Honeycomb indicator film responding to freshness of fresh meat and preparation method and application thereof

Abstract

The invention discloses a honeycomb indicator film that responds to the freshness of fresh meat, a preparation method thereof, and an application thereof. The honeycomb indicator film that responds to the freshness of fresh meat uses carboxymethyl cellulose and polyvinyl alcohol as a membrane matrix, uses at least one of cyanidin-3-glucoside and delphinidin-3-glucoside as a freshness indicator, and has a three-dimensional honeycomb spatial structure, wherein the mass ratio of the freshness indicator to the membrane matrix is ​​3.0% to 7.5%, and the detection limit of the honeycomb indicator film that responds to the freshness of fresh meat in response to ammonia is 2.5 ppm. The honeycomb indicator film that responds to the freshness of fresh meat of the present invention has a rapid acid-base response and a wide range, exhibits excellent antioxidant properties, can effectively and quickly monitor the freshness of fresh meat, is suitable for monitoring the freshness of fresh agricultural and livestock products, and can intuitively identify changes in the freshness of fresh agricultural and livestock products such as meat, especially in the warehousing and sales links.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and more specifically to a honeycomb indicator film that responds to the freshness of fresh meat, and a preparation method and application thereof. Background Art

[0002] Meat has long been a core source of human nutrition, providing protein, minerals and vitamins. However, meat quality and safety are highly dependent on the hygiene and temperature control of the cold chain process, including production, storage, sales and transportation. Unfortunately, many consumers doubt the freshness and quality of meat products that have gone through this process. During transportation and storage, the proteins and lipids of meat and seafood are easily decomposed by the action of microorganisms and enzymes, producing various metabolites. These metabolites include CO 2 , H 2 S, amines and volatile NH 3 , which can cause changes in the color and pH of meat products, or even deterioration. Indicators such as pH value, color, and volatile basic nitrogen (TVB-N) are often used as indicators to measure the freshness of meat products. Although the freshness of meat and seafood can be accurately analyzed by chromatographic instruments, electrochemistry, and bioactive ingredients, their application in real-time monitoring is limited due to their high operating costs. Visual intelligent pH indicator membranes have attracted widespread attention in the fields of environmental monitoring, food safety, and biomedicine due to their advantages such as simple operation, high sensitivity, and good degradability.

[0003] The indicator film presents continuous color change during food processing and storage, which helps to improve the safety and utilization of food. Many smart indicator films based on anthocyanins and natural or biocompatible polymers have been developed, and the resulting materials have been shown to be sensitive to different pH or NH 3 It reacts to acidic and alkaline solutions. However, the pH range of meat spoilage is between 5.5-7.0. The indicator membranes currently developed are all wide-range response indicators, and cannot respond sensitively during the pH change of fresh meat between 5.5-7.0 after acid removal, and the response color cannot be visually distinguished. At the same time, the adsorption capacity of volatile gases produced by the decrease in freshness is weak, and the RGB response difference is small. Therefore, in response to this problem, it is particularly important to develop a new type of effective indicator membrane. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a honeycomb indicator film that responds to the freshness of fresh meat. The honeycomb indicator film can sensitively respond to the change of pH of fresh meat between 5.5 and 7.0 after acid discharge, has a fast response time of as short as 1 s, has excellent antioxidant properties, can quickly monitor and indicate the freshness of fresh meat, and is suitable for visual identification of freshness changes of fresh meat and other meats during storage and sales. The honeycomb indicator film that responds to the freshness of fresh meat has a detection limit of 2.5 ppm for ammonia, and has good adsorption and retention capabilities for ammonia and hydrochloric acid volatile gases.

[0006] Another object of the present invention is to provide a method for preparing a honeycomb indicator film that responds to the freshness of fresh meat, which uses carboxymethyl cellulose and polyvinyl alcohol as membrane matrices, uses EDC / NHS as a carboxyl activation system for carboxyl activation, and uses cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G) or complex anthocyanins (C3G+D3G) as freshness indicators.

[0007] Another object of the present invention is to provide an application of a honeycomb indicator film responsive to the freshness of fresh meat in monitoring the freshness of fresh agricultural and livestock products, wherein the fresh agricultural and livestock products are pork, beef, mutton, chicken, goose, duck, shrimp or crab, and the honeycomb indicator film responsive to the freshness of fresh meat is placed in the packaging of the fresh agricultural and livestock products and does not contact the fresh agricultural and livestock products.

