Fluorescent probe for identifying biogenic amine as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for identifying biogenic amine as well as a preparation method and application of the fluorescent probe, and relates to the technical field of biogenic amine detection. According to the invention, phenothiazine with high fluorescence quantum yield is used as a fluorophore to ensure that the probe has an obvious fluorescence signal to facilitate visual detection, an amine substance recognition group 2, 4-thiazolidinedione is introduced to synthesize a novel OFF-ON type fluorescent probe, and the fluorescent probe is rapid in response to biogenic amine and low in detection limit; the prepared sensing label loaded with the fluorescent probe realizes lossless visual monitoring of the freshness of the fish meat, and has obvious sunlight and fluorescence color signal changes.

Description

Technical Field

[0001] The present invention relates to the technical field of biogenic amine detection, and in particular to a fluorescent probe for identifying biogenic amines, and a preparation method and application thereof. Background Art

[0002] Food safety has become an important factor affecting human health and has attracted worldwide attention. Improper handling of food during production, transportation, storage, and sales will cause the quality of food to deteriorate and eventually lead to food corruption. Especially for protein-rich foods, the action of endogenous enzymes and microorganisms in the food will cause amino acids to degrade and form toxic biogenic amines. Biogenic amines not only affect the quality of food, but also endanger human health and cause various diseases, such as headaches, diarrhea, etc. In severe cases, they can even lead to shock or death.

[0003] Salmon is loved by people because of its plump meat, rich in high-quality polyunsaturated fatty acids such as DHA and EPA, trace elements and a large amount of high-quality protein. However, salmon and other fish foods are one of the most easily perishable foods due to their soft tissue, high water and protein content. Therefore, in order to ensure human health, it is crucial to accurately assess the freshness of fish meat.

[0004] At present, traditional methods for evaluating the freshness of fish meat include total volatile basic nitrogen (TVB-N), total viable count (TVC), pH value, thiobarbituric acid value (TBA), etc. These methods require contact and destruction of samples, and require complex pretreatment steps. Although new detection methods such as electrochemistry, chromatography, and biosensor arrays can judge the freshness of fish meat by directly detecting the content of biogenic amines, they usually require expensive and complex equipment and cumbersome operations, which severely limits their applicability in real-time monitoring of fish meat freshness. Therefore, the development of simple, non-contact, rapid, and accurate methods for detecting the freshness of fish meat is crucial for food safety supervision and human health protection.

[0005] In recent years, small molecule fluorescent probes have been widely used in environmental monitoring and food safety analysis due to their advantages of non-destructive detection, simplicity, high efficiency, and sensitivity. In this regard, the inventor believes that how to design a fluorescent probe with a rapid response to biogenic amines and a low detection limit, and to achieve visual detection of biogenic amines, is a technical problem that needs to be solved urgently by technicians in this field.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In view of the above technical problems, the embodiments of the present invention provide a fluorescent probe for identifying biogenic amines and a preparation method and application thereof to solve the problems raised in the above background technology.

[0008] A fluorescent probe, characterized in that the structural formula is as follows: ; The above fluorescent probe was named MPZ.

[0009] A method for preparing a fluorescent probe as claimed in claim 1, comprising the following steps: S21: Compound a, 2,4-thiazolidinedione and piperidine were added to dry ethanol and heated under reflux for 4 h for reaction; S22: After the heating reflux reaction is completed, the precipitate is filtered and washed with ethanol to prepare the fluorescent probe; Wherein, the structural formula of compound a is as follows: .

[0010] Preferably, the synthetic route of the fluorescent probe is as follows: .

[0011] An application of the fluorescent probe as described above in the recognition of biogenic amines.

[0012] Specifically, the biogenic amine includes any one of cyclohexanediamine, diethylamine, n-propylamine, triethylamine, ethylamine, spermine, cadaverine, putrescine, 2-phenylethylamine, tyramine, trimethylamine, dimethylamine, ammonia water and hydrazine.

[0013] Preferably, the fluorescent probe is used to detect biogenic amines in fish meat for evaluating the freshness of the fish meat.

[0014] A sensor label comprises filter paper soaked with the fluorescent probe as described above.

