A KGM-anthocyanin membrane, its preparation method and application

Abstract

This invention relates to the field of food packaging films, and discloses a KGM-anthocyanin film, its preparation method, and its application. The method includes: crushing berries to obtain dried berry powder, adding methanol-formic acid solution, repeatedly extracting multiple times, combining the extracts, centrifuging to remove impurities, collecting the supernatant, and obtaining a crude berry anthocyanin extract; using XDA-7HP as the medium, performing gradient elution with different mobile phases to separate and purify the crude berry anthocyanin extract, concentrating under reduced pressure, and freezing under vacuum to obtain a lyophilized berry anthocyanin powder; preparing a KGM emulsion, and stirring in a water bath to obtain a membrane matrix; adding the lyophilized berry anthocyanin powder to a lyophilized anthocyanin solution, adding it to the membrane matrix, and performing a molding process to obtain a KGM-anthocyanin film with added anthocyanins. This invention solves the problems of existing single KGM films, such as weak pH sensitivity, weak antioxidant capacity, unsatisfactory antibacterial effect, weak resistance to external shocks, and inability to achieve real-time monitoring of food freshness and extend food shelf life.

Description

Technical Field

[0001] This invention relates to the field of food packaging films, and particularly to a KGM-anthocyanin film, its preparation method, and its application. Background Technology

[0002] Food packaging is crucial in the food industry, and selecting appropriate packaging is key to ensuring food quality and freshness. Traditional edible packaging, based on petroleum derivatives, suffers from drawbacks such as poor degradation, poor mechanical properties, and significant pollution. To address this issue, edible packaging materials based on natural polymers, such as polysaccharides, starches, and proteins, have received widespread attention. Their use ensures food integrity and safety, effectively extends shelf life, and prevents the migration of harmful polymers from traditional packaging materials into food, thereby reducing food safety risks.

[0003] Currently, KGM is one of the most widely used food packaging matrix materials. It is a natural, high-molecular-weight soluble dietary fiber. Its molecular chain is composed of D-glucose and D-mannose residues linked by β-1,4-glycosidic bonds in a molar ratio of 1:1.5 or 1:1.6. The mannose backbone contains a branched structure with β-1,3-glycosidic bonds at the C3 position, and there are 3 branches on 32 sugar residues. It has high viscosity, biodegradability, and biocompatibility, thus possessing great potential and application value in extending the shelf life of food. However, food packaging materials developed using KGM alone are easily soluble in water and have disadvantages such as poor mechanical properties, barrier properties, antibacterial and antioxidant activities. In particular, they cannot reflect the freshness index of food in a timely manner. To solve this problem, Chang et al. double-modified KGM and introduced nano-SiO2 to form a composite membrane structure, which improved the mechanical properties, hydrophobicity, and antioxidant properties of the membrane. Besides modifying KGM, some researchers have reported that the performance of KGM membranes can be improved by adding natural / synthetic polymers. For example, the addition of cellulose nanocrystals (CNC) can improve the physical, mechanical, and gas barrier properties of the membrane. Adding honeysuckle leaf extract (HLE) to a composite edible film of KGM and gelatin can enhance the mechanical properties and antibacterial ability of the film. Furthermore, antibacterial edible coatings have already been used in the food industry, but single edible coatings can no longer meet the demands for food preservation. Therefore, finding multifunctional edible coatings with antibacterial, antioxidant, and pH-indicating properties is an urgent problem to be solved.

[0004] Anthocyanins, as a class of natural products with multiple functional activities, not only possess antioxidant and antibacterial capabilities but also exhibit corresponding colors in response to pH values. Specifically, the system appears red when pH < 2; transitions from red to colorless at pH 3-6; gradually turns purple as pH rises to 7-8; turns blue at 8-10; and becomes yellow when pH > 11. Based on these color characteristics, ZANG et al. developed a pH indicator membrane based on a gelatin / polyvinyl alcohol matrix and mulberry pomace anthocyanin extract to monitor the spoilage of dace (Cirrhina molitorella) during storage. The results showed that during the 24-hour observation period, the pH of the dace increased due to the production of total volatile basic nitrogen (TVB-N) during spoilage, and the color of the composite membrane changed accordingly, showing a good dependence on the change in TVB-N. Zhai et al. also observed similar results, finding that when hibiscus anthocyanins were used as indicators to indicate changes in the freshness of fish, the color of the indicator film gradually changed from red to light blue as the degree of spoilage of the fish deepened. It is important to note that, in addition to being a pH indicator, anthocyanins can also be used as natural antioxidants and antibacterial agents to prevent the oxidation of food components and the growth of microorganisms, thereby extending the shelf life of food. For example, adding *Phyllostachys edulis* anthocyanins to a chitosan / methylcellulose composite solution to create a food packaging film can inhibit the growth of *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Escherichia coli* in food. Furthermore, SUN et al. found that the addition of mulberry anthocyanins can improve the antioxidant activity of KGM / chitosan composite films and exhibit good inhibitory activity against *Escherichia coli* and *Staphylococcus aureus*, thus extending the shelf life of food. Therefore, anthocyanins can not only improve the mechanical properties of natural polymer membranes and endow them with antioxidant and antibacterial properties, but also change their color during food storage, thus visually reflecting the spoilage of food. They have the potential to extend the shelf life of goods and monitor the freshness index of food.

[0005] However, anthocyanins from different plant sources exhibit significant differences in composition, resulting in varying pH sensitivities, antioxidant capacities, and antibacterial properties. This presents considerable challenges for developing anthocyanin-containing food packaging films and their applications. Therefore, this study used KGM as a matrix to prepare four types of KGM-anthocyanin films by adding blueberry anthocyanin (BBA), black goji berry anthocyanin (BWA), honeysuckle berry anthocyanin (BHA), and blackcurrant anthocyanin (BCA). The color characteristics, antioxidant and antibacterial properties, and mechanical properties of the prepared films were investigated. Fourier transform infrared spectroscopy and DSC thermal stability analysis were conducted to explore the binding mechanism of KGM with each anthocyanin, aiming to develop KGM-anthocyanin films suitable for food preservation. Summary of the Invention

[0006] This invention aims to provide a KGM-anthocyanin membrane, its preparation method, and its application, in order to solve the problems of weak pH sensitivity, weak antioxidant capacity, unsatisfactory antibacterial effect of KGM food packaging materials, weak resistance to external impact, and inability to achieve real-time monitoring of food freshness in existing technologies.

