A Nile blue dye and its preparation method, a preparation method of a packaging film based on the prepared dye, and the uses of the dye and the packaging film

Abstract

The present invention discloses a Nile blue dye and its preparation method, a preparation method of a packaging film based on the prepared dye, and the uses of the dye and the packaging film, belonging to the technical field of food monitoring and preservation. Based on the Nile blue parent structure, by introducing different heavy atoms at the meso position and introducing alkyl chains with different chain lengths at the amino position, photosensitive dyes with various pH responses and photodynamic therapy capabilities are constructed. By comparing various heavy atom substitution structures, Dye I and Dye II of the present invention are both sensitive to the pH of the solution to a certain extent, and the thio-dye packaging film and the seleno-dye packaging film both exhibit excellent antibacterial ability against Escherichia coli. The Dye I packaging film and the Dye II packaging film of the present invention both have excellent antibacterial activity against Escherichia coli. Both can achieve visual detection of the decay process of fresh shrimps and preservation of bananas.

Description

Technical Field

[0001] The present invention belongs to the technical field of food monitoring and preservation. Specifically, it relates to a Nile blue dye and a preparation method thereof, and also relates to a preparation method of a packaging film based on the prepared dye, and further relates to the application of the dye and the packaging film in the fields of food dynamic visualization detection and antibacterial preservation. Background Art

[0002] With the development of the economy and the increasing improvement of living standards, consumers have begun to pursue high-quality foods with higher cleanliness and better freshness, and the research and development of food packaging science and technology to meet the needs of consumers is very urgent. Traditional packaging methods usually only provide static information such as production date and shelf life. Since these static information cannot convey the real-time status and dynamic changes of food freshness, their reference value for consumers to judge food freshness is limited.

[0003] By real-time monitoring the freshness of packaged foods during transportation, storage, and sales, intelligent packaging can effectively solve the above problems. The application of intelligent packaging not only improves the quality and safety of foods, but also meets the higher requirements of consumers for food safety, and is an important innovation area for the packaging industry in the future.

[0004] During the process of food spoilage, the chemical environment inside the food changes, affecting its pH value. In addition, foods are prone to microbial contamination at all stages. Proteins in meat and seafood will decompose into alkaline volatile nitrogen compounds (such as ammonia, trimethylamine, and dimethylamine, etc.), resulting in further changes in pH value. Therefore, real-time monitoring of the changes in the pH value of foods throughout the supply chain can effectively track the freshness and spoilage of packaged foods.

[0005] Currently, various methods and technologies reported for pH sensing and imaging include potentiometric titration, microelectrode sensing, and absorption spectroscopy, etc. However, these technologies require relatively professional technical knowledge and are usually operated by technicians in the laboratory, and cannot meet the needs of real-time detection of the freshness of fish and shrimp foods in daily life. As an alternative to traditional detection methods, fluorescent pH-responsive sensors have been used for pH detection and visualization in the fields of optics and biomedicine. Natural pH pigments such as anthocyanins, curcumin, and alizarin have been widely reported as intelligent films for food packaging due to their visible color changes and non-toxic characteristics. Among them, more reports are about using anthocyanins from different sources as food freshness indicators, such as purple sweet potatoes, butterfly pea flowers, purple cabbages, red cabbages, etc. However, anthocyanins are prone to degradation and color change under the action of environmental factors such as light, temperature, and metal ions, affecting their pH indicating ability. In addition, the poor stability of anthocyanins greatly limits their application in intelligent food packaging.

[0006] The PCT patent application with the publication number WO2017007552A1 discloses a "pH-sensitive nanoparticle for detecting and preventing food spoilage", which involves the technical problems of food pH-sensitive detection and preventing food spoilage. However, it has the following main defects: First, the structure of its pH-sensitive nanoparticles is complex, the synthesis and purification are difficult, and the repeatability is poor. It requires the aid of exogenous hydrophobic surfactants as indicators to indicate or prevent food spoilage. Second, the nanoparticle uses Nile red dye as an exogenous hydrophobic surfactant, and uses its fluorescence signal to indicate the change of environmental pH, and then realizes the monitoring of food spoilage. It needs to be operated with the aid of additional large instruments such as fluorescence spectrometers, and the process is complex. Secondly, it cannot achieve real-time dynamic visual monitoring and cannot achieve the purpose of real-time visual monitoring with the naked eye. Third, the nanoparticle uses Nile red dye as an exogenous hydrophobic surfactant. According to the change of environmental pH, the relative change in the amount of Nile red released is used to indirectly indicate the food spoilage situation according to the change of the red depth in the solution, and the color recognition is limited, which is easy to cause confusion. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a Nile blue dye and its preparation method, a preparation method of a packaging film based on the prepared dye, and the uses of the dye and the packaging film, to explore and develop a dye with high sensitivity, good stability and good biocompatibility, which is used on the one hand to monitor the pH change during food storage, and on the other hand to extend the food preservation time.