[0015] A method for preparing the sensor tag as described above comprises the following steps: S61: Cut the filter paper into circular slices with a diameter of 1 cm; S61: Soak the circular thin piece of filter paper in a fluorescent probe stock solution with a concentration of 1 mM for 10 minutes, and dry it at 40° C. for 3 h to prepare the sensor label.

[0016] An application of the sensor tag as described above in the evaluation of fish freshness.

[0017] Preferably, the sensor tag changes color under both sunlight and fluorescence after detecting fish meat, and the color change can be used as a detection indicator for evaluating the freshness of fish meat.

[0018] A freshness level indicator card, wherein a first color colorimetric area and a second color colorimetric area are provided on the freshness level indicator card; the first color colorimetric area includes a plurality of colors displayed under sunlight after the above-mentioned sensor tag detects the fish meat, and the second color colorimetric area includes a plurality of colors displayed under fluorescence after the above-mentioned sensor tag detects the fish meat; the color changes in the first color colorimetric area and the second color colorimetric area are used to indicate different levels of freshness of the fish meat.

[0019] Preferably, the first color colorimetric area is provided with a yellow colorimetric area, a light pink colorimetric area and a pink colorimetric area; the yellow colorimetric area indicates that the fish meat is at the first level of freshness and is a superior product, the light pink colorimetric area indicates that the fish meat is at the second level of freshness and is a qualified product, and the pink colorimetric area indicates that the fish meat has been corrupted and deteriorated and is inedible; The second color comparison area is provided with an orange-red colorimetric area, a purple colorimetric area and a gray-blue colorimetric area; the orange-red colorimetric area indicates that the fish meat is at the first level of freshness and is a superior product, the purple colorimetric area indicates that the fish meat is at the second level of freshness and is a qualified product, and the gray-blue colorimetric area indicates that the fish meat has been rotten and deteriorated and is inedible.

[0020] A fluorescent probe for identifying biogenic amines, a preparation method thereof, and an application thereof provided in an embodiment of the present invention have the following beneficial effects: the present invention uses phenothiazine with a high fluorescence quantum yield as a fluorophore to ensure that the probe has an obvious fluorescence signal for easy visual detection, introduces an amine substance recognition group 2,4-thiazolidinedione, and synthesizes a new "OFF-ON" type fluorescent probe MPZ. The fluorescent probe MPZ responds quickly (7 s) to biogenic amines in an EtOH / H2O (4 / 6, v / v) solution, has a low detection limit (0.72 μM), and emits red fluorescence (620 nm); the prepared sensor tag (MPZ / FPS) loaded with MPZ realizes non-destructive visual monitoring of the freshness of fish meat, and has obvious changes in daylight and fluorescence color signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The UV-visible absorption and fluorescence spectra of the probe MPZ (10 μM) in EtOH / H2O (4 / 6, v / v) solution after adding various biogenic amines (50 μM); Among them, biogenic amines include 1: cyclohexanediamine, 2: diethylamine, 3: n-propylamine, 4: triethylamine, 5: ethylamine, 6: spermine, 7: cadaverine, 8: putrescine, 9: 2-phenylethylamine, 10: tyramine, 11: trimethylamine, 12: dimethylamine, 13: ammonia, 14: hydrazine; Figure 1 a is the UV-visible absorption spectrum, inset: color change in sunlight; Figure 1b is the fluorescence spectrum, inset: fluorescence color change; Figure 1 c is the fluorescence response of MPZ to different concentrations of Cad (0-50 μM), inset: the linear relationship between Cad concentration (0-50 μM) and the fluorescence intensity of MPZ at 620 nm; Figure 1 d is the fluorescence intensity changes of probe MPZ and MPZ + Cad at different pH; Figure 1 e is the relationship between fluorescence color and time after Cad (50 μM) was added to the probe MPZ (10 μM); Figure 2 This is a diagram showing the results of studying the reaction mechanism of MPZ in response to Cad; in, Figure 2 a is the inferred reaction mechanism of MPZ and Cad; Figure 2 b is the UV spectra of MPZ + Cad, MPZ + NaOH, and MPZ; Figure 2 c is the fluorescence spectra of MPZ + Cad, MPZ + NaOH, and MPZ; Figure 2 d is the MPZ and MPi frontier molecular orbital energy diagram calculated based on B3LYP / 6-31G; Figure 3 a is the preparation process of sensor tag MPZ / FPS; Figure 3 b is the colorimetric and fluorescence responses of MPZ / FPS to different amine vapors (1: MPZ, 2: cyclohexanediamine, 3: diethylamine, 4: n-propylamine, 5: triethylamine, 6: ethylamine, 7: spermine, 8: cadaverine, 9: putrescine, 10: 2-phenylethylamine, 11: tyramine, 12: trimethylamine, 13: dimethylamine, 14: ammonia, 15: hydrazine, 50 mM); Figure 4 The results of fluorescence monitoring of MPZ / FPS stability for 7 consecutive days; Figure 5 The fluorescence (R+G) / B value of MPZ / FPS for 7 consecutive days Figure 6 The daylight and fluorescence color changes of MPZ / FPS during fish storage at 4°C; Figure 7 a is the changing trend of TVB-N value and pH value in salmon meat stored at 4°C over time; Figure 7 b shows the changing trend of TVC and TBA values ​​in salmon meat stored at 4°C over time; Figure 7c is the daylight and fluorescence color changes of MPZ / FPS stored at 4°C within 8 days; Figure 8 This is a diagram of a freshness level indicator card. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0023] In view of the above technical problems, the embodiments of the present invention provide a fluorescent probe for identifying biogenic amines and a preparation method and application thereof to solve the problems raised in the above background technology.

