Preparation method and application of a porous and transparent meat freshness ratio type fluorescent indicator film

Abstract

The present application belongs to the field of intelligent indicating packaging of food, and particularly relates to a preparation method of a porous and transparent meat freshness ratio fluorescent indicating film and application thereof. The present application first synthesizes red fluorescent carbon quantum dots through a one-step hydrothermal method, then constructs a double-emission fluorescent probe with yellow-green fluorescent fluorescein, embeds the fluorescent probe in hydrophobic fibers through electrospinning technology to form a film, and finally prepares a porous and transparent meat freshness ratio fluorescent indicating film by applying mechanical pressure to the film. The indicating film prepared by the present application has high mechanical strength, high transparency and high fluorescence intensity, and the color change generated is more easily observed by the naked eye, so that consumers can determine the freshness of meat by observing the color change visible to the naked eye. Moreover, the indicating film has stronger flexibility and is suitable for the packaging field. In addition, the operation method and process of the present application are simple, the raw material cost is low, the raw material is easy to obtain and the industrialized production is easy to realize, so the present application has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of intelligent food packaging, specifically relating to the preparation method and application of a porous and transparent fluorescent indicator film for meat freshness ratio. Background Technology

[0002] Foods rich in protein, such as meat, poultry, and fish, are prone to spoilage, and their freshness decreases as the degree of spoilage increases. From the perspective of food safety and shelf life, freshness detection is of great significance. Traditional freshness detection methods mostly cannot display the freshness of meat in real time, making it difficult to meet the needs of retailers or consumers for visual assessment. In recent years, rapid non-destructive food testing technologies have received widespread attention due to their non-destructive nature, low cost, ease of operation, and high accuracy. During storage, the proteins in meat undergo biochemical changes due to the decomposition by proteolytic enzymes, producing volatile biogenic amines and other gases. These gases become one of the markers for freshness assessment. Fluorescent sensors, due to their advantages of high sensitivity, high selectivity, real-time detection, and ease of operation, have become a research hotspot in recent years, with the development of fluorescent sensors sensitive to these volatile gases being a key research area. Ratio-modified fluorescent sensors, in particular, have self-calibration capabilities, which not only enhance the sensor's ability to resist interference from external environmental factors but also typically exhibit multiple color changes, facilitating visual observation.

[0003] To enhance the sensitivity of fluorescent sensors to spoilage gases from meat and fish, many studies have loaded fluorescent probes into porous films. Electrospinning technology can prepare porous nanofiber membranes with high specific surface area under mild conditions. The porous microstructure and the high specific surface area of ​​the nanofibers are highly advantageous for gas sensing. Current research reports the use of electrospinning technology to prepare fluorescent films for indicating the freshness of meat and fish. The polymer used for film formation in electrospinning is a crucial factor affecting sensor performance. To prevent the fluorescent probe from dissolving in the film during high humidity applications, hydrophobic polymers are typically used. However, the inherent characteristic of hydrophobic polymers is the high packing density of polymer chains and numerous crystalline regions after film formation, resulting in poor transparency of the hydrophobic polymer spun film fibers, with most of the film appearing nearly white. This inherent phenomenon makes it difficult to detect or even observe the fluorescence of the fluorescent probe within the fibers in practical applications of hydrophobic polymer electrospun fluorescent films, thus reducing the sensor's sensitivity to gas detection. Therefore, developing highly transparent electrospun thin films to improve the fluorescence intensity of observed fluorescent probes is of great significance.

[0004] There are two main categories of methods for preparing transparent electrospun fiber membranes. One method involves filling the fiber membrane with a polymer to create a transparent nanocomposite material; however, this method can damage the fiber structure. The other method involves reducing the thickness of the electrospun fiber membrane; the thinner the membrane, the higher its light transmittance. However, this nanomembrane suffers from poor mechanical properties and requires stringent preparation conditions. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to solve one of the aforementioned problems: providing a method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio and its application. First, red fluorescent carbon quantum dots are synthesized via a one-step hydrothermal method. Then, a dual-emission fluorescent probe is constructed with a yellow-green fluorescent fluorescein. The fluorescent probe is semi-embedded in hydrophobic fibers using electrospinning technology to form a film. Finally, mechanical pressure is applied to the film to prepare a porous and transparent portable indicator tag. This indicator tag responds to volatile biogenic amines, thereby enabling the monitoring of meat freshness.