[0007] To achieve the above objectives, the present invention provides the following method:

[0008] The present invention provides a method for preparing a KGM-anthocyanin membrane:

[0009] S1: After harvesting ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants, dry them in a vacuum freeze dryer to obtain dried fruits. Crush the dried fruits to obtain berry powder. Weigh the berry powder, place it in an Erlenmeyer flask, add methanol formic acid solution, seal the flask with plastic wrap, and repeatedly extract it at room temperature and in the dark. Combine the obtained extracts and centrifuge them to remove fruit residue, protein, and polysaccharide impurities. Collect the supernatant, which is the anthocyanin extract of each berry. Store it in a -18℃ refrigerator in the dark for later use.

[0010] S2: Using XDA-7HP as the medium, gradient elution was performed sequentially with distilled water and 40% ethanol aqueous solution as the mobile phase to separate and purify the anthocyanin extracts of each berry. The ethanol eluent was collected, concentrated under reduced pressure, frozen under vacuum, and dried to obtain the lyophilized powder of each berry anthocyanin.

[0011] S3: Weigh KGM powder and dissolve it in distilled water with added glycerin, and continuously stir in a water bath to prepare KGM emulsion;

[0012] S4: The prepared KGM emulsion is stirred for 0.5 h in a water bath at 60 °C to serve as a membrane matrix; the lyophilized anthocyanin powders of each berry from step S2 are weighed and prepared into a 6 mg / mL anthocyanin solution, which is then added to the membrane matrix at a ratio of 1 ml: 100 g and stirred thoroughly to obtain a KGM-anthocyanin mixed emulsion.

[0013] S5: The KGM-anthocyanin mixed emulsion is inverted in a petri dish to spread it evenly and then dried. The total KGM content in the resulting sample is kept at 1.0g to obtain a KGM-anthocyanin membrane.

[0014] Preferably, the step of harvesting ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants and drying them in a vacuum freeze dryer to obtain dried fruit, and then pulverizing the dried fruit to obtain berry powder, includes: harvesting ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants and drying them in a vacuum freeze dryer to obtain dried fruit; pulverizing the dried fruit and collecting the residue through an 80-mesh sieve to obtain berry powder; and storing the berry powder in a -18°C refrigerator.

[0015] Preferably, the concentration-to-volume ratio of the methanol-formic acid solution is 98:2.

[0016] Preferably, the steps of weighing the dried berry powder, placing it in an Erlenmeyer flask, adding a methanol-formic acid solution, sealing the flask opening with plastic wrap, and repeatedly extracting the berry powder at room temperature and in the dark include: weighing the dried berry powder, placing it in an Erlenmeyer flask, adding a methanol-formic acid solution with a volume ratio of 98:2, sealing the flask opening with plastic wrap, and extracting the berry powder at room temperature and in the dark for 8-10 hours. After repeating the extraction three times, the resulting extracts are combined.

[0017] Preferably, the step of combining the obtained extracts and then centrifuging them includes: combining the obtained extracts and centrifuging them at 3000g for 10min at 4°C.

[0018] Preferably, the step of weighing KGM powder, dissolving it in distilled water with added glycerin, and continuously stirring in a water bath to prepare KGM emulsion includes: weighing KGM powder, dissolving it in distilled water with added glycerin (the glycerin content is 1%), and continuously stirring in a water bath at 60°C for 2 hours to prepare KGM emulsion.

[0019] Preferably, the mass ratio of the lyophilized berry anthocyanin powder to the membrane matrix is ​​6:100.

[0020] Preferably, the step of inverting the KGM-anthocyanin mixed emulsion in a petri dish to spread it evenly and drying it to maintain the total KGM content in the obtained sample at 1.0g to obtain the KGM-anthocyanin membrane includes: inverting the KGM-anthocyanin mixed emulsion in a petri dish, drying it at 35°C for 48h, maintaining the total KGM content in the obtained sample at 1.0g to obtain the KGM-anthocyanin membrane, and storing the KGM-anthocyanin membrane at 25°C and 4.0% relative humidity for later use.

[0021] The present invention provides a KGM-anthocyanin membrane prepared according to the preparation method of the KGM-anthocyanin membrane described above.

[0022] The present invention provides an active packaging film for extending the shelf life of food and its application as a smart packaging film for real-time monitoring of food freshness, based on the KGM-anthocyanin film described above.

[0023] The beneficial effects of this invention are as follows: By adding different anthocyanins to a KGM matrix, this invention successfully prepared an active intelligent packaging film. Due to the hydrogen bonds formed between the anthocyanins and KGM, the film's pH sensitivity, antioxidant activity, antibacterial ability, and mechanical strength are improved. Therefore, the KGM-anthocyanin film can not only serve as an active packaging film to extend the shelf life of food, but also as an intelligent packaging film to monitor the freshness of food in real time. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0025] Figure 1 A schematic flowchart illustrating the steps of a method for preparing a KGM-anthocyanin membrane according to an embodiment of the present invention;

[0026] Figure 2 HPLC chromatograms of anthocyanins from blueberry, blackcurrant, honeysuckle, and black goji berry provided in embodiments of the present invention (detection wavelength 525 nm).

[0027] Figure 3 Physicochemical property diagrams of blueberry anthocyanins (BBA), blackcurrant anthocyanins (BCA), honeysuckle anthocyanins (BHA), and black goji berry anthocyanins (BWA) provided in embodiments of the present invention, wherein: Figure 3 A represents the color characteristics of BBA, BCA, BHA, and BWA under pH conditions ranging from 2 to 13. Figure 3 B represents the UV-Vis absorption spectra of BBA, BCA, BHA, and BWA under pH conditions of 2-13. Figure 3 C is a comparison chart of the antioxidant capacity of BBA, BCA, BHA, and BWA; Figure 3 D is a comparison chart of the antibacterial abilities of BBA, BCA, BHA, and BWA against Escherichia coli and Staphylococcus aureus.