[0008] The present invention adopts the following technical solutions:

[0009] A Nile blue dye, the structural formula of the Nile blue dye is as follows:

[0010] 。

[0011] The preparation method of the Nile blue dye includes the following steps:

[0012] (1) Add 1-naphthylamine, K 2 CO 3 and iodoethane into ethanol, heat under reflux and stir; remove the solvent, and the crude product is eluted and purified to obtain compound B;

[0013] (2) Dissolve 3-iodoaniline and K 2 CO 3 in acetonitrile, add iodoethane, and use N 2 protection, heat and reflux and stir to react; after the reaction is completed, cool, remove the solvent, and the crude product is eluted and purified to obtain compound C;

[0014] (3) Place compound C, selenium powder, dimethyl sulfoxide, copper oxide and potassium hydroxide in a container, and use N2 Protect the heating and reflux reaction; extract after suction filtration, dry and remove the solvent; elute and purify the crude product to obtain compound D;

[0015] (4)In an ice bath, add hydrochloric acid to compound D while stirring, and then add NaNO 2 , and then stir in the ice bath; extract, dry, remove the solvent, and elute and purify the crude product to obtain compound E;

[0016] (5)Dissolve compound E and compound B prepared in step (1) in trifluoroethanol, heat and stir, and use N 2 Protect the heating and reflux reaction; after the reaction stops, cool, add hydrochloric acid to adjust the pH of the solution to acidic; extract, dry, purify, and rotary evaporate to form a powder to obtain Nile blue dye for food dynamic visualization detection and antibacterial preservation.

[0017] Furthermore, in the said step (1), the molar ratio of 1-naphthylamine:K 2 CO 3 : iodoethane is 1:(1 - 1.5):(1 - 1.5); the heating and reflux condition is to react at 80 - 90 °C for 2 - 6 hours; in the said step (2), the molar ratio of 3-iodoaniline:K 2 CO 3 : iodoethane is 1:(2 - 3):(2 - 3); the reaction condition of heating and refluxing is to react at 80 - 85 °C for 14 - 18 hours; in the said step (3), the molar ratio of compound C, selenium powder, copper oxide and potassium hydroxide is 1:(1.5 - 2.5):(0.4 - 0.6):(6 - 8); the heating and reflux condition is to react at 95 - 105 °C for 10 - 14 hours; the reaction time of stirring in the ice bath in the said step (4) is 0.3 - 4 hours; in the said step (5), the molar ratio of compound E and compound B is 1:(2 - 4); the heating and reflux reaction condition is to react at 85 - 95 °C for 1 - 3 hours; the acidification condition is pH 2 - 3.

[0018] A preparation method of a packaging film based on the said Nile blue dye, comprising the following steps:

[0019] Disperse the said Nile blue dye in a dimethyl sulfoxide solution, and ultrasonically treat it to form a uniform dispersion liquid to obtain a mother liquor; dissolve sodium alginate in water by heating; dissolve the said mother liquor in the sodium alginate solution, stir, let stand, and then pour it into a mold and dry to obtain a packaging film of Nile blue dye for food dynamic visualization detection and antibacterial preservation.

[0020] Furthermore, the mass ratio of Nile blue dye to sodium alginate is 1:(450 - 550); the heating and dissolving condition is 45 - 55 °C for 3 - 6 h; the film-forming temperature is 45 - 55 °C.

[0021] A method for preparing a packaging film of Nile blue dye prepared by the described method for preparing Nile blue dye, comprising the following steps:

[0022] Disperse the Nile blue dye in a dimethyl sulfoxide solution, and perform ultrasonic treatment to form a uniform dispersion to obtain a mother liquor; dissolve sodium alginate in water by heating; dissolve the mother liquor in the sodium alginate solution, stir, let stand, and then pour it into a mold and dry to obtain a packaging film of Nile blue dye for dynamic visual detection and antibacterial preservation of food.

[0023] Furthermore, the mass ratio of Nile blue dye to sodium alginate is 1:(450 - 550); the heating and dissolving conditions are 45 - 55 °C for 3 - 6 h; the film-forming temperature is 45 - 55 °C.