[0006] To achieve the above objectives, the specific steps are as follows:

[0007] Step 1: Synthesis of red fluorescent carbon quantum dots;

[0008] Step 1.1: Dissolve citric acid monohydrate in ultrapure water and sonicate to obtain citric acid monohydrate solution; then, under stirring conditions, add neutral red powder to citric acid monohydrate solution to obtain purple-red mixed solution; transfer the mixed solution to a high-pressure reactor and react at a certain temperature for a period of time, then cool to room temperature to obtain reaction solution;

[0009] Step 1.2: After centrifuging the reaction solution obtained in Step 1.1, filter it through a microporous membrane to obtain the filtrate; after dialysis, the obtained filtrate yields a carbon dot solution; finally, the product obtained by vacuum freeze-drying is the red fluorescent carbon quantum dots.

[0010] Step 2: Construction of dual-emission fluorescent probe;

[0011] Step 2.1: Dissolve fluorescein isothiocyanate in anhydrous ethanol, and sonicate to obtain fluorescein isothiocyanate ethanol solution; then dissolve the red fluorescent carbon quantum dots obtained in step 1 in water, and sonicate to obtain carbon dot solution.

[0012] Step 2.2: Mix the isothiocyanate ethanol solution from Step 2.1 with the carbon dot solution. The resulting mixed solution is the dual-emission fluorescent probe solution.

[0013] Step 3: Preparation of electrospinning solution

[0014] Step 3.1: Dissolve the hydrophobic polymer in one or more organic solvents to obtain a hydrophobic polymer solution. Heat the polymer solution to a certain temperature and stir to dissolve it. Then, defoam it by ultrasound to obtain a transparent spinning solution. The organic solvent is one or a mixture of two of N,N-dimethylformamide, ethanol, tetrahydrofuran, dichloromethane, acetone, and dimethyl sulfoxide. The hydrophobic polymer is any one of polyvinylidene fluoride, polycaprolactone, polyurethane, and polyacrylonitrile.

[0015] Step 3.2: Add the dual-emission fluorescent probe solution obtained in step 2 to the transparent spinning solution obtained in step 3.1, stir and mix evenly, and then obtain an electrospinning solution containing fluorescent probe after ultrasonic defoaming treatment.

[0016] Step 4: Preparation of transparent electrospun film;

[0017] Electrospinning was performed using an electrospinning solution containing a fluorescent probe to obtain an electrospinned membrane. The electrospinned membrane was then placed in a press and maintained at a certain pressure for a certain time to obtain a transparent electrospinned membrane, which is a porous and transparent fluorescent indicator membrane for meat freshness ratio.

[0018] Preferably, in step 1.1, the concentration of the citric acid monohydrate solution is 0.5-1.5 mol / L; the concentration of neutral red in the purple-red mixed solution is 0.5-1.5 mmol / L; the reaction temperature is 140-220℃, and the reaction time is 2-6 h.

[0019] Preferably, the centrifugation speed in step 1.2 is 8000-11000 rpm, and the time is 10-30 min; the pore size of the microporous filter membrane is 0.22 μm; and the dialysis step is: dialysis for 12 h using a dialysis bag with a molecular weight cutoff of 500.

[0020] Preferably, in step 2.1, the concentration of the fluorescein isothiocyanate ethanol solution is 0.05-0.15 mg / mL; and the concentration of the red fluorescent carbon dot solution is 0.05-0.09 g / mL.

[0021] Preferably, in step 2.2, the mass ratio of fluorescein isothiocyanate in the ethanol solution to red fluorescent carbon dots in the carbon dot solution is 1:(200-400).

[0022] Preferably, the mass concentration of the hydrophobic polymer solution in step 3.1 is 15%-20%; when the organic solvent is a mixture of acetone and N,N-dimethylformamide, the volume ratio is 1:4; when the organic solvent is a mixture of acetic acid and ethanol, the volume ratio is 4:1; when the organic solvent is a mixture of dichloromethane and dimethyl sulfoxide, the volume ratio is 7:3; when the organic solvent is a mixture of tetrahydrofuran and N,N-dimethylformamide, the volume ratio is 2:3; the mixture is heated to a certain temperature of 25-45℃ and stirred for 2-5 hours.

[0023] Preferably, in step 3.2, the volume ratio of the dual-emission fluorescent probe solution to the electrospinning solution is 1:(10-30); the stirring time is 0.5-1.5h; and the ultrasonic treatment time is 20-50min.

[0024] Preferably, the electrospinning process parameters in step 4 are as follows: voltage 15-20kV, temperature 30-40℃, relative humidity 35-75%, spinning distance 15-25cm, spinning solution flow rate 0.0005-0.0015mm / s, roller speed 20-50rpm, spinning time 6-12h; pressure 0-35MPa, time 10-30min.

[0025] The porous and transparent meat freshness ratio fluorescent indicator film is used for meat freshness detection. The specific steps are as follows:

[0026] Fresh meat is placed in a food storage container. A fluorescent ratio-type meat freshness indicator film, cut to size as needed, is sealed together with the meat and stored in an environment of 0-4℃. As the storage time of the meat increases, the volatile amine gases in the packaging environment increase (meat products contain a large amount of protein, which decomposes during storage due to external microbial contamination, producing various volatile amines such as ammonia, dimethylamine, and trimethylamine). The fluorescence color of the indicator film changes from red to orange and then to yellow. At the same time, the volatile basic nitrogen content of the meat is measured at regular intervals, and the corresponding changes in the fluorescence color of the indicator film under ultraviolet light are recorded. The obtained color difference value is correlated with the volatile basic nitrogen value of the meat, thereby judging the freshness of the meat based on the change in fluorescence color, achieving visual detection.

[0027] The beneficial effects of this invention are:

[0028] (1) The present invention synthesizes red fluorescent carbon quantum dots in one step by hydrothermal method. Red fluorescent carbon quantum dots are used as internal references and yellow-green fluorescein is used as an indicator. The fluorescein forms a ratiometric fluorescent probe and has a significant response to amine gases. The method is simple and easy to operate.

[0029] (2) This invention utilizes hydrophobic polymers to obtain a convenient solid fluorescent film through electrospinning film formation technology. It is a nano-scale fiber with a larger specific surface area and higher porosity compared to traditional films. At the same time, by using electrospinning technology to fix the fluorescent probe in the solid film, the carbon quantum dots are prevented from quenching due to aggregation, thereby significantly improving its stability, making it more sensitive to gases, and greatly improving its practicality.

[0030] (3) The present invention prepares an electrospun fluorescent indicator film with high mechanical strength and high transparency by mechanical pressing. It has high fluorescence intensity, is easier to observe with the naked eye, is more flexible, and has better indication properties, making it suitable for the packaging field.

[0031] (4) The present invention provides a safe and stable ratio-type fluorescent indicator film for meat freshness. It utilizes the reaction of the ester group on the fluorescein with volatile amines produced by meat spoilage to cause a change in fluorescence color to monitor meat freshness. By establishing a correlation between the fluorescence color change of the indicator film and the quality indicators of the meat, the freshness of the meat can be determined by observing the visible color change, thus ensuring quality and safety.

[0032] (5) The operation method and process of the present invention are simple, the raw materials are inexpensive and easy to obtain, and it is easy to realize industrial production, with broad application prospects. Attached Figure Description

[0033] Figure 1 This is a transmission electron microscope image of the carbon quantum dots prepared in Example 1.

[0034] Figure 2 The difference in fluorescence ratio at 519 nm and 610 nm before and after the addition of different volumes of luciferin solution in Example 1: R0 is the initial F 519 / F 610 R1 is F after the reaction of ammonia water. 519 / F 610 .

[0035] Figure 3 Scanning electron microscope images of ratio fluorescent electrospun film (A) and transparent electrospun film (B) in Example 1, and a comparison image (C) of the electrospun film before and after mechanical pressing.

[0036] Figure 4 To screen the most suitable fluorescent probe for different concentrations of ammonia (μM) in Example 1: where A is the fluorescence spectrum change of the fluorescent probe reacting with different concentrations of ammonia, and B is the change of the ratio of fluorescence value at 519nm to 610nm with the concentration of ammonia.

[0037] Figure 5 The images show the indicator membrane prepared in Example 1 before and after its reaction with ammonia, as well as the L*, a*, and b* values. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1:

[0044] Step 1: Synthesis of red fluorescent carbon quantum dots

[0045] Citric acid monohydrate was dissolved in ultrapure water and sonicated to obtain a 1 mol / L citric acid monohydrate solution. Neutral red powder was slowly added to the citric acid monohydrate solution under stirring to obtain a purple-red mixed solution. The molar ratio of neutral red to citric acid was 1:1000. The mixed solution was transferred to a Teflon high-pressure reactor and reacted at 180°C for 4 hours. After cooling to room temperature, the solution was centrifuged at 10000 rpm for 15 minutes to remove insoluble precipitates. The supernatant was filtered through a 0.22 μM aqueous microporous membrane, and the filtrate was dialyzed for 12 hours using a dialysis bag with a molecular weight cutoff of 500 to remove unreacted substances. Finally, the purified carbon dot solution was dried using a vacuum freeze dryer for 12 hours to obtain carbon quantum dot powder. Figure 1 The image shown is a transmission electron microscope (TEM) schematic of red fluorescent carbon quantum dots.

[0046] Step 2: Construction of dual-emission fluorescent probe;

[0047] Fluorescein isothiocyanate was dissolved in anhydrous ethanol to obtain a fluorescein isothiocyanate ethanol solution with a concentration of 0.1 mg / mL. The dried carbon quantum dot powder from step 1 was dissolved in ultrapure water and sonicated to obtain a carbon dot solution with a concentration of 1 mg / mL.

[0048] Take 1 mL of carbon dot solution and add 5, 10, 20, 30, 40, and 50 μL of fluorescein isothiocyanate ethanol solution, respectively. Figure 2 The solution was prepared by mixing the fluorescein solution (abbreviated as fluorescein solution) to obtain a mixed solution; then it was reacted with 150 μL of 0.1 M ammonia solution and the fluorescence ratio before and after the reaction was detected.

[0049] like Figure 2 As shown, when the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution is 1:250, the difference in fluorescence ratio before and after the reaction is the largest. Therefore, it is indicated that when the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution is 1:250, the constructed dual-emission fluorescent probe exhibits the most significant color change. This solution was used as the final dual-emission fluorescent probe solution for subsequent experiments.

[0050] The reaction steps of the fluorescent probe with ammonia solutions of different concentrations were as follows: 1 mL of carbon dot solution was mixed with 40 μL of fluorescein isothiocyanate solution (the mass ratio of fluorescein isothiocyanate to carbon quantum dots in the carbon dot solution was 1:250), and then reacted with ammonia solutions of 20, 40, 60, 80, 100, 140, 180, 220, 260, 300, and 340 μM, respectively. The fluorescence spectra after the reaction are shown below. Figure 4 As shown, with the continuous increase of ammonia concentration, the emission peak of the fluorescent probe rises at 519 nm and remains basically unchanged at 610 nm. Figure 4A), the fluorescence ratio value is linearly related to the concentration of ammonia. Figure 4 B).

[0051] Step 3: Preparation of electrospinning solution

[0052] Dissolve 1.5 g of polyvinylidene fluoride powder in 10 mL of a two-system solution (acetone: N,N-dimethylformamide = 1:4, v / v), stir at 40 °C for 3 h until it becomes clear and transparent, and obtain a polyvinylidene fluoride solution; add 0.5 mL of the dual-emission fluorescent probe solution screened in step 2, stir magnetically for 30 min, and sonicate at room temperature for 30 min to defoam and disperse evenly, to obtain the final polyvinylidene fluoride electrospinning solution containing the fluorescent probe.

[0053] Step 4: Preparation of transparent electrospun film

[0054] The polyvinylidene fluoride solution containing the fluorescent probe obtained in step 3 was injected into a 10 mL disposable syringe to eliminate air bubbles. Electrospinning was then performed using an electrospinning machine. The corresponding technical parameters were: a voltage of 15 kV applied by the high-voltage power supply, a receiving distance of 15 cm, a solution propulsion rate of 0.001 mm / s, a spinning temperature of 30 °C, and a relative humidity of 55%. The electrospun film was obtained by collecting the solution on silicone paper as the receiving substrate using a rotating drum at a speed of 20 rpm for 10 hours. Figure 3 Figure A shows the electron microscope structure of the ratio fluorescence indicator film;

[0055] The electrospun membrane was placed in a press and maintained at 35 MPa for 10 minutes to obtain a transparent electrospun membrane (i.e., a porous and transparent fluorescent indicator membrane for meat freshness ratio); For example Figure 3 Figure B shows the electron microscope structure of a transparent electrospun membrane. It can be seen that the fibers of the electrospun membrane become denser after mechanical pressing, but still retain some porosity. Figure 3 As shown in Figure C, the increased transparency of the electrospun film after mechanical pressing is visible to the naked eye.

[0056] Fluorescence images of the prepared transparent electrospun fibers before and after reaction with ammonia gas are shown below. Figure 5 As shown, the color of the indicator membrane changes from red to yellow, with the a* value of the indicator membrane changing from 33.65 to 10.26 and the b* value changing from 0.26 to 17.8.

[0057] The transparent fluorescent electrospun membrane prepared in this embodiment was used to determine the freshness of pork. The color of the membrane was recorded every 12 hours, and its L*, a*, b* values ​​were measured, and the color difference value ΔE was calculated according to Formula 1.

[0058] When pork spoils, the fluorescent color of the indicator membrane changes from red to orange and then to yellow. The color change of the membrane is shown in Table 1.

[0059]

[0060] Where ΔL*=L*-L0*, Δa=a*-a0*, Δb=b*-b0*; L*, a*, and b* are the color parameters of the indicator film after a certain storage time. L0*, a0*, and b0* are the initial color parameters of the indicator film.

[0061] Table 1. Changes in volatile basic nitrogen content of pork during storage and changes in the color value of the indicator film.

[0062]

[0063] As the storage time of pork increases, the color difference value changes more significantly. The fluorescence color is red on the first and second days. From the third to the fourth day, the fluorescence color gradually changes from red to orange, indicating that the meat products are starting to become less fresh, but are still within the edible range. By the fifth and sixth days, the meat products are rotting, and the fluorescence color gradually changes from orange to yellow. Taking the meat products on the sixth day of rotting as an example, the a* value decreased by 21.10 compared to the first day, and the b* value increased by 12.15, indicating that the green color intensity of the indicator film increased significantly, thus changing the fluorescence color from orange to yellow, achieving visual detection.

[0064] Example 2:

[0065] Step 1: Synthesis of red fluorescent carbon quantum dots

[0066] Citric acid monohydrate was dissolved in ultrapure water and sonicated to obtain a 0.5 mol / L citric acid monohydrate solution. Neutral red powder was slowly added to the citric acid monohydrate solution under stirring to obtain a purple-red mixed solution. The molar ratio of neutral red to citric acid was 1:1000. The mixed solution was transferred to a Teflon high-pressure reactor and reacted at 160℃ for 5 h. After the reaction, the solution was cooled to room temperature and centrifuged at 8000 rpm for 20 min to remove insoluble precipitates. The supernatant was filtered through a 0.22 μM aqueous microporous membrane, and the filtrate was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 500 to remove unreacted substances. Finally, the purified carbon dot solution was dried in a vacuum freeze dryer for 12 h to obtain carbon quantum dot powder.

[0067] Step 2: Construction of dual-emission fluorescent probe;

[0068] Fluorescein isothiocyanate was dissolved in anhydrous ethanol to obtain a fluorescein isothiocyanate ethanol solution with a concentration of 0.1 mg / mL. The dried carbon quantum dot powder from step 1 was dissolved in ultrapure water and sonicated to obtain a carbon dot solution with a concentration of 0.5 mg / mL.

[0069] 1 mL of carbon dot solution was added to 5, 10, 20, 30, 40, and 50 μL of fluorescein isothiocyanate ethanol solution, respectively, and then reacted with 150 μL of 0.1 M ammonia solution. The largest difference in fluorescence ratio before and after the reaction was observed when the volume of the fluorescein isothiocyanate ethanol solution was 50 μL, i.e., the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution was 1:200. Therefore, the constructed dual-emission fluorescent probe exhibited the most significant color change when the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution was 1:200. This solution was used as the final dual-emission fluorescent probe solution for subsequent experiments.

[0070] Step 3: Preparation of electrospinning solution

[0071] Dissolve 1.8 g of polyurethane powder in 10 mL of a two-system solution (tetrahydrofuran: N,N-dimethylformamide = 7:3, v / v), stir at 30 °C for 4 h until it becomes clear and transparent, and obtain a polyurethane solution; add 1 mL of the dual-emission fluorescent probe solution screened in step 2, stir magnetically for 30 min, and sonicate at room temperature for 30 min to defoam and disperse evenly, to obtain the final polyurethane electrospinning solution containing the fluorescent probe.

[0072] Step 4: Preparation of transparent electrospun film

[0073] The polyvinylidene fluoride solution containing the fluorescent probe obtained in step 3 was injected into a 10 mL disposable syringe to eliminate air bubbles. Electrospinning was then performed using an electrospinning machine. The corresponding technical parameters were: 18 kV applied by a high-voltage power supply, a receiving distance of 20 cm, a solution propulsion rate of 0.0008 mm / s, a spinning temperature of 35 °C, and a relative humidity of 45%. Silicone paper was used as the receiving substrate, and the solution was collected by a rotating drum at 30 rpm. The electrospun membrane obtained after 12 hours of spinning was the ratiometric fluorescent indicator membrane. The electrospun membrane was then placed in a press and maintained at 25 MPa for 15 minutes to obtain a transparent electrospun membrane.

[0074] The transparent fluorescent electrospun membrane prepared in this embodiment was used to determine the freshness of beef. The color of the membrane was recorded every 12 hours, and its L*, a*, b* values ​​were measured, and the color difference value ΔE was calculated according to Formula 1.

[0075] The fluorescent color of the indicator membrane is red when the beef is initially fresh; as the beef deteriorates from fresh to stale and eventually spoils, the fluorescent color of the indicator membrane changes from red to orange and then to yellow.

[0076] Example 3:

[0077] Step 1: Synthesis of red fluorescent carbon quantum dots

[0078] Citric acid monohydrate was dissolved in ultrapure water and sonicated to obtain a 1.5 mol / L citric acid monohydrate solution. Under stirring, 0.0042 g of neutral red powder was slowly added to the citric acid monohydrate solution, resulting in a purplish-red mixed solution. The molar ratio of neutral red to citric acid was 1:1000. The mixed solution was transferred to a Teflon high-pressure reactor and reacted at 200℃ for 3 h. After cooling to room temperature, the solution was centrifuged at 11000 rpm for 10 min to remove insoluble precipitates. The supernatant was filtered through a 0.22 μM aqueous microporous membrane, and the filtrate was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 500 to remove unreacted matter. Finally, the purified carbon dot solution was dried in a vacuum freeze dryer for 12 h to obtain carbon quantum dot powder.

[0079] Step 2: Construction of dual-emission fluorescent probe;

[0080] Fluorescein isothiocyanate was dissolved in anhydrous ethanol to obtain a fluorescein isothiocyanate ethanol solution with a concentration of 0.15 mg / mL; the dried carbon quantum dot powder from step 1 was dissolved in ultrapure water and sonicated to obtain a carbon dot solution with a concentration of 1.5 mg / mL.

[0081] 1 mL of carbon dot solution was added to 5, 10, 20, 30, 40, and 50 μL of fluorescein isothiocyanate ethanol solution, respectively, and reacted with 150 μL of 0.1 M ammonia solution. The largest difference in fluorescence ratio before and after the reaction was observed when the volume of the fluorescein isothiocyanate ethanol solution was 30 μL, i.e., the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution was 1:333. Therefore, the constructed dual-emission fluorescent probe exhibited the most significant color change when the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution was 1:333. This solution was used as the final dual-emission fluorescent probe solution for subsequent experiments.

[0082] Step 3: Preparation of electrospinning solution

[0083] Dissolve 2.0 g of polycaprolactone powder in 10 mL of a two-system solution (acetic acid: ethanol = 4:1, v / v), stir at 25 °C for 5 h until it becomes clear and transparent, and obtain a polycaprolactone solution; add 4 mL of the most suitable fluorescent probe selected in step 2, stir magnetically for 30 min, and sonicate at room temperature for 30 min to defoam and disperse evenly, to obtain the final polycaprolactone electrospinning solution containing the fluorescent probe.

[0084] Step 4: Preparation of transparent electrospun film

[0085] The polycaprolactone solution containing the fluorescent probe obtained in step 3 was injected into a 10 mL disposable syringe to eliminate air bubbles. Electrospinning was then performed using an electrospinning machine. The corresponding technical parameters were: a voltage of 20 kV applied by a high-voltage power supply, a receiving distance of 25 cm, a solution propulsion rate of 0.0012 mm / s, a spinning temperature of 40 °C, and a relative humidity of 65%. Silicone paper was used as the receiving substrate, and the solution was collected by a rotating drum at a speed of 50 rpm. The resulting electrospinned membrane after 8 hours of spinning was the ratiometric fluorescent indicator membrane. The electrospinned membrane was then placed in a press and maintained at 15 MPa for 20 minutes to obtain a transparent electrospinned membrane.

[0086] The transparent fluorescent electrospun membrane prepared in this embodiment was applied to determine the freshness of chicken meat. The color of the membrane was recorded every 12 hours, and its L*, a*, and b* values ​​were measured, along with the color difference value ΔE calculated according to Formula 1. Initially, the fluorescent color of the indicator membrane was red when the chicken meat was fresh; as the chicken meat spoiled, the fluorescent color of the indicator membrane turned yellow.

[0087] Example 4:

[0088] Step 1: Synthesis of red fluorescent carbon quantum dots

[0089] 2.1014 g of citric acid monohydrate was dissolved in 10 mL of ultrapure water and sonicated to obtain a 1 mol / L citric acid monohydrate solution. Under stirring, 0.0029 g of neutral red powder was slowly added to the citric acid monohydrate solution, resulting in a purplish-red mixed solution. The molar ratio of neutral red to citric acid was 1:1000. The mixed solution was transferred to a Teflon high-pressure reactor and reacted at 180 °C for 4 h. After cooling to room temperature, the solution was centrifuged at 9000 rpm for 20 min to remove insoluble precipitates. The supernatant was filtered through a 0.22 μM aqueous microporous membrane, and the filtrate was dialyzed for 12 h using a dialysis bag with a molecular weight cutoff of 500 to remove unreacted substances. Finally, the purified carbon dot solution was dried in a vacuum freeze dryer for 12 h to obtain a purplish-red powder. Figure 1 The image shown is a transmission electron microscope (TEM) schematic of red fluorescent carbon quantum dots.

[0090] Step 2: Construction of dual-emission fluorescent probe;

[0091] Dissolve 1 mg of fluorescein isothiocyanate in 10 mL of anhydrous ethanol to obtain an ethanol solution of fluorescein isothiocyanate with a concentration of 0.1 mg / mL. Dissolve 10 mg of the dried carbon dot powder from step 1 in 10 mL of ultrapure water and sonicate to obtain a carbon dot solution with a concentration of 1 mg / mL.

[0092] Add 5, 10, 20, 30, 40, and 50 μL of fluorescein isothiocyanate ethanol solution to 1 mL of carbon dot solution, and then react with 150 μL of 0.1 M ammonia solution. The largest difference in fluorescence ratio before and after the reaction was observed when the volume of the fluorescein isothiocyanate ethanol solution was 40 μL, i.e., the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution was 1:250. Therefore, the constructed dual-emission fluorescent probe exhibited the most significant color change when the mass ratio of fluorescein isothiocyanate in the ethanol solution to carbon quantum dots in the carbon dot solution was 1:250. This solution was used as the final dual-emission fluorescent probe solution for subsequent experiments.

[0093] Step 3: Preparation of electrospinning solution

[0094] Dissolve 1.8 g of polyacrylonitrile powder in 10 mL of a two-system solution (tetrahydrofuran: N,N-dimethylformamide = 2:3, v / v), stir at 45 °C for 2 h until it becomes clear and transparent, and obtain a polyacrylonitrile solution; add 0.5 mL of the optimal fluorescent probe selected in step 2, stir magnetically for 1 h, and sonicate at room temperature for 40 min to defoam and disperse evenly, to obtain the final polyacrylonitrile electrospinning solution containing the fluorescent probe.

[0095] Step 4: Preparation of transparent electrospun film

[0096] The polyacrylonitrile solution containing the fluorescent probe obtained in step 3 was injected into a 10 mL disposable syringe to eliminate air bubbles. Electrospinning was then performed using an electrospinning machine. The corresponding technical parameters were: a voltage of 16 kV applied by a high-voltage power supply, a receiving distance of 15 cm, a solution propulsion rate of 0.001 mm / s, a spinning temperature of 35 °C, and a relative humidity of 40%. Silicone paper was used as the receiving substrate, and the solution was collected by a rotating drum at a speed of 30 rpm. The resulting electrospinned membrane after 10 hours of spinning was the ratiometric fluorescent indicator membrane. The electrospinned membrane was then placed in a press and maintained at 5 MPa for 30 minutes to obtain a transparent electrospinned membrane.

[0097] The transparent fluorescent electrospun membrane prepared in this embodiment was applied to determine the freshness of pork. The color of the membrane was recorded every 12 hours, and its L*, a*, and b* values ​​were measured, as well as the color difference value ΔE was calculated according to Formula 1. The fluorescent color of the indicator membrane corresponding to the initial fresh pork state was red; as the pork changed from fresh to stale and eventually spoiled, the fluorescent color of the indicator membrane changed from red to orange and then to yellow.

[0098] In summary, in practical applications, as the freshness of pork, beef, and chicken decreases, the color of the indicator film gradually changes from red to orange and finally to yellow, clearly and accurately indicating the change in meat freshness from fresh to not fresh and even to spoilage. The color change provides a direct understanding of the meat's freshness and confirms its current condition.

[0099] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio, characterized in that, Includes the following steps: Step 1: Synthesis of red fluorescent carbon quantum dots; Step 1.1: Dissolve citric acid monohydrate in ultrapure water and sonicate to obtain citric acid monohydrate solution; then, under stirring conditions, add neutral red powder to the citric acid monohydrate solution to obtain a purple-red mixed solution. The mixed solution was transferred to a high-pressure reactor for reaction, and then cooled to room temperature to obtain the reaction solution. Step 1.2: After centrifuging the reaction solution obtained in Step 1.1, filter it through a microporous membrane to obtain the filtrate; after dialysis, the obtained filtrate yields a carbon dot solution; finally, the product obtained by vacuum freeze-drying is the red fluorescent carbon quantum dots. Step 2: Construction of dual-emission fluorescent probe; Step 2.1: Dissolve fluorescein isothiocyanate in anhydrous ethanol, and sonicate to obtain fluorescein isothiocyanate ethanol solution; then dissolve the red fluorescent carbon quantum dots obtained in step 1 in water, and sonicate to obtain carbon dot solution. Step 2.2: Mix the fluorescein isothiocyanate ethanol solution from Step 2.1 with the carbon dot solution. The resulting mixed solution is the dual-emission fluorescent probe solution. Step 3: Preparation of electrospinning solution Step 3.1: Dissolve the hydrophobic polymer in one or more organic solvents to obtain a hydrophobic polymer solution. Heat and stir the polymer solution to dissolve it, and then defoam it by ultrasound to obtain a transparent spinning solution. The organic solvent is one or a mixture of two of N,N-dimethylformamide, ethanol, acetic acid, tetrahydrofuran, dichloromethane, acetone, and dimethyl sulfoxide. The hydrophobic polymer is any one of polyvinylidene fluoride, polycaprolactone, polyurethane, and polyacrylonitrile. Step 3.2: Add the dual-emission fluorescent probe solution obtained in step 2 to the transparent spinning solution obtained in step 3.1, stir and mix evenly, and then obtain an electrospinning solution containing fluorescent probe after ultrasonic defoaming treatment. Step 4: Preparation of transparent electrospun film; Electrospinning was performed using an electrospinning solution containing a fluorescent probe to obtain an electrospinned membrane. The electrospinned membrane was then placed in a press, and after setting and maintaining the pressure, a transparent electrospinned membrane was obtained, which is a porous and transparent fluorescent indicator membrane for meat freshness ratio.

2. The method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, In step 1.1, the concentration of citric acid monohydrate solution is 0.5-1.5 mol / L; the concentration of neutral red in the purple-red mixed solution is 0.5-1.5 mmol / L; the reaction temperature is 140-220℃, and the reaction time is 2-6 h.

3. The method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, In step 1.2, the centrifugation speed is 8000-11000 rpm and the time is 10-30 min; the pore size of the microporous filter membrane is 0.22 μm; the dialysis step is: dialysis for 12 h using a dialysis bag with a molecular weight cutoff of 500.

4. The method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, In step 2.1, the concentration of the fluorescein isothiocyanate ethanol solution is 0.05-0.15 mg / mL; the concentration of the carbon dot solution is 0.05-0.09 g / mL.

5. The method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, In step 2.2, the mass ratio of fluorescein isothiocyanate in the ethanol solution to red fluorescent carbon dots in the carbon dot solution is 1:(200-400).

6. A method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, In step 3.1, the mass concentration of the hydrophobic polymer solution is 15%-20%; when the organic solvent is a mixture of acetone and N,N-dimethylformamide, the volume ratio is 1:4; when the organic solvent is a mixture of acetic acid and ethanol, the volume ratio is 4:1; when the organic solvent is a mixture of dichloromethane and dimethyl sulfoxide, the volume ratio is 7:3; when the organic solvent is a mixture of tetrahydrofuran and N,N-dimethylformamide, the volume ratio is 2:3; the heating temperature is 25-45℃, and the stirring time is 2-5 h.

7. A method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, In step 3.2, the volume ratio of the dual-emission fluorescent probe solution to the electrospinning solution is 1:(10~30); the stirring time is 0.5-1.5 h; and the ultrasonic treatment time is 20-50 min.

8. A method for preparing a porous and transparent fluorescent indicator film for meat freshness ratio according to claim 1, characterized in that, The electrospinning process parameters in step 4 are as follows: voltage 15-20 kV, temperature 30-40℃, relative humidity 35-75%, spinning distance 15-25 cm, solution propulsion rate 0.0005-0.0015 mm / s, roller speed 20-50 rpm, spinning time 6-12 h; pressure 5-35 MPa, and holding time 10-30 min.

9. A method for detecting meat freshness using a porous and transparent meat freshness ratio fluorescent indicator membrane prepared according to any one of claims 1-8, characterized in that, The specific steps are as follows: Place fresh meat in a food storage container, cut a fluorescent ratio type meat freshness indicator film to the required size, seal it together with the meat, and store it in an environment of 0-4℃. As the storage time of the meat increases, the volatile amine gas in the packaging environment increases, and the fluorescence color of the indicator film changes from red to orange and then to yellow. At the same time, the volatile basic nitrogen content of the meat is measured, and the corresponding change in the fluorescence color of the indicator film under ultraviolet light is recorded. The obtained color difference value is correlated with the volatile basic nitrogen value of the meat, so as to judge the freshness of the meat based on the change in fluorescence color and achieve visual detection.

Images