A tea polyphenol rapid detection test paper and a preparation method thereof

Abstract

The application discloses a tea polyphenol rapid detection test paper, which comprises a hydrophobic region and a hydrophilic region, wherein the hydrophilic region is sequentially provided with a sample injection region, a reaction region and a detection region in the order of sample migration, and the detection region is closely attached to the hydrophobic region; the reaction region is fixed with an alkaline substance, and the detection region is fixed with a metal salt. The application further discloses a preparation method of the test paper and a method for rapidly detecting the tea polyphenol content by using the test paper. The tea polyphenol rapid detection test paper is suitable for on-site detection, has rapid reaction, and can complete the detection in only 2 minutes, is simple to operate, can complete the tea polyphenol content detection in a short time, does not need professional instruments and reagents, is convenient to operate, can meet the demand of the tea industry chain for rapid identification of the tea polyphenol content, can preliminarily and rapidly identify the quality of green tea, and has great popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis methods, specifically relating to a rapid test strip for detecting tea polyphenol content and its preparation method. Background Technology

[0002] Tea polyphenols are the most abundant functional components in tea, possessing various functions such as anti-oxidation, anti-inflammation, antibacterial properties, and regulation of metabolic diseases, and are directly related to tea quality. Excessive tea polyphenol content results in a bitter tea, while low content leads to a bland taste. Therefore, its content is one of the important indicators for judging tea quality.

[0003] The current national standard GB / T 8313-2018 specifies the use of the Folin-Ciocalteu method to determine the polyphenol content in tea. The experimental principle is that under alkaline conditions, the phosphomolybdic acid and phosphotungstic acid in the Folin-Ciocalteu reagent can be reduced by the phenolic hydroxyl groups in tea polyphenols, generating a blue mixture of molybdenum blue and tungsten blue. This mixture has a maximum absorption peak at a wavelength of 765 nm. Although the Folin-Ciocalteu method is simple in principle and low in cost, it has significant limitations: it consumes a large amount of chemical reagents, the operation procedure is cumbersome, and the colorimetric reaction of Folin-Ciocalteu tea polyphenols requires more than 2 hours, making it unsuitable for rapid on-site detection of tea samples.

[0004] Therefore, developing a simple, rapid, and sensitive method for detecting tea polyphenols is of great significance.

[0005] Patent application 202311032450.4 discloses a rapid detection method for tea polyphenol content. This method utilizes the principle of color change due to self-assembly of tea polyphenols under alkaline conditions, detects the absorbance value at a wavelength of 300~400nm, and then detects the concentration of tea polyphenols.

[0006] However, this patent requires the use of a spectrophotometer to detect absorbance values ​​and the preparation of a standard curve for quantitative analysis. Therefore, in practical applications, it necessitates carrying specialized instruments and accompanying test tubes and other experimental equipment, resulting in poor portability and making it difficult to meet the actual needs of rapid on-site testing. Therefore, developing a simple, instrument-free, and instantly interpretable rapid test strip for tea polyphenols has significant practical importance and application value. Summary of the Invention

[0007] In view of the problems existing in the current tea polyphenol detection technology, the purpose of this invention is to provide a rapid test strip for tea polyphenol content, which can detect samples in batches quickly, and the method is simple and the results are accurate.

[0008] The technical solution adopted in this invention is:

[0009] A rapid test strip for tea polyphenols includes a hydrophobic region and a hydrophilic region. The hydrophilic region is arranged in sequence with an injection area, a reaction area, and a detection area according to the sample migration order. The detection area is in close contact with the hydrophobic region. An alkaline substance is fixed in the reaction area, and a metal salt is fixed in the detection area.

[0010] Furthermore, the rapid test strip for tea polyphenols is strip-shaped, and along the length of the strip, there are sequentially arranged sample injection area, reaction area, detection area, and hydrophobic area. The sample injection area, reaction area, and detection area are all hydrophilic areas, and the detection area is in close contact with the hydrophobic area.

[0011] The sample injection area, reaction area, detection area, and hydrophobic area can be of any shape, such as rectangle, square, circle, or irregular shape, and are preferably strip-shaped.

[0012] Furthermore, the reaction zone and the detection zone are spaced a certain distance apart and are not in close contact.

[0013] Furthermore, the alkaline substance fixed in the reaction zone is sodium hydroxide, sodium bicarbonate, or potassium bicarbonate, preferably NaOH.