[0024] The described Nile blue dye is applied to dynamic visual detection and antibacterial preservation of food.

[0025] The Nile blue dye prepared by the described method for preparing Nile blue dye is applied to dynamic visual detection and antibacterial preservation of food.

[0026] The packaging film prepared by the described method for preparing the packaging film is applied to dynamic visual detection and antibacterial preservation of food.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Nile blue is a kind of benzophenoxazine dye, which is usually used for in vitro tissue staining imaging and photodynamic therapy of RNA, lysosomes or lipids. Due to its excellent photophysical properties, stability and good biosafety, it has become a research hotspot in recent years. The photodynamic antimicrobial technology (PDAT) with Nile blue dye as the core has been widely reported. The mechanism of PDAT is that under the excitation of a specific wavelength light source, the photosensitizer jumps from the ground state (S 0 ) to the singlet excited state (S 1 ), and S 1 undergoes intersystem crossing (ISC) to the triplet excited state (T 1 ), transfers the energy directly or indirectly to oxygen, and then generates reactive oxygen species (ROS). The generated ROS will interact with cells, causing the leakage of cytoplasmic contents and ultimately leading to the death of microorganisms. Therefore, by regulating the relationship between the structure and properties of Nile blue dyes and applying them to intelligent food monitoring, it is a promising and effective technical path to achieve food preservation and dynamic visual detection.

[0029] Based on the Nile blue parent structure, the present invention constructs photosensitizing dyes with various pH responses and photodynamic therapy capabilities by introducing different heavy atoms at the meso position and alkyl chains with different chain lengths at the amino position. By comparing various heavy-atom substitution structures, it is found that thio dyes (Dye I of the present invention) and seleno dyes (Dye II of the present invention) are both sensitive to the pH of the solution, and the packaging films of thio dyes and seleno dyes both exhibit excellent antibacterial ability against Escherichia coli. Further, the seleno dye is more sensitive to the pH of the solution and starts to change color at pH 8; compared with the packaging film of Dye I of the present invention, the packaging film of Dye II has stronger antibacterial activity against Escherichia coli.

[0030] The present invention respectively dopes Dye I and Dye II with sodium alginate as the substrate, and uses the casting method to prepare the packaging film of Dye I and the packaging film of Dye II. The packaging films of Dye I and Dye II are respectively used for visual detection tests on fresh shrimps as the storage time extends. It is found that the packaging film of Dye I starts to have obvious color changes on the 3rd day, and the packaging film of Dye II has obvious color changes on the 2nd day. The packaging films of Dye I and Dye II both have the effect of preserving bananas. Among them, the packaging film of Dye II has a longer banana preservation time compared with the packaging film of Dye I. The Nile blue dyes and packaging films of the present invention can achieve more efficient and rapid pH-sensitive detection and photo-bacteriostatic and anti-corrosion. Description of the Drawings

[0031] Figure 1 It is a diagram of the color change situation and ultraviolet absorption spectrum of Nile blue-based photosensitizing Dye I obtained in Example 5 of the present invention in solutions with different pH values.

[0032] Figure 2 It is a diagram of the color change situation and ultraviolet absorption spectrum of Nile blue-based photosensitizing Dye II obtained in Example 5 of the present invention in solutions with different pH values.

[0033] Figure 3 It is a diagram of the color change situation of the packaging film of Nile blue-based photosensitizing Dye I and the packaging film of Nile blue-based photosensitizing Dye II obtained in Example 6 of the present invention in solutions with different pH values. In the figure, Film I represents the packaging film based on Dye I, and Film II represents the packaging film based on Dye II.

[0034] Figure 4 It is a diagram of the color change situation of the packaging film of Nile blue-based photosensitizing Dye I and the packaging film of Nile blue-based photosensitizing Dye II obtained in Example 7 of the present invention in various volatile basic nitrogen solutions. In the figure, Film I represents the packaging film based on Dye I, and Film II represents the packaging film based on Dye II.

[0035] Figure 5 It is a diagram of the antibacterial performance test of Nile blue-based photosensitizing Dye I and Nile blue-based photosensitizing Dye II against Staphylococcus aureus obtained in Example 8 of the present invention.

[0036] Figure 6 This is a graph showing the antibacterial performance test of Nile blue - type photosensitive dye Ⅰ packaging film and Nile blue - type photosensitive dye Ⅱ packaging film obtained in Example 9 of the present invention against Escherichia coli. In the figure, film Ⅰ represents the packaging film based on dye Ⅰ, and film Ⅱ represents the packaging film based on dye Ⅱ.

