into the linear relationship obtained in step 2.2) to obtain the corresponding Vc equivalent concentration. Multiply this by the dilution factor of 50 to obtain the final Vc equivalent concentration of the test sample, which also determines the total antioxidant capacity of the test sample.

[0095] The linear relationship obtained in step 2.2) is as follows Figure 6 shown.

[0096] The method of the present invention was used to detect the test samples (black tea, Pu'er tea and a certain tea), and the total antioxidant capacity H value and Vc content obtained were shown in Table 3 below:

[0097] Table 3 H value and Vc equivalent of total antioxidant capacity of standard samples and test samples in Example 3

[0098]

[0099] like Figure 7 As shown, since the actual sample dilution factor is 50, the total antioxidant capacity of each sample to be tested can be calculated according to the working curve: black tea is 2.26mmol / L, Pu'er is 1.36mmol / L, and a certain tea is 2.63mmol / L (Vc equivalent).

[0100] Example 4

[0101] A multicolor detection kit for the total antioxidant capacity of beverages, comprising the following solutions:

[0102] Color development solution A: includes 2000 μM TMB, 1000 nM HRP, 1000 mM sodium carbonate-sodium bicarbonate buffer solution with a pH of 10, and 10% by mass of a surfactant Triton X100.

[0103] Detection solution B: contains 2000 μM hydrogen peroxide, pH 10, and 1000 mM sodium carbonate-sodium bicarbonate buffer solution.

[0104] Standard samples: including ascorbic acid (Vc) at concentrations of 0 μM, 25 μM, 50 μM, and 75 μM.

[0105] A digital quantification method for a multicolor detection kit for the total antioxidant capacity of beverages comprises the following steps:

[0106] Step 1: Take equal volumes of colorimetric solution A and detection solution B and add them to a centrifuge tube or a larger container. React at room temperature for 10 minutes to obtain detection solution C.

[0107] Step 2: Mix equal volumes of the standard sample and detection solution C, perform color development, and obtain a linear relationship between the antioxidant capacity and the H value.

[0108] 2.1) Add equal volumes of four standard samples of different concentrations to four containers. Add detection solution C to each container, where the volume of detection solution C added is the same as that of each standard sample. Incubate at room temperature for 10 minutes to develop color.

[0109] 2.2) Take photos of the four color development results from the test kit using a mobile phone. Use imaging software to obtain the four H values ​​of the color development results. Use the Vc concentration of the standard sample as the X-axis and the H value as the Y-axis to fit the linear relationship between the H value and the Vc concentration, where the Vc concentration corresponds to the total antioxidant capacity.

[0110] Step 3: Obtain the total antioxidant capacity of the sample to be tested

[0111] 3.1) Dilute the sample solution (black tea, Pu'er tea, and a certain tea) 50-fold and add the diluted sample solution to the same container as in step 2.1). Then add detection solution C, where the volumes of detection solution C and the sample solution are the same as in step 2.1). Incubate at room temperature for 10 minutes to develop color. TMB in detection solution C 2+ It can also be reduced by reducing substances in the sample solution to be tested, showing color.

[0112] 3.2) Visually compare the color of the diluted sample in step 3.1) with the four colors in step 2.1) to roughly determine that the H value in step 3.1) is within the color development range of 0 to 75 μM Vc, and the appropriate dilution factor is 50.

[0113] 3.3) Use a mobile phone to photograph the color development results of each of the three test sample solutions. Use imaging software to obtain the H value of the color development results. Substitute the obtained H value into the linear relationship obtained in step 2.2) to obtain the corresponding Vc equivalent concentration. Multiply this by the dilution factor of 50 to obtain the final Vc equivalent concentration of the test sample, which also determines the total antioxidant capacity of the test sample.

[0114] The linear relationship obtained in step 2.2) is as follows Figure 8 shown.

[0115] The method of the present invention was used to detect the test samples (black tea, Pu'er tea and a certain tea), and the total antioxidant capacity H value and Vc content obtained were shown in Table 4 below:

[0116] Table 4 H value and Vc equivalent of total antioxidant capacity of standard samples and test samples in Example 4

[0117]

[0118] like Figure 9 As shown, since the actual sample dilution factor is 50, the total antioxidant capacity of each sample to be tested can be calculated according to the working curve: black tea is 2.22mmol / L, Pu'er is 1.34mmol / L, and a certain tea is 2.58mmol / L (Vc equivalent).