[0014] The alkaline substance in the reaction zone is generally fixed in the following way: an aqueous solution of the alkaline substance is coated on the reaction zone, and after drying, the alkaline substance is fixed in the reaction zone.

[0015] The concentration of the aqueous solution of the alkaline substance is preferably 1~5 mol / L.

[0016] Furthermore, the metal salt is a soluble iron salt, gold salt, silver salt, zinc salt, copper salt, or magnesium salt. The metal salt is preferably a soluble iron salt, and more preferably ferric chloride.

[0017] The metal salt in the detection area is generally fixed in the following way: an aqueous solution of the metal salt is coated on the detection area, and after drying, the metal salt is fixed in the detection area.

[0018] The concentration of the aqueous solution of the metal salt is preferably 0.1~0.3 mol / L.

[0019] The substrate of the rapid test strip for tea polyphenols is a hydrophilic paper-based material, which may be one of filter paper, chromatography paper, nitrocellulose membrane, or glass cellulose paper.

[0020] Furthermore, the present invention also provides a method for preparing a rapid test strip for tea polyphenols, comprising the following steps:

[0021] (1) Cover the wax material with heat and penetrate it into the hydrophilic paper-based material to form a hydrophobic area;

[0022] (2) Apply an aqueous solution of an alkaline substance to the reaction area and an aqueous solution of a metal salt to the detection area. The detection area and the hydrophobic area are in close contact. Dry, fix the alkaline substance and the metal salt, and cut to obtain the rapid test paper for tea polyphenols.

[0023] Furthermore, in step (1), the following method is preferred:

[0024] Hydrophobic regions are drawn on a hydrophilic paper-based material using methods such as hand-drawing, printing, or engraving. The material is then heated to 110-130℃ and held for 10-30 minutes (preferably 20 minutes) to melt the wax and allow it to penetrate to the back side, forming a completely hydrophobic area. This hydrophobic area is in close contact with the detection area to prevent metal ions from reacting with tea polyphenols to form nanoparticles, which could then migrate during chromatography and affect the color development of the detection line.

[0025] For faster and more convenient processing, a wax printer is typically used to print wax onto the designed hydrophobic area.

[0026] Furthermore, in step (2), the aqueous solution of alkaline substance can be drawn on the reaction area of ​​the hydrophilic paper-based material by hand-drawing, printing, special instruments, etc., and the aqueous solution of metal salt can be drawn on the detection area of ​​the hydrophilic paper-based material by hand-drawing, printing, special instruments, etc.

[0027] To more accurately control the reagent volume, it is preferable to use a streak plotter.

[0028] Specifically, this invention uses a scribing apparatus to scribble an aqueous solution of an alkaline substance at a rate of 0.1~2 μL / cm in the reaction zone, and after drying, fixes the alkaline substance onto a paper base. Similarly, it uses a scribing apparatus to scribble an aqueous solution of a metal salt at a rate of 0.1~1 μL / cm in the detection zone, and after drying, fixes the metal salt onto a paper base.

[0029] Furthermore, in a preferred embodiment, a 4 mol / L sodium hydroxide solution is drawn in the reaction area using a scribing apparatus at a rate of 0.1–2 μL / cm, preferably 1 μL / cm, and the NaOH is then fixed onto the paper substrate after drying. Similarly, a 0.2 mol / L ferric chloride solution is drawn in the detection area using a scribing apparatus at a rate of 0.1–1 μL / cm, preferably 0.5 μL / cm, and the ferric chloride is then fixed onto the paper substrate after drying.

[0030] Lines drawn with sodium hydroxide solution can also be called alkali lines.

[0031] Furthermore, in step (2), the drying is preferably carried out at 60~80℃ for 1~3 min.

[0032] Drying removes moisture from the solution, ensuring that the alkali and metal salts are completely fixed within the paper base. Metal ions are less prone to oxidation under dry conditions, effectively extending the shelf life of the test paper.

[0033] In the test strip of this invention, the color of the detection area varies depending on the metal ion; when ferric chloride is fixed in the detection area, it appears pale yellow.

[0034] This invention utilizes the principle of metal self-assembly of tea polyphenols to generate colorimetric nanoparticles, and combines it with rapid test strips to design a rapid test strip for tea polyphenols.

[0035] Tea polyphenol rapid test strips generally need to be stored in a dry, light-proof, and sealed container to prevent oxidation of metal salts.

[0036] This invention also provides the application of rapid test strips for tea polyphenols in the rapid detection of tea polyphenol content.