[0028] Figure 4 The physicochemical properties of the KGM-anthocyanin membrane solution provided in the embodiments of the present invention are shown in the following diagram: Figure 4 A represents the color characteristics of KGM-BBA, KGM-BCA, KGM-BHA, and KGM-BWA membrane solutions under pH conditions ranging from 2 to 13. Figure 4B represents the UV-Vis absorption spectra of KGM-BBA, KGM-BCA, KGM-BHA, and KGM-BWA membrane solutions under pH conditions of 2-13. Figure 4 C is a comparison chart of the antioxidant capacity of KGM-BBA, KGM-BCA, KGM-BHA, and KGM-BWA membrane solutions; Figure 4 D is a comparison of the antibacterial abilities of KGM-BBA, KGM-BCA, KGM-BHA, and KGM-BWA membrane solutions against Escherichia coli and Staphylococcus aureus.

[0029] Figure 5 Fourier transform infrared (FT-IR) and differential scanning calorimetry (DSC) spectra of the KGM-anthocyanin membrane and membrane solution provided in this embodiment of the invention, wherein: Figure 5 A and 5B are FTIR and DSC images of KGM-BBA, KGM-BCA, KGM-BHA, KGM-BWA membranes and KGM emulsion alone, respectively. Figure 5 C and 5D are FTIR and DSC diagrams of KGM-BBA, KGM-BCA, KGM-BHA, and KGM-BWA membrane solutions after storage at 4°C for 48 hours, respectively. Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this invention are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or end that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or ends.

[0032] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0033] Currently, KGM is one of the most widely used food packaging matrix materials. It is a natural, high-molecular-weight soluble dietary fiber. Its molecular chain is composed of D-glucose and D-mannose residues linked by β-1,4-glycosidic bonds in a molar ratio of 1:1.5 or 1:1.6. The mannose backbone contains a branched structure with β-1,3-glycosidic bonds at the C3 position, and there are 3 branches on 32 sugar residues. It has high viscosity, biodegradability, and biocompatibility, thus possessing great potential and application value in extending the shelf life of food. However, food packaging materials developed using KGM alone are easily soluble in water and have disadvantages such as poor mechanical properties, barrier properties, antibacterial and antioxidant activities. In particular, they cannot reflect the freshness index of food in a timely manner. To solve this problem, Chang et al. double-modified KGM and introduced nano-SiO2 to form a composite membrane structure, which improved the mechanical properties, hydrophobicity, and antioxidant properties of the membrane. Besides modifying KGM, some researchers have reported that the performance of KGM membranes can be improved by adding natural / synthetic polymers. For example, the addition of cellulose nanocrystals (CNC) can improve the physical, mechanical, and gas barrier properties of the membrane. Adding honeysuckle leaf extract (HLE) to a composite edible film of KGM and gelatin can enhance the mechanical properties and antibacterial ability of the film. Furthermore, antibacterial edible coatings have already been used in the food industry, but single edible coatings can no longer meet the demands for food preservation. Therefore, finding multifunctional edible coatings with antibacterial, antioxidant, and pH-indicating properties is an urgent problem to be solved.

[0034] Anthocyanins, as a class of natural products with multiple functional activities, not only possess antioxidant and antibacterial capabilities but also exhibit corresponding colors in response to pH values. Specifically, the system appears red when pH < 2; transitions from red to colorless at pH 3-6; gradually turns purple as pH rises to 7-8; turns blue at 8-10; and becomes yellow when pH > 11. Based on these color characteristics, ZANG et al. developed a pH indicator membrane based on a gelatin / polyvinyl alcohol matrix and mulberry pomace anthocyanin extract to monitor the spoilage of dace (Cirrhina molitorella) during storage. The results showed that during the 24-hour observation period, the pH of the dace increased due to the production of total volatile basic nitrogen (TVB-N) during spoilage, and the color of the composite membrane changed accordingly, showing a good dependence on the change in TVB-N. Zhai et al. also observed similar results, finding that when hibiscus anthocyanins were used as indicators to indicate changes in the freshness of fish, the color of the indicator film gradually changed from red to light blue as the degree of spoilage of the fish deepened. It is important to note that, in addition to being a pH indicator, anthocyanins can also be used as natural antioxidants and antibacterial agents to prevent the oxidation of food components and the growth of microorganisms, thereby extending the shelf life of food. For example, adding *Phyllostachys edulis* anthocyanins to a chitosan / methylcellulose composite solution to create a food packaging film can inhibit the growth of *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Escherichia coli* in food. Furthermore, SUN et al. found that the addition of mulberry anthocyanins can improve the antioxidant activity of KGM / chitosan composite films and exhibit good inhibitory activity against *Escherichia coli* and *Staphylococcus aureus*, thus extending the shelf life of food. Therefore, anthocyanins can not only improve the mechanical properties of natural polymer membranes and endow them with antioxidant and antibacterial properties, but also change their color during food storage, thus visually reflecting the spoilage of food. They have the potential to extend the shelf life of goods and monitor the freshness index of food.

[0035] However, anthocyanins from different plant sources exhibit significant differences in composition, resulting in varying pH sensitivities, antioxidant capacities, and antibacterial properties. This presents considerable challenges for developing anthocyanin-containing food packaging films and their applications. Therefore, this study used KGM as a matrix to prepare four types of KGM-anthocyanin films by adding blueberry anthocyanin (BBA), black goji berry anthocyanin (BWA), honeysuckle berry anthocyanin (BHA), and blackcurrant anthocyanin (BCA). The color characteristics, antioxidant and antibacterial properties, and mechanical properties of the prepared films were investigated. Fourier transform infrared spectroscopy and DSC thermal stability analysis were conducted to explore the binding mechanism of KGM with each anthocyanin, aiming to develop KGM-anthocyanin films suitable for food preservation.

[0036] This invention aims to provide a KGM-anthocyanin membrane, its preparation method, and its application, in order to solve the problems of weak pH sensitivity, weak antioxidant capacity, unsatisfactory antibacterial effect of KGM food packaging materials, weak resistance to external impact, and inability to achieve real-time monitoring of food freshness in existing technologies.