[0119] The above-described embodiments merely express the implementation methods of the present invention, but should not be understood as limiting the scope of the patent of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A digital quantification method for a multicolor detection kit for total antioxidant capacity of beverages, characterized in that: The digital quantification method comprises the following steps: Step 1: Take equal volumes of colorimetric solution A and detection solution B and add them to a centrifuge tube or a larger container. Incubate at room temperature for 1-10 minutes to obtain detection solution C. Step 2: Mix equal volumes of the standard sample and the test solution C, perform color development, and obtain a linear relationship between the antioxidant capacity, i.e., Vc equivalent, and the color development H value; 2.1) Add equal volumes of four standard samples of varying concentrations to four containers. Add Detection Solution C to each container, with the volume of Detection Solution C equal to that of each standard sample. Incubate at room temperature for 1-10 minutes to develop color. 2.2) Take photos of the four color development results from the test kit using a mobile phone. Use imaging software to obtain the four H values ​​of the color development results. With the concentration of the standard sample Vc as the X-axis and the H value as the Y-axis, a linear relationship between the H value and the Vc concentration is obtained, where the Vc concentration corresponds to the total antioxidant capacity. Step 3, obtaining the total antioxidant capacity of the sample to be tested; 3.1) Dilute the sample to be tested N-fold. Add the diluted sample solution to the same container as in step 2.1) and then add detection solution C, where the volumes of detection solution C and the sample solution to be tested are the same as in step 2.1). Incubate at room temperature for 1-10 minutes to develop color. 3.2) Determine the appropriate dilution factor N by visual comparison. Compare the color of the diluted sample solution in step 3.1) with the four colors obtained in step 2.1) to preliminarily determine whether the H value of the diluted sample solution in step 3.1) is within the color development range of 0-75 μM Vc. If the color is pure blue or light blue, the H value is directly greater than the color development H value of 75 μM Vc. Increase the dilution factor N and repeat steps 3.1) and 3.

2. If the color is between yellow-green and blue, the H value is determined to be within the color development range of 0-75 μM Vc and proceed to step 3.

3. 3.3) Take a photo of the color development result of the sample solution using your mobile phone and use imaging software to obtain the H value of the color development result: 3.3.1) If the H value is within the color development H value range of 0-75 μM Vc, substitute this H value into the linear relationship obtained in step 2.2) to obtain the corresponding Vc equivalent concentration. Multiply this value by the dilution factor N to obtain the final Vc equivalent concentration of the sample to be tested, thus obtaining the total antioxidant capacity of the sample to be tested. 3.3.2) If the H value is greater than the color-developing H value of 75 μM Vc, return to step 3.1) and further dilute the diluted sample solution to ensure that the color-developing H value of the sample solution is within the color-developing H value range of 0-75 μM Vc. Then repeat step 3.3.1) to obtain the total antioxidant capacity of the sample. The multicolor detection kit for total antioxidant capacity of beverages includes the following solutions: Color development solution A: including 100-2000 μM 3,3',5,5'-tetramethylbenzidine, 1-1000 nM horseradish peroxidase, a buffer solution with a pH of 4-10 and a concentration of 100-1000 mM, and a surfactant with a mass fraction of 0.1%-10.0%; Detection solution B: includes 100-2000 μM oxidant, 100-1000 mM buffer solution, pH 4-10; Standard samples: including ascorbic acid (Vc) at concentrations of 0 μM, 25 μM, 50 μM, and 75 μM.

2. The digital quantification method of a multicolor detection kit for total antioxidant capacity of beverages according to claim 1, characterized in that: The buffer solution in the color developing solution A includes acetic acid-sodium acetate buffer solution, Tris-HCl buffer solution, PBS buffer solution or sodium carbonate-sodium bicarbonate buffer solution.

3. The digital quantification method of a multicolor detection kit for total antioxidant capacity of beverages according to claim 1, characterized in that: The surfactant in the color developing solution A includes Tween 20, Tween 60, Tween 80 or Triton X100.

4. The digital quantification method of a multicolor detection kit for total antioxidant capacity of beverages according to claim 1, characterized in that: The oxidant in the detection solution B includes carbamide peroxide or hydrogen peroxide.

5. The digital quantification method of a multicolor detection kit for total antioxidant capacity of beverages according to claim 1, characterized in that: The buffer solution in the detection solution B includes acetic acid-sodium acetate buffer solution, Tris-HCl buffer solution, PBS buffer solution or sodium carbonate-sodium bicarbonate buffer solution.

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