[0037] Figure 7 This is a graph showing the visual detection of Nile blue - type photosensitive dye Ⅰ packaging film and Nile blue - type photosensitive dye Ⅱ packaging film during the spoilage process of fresh shrimp obtained in Example 10 of the present invention. In the figure, film Ⅰ represents the packaging film based on dye Ⅰ, and film Ⅱ represents the packaging film based on dye Ⅱ.

[0038] Figure 8 This is a graph showing the preservation of bananas by Nile blue - type photosensitive dye Ⅰ packaging film and Nile blue - type photosensitive dye Ⅱ packaging film obtained in Example 11 of the present invention. In the figure, film Ⅰ represents the packaging film based on dye Ⅰ, and film Ⅱ represents the packaging film based on dye Ⅱ. Detailed implementation manners

[0039] The following further illustrates the detailed implementation manners of the present invention in combination with examples. It should be noted that the detailed implementation manners described here are only for explaining and interpreting the present invention, and are not used to limit the protection scope of the present invention.

[0040] Example 1 (as a comparative example) Preparation example of Nile blue - type photosensitive dye Ⅰ (abbreviated as dye Ⅰ).

[0041] (1) In a reaction vessel, N,N -Diethyl - p - phenylenediamine (1.64 g, 10 mmol) was added to an aqueous solution of 20 mL of aluminum sulfate (4.11 g, 12 mmol), and continuous stirring was carried out. Sodium thiosulfate (4.42 g, 28 mmol) and zinc chloride (1.74 g, 12.78 mmol) were added successively. The reaction vessel was placed in an ice bath, and within 20 min, about 8 mL of an aqueous solution of potassium dichromate (0.98 g, 3.36 mmol) was added. After continuous stirring in the ice bath for 2 h, the obtained product was washed with acetone and filtered. The obtained crude product was refluxed in 24 mL of methanol, filtered to obtain a dark - gray solid, and after drying, a compound A with the following structure was obtained:

[0042] .

[0043] (2) 1-Naphthylamine (0.89 g, 6.25 mmol), potassium carbonate (1.05 g, 7.58 mmol) and iodoethane (1.04 g, 6.61 mmol) were added to 12 mL of ethanol, and the mixture was refluxed and stirred at 85 °C for 4 h. The solvent was removed by distillation under reduced pressure, and the crude product was eluted with petroleum ether and ethyl acetate as eluents, and purified by silica gel column chromatography to obtain compound B in the form of a yellow oil and having the following structure:

[0044] .

[0045] (3) Compound A (298 mg, 1 mmol) and compound B (308 mg, 1.8 mmol) were added to 25 mL of methanol, and the mixture was refluxed and stirred at 70 °C. Then, silver carbonate (606 mg, 2.2 mmol) was slowly added to the reflux reaction. The mixture was further heated for 30 min until the reaction mixture turned dark blue, and then cooled to room temperature. The solvent was removed by distillation under reduced pressure to obtain a dark blue crude product. The crude product was dissolved in 25 mL of dichloromethane, and extracted three times with saturated aqueous sodium carbonate solution (3×100). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, acidified with concentrated hydrochloric acid (0.4 mL) and concentrated. The product was purified by silica gel column chromatography using methanol and dichloromethane as eluents to obtain dye I having the following structural formula:

[0046] .

[0047] Example 2 Preparation example of Nile blue-based photosensitive dye II (abbreviated as dye II).

[0048] (1) 3-Iodoaniline (1 g, 5 mmol) and K 2 CO 3 (1.6 g, 11.5 mmol) were dissolved in 30 mL of MeCN (acetonitrile), and iodoethane (1.8 g, 11.5 mmol) was added dropwise. The reaction was carried out under N 2 protection and refluxed and stirred at 82 °C in an oil bath for 16 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction progress was monitored by thin layer chromatography (TLC). Silica gel powder was added, and the mixture was evaporated to a powder in a vacuum rotary evaporator. Finally, it was purified by column chromatography (PE:EtOAc = 20:1) to obtain an orange-yellow oily compound C having the following structural formula:

[0049] .

[0050] (2) Place compound C (1.1 g, 4 mmol), selenium powder (0.632 g, 8 mmol), dimethyl sulfoxide (15 mL), copper oxide (0.152 g, 2 mmol) and potassium hydroxide (1.692 g, 28.8 mmol) in a container, protect it with N 2 and place the container in an oil bath. React under reflux at 100 °C for 12 hours. Place filter paper in a Buchner funnel and perform vacuum filtration on the solution. The filtered solution is grass green. Then perform multiple extractions with an EtOAc / saturated brine system, take the upper organic phase to obtain an orange-red solution. Monitor the reaction of the obtained solution by TLC, and observe the formation of the target product and the remaining raw materials in the product. Use a vacuum rotary evaporator to remove the solvent, add silica gel powder and rotary evaporate to form a powder, and purify it by column chromatography (PE:DCM = 2:1) to obtain compound D with the following structural formula:

[0051] .

