Fluorescent tag for non-destructive visual monitoring of freshness of fish and meat food products and method for its preparation

Abstract

The application discloses a fluorescent label for nondestructive visual monitoring of freshness of fish and meat food and a preparation method thereof. The fluorescent label is characterized in that a key fluorescent substance JDCN specifically interacts with volatile biogenic amines generated in the meat spoilage process, color and fluorescence double optical signal changes occur, the biogenic amine content is judged according to the visual optical characteristics, and thus the freshness of the fish and meat food is monitored. The application has the characteristics of simple preparation, low cost, high sensitivity, high selectivity, fast visual response, and the like, can in-situ rapidly detect the volatile biogenic amines generated in the spoilage of the fish and meat food, provides a simple, convenient and effective means for the rapid visual detection of the freshness of the fish and meat food, is expected to become a preferred method for rapid detection of the content of the volatile biogenic amines in the fish and meat food, provides a powerful technical guarantee for self-monitoring of food manufacturers and market supervision, has great application prospect, and is of great popularization significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food detection, and particularly relates to a fluorescent tag for non-destructive visual monitoring of freshness of fish and meat food and a preparation method thereof. BACKGROUND

[0002] Food quality and safety are closely related to public health. According to statistics of the World Health Organization (WHO), the intake of spoiled food can cause more than 200 diseases such as diarrhea and cancer, and the problem of food quality and safety has become one of the issues that people are increasingly concerned about. Seafood and meat food are indispensable, delicious and nutritious delicacies in our daily diet, which contain rich protein, fat and vitamins. Due to the contamination of external microorganisms and the decomposition of internal enzymes, seafood and meat food are easily spoiled during storage and transportation, which not only leads to the decrease of quality and loss of nutritional components of seafood and meat food, but also produces some substances harmful to human health, causing a series of discomfort reactions such as abdominal pain, diarrhea, fever, vomiting, sweating and rapid heartbeat. Even during the cold storage process, the microorganisms in seafood and meat food can grow rapidly and decompose to produce various biogenic amines such as cadaverine, putrescine, histamine, tyramine, tryptamine, spermine and spermidine, resulting in changes in the meat quality of seafood and meat food. Among them, cadaverine and putrescine have high volatility, can be inhaled, ingested or absorbed through the skin, and have strong irritation to the eyes, mucous membranes, skin and respiratory tract. Inhaling volatile biogenic amines can cause inflammation, spasm, edema, pulmonary edema or chemical pneumonia, and even death. Therefore, the content of volatile cadaverine and putrescine is the most significant indicator of food freshness, and some countries have tried to give the limit standard according to the characteristics of different foods. Therefore, in order to protect the quality and safety of meat food, it is necessary to develop a simple and effective means to detect the freshness of fish and meat food.

[0003] So far, the methods for detecting biogenic amines include gas chromatography-mass spectrometry, electrochemical method, spectrophotometry and liquid chromatography. However, these methods still have some deficiencies. For example, the sample preparation process is complex, expensive instruments are needed, the sensitivity is low, and on-site visual detection cannot be achieved. In recent years, fluorescent probe technology has been evaluated as a powerful tool for detecting biogenic amines due to its high sensitivity, high selectivity, real-time detection and simple operation. Since biogenic amines themselves cannot emit fluorescence, the determination of biogenic amines by fluorescence spectrophotometry is mainly based on the reaction between the reaction site of the fluorescent probe and the amino group of biogenic amines under certain conditions, to generate a product different from the fluorescence properties of the fluorescent probe itself. The content of biogenic amines can be detected according to the change of fluorescence and color double signals. Considering the importance of food safety, it is very important and challenging to develop a new fluorescent probe for rapid, high-sensitivity visual determination of the content of volatile biogenic amines in fish and meat food.

[0004] Therefore, it is urgent to develop a simple and low-cost detection method to detect volatile biogenic amines in fish and meat. SUMMARY

[0005] The technical problem solved by the present application is to provide a fluorescent tag for non-destructive visual monitoring of freshness of fish and meat food, which has stable structure, excellent performance and high sensitivity, and a preparation method thereof, so as to facilitate on-site rapid visual detection.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] The compound JDCN is applied in non-destructive visual monitoring of food freshness, and the structural formula of the compound JDCN is

[0008]

[0009] The food is fish or meat.

[0010] The compound JDCN is used as a fluorescent probe for non-destructive visual monitoring of food freshness.

