A high-strength, high-pH-sensitive food freshness indicator film and its preparation method and application

Abstract

This invention belongs to the field of colloidal materials technology, and particularly relates to a high-strength, high-pH-sensitive food freshness indicator film, its preparation method, and its application. This invention utilizes a matrix cross-linked with bovine bone gelatin, chitosan, and citric acid, and incorporates a blueberry anthocyanidin ferrous ion chelate into the film. This improves the performance of traditional protein-polysaccharide films, achieving excellent mechanical properties, water vapor barrier properties, oxygen barrier properties, antioxidant capacity, and antibacterial properties. It also enhances the pH sensitivity of blueberry anthocyanins, significantly increasing the film's sensitivity to food freshness.

Description

Technical Field

[0001] The present invention belongs to the technical field of colloid materials, and in particular relates to a high-strength and high-pH-sensitive food freshness indicator film, a preparation method and application thereof. Background Art

[0002] With the rapid development of the food industry, there is growing interest in advanced smart packaging, which often incorporates external or internal indicators to provide real-time information on the status and quality of food or to demonstrate product freshness to consumers. Smart food packaging offers advantages such as maintaining food quality and safety, enabling safe storage, reducing economic losses, and facilitating product marketing.

[0003] Meat, as an indispensable ingredient in our daily diet, is susceptible to microbial contamination during storage, resulting in quality degradation and even food safety issues. With consumers paying increasing attention to food safety, monitoring and maintaining the freshness of animal-derived foods without affecting their quality is a current issue that needs to be addressed. Generally speaking, characteristic metabolites of total volatile base nitrogen (TVB-N), including dimethylamine, trimethylamine, and ammonia, are produced during the process of corruption and deterioration, resulting in an alkaline environment in the food packaging environment. Therefore, the use of pH-sensitive colorimetric films to monitor the freshness of animal-derived foods, especially food freshness indicator films made from plant-derived pH-sensitive substances - anthocyanins, are favored due to their non-toxic and environmentally friendly properties.

[0004] Anthocyanins are easily released and degraded quickly in conventional matrices, so it is crucial to select a suitable matrix for them. Natural polymers, due to their unique structure and physical properties, have excellent safety, availability, biocompatibility and biodegradability, and have become a viable raw material for the development of sustainable food freshness indicator materials. However, conventional ordinary anthocyanins do not respond quickly to pH color, and lack color richness and indication precision. Conventional anthocyanin pH indicator films have disadvantages such as poor stability and low ammonia sensitivity, which limit the further development and utilization of anthocyanin pH indicator films.

[0005] Bovine bone gelatin has excellent biocompatibility, making it suitable for direct contact with food and organisms without toxic side effects. It is biodegradable by microorganisms in the natural environment, reducing the environmental burden of packaging waste. Its biocompatibility as a packaging material holds broad application prospects. However, bovine bone gelatin has poor mechanical properties, making it difficult to form into films.

[0006] Therefore, how to develop an environmentally friendly food freshness indicator film with good mechanical properties and pH sensitivity is an urgent issue to be solved in food safety management. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention aims to propose a high-strength and high-pH-sensitive food freshness indicator film, its preparation method and application. By cross-linking bovine bone gelatin, chitosan and citric acid into a matrix and incorporating blueberry anthocyanin ferrous ion chelate into the film, the performance of the traditional protein-polysaccharide film is improved, and excellent mechanical properties, water vapor barrier, oxygen barrier, antioxidant capacity and antibacterial properties are achieved; the sensitivity of blueberry anthocyanin to pH is enhanced, and the film's sensitivity to food freshness is significantly improved.

[0008] To achieve the above object, the present invention provides a high-strength and high-pH-sensitive food freshness indicator film, the indicator film comprising: bovine bone gelatin, chitosan, a cross-linking agent, blueberry anthocyanin and Fe 2+ ;

[0009] Furthermore, the mass ratio of bovine bone gelatin:chitosan:crosslinking agent is 1.8-2.2:0.8-1.2:0.2-0.4;

[0010] Furthermore, the mass ratio of bovine bone gelatin:chitosan:crosslinking agent is 2:1:0.3.

[0011] Furthermore, the Fe in the indicator film 2+ Added in the form of FeSO4·7H2O;

[0012] The mass ratio of blueberry anthocyanins to FeSO4·7H2O is 0.10-0.14:0.04-0.06;

[0013] Furthermore, the mass ratio of blueberry anthocyanin to FeSO4·7H2O is 0.12:0.05.

[0014] Furthermore, the cross-linking agent is one or more of tannic acid, genipin, citric acid, proanthocyanidin or ferulic acid.

[0015] Chitosan, as a packaging material, offers numerous advantages, including biodegradability, environmental friendliness, biocompatibility, safety, excellent mechanical properties, good gas and moisture barrier properties, and versatility and customizability, enhancing the comprehensive performance of bovine bone gelatin for film formation. Bovine bone gelatin-chitosan constructs achieve suitable characteristics when using higher concentrations of bovine bone gelatin, although small amounts of chitosan are sufficient to achieve the desired mechanical properties while maintaining cell adhesion and proliferation. However, bovine bone gelatin itself forms a fibrous structural scaffold with poor mechanical properties. Furthermore, due to its high hygroscopicity, it easily swells and dissolves when in contact with high-moisture foods, limiting its direct application in food packaging. Therefore, chemical or physical methods, such as cross-linking agents or combination with other biopolymers, have been used to improve these properties. Cross-linking reduces the mobility of gelatin chains, improving dimensional stability, water and heat resistance, barrier properties, and mechanical properties.