[0008] In order to achieve these purposes and other advantages according to the present invention, a honeycomb indicator film responsive to the freshness of fresh meat is provided, which uses carboxymethyl cellulose and polyvinyl alcohol as film matrices, and uses at least one of cyanidin-3-glucoside and delphinidin-3-glucoside as a freshness indicator, and the honeycomb indicator film responsive to the freshness of fresh meat has a three-dimensional honeycomb spatial structure, wherein the mass ratio of the freshness indicator to the film matrix is ​​3.0% to 7.5%, and the detection limit of the honeycomb indicator film responsive to the freshness of fresh meat in response to ammonia is 2.5 ppm.

[0009] The present invention is further implemented by a method for preparing the honeycomb indicator film responsive to the freshness of fresh meat, the method comprising the following steps:

[0010] Step 1: Under gradient temperature, carboxymethyl cellulose and polyvinyl alcohol are respectively dissolved in phosphate buffer to prepare an indicator film precursor solution, wherein the mass volume ratio of polyvinyl alcohol to phosphate buffer is (60-80) mg:1 mL, and the mass volume ratio of carboxymethyl cellulose to phosphate buffer is (2-3) mg:1 mL;

[0011] Step 2: Carboxyl activating the indicator film precursor solution with a carboxyl activating agent, wherein the amount of the carboxyl activating agent added is 3% to 5% of the indicator film precursor solution;

[0012] Step 3: adding the freshness indicator to the solution after carboxyl activation in step 2, performing esterification coupling, and freeze-drying to prepare a honeycomb indicator film that responds to the freshness of fresh meat.

[0013] Preferably, in step three, the freshness indicator is a composite anthocyanin formed by cyanidin-3-glucoside and delphinidin-3-glucoside, wherein the mass ratio of cyanidin-3-glucoside to delphinidin-3-glucoside is 2:1.

[0014] Preferably, step one specifically includes: first dissolving polyvinyl alcohol in a phosphate buffer solution with a pH of 7.2 at 90°C, and stirring and dissolving for 30 min; after the polyvinyl alcohol is completely dissolved, gradually lowering the temperature to 60°C, adding carboxymethyl cellulose, and stirring and dissolving for 240 min to prepare an indicator film precursor solution.

[0015] Preferably, the mass ratio of polyvinyl alcohol to carboxymethyl cellulose is 23:1.

[0016] Preferably, in step 2, the carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the carboxyl activator is 1:1, the carboxyl activation temperature is 45°C, the activation time is 30 min, and the concentration of the carboxyl activator is 0.5 mM.

[0017] Preferably, in step three, freeze-drying specifically comprises: freezing at -80°C, thawing at room temperature, freezing and thawing repeatedly three times, and finally freeze-drying at -80°C for 36 h.

[0018] The present invention is further realized by using a honeycomb indicator film responsive to the freshness of fresh meat in monitoring the freshness of fresh agricultural and livestock products, wherein the fresh agricultural and livestock products include but are not limited to pork, beef, mutton, chicken, goose, duck, shrimps, and crabs.

[0019] Preferably, the honeycomb indicator film responsive to the freshness of fresh meat is placed in the fresh agricultural and livestock product packaging and does not contact the fresh agricultural and livestock products.

[0020] The present invention has at least the following beneficial effects:

[0021] The preparation method of the honeycomb indicator film responding to the freshness of fresh meat of the present invention comprises the following steps: esterification coupling is used to form a 3D combination with an indicator film matrix carboxymethyl cellulose / polyvinyl alcohol as a membrane matrix, and cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G) or composite anthocyanin (C3G+D3G) is used as a freshness indicator. The formed three-dimensional honeycomb spatial structure can increase the rapid contact response between the acid-base volatile substances and the freshness indicator. The honeycomb indicator film responding to the freshness of fresh meat can realize a sensitive response during the change of the pH value of fresh meat between 5.5 and 7.0 after acid removal, and the response is fast and the response time is as short as 1 s, the change of E value of the honeycomb indicator film responding to the freshness of fresh meat after response is positively correlated with the change law of the freshness index of fresh meat. The freshness of fresh meat can be quickly and visually identified and monitored. The difference in response color can be visually identified. A comprehensive evaluation was conducted from the aspects of preparation stability, indication performance, response effect, etc. The honeycomb indicator film responding to the freshness of fresh meat has excellent indication performance and excellent antioxidant properties. It is suitable for the visual identification of freshness changes of fresh meat and other meats during warehousing and sales, providing a new and effective antibacterial packaging for the food industry.