[0024] 1. Experimental 1.1. Synthesis of the MPZ probe Compound a (3.5 mmol, 1.00 g), 2,4-thiazolidinedione (2 mmol, 0.82 g) and piperidine (0.5 mL) were added to dry ethanol (15 mL) and heated under reflux for 4 h. After the reaction was completed, the precipitate was filtered and washed with ethanol three times to obtain a red solid (0.68 g, 50.4%). Mp 288.4-289.3 °C, QY=0.01% (in EtOH / H2O =4 / 6). 1H NMR (400 MHz, DMSO- d 6 ) δ 12.48 (s, 1H), 7.83 (s, 1H), 7.22 (t, J =8.4 Hz, 6.8 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 7.03(s, 1H), 13C NMR (101 MHz, DMSO- d 6) δ168.04, 167.64, 158.94, 148.47, 142.76, 127.82, 127.18, 126.14, 125.46,123.33, 122.65, 120.12, 116.20, 115.49, 113.68, 99.82, 56.17, 41.64, 12.75.HRMS (ESI - ) m / z, calculated for C 19 H 15 N2O3S2 - [MH] - calcd: 383.0530, found: 383.0515.

[0025] 1.2. Preparation for spectral testing Weigh 3.84 mg of probe MPZ, dissolve it in DMSO to make a 1 mM stock solution, and then dilute it with EtOH / H2O (v / v = 4 / 6) to a final concentration of 10 μM before use. The amine solution required for the experiment was prepared with double distilled water (1: cyclohexanediamine, 2: diethylamine, 3: n-propylamine, 4: triethylamine, 5: ethylamine, 6: spermine, 7: cadaverine, 8: putrescine, 9: 2-phenylethylamine, 10: tyramine, 11: trimethylamine, 12: dimethylamine, 13: ammonia, 14: hydrazine, 50 mM). The prepared ion solution was stored in the refrigerator and diluted to the required concentration when used. Test solution: EtOH / H2O (v / v = 4 / 6, pH = 7.4); Test conditions: λex = 410 nm, λem = 620 nm, slit = 5 / 10.

[0026] 1.3. Preparation of filter paper (MPZ / FPS) sensor tags The filter paper (FPS) was cut into circular sheets with a diameter of 1 cm. Then, the FPS was immersed in a probe stock solution with a concentration of 1 mM for 10 minutes and dried at 40°C for 3 h to obtain the MPZ / FPS sensor label.

[0027] 1.4. Application of sensor tags (MPZ / FPS) in monitoring salmon freshness Fresh salmon were purchased from Jinzhou Aquatic Market, quickly transported to the laboratory for cutting and packaging into transparent culture dishes or lunch boxes. The sensor tag (MPZ / FPS) was placed in the culture dish or lunch box containing fresh fish meat. The experiment was carried out at 4°C (refrigerated). Under refrigerated conditions, the TVB-N, pH, TBA and TAC values ​​of salmon meat were measured every 24 hours using the national standard method, and the daylight and fluorescence changes of MPZ / FPS were recorded.