[0016] For the cross-linking of bovine bone gelatin and chitosan, the traditional chemical cross-linking agent - aldehyde (one of the most widely used compounds is glutaraldehyde) has a good effect, but it will produce cytotoxicity when the aldehyde concentration is higher than 10%; in addition, the unreacted cross-linking agent in the scaffold is prone to the risk of toxic products, and the aldehyde as a cross-linking agent also limits the mechanical strength after cross-linking. In order to overcome this problem, the applicant pioneered the use of natural organic acids such as tannic acid, genipin, citric acid, proanthocyanidins or ferulic acid as cross-linking agents. For example, citric acid, as a naturally occurring organic acid, is widely present in citrus fruits. It is environmentally friendly and non-toxic. It contains three carboxyl groups in the molecule and can react with bovine bone gelatin and chitosan groups (such as amino and hydroxyl groups) to form a stable cross-linked structure, thereby locking the blueberry anthocyanidins more firmly in the cross-linked matrix.

[0017] At the same time, the applicants identified ferrous ions, which can form stable colored complexes with blueberry anthocyanins, improving the color response of food freshness indicator membranes. Compared to indicator membranes immobilized with ferrous ions, membranes immobilized with ferric blueberry anthocyanin chelates generally exhibit a deeper, more intense blue color, making it difficult to detect meat spoilage. However, the color of ferrous ions chelated with blueberry anthocyanins remains virtually unchanged. Therefore, trace amounts of ferrous ions were used to chelate blueberry anthocyanins, combined with a bovine bone gelatin-citric acid-chitosan system, to create a pH-sensitive indicator membrane.

[0018] It can be seen that the addition of chitosan and citric acid can significantly enhance the mechanical properties of bovine bone gelatin, making it an excellent film-forming matrix, but the mechanism is currently unclear. Furthermore, due to the possibility that free citric acid may destroy the ferrous ion blueberry anthocyanin chelate, it is unknown whether the ferrous ion blueberry anthocyanin chelate can be incorporated into the matrix to form a film, nor whether it can improve the sensitivity of the reaction to ammonia during meat spoilage.

[0019] In a second aspect, the present invention provides a method for preparing the indicator film, comprising the following steps:

[0020] S1. Bovine bone gelatin and chitosan were dissolved in 1% acetic acid, homogenized, and then citric acid was added to prepare a substrate;

[0021] S2. Add FeSO4·7H2O and blueberry anthocyanin to deionized water to prepare pH-sensitive pigment;

[0022] S3. The pH-sensitive pigment prepared in S2 is poured into the substrate prepared in S1 and stirred to obtain an indicator solution;

[0023] S4. Pour the indicator solution prepared in S3 into a film, and dry it in a dark environment under controlled temperature and humidity to form the high-strength and high-pH-sensitive food freshness indicator film.

[0024] Furthermore, in step S1, the homogenization condition is 8000-12000 rpm, 8-15 min.

[0025] Furthermore, the homogenization condition is 8000 rpm, 8500 rpm, 9000 pm, 9500 pm, 10000 pm, 10500 pm, 11000 pm, 11500 pm or 12000 rpm.

[0026] Furthermore, the homogenization time is 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0027] Furthermore, in step S2, FeSO4·7H2O and blueberry anthocyanidin were added to deionized water at a ratio of 50 mg / 10 mL deionized water and 0.12 g / 10 mL deionized water, respectively.

[0028] Furthermore, step S2 also includes an ultrasonic step in a dark environment, and the ultrasonic conditions are 50-70 kHz and 8-12 minutes.

[0029] Furthermore, the ultrasonic frequency is 50, 55, 60, 65 or 70 kHz;

[0030] Furthermore, the ultrasonic time is 8 min, 9 min, 10 min, 11 min or 12 min.

[0031] Furthermore, step S3 also includes an ultrasonic debubbling step.

[0032] Furthermore, the drying condition in step S4 is 50% relative humidity and 22-28° C. in the dark for 36 hours.

[0033] In a third aspect, the present invention provides an application of the indicator film described in the first aspect, or the indicator film prepared by the preparation method described in the second aspect, wherein the application comprises at least one of the following:

[0034] (1) Application as food packaging material;

[0035] (2) Application in indicating food freshness.

[0036] Furthermore, the pH of the food changes during the spoilage process.

[0037] Furthermore, the food is meat, such as pork, beef, mutton, chicken, fish, and donkey meat.

[0038] In a fourth aspect, the present invention provides chelated Fe 2+ Application of blueberry anthocyanins in improving the performance of protein-polysaccharide polymer substrates, the application comprising at least one of the following:

[0039] (1) Improve the structural density of protein-polysaccharide polymer matrix;

[0040] (2) improving the water and oxygen barrier properties of protein-polysaccharide polymer substrates;

[0041] (3) improving the antibacterial properties of protein-polysaccharide polymer substrates;

[0042] (4) Improve the pH sensitivity of protein-polysaccharide polymer matrix.

[0043] Furthermore, the protein-polysaccharide polymer matrix is a bovine bone gelatin-chitosan matrix.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] The present invention uses bovine bone gelatin, chitosan and citric acid to cross-link into a matrix and incorporates blueberry anthocyanin ferrous ion chelate into the membrane. First, it successfully and innovatively realizes the combination of anthocyanin ferrous ion chelate with the citric acid cross-linked matrix, ensuring that the chelate is not destroyed and is successfully integrated into the matrix; second, it improves the performance of traditional protein-polysaccharide membranes, achieving excellent mechanical properties, water vapor barrier, oxygen barrier, antioxidant capacity and antibacterial properties; third, it enhances the sensitivity of blueberry anthocyanin to pH, which significantly improves the membrane's sensitivity to food freshness (especially ammonia vapor in meat spoilage). BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The mechanical properties of the BGCSCA membrane under different chitosan addition amounts and different citric acid addition amounts in Example 1, wherein a is the mechanical properties of the indicator membrane under different chitosan addition amounts, and b is the mechanical properties of the BGCSCA membrane under different citric acid addition amounts.

[0047] Figure 2 The light transmittance of the BGCSCA film under different chitosan addition amounts and different citric acid addition amounts in Example 1, wherein a is the light transmittance of the BGCSCA film under different chitosan addition amounts, and b is the light transmittance of the BGCSCA film under different citric acid addition amounts.

[0048] Figure 3 The sensitivity of the BGCSCA film, BGCSCAA film and BGCSCAAF film with different FeSO4 addition amounts to ammonia in Example 3, where a is the color change of each group of indicator films, and b is the SRGB value (sensitivity).