[0022] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison diagram of the color response sensitivity of C3G and D3G at different pH values ​​of the present invention;

[0024] Figure 2 This is a comparison diagram of the color response sensitivity of the composite anthocyanins formed by C3G and D3G at different pH values ​​in Example 1 of the present invention;

[0025] Figure 3 The figure is the result of measuring the elastic properties of the indicator film precursor solution prepared with different mixing ratios of PVA and CMC;

[0026] Figure 4 This is a graph showing the stability evaluation results of the spectral response of the negative film pre-liquid prepared in Examples 1 to 3 of the present invention;

[0027] Figure 5 This is a graph showing the sensitivity evaluation results of the pre-membrane solution prepared in Examples 1 to 3 of the present invention in response to pH values ​​of 4.6-7.8;

[0028] Figure 6 The SEM images of the honeycomb indicator films responsive to the freshness of fresh meat prepared in Examples 1 to 3 of the present invention;

[0029] Figure 7The cytotoxicity fluorescence display diagram of the honeycomb indicator film responding to the freshness of fresh meat prepared in Examples 1 to 3 of the present invention;

[0030] Figure 8 The cytotoxicity bar graph of the honeycomb indicator film responsive to the freshness of fresh meat prepared in Examples 1 to 3 of the present invention;

[0031] Fig. 9 This is a graph showing the XPS evaluation results of the honeycomb indicator film for responding to the freshness of fresh meat loaded with cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G) or composite anthocyanins prepared in Examples 1 to 3 of the present invention;

[0032] Fig.10 This is a graph showing the thermal stability evaluation results of the honeycomb indicator films responsive to the freshness of fresh meat prepared in Examples 1 to 3 of the present invention;

[0033] Fig.11 This is a graph showing the results of evaluating the sensitivity of the honeycomb indicator membranes for responding to the freshness of fresh meat prepared in Examples 1 to 3 of the present invention to acid and alkaline gases;

[0034] Fig.12 A color response result diagram of the honeycomb indicator film for indicating the freshness of fresh meat prepared in Examples 1 to 3 of the present invention indicating the change in the freshness of pork during the storage period;

[0035] Fig.13 This is a graph showing the correlation evaluation results between the color change of the honeycomb indicator film responsive to the freshness of fresh meat prepared in Examples 1 to 3 of the present invention and the changes in the total bacterial colony count (TVC) and volatile basic nitrogen (TVB-N) of pork during storage. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0037] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0038] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0039] Main chemical reagents: cyanidin-3-glucoside (C3G), delphinidin-3-glucoside (D3G); carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), all purchased from Shanghai McLean Biochemical Technology Co., Ltd.; amino-heptapolyethylene glycol-amino (Amino-PEG7-amine), purchased from Xi'an Qiyue Biotechnology Co., Ltd., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, biological grade, 99%) and N-hydroxysuccinimide (NHS, biological grade, ≥98%), purchased from Shanghai Yuanye Biochemical Technology Co., Ltd.

[0040] Main instruments and equipment: Physical MCR 502 rheometer, purchased from Anton Paar, Austria; RE-10D rotary evaporator, purchased from Zhengzhou Keda Machinery Instrument Equipment Co., Ltd., China; Alpha1-2 LDplus freeze dryer, purchased from Martin Christ, Germany.

[0041] Example 1

[0042] A method for preparing a honeycomb indicator film that responds to the freshness of fresh meat comprises the following steps:

[0043] Step 1: First, dissolve 2.3 g of PVA in 30 mL of phosphate buffer (0.1 mM, pH 7.2) at 90°C, stir and dissolve for 30 min, and after the PVA is completely dissolved, gradually lower the temperature to 60°C, add 0.1 g of CMC, and stir and dissolve for 240 min to prepare an indicator film precursor solution;

[0044] Step 2: The temperature of the indicator film precursor solution was lowered to 45°C, and 0.5 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5 mg / mL) and 0.5 mL of N-hydroxysuccinimide (5 mg / mL) were added as carboxyl activators for carboxyl activation. The reaction was activated at 60°C for 30 min, followed by continuous stirring for 240 min, and then the temperature was lowered to 25°C. Ultrasonic treatment was performed at a power of 480 W for 30 minutes. After ultrasonic treatment, it was allowed to stand for 2 hours, 0.15 g of C3G dry powder was added, and it was stirred for 2 h to obtain a pre-membrane solution. The pre-membrane solution was vacuum freeze-dried at -80°C for 36 hours to prepare a honeycomb indicator film that responds to the freshness of fresh meat, recorded as CMC / PVA@C3G, and the dried honeycomb indicator film that responds to the freshness of fresh meat was placed in a 4°C refrigerator for storage.