[0037] Furthermore, the present invention also provides a method for rapidly detecting tea polyphenol content using a rapid test strip for tea polyphenols, the method being as follows:

[0038] (1) The tea sample to be tested is brewed and extracted with boiling water to obtain tea soup. The tea soup or the tea beverage sample to be tested is diluted to obtain a colorless diluted solution. The diluted solution is used as the sample to be tested.

[0039] (2) The sample to be tested is added to the injection area. After 1-3 minutes, the color of the detection area is observed and semi-quantitative or quantitative analysis is performed.

[0040] The semi-quantitative analysis is as follows:

[0041] The detection area turns brown, and the color is compared with the standard colorimetric card. The tea polyphenol content is then read according to the standard colorimetric card.

[0042] A standard colorimetric card can be obtained as follows:

[0043] Prepare standard solutions of tea polyphenols of different concentrations, and detect them with rapid test strips of tea polyphenols. After the detection area develops color, collect color photos of the standard solutions of tea polyphenols of different concentrations, and convert them into tea polyphenol content according to the dilution ratio in step (1). Establish a standard colorimetric card corresponding to different tea polyphenol contents and colors.

[0044] The quantitative analysis is as follows:

[0045] The color image of the detection area is acquired, the gray value is extracted, and the tea polyphenol concentration of the sample is calculated based on the standard curve of tea polyphenol concentration and gray value. The tea polyphenol content in the sample is then calculated.

[0046] The standard curve can be obtained using the following method:

[0047] Distilled water without tea polyphenols was tested with a rapid tea polyphenol test strip as a blank control. Tea polyphenol standard solutions of different concentrations were prepared and tested with the rapid tea polyphenol test strip. After the test area developed color, color photos of the tea polyphenol standard solutions of different concentrations were collected. The gray value of the blank control was used as the gray value calibration baseline. The gray value of the standard solution after color development was extracted. A standard curve was plotted with tea polyphenol concentration as the abscissa and gray value as the ordinate.

[0048] Digital electronic imaging devices, such as cameras and scanners, can generally be used to capture color photographs of tea polyphenol standard solutions.

[0049] Grayscale values ​​can be extracted using various image processing software, such as MATLAB, ImageJ, Photoshop, and mobile image processing software.

[0050] The amount of sample added is generally 20~100μL, preferably 40μL.

[0051] Furthermore, the dilution factor of the tea infusion or the tea-containing beverage sample to be tested is 10 to 80 times, preferably 10 to 50 times.

[0052] More preferably, when using semi-quantitative analysis, the dilution factor of the tea infusion or the tea-containing beverage sample to be tested is 10 times, and when using quantitative analysis, the dilution factor of the tea infusion or the tea-containing beverage sample to be tested is 50 times.

[0053] Furthermore, the volume of boiling water used is 40-60 mL / g based on the mass of the tea sample to be tested, preferably 50 mL / g.

[0054] The volume of boiling water used is 40-60 mL / g based on the mass of the tea sample to be tested, preferably 50 mL / g.

[0055] The steeping and extraction time is generally 3 to 10 minutes, preferably 4 minutes. After steeping and extraction, filter to obtain the filtrate, which is the tea soup.

[0056] The tea sample to be tested can be extracted by soaking in boiling water at room temperature and pressure.

[0057] Furthermore, the tea sample to be tested is steeped and extracted with boiling water 1 to 2 times, preferably twice, because after two steeping and extractions, all the soluble tea polyphenols have been extracted from the dry tea.

[0058] Generally, the tea is first steeped in boiling water and the resulting infusion is filtered out to form the first sample solution. Then, it is steeped a second time in boiling water and the resulting infusion is filtered out to form the second sample solution. The first and second sample solutions are then mixed to form the final tea infusion sample solution. This mixture is subsequently diluted for testing.

[0059] The tea samples to be tested can be any variety of tea leaves, tea bags, tea powder, etc. from the six major tea categories. Tea-containing beverage samples can be commercially available tea beverages, which are liquid and therefore only require dilution.

[0060] Dilute the tea infusion or tea-containing beverage sample with distilled water. The purpose of dilution is to reduce the color of the tea infusion or tea-containing beverage sample to colorless. Generally, green tea infusion can be diluted more than 10 times to achieve colorlessness. When using a standard colorimetric card for colorimetric comparison, a 10-fold dilution is preferred. For quantitative analysis using software for photographic detection, a 50-fold dilution is preferred. This facilitates subsequent characterization of tea polyphenol content by observing the intensity of color changes in the detection area.