[0049] Figure 4 are the Fourier infrared spectra and X-ray diffraction patterns of each group of indicator films, where a and c are Fourier infrared spectra, and b and d are X-ray diffraction patterns.

[0050] Figure 5 Electron microscope images of the surface morphology of each group of indicator membranes.

[0051] Figure 6 AFM images of the surface roughness of the indicator films of each group.

[0052] Figure 7 The thermal properties of each group of indicator films are shown in Figure 2, where a is the TGA curve and b is the DTG curve.

[0053] Figure 8 are the test results of vapor permeability, oxygen permeability and water contact angle of each group of indicator films, where a is the vapor permeability of the indicator film, b is the oxygen permeability of the indicator film, and c is the test result of the water contact angle of the indicator film.

[0054] Figure 9 are the antioxidant capacity test results of each group of indicator membranes, where a is the DPPH free radical scavenging rate and b is the ABTS free radical scavenging rate.

[0055] Figure 10 are the antibacterial performance test results of each group of indicator films, where a is the inhibition zone area of Escherichia coli and Staphylococcus aureus, and b is the inhibition zone photos of Escherichia coli and Staphylococcus aureus.

[0056] Figure 11 The blueberry anthocyanin release from the indicator membranes of each group.

[0057] Figure 12 Monitor the color development of meat freshness for each group of indicator films. DETAILED DESCRIPTION

[0058] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0059] The terms or abbreviations in the present invention are as follows:

[0060] The abbreviation "BGCS membrane" refers to the gelatin chitosan indicator membrane;

[0061] The abbreviation "BGCSCA membrane" refers to the gel chitosan citric acid indicator membrane;

[0062] The abbreviation "BGCSCAA membrane" refers to the gel chitosan citrate blueberry anthocyanin;

[0063] The abbreviation "BGCSCAAF membrane" refers to the chitosan citrate blueberry anthocyanidin ferrous sulfate heptahydrate membrane;

[0064] The term "S RGB " represents the color change efficiency of the film, which is measured by S RGB The degree of discoloration of the film can be determined when the ammonia vapor concentration changes over a consistent time period.

[0065] Performance testing methods

[0066] 1. Fourier transform infrared spectroscopy

[0067] Fourier transform infrared (FTIR) spectra of the dried indicator films were recorded using a Cary 630 FTIR spectrometer (Agilent Technologies, USA). The dried sample was ground into a powder and then compressed into discs for testing. The tablets were compressed and secured under a test needle, and interactions between the matrix components were analyzed using FTIR spectroscopy (4000-500 cm⁻¹).

[0068] 2. X-ray diffraction (XRD)

[0069] The powder samples were ground to pass through a 200 mesh sieve, and the indicator film samples were cut into strips (15 mm × 15 mm) for measurement. The crystalline phase of the samples was studied using an XRD diffractometer (X'Pert PRO MPD, Panaco, The Netherlands) at a scanning speed of 10° / min in the 2θ range from 5° to 50°.

[0070] 3. Thermogravimetric analysis (TGA)

[0071] Thermogravimetric analysis of the CF and C-ATH indicator films was performed using a TGA analyzer (DuPont Instrument 2950 Thermogravimetric Analyzer) with a heating rate of 2°C min-1 and a temperature range of 25-500°C.

[0072] 4. Scanning electron microscopy (SEM) and atomic force microscopy (AFM)

[0073] The cross sections and surfaces of the membranes were subjected to SEM analysis using a scanning electron microscope (5136SB, TESCAN, Czech Republic) at an accelerating voltage of 5 kV.

[0074] The surface roughness of each set of indicator film samples was studied using an atomic force microscope (NX10, Park, South Korea). For this purpose, a portion of the indicator film sample of 10 μm × 10 μm was used, and the roughness values were evaluated as root mean square roughness (Rq) and average roughness (Ra).

[0075] 5. Water vapor permeability (WVP) and oxygen permeability (OP)

[0076] The water vapor permeability (WVP) of the membranes was measured using a modified ASTM E96-00 gravimetric method. Each membrane sample (5 cm diameter) was used to seal 40 g of anhydrous silica gel in a centrifuge cup. The cup was then stored in a desiccator at 20°C containing distilled water. The cup was weighed daily for 5 days. Three replicate measurements were performed for each membrane type.

[0077] Test the oxygen permeability of the indicator film. Prepare a test tube filled with 5.0 g of an oxygen scavenger mixture (consisting of sodium chloride, iron powder, and activated carbon in a ratio of 1.5:0.5:1.0). Cover the test tube with the film and seal it with ethanol-based glue. After recording the initial weight, store the test tube at room temperature in the dark for 7 days. Weigh the centrifuge tube every 24 hours and calculate the oxygen permeability using the formula

[0078] 6. Water contact angle test (WCA)

[0079] The water contact angles of different indicator films were measured using an optical contact angle meter (Dataphysics OCA20, Dataphysics, Germany). A small drop of water was placed on the surface of the indicator film mounted on a horizontal plate, and an optical image of the droplet was immediately captured. The contact angle values were calculated using ImageJ software. Three replicate measurements were performed at different locations on each sample.

[0080] 7. Antioxidation of indicator membrane

[0081] The DPPH radical scavenging activity of the indicator membranes was determined, with some modifications. Each indicator membrane (20 mg) was placed in a 4 mL, 0.2 mM DPPH ethanol solution for 30 minutes. The absorbance was then measured at 517 nm using a UV-visible spectrophotometer (L003, Shanghai Jinghua Technology Instrument Co., Ltd.). The ABTS radical cation scavenging activity was determined by placing each indicator membrane (40 mg) in a 4 mL ABTS+ solution for 5 minutes. The absorbance of the solution was then measured at 734 nm.