[0045] Example 2

[0046] Step 1: First, dissolve 2.3 g of PVA in 30 mL of phosphate buffer (0.1 mM, pH 7.2) at 90°C, stir and dissolve for 30 min, and after the PVA is completely dissolved, gradually lower the temperature to 60°C, add 0.1 g of CMC, and stir and dissolve for 240 min to prepare an indicator film precursor solution;

[0047] Step 2: The temperature of the indicator film precursor solution was lowered to 45°C, and 0.5 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5 mg / mL) and 0.5 mL of N-hydroxysuccinimide (5 mg / mL) were added as carboxyl activators for carboxyl activation. The reaction was carried out at 60°C for 30 min, followed by continuous stirring for 240 min, and then the temperature was lowered to 25°C. Ultrasonic treatment was carried out at a power of 480 W for 30 minutes. After ultrasonic treatment, it was allowed to stand for 2 hours, 0.15 g of D3G dry powder was added, and it was stirred for 2 h to obtain a pre-membrane solution. The pre-membrane solution was vacuum freeze-dried at -80°C for 36 hours to prepare a honeycomb indicator film that responds to the freshness of fresh meat, recorded as CMC / PVA@D3G, and the dried honeycomb indicator film that responds to the freshness of fresh meat was placed in a 4°C refrigerator for storage.

[0048] Example 3

[0049] A method for preparing a honeycomb indicator film that responds to the freshness of fresh meat comprises the following steps:

[0050] Step 1: First, dissolve 2.3 g of PVA in 30 mL of phosphate buffer (0.1 mM, pH 7.2) at 90°C, stir and dissolve for 30 min, and after the PVA is completely dissolved, gradually lower the temperature to 60°C, add 0.1 g of CMC, and stir and dissolve for 240 min to prepare an indicator film precursor solution;

[0051] Step 2: The temperature of the indicator film precursor solution was lowered to 45°C, and 0.5 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5 mg / mL) and 0.5 mL of N-hydroxysuccinimide (5 mg / mL) were added as carboxyl activators for carboxyl activation. The reaction was carried out at 60°C for 30 min, followed by continuous stirring for 240 min, and then the temperature was lowered to 25°C. Ultrasonic treatment was carried out at a power of 480 W for 30 minutes. After ultrasonic treatment, it was allowed to stand for 2 hours, 0.05 g of D3G and 0.1 g of C3G mixed powder were added, and the mixture was stirred for 2 h to obtain a pre-membrane solution. The pre-membrane solution was vacuum freeze-dried at -80°C for 36 hours to prepare a honeycomb indicator film that responds to the freshness of fresh meat, recorded as CMC / PVA@D3G+C3G, and the dried honeycomb indicator film that responds to the freshness of fresh meat was placed in a 4°C refrigerator for storage.

[0052] Performance test

[0053] 1. Anthocyanin response color change and spectral curve:

[0054] The composite anthocyanin solutions of C3G, D3G or their mixture at different pH values ​​(2.0-13.0) were measured by UV-visible spectrophotometer (SuPerMax 3000FA, Flash, China) in the wavelength range of 250-700 nm, and the color changes of RGB were recorded by photography. Figure 1 A is 0.15 g of C3G dry powder dissolved in 30 mL of buffer solution to form solutions of different pH values. Figure 1 Where B is 0.15 g of D3G dry powder dissolved in 30 mL of buffer solution to form solutions of different pH values; Figure 2 The mixed dry powder of C3G and D3G was dissolved in 30 mL of buffer solution to form solutions with different pH values, wherein the amount of C3G in the complex anthocyanins was 0.10 g and the amount of D3G was 0.05 g. For solutions with the same pH value, the 30 mL of buffer solution used was the same.

[0055] The results of the test are as follows Figure 1 and Figure 2 As shown by Figure 1 It can be seen that the complex anthocyanins formed by C3G, D3G or a mixture of the two have different responses to pH values ​​of 2 to 13, and the corresponding pH values ​​can be well distinguished based on color changes. The color of C3G responding to different pH values ​​( Figure 1 Middle A) Color change compared to D3G ( Figure 1 B) is easier to distinguish. Figure 2 As shown, compared with the color changes of C3G and D3G, the complex anthocyanins ( Figure 2) The colors corresponding to different pH values ​​are stronger, and the color changes are easier to distinguish. By responding to the volatile acidic and alkaline substances produced during the storage of fresh meat, it is helpful to build a freshness prediction model based on the color response under the changes of volatile acidic and alkaline gases.

[0056] 2. Storage modulus and loss modulus:

[0057] The elastic properties of the prepared indicator film precursor solutions with different mixing ratios of PVA and CMC were investigated. The indicator film precursor solutions were prepared according to step 1 of Example 1, wherein the mass ratios of PVA and CMC were as follows: 2.35 g PVA and 0.05 g CMC, 2.3 g PVA and 0.1 g CMC, 2.2 g PVA and 0.2 g CMC, 2.1 g PVA and 0.3 g CMC, and 2.0 g PVA and 0.4 g CMC. 2 mL of the indicator film precursor solution was placed in the sample plate of a Physical MCR 702 multi-drive rheometer (Anntonpeer, Graz, Austria) under a shear stress of 0 to 500 s -1 , storage modulus, loss modulus and damping factor with angular frequencies between 0 and 100 s -1 Test inside.

[0058] The results of the test are as follows Figure 3 As shown, Figure 3 A in the figure is the shear stress-shear rate measurement result diagram, Figure 3 B is the storage modulus-angular frequency measurement result diagram, Figure 3 C in the figure is the loss modulus-angular frequency measurement result diagram, Figure 3 D in the figure is the damping factor-angular frequency measurement result diagram. Storage modulus and loss modulus are important parameters of material mechanical properties and play an important role in material science and engineering. Storage modulus refers to the elastic energy that a material can store when subjected to force, while loss modulus refers to the energy that a material can consume when subjected to force. Figure 3 The results showed that the storage modulus and loss modulus of the indicator membrane precursor solutions in each group showed an increasing trend. Compared with the other groups, the indicator membrane precursor solution prepared by mixing 2.3 g PVA and 0.1 g 0.1CMC had a higher storage modulus and a lower loss modulus, indicating that the honeycomb indicator membrane responding to the freshness of fresh meat prepared by this indicator membrane precursor solution had better elastic properties.

[0059] 3. Stability fluorescence spectroscopy determination:

[0060] The response stability fluorescence spectrometry of the pre-membrane solutions prepared in Examples 1 to 3 was measured: the response spectrum sensitivity of the pre-membrane solutions prepared in Examples 1 to 3 was measured in the wavelength range of 250-800 nm.

[0061] The results of the test are as follows Figure 4 As shown by Figure 4 It can be seen that the pre-membrane solution ( Figure 4 B), the pre-membrane solution loaded with only D3G prepared in Example 2 ( Figure 4 The light absorption peak between 500 nm and 600 nm is not obvious, and the absorption peak shifts forward with the increase of anthocyanin loading concentration. Therefore, loading concentration becomes an important factor affecting the response stability of the honeycomb indicator membrane that responds to the freshness of fresh meat. Compared with the response sensitivity of monomer anthocyanin, it can be seen that the membrane pre-liquid ( Figure 4 The absorption peak of (C) is relatively stable, and the peak is evenly distributed at 540 nm.

[0062] 4. Color change of pre-membrane solution in response to narrow spectrum pH value:

[0063] The pH response was measured using the pre-membrane solution prepared in Example 3. The results are as follows: Figure 5 As shown by Figure 5 It can be seen that the color of the pre-membrane solution prepared in Example 3 changes from light pink to purple, blue, gray, and finally to yellow-green in response to pH values ​​of 4.6, 5.0, 5.4, 5.8, 6.2, 6.6, 7.0, 7.4, and 7.8, and the colors corresponding to different pH values ​​can be distinguished more significantly. Therefore, the honeycomb indicator film responding to the freshness of fresh meat prepared in Example 3 has a sensitive indication effect.

[0064] 5. Apparent determination of honeycomb indicator film in response to freshness of fresh meat:

[0065] The morphology of the honeycomb indicator films responsive to the freshness of fresh meat prepared in Examples 1 to 3 was analyzed by SEM (EVO-LS10, Carl Zeiss, Germany). The measurement results are as follows: Figure 6 As shown by Figure 6 It can be seen that anthocyanins have a certain influence on the appearance of the honeycomb indicator film that responds to the freshness of fresh meat. The honeycomb indicator film that responds to the freshness of fresh meat loaded with only D3G prepared in Example 2 ( Figure 6 A) and a honeycomb indicator film for responding to the freshness of fresh meat loaded with only C3G prepared in Example 1 ( Figure 6 The micropores in B are relatively large; while the honeycomb indicator film ( Figure 6 The micropores in (C) are more uniform and have smaller pore sizes. Under the same conditions, the surface area of ​​the honeycomb indicator membrane that responds to the freshness of fresh meat is increased, which is more conducive to increasing the adsorption of volatile gases.

[0066] 5. Biocompatibility determination of honeycomb indicator membrane responding to the freshness of fresh meat:

[0067] RPMI 1640 medium was used, supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin and 1% streptomycin (50 mg / mL), and the concentration of human colon cancer cells (LOVO cells) was adjusted to 5×10 4 CFU / mL. The cell suspension was pipetted to 100 μL in sequence and added to a 96-well plate (the number of cells per well was about 5000 at this time). Then 1, 2 and 3 mg of CMC / PVA@D3G prepared in Example 2, CMC / PVA@C3G prepared in Example 1, and CMC / PVA@C3G+D3G prepared in Example 3 were taken, irradiated with ultraviolet light for 30 min, and placed in a 96-well culture plate of LOVO cells. Culture medium containing 0.5 mg / mL MTT was added to the culture plate, 200 μL of culture medium solution was prepared in each well, and the culture was incubated at 5% CO 2 , 37℃ constant humidity incubator for 3-5 h. Untreated LOVO cells were used as control. After the blue-purple crystals were formed, the film was removed and the original medium was gently aspirated. After adding DMSO to each well, the 96-well plate was gently shaken at room temperature for 5 min to completely dissolve the crystals. Then, the 96-well plate was quickly placed in the Varioskan LUX multi-function microplate reader (Thermo Fisher scientific). The OD value was read at 490 nm.

[0068] The results of the test are as follows Figure 7 and Figure 8 As shown, Figure 7 A is the cell fluorescence display of the blank control group. Figure 7 Figure B is the cytotoxicity fluorescence display of CMC / PVA@D3G prepared in Example 2. Figure 7 Figure C is the cytotoxicity fluorescence display of CMC / PVA@C3G prepared in Example 1. Figure 7 D is the cytotoxicity fluorescence display diagram of CMC / PVA@C3G+D3G prepared in Example 3. Figure 8 A is a bar graph showing the cytotoxicity of CMC / PVA@D3G prepared in Example 2. Figure 8 B is the cytotoxicity bar graph of CMC / PVA@C3G prepared in Example 1, and the cytotoxicity bar graph of CMC / PVA@C3G+D3G prepared in Example 3. Figure 7 and Figure 8 It can be seen that the VOVO cell activity shows that the honeycomb indicator film responding to the freshness of fresh meat of the present invention is safe.

[0069] 6. XPS spectrum measurement of honeycomb indicator film responding to the freshness of fresh meat:

[0070] The honeycomb indicator films responsive to the freshness of fresh meat prepared in Examples 1 to 3 were analyzed using a fully automatic focusing scanning micro-area photoelectron spectrometer (PHIQuanteraⅡ, Nano Surface Analysis Instruments Co., Japan). The analysis results are as follows: Fig. 9 As shown, Fig. 9 A in the figure is the XPS spectrum measurement combination diagram of CMC / PVA, CMC / PVA@C3G, CMC / PVA@D3G and CMC / PVA@C3G+D3G. Fig. 9 B is the XPS spectrum measurement diagram of CMC / PVA@D3G prepared in Example 2. Fig. 9 Figure C is the XPS spectrum measurement diagram of CMC / PVA@C3G prepared in Example 1. Fig. 9 D is the XPS spectrum measurement diagram of CMC / PVA@C3G+D3G prepared in Example 3. Fig. 9 It can be seen that the CMC / PVA@D3G prepared in Example 2, the CMC / PVA@C3G prepared in Example 1, and the CMC / PVA@C3G+D3G prepared in Example 3, due to the addition of anthocyanins, the content of phenolic hydroxyl groups in the indicator membrane is increased, so that the indicator membrane has certain antibacterial and antioxidant properties; at the same time, it also makes the indicator membrane have protonation and protonation freedom, laying a good foundation for rapid color response.

[0071] 7. X-ray diffraction (XRD):

[0072] The honeycomb indicator films responsive to the freshness of fresh meat prepared in Examples 1 to 3 were analyzed using a Bruker D8 Advance X-ray diffraction analyzer. The voltage was 20 kV, the current was 5 mA, Kα (λ=1.5418 Å), the scanning rate was 4 ° / min, the step length was 0.02°, and the scanning range was 5°~40°. The measurement results are shown in FIG. Fig.10 As shown in A.

[0073] 8. Thermal stability analysis:

[0074] In a nitrogen atmosphere (gas flow rate 15 mL / min), the temperature was gradually heated from room temperature to 600°C at a heating rate of 10°C / min, and the thermal stability change parameters of the honeycomb indicator films responsive to the freshness of fresh meat prepared in Examples 1 to 3 were measured using a TGA Q50 (TA, USA) thermogravimetric analyzer. The measurement results are as follows: Fig.10 As shown in B and C.

[0075] Depend on Fig.10It can be seen that loading anthocyanins reduces the crystallinity of the indicator film, making the film have better toughness and better maintaining its integrity during use. The honeycomb indicator films responding to the freshness of fresh meat prepared in Examples 1 to 3 have little effect on thermal stability and maintain good thermal stability as a whole.

[0076] 9. Ammonia and hydrochloric acid sensitivity test:

[0077] The CMC / PVA@C3G+D3G prepared in Example 3 was cut into discs with a diameter of 5 mm and then placed at a distance of 2.5×10 5 ppm ammonia or 37 wt% hydrochloric acid container (20 mL) at 2 cm at 25 °C for 5 seconds. The appearance of the honeycomb indicator film in response to the freshness of the raw meat was recorded at 0, 1, 2, 3, 4, and 6 seconds, and then measured by a UV-visible spectrophotometer (Shimadzu UV-3600 Plus) in the wavelength range of 350-700 nm.

[0078] Color measurement: The surface color of the honeycomb indicator film (CMC / PVA@C3G+D3G) loaded with D3G+C3G prepared in Example 3 and responsive to the freshness of fresh meat prepared in Example 3 was measured using a colorimeter (CM-5, Konica Minolta, Japan) calibrated with a standard white plate (L = 96.59, a = 0.13, b = 0.11). , , ). The total color difference (ΔE) is calculated as follows:

[0079]

[0080] The results of the test are as follows Fig.11 As shown, Fig.11 A is an ultraviolet spectrum of the honeycomb indicator film loaded with D3G+C3G prepared in Example 3 that responds to the freshness of fresh meat to ammonia over time, Fig.11 B is an ultraviolet spectrum of the response of the honeycomb indicator film loaded with D3G+C3G prepared in Example 3 to the freshness of fresh meat to HCl gas over time, Fig.11 C is a color change diagram of the honeycomb indicator film loaded with D3G+C3G prepared in Example 3 in response to ammonia over time, Fig.11 D is a color change diagram of the honeycomb indicator film loaded with D3G+C3G prepared in Example 3 in response to HCl gas over time. Fig.11It can be seen that the CMC / PVA@C3G+D3G prepared in Example 3 has obvious color differences in response to ammonia and hydrochloric acid, the detection limit of response to ammonia is 2.5 ppm, and the light absorption intensity at different response times is easy to distinguish, indicating that the honeycomb indicator film loaded with D3G+C3G that responds to the freshness of fresh meat has better adsorption and retention capabilities for ammonia and hydrochloric acid volatile gases. Fig.12 A represents pork stored at 25℃ for 16 hours. Fig.12 B represents pork stored at 25°C for 32 hours, C represents pork stored at 25°C for 48 hours, and D represents pork stored at 25°C for 60 hours. When testing the freshness of fresh agricultural and livestock products, the honeycomb indicator film responsive to the freshness of fresh meat of the present invention is placed in the packaging of fresh agricultural and livestock products and does not contact the fresh agricultural and livestock products. Fig.12 The color response of the honeycomb indicator film loaded with D3G+C3G and responsive to the freshness of fresh meat prepared in Example 3 to indicate the change in freshness of pork during storage shows that the color change of the honeycomb indicator film loaded with D3G+C3G and responsive to the freshness of fresh meat can obviously distinguish the change in the freshness of fresh pork during storage. Fig.13 A and B are the result graphs of the correlation evaluation of the changes in the total bacterial count (TVC) and volatile basic nitrogen (TVB-N) content of meat conventionally placed at room temperature 25°C by the honeycomb indicator film loaded with D3G+C3G prepared in Example 3 in response to the freshness of fresh meat. Fig.13 C and D are the results of the correlation evaluation of the changes in the total bacterial count (TVC) and volatile basic nitrogen (TVB-N) content of meat placed at room temperature 4°C by the honeycomb indicator film loaded with D3G+C3G prepared in Example 3. Fig.13 It can be seen that CMC / PVA@D3G+C3G has a sensitive monitoring and indicating effect on meat stored at room temperature of 25℃ and 4℃, and has a significant correlation with the changes in the total bacterial count (TVC) and volatile basic nitrogen (TVB-N) content of pork during storage. The correlation with TVC of pork stored at 4℃ is as high as 0.9861, indicating that the color change of the indicator film can significantly predict the changes in TVC and TVB-N content of meat during storage, indicating that CMC / PVA@D3G+C3G has an excellent monitoring and indicating effect on the freshness changes of fresh meat.

[0081] In the prior art, for example, PVA / ARC film prepared by polyvinyl alcohol ( Carbohydrate Polymers. 233(2020) 115859) has a pH response range of 5 to 12. In solutions with pH = 7, 10 and 12, the color changes from yellow to brick red to purple. It has a wide response range, and its sensitivity is poor during the pH change of 5.5-7.0 in fresh meat after acid removal. The colorimetric membrane ( Food Packaging and Shelf Life . 28 (2021) 100641), its color is purple as a whole when the pH changes from 5.5 to 7.0, and the color change difference is not large, and the difference cannot be visually distinguished. pH sensitive food packaging film prepared from konjac glucomannan, hydroxypropyl methylcellulose and mulberry extract ( International Journal of Biology Macromolecules 172 (2021) 515–523), its pH response range is 2 to 12, and its color change difference is not large during the change from 5.5 to 7.0, and the whole is brown, which cannot achieve visual identification of difference. Indicator membrane made of polyvinyl alcohol, methyl cellulose and black wolfberry anthocyanin ( Food Packaging and Shelf Life 34 (2022) 100939), its pH response range is relatively wide, and the color change difference is not large during the change from 5.5 to 7.0, showing light brown and light purple, and the difference cannot be visually distinguished. pH indicator prepared from polyvinyl alcohol, ricin and purple potato anthocyanin ( Carbohydrate Polymers 314 (2023) 120882), which has a wide pH response range (2-12). During the change from 5.5 to 7.0, pH=5 is light pink, pH=6 is pink, and pH=7 is purple. The color change difference is not large, and rapid visual identification cannot be achieved.

[0082] The present invention aims to solve the problems that the pH response range of existing indicator films is relatively wide, and they cannot achieve sensitive response when the pH of fresh meat changes between 5.5 and 7.0 after acid drainage, and the response color cannot be visually distinguished by difference. By optimizing the components and mixing ratio of the membrane precursor solution and the components and mixing ratio of anthocyanins, a honeycomb indicator film that responds to the freshness of fresh meat is prepared through the preparation process. The honeycomb indicator film achieves sensitive response when the pH of fresh meat changes between 5.5 and 7.0 after acid drainage, and visually distinguishes by difference.

[0083] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a honeycomb indicator film that responds to the freshness of fresh meat, characterized in that: The following steps are involved: Step 1: Under gradient temperature, carboxymethyl cellulose and polyvinyl alcohol are respectively dissolved in phosphate buffer to prepare an indicator film precursor solution, wherein the mass volume ratio of polyvinyl alcohol to phosphate buffer is (60-80) mg:1 mL, the mass volume ratio of carboxymethyl cellulose to phosphate buffer is (2-3) mg:1 mL, and the mass ratio of polyvinyl alcohol to carboxymethyl cellulose is 23:1; Step 2: Carboxyl activating the indicator film precursor solution with a carboxyl activating agent, wherein the amount of the carboxyl activating agent added is 3% to 5% of the indicator film precursor solution; Step 3: adding the freshness indicator to the solution after carboxyl activation in step 2, performing esterification coupling, and freeze-drying to prepare a honeycomb indicator film that responds to the freshness of fresh meat; Among them, the honeycomb indicator film that responds to the freshness of fresh meat has carboxymethyl cellulose and polyvinyl alcohol as membrane matrices, and a complex anthocyanin formed by cyanidin-3-glucoside and delphinidin-3-glucoside in a mass ratio of 2:1 as a freshness indicator, and the mass ratio of the freshness indicator to the membrane matrix is ​​3.0% to 7.5%.

2. The method according to claim 1, characterized in that Step one specifically includes: firstly dissolving polyvinyl alcohol in a phosphate buffer solution with a pH value of 7.2 at 90°C, and stirring and dissolving for 30 minutes; after the polyvinyl alcohol is completely dissolved, gradually lowering the temperature to 60°C, adding carboxymethyl cellulose, and stirring and dissolving for 240 minutes to prepare an indicator film precursor solution.

3. The method according to claim 2, characterized in that In step 2, the carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the carboxyl activator is 1:1, the carboxyl activation temperature is 45°C, the activation time is 30 min, and the concentration of the carboxyl activator is 0.5 mM.

4. The method according to claim 1, characterized in that In step three, freeze-drying specifically includes: freezing at -80°C, thawing at room temperature, freezing and thawing repeatedly three times, and finally freeze-drying at -80°C for 36 h.

5. A honeycomb indicator film responsive to the freshness of fresh meat prepared by the method according to any one of claims 1 to 4.

6. Use of the honeycomb indicator film responsive to the freshness of fresh meat as claimed in claim 5 in monitoring the freshness of fresh agricultural and livestock products, wherein: Fresh agricultural and livestock products include pork, beef, mutton, chicken, goose, duck, shrimp or crab.

7. The use as claimed in claim 6, wherein the honeycomb indicator film responding to the freshness of fresh meat is placed in the packaging of fresh agricultural and livestock products and does not contact the fresh agricultural and livestock products.

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