[0061] Furthermore, the standard curve was validated using the method, and the linear relationship of the tea polyphenol standard curve was good (R > 0.850), and the quadratic relationship was good (R > 0.900). Therefore, the quantitative calculation of the tea polyphenol content in the sample can be simplified using the following method:

[0062] Standard curve formula:

[0063] Based on the gray values ​​of the sample to be tested and the standard solution of tea polyphenols, the percentage content of tea polyphenols in the sample is calculated using the following formula:

[0064] 100%

[0065] in, The content of tea polyphenols in the sample is expressed as % (%). The gray value of tea polyphenols in the sample to be tested; The concentration of the standard solution is expressed in μg / mL. The total volume of the tea infusion or the tea-containing beverage sample to be tested in step (1) is expressed in mL. The mass of the tea sample to be tested is expressed in grams. is the gray value of the tea polyphenol standard solution; n is the dilution factor of the tea soup in step (1); 1000000 is the conversion factor between g and μg.

[0066] Furthermore, for the tea sample to be tested, step (1) is preferably performed as follows:

[0067] (1) Accurately weigh 3g of the tea sample to be tested (usually accurate to 0.01g) into the tasting cup, add 150mL of boiling water, soak for 4min, filter out the tea soup as the first sample solution, then add 150mL of boiling water and soak for 4min, filter out the tea soup as the second sample solution, mix the first sample solution and the second sample solution to obtain the tea soup sample solution, dilute with distilled water 50 times to obtain the diluted solution for later use.

[0068] The simplified formula for calculating the polyphenol content of the tea sample at this time can be:

[0069]

[0070] By comparing the color development of the detection area with the standard colorimetric card or by using a mobile app to take a picture and analyze the shade of brown (such as extracting gray values), the content of tea polyphenols in the sample can be determined.

[0071] The detection temperature is 10-45℃, preferably 15-45℃, and more preferably 25-45℃.

[0072] In this invention, the reaction time is typically 1 to 240 seconds. Generally, the higher the reaction temperature, the shorter the reaction time.

[0073] Preferably, the reaction time is 10 to 180 seconds, and more preferably 30 to 120 seconds.

[0074] In this invention, after the sample is added, the color of the detection area changes from light yellow (the color of ferric chloride) to light brown. As the reaction proceeds, oxidative self-assembled nanoparticles are gradually formed, and the color of the detection line gradually changes from yellow to brown. The higher the content of tea polyphenols in the sample, the darker the color. Generally, the color begins to stabilize after 30 seconds of reaction.

[0075] Generally, colorimetric analysis is performed 2 minutes after sample addition.

[0076] The principle of the reaction in this invention is as follows: After the diluent is added dropwise to the sample injection zone, it moves with the chromatography process. The tea polyphenols in the sample first come into contact with the alkaline substances (such as sodium hydroxide) in the reaction zone and undergo a self-assembly reaction, turning yellow to brown (there is no obvious color change in the reaction zone when the tea polyphenol concentration is <0.06 mg / mL). The self-assembled nanoparticles generated continue to move with the reaction solution and further undergo a self-assembly reaction with the metal salt in the detection zone, generating darker brown nanoparticles. The detection zone and the hydrophobic zone are in close contact, at which point the reaction solution cannot continue to move, thus enriching in the detection zone, which turns brown. The darker the color, the higher the tea polyphenol content.

[0077] This invention utilizes the principle of in-situ self-assembly of tea polyphenols with metals under alkaline conditions to generate colorimetric nanoparticles, providing a simple, rapid, and environmentally friendly rapid test strip and method for detecting tea polyphenols in tea infusions and beverages. Compared with existing technologies, this invention has the following advantages:

[0078] 1) The method of this invention requires no experimental reagents, avoiding the cumbersome preparation of multiple reagents, and is simple, fast, and environmentally friendly. No testing instruments are needed; only a test strip is required, making it convenient and quick.

[0079] 2) Compared with the detection method of tea polyphenols in the national standard, the method of this invention is rapid and can complete the detection in only 2 minutes. It is easy to operate and can be used for rapid on-site detection. Moreover, a large number of samples can be processed in a single experiment, which greatly shortens the experimental time and improves the detection efficiency. It is suitable for rapid on-site detection of tea samples or tea beverages.