[0082] 8. Antibacterial properties of indicator film

[0083] Escherichia coli and Staphylococcus aureus were inoculated into culture medium for activation. Subsequently, equal amounts of each indicator membrane solution were added to sterile filter paper discs of the same size (10 mm). The filter paper discs containing the different indicator membrane solutions were then placed on agar plates inoculated with Escherichia coli and Staphylococcus aureus and incubated at 37°C for 24 hours. The area of the inhibition zone was then measured.

[0084] 9. Application test of pH indicator membrane on pork

[0085] Fresh pork (3 cm × 3 cm × 1 cm) was placed in a sealed storage container (4 cm × 4 cm × 4 cm). Indicator films BGCS, BGCSCA, BGCSCAA, and BGCSAAF were cut into 2 cm × 2 cm pieces and affixed to the top of the storage container. The container was then placed in an incubator at 25°C and stored for 2 days. Images were captured daily using a camera (Sony, Japan) mounted on the top of the light box. Additionally, the pork mass loss, pH, and TVB-N were measured daily. Changes in pork pH were measured. Pork (5 g) was minced, 50 mL of deionized water was added, and the pH of the solution was measured using a handheld pH meter (Solids Pro-ISM, Mettler-Toledo, Switzerland). The volatile basic nitrogen (TVB-N) content in the pork was determined. The weight loss rate of the pork, changes in pH and TVB-N, and color changes of the indicator films were recorded.

[0086] Example 1 Preparation of BGCSCA membrane and optimization of CS and CA concentrations

[0087] Bovine bone gelatin (BG) and chitosan were dissolved in acetic acid (1% v / v) and homogenized at 10,000 rpm for 10 minutes at room temperature. Citric acid was then added and stirred on a magnetic stirrer for 0.5 hours. Finally, 23 g of the indicator membrane solution was poured into a 7 cm diameter glass Petri dish and dried in a fume hood at 50% relative humidity and 25°C in the dark for 36 hours to form a BGCSCA membrane. The amounts of BG, CS, and CA added to each group were as follows:

[0088] (1) 10% CS group: BG 2.0 g, CS 0.2 g, CA 0 g;

[0089] (2) 20% CS group: BG 2.0 g, CS 0.4 g, CA 0 g;

[0090] (3) 30% CS group: BG 2.0 g, CS 0.6 g, CA 0 g;

[0091] (4) 40% CS group: BG 2.0 g, CS 0.8 g, CA 0 g;

[0092] (5) 50% CS group: BG 2.0 g, CS 1.0 g, CA 0 g;

[0093] (6) 0% CA group: BG 2.0 g, CS 1.0 g, CA 0 g;

[0094] (7) 2.5% CA group: BG 2.0 g, CS 1.0 g, CA 0.075 g;

[0095] (8) 5.0% CA group: BG 2.0 g, CS 1.0 g, CA 0.15 g;

[0096] (9) 7.5% CA group: BG 2.0 g, CS 1.0 g, CA 0.225 g;

[0097] (10) 10.0% CA group: BG 2.0 g, CS 1 g, CA 0.3 g.

[0098] The percentages in the group names of groups (1) to (5) represent the ratio of the amount of CS added to the amount of BG added; the percentages in the group names of groups (6) to (10) represent the ratio of the amount of CA added to the amount of (BG+CS) added.

[0099] Mechanical properties and light transmittance are important indicators of biopolymer indicator films. Indicator films made solely from bovine bone gelatin (collagen) are prone to rapid degradation and exhibit poor mechanical properties. To improve the mechanical strength of bovine bone gelatin indicator films, the applicant mixed bovine bone gelatin with chitosan during the acid treatment process, leveraging chitosan's excellent stability and biocompatibility.

[0100] like Figure 1 As shown in Figure a, the effects of chitosan and citric acid on the mechanical strength of bovine bone gelatin were evaluated using maximum tensile force, tensile strength, elongation at break, and Young's modulus tests. As can be seen, with increasing chitosan proportions, the maximum tensile force, tensile strength, and elongation at break of the indicator film significantly increased, while Young's modulus significantly decreased.

[0101] Figure 2Figure a shows that the transmittance of the indicator membrane decreases with increasing chitosan content, with little difference between the 40wt% CS and 50wt% CS groups. This suggests that the addition of chitosan leads to decreased transmittance and insufficient stability, making the indicator membrane potentially unsuitable for use in the food industry. To address these issues, the crosslinking agent citric acid was added to the bovine bone gelatin and chitosan mixture during the acid treatment process to improve the performance of the indicator membrane. To ensure that the pH indicator membrane possessed both strong mechanical strength and high transmittance, a 2:1 mass ratio of bovine bone gelatin to chitosan was selected for use in the 50wt% CS group in subsequent examples.

[0102] like Figure 1 As shown in Figure b, the addition of citric acid (CA) gradually increased the maximum tensile strength and elongation at break. When CA reached 10%, the maximum tensile strength was significantly higher than that of the other groups, and the elongation at break was significantly higher than that of the 0% CA, 2.5% CA, and 5% CA groups. Concurrently, the tensile strength gradually decreased, with the 10% CA group showing a significant decrease compared to the 0% CA, 2.5% CA, and 5% CA groups. This trend can be attributed to citric acid acting as a plasticizer and crosslinker within the biopolymer matrix. Young's modulus increased significantly at 2.5% CA and then gradually decreased. The 10% CA group maintained a higher modulus than the 0% CA group (P < 0.05). The rapid increase in Young's modulus observed in the 2.5% CA group may be attributed to the strong water-retention capacity of citric acid, which enhances the flexibility of the indicator film. However, when the citric acid content exceeds 2.5%, the residual free citric acid acts as a plasticizer, reducing the flexibility of the indicator film.

[0103] like Figure 2 As shown in Figure b, the transmittance of the 2.5% CA, 5% CA, 7.5% CA, and 10% CA groups was significantly higher than that of the 0% CA group (P < 0.05). Furthermore, it can be seen that the transmittance curves of the four groups (7) to (10) within the wavelength range of 200 nm to 800 nm are very similar. To enhance mechanical strength and maximize transmittance, the 10% CA group was selected as the substrate for the pH indicator film.