[0028] The determination of TVB-N value shall refer to GB / T 18108-2019, the determination of TVC value shall refer to GB 4789.2-2022, the determination of TBA value shall refer to GB 5009.181-2016, and the pH value shall be determined using a pH meter.

[0029] 2. Results and Discussion 2.1. Spectral characteristics of MPZ for detecting biogenic amines In order to study the sensing properties of probe MPZ to biogenic amines, UV-visible absorption and fluorescence spectroscopy tests were carried out in EtOH / H2O (v / v = 4 / 6) system solution. Figure 1 As shown in a, MPZ has a weak absorption peak at 437 nm. When different amines are added to the MPZ solution, the absorption peak at 437 nm blue-shifts to 416 nm and is significantly enhanced. At the same time, the color of the solution changes from colorless to yellow ( Figure 1 aInset).

[0030] The addition of amine solution also caused a significant change in the fluorescence spectrum of MPZ. Figure 1 As shown in Figure b, MPZ has a very weak emission peak at 640 nm. When different amines are added, the emission peak at 640 nm blue-shifts to 620 nm, and the fluorescence intensity is greatly enhanced. The fluorescence color of the solution changes from weak dark red to bright orange-red ( Figure 1 b (inset). The results show that the probe MPZ has a good response to biogenic amines and has obvious colorimetric and fluorescence changes, which can realize the naked eye detection of biogenic amines.

[0031] Aquatic products produce a large amount of biogenic amines during the spoilage process. In the late storage period of salmon, the contents of cadaverine (Cad) and putrescine increased by 32.65 and 15.81 times, respectively, which are much higher than the changes of other biogenic amines. Therefore, as representative biogenic amines, the changes in the contents of cadaverine and putrescine can be used to evaluate the freshness of salmon. According to the spectral test results, cadaverine was finally selected for titration experiments to study the response sensitivity of MPZ to Cad. Figure 1As shown in Figure c, as the concentration of cadaverine increases, the fluorescence intensity of the probe MPZ at 620 nm gradually increases. When the Cad concentration is 50 μM, the fluorescence intensity reaches a stable state, and the fluorescence quantum yield changes from 0.01% to 5.49%. From the fluorescence titration data, it can be seen that there is a good linear relationship between the change in MPZ fluorescence intensity and the concentration of Cad (0-50 μM) (Y = 238.72 + 102.03X, R 2 = 0.999), according to the equation LOD = 3S / K, the detection limit of probe MPZ for Cad was calculated to be 0.72 mM ( Figure 1 c inset). This indicates that MPZ has a higher response sensitivity to Cad.

[0032] In addition, different pH environments may affect the detection of Cad by MPZ. In order to explore the pH applicable range of MPZ, the fluorescence changes of MPZ and MPZ + Cad in different pH system solutions were tested. Figure 1 As shown in Figure d, MPZ can produce a good recognition effect on Cad in the pH range of 4-7. Then, we explored the response time of MPZ to Cad. Figure 1 As shown in Figure e, after adding Cad (50 M), the fluorescence color of MPZ completely changed from dark red to orange red within 7 s. The results show that MPZ can quickly detect Cad.

[0033] 2.2. Sensing mechanism Based on the above experimental results, the reaction mechanism of the probe MPZ in response to Cad was studied. After the pH reached 10, the fluorescence intensity of MPZ and MPZ + Cad was almost the same. Therefore, we speculate that the addition of alkaline Cad and inorganic base will cause the H proton on 2,4-thiazolidinedione to be removed ( Figure 2 a), which resulted in significant changes in the UV and fluorescence spectra of MPZ. To verify this hypothesis, a high-resolution mass spectrometry test was performed on MPZ after Cad was added. The mass spectrometry test showed that MPZ + Cad produced a mass peak at m / z = 383.0541, which was the target product [MH] - The corresponding peak (theoretical value is 383.0530). In addition, the UV and fluorescence spectra of MPZ + Cad and MPZ + NaOH solutions at the same pH (9.94) were tested, and the daylight and fluorescence color changes were recorded. Figure 2 b, 2c, there is no obvious difference between the UV and fluorescence spectra of the two, and the color changes of sunlight and fluorescence are consistent ( Figure 2b, inset in 2c). The above experimental results confirm that the addition of Cad causes the H proton of MPZ to be removed, resulting in a deprotonation reaction. In addition, due to the differences in the alkalinity of different amines, their deprotonation abilities are different, which leads to differences in UV and fluorescence spectra.