[0080] 3) Low production cost. The preparation process of this invention does not use relatively expensive auxiliary materials such as precious metals, metal framework structures, nucleic acids, and proteins, and the production process does not require large-scale precision instruments and equipment.

[0081] The rapid test strip for tea polyphenols of this invention is suitable for on-site testing, meeting the needs of the tea industry chain for rapid identification of tea polyphenol content. It can perform preliminary and rapid identification of green tea quality, completing the detection of tea polyphenol content in a short time without the need for professional instruments. It is easy to operate and provides a practical solution for rapid on-site screening of tea quality, effectively complementing traditional methods. This invention is low-cost, easy to use, and has significant potential for widespread application. Attached Figure Description

[0082] Figure 1 These are comparative photographs showing the color changes of tea infusion, tea polyphenols, theanine, and caffeine under alkaline and metal salt conditions.

[0083] Figure 2 This is the standard curve prepared using tea polyphenol test strips in Example 3.

[0084] Figure 3 This is a standard curve of gallic acid standard in the Folin-Ciocalteu detection method of Example 4.

[0085] Figure 4 These are images showing the process of testing the total polyphenol content of samples using both the test strip method and the national standard method. Figure 4 The top images are photos of the tea sample and the first and second infusions of tea, respectively; the bottom image is a photo of the test solution after color development using the test paper of Example 3 and absorbance testing using the national standard method of Folin-Ciocalteu in Example 4.

[0086] Figure 5 This is a diagram illustrating the preparation process of a rapid test strip for tea polyphenols.

[0087] Figure 6 This is a schematic diagram of the structure of the rapid test strip for tea polyphenols of the present invention.

[0088] Figure 7 It is a color chart comparing the content of tea polyphenols. Detailed Implementation

[0089] The technical solution of the present invention will now be further described with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0090] Example 1

[0091] Accurately weigh 3.00g of tea sample into a 150mL tasting cup, add 150mL of 100℃ boiling water (1:50, m / v), steep for 4min, and filter out the tea soup; dilute the tea soup 10 times for later use.

[0092] Prepare solutions of tea polyphenols, theanine, and caffeine at a concentration of 0.1 mg / ml.

[0093] Take 1 mL of diluted tea infusion, tea polyphenols, theanine, and caffeine solutions and place them separately in centrifuge tubes. Add 10 μL of 1 mol / L NaOH solution. The color development is as follows: Figure 1 As shown in the second line.

[0094] Add 5 μL of 0.02 mol / L FeCl3 solution, and the color development is as follows. Figure 1 As shown in the third line.

[0095] The colorimetric reactions of tea infusion, tea polyphenols, and theanine and caffeine (the main substances in tea infusion besides tea polyphenols) are as follows: Figure 1 As shown, the results indicate that both the tea infusion and tea polyphenols undergo self-assembly reactions to form colored nanomaterials with obvious color development: the infusion turns yellow after the addition of sodium hydroxide and brown after the addition of ferric chloride.

[0096] Theanine and caffeine showed no obvious reaction.

[0097] Therefore, the color change of tea soup is related to the content of tea polyphenols and is not affected by other substances in the tea soup.

[0098] Example 2

[0099] The preparation process of the rapid test strip for tea polyphenols is as follows: Figure 5 As shown.

[0100] 1. Using a wax printer, print gray wax onto hydrophilic color paper according to the designed hydrophobic region pattern, such as... Figure 5 As shown in Figure a.

[0101] 2. Heat at 110℃ or above for 20 minutes to melt the wax and allow it to penetrate the paper, forming a hydrophobic barrier, such as... Figure 5 As shown in Figure b. The gray wax used in the example turns green upon heating.

[0102] 3. Using a scribing apparatus, draw an alkali line on paper with 4 mol / L NaOH solution at a rate of 1 μL / cm; draw a metal line on paper with 0.2 mol / L ferric chloride solution at a rate of 0.5 μL / cm. Figure 5As shown in diagram c, the alkali line and the metal line are spaced a certain distance apart. The metal line is in close contact with the hydrophobic line.

[0103] 4. Dry the test paper at above 50℃ for 2 minutes to fix sodium bicarbonate and ferric chloride onto the paper base, such as... Figure 5 As shown in Figure d.

[0104] 5. Cut the large strip of test paper into strips 0.5cm wide. The test strip is now complete. Figure 5 As shown in Figure e.