[0104] Therefore, in the subsequent examples, the mass ratio of BG:CS:CA was 2:1:0.3 as the concentration of bovine collagen, chitosan and citric acid.

[0105] Example 2 Preparation of BGCSCAA membrane

[0106] Bovine bone gelatin and chitosan were dissolved in acetic acid (1% v / v) and homogenized at 10,000 rpm for 10 minutes at room temperature. Citric acid was then added and stirred on a magnetic stirrer for 0.5 hours. Blueberry anthocyanins were added to deionized water at a ratio of 0.12 g / 10 mL of deionized water. The mixture was ultrasonically treated at 60 kHz for 10 minutes in the dark. The mixture was then poured into the gelatin-chitosan matrix and stirred for another 10 minutes. The mixture was then ultrasonically removed at 60 kHz. Finally, 23 g of the indicator membrane solution was poured into a 7 cm diameter glass Petri dish and dried in a fume hood at 50% relative humidity and 25°C for 36 hours to form a BGCSCAA membrane.

[0107] Example 3 Preparation of BGCSCAAF membrane and Fe 2+ Optimization of ion concentration

[0108] Bovine bone gelatin and chitosan were dissolved in acetic acid (1% v / v) and homogenized at 10,000 rpm for 10 minutes at room temperature. Citric acid was then added and stirred on a magnetic stirrer for 0.5 hours. Ferrous sulfate heptahydrate and blueberry anthocyanidins were added to deionized water at a ratio of 50 mg / 10 mL and 0.12 g / 10 mL, respectively. The mixture was ultrasonically treated at 60 kHz for 10 minutes in the dark. The mixture was then poured into the gelatin-chitosan matrix and stirred for another 10 minutes. Bubbles were then removed by ultrasonication at 60 kHz. Finally, 23 g of the indicator membrane solution was poured into a 7 cm diameter glass Petri dish and dried in a fume hood at 50% relative humidity and 25°C for 36 hours to form a BGCSCAAF membrane.

[0109] Figure 3 The sensitivity of BGCSCA, BGCSCAA, and indicator films containing 0.01%, 0.03%, and 0.05% FeSO4 to ammonia is shown. The BGCSCA indicator film did not show any color change, which is attributed to the absence of color-changing substances in the indicator film. In contrast, the BGCSCAA indicator film and the 0.01% FeSO4 indicator film showed no color change. 2+ , 0.03% Fe 2+ and 0.05% Fe 2+ The indicator films showed obvious color changes, showing similar rose-red tones at 0 minutes. 2+ , 0.03% Fe 2+ and 0.05% Fe 2+ The indicator film begins to change from rose red to purple, which contains 0.05% Fe 2+ The indicator film shows the most obvious color change visible to the naked eye, and its S RGBThe value (sensitivity) was 21.77% higher than that of the other four groups. 2+ , 0.03% Fe 2+ , 0.05% Fe 2+ The BGCSCAA group and the BGCSCAA group have turned purple, and the depth of the purple increases with the Fe 2+ The color darkens significantly with increasing concentration. 2+ group (36.85%), 0.03% Fe 2+ group (33.03%), 0.05% Fe 2+ The SRGB values of the group (44.12%) were higher than those of the BGCSCAA group (24.40%), indicating that the purple color was deeper. 2+ The SRGB value of the group was the highest among these groups, which was associated with the deepest purple color observed. At 30 min, the purple color of the BGCSCAA group deepened further (37.79%), and the 0.01% Fe 2+ The purple color of the group was more obvious (45.78%), and the 0.03% Fe 2+ The color of the group changes to blue-purple (50.39%), 0.05% Fe 2+ The group has turned dark gray, and the corresponding SRGB value is still 0.05% Fe 2+ At 40 minutes, the BGCSCAA group was still purple (47.23%), and the 0.01% Fe 2+ The group showed a blue-purple trend (54.67%), 0.03% Fe 2+ The group has turned blue-purple (58.84%), 0.05% Fe 2+ The group has turned dark gray. Correspondingly, the SRGB value is still the highest in the 0.05% Fe2+ group (55.61%). 2+ As the concentration increases, the response efficiency of the indicator membrane to ammonia increases. 0.05% Fe 2+ The group responded to ammonia more quickly than the other four groups within 0-40 minutes. Therefore, 0.05% Fe 2+ The indicator membrane of the group was used for further characterization tests and was named BGCSCAAF.

[0110] Performance Test Example 1: Using Fourier Transform Infrared Spectroscopy and X-ray Diffraction to Explore the Formation Mechanism of the Indicator Film

[0111] like Figure 4 a. Figure 4Figure c shows the Fourier transform infrared spectra of the BGCS, BGCSCA, BGCSCAA, and BGCSCAAF membranes. When gelatin (BG) is complexed with chitosan (CS), its waveform exhibits characteristic absorption bands at 1250-1750 cm⁻¹ similar to those of BG, but the peaks at 950-1150 cm⁻¹ are similar to those of CS, with a slight increase in the peak intensity between 1551 and 1246 cm⁻¹. This suggests that the -OH and -NH₂ groups in BG can form hydrogen bonds with those in CS.

[0112] The characteristic amide I absorption band (1600-1700 cm⁻¹) undergoes significant changes after the incorporation of citric acid (CA), with a new absorption peak appearing at 1669 cm⁻¹ and a decrease in the intensity of the absorption band at 1651 cm⁻¹. This is attributed to the crosslinking of CA with CS or BG, which reduces the number of free C=O bonds and forms new ester bonds. The characteristic amide II absorption band (1551 cm⁻¹) broadens and decreases in intensity. This broadening and decrease in intensity during the crosslinking reaction between chitosan and collagen may indicate the formation of new crosslinks, which affect the NH and CN vibrations. Furthermore, a characteristic absorption band at 3263 cm⁻¹ was observed for the BGCS group, while a shift to 3278 cm⁻¹ for BGCSCA is likely due to the reaction of the amino groups on CS and BG with CA. The chemical crosslinking of the amino groups on CS and BG with CA results in significant shifts in this region, suggesting that CA disrupts the structural order associated with the triple helical structure of BG.

