Fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables and application of fluorescent test paper

Abstract

The invention discloses fluorescent test paper for visually detecting methyl mercaptan in fresh-cut brassicaceous vegetables, which takes a hydrophobic paper base as a carrier and is loaded with an HRP modified gold nanoparticle and AOX enzyme compound. The invention also discloses a method for visually detecting the methyl mercaptan in the fresh-cut cruciferous vegetables by using the fluorescent test paper, and a kit comprising the fluorescent test paper and used for visually detecting the methyl mercaptan in the fresh-cut cruciferous vegetables. The fluorescent test paper disclosed by the invention can be used for quickly and visually carrying out qualitative and semi-quantitative detection on methyl mercaptan and dividing the freshness of vegetables. The visual detection method is convenient, efficient and low in cost and can be used for real-time monitoring and field application, only one piece of fluorescent test paper needs to be arranged in the vegetable packaging process, sampling and package opening are not needed during detection, nondestructive detection can be conducted, the detection efficiency is greatly improved, the method can be used for preservation and quality monitoring of the fresh-cut brassicaceous vegetables, and the method is suitable for popularization and application. Powerful technical support can be provided for the quality guarantee of vegetable products.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid food detection, and particularly relates to a fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables and an application thereof. Background Art

[0002] Methyl mercaptan, a volatile sulfur compound with a strong odor, is one of the main volatile components in cruciferous vegetables such as broccoli. Its release is closely related to the freshness and storage conditions of the broccoli and often precedes yellowing. Methyl mercaptan release often causes an off-flavor in cruciferous vegetables, affecting their quality and marketability.

[0003] Methyl mercaptan, the precursor of which is S-methyl-L-cysteine sulfoxide, is an important nutrient in cruciferous vegetables. It not only plays a key role in the growth and metabolism of vegetables but also exhibits biological activities such as anti-cancer, promoting intestinal health, and supporting the immune system. It has significant health benefits and is beneficial for maintaining human health. Excessive release of methyl mercaptan not only causes an off-flavor in broccoli but also may reduce its nutritional value. Therefore, measuring the methyl mercaptan content in fresh-cut cruciferous vegetables is not only an effective way to monitor off-flavor production but also a means to assess nutrient loss. This is important for ensuring the quality of fresh-cut cruciferous vegetables and extending their shelf life.

[0004] The detection methods of methyl mercaptan in the prior art mainly rely on high-precision analysis technologies such as gas chromatography and mass spectrometry. Although these methods have advantages in terms of accuracy, they have many limitations: they rely on expensive and complex instruments and equipment, the analysis process is time-consuming, and professional personnel are required to operate and maintain it, which makes it difficult to meet the needs of rapid and on-site detection. To overcome this limitation, fluorescent paper-based detection technology has emerged and has gradually become a promising alternative. Fluorescence detection methods have emerged as an emerging alternative with high sensitivity and selectivity, but there are still problems such as high equipment requirements, fluorescence interference, and complex operation, which to a certain extent limit their application in rapid detection. Based on this, it is particularly important to develop a simple, fast and convenient method for detecting methyl mercaptan that can meet the needs of real-time monitoring and on-site application.

[0005] Existing technologies primarily rely on high-precision analytical methods such as gas chromatography and mass spectrometry for detecting methyl mercaptan. While these methods offer excellent accuracy, they suffer from numerous limitations: they rely on expensive and complex instrumentation, the analysis process is time-consuming, and they require high levels of operator expertise, making them difficult to apply to rapid on-site testing scenarios.

[0006] Fluorescence detection technology has emerged as an emerging alternative. It offers high sensitivity and selectivity, along with convenient visualization. Test results are presented as fluorescent signals, allowing for direct visual inspection of signal strength. This allows for rapid qualitative and even semi-quantitative results, eliminating the need for complex instrumentation and reducing testing costs and operational complexity. Furthermore, the simple and rapid detection process, with a rapid response and no need for complex pre-processing or lengthy analysis, makes it particularly suitable for on-site, immediate testing.

[0007] However, there are still some problems with fluorescence detection: some methods have high requirements for equipment to detect fluorescence signals and are easily affected by fluorescence interference. On-site sampling is more troublesome and quantitative operations are more complicated. These factors restrict its widespread application in the field of rapid detection.