[0037] like Figure 1 As shown in the figure, a specific embodiment of the present invention provides a method for preparing a KGM-anthocyanin membrane, comprising the following steps:

[0038] S1: After harvesting ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants, dry them in a vacuum freeze dryer to obtain dried fruits. Crush the dried fruits to obtain berry powder. Weigh the berry powder and place it in an Erlenmeyer flask. Add methanol and formic acid solution, seal the flask with plastic wrap, and repeat the extraction multiple times under room temperature and light-proof conditions. Combine the obtained extracts and centrifuge to remove fruit residue, protein, and polysaccharide impurities. Collect the supernatant, which is the anthocyanin extract of each berry. Store it in a -18℃ refrigerator in the dark for later use.

[0039] In this embodiment of the invention, ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants are harvested and dried in a vacuum freeze dryer to obtain dried fruit. The dried fruit is then pulverized and the residue is collected through an 80-mesh sieve to obtain berry powder. The berry powder is stored in a -18°C refrigerator. The concentration-to-volume ratio of methanol to formic acid solution is 98:2. The berry powder is weighed and placed in an Erlenmeyer flask. Methanol to formic acid solution is added at a volume ratio of 98:2. The flask is sealed with plastic wrap and extracted at room temperature and in the dark for 8-10 hours. The extraction is repeated 3 times. The resulting extracts are then combined. The combined extracts are centrifuged at 3000g for 10 minutes at 4°C.

[0040] S2: Using XDA-7HP as the medium, gradient elution was performed sequentially with distilled water and 40% ethanol aqueous solution as the mobile phase to separate and purify the anthocyanin extracts of each berry. The ethanol eluent was collected, concentrated under reduced pressure, frozen under vacuum, and dried to obtain lyophilized powders of each berry anthocyanin.

[0041] S3: Weigh KGM powder and dissolve it in distilled water with added glycerin. Stir continuously in a water bath to prepare KGM emulsion.

[0042] In this embodiment of the invention, KGM powder was weighed and dissolved in distilled water with added glycerin, the glycerin content being 1%, and KGM emulsion was prepared by continuous stirring in a water bath at 60°C for 2 hours.

[0043] S4: The prepared KGM emulsion was stirred for 0.5 h in a water bath at 60 °C to serve as the membrane matrix; the lyophilized anthocyanin powders from each berry in step S2 were weighed and prepared into a 6 mg / mL anthocyanin solution, which was then added to the membrane matrix at a ratio of 1 ml: 100 g and stirred thoroughly to obtain the KGM-anthocyanin mixed emulsion.

[0044] In this embodiment of the invention, the mass ratio of lyophilized berry anthocyanin powder to membrane matrix is ​​6:100.

[0045] S5: Invert the KGM-anthocyanin mixed emulsion into a petri dish to spread it evenly, and dry it. Keep the total KGM content in the obtained sample at 1.0g to obtain the KGM-anthocyanin membrane.

[0046] In this embodiment of the invention, the KGM-anthocyanin mixed emulsion was inverted in a petri dish and dried at 35°C for 48 hours. The total KGM content in the resulting sample was kept at 1.0g to obtain a KGM-anthocyanin membrane. The KGM-anthocyanin membrane was then stored at 25°C and 4.0% relative humidity for later use.

[0047] The present invention provides a KGM-anthocyanin membrane prepared according to the aforementioned method for preparing a KGM-anthocyanin membrane.

[0048] The present invention provides an active packaging film for extending the shelf life of food and its application as a smart packaging film for real-time monitoring of food freshness, based on the KGM-anthocyanin film described above.

[0049] Example 1

[0050] 1. Extraction and purification of four types of berry anthocyanins

[0051] Ripe blueberries, blackcurrants, honeysuckle berries, and black goji berries were all hand-picked and then placed in a vacuum freeze dryer (LGJ-18C type, Beijing Sihuan Technology Co., Ltd.). After pulverization, the residue was collected by passing it through an 80-mesh sieve and stored at -18℃. Anthocyanin extraction was performed using the method of Zhang et al., with appropriate adjustments. 1.5g of dried black goji berry powder was accurately weighed and placed in a 50mL Erlenmeyer flask. 15mL of methanol-formic acid solution (98:2, v:v) was added, and the flask was sealed with plastic wrap. Extraction was carried out at room temperature and in the dark for 8 hours. The extraction was repeated three times under the same conditions. The extracts were combined and centrifuged at 3000g for 10 minutes at 4℃ to remove fruit residue, protein, polysaccharides, and other impurities. The supernatant was collected and stored at -18℃ in the dark for later use.

[0052] Sample purification was performed using column chromatography with XDA-7HP as the medium. Gradient elution was carried out sequentially with distilled water and 10% ethanol aqueous solution, 40% ethanol aqueous solution, and 70% ethanol aqueous solution as mobile phases to separate and purify the crude extract of berry anthocyanins. Eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and frozen under vacuum to obtain lyophilized powder of berry anthocyanins.

[0053] 1. Composition of four types of berry anthocyanins

[0054] HPLC / MS was used to obtain the chromatographic and mass spectrometric information of four berry anthocyanins (BBA, BCA, BHA, and BWA), and their anthocyanin components were identified and analyzed by comparing them with previously published related results. The results are shown in [Figure number missing]. Figure 2 Table 1 shows the determination results of the four anthocyanin components during the implementation of this invention:

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Blueberries contain 13 anthocyanins, blackcurrants contain 4, honeysuckle berries contain 5, and black goji berries contain 13. The composition of these anthocyanins varies considerably. For example, the most abundant anthocyanin in BBA is peonidin-3-arabinoside (25.02%), followed by delphinidin-3-glucoside (19.72%), then cyanidin-3-arabinoside, delphinidin-3-arabinoside, and cyanidin-3-galactoside (10.50% and 10.13%, respectively). The four anthocyanins detected in BCA are delphinidin-3-glucoside, delphinidin-3-rutin glycoside, cyanidin-3-glucoside, and cyanidin-3-rutin glycoside, each accounting for approximately 25%. Cyanidin-3-glucoside is the most abundant in BHA, reaching approximately 85.64%. BWA had the highest content of petunia-3-rutinoside (trans-coumaroyl)-5-glucoside, accounting for 64.08% of the total anthocyanins. Further analysis revealed that the anthocyanins in blackcurrants, blueberries, and honeysuckle berries were almost entirely monosaccharide anthocyanins, with only BHA containing one type of 3,5-disaccharide anthocyanin. In contrast, all identified anthocyanins in black goji berries were 3,5-disaccharide anthocyanins, including eight acylated anthocyanins. This highlights the unique characteristics of anthocyanins in black goji berries.