[0113] After the addition of blueberry anthocyanin (A), the characteristic peaks of the amide group absorption band (between 1500-1600 and 1600-1700 cm^-1) shifted significantly to the left, and the characteristic peak at the 3278 cm-1 absorption band attributed to the OH vibration of BG / CS shifted to the right. This is attributed to the presence of multiple hydroxyl groups in the A molecule, which can act as hydrogen bond donors and form hydrogen bonds with the carbonyl (C=O) oxygen atom and nitrogen atom (NH) in the amide bond as hydrogen bond acceptors, causing the characteristic peak at the 3278 cm-1 absorption band to shift to the right, thereby changing the electron cloud density of the carbonyl group and causing the amide band to shift to the left.

[0114] After the addition of Fe2SO4·7H2O, the characteristic absorption peaks of blueberry anthocyanins, the absorption peaks of hydroxyl (-OH) (around 3300 cm-1) and amide (C=O) (1600-1700 cm-1) vibration, were significantly reduced, indicating that Fe 2+ The chelation with A changes the electron distribution of A molecule, thereby reducing its vibration frequency. The presence of CA may compete with the hydroxyl group or other coordination groups in A for Fe 2+coordination, which may destroy the original Fe 2+ It is worth mentioning that no new absorption peak was found at the metal-oxygen (Fe-O) coordination bond absorption band position (400-600cm-1), which proves that Fe 2+ -A chelate can stably exist in the system to produce a highly sensitive pH color reaction.

[0115] like Figure 4 b. Figure 4 Figure d shows the X-ray diffraction patterns of the samples. Chitosan's diffraction peaks at 2θ = 12° and 20° reflect the specific interplanar spacing within its crystal structure. The diffraction peak of gelatin at approximately 2θ = 8.5° is associated with the ordered triple-helical structure of proteins. The X-ray diffraction spectrum of blueberry anthocyanins exhibits a broad peak at ~18°, indicating an amorphous state. The multimodal patterns of citric acid and ferrous sulfate heptahydrate may be due to the presence of multiple unit cells or different crystal morphologies. The small peak at 2θ = 18° in the BGCS group is likely due to hydrogen bonding between collagen and chitosan during mixing, resulting in a new crystal structure or slight changes in the lattice, consistent with the conclusions from Fourier transform infrared spectroscopy. The BGCSCA group exhibits distinct peaks at 2θ = 8° and 12°, with the peak at 2θ = 18° also becoming stronger. The obvious peaks at 2θ=8° and 12° are due to the fact that the addition of citric acid affects the crystallinity or crystal structure of chitosan and collagen, respectively, resulting in changes in the shape or intensity of these peaks; and the enhanced peak intensity at 2θ=18° is attributed to the introduction of new chemical structures or complex formation by the addition of citric acid. The changes in these three peaks therefore once again prove the conclusion that CA is successfully cross-linked with CS and BG in Fourier transform infrared spectroscopy analysis. Due to the addition of A, the peaks of BGCSCAA at 2θ=12° and 18° are weakened, which is because the hydrogen bonds between chitosan and blueberry anthocyanins destroy the interaction between the original polymers. With the addition of Fe 2+ With the addition of -A chelate, the amorphous part in the BGCSCAAF group increased, the overall crystallinity decreased, and the intensity of the crystalline peak in the XRD pattern also weakened accordingly.

[0116] Depend on Figure 4 It can be seen that citric acid successfully cross-linked chitosan and bovine bone gelatin, and the blueberry anthocyanin ferrous ion chelate was not destroyed by the residual citric acid after addition, and the blueberry anthocyanin ferrous ion chelate was successfully embedded in it.

[0117] Performance Test Example 2 Surface Morphology Observation of the Film

[0118] The surface morphology of the film observed by SEM is shown in Figure 5As shown in . The BGCS membrane shows a dense and smooth surface and cross-section, confirming that collagen and chitosan have good compatibility and interaction. A porous structure can be observed on the surface and cross-section of the BGCSCA membrane. These porous structures are formed due to the competition between citric acid and collagen and chitosan during the cross-linking process, resulting in intramolecular dehydration, which is consistent with the FTIR analysis study. In addition, the negatively charged citrate is also an important reason for the mineralization of collagen to form this amorphous porous structure. At the same time, this multi-level structure also gives the BGCSCA membrane excellent mechanical properties. The surface and cross-section of the BGCSCAA membrane are denser than those of the BGCSCA membrane, which may be due to the addition of A making the pore structure on its surface slightly smoother, indicating that A is compatible with the polymer matrix of the BGCSCA membrane. However, Fe 2+ The addition of BGCSCAAF membrane had almost no effect on the surface and cross-sectional morphology compared with BGCSCAA membrane under SEM observation.

[0119] like Figure 6 As shown in Figure 2, the surface roughness of the indicator film was evaluated using AFM images. With the addition of CA, the roughness of the indicator film gradually increased (Ra and Rq), while the roughness decreased (Ra and Rq) with the addition of A. 2+ The addition of the roughness slightly increases (Ra and Rq), but the specific surface area is greatly increased, which may lead to more obvious contact with ammonia vapor. This is basically consistent with our scanning electron microscopy observations.