[0052] (3) Add compound D (0.36 g, 0.8 mmol) to a 25 mL container, add 1 mol / L HCl, add a magnetic stir bar to the container and place the container in a magnetic stirrer with an ice bath. While stirring, dropwise add 1.2 mL of NaNO 2 , after the addition is complete, stir in the ice bath for 0.5 h, and observe that the solution is dark red. Perform multiple extractions with an EtOAc / saturated brine system, take the upper organic phase for TLC detection of the reaction, and obtain the mobile phase of the column chromatography suitable for the target product. Then use anhydrous Na 2 SO 4 for overnight drying, use a vacuum rotary evaporator to remove the solvent, add silica gel powder and rotary evaporate to form a powder, and purify it by column chromatography (MeOH:DCM = 1:20) to obtain a red powder compound E with the following structural formula:

[0053] .

[0054] (4) In a container, dissolve compound E (102.8 mg, 0.2 mmol) and compound B prepared in Example 1 (321.6 g, 0.6 mmol) in 5 mL of trifluoroethanol, add a magnetic stir bar and place the container in a magnetic stirrer with an oil bath, protect it with N 2Protect and reflux at 90 °C for 2 h. After the reaction stopped, the solution was observed to be dark red. After natural cooling, 1 mL of 1M HCl was added for acidification, and the pH was detected with pH test paper to be 2 - 3. It was extracted multiple times with a DCM / saturated brine system, and the lower organic phase was taken and dried overnight with anhydrous sodium sulfate. Silica gel powder was added and rotary evaporated to form a powder, and column chromatography (methanol:dichloromethane = 1:10) was used for purification to obtain the product dye II in the form of a blue powder with the following structural formula:

[0055] .

[0056] Example 3 (as a comparative example) Preparation example of the packaging film based on Dye I.

[0057] Disperse the Dye I (3.581 mg) prepared in Example 1 in 3 mL of dimethyl sulfoxide solution, and ultrasonically treat it for 1 min to form a uniform dispersion to obtain mother liquor I. Dissolve 0.5 g of sodium alginate in 50 mL of deionized water, heat at 50 °C for 4 h. After it was completely dissolved, 833.3 μL of mother liquor I was dissolved in 50 mL of sodium alginate solution (10 mg / mL), stirred for 30 min, left to stand overnight, poured into a mold to form a film, and the mold was placed in an oven and heated at 50 °C for 48 h to obtain the packaging film based on Dye I.

[0058] Example 4 Preparation example of the packaging film based on Dye II.

[0059] Disperse the Dye II (3.684 mg) prepared in Example 2 in 3 mL of dimethyl sulfoxide solution, and ultrasonically treat it for 1 min to form a uniform dispersion to obtain mother liquor II. Dissolve 0.5 g of sodium alginate in 50 mL of deionized water, heat at 50 °C for 4 h. After it was completely dissolved, 833.3 μL of mother liquor II was dissolved in 50 mL of sodium alginate solution (10 mg / mL), stirred for 30 min, left to stand overnight, poured into a mold to form a film, and the mold was placed in an oven and heated at 50 °C for 48 h to obtain the packaging film based on Dye II.

[0060] Example 5 Characterization example of the color change of the solution based on Dye I and the solution based on Dye II in solutions with different pH values.

[0061] Perform color and ultraviolet absorption spectrum analysis on the Dye I prepared in Example 1 and the Dye II prepared in Example 2 in solutions with different pH values, and respectively obtain Figure 1 and Figure 2 the color and ultraviolet absorption spectrum diagrams shown.

[0062] From Figure 1It can be seen that the solution of Dye I is blue when the pH value is between 2 and 9, turns from blue to purple at pH 10, and finally shows pink at pH 11, and the pink color gradually deepens with the increase of pH. From Figure 2 It can be seen that the solution of Dye II is blue when the pH value is between 2 and 8. When the pH value is 9, the solution color changes from blue to light purple, and when the pH value is between 10 and 12, the pink color gradually deepens.