[0011] The preparation method of the fluorescent probe is as follows:

[0012]

[0013] The preparation method of the fluorescent probe comprises the following steps:

[0014] <1> Preparation of intermediate compound 4JD-CHO: Compound 1, 1, 3-ethyl malonate and anhydrous ethanol are sequentially added into a three-necked round-bottom flask, and then two drops of piperidine are added to the mixture; the reaction mixture is heated and stirred under reflux at 80℃ in a nitrogen atmosphere for 3-5 hours, and compound 2 is obtained after the reaction is completed; compound 2 is subjected to deprotection of ethyl formate group in the presence of hydrochloric acid and glacial acetic acid to obtain compound 3; compound 3, dimethylformamide and phosphorus oxychloride are added into a three-necked round-bottom flask, and then heated and stirred under reflux at 60℃ in a nitrogen atmosphere for 3-5 hours to obtain compound 4;

[0015] <2> Preparation of target compound 5JDCN: malononitrile and compound 4 are dissolved in 8 mL of anhydrous ethanol in a three-necked round-bottom flask, and then two drops of piperidine are added to the mixture solution; the mixture solution is stirred at 80℃ in a nitrogen atmosphere for 0.5 h, and the reaction is monitored in real time by TLC; after compound 4 is completely reacted, nitrogen is released to reduce the pressure, the solvent is evaporated, the precipitate is washed with anhydrous ethanol twice, and finally purple-black solid JDCN is obtained.

[0016] In step (1), the molar ratio of compound 1 to ethyl 1,3-propanedioate is 1-5, and the molar ratio of compound 3 to phosphorus oxychloride is 1-5.

[0017] In step (2), the molar ratio of compound 4 to malononitrile is 1-1.2.

[0018] A fluorescent label for non-destructive visual monitoring of food freshness, which is loaded with the above fluorescent probe.

[0019] The fluorescent label according to claim 8 is prepared by immersing a filter paper strip in a solution of the fluorescent probe and then taking it out and drying.

[0020] A detection method for non-destructive visual monitoring of food freshness, which places the above fluorescent label in a sealed container containing a fish or meat sample.

[0021] In view of the problem that the freshness of fish, meat and other food cannot be detected on site quickly and non-destructively, the inventors use compound JDCN as a fluorescent probe for non-destructive visual monitoring of food freshness and prepare a fluorescent label, and accordingly establish a corresponding preparation method and detection method. The fluorescent label undergoes specific interaction with volatile biogenic amines produced in the meat spoilage process through the key fluorescent substance JDCN, and undergoes color and fluorescence double optical signal changes, and the biogenic amine content is determined according to the two visual optical characteristics, so as to monitor the freshness of fish and meat food. The present application has the characteristics of simple preparation, low cost, high sensitivity and selectivity, and rapid visual response, and can detect the volatile biogenic amines produced in the spoilage of fish and meat food in situ quickly, and provides a simple, convenient and effective means for rapid visual detection of the freshness of fish and meat food, and is expected to become the preferred method for rapid detection of the content of volatile biogenic amines in fish and meat food, and can provide strong technical support for self-monitoring of food manufacturers and market supervision, has great application prospect, and has great popularization significance.

[0022] The working principle of the present application is as follows: the key fluorescent substance JDCN in the fluorescent label has a clear absorption peak at 550 nanometers, and a new absorption peak appears at 440 nanometers after the addition of cadaverine; with the increase of the concentration of cadaverine, the original absorption peak of the fluorescent label gradually weakens and the absorption peak at 440 nanometers gradually strengthens, and a clear color change from light red to light yellow can be observed. In addition, under the excitation of excitation light, the probe has obvious green fluorescence at about 604 nanometers. When cadaverine is added, the recognition group combines with the amino group, which destroys the original electron transfer process of the probe molecule, so that the red fluorescence of the probe at about 604 nanometers is weakened and the light green fluorescence at about 504 nanometers is enhanced, thereby realizing the ratio type fluorescence detection of cadaverine. In the ratio type detection recognition event, the influence of external factors on the fluorescence signal intensity of the two is consistent, so the external factors are difficult to affect the ratio of the two fluorescence intensities, and this mode can greatly reduce the influence of external factors and improve the detection precision and accuracy. Therefore, compared with the traditional detection method, the present application can realize high-sensitivity visual monitoring of the freshness of fish and meat. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is a one-dimensional hydrogen nuclear magnetic spectrum of the key fluorescent substance of the fluorescent label in Example 1, the abscissa is the chemical shift, and the ordinate is the signal intensity.