[0120] Performance Test Example 3 Thermal Performance Test of Indicator Film

[0121] The thermal properties of the indicator film reflect its ability to resist decomposition at high temperatures. Thermogravimetric analysis was performed to study the thermal behavior of BG after reacting with CS, CA, A and Fe2SO4·7H2O. The TGA and DTG curves are shown in Figure 2. Figure 7All blends exhibited similar behavior, with three main stages. For temperatures below 150°C, weight loss was attributed to water absorption, approximately 5%. Electrostatic interactions and hydrogen bonding between CS and BG enhanced the thermal stability of the membrane. Similar behavior was observed for the BGCSCA, BGCSCAA, and BGCSCAAF groups crosslinked with CA, with weight loss further reduced during this stage. The second stage (240-250°C) was dominated by CA decomposition and thermal degradation of -NH2. The -NH2 and CA contents of the G group during this stage were significantly lower than those of the other groups, resulting in lower mass loss than all other groups. The third stage (190-800°C) was primarily attributed to mass loss caused by the decomposition of biopolymers such as BG, CS, and A. At 800°C, BGCSCAAF exhibited the lowest weight loss (24.97%), followed by BGCSCAA (23.35%). BGCSCA (21.80%) and BGCS (21.63%) were almost identical, both exceeding BG (17.57%). The fact that the weight loss of BGCS (21.63%) and BGCSCA (21.80%) is lower than that of BG shows that the electrostatic force and hydrogen bond between BG and CS may be the key to the difference in mass loss at this temperature. The cross-linked structure formed by CA was almost completely destroyed with the decomposition of CA in the second stage, which also explains why the weight loss rate of BGCSCA was higher than that of BGCS before 650°C. The fact that BGCSCAAF (24.97%) and BGCSCAA (23.35%) in the third stage have always been higher than the other three groups may be because A changed the crystal structure of BGCSCA, making it show better geothermal stability. The fact that BGCSCAAF (24.97%) is higher than BGCSCAA (23.35%) may be due to Fe 2+ The -A chelate has higher thermal stability, which enhances the stability of the thermal decomposition residue and thus may show improved thermal stability in thermogravimetric analysis (TGA).

[0122] In addition, the DTG curve is as follows Figure 7 As shown in b. With the addition of CA, the main degradation peak of the indicator film moves towards the high temperature direction. This shows that the addition of CA to the cross-linking of BG and CS successfully improves the thermal stability of the indicator film. When A is added with BGCSCAA (313.3℃), the main degradation peak of the indicator film moves towards the low temperature direction (294.3℃), which may be due to its antioxidant properties and the possible change of the decomposition path, which accelerates the rate of the decomposition process. This is consistent with the study. 2+ After chelation, the main degradation peak temperature of BGCSCAAF (314.4 °C) was significantly higher than that of BGCSCAA (313.3 °C), which may be due to the fact that a higher temperature is required to break this stable structure during the heating process, which is consistent with the TGA results.

[0123] Performance Test Example 4 Water Vapor Permeability, Oxygen Permeability and Water Contact Angle Test

[0124] Indicates the WVP value of the membrane. Figure 8 As shown in Figure 2, FTIR results indicate that hydrogen bonds are formed between A and the hydrophilic groups of BGCSCA, reducing the free hydroxyl groups. This reduction in free hydrophilic groups leads to a decrease in water absorption, which in turn reduces the WVP of the membrane. In addition, the phenolic compounds present in blueberry anthocyanins physically interact with BGCSCA, acting as bridges between polysaccharide chains, resulting in the formation of a dense structure as shown in the scanning electron microscopy images, thereby reducing the WVP of the membrane. Figure 8 As shown in the figure, the WVP, OP and WCA of BGCSCAAF are the highest, which are significantly better than those of BGCSCA film and BGCSCAA film. This proves that blueberry anthocyanin chelating ferrous ions can effectively improve the barrier properties of the substrate, while giving the indicator film color development properties, it also improves the basic performance of the indicator film, making it more suitable as a packaging material.

[0125] Performance Test Example 5: Antioxidant Performance Test of the Membrane

[0126] The antioxidant capacity of each group of indicator membranes was evaluated by DPPH and ABTS free radical scavenging tests. Figure 9 As shown in Figure 2 , the DPPH and ABTS free radical scavenging rates of the G indicator membrane were 15.92% and 23.40%, respectively. The addition of chitosan (CS) significantly enhanced (P < 0.05) the free radical scavenging ability of the indicator membrane. In dilute acid solution, chitosan contains hydroxyl and amino groups within its repeating units, as well as free amino (NH2) groups that can react with free radicals to form stable macromolecular complexes. The addition of citric acid further increased the DPPH free radical scavenging rate to 35.22%, and the ABTS free radical scavenging rate significantly increased (P < 0.05) to 33.51%. This improvement is attributed to the disruption of the original intermolecular and intramolecular hydrogen bonds within the chitosan molecule and the introduction of reactive carboxyl groups. The hydrogen atoms in these carboxyl groups can combine with free radicals to form stable species, thereby scavenging hydroxyl radicals. With the addition of blueberry anthocyanins, the DPPH radical scavenging rate was significantly increased (P<0.05) to 61.63%, and the ABTS radical scavenging rate increased to 56.32%. This enhancement is due to the excellent antioxidant capacity of A, including acting as a reducing agent, hydrogen donor, singlet oxygen quencher, and metal chelator. It is worth noting that the observed ABTS radical scavenging rate was lower than the DPPH radical scavenging rate. It can be seen that the A-Fe 2+ The indicator film not only exhibits pH responsiveness but also has significant free radical quenching ability.

[0127] Performance Test Example 6 Antibacterial Performance Test of Membrane

[0128] As a potential antimicrobial agent, the antimicrobial performance of membrane materials is a key indicator for evaluating the quality of functional packaging. Therefore, a comparative study was conducted on the antimicrobial performance of various biopolymer indicator membranes against Escherichia coli and Staphylococcus aureus. Figure 10 As shown in the photographs, among the four groups of samples, the BGCSCAA group exhibited the strongest antibacterial activity, while the BGCS group exhibited the weakest. This effect is attributed to the inherent positive charge of CS, which binds to the negatively charged bacterial cell membrane, disrupting the bacterial cell structure. This disruption enables CS to penetrate the cells and interfere with bacterial metabolism and gene expression. The BGCSCA group exhibited significantly enhanced antibacterial activity against Escherichia coli, attributed to the antibacterial properties of some free ca. With the addition of A, the antibacterial capacity of the BGCSCAA group membranes was significantly enhanced (P < 0.05). This enhancement was attributed to A's ability to reduce bacterial TCA cycle activity and energy transfer, thereby inhibiting bacterial growth and reproduction. Furthermore, the high amount of polyphenols in A disrupts the cell membrane structure, leading to nucleic acid leakage and subsequent bacterial inhibition. After the addition of FeSO4·7H2O, the inhibition zone of the BGCSCAAF group membrane decreased slightly, but remained stronger than that of the membrane without A. FeSO4·7H2O is not a conventional antibacterial agent. It is speculated that the reduction in the inhibition zone may be due to its stronger binding force with A, which reduces the diffusion of blueberry anthocyanins and thus narrows the inhibition range. This observation is consistent with the test results of A release ( Figure 11 ) are consistent.