[0105] A schematic diagram of the structure of the rapid test strip for tea polyphenols of the present invention is shown below. Figure 6 As shown, it includes a hydrophobic region 1 and a hydrophilic region. The hydrophilic region is arranged in sequence according to the sample migration order, including an injection region 4, a reaction region 3, and a detection region 2. The detection region 2 is in close contact with the hydrophobic region 1. The reaction region 3 is fixed with an alkaline substance such as NaOH, and the detection region 2 is fixed with a metal salt such as ferric chloride.

[0106] Furthermore, the rapid test strip for tea polyphenols is strip-shaped, and along the length of the strip, there are sequentially arranged sample injection area 4, reaction area 3, detection area 2, and hydrophobic area 1. Sample injection area 4, reaction area 3, and detection area 2 are all hydrophilic areas, and the detection area 2 is in close contact with the hydrophobic area 1.

[0107] Furthermore, the reaction zone 3 and the detection zone 2 are spaced a certain distance apart and are not in close contact.

[0108] Example 3

[0109] The polyphenol content of tea samples was detected using tea polyphenol test strips.

[0110] 1) Sample preparation

[0111] After two infusions, all soluble tea polyphenols in the dry tea have been extracted. Therefore, the total polyphenol content in the tea is determined by mixing the two infusions. Specifically, the tea sample is infused twice with boiling water. 3.00g of tea sample is accurately weighed into a 150mL tasting cup, and 150mL of 100℃ boiling water (1:50, m / v) is added. The mixture is infused for 4 minutes, and the resulting infusion is filtered out as the first infusion. Then, another 150mL of 100℃ boiling water is added, and the mixture is infused for another 4 minutes. This second infusion is then filtered out as the second infusion. The first and second infusion solutions are then mixed to obtain the final tea infusion sample solution.

[0112] 2) Preparation of standard solutions

[0113] A blank sample solution was obtained using distilled water without tea polyphenols, following the method in step 1), as a blank control. Different concentrations of tea polyphenol standard solutions (20, 40, 60, 80, 100 μg / mL) were prepared using distilled water. 40 μL of each solution was added to the sample application area of ​​the test strip. After reacting at room temperature for 2 minutes, a photograph was taken, and the grayscale value was extracted. A standard curve of the tea polyphenol standard was plotted with concentration on the x-axis and grayscale value on the y-axis.

[0114] Colorized photographs of the standard curve, linear equations such as Figure 2 As shown.

[0115] 3) Sample testing

[0116] Dilute the above tea sample solution 50 times with distilled water, then add 40 μL to the sample application area of ​​the test strip. After reacting at room temperature for 2 minutes, analyze the color intensity of the detection line. Generally, tea polyphenols change color immediately upon contact with the detection metal line; to ensure complete reaction and color stability, analyze after 2 minutes.

[0117] 4) Quantitative

[0118] Based on the gray value of the sample solution and the standard curve of the tea polyphenol standard, the concentration of tea polyphenols in the sample solution is calculated, which is then converted to the concentration of tea polyphenols in the tea infusion, and further converted to the content of tea polyphenols in the tea sample.

[0119] The method for detecting the polyphenol content in tea samples is shown in Table 1.

[0120] Table 1. Proportion of tea polyphenols in tea infusion determined by tea polyphenol test strips 1 Biluochun 1.0575 10.57 2 Longjing green tea 0.5722 5.72 3 Huangshan Maofeng 0.4439 4.44

[0121] Example 4:

[0122] The accuracy of the test strip detection method and the national standard method for determining tea polyphenol content was compared. The same tea sample was subjected to pretreatment and determination using a rapid method (see Example 2); and the Folin-Ciocalteu method was used for determination (the tea infusion method and rapid detection method were treated the same). The specific methods are as follows:

[0123] 1) Reagent preparation

[0124] To prepare a 10% Folin-Ciocalteu solution: Dissolve 10 mL of Folin-Ciocalteu in 90 mL of water, for a total volume of 100 mL. Add the prepared Folin-Ciocalteu solution to a 500 mL beaker and leave it uncovered for 2 hours.

[0125] To prepare a 7.5% anhydrous Na2CO3 solution: dilute 37.5g of anhydrous Na2CO3 to 500mL.

[0126] 2) Sample preparation

[0127] Weigh 3g of dry tea and put it into the tasting cup. Add 150mL of boiling water at 100℃ and steep for 4 minutes. Filter out the tea soup as the first test sample. Add another 150mL of boiling water at 100℃ and steep for 4 minutes. The resulting tea soup is the second test sample. Mix the first and second infusion solutions to obtain the tea test sample solution.