[0024] Figure 2 Figure 2 is a one-dimensional carbon nuclear magnetic spectrum of the key fluorescent substance of the fluorescent label in Example 1, the abscissa is the chemical shift, and the ordinate is the signal intensity.

[0025] Figure 3 Figure 3 is a fluorescence coloration result diagram of the key fluorescent substance of the fluorescent label in Example 1 to different concentrations of cadaverine, in which: (A) is a fluorescence CIE1931 coloration coordinate diagram of the key fluorescent substance to different concentrations of cadaverine (0-20 μM); (B) is a diagram of the fluorescence intensity ratio of the solution at 504 nm and 604 nm with time after 20 μM cadaverine is added to the key fluorescent substance solution (10 μM); (C) is a photo of the fluorescence of the solution with time after 20 μM cadaverine is added to the key fluorescent substance solution (10 μM).

[0026] Figure 4 Figure 4 is a result diagram of the fluorescent label of the present application applied to the detection of various substances, in which: (A) is a colorimetric and fluorescence coloration photo of the fluorescent label to different concentrations of cadaverine; (B) is a standard curve of the fluorescence color of the fluorescent label with the concentration change of cadaverine; (C) is a colorimetric and fluorescence coloration result diagram of the fluorescent label to ammonia, formaldehyde, formic acid, sulfur dioxide, tyramine, histamine, putrescine and cadaverine (20 μM); (D) is a fluorescence coloration chroma numerical value diagram of the fluorescent label to the various substances in (C).

[0027] Figure 5 Figure 1 is a chart of the determination results of the freshness of beef samples by the fluorescent tag of the present application at different temperatures (-20℃, 0℃, 25℃) for different times, in which: A is the determination results of the fluorescent color change of beef samples at 365 nm ultraviolet light at three temperatures, B, C, and D are respectively the line graphs of the cadaverine concentration change with time at three temperatures. DETAILED DESCRIPTION

[0028] Example 1 Preparation of the key chemical substance of the fluorescent tag

[0029] The chemical formula of the key chemical substance (target compound 5, JDCN) of the fluorescent tag for non-destructive visual monitoring of the freshness of fish and meat food according to the present application is C 19 H 15 N3O2, and the molecule has the following structure:

[0030]

[0031] The specific synthesis route of the target compound 5 is as follows:

[0032]

[0033] The specific preparation steps of the target compound 5 are as follows:

[0034] <1> Preparation of intermediate compound 4 (JD-CHO): Compound 1 (434 mg, 2.0 mmol), diethyl malonate (640 mg, 4.0 mmol), and 20 mL of anhydrous ethanol (99.5%) were sequentially added to a three-necked round-bottom flask, and then 0.1 mL of piperidine was added to the mixture. The reaction mixture was heated to reflux under a nitrogen atmosphere at 80℃ and stirred for 3-5 hours. After the reaction was completed, column chromatography (PE:EA=3:1) was used for purification to obtain compound 2 (480 mg, yield: 76%). Compound 2 (480 mg, 1.52 mmol), hydrochloric acid (37%, 4 mL), and glacial acetic acid (99.5%, 4 mL) were sequentially added to a three-necked round-bottom flask, and the reaction mixture was heated to reflux under a nitrogen atmosphere at 100℃ and stirred for 10-12 hours. After the reaction was completed, the precipitate was washed with anhydrous ethanol (99.5%) by vacuum filtration, and column chromatography (petroleum ether: ethyl acetate=5:1) was used for purification to obtain compound 3 (330 mg, yield: 90%). Dimethylformamide (DMF, 6 mL) and phosphorus oxychloride (POCl3, 6 mL) were sequentially added to a three-necked round-bottom flask under ice bath conditions, and the reaction mixture was stirred under a nitrogen atmosphere for 1-2 hours. After the reaction was completed, compound 3 (330 mg, 1.36 mmol) was continuously added to the flask, and then the reaction mixture was heated to reflux under a nitrogen atmosphere at 60℃ and stirred for 3-5 hours to obtain compound 4 (348 mg, yield: 95%).