[0034] In order to further study the fluorescence change mechanism of MPZ in response to Cad, we performed quantum chemical calculations using density functional theory (DFT) and time-dependent density functional theory (TD-DFT). Figure 2 As shown in Figure d, the frontier molecular orbital (FMO) on the highest occupied molecular orbital (HOMO) of MPZ is mainly located in the phenothiazine part, while the FMO on the lowest unoccupied molecular orbital (LUMO) is mainly distributed on the 2,4-thiazolidinedione group. After photoexcitation, the electrons transition from LUMO back to HOMO, accompanied by complete intramolecular charge transfer from 2,4-thiazolidinedione group to phenothiazine, and the photoinduced electron transfer (PET) process occurs, thereby causing fluorescence quenching through non-radiative transitions. The FMO on the HOMO and LUMO of MPi is distributed throughout the molecular skeleton, and the excited state S1 state is a localized transition state. After photoexcitation, the electrons can directly fall back from LUMO to HOMO through radiative transitions, thereby causing fluorescence to turn on.

[0035] 2.3. Response of MPZ / FPS to biogenic amine vapor In order to non-destructively monitor the freshness of fish meat, we placed MPZ / FPS in vials containing different biogenic amines (50 mM) for fumigation (e.g. Figure 3 As shown in a). Through the simulated fish spoilage environment, the practical application value of the sensor tag MPZ / FPS was explored. Figure 3 As shown in b, MPZ / FPS showed obvious changes in sunlight and fluorescence color for most biogenic amines, indicating that MPZ / FPS can detect gaseous biogenic amines. In addition, the stability of MPZ / FPS was tested through 7 days of continuous monitoring, and it was found that there was no obvious change in the fluorescence color of MPZ / FPS ( Figure 4 ), and the fluorescence (R+G) / B value of MPZ / FPS did not fluctuate significantly ( Figure 5 ). The above results show that MPZ / FPS has good stability and has the potential to be used as a portable tool for monitoring fish freshness.

[0036] 2.4. Monitoring of salmon freshness To further evaluate the practical application capability of the sensor tag MPZ / FPS for monitoring fish freshness, the daylight and fluorescence color changes of MPZ / FPS during fish storage at 4°C were recorded ( Figure 6At the same time, in order to accurately determine the corresponding relationship between the color change trend of the sensor tag and the freshness of the fish, four indicators of fish freshness, TVB-N, TBA, TVC, and pH, were selected as the basis for judgment. Figure 7 As shown in Figures 7a and 7b, as the storage time increases, the TVB-N, TBA, and TVC values ​​gradually increase. The increase in TVB-N indicates that the content of ammonia produced by the decomposition of protein in the fish meat has increased; the increase in TBA indicates that the unsaturated fatty acids in the fish meat are being continuously oxidized; the increase in TVC indicates that the microorganisms in the fish meat are constantly multiplying. The pH shows a trend of first decreasing and then increasing. This is because the fish meat will decompose to produce lactic acid in the early stage of storage. The accumulation of lactic acid will cause the pH to decrease. After a period of time, the protein will decompose in large quantities, and the alkaline substances such as ammonia produced will cause the pH to rise. The changes in the four indicators all indicate that the quality of the fish meat is constantly declining.