[0128] 3) Sample pretreatment

[0129] The sample solution was diluted 100 times with distilled water. 1 mL of the solution was then added sequentially to 5 mL of 10% Folin-Ciocalteu solution and 4 mL of 7.5% anhydrous Na₂CO₃ solution. The mixture was shaken well and reacted at room temperature for 1 hour before being used for tea polyphenol content analysis. The blank solution was prepared by adding 1 mL of distilled water, with all other steps identical.

[0130] 4) Preparation of standard solutions

[0131] A blank sample solution was obtained using gallic acid-free distilled water following the method in step 2). The standard solution was diluted with the blank sample solution to a volume ratio of at least 95%, yielding matrix-matched standard solutions (10, 20, 30, 40, 50 μg / mL). 5 mL of 10% Folin-Ciocalteu solution and 4 mL of 7.5% anhydrous Na₂CO₃ solution were added to the standard solutions, and the mixture was shaken well and reacted at room temperature for 1 hour before being used for tea polyphenol content analysis.

[0132] 5) Ultraviolet spectrophotometer analysis

[0133] The OD values ​​of each sample were measured at OD 725 nm using a UV spectrophotometer.

[0134] Images showing the process of detecting total polyphenol content in samples using test strips and the national standard method are shown below. Figure 4 As shown, Figure 4 The top images are photos of the tea sample and the first and second infusions of tea, respectively; the bottom image is a photo of the test solution after color development using the test paper of Example 3 and absorbance testing using the national standard method of Folin-Ciocalteu in Example 4.

[0135] 6) Quantitative

[0136] Based on the OD values ​​of the standard, a standard curve for gallic acid standard was plotted, as follows: Figure 3 As shown in Table 2, the concentrations of tea polyphenols in the sample solution and tea infusion were calculated based on the OD value of the sample solution and the standard curve of gallic acid standard, and the tea polyphenol content in the tea sample was obtained by conversion. The results obtained by the two methods are shown in Table 2.

[0137] Table 2 Comparison of tea polyphenol content in tea samples detected by national standard method and test strips.

[0138] The results showed that the test strip method of this invention yielded similar results to the national standard method, with no significant difference between the two.

[0139] Example 5:

[0140] The addition and recovery test was conducted using the test strip detection method and the national standard method for Folin-Ciocalteu. Green tea infusion was used as the matrix, and the addition level of the standard (gallic acid and tea polyphenols) was 1 times the content of tea polyphenols in the tea infusion matrix. The spiked recovery rate was calculated, and the results are shown in Table 3.

[0141] Table 3 Results of the test strips and the national standard method for adding and recovering the reagents.

[0142] Example 6

[0143] Preparation process of semi-quantitative colorimetric card for tea polyphenols.

[0144] 1) Scale interval division

[0145] GB / T 14456.1 Green Tea: Basic Requirements recommends a polyphenol content of ≥11% as acceptable. Previous studies tested polyphenols in 40 tea samples, with an average content (μ) of 12.8% and a standard deviation (σ) of 4.6%. Based on these data, calibration intervals were defined, and the results are shown in Table 4.

[0146] Table 4. Content Range of Colorimetric Card

[0147] 2) Extract color data

[0148] Different concentrations of tea polyphenol standard solutions (0, 0.8, 1.2, 1.6, 1.8, 2.0 mg / mL) were prepared using distilled water without tea polyphenols. After diluting 10 times, 40 μL of each solution was added to the sample application area of ​​the test strip. The solution was reacted at room temperature for 2 min, and then photographed. The mean values ​​of R (red) / G (green) / B (blue) of the RGB color system were extracted.

[0149] The concentration of the tea polyphenol standard solution is converted to the tea polyphenol content in tea leaves as the color chart scale. The conversion formula is as follows:

[0150]

[0151] 3) Color calibration

[0152] Use graphic design software (such as Photoshop) to input the concentration and corresponding R / G / B values ​​into the fill color. Then, in the HSB color system, fix the B value (brightness) and adjust the color by modifying the H value (hue) and S value (saturation) to make it closely resemble the actual test paper color seen by the naked eye. Additionally, color calibration must be performed in a well-lit environment. The resulting standard colorimetric chart is shown below. Figure 7 As shown.

Claims

1. A rapid test strip for tea polyphenols, characterized in that... The test strip includes a hydrophobic region and a hydrophilic region. The hydrophilic region is arranged in sequence with an injection area, a reaction area, and a detection area according to the sample migration order. The detection area is in close contact with the hydrophobic region. An alkaline substance is fixed in the reaction area, and a metal salt is fixed in the detection area.

2. The rapid test strip for tea polyphenols as described in claim 1, characterized in that... The rapid test strip for tea polyphenols is strip-shaped, and along the length of the strip, it is sequentially provided with a sample injection area, a reaction area, a detection area, and a hydrophobic area. The sample injection area, reaction area, and detection area are all hydrophilic areas, and the detection area is in close contact with the hydrophobic area.

3. The rapid test strip for tea polyphenols as described in claim 1, characterized in that... The reaction zone and the detection zone are separated by a certain distance.

4. The rapid test strip for tea polyphenols as described in claim 1, characterized in that... The alkaline substance fixed in the reaction zone is sodium hydroxide, sodium bicarbonate, or potassium bicarbonate; the metal salt fixed in the detection zone is a soluble iron salt, gold salt, silver salt, zinc salt, copper salt, or magnesium salt.

5. The rapid test strip for tea polyphenols as described in claim 4, characterized in that... The alkaline substance in the reaction zone is fixed in the following way: an aqueous solution of the alkaline substance is coated on the reaction zone, and after drying, the alkaline substance is fixed in the reaction zone; the concentration of the aqueous solution of the alkaline substance is 1~5 mol / L; The metal salt in the detection area is fixed in the following way: an aqueous solution of the metal salt is coated on the detection area, and after drying, the metal salt is fixed in the detection area; the concentration of the aqueous solution of the metal salt is 0.1~0.3 mol / L.

6. The rapid test strip for tea polyphenols as described in claim 1, characterized in that... The substrate of the rapid test strip for tea polyphenols is a hydrophilic paper-based material, which is one of filter paper, chromatography paper, nitrocellulose membrane, and glass cellulose paper.

7. The method for preparing the rapid test strip for tea polyphenols as described in any one of claims 1 to 6, characterized in that... The method includes the following steps: (1) Cover the wax material with heat and penetrate it into the hydrophilic paper-based material to form a hydrophobic area; (2) Apply an aqueous solution of an alkaline substance to the reaction area and an aqueous solution of a metal salt to the detection area. The detection area and the hydrophobic area are in close contact. Dry, fix the alkaline substance and the metal salt, and cut to obtain the rapid test paper for tea polyphenols.

8. The application of the rapid test strip for tea polyphenols as described in any one of claims 1 to 6 in the rapid detection of tea polyphenol content.

9. A method for rapidly detecting tea polyphenol content using the rapid test strip for tea polyphenols as described in any one of claims 1 to 6, characterized in that... The method is as follows: (1) The tea sample to be tested is brewed and extracted with boiling water to obtain tea soup. The tea soup or the tea beverage sample to be tested is diluted to obtain a colorless diluted solution. The diluted solution is used as the sample to be tested. (2) The sample to be tested is added to the injection area. After 1-3 minutes, the color of the detection area is observed and semi-quantitative or quantitative analysis is performed.

10. The method as described in claim 9, characterized in that... The semi-quantitative analysis is as follows: The detection area of ​​the sample turns brown, and the color is compared with the standard colorimetric card. The content of tea polyphenols is then read according to the standard colorimetric card. A standard colorimetric card can be obtained as follows: Prepare standard solutions of tea polyphenols of different concentrations, and detect them with rapid test strips of tea polyphenols. After the detection area develops color, collect color photos of the standard solutions of tea polyphenols of different concentrations, and convert them into tea polyphenol content according to the dilution ratio in step (1). Establish a standard colorimetric card corresponding to different tea polyphenol contents and colors. The quantitative analysis is as follows: The color image of the detection area of ​​the sample to be tested is acquired, the gray value is extracted, and the concentration of tea polyphenols in the sample is calculated based on the standard curve of tea polyphenol concentration and gray value. The tea polyphenol content in the sample is then calculated. The standard curve can be obtained using the following method: Distilled water without tea polyphenols was tested with a rapid tea polyphenol test strip as a blank control. Tea polyphenol standard solutions of different concentrations were prepared and tested with the rapid tea polyphenol test strip. After the test area developed color, color photos of the tea polyphenol standard solutions of different concentrations were collected. The gray value of the blank control was used as the gray value calibration baseline. The gray value of the standard solution after color development was extracted. A standard curve was plotted with tea polyphenol concentration as the abscissa and gray value as the ordinate.

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