[0008] In view of this, it is extremely urgent to develop a simple, rapid and economical method for detecting methyl mercaptan to meet the needs of real-time monitoring and on-site applications. Summary of the Invention

[0009] To solve the above technical problems, the purpose of the present invention is to provide a fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, and to provide its preparation method and application. The fluorescent test paper of the present invention can be used for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables. The detection method is convenient, efficient, low-cost and does not require complex equipment, which is convenient for supervisory departments and consumers to quickly judge the freshness of fresh-cut cruciferous vegetables.

[0010] The technical solution adopted in the present invention is:

[0011] A fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, wherein the fluorescent test paper uses a hydrophobic paper substrate as a carrier and is loaded with HRP-modified gold nanoparticles (HRP-AuNCs) and an AOX enzyme complex;

[0012] HRP refers to horseradish peroxidase.

[0013] Furthermore, the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables can be prepared according to the following method:

[0014] The hydrophobic paper substrate is used as a carrier, a mixture of HRP-modified gold nanoparticles and AOX enzyme is added dropwise, and the mixture is dried to prepare the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

[0015] Fluorescent test paper needs to be stored sealed and away from light.

[0016] Drying is generally done at room temperature for 1 to 2 hours.

[0017] The mixture of HRP-modified gold nanoparticles and AOX enzyme is prepared by mixing HRP-AuNCs solution and AOX enzyme dilution solution in a volume ratio of 1:1 to 6;

[0018] In the HRP-AuNCs solution, the concentration of HRP-modified gold nanoparticles is 12-60 μM (preferably 30-40 μM), and the solvent is water.

[0019] In the AOX enzyme diluent, the solvent is PBS buffer, and the activity concentration of the AOX enzyme is 0.1 to 3 U / mL, preferably 0.2 to 0.5 U / mL.

[0020] Furthermore, the 50-250 U / mL AOX enzyme solution can be diluted 100-300 times to obtain an AOX enzyme dilution solution.

[0021] Furthermore, the HRP-AuNCs solution was prepared by biomineralization under light-proof conditions, and the specific steps were as follows:

[0022] Under light-proof conditions, a 1-2 mM aqueous solution of tetrachloroauric acid and an 8-15 mg / mL aqueous solution of horseradish peroxidase were mixed in a volume ratio of 1:1 and vigorously stirred at 37°C for 10-30 minutes. Then, a 0.5-2 M aqueous solution of sodium hydroxide was added dropwise in a volume ratio of 0.1-0.2:1 between the aqueous sodium hydroxide solution and the aqueous tetrachloroauric acid solution. The mixture was stirred at 37°C for 8-12 hours. The reaction solution changed from reddish brown to dark green, thereby obtaining an HRP-AuNCs solution. The HRP-AuNCs solution was stored in the dark at room temperature and diluted to 12-60 μM before use.

[0023] Furthermore, the hydrophobic paper base is a paper base material with a hydrophobically modified surface, or a plastic composite paper made of a hydrophobic polymer, such as PTFE composite paper, FEP composite paper, PE composite paper, and HDPE composite paper.

[0024] Furthermore, the volume of the mixed solution of HRP-modified gold nanoparticles and AOX enzyme added dropwise is 1 to 3 μL.

[0025] The present invention also provides a method for preparing a fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, the method comprising the following steps:

[0026] (1) Under light-proof conditions, 1-2 mM aqueous solution of tetrachloroauric acid and 8-15 mg / mL aqueous solution of horseradish peroxidase were mixed in a volume ratio of 1:1, stirred vigorously at 37°C for 10-30 minutes, and then 0.5-2 M aqueous solution of sodium hydroxide was added dropwise. The volume ratio of the aqueous solution of sodium hydroxide to the aqueous solution of tetrachloroauric acid was 1:1.

[0027] 0.1-0.2:1, stirred at 37°C for 8-12 h, the reaction solution changed from reddish brown to dark green, and the HRP-AuNCs solution was prepared. Stored in the dark at room temperature, diluted to 12-60 μM before use;

[0028] (2) Mixing 12-60 μM HRP-AuNCs solution with AOX enzyme dilution solution at a volume ratio of 1:1-6 to prepare a mixture of HRP-modified gold nanoparticles and AOX enzyme;

[0029] In the AOX enzyme diluent, the solvent is PBS buffer, and the activity concentration of the AOX enzyme is 0.1 to 3 U / mL, preferably 0.2 to 0.5 U / mL.