[0129] Performance Test Example 7: Blueberry Anthocyanin Release Test

[0130] The interaction between the BGCSA matrix and blueberry anthocyanin (A) was investigated through release experiments. The release rates of the pigment from the color indicator film were measured using 10%, 50%, and 95% aqueous solutions and 95% ethanol solutions. The results are shown in the figure. A released fastest in 10% ethanol solution, followed by water, 50%, and 95% ethanol solutions. This is primarily due to the polarity of the water-soluble blueberry anthocyanin. The pigment release rate from the color indicator film was affected by the type and polarity of the food simulant, its swelling degree, and the solubility of the film. The high release rates of A in water and 10% ethanol solutions were attributed to the high water solubility of the film, the solubility of the anthocyanin in water, and the hydrophilicity of the biopolymer composite film. BGCSCAAF released the slowest of the four states, indicating that the blueberry anthocyanin ferrous ion chelate is most stable within the cross-linked matrix.

[0131] Table 1 Film thickness, moisture content, and water solubility

[0132]

[0133] Different letter superscripts indicate significant differences between groups in the same column (P < 0.05).

[0134] Pork protein is easily broken down by bacteria and mold, producing various volatile nitrogen-containing compounds such as ammonia and amines. Because collagen and chitosan are hydrophilic and highly water-soluble compounds, an indicator label was affixed to the top of the sealed box containing the pork to prevent direct contact with food, as shown in the figure. The TVB-N value, pH, and weight are shown in the table. On the first day, the indicator label in the BGCSCAA group showed little change, while the indicator label in the BGCSCAA group changed from rose red to a deep red, visible to the naked eye. The volatile basic nitrogen content of the pork increased to 14.41 ± 1.51 mg / 100 g, still below the limit for pork stipulated in the Chinese standard GB 2707-2016 (TVB-N < 15 mg / 100 g), indicating that the pork was suitable for consumption. The pH increased to 7.83 ± 0.14, likely due to the production of alkaline substances by microbial degradation of protein. On the second day, the indicator label in the GCSCAA group showed slight changes, although not as significant as the changes observed in the GCSCAAF group on the first day. However, the color of the indicator label in the BGCSCAAF group changed from deep red to deep purple, a clear change attributable to the degradation of blueberry anthocyanins caused by protonation. TVB-N and pH values increased significantly (p < 0.05), reaching 24.19 ± 1.02 mg / 100 g and 8.18 ± 0.14, respectively. The TVB-N content exceeded the limit for pork specified in the Chinese standard GB 2707-2016 (TVB-N < 15 mg / 100 g), making it unsuitable for consumption according to Chinese standards. The weight of the pork decreased significantly on both the first and second days compared to the previous day (p < 0.05). By the second day, the weight had dropped to 23.27 ± 1.69 g. This decrease was due to water evaporation from the pork and microbial growth, which consumed a significant portion of the dry matter on the pork surface. These changes, consistent with the observed increases in TVB-N and pH, indicate progressive spoilage during storage, demonstrating that the BGCSCAAF group is more sensitive than the BGCSCAA group for monitoring meat freshness. The red color of the BGCSCAAF indicator label indicates the freshness of pork, while a dark purple color indicates that the pork has deteriorated. Therefore, the BGCSCAAF film is suitable as a pork freshness indicator film.

[0135] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. Chelated Fe 2+ The application of blueberry anthocyanidins in improving the performance of protein-polysaccharide polymer substrates is characterized in that: The applications include: Improving the water and oxygen barrier properties of the protein-polysaccharide polymer matrix, or improving both the structural density and the water and oxygen barrier properties of the protein-polysaccharide polymer matrix; The protein-polysaccharide polymer matrix is a bovine bone gelatin-chitosan matrix; The application comprises the following steps: cross-linking bovine bone gelatin, chitosan and citric acid into a matrix and incorporating blueberry anthocyanin ferrous ion chelate into the matrix to form a film. S1. Bovine bone gelatin and chitosan were dissolved in 1% acetic acid, homogenized, and then citric acid was added to prepare a substrate; the mass ratio of bovine bone gelatin: chitosan: citric acid was 1.8-2.2:0.8-1.2:0.2-0.4; S2. Adding FeSO4·7H2O and blueberry anthocyanidin to deionized water to prepare a pH-sensitive pigment; the mass ratio of blueberry anthocyanidin to FeSO4·7H2O is 0.10-0.14:0.04-0.06; S3. The pH-sensitive pigment prepared in S2 was poured into the substrate prepared in S1 and stirred to obtain an indicator solution; S4. Pour the indicator solution prepared in S3 into a film and dry it in a dark environment under controlled temperature and humidity.

2. The use according to claim 1, characterized in that The mass ratio of the bovine bone gelatin:chitosan:citric acid is 2:1:0.

3.

3. The use according to claim 1, characterized in that The mass ratio of blueberry anthocyanins to FeSO4·7H2O is 0.12:0.

05.

4. The use according to claim 1, characterized in that In step S1, the homogenization condition is 8000-12000 rpm, 8-15 min.

5. The use according to claim 1, characterized in that In step S2, FeSO4·7H2O and blueberry anthocyanidin were added to deionized water at a ratio of 50 mg / 10 mL deionized water and 0.12 g / 10 mL deionized water, respectively.

6. The use according to claim 1, characterized in that Step S2 also includes an ultrasonic step in a dark environment, and the ultrasonic conditions are 50-70 kHz and 8-12 minutes.

7. The use according to claim 1, characterized in that The drying conditions in step S4 are 50% relative humidity, 22-28° C. in the dark for 28-40 hours.

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