[0061] 3. Color characteristics and antioxidant and antibacterial effects of four types of berry anthocyanins

[0062] (1) Color characteristics: The color changes of BBA, BCA, BHA, and BWA under different pH conditions are as follows Figure 3 As shown in Figure A, the figure reveals that the four anthocyanins exhibit significant and distinct phased color differences under different pH conditions. They generally show a common characteristic of "stable red hue in acidic conditions, fading in neutral conditions, and transitioning to brown / blue / yellow hues in alkaline conditions," but the specific color evolution pathways of each anthocyanin differ significantly. Specifically, BBA, under acidic conditions at pH 2-6, gradually lightens from a bright magenta color to light pink and pale pink as pH increases, eventually becoming almost colorless at pH 6. Upon increasing pH to 7, the system begins to show obvious browning, with the solution exhibiting a grayish-brown hue. The degree of browning deepens with increasing pH, reaching its peak at pH 9, where the solution is dark grayish-brown, while a faint blue undertone can still be observed within the pH 7-9 range. When the pH further increases to 10-13, the predominantly brown undertone gradually fades, the original blue hue completely disappears, and the solution eventually transforms into a yellow hue that gradually deepens. The color changes of BCA and BHA under acidic conditions are quite consistent, both exhibiting a fading process from deep to light red as pH increases: BCA is a bright orange-red at pH 2-3, while BHA is a deeper orange-red. Both gradually transition to light pink and pale pink as pH increases (4-6), becoming nearly colorless at pH 6. After pH 7, both begin to brown, with the solution turning light grayish-brown. BCA browns faster, reaching its peak at pH 8, resulting in a dark grayish-brown solution, while BHA reaches its highest browning degree at pH 9. Both also exhibit a slight bluish undertone during the browning stage at pH 7-9. Beyond pH 9, their color evolution differs: BHA solutions gradually turn light yellow, deepening with increasing pH, reaching a clear deep yellow at pH 13; while BCA's color change in the pH 9-12 range is more complex, alternating between brown undertones and lighter shades with no clear pattern, until it reaches a clear yellow consistent with BHA at pH 13. Unlike the previous three, BWA exhibits clear and stable phased color changes: at pH 2-6, the solution gradually fades from a bright magenta to a light pink and pale pink as the pH increases, becoming nearly colorless at pH 6; in the pH 7-9 range, the solution displays a stable blue hue, gradually transitioning from a light purplish-blue to a deep sapphire blue as the pH increases, with a distinct blue characteristic and no obvious browning interference; at pH 10-13, the blue hue of the solution completely fades, turning into a clear yellow, and the yellow gradually deepens as the pH increases, with no overall browning phenomenon, and the color evolution path is clear and more reversible.

[0063] Correspondingly, the absorbance values ​​of the four anthocyanins under different pH conditions are shown in the figure. Figure 3B. As can be seen from the figure, the spectral changes of the four anthocyanins all exhibit typical pH response characteristics: under acidic conditions of pH 2-5, they all exist in the form of stable flavonoid cations, with strong characteristic absorption peaks in the visible light region of 490-540 nm, and the absorbance is higher and the peak shape is more stable as the pH is lower; as the pH increases to neutral to weakly alkaline (pH=6-8), the intensity of the characteristic peaks decreases significantly, gradually transforming into the colorless methanol pseudobase / chalcone form; under strong alkaline conditions (pH=9-13), a weak absorption band appears near 600-650 nm, corresponding to the formation of quinone bases, but the overall absorbance is much lower than under acidic conditions, and the curve tends to flatten with further increase in pH, reflecting the intensified degradation under alkaline conditions. Nevertheless, the spectral changes of each anthocyanin showed significant differences: BCA had the sharpest acidic characteristic peak, was most sensitive to pH changes, and exhibited the fastest peak intensity decay under neutral conditions; BHA had the highest absorbance under acidic conditions and showed slightly better stability in neutral environments; BBA formed a distinct quinone base absorption peak near 600 nm under weakly alkaline conditions; and BWA, in particular, showed the most significant spectral redshift effect, with the absorption peak shifting towards longer wavelengths as pH increased, and it still retained strong colorimetric ability under alkaline conditions, making it the most stable of the four under alkaline conditions. These observations are consistent with... Figure 3 The color observation results in A were consistent. Therefore, all four anthocyanins follow the general rule of "stable color development in acidic environments, fading in neutral environments, and color change + degradation in alkaline environments." However, due to differences in monomer composition, BWA shows the best adaptability to alkaline environments, which can provide direct evidence for its application in food systems with different pH values.

[0064] (2) Antioxidant activity: The antioxidant activities of the four anthocyanins are shown in [reference needed]. Figure 3C. In the figure, AP and TP represent apple polyphenols and tea polyphenols, respectively. Both have good antioxidant activity and were therefore selected as positive controls in the experiment. The figure shows significant differences among the DPPH, ABTS, and FRAP systems, with the overall trend showing the highest activity in the ABTS system, followed by DPPH, and relatively lower activity in FRAP. In the DPPH free radical scavenging system, BHA had the highest activity, significantly higher than the other anthocyanins and AP and TP, followed by BBA. BCA and AP had comparable activities, both significantly higher than BWA and TP, with TP having the lowest activity. In the ABTS free radical scavenging system, all samples showed strong scavenging ability, with BWA showing the highest activity, followed by BCA, BHA, BBA, TP, and AP in that order. The activities of each anthocyanin were close to or higher than the positive control. In the FRAP reducing power system, AP had significantly higher reducing power than all anthocyanins. BCA had the highest activity among the four anthocyanins, followed by BBA and BHA, with BWA having the lowest activity. TP's reducing power was only slightly higher than BWA. In summary, the antioxidant activities of the four anthocyanins varied across different evaluation systems: BWA showed a significant advantage in the ABTS system, BHA performed best in the DPPH system, and BCA exhibited the highest activity in the FRAP system. Overall, the ABTS scavenging activity of the anthocyanin samples was generally superior to that of the positive control, while the reducing power of FRAP was weaker than that of AP. This indicates that their antioxidant activity is closely related to the evaluation system, and the activity of different anthocyanins under different mechanisms of action is specific.