[0063] Conclusion: For both Dye I and Dye II, as the pH increases, the solution color changes from blue through purple and finally shows pink, and the pink color gradually deepens with the increase of pH. Further, Dye II is more sensitive to pH than Dye I, and the color change occurs at pH 9.

[0064] Example 6: Characterization example of the color change of Dye I packaging film and Dye II packaging film in solutions with different pH values.

[0065] The color changes of the Dye I packaging film prepared in Example 3 and the Dye II packaging film prepared in Example 4 in solutions with different pH values were tested. The Dye I packaging film and the Dye II packaging film were respectively immersed in buffer solutions with a pH value ranging from 4 to 13 for 5 minutes. The color changes of the packaging films were captured using a mobile phone camera to obtain Figure 3 Schematic diagrams showing the color changes of the Dye I packaging film and the Dye II packaging film at different pH values.

[0066] From Figure 3 it can be known that when the solution pH value is between 4 and 9, the color of the Dye I packaging film is blue. When the solution pH value is between 10 and 11, the color of the Dye I packaging film changes from grayish blue to purple, and finally shows pink at pH 12, and the pink color gradually deepens with the increase of pH. When the solution pH value is between 4 and 8, the color of the Dye II packaging film is blue. When the solution pH value is between 9 and 10, the color of the Dye II packaging film changes from grayish blue to light purple, and finally shows pink at pH 11, and the pink color gradually deepens with the increase of pH.

[0067] Conclusion: For both the Dye I packaging film and the Dye II packaging film, as the pH increases, the solution color changes from blue through grayish blue and purple and finally shows pink, and the pink color gradually deepens with the increase of pH. Further, the Dye II packaging film is more sensitive to pH response, and the color change occurs at pH 9.

[0068] Example 7: Characterization example of the color change of Dye I packaging film and Dye II packaging film in various volatile basic nitrogen.

[0069] The dye I packaging film prepared in Example 3 and the dye II packaging film prepared in Example 4 were respectively placed in various volatile basic nitrogen environments to detect the color change of the film. Specifically, the dye I packaging film and the dye II packaging film were respectively placed in 10 mL of solutions containing ammonia, dimethylamine, and trimethylamine (2 mol / L). The color change was recorded every 2 minutes using a mobile phone camera for a total of 10 minutes. The Figure 4 figure showing the color change of the Nile blue-based photosensitive dye I packaging film and the Nile blue-based photosensitive dye II packaging film in various volatile basic nitrogen solutions.

[0070] As Figure 4 , in the ammonia water solution, the dye I packaging film changed from blue to purple within 6 minutes and then to pink at 8 minutes; in the dimethylamine solution, the dye I packaging film changed from blue to purple within 4 minutes and then to pink at the 6th minute; in the trimethylamine solution, the dye I packaging film immediately turned pink after 2 minutes. For the dye II packaging film, in the three volatile basic nitrogen solutions, it quickly changed from blue to pink within 2 minutes, indicating that both the dye I packaging film and the dye II packaging film have sensitivity and universality. However, the dye II packaging film has better sensitivity and universality.

[0071] Example 8 Characterization example of the antibacterial test of dye I and dye II against Staphylococcus aureus.

[0072] First, pick a single colony of Staphylococcus aureus and transfer it to a conical flask containing LB medium (20 mL), and culture it on a shaker for 12 h (rotation speed: 150 rpm / min, temperature: 37 °C); then, centrifuge the obtained fresh bacterial suspension at 5000 rpm / min for 5 min, discard the supernatant, redisperse the precipitated bacteria into sterile PBS solution, and continue to centrifuge and wash twice at the same rotation speed; finally, directly dilute the bacteria with PBS solution to 10 3 cfu / mL for standby.

[0073] Add PBS, dye I, and dye II to 3 mL of the standby bacterial solution (10 3 cfu / mL) respectively to make their concentrations 50 μM. The PBS-treated one was set as the control group, the PBS-treated bacterial solution with light was set as the light group, the bacterial solutions treated with dye I and dye II were respectively set as the dye I group and the dye II group, and the bacterial solutions treated with dye I and dye II with light were respectively set as the dye I + light group and the dye II + light group. Among them, the light group was incubated in a 37 °C shaker after adding the dye, and then irradiated with a 650 nm xenon lamp for 10 minutes (light dose: 50 mW / cm 2), and then incubated in a shaker at 37°C for 12 hours. 0.1 mL of the bacterial solution from each group was spread on an agar plate and incubated in an incubator at 37°C for 24 hours to obtain Figure 5 The test diagrams of the antibacterial properties of Nile blue-based photosensitizing dye Ⅰ and Nile blue-based photosensitizing dye Ⅱ against Staphylococcus aureus as shown.

[0074] As can be seen from Figure 5 : Taking the PBS treatment group as the control group, the group with only light irradiation had no obvious antibacterial activity, and the dark treatment of the dye Ⅰ solution and the dye Ⅱ solution also did not show obvious antibacterial activity. The inhibition efficiencies of the light-irradiated groups of the dye Ⅰ solution and the dye Ⅱ solution against Staphylococcus aureus were 74.8% and 96.3% respectively after light irradiation. The above experiments show that both dye Ⅰ and dye Ⅱ exhibit significant antibacterial abilities against Staphylococcus aureus. Further, compared with dye Ⅰ, dye Ⅱ has stronger antibacterial activity against Staphylococcus aureus.

[0075] Example 9 Characterization example of the use of the packaging film of dye Ⅰ and the packaging film of dye Ⅱ for antibacterial test of Escherichia coli.

[0076] First, pick a single Escherichia coli colony and transfer it to a conical flask containing LB medium (20 mL), and culture it on a shaker for 12 h (rotation speed: 150 rpm / min, temperature: 37°C); then, centrifuge the obtained fresh bacterial suspension at 5000 rpm / min for 5 min, discard the supernatant, redisperse the precipitated bacteria in a sterile PBS solution, and continue to centrifuge and wash twice at the same rotation speed; finally, directly dilute the bacteria with PBS solution to 10 6 cfu / mL for standby.

[0077] Apply 0.1 mL (10 6 cfu / mL) of the bacterial solution on an agar plate. Cut the packaging film of dye Ⅰ and the packaging film of dye Ⅱ into squares with a side length of 1.0 cm respectively, attach them to the middle position of the agar plate, incubate in an incubator at 37°C for 2 hours, and then irradiate with a 650 nm xenon lamp for 10 minutes (light dose: 50 mW / cm 2 ), and incubate in an incubator at 37°C for 24 hours. Measure the diameter of the inhibition zone produced to obtain Figure 6 The test diagrams of the antibacterial properties of the packaging film of Nile blue-based photosensitizing dye Ⅰ and the packaging film of Nile blue-based photosensitizing dye Ⅱ against Escherichia coli as shown.

[0078] As can be seen from Figure 6It can be seen that taking no treatment as the control group, the antibacterial activity of the group with only light irradiation can be ignored, and the antibacterial activity of the dark treatment of the packaging film of Dye I and the packaging film of Dye II can be ignored. The inhibition efficiencies of the packaging film of Dye I and the packaging film of Dye II against Escherichia coli under light conditions are 50.0% and 52.4% respectively. The above experiments show that both the packaging film of Dye I and the packaging film of Dye II exhibit obvious antibacterial ability against Escherichia coli. However, compared with the packaging film of Dye I, the packaging film of Dye II has stronger antibacterial activity against Escherichia coli.

[0079] Example Ten: Characterization Example of Using the Packaging Film of Dye I and the Packaging Film of Dye II for Visual Detection of the Freshness of Fresh Shrimp.

[0080] The packaging film of Dye I prepared in Example Three and the packaging film of Dye II prepared in Example Four were respectively used for visual detection of the freshness of fresh shrimp. First, the fresh shrimp samples (10 ± 1.0 g) were placed in a transparent culture dish (Φ 90 mm), and a rectangular packaging film of Dye I and a packaging film of Dye II (length: 1.5 cm, width: 0.8 cm) were pasted on the inner side of the culture dish cover. Subsequently, the fresh shrimp samples were stored at 25 ± 1 °C, and the color changes of the indicator film were regularly recorded using the camera function of a smartphone to obtain Figure 7 Visual detection diagram of the freshness of fresh shrimp.

[0081] From Figure 7 It can be seen that with the spoilage of the fresh shrimp, the color of the sodium alginate film did not change, while both the packaging film of Dye I and the packaging film of Dye II changed from blue to pink. Among them, the packaging film of Dye II changed from blue-green to purple on the second day, and the packaging film of Dye I changed from blue to light purple on the third day, which indicates that both the packaging film of Dye I and the packaging film of Dye II respond to pH. Among them, the packaging film of Dye II responds more sensitively.

[0082] Example Eleven: Characterization Example of the Preservation of Bananas by the Packaging Film of Dye I and the Packaging Film of Dye II.

[0083] The packaging film of Dye I prepared in Example Three and the packaging film of Dye II prepared in Example Four were respectively used for banana preservation. The bananas were wrapped with sodium alginate film, the packaging film of Dye I, and the packaging film of Dye II respectively, and the dark treatment of the sodium alginate film, the dark treatment of the packaging film of Dye I, the dark treatment of the packaging film of Dye II, the light treatment of the packaging film of Dye I, and the light treatment of the packaging film of Dye II were set respectively, with being placed in the natural environment as the control group to obtain Figure 8 The diagram of the banana preservation process shown.

[0084] From Figure 8It can be seen that, compared with the natural environment, the preservation period of bananas is effectively extended after being wrapped with the packaging film. The appearance of the packaging film of Dye I after illumination can still remain intact on the 4th day and starts to rot on the 6th day; the appearance of the packaging film of Dye II after illumination can still remain intact on the 6th day and starts to rot on the 8th day. These phenomena indicate that both the illumination groups of the packaging films of Dye I and Dye II show obvious fresh-keeping effects. Further, the preservation period of the packaging film of Dye II is longer.

[0085] Among the reagents used in the embodiments of the present invention, all reagents except those for which the preparation methods are provided are commercially available.

Claims

1. A method for preparing a packaging film based on Nile blue dye, characterized in that The following steps are involved: The Nile blue dye is dispersed in a dimethyl sulfoxide solution, and ultrasonically treated to form a uniform dispersion to obtain a mother solution; sodium alginate is dissolved in water and heated to dissolve; the mother solution is dissolved in a sodium alginate solution, stirred, allowed to stand, poured into a mold, and dried to obtain a packaging film of the Nile blue dye for dynamic visualization detection of food and antibacterial preservation; The Nile blue dye structural formula is as follows: 。 2. The method for preparing a packaging film according to claim 1, characterized in that: The mass ratio of Nile blue dye to sodium alginate is 1:(450-550); the heating dissolution conditions are 45-55 ℃, 3-6 h; and the film forming temperature is 45-55 ℃.

3. The method for preparing the packaging film according to claim 1 or 2, characterized in that The Nile blue dye preparation comprises the following steps: (1) 1-naphthylamine, K2CO3 and iodoethane are added to ethanol, heated under reflux and stirred; the solvent is removed, and the crude product is eluted and purified to obtain compound B with the following structure: ； (2) 3-iodoaniline and K2CO3 were dissolved in acetonitrile, iodoethane was added, and the mixture was heated under N2 protection and stirred under reflux to react; after the reaction was completed, the mixture was cooled and the solvent was removed, and the crude product was eluted and purified to obtain compound C with the following structure: ； (3) Compound C, selenium powder, dimethyl sulfoxide, copper oxide and potassium hydroxide are placed in a container, heated and refluxed under N2 protection; filtered and extracted, dried and the solvent is removed; the crude product is eluted and purified to obtain a compound D with the following structure: ； (4) Add hydrochloric acid to compound D while stirring in an ice bath, then add NaNO2 while stirring, and stir in an ice bath again; extract, dry, remove the solvent, and elute and purify the crude product to obtain compound E with the following structure: ； (5) Compound E and compound B prepared in step (1) are dissolved in trifluoroethanol, heated and stirred, and refluxed under N2 protection; after the reaction stops, the solution is cooled and hydrochloric acid is added to adjust the pH of the solution to acidic; extraction, drying, purification, and rotary evaporation are performed to obtain Nile blue dye for dynamic visualization detection of food and antibacterial preservation.

4. The method for preparing a packaging film according to claim 3, characterized in that: In the step (1), the molar ratio of 1-naphthylamine: K2CO3: iodine ethane is 1: (1-1.5): (1-1.5); the heating reflux condition is 80-90°C for 2-6 hours; in the step (2), the molar ratio of 3-iodoaniline: K2CO3: iodine ethane is 1: (2-3): (2-3); the heating and reflux reaction conditions are 80-85°C for 14-18 hours; in the step (3), the molar ratio of compound C, selenium powder, copper oxide and potassium hydroxide is 1: (1.5-2.5): (0.4-0.6): (6-8); the heating and reflux conditions are 95-105°C for 10-14 hours; in the step (4), the reaction time of ice bath stirring is 0.3-4 hours; in the step (5), the molar ratio of compound E to compound B is 1: (2-4); the heating reflux reaction conditions are 85-95 ℃ reaction for 1-3 hours; the acidification condition is pH 2-3.

5. The Nile blue dye with the following structural formula is used for dynamic visual detection and antibacterial preservation of food: 。 6. The packaging film prepared by the method for preparing the packaging film according to any one of claims 1 to 4 is used for dynamic visualization detection and antibacterial preservation of food.

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