[0030] (3) Using a hydrophobic paper substrate as a carrier, 1 to 3 μL of a mixture of HRP-modified gold nanoparticles and AOX enzyme was added dropwise and dried to prepare the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

[0031] The present invention also provides application of the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables in visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

[0032] Furthermore, the application method is:

[0033] Fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables and the fresh-cut cruciferous vegetables are sealed and stored in a transparent film. Under ultraviolet light with a wavelength of 250 to 365 nm, the color change of the fluorescent test paper is observed to perform qualitative analysis, semi-quantitative analysis or quantitative analysis.

[0034] The present invention also provides a method for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, which comprises:

[0035] Fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables and the fresh-cut cruciferous vegetables are sealed and stored in a transparent film. Under ultraviolet light with a wavelength of 250 to 365 nm, the color change of the fluorescent test paper is observed to perform qualitative analysis, semi-quantitative analysis or quantitative analysis.

[0036] The fluorescent test paper is located in a transparent film package, with the side loaded with the complex of HRP-modified gold nanoparticles and AOX enzyme facing outward. The fluorescent test paper can be directly observed through the transparent film, and the other side is in direct contact with the vegetables.

[0037] The qualitative analysis means that under ultraviolet light irradiation, the color of the fluorescent spot on the test paper changes from the original pink to pink-white or pink-purple, and further to purple, indicating that methyl mercaptan is generated.

[0038] Semi-quantitative analysis involves observing the color of the fluorescent spot on a test paper under ultraviolet light, comparing it with a standard colorimetric chart, and reading the concentration range of methyl mercaptan based on the standard colorimetric chart. This can be used to determine the freshness of vegetables.

[0039] The standard colorimetric card can be obtained as follows:

[0040] Fluorescent test paper for visual detection of methyl mercaptan in fresh-cut cruciferous vegetables is placed in a closed reaction vessel with constant temperature and humidity. Then, methyl mercaptan gas samples of different concentrations are injected and reacted for 20 to 40 minutes. Under ultraviolet light with a wavelength of 250 to 365 nm, fluorescence color images at different methyl mercaptan concentrations are collected, and a standard colorimetric card corresponding to different concentrations and fluorescence colors is established.

[0041] Generally, digital electronic imaging equipment, such as cameras and scanners, can be used to capture fluorescent color images.

[0042] The quantitative analysis refers to collecting the fluorescence color image on the test paper under ultraviolet light, extracting the RGB value of the fluorescence color, and calculating the concentration of methyl mercaptan based on a standard curve of methyl mercaptan concentration and RGB value.

[0043] The standard curve can be obtained as follows:

[0044] Fluorescent test paper for visual detection of methyl mercaptan in fresh-cut cruciferous vegetables was placed in a closed reaction vessel at constant temperature and humidity. Methyl mercaptan gas samples of different concentrations were then injected and allowed to react for 20 to 40 minutes. Under ultraviolet light irradiation with a wavelength of 250 to 365 nm, fluorescence color images at different methyl mercaptan concentrations were collected, and the RGB values of the fluorescence colors were extracted. A standard curve was plotted with the methyl mercaptan concentration as the horizontal axis and the ratio of the R value to the B value as the vertical axis.

[0045] Image processing software such as PHOTOSHOP can be used to extract the RGB value of the fluorescent color.

[0046] The preferred wavelength of ultraviolet light is 254 nm.

[0047] Furthermore, the humidity in the sealed reaction vessel at a constant temperature and humidity is 50% to 80% relative humidity, and the humidity is constant during the reaction. Humidity has little effect on the fluorescence quenching reaction and color change.

[0048] The temperature of the sealed reaction vessel with constant temperature and humidity is within the range of 4 to 37° C., and the temperature is kept constant during the reaction.

[0049] By monitoring the changes in methyl mercaptan content, the freshness of fresh-cut cruciferous vegetables can be detected.

[0050] Specifically, the present invention also provides a kit for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, which includes fluorescent test paper and a standard colorimetric card for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

[0051] The kit may further comprise a 254 nm ultraviolet lamp.

[0052] The present invention also provides a kit for visually detecting the freshness of fresh-cut cruciferous vegetables. The kit comprises fluorescent test paper for visually detecting methyl mercaptan in the fresh-cut cruciferous vegetables and a standard colorimetric card.

[0053] The present invention also provides a method for visually detecting the freshness of fresh-cut cruciferous vegetables, which comprises:

[0054] Fluorescent test paper for visual detection of methyl mercaptan in fresh-cut cruciferous vegetables is stored in a transparent film-sealed package. Under ultraviolet light with a wavelength of 250-365 nm, the color change of the fluorescent test paper is observed and compared with a standard colorimetric card. The concentration range of methyl mercaptan is read according to the standard colorimetric card to classify the freshness;

[0055] The standard colorimetric card is obtained as follows:

[0056] Fluorescent test paper for visual detection of methyl mercaptan in fresh-cut cruciferous vegetables was placed in a closed reaction vessel at a constant temperature and humidity. Methyl mercaptan gas samples of varying concentrations were then injected and allowed to react for 20 to 40 minutes. Under ultraviolet light irradiation at a wavelength of 250 to 365 nm, fluorescence color images were collected at varying methyl mercaptan concentrations, and a standard colorimetric card corresponding to different concentrations and fluorescence colors was established. Freshness was then classified according to methyl mercaptan concentration as follows: 0 to 20 ppm for freshness, 20 to 50 ppm for moderate freshness, and above 50 ppm for not freshness and the presence of spoilage.

[0057] The present invention can more accurately evaluate the freshness of fresh-cut cruciferous vegetables through semi-quantitative analysis and quantitative analysis, thereby further improving the monitoring accuracy of the quality of fresh-cut cruciferous vegetables.

[0058] The present invention designs and synthesizes a complex of HRP-modified gold nanoparticles (HRP-AuNCs) and AOX enzyme on a paper-based surface, and uses the complex to monitor the concentration of methyl mercaptan. The principle is that HRP-AuNCs turn pink under ultraviolet light, and the AOX enzyme can react with methyl mercaptan to release hydrogen peroxide (H2O2). The hydrogen peroxide quenches the fluorescence of HRP-AuNCs, thereby monitoring the concentration change of methyl mercaptan through the fluorescence quenching effect.

[0059] The present invention develops a kind of fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, which can quickly visualize methyl mercaptan and carry out qualitative and semi-quantitative detection, and can be combined with electronic imaging equipment and image processing software to carry out quantitative detection, thus distinguishing the freshness of vegetables, the higher the methyl mercaptan content, the less fresh the vegetables are. Fluorescent test paper, test kit and visual detection method of the present invention, effectively overcome the problems such as the complicated pre-treatment and instrument and equipment condition requirements of existing detection methods, visual detection method is convenient, efficient and low cost, only needs to be built-in a fluorescent test paper when vegetables are packaged, no sampling is required during detection, no packaging is needed, non-destructive testing can be carried out, greatly improving detection efficiency.

[0060] The fluorescent test paper of the present invention is suitable for real-time monitoring and on-site application, and is particularly suitable for the preservation and quality monitoring of fresh-cut cruciferous vegetables, and can provide strong technical support for the quality assurance of vegetable products. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the standard curve of methyl mercaptan concentration.

[0062] Figure 2 It is a colorimetric card for the standard concentration of methyl mercaptan.

[0063] Figure 3 The color change of fluorescent test paper after fresh-cut broccoli is observed at different storage times.

[0064] Figure 4 The figure shows the line graph of methyl mercaptan content in fresh-cut broccoli at different storage times detected by two methods. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0066] Example 1

[0067] Step 1: Preparation of HRP-AuNCs

[0068] HRP-AuNCs were prepared by biomineralization under light-shielding conditions. The specific steps are as follows:

[0069] (1) 1 mL of tetrachloroauric acid aqueous solution (1.5 mM) and 1 mL of horseradish peroxidase aqueous solution (10 mg / mL) were vigorously stirred at 37°C for 20 min.

[0070] (2) 100 μL of sodium hydroxide (1 M) aqueous solution was added to the above mixed solution, and then stirred at 37 °C for 10 h until the solution changed from reddish brown to dark green. The prepared HRP-AuNCs solution (HRP was 120 μM) was stored at room temperature in the dark.

[0071] Step 2:

[0072] 4 μL of AOX enzyme (50 U / mL) was diluted with 1000 μL of PBS buffer (0.01 M, pH 7.2-7.4) to prepare an AOX enzyme dilution solution.

[0073] 25 μL of HRP-AuNCs solution (36 μM) and 150 μL of AOX enzyme diluent were mixed to prepare a mixture of HRP-modified gold nanoparticles and AOX enzyme.

[0074] Step 3:

[0075] Use a pipette to transfer 1 μL of the mixture of HRP-modified gold nanoparticles and AOX enzyme, drop it onto the hydrophobic PTFE composite paper substrate, and dry it at room temperature for 1 hour to prepare the fluorescent test paper, which is then stored in a sealed and light-proof condition.

[0076] Step 4: Make the song

[0077] Above-mentioned test paper is placed in the reaction box of constant humidity airtight (60% relative humidity), take methyl mercaptan 0,10,20,30,410,50,60,70,80,90,100ppm of different concentrations, be placed in sealed bottle, put into reaction box, uncap and heat, ensure that methyl mercaptan fully volatilizes, constant temperature 37 DEG C in reaction box, cultivate balance 30min, observe test paper color change under ultraviolet lamp 254nm. Camera takes pictures and obtains image, utilizes image processing software to identify the RGB value of fluorescence color in extraction photo, R, G, B value and methyl mercaptan concentration are carried out fitting curve, after multiple fitting, find that the ratio of R / B is inversely proportional to methyl mercaptan concentration, therefore take methyl mercaptan concentration as horizontal coordinate, ordinate is R / B ratio, draws standard curve, as shown in Figure 2. Figure 1 As shown, the linear equation is y = -0.0056*x + 1.110 (R 2 =0.99321).

[0078] According to the above method, 20, 40, 60, and 80 ppm of methyl mercaptan were spiked and reacted with the fluorescent test paper. The operating steps were the same as above. After acquiring the image, the R / B value was extracted and substituted into the standard curve to obtain the detected concentration and calculate the recovery rate. This was repeated three times in parallel. The results are shown in Table 1 below.

[0079] Table 1

[0080]

[0081] It can be seen that the quantitative analysis accuracy of the method of the present invention is high.

[0082] Example 2

[0083] Make a color chart.

[0084] According to step 4 of Example 1, the reaction temperature was 37°C, and the fluorescent test paper was placed at methyl mercaptan concentrations of 0, 10, 20, 30, 410, 50, 60, 70, 80, 90, and 100 ppm for reaction for 30 min. Under 254 nm ultraviolet light, a camera was used to take pictures, and a colorimetric card corresponding to different concentrations and fluorescent colors was established, as shown in FIG. Figure 2 shown. Figure 2 It shows that as the concentration of methyl mercaptan increases, the fluorescent color changes from pink to pink-purple and then to purple. The freshness of vegetables can be divided based on this. 0-20ppm is considered fresh, 20-50ppm is medium freshness, and above 50ppm is considered not fresh and is rotten.

[0085] What I want to explain is that different temperatures have a certain impact on color. During quantitative analysis, it is generally possible to establish standard curves at different temperatures, and compare and calculate based on the temperature at which the sample is tested with the standard curve at that temperature.

[0086] In semi-quantitative analysis, subjective errors are difficult to avoid when visually observing changes in fluorescence color. The differences between colorimetric charts at different temperatures may be beyond the range of the naked eye, so colorimetric charts at different temperatures are generally not developed. Standard colorimetric charts are generally prepared and validated at room temperature or 37°C. Sample storage or testing temperatures can be flexibly selected within a range of 4 to 37°C. During actual colorimetric analysis, direct comparison with the standard colorimetric chart is sufficient, without being limited by the temperature at which the colorimetric chart was developed.

[0087] Example 3 Actual sample detection

[0088] Fresh-cut broccoli was sealed in transparent plastic wrap and placed inside a fluorescent test strip. The side loaded with the HRP-modified gold nanoparticle-AOX enzyme complex faced outward, allowing for direct observation through the plastic wrap. The other side was in contact with the broccoli. The strip was then stored in a refrigerator at 4°C for 15 days. Every three days, the test strip was illuminated with a 254nm UV lamp to obtain a fluorescence image and observe any color changes.

[0089] The results are as follows Figure 3 As shown, Figure 3 The fluorescence color changes from pink to pink-purple and then to purple. On the third day, the methyl mercaptan concentration is about 0-10 ppm according to the color card, indicating freshness. On the sixth and ninth days, the methyl mercaptan concentration is about 30-40 ppm according to the color card, indicating medium freshness. On the 12th and 15th days, the methyl mercaptan concentration is about 50-60 ppm according to the color card, indicating that it is not fresh. The line graph of the change of methyl mercaptan concentration of the sample over time is shown as follows. Figure 4 shown.

[0090] GC-MS detection

[0091] Fresh-cut broccoli was sealed and stored in a refrigerator at 4°C for 15 days. Every 3 days, 1.5 g of fresh-cut broccoli was taken as a sample for testing. 1.5 g of sample and 2 mL of distilled water were placed in a glass vial (15 mL). Then, N-nonane was added as an internal standard. Volatile compounds were extracted using headspace solid phase microextraction (HS-SPME) and detected using GC-MS. The volatile compound content was calculated by comparing the peak area with the peak area of the internal standard, and the results were expressed as μg kg - 1 FW. The experiment was run twice, and samples were prepared in duplicate and triplicate, respectively.

[0092] The content of methyl mercaptan in the volatile compounds was converted into ppm, and the methyl mercaptan content at different storage times was plotted, as shown in FIG. Figure 4 shown. Figure 4 The results of the fluorescence test paper and the GC-MS test results are similar and have the same trend, which proves that the semi-quantitative analysis results of the fluorescence test paper of the present invention are accurate and reliable.

Claims

1. A fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, wherein the fluorescent test paper is based on a hydrophobic paper substrate and loaded with HRP-modified gold nanoparticles and an AOX enzyme complex.

2. The fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables as claimed in claim 1, characterized in that Prepared as follows: The hydrophobic paper substrate is used as a carrier, a mixture of HRP-modified gold nanoparticles and AOX enzyme is added dropwise, and the mixture is dried to prepare the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

3. The fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables as claimed in claim 2, characterized in that The mixture of HRP-modified gold nanoparticles and AOX enzyme is prepared by mixing HRP-AuNCs solution and AOX enzyme dilution solution in a volume ratio of 1:1 to 6; In the HRP-AuNCs solution, the concentration of HRP-modified gold nanoparticles is 12 to 60 μM, and the solvent is water; In the AOX enzyme diluent, the solvent is PBS buffer, and the activity concentration of the AOX enzyme is 0.1-3 U / mL; The HRP-AuNCs solution was prepared in the dark according to the following steps: Under light-proof conditions, a 1-2 mM aqueous solution of tetrachloroauric acid and an 8-15 mg / mL aqueous solution of horseradish peroxidase were mixed in a volume ratio of 1:1 and vigorously stirred at 37°C for 10-30 minutes. Then, a 0.5-2 M aqueous solution of sodium hydroxide was added dropwise in a volume ratio of 0.1-0.2:1 between the aqueous sodium hydroxide solution and the aqueous tetrachloroauric acid solution. The mixture was stirred at 37°C for 8-12 hours. The reaction solution changed from reddish brown to dark green to obtain an HRP-AuNCs solution. The HRP-AuNCs solution was stored in the dark at room temperature and diluted to 12-60 μM before use.

4. The fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables as claimed in claim 1, characterized in that The hydrophobic paper base is a paper base material with a hydrophobic surface modification, or a plastic composite paper made of a hydrophobic polymer.

5. The method for preparing a fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables according to any one of claims 1 to 4, characterized in that The method comprises the following steps: (1) Under light-proof conditions, a 1-2 mM aqueous solution of tetrachloroauric acid and an 8-15 mg / mL aqueous solution of horseradish peroxidase were mixed in a volume ratio of 1:1 and vigorously stirred at 37°C for 10-30 minutes. Then, a 0.5-2 M aqueous solution of sodium hydroxide was added dropwise, and the volume ratio of the aqueous solution of sodium hydroxide to the aqueous solution of tetrachloroauric acid was 0.1-0.2:

1. The mixture was stirred at 37°C for 8-12 hours. The reaction solution changed from reddish brown to dark green, thereby obtaining an HRP-AuNCs solution. The solution was stored in the dark at room temperature and diluted to 12-60 μM before use. (2) Mixing 12-60 μM HRP-AuNCs solution with AOX enzyme dilution solution at a volume ratio of 1:1-6 to prepare a mixture of HRP-modified gold nanoparticles and AOX enzyme; In the AOX enzyme diluent, the solvent is PBS buffer, and the activity concentration of the AOX enzyme is 0.1-3 U / mL; (3) Using a hydrophobic paper substrate as a carrier, 1 to 3 μL of a mixture of HRP-modified gold nanoparticles and AOX enzyme was added dropwise and dried to prepare the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

6. Use of the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables according to any one of claims 1 to 4 in visually detecting methyl mercaptan in fresh-cut cruciferous vegetables.

7. A method for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, characterized in that The method is: The fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables according to any one of claims 1 to 4 and the fresh-cut cruciferous vegetables are sealed and stored in a transparent film, and the color change of the fluorescent test paper is observed under ultraviolet light with a wavelength of 250 to 365 nm to perform qualitative analysis, semi-quantitative analysis or quantitative analysis.

8. The method according to claim 7, wherein The qualitative analysis refers to the fact that under ultraviolet light, the color of the fluorescent spot on the test paper changes from the original pink to pink-white or pink-purple or purple, indicating the formation of methyl mercaptan; The semi-quantitative analysis refers to observing the color of the fluorescent spot on the test paper under ultraviolet light, comparing it with a standard colorimetric card, and reading the concentration range of methyl mercaptan according to the standard colorimetric card; The standard colorimetric card is obtained as follows: Fluorescent test paper for visual detection of methyl mercaptan in fresh-cut cruciferous vegetables was placed in a sealed reaction vessel at a constant temperature and humidity. Methyl mercaptan gas samples of varying concentrations were then injected and allowed to react for 20 to 40 minutes. Under ultraviolet light irradiation at a wavelength of 250 to 365 nm, fluorescence color images were collected at different methyl mercaptan concentrations to establish a standard colorimetric chart corresponding to different concentrations and fluorescence colors. The quantitative analysis refers to collecting the fluorescence color image on the test paper under ultraviolet light, extracting the RGB value of the fluorescence color, and calculating the concentration of methyl mercaptan based on the standard curve of methyl mercaptan concentration and RGB value; The standard curve was obtained as follows: Fluorescent test paper for visual detection of methyl mercaptan in fresh-cut cruciferous vegetables was placed in a closed reaction vessel at constant temperature and humidity. Methyl mercaptan gas samples of different concentrations were then injected and allowed to react for 20 to 40 minutes. Under ultraviolet light irradiation with a wavelength of 250 to 365 nm, fluorescence color images at different methyl mercaptan concentrations were collected, and the RGB values of the fluorescence colors were extracted. A standard curve was plotted with the methyl mercaptan concentration as the horizontal axis and the ratio of the R value to the B value as the vertical axis.

9. A kit for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables, comprising the fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables according to any one of claims 1 to 4 and a standard colorimetric card.

10. A method for visually detecting the freshness of fresh-cut cruciferous vegetables, characterized in that The method is: The fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables according to any one of claims 1 to 4 is sealed and stored with the fresh-cut cruciferous vegetables in a transparent film, and the color change of the fluorescent test paper is observed under ultraviolet light with a wavelength of 250 to 365 nm, and the color change is compared with a standard colorimetric card. The concentration range of methyl mercaptan is read according to the standard colorimetric card to classify the freshness; The standard colorimetric card is obtained as follows: The fluorescent test paper for visually detecting methyl mercaptan in fresh-cut cruciferous vegetables according to any one of claims 1 to 4 is placed in a closed reaction vessel with constant temperature and humidity, and then methyl mercaptan gas samples of different concentrations are injected, reacting for 20 to 40 minutes, and collecting fluorescence color images at different methyl mercaptan concentrations under ultraviolet light irradiation with a wavelength of 250 to 365 nm, and establishing a standard colorimetric card corresponding to different concentrations and fluorescence colors; and the freshness is divided according to the concentration of methyl mercaptan as follows: 0 to 20 ppm is fresh, 20 to 50 ppm is moderately fresh, and above 50 ppm is not fresh and is spoiled.

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