[0035] <2> Preparation of target compound 5 (JDCN): Propiolonitrile (79 mg, 1.2 mmol) and compound 4 (JD-CHO) (269 mg, 1.0 mmol) were dissolved in 8 mL of anhydrous ethanol (99.5%) in a three-necked round-bottom flask, and then 0.1 mL of piperidine was added to the mixture solution. The mixture solution was stirred at 80°C under a nitrogen atmosphere for 0.5 h, and the reaction was monitored in real time by TLC. After compound 4 (JD-CHO) was completely reacted, the precipitate was filtered under reduced pressure, washed with anhydrous ethanol (99.5%) twice (2.5 mL x 2), and finally obtained as a purple-black solid JDCN (301 mg, yield: 95%).

[0036] The one-dimensional hydrogen nuclear magnetic spectrum and one-dimensional carbon nuclear magnetic spectrum of JDCN are shown in Figure 1 and Figure 2 .

[0037] 1H NMR (500 MHz, DMSO-d6) δ 8.47 (s, 1H), 7.93 (s, 1H), 7.23 (s, 1H), 3.45 (q, J = 5.0 Hz, 4H), 2.73 (t, J = 6.5 Hz, 4H), 1.90 (h, J = 8.0, 7.0 Hz, 4H).

[0038] 13C NMR (126 MHz, CDCl3) δ 160.6, 153.3, 152.2, 150.8, 143.5, 128.6, 121.0, 115.2, 114.5, 109.0, 108.6, 106.5, 74.2, 50.9, 50.4, 27.2, 20.8, 19.8, 19.7.

[0039] Example 2 Fluorescent color development of the key fluorescent substance pair on cadaverine

[0040] As shown in Figure 3 , the JDCN solution itself has very bright red fluorescence, and after the addition of cadaverine, the local area of the probe solution gradually shows green color, and the green fluorescence gradually increases with time; when cadaverine is added for 60 seconds, the probe solution completely shows green fluorescence, and the fluorescence intensity reaches the maximum. Therefore, the key fluorescent substance pair has very good color development effect on cadaverine, and the color development speed is fast, the color change is obvious, which is conducive to naked eye identification of cadaverine, simple and fast, and has good application prospect.

[0041] Example 3 Comparison of fluorescent detection of the key fluorescent substance pair on various analytes

[0042] 3.1 Preparation method of fluorescent label

[0043] Whatman filter paper was cut into strips (size: 0.8 cm x 0.8 cm, size: 0.8 x 2.0 cm), and a JDCN solution (1.0 mM) was prepared with anhydrous ethanol. The strip filter paper was soaked in the key fluorescent substance JDCN solution (1.0 mM) for 25 min, taken out and dried, and a fluorescent tag was prepared.

[0044] 3.2 Method for using the fluorescent tag

[0045] The fluorescent tag was attached to the inner cover of the container, different concentrations of cadaverine or samples to be tested were added in the container (glass bottle volume 7 mL), and the container was placed in a thermostat at 25°C. After 30 minutes, the fluorescent tag was taken out, and a fluorescent photo was taken with a smartphone under a handheld 365 nm ultraviolet lamp.

[0046] 3.3 Application of the fluorescent tag

[0047] The fluorescent tag was used to evaluate the fluorescence detection of cadaverine and various analytes, and the response under white light and 365 nm ultraviolet light was evaluated, and the results are shown in Figure 4 Figure 4 (A) and (B) are the color and fluorescence changes of the fluorescent tag in the presence of different concentration gradients of cadaverine vapor. It can be seen that as the concentration of cadaverine increases, the fluorescent color of the fluorescent tag under 365 nm gradually changes from red to green, and the fluorescence intensity ratio has a clear linear relationship with the concentration of cadaverine. Figure 4 (C) and (D) are the fluorescence response of the fluorescent tag to the vapor of various common biological amines and their interferents. It can be seen that the fluorescent tag only shows a clear red to green fluorescence transition for cadaverine, and no obvious fluorescence change for other various analytes. Therefore, the fluorescent tag has good specific recognition and practical application for biological amine-cadaverine under daylight and 365 nm ultraviolet light.