[0065] (3) Antibacterial properties: All four anthocyanins showed certain antibacterial activity against Escherichia coli and Staphylococcus aureus, and there were significant differences among different samples. Figure 3 D). Overall, BBA exhibited the strongest antibacterial activity, significantly higher than BCA, BHA, and BWA; while BCA, BHA, and BWA showed no significant difference in antibacterial effects against the two pathogenic bacteria, with similar activity levels. Furthermore, all anthocyanins and positive controls showed a generally consistent antibacterial trend against Escherichia coli and Staphylococcus aureus, without demonstrating significant species specificity.

[0066] 4. Preparation of KGM-anthocyanin emulsion, its color characteristics, and its antioxidant and antibacterial effects.

[0067] (1) Preparation of KGM-anthocyanin emulsion: Weigh 70g of KGM powder and dissolve it in 700mL of distilled water with added glycerol (1%). Stir continuously in a water bath at 60℃ for 2h for later use. Preparation of mixed emulsion: Stir the prepared KGM emulsion in a water bath at 60℃ for 0.5h to obtain the membrane matrix; Weigh 0.6g of each anthocyanin powder and dissolve it in a 100mL volumetric flask. Take 1mL of anthocyanin solution with a concentration of 6mg / mL and add it to 100g of membrane matrix for later use to obtain KGM-anthocyanin emulsion emulsion.

[0068] (2) Color characteristics of KGM-anthocyanin emulsions: The color characteristics of the four KGM-anthocyanin emulsions are shown in the figure. Figure 4 A. Combining the color characteristics of individual anthocyanins ( Figure 4 A) It was found that compared with pure anthocyanins, the anthocyanin emulsions prepared by mixing with KGM showed significantly improved color stability under different pH conditions, while retaining the pH response characteristics of anthocyanins themselves. Specifically, the fading process of the KG+BBA emulsion was slowed at pH 2-6, retaining a slight pink color even at pH 6. Browning was significantly inhibited in the pH range of 7-9, without the dark grayish-brown color found in pure BBA, and uniformly transitioned to a clear yellow at pH 10-13. The KG+BCA emulsion eliminated the color confusion of pure BCA in the pH range of 9-12, maintaining a stable orange-red hue under acidic conditions. The browning rate was significantly reduced in the pH range of 7-9, and the color transitioned uniformly to yellow with increasing pH after pH > 9. The KG+BHA emulsion showed the most significant improvement in color stability, retaining the orange-red hue for longer under acidic conditions, significantly slowing down the browning rate in the pH range of 7-9, and exhibiting a smoother overall color transition. KG+BWA emulsion retains the original phased color changes. The magenta tone is brighter and more stable at pH 2-6, the blue tone transition is softer at pH 7-9 with no browning interference, and the yellow color changes are uniform and the deepening trend is stable at pH 10-13.

[0069] Furthermore, the absorbance characteristics of the four KGM-anthocyanin emulsions are shown in [the table below]. Figure 4B. Compared with pure anthocyanins, the UV-Vis absorption spectral characteristics of KGM-anthocyanin emulsion under different pH conditions changed significantly, and the overall stability was significantly improved. For KG+BBA emulsion, under acidic conditions (pH 2-6), the original characteristic absorption peak (approximately 510-520 nm) was stronger and sharper, and the decay rate with increasing pH was significantly slower, maintaining strong characteristic absorption even at pH=6; under neutral and weakly alkaline conditions (pH 7-9), the spectral baseline rise and broadening caused by browning and degradation in the original pure BBA were significantly suppressed, and the trend of characteristic peak disappearance was delayed; under strongly alkaline conditions (pH 10-13), the spectrum was flatter, and no obvious impurity peaks were observed, indicating that KGM effectively inhibited its degradation and browning. In KG+BCA emulsions, the stability of the characteristic absorption peak (approximately 520 nm) under acidic conditions (pH 2-6) is significantly improved, with a more concentrated peak shape and a significantly slower rate of absorbance decrease with increasing pH. Within the pH 7-9 range, the spectral disorder caused by browning in the original pure BCA is eliminated, and the disappearance of the characteristic peak is smoother. After pH > 9, the spectral changes are more uniform, and the absorbance baseline under strongly alkaline conditions is more stable without obvious irregular fluctuations. The spectral stability of KG+BHA emulsions is most significantly improved. Under acidic conditions (pH 2-6), the characteristic absorption peak (approximately 520-530 nm) has high intensity and reduced peak broadening. As the pH increases to the neutral and weakly alkaline range, the absorbance decay rate is significantly slower than that of pure BHA. A certain intensity of characteristic absorption can still be retained at pH 7-9, indicating that KGM delays its conversion to a colorless form. The spectrum under strongly alkaline conditions is also smoother, and the interference peaks of degradation products are significantly reduced. The KG+BWA emulsion, while retaining the original pH response characteristics, enhances spectral stability: the characteristic absorption peaks (approximately 520-530 nm) under acidic conditions (pH 2-6) are more stable and sharper; the blue characteristic absorption (approximately 580-620 nm) in the pH 7-9 range is more pronounced and lasts longer, with no obvious interference from other peaks, indicating that KGM stabilizes its quinone base structure; the spectrum under strongly alkaline conditions is also smoother, with no obvious absorption peaks from degradation products. Therefore, the KGM emulsion system effectively delays the fading, browning, and degradation processes of anthocyanins under neutral and alkaline environments, resulting in smoother color changes and more stable spectral responses across a wide pH range, significantly improving its adaptability in complex systems.