[0037] According to GB / T 18108-2019 "National Food Safety Standard for Fresh Marine Fish", when the TVB-N content is < 15 mg / 100 g, the fish meat is of the first-grade freshness and is a superior product; when the TVB-N content is within the range of 15-30 mg / 100 g, the fish meat is of the second-grade freshness and is a qualified product and can still be eaten; when the TVB-N content is > 30 mg / 100 g, it means that the fish meat is corrupt and cannot be eaten. Figure 7 As shown in c, on the first day, MPZ / FPS appeared yellow in sunlight and showed orange-red fluorescence under 365 nm ultraviolet light. At this time, the TVB-N value was 4.2 mg / 100 g, and the fish meat was at the first-level freshness, which was a superior product; on the fifth day, the sunlight color and fluorescent color of MPZ / FPS were light pink and purple, respectively. At this time, TVB-N was 15.4 mg / 100 g, and the fish meat was at the second-level freshness, which was a qualified product and could still be eaten; on the seventh day, the sunlight color and fluorescent color of MPZ / FPS were pink and gray-blue, respectively. At this time, TVB-N reached 30.8 mg / 100 g, and the fish meat had already spoiled and was inedible. In addition, on the seventh day, the TVC and TBA values ​​were 7.02 lg (cfu / g), 1.10 mg MDA / kg ( Figure 7 b), both exceeded their respective maximum safety limits (7.0 lg (cfu / g) and 1.0 mg MDA / kg), which fully indicates that the fish meat has been spoiled and inedible on the seventh day. Based on the good correspondence between the colorimetric and fluorescence dual change trends of MPZ / FPS and the freshness of fish meat, a freshness grade indicator card was designed ( Figure 8 ), which is used to help consumers and merchants judge the freshness level of fish.

[0038] In summary, we have successfully synthesized a new fluorescent probe MPZ, which can rapidly detect biogenic amines in EtOH / H2O (v / v = 4 / 6, pH = 7.4) system via daylight colorimetry and fluorescence dual channels; the prepared sensor label MPZ / FPS can be used for real-time monitoring of fish freshness.

[0039] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A fluorescent probe, characterized in that The structural formula is as follows: 。 2. A method for preparing a fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: S21: Compound a, 2,4-thiazolidinedione and piperidine were added to dry ethanol and heated under reflux for 4 h for reaction; S22: After the heating reflux reaction is completed, the precipitate is filtered and washed with ethanol to prepare the fluorescent probe; Wherein, the structural formula of compound a is as follows: 。 3. Use of the fluorescent probe as claimed in claim 1 in the recognition of biogenic amines.

4. The use of the fluorescent probe according to claim 3 in the identification of biogenic amines, characterized in that: The fluorescent probe is used to detect biogenic amines in fish meat so as to evaluate the freshness of the fish meat.

5. A sensor tag, characterized in that: The invention comprises filter paper soaked with the fluorescent probe as claimed in claim 1.

6. A method for preparing a sensor tag as claimed in claim 5, characterized in that: The following steps are involved: S61: Cut the filter paper into circular slices with a diameter of 1 cm; S61: Soak the circular thin piece of filter paper in a fluorescent probe stock solution with a concentration of 1 mM for 10 minutes, and dry it at 40° C. for 3 h to prepare the sensor label.

7. Use of the sensor tag as claimed in claim 5 in the evaluation of fish freshness.

8. The use of the sensor tag according to claim 5 in the evaluation of fish freshness, characterized in that: After the sensor tag detects fish meat, the color changes under both sunlight and fluorescence, and the color change can be used as a detection index for evaluating the freshness of fish meat.

9. A freshness level indicator card, characterized in that: The freshness level indicator card is provided with a first color colorimetric area and a second color colorimetric area; the first color colorimetric area includes a plurality of colors displayed by the sensor tag under sunlight after detecting the fish meat, and the second color colorimetric area includes a plurality of colors displayed by the sensor tag under fluorescence after detecting the fish meat; the color changes in the first color colorimetric area and the second color colorimetric area are used to indicate different levels of freshness of the fish meat; the sensor tag is the sensor tag described in claim 5.

10. The freshness level indicator card according to claim 9, characterized in that: The first color comparison area is provided with a yellow color comparison area, a light pink color comparison area and a pink color comparison area; the yellow color comparison area indicates that the fish meat is at the first level of freshness and is a superior product, the light pink color comparison area indicates that the fish meat is at the second level of freshness and is a qualified product, and the pink color comparison area indicates that the fish meat has been corrupted and deteriorated and is not edible; The second color comparison area is provided with an orange-red colorimetric area, a purple colorimetric area and a gray-blue colorimetric area; the orange-red colorimetric area indicates that the fish meat is at the first level of freshness and is a superior product, the purple colorimetric area indicates that the fish meat is at the second level of freshness and is a qualified product, and the gray-blue colorimetric area indicates that the fish meat has been rotten and deteriorated and is inedible.

Images