[0048] Example 4

[0049] The fluorescent tag was used to determine the freshness of beef samples in a closed dish at different temperatures (-20°C, 0°C and 25°C) for different storage times. The meat samples were purchased from a supermarket. The meat samples were placed in a glass culture dish (80 mL), and the fluorescent tag JDCN was fixed on the inner cover of the culture dish. The cover was put back on the culture dish and sealed with tape. The meat samples were stored at 25°C for 0, 4, 8, 12, 16, 24 and 36 hours, respectively. The fluorescence pictures of these fluorescent tags under 365 nm ultraviolet light were recorded with a mobile phone (Mi 11 Pro), and then the RGB (red, green, blue) values were read out with APP software (Dragon RGB). The G / R ratio was plotted against the cadaverine concentration to obtain a calibration curve.

[0050] The results are shown in Figure 5 ​As shown, the fluorescence tag shows good practical applicability and can realize on-site visual detection of the freshness of beef samples.

[0051] In addition, the applicant's prior patent application "Fluorescent probe for rapid non-destructive detection of food freshness and preparation method thereof" (patent application number 2022107602707, application date June 29, 2022) reports a compound Cl-BODIPY as a fluorescent probe, and its structural formula is:

[0052]

[0053] The Cl-BODIPY fluorescent probe has a distinct absorption peak at 499 nanometers. When cadaverine is added, a new absorption peak appears at 395 nanometers; as the concentration of cadaverine increases, the original absorption peak of the probe gradually weakens and the absorption peak at 395 nanometers gradually strengthens, and a clear color change from green to light yellow can be observed. In addition, under excitation light excitation, the probe has distinct green fluorescence at around 512 nanometers. When cadaverine is added, the recognition group binds with the amino group, disrupting the original electron transfer process of the probe molecule, causing the green fluorescence of the probe at around 512 nanometers to weaken and the blue fluorescence at around 452 nanometers to strengthen, thereby achieving ratio-type fluorescence detection of cadaverine.

[0054] Compared with Cl-BODIPY, the fluorescent probe has strong reaction specificity for biological amines, no detection interference Figure 3 ), and the response speed of the fluorescent probe is fast, the fluorescence color change is more significant (from red to green), which is conducive to visual detection.

Claims

1. Use of the compound JDCN for monitoring the freshness of food products without loss of visibility, characterized in that: The structural formula of the compound JDCN is The food is fish or meat; The compound JDCN is used as a fluorescent probe to non-destructively visually monitor the freshness of food.

2. The method for preparing the fluorescent probe according to claim 1, characterized in that According to the following synthetic route:

3. The method of claim 2, wherein The method comprises the following steps: <1> Preparation of intermediate compound 4JD-CHO: compound 1, ethyl 1,3-propanedioate and anhydrous ethanol are sequentially added into a three-necked round-bottom flask, and then two drops of piperidine are added to the mixture; the reaction mixture is heated and stirred under reflux at 80 DEG C in a nitrogen atmosphere for 3-5 hours, and compound 2 is obtained after the reaction is completed; compound 2 is subjected to removal of ethyl formate under the action of hydrochloric acid and glacial acetic acid to obtain compound 3; compound 3, dimethylformamide and phosphorus oxychloride are added into a three-necked round-bottom flask, and then heated and stirred under reflux at 60 DEG C in a nitrogen atmosphere for 3-5 hours to obtain compound 4; <2> Preparation of target compound 5JDCN: malononitrile and compound 4 are placed in a three-necked round-bottom flask and dissolved in 8 mL of anhydrous ethanol, and then two drops of piperidine are added to the mixture solution; the mixture solution is stirred at 80 DEG C in a nitrogen atmosphere for 0.5 h, and the reaction is monitored in real time by TLC; after compound 4 is completely reacted, nitrogen is released to reduce the pressure, the solvent is evaporated, the precipitate is washed with anhydrous ethanol twice, and finally purple-black solid JDCN is obtained.

4. The method of claim 3, wherein In step (1), the molar ratio of compound 1 to ethyl 1,3-propanedioate is 1-5, and the molar ratio of compound 3 to phosphorus oxychloride is 1-5.

5. The method of claim 3, wherein In step (2), the molar ratio of compound 4 to malononitrile is 1-1.

2.

6. A fluorescent label for non-destructively visually monitoring the freshness of food, which is loaded with the fluorescent probe according to claim 1.

7. The fluorescent tag of claim 6, wherein The fluorescent label is prepared by the following operation: a filter paper strip is soaked in a solution of the fluorescent probe, and then taken out and dried.

8. A method of detecting freshness of food by non-destructive visual inspection, characterized in that The fluorescent label according to claim 6 is placed in a sealed container containing a fish or meat sample.

Images