[0070] (3) Antioxidant activity of KGM-anthocyanin emulsions: The antioxidant activity of the four KGM-anthocyanin emulsions is shown in the figure. Figure 4C. Compared with pure anthocyanins, the four anthocyanin emulsions maintained good antioxidant activity, but the order and performance of activity varied in different systems. Overall, the ABTS system showed the highest activity, followed by the DPPH system, while FRAP had relatively low reducing power. Specifically, in the DPPH free radical scavenging system, the KGM+BBA emulsion showed significantly the highest activity, followed by KGM+BHA, with KGM+BWA and KGM+BCA showing decreasing activity in that order. Compared with pure anthocyanins, KGM enhanced the DPPH scavenging activity of BBA while relatively decreasing the activity of BHA. In the ABTS free radical scavenging system, the KGM+BCA and KGM+BWA emulsions showed significantly the highest activity, with no significant difference between the two, followed by KGM+BHA, and then KGM+BBA, with overall activity levels remaining high. Compared with pure anthocyanins, BCA retained its ABTS activity advantage, while BWA's activity remained at a high level. In the FRAP reducing power system, the reducing power of KGM+BCA emulsion was significantly higher than the other three, followed by KGM+BWA, while KGM+BBA and KGM+BHA showed the lowest activity with no significant difference. Compared with pure anthocyanins, KGM did not significantly reduce the reducing power of anthocyanins, and the advantages of BCA were still evident. Overall, KGM did not destroy the antioxidant activity of anthocyanins and even had a stabilizing or enhancing effect on activity in some systems, which may be related to the enhanced structural stability and reduced degradation of anthocyanins after encapsulation. At the same time, the differences in activity of different anthocyanins in various antioxidant systems still existed after mixing. KGM+BCA showed strong activity in both ABTS and FRAP systems, while KGM+BBA showed a significant advantage in the DPPH system, indicating that KGM, while preserving the antioxidant properties of anthocyanins, also had a certain regulatory effect on their activity.

[0071] (4) Antibacterial properties of KGM-anthocyanin emulsions: The antibacterial properties of the four types of KGM-anthocyanin emulsions are shown in [the table below]. Figure 4 D.

[0072] Compared to pure anthocyanins, the four KGM-anthocyanin emulsions still exhibited antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*, with an overall antibacterial trend consistent with pure anthocyanins, although the activity levels differed somewhat: KGM+BBA showed significantly the highest antibacterial activity against both pathogens, while KGM+BCA, KGM+BHA, and KGM+BWA showed no significant difference in antibacterial activity. Compared to pure anthocyanins, the overall antibacterial activity of the four KGM-anthocyanin emulsions decreased slightly, but BBA's antibacterial advantage remained prominent, not changing its ranking among the four anthocyanins, indicating that the KGM-anthocyanin emulsions did not destroy the antibacterial activity of anthocyanins, but only weakened its intensity. Furthermore, all emulsions showed slightly stronger antibacterial effects against *Staphylococcus aureus* than against *Escherichia coli*, and the antibacterial trends against both bacteria were consistent, showing no obvious species specificity. Therefore, KGM-anthocyanin emulsion has little impact on the antibacterial function of anthocyanins, and this series of emulsions still has good antibacterial application potential.

[0073] 5. Binding characteristics of KGM and anthocyanins

[0074] (1) FTIR analysis: The FTIR spectra of the four KGM-anthocyanin emulsions all retained the main characteristic absorption bands of pure KGM, accompanied by significant shifts and intensity changes in the characteristic peaks. Figure 5 A). Coverage The broad OH stretching vibration band further broadened and redshifted to lower wavenumbers, confirming the formation of extensive hydrogen bonds between the anthocyanin phenolic hydroxyl groups and the hydroxyl groups on the KGM backbone. Meanwhile, approximately The weakening and slight shift of the C=O stretching vibration peak at the KGM site indicates that the carbonyl group of anthocyanins also participates in hydrogen bond crosslinking with KGM. Notably, the four anthocyanins exhibit significantly different peak shift amplitudes, revealing differentiated binding affinity and interaction strength between anthocyanins with different structures and the KGM chain. These spectral changes demonstrate that KGM and anthocyanins primarily form non-covalent complexes through hydrogen bonds, and that this molecular assembly does not alter the basic chemical framework of any component, providing molecular-level evidence for the excellent antioxidant and antibacterial activities of the composite emulsion.

[0075] (2) DSC analysis: The complexes formed by KGM and different anthocyanins significantly altered the thermal properties of the emulsions, which can be seen from the DSC thermal analysis charts of the four KGM-anthocyanin emulsions ( Figure 5 B) was confirmed. Specifically, pure KGM exhibits a distinct endothermic peak upon heating, corresponding to a phase transition process involving chain disentanglement and hydration network dissociation. After the addition of anthocyanins, the endothermic peak temperature of the KGM+BWA emulsion increased, while that of the KGM+BBA emulsion decreased; the endothermic peak temperatures of the other samples were comparable to those of the pure KGM emulsion. Figure 5B), whose enthalpy change trend is consistent with the change in endothermic peak temperature. These results confirm the reorganization of intermolecular interactions, indicating that anthocyanins reshape the intrinsic interchain interaction network of KGM: the intermolecular hydrogen bonds formed between anthocyanins and the hydroxyl / sulfate groups on the KGM backbone weaken the intrinsic interchain forces of KGM, thereby increasing the phase transition threshold temperature. The differences in the degree of thermoregulation caused by different anthocyanins stem from their different binding affinities to KGM. Among the anthocyanins, only BWA can improve the thermal stability of the formed composite emulsion and make the heat flow curve show a more stable baseline ( Figure 5 (B) indicates that the encapsulated BWA restricts the thermal migration of the KGM chain, thereby enhancing the structural thermal stability of the entire system. This is attributed to the fact that BWA belongs to acylated-3,5-disaccharide-based anthocyanins, which inherently possess excellent stability.

[0076] 6. Stability of KGM-Anthocyanin Emulsion

[0077] By combining FTIR and DSC techniques, the structural and thermal changes of the composite emulsion after 48 hours of refrigerated storage at 4°C were systematically revealed. Figure 5 C and 5D); the differences between the stored emulsion and the fresh sample further elucidate the effect of low-temperature storage on the KGM-anthocyanin supramolecular complex. From the perspective of FTIR analysis ( Figure 5 C), the overall spectral characteristics of the refrigerated emulsion still match the characteristic absorption bands of pure KGM, but The broad OH stretching vibration peak shows a significant blue shift (dispersion shift) towards higher wavenumbers, while... The intensity of the C=O stretching vibration peak at the chromatogram was significantly reduced. These spectral changes indicate that low-temperature treatment partially disrupted the hydrogen bond cross-linking network between KGM and anthocyanins. The weakened binding force led to the partial dissociation of anthocyanin molecules from the KGM matrix, which provides a reasonable explanation for the slight decrease in antioxidant and antibacterial activity of the emulsion compared to free anthocyanins. The DSC thermogram of the refrigerated emulsion showed significant thermodynamic changes (…). Figure 5D), which is mainly reflected in the behavior of glass transition temperature (Tg) and dehydration enthalpy (ΔH); while the distinct high-temperature degradation peak observed in the fresh emulsion completely disappears within the test range. All stored emulsions exhibit a distinct endothermic peak in the range of 91-94°C, which is attributed to the evaporation of tightly bound water molecules in the KGM network; there are significant differences in the dehydration enthalpy values among different formulations. The KGM+BWA system retains the highest bound water content, further confirming that BWA forms abundant hydrogen bonds with KGM to immobilize water molecules. More importantly, cold storage treatment induces a distinct glass transition step in all emulsion samples, which does not occur in fresh emulsions. The glass transition temperature (Tg) values are in the following order: KGM+BBA (100.6°C) < KGM+BCA (113.3°C) < pure KGM (115.0°C) < KGM+BHA (116.6°C) < KGM+BWA (132.3°C). Low-temperature storage promotes the rearrangement of KGM segments and KGM-anthocyanin hydrogen bonds: the strong cross-linking effect of BWA significantly increases the energy barrier for chain motion, thus achieving the highest Tg value. The above results collectively indicate that low-temperature storage remodels the supramolecular structure of the composite emulsion by disrupting part of the hydrogen bonds and triggering the glass transition. Although the hydrogen bond complexes partially dissociate during cold storage, the slow release of anthocyanins from the KGM matrix helps maintain the long-term antioxidant and antibacterial properties of the packaging film during the cold preservation of food.

[0078] 7. Mechanical properties of KGM-anthocyanin films

[0079] The analysis results of the viscosity of the KGM-anthocyanin emulsion and the mechanical properties of the film prepared therefrom are shown in Table 2. Table 2 presents the analysis results of the viscosity of the KGM-anthocyanin emulsion and the mechanical properties of the film prepared therefrom during the implementation of the present invention:

[0080]

[0081] As shown in Table 2, there was no significant difference in viscosity between the pure KGM emulsion and the four KGM-anthocyanin composite emulsions, indicating that the addition of berry anthocyanins did not alter the rheological behavior of the KGM emulsion. Therefore, these composite coatings remain suitable for conventional processing techniques such as coating and infiltration. Further mechanical evaluation of the films prepared for simulated real-world applications revealed significant performance differences among the materials: compared to pure KGM films, the elongation at break of KGM+BBA and KGM+BCA films was significantly improved; KGM+BWA and KGM+BHA films also showed slight improvements (Table 2). Furthermore, the tensile strength of all composite films was comparable to that of pure KGM films, with enhanced hardness, brittleness, yield strength, and elasticity (Table 2). Notably, the KGM-BBA film showed the most significant improvement in hardness and brittleness. Meanwhile, the moisture content (MC) of all four KGM-anthocyanin films was lower than that of pure KGM film (22.02%), ranging from 19.51% to 22.43%. This indicates that the addition of berry anthocyanins reduced the water retention capacity of the KGM matrix. This phenomenon can be attributed to the intermolecular hydrogen bond cross-linking structure formed between KGM and anthocyanin molecules, which inhibited water-induced matrix swelling. Therefore, the swelling degree (water absorption rate) of all four films was significantly lower than that of pure KGM film; among them, KGM-BHA film had the lowest swelling value, only 2.28% (Table 2). The lower swelling degree is beneficial to the application of KGM-anthocyanin films because pure KGM film undergoes severe and uncontrollable swelling when in contact with water, which is particularly unfavorable in humid environments. In addition, the water vapor permeability (WVP) of pure KGM film was significantly lower. The WVP values ​​of the four anthocyanin-containing films were similar, all ranging from 15.76 to... The results (between the two values) indicate that the addition of berry anthocyanins weakens the water vapor barrier properties of KGM-based films, with the KGM-BHA film exhibiting the worst moisture barrier properties and the highest WVP value (Table 2). The increase in WVP value is mainly due to the additional hydrophilic hydroxyl groups of anthocyanins and the looser polysaccharide network structure; despite this, these films are still suitable for packaging active fresh foods (such as strawberries and cherries) that require moderate moisture exchange.

[0082] The beneficial effects of this invention are as follows: By adding different anthocyanins to a KGM matrix, this invention successfully prepared an active intelligent packaging film. Due to the hydrogen bonds formed between the anthocyanins and KGM, the film's pH sensitivity, antioxidant activity, antibacterial ability, and mechanical strength are improved. Therefore, the KGM-anthocyanin film can not only serve as an active packaging film to extend the shelf life of food, but also as an intelligent packaging film to monitor the freshness of food in real time.

[0083] The above descriptions are merely embodiments of the present invention. Commonly known technical solutions or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a KGM-anthocyanin membrane, characterized in that, The method includes: S1: After harvesting ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants, dry them in a vacuum freeze dryer to obtain dried fruits. Crush the dried fruits to obtain berry powder. Weigh the berry powder, place it in an Erlenmeyer flask, add methanol formic acid solution, seal the flask with plastic wrap, and repeatedly extract it at room temperature and in the dark. Combine the obtained extracts and centrifuge them to remove fruit residue, protein, and polysaccharide impurities. Collect the supernatant, which is the anthocyanin extract of each berry. Store it in a -18℃ refrigerator in the dark for later use. S2: Using XDA-7HP as the medium, gradient elution was performed sequentially with distilled water and 40% ethanol aqueous solution as the mobile phase to separate and purify the anthocyanin extracts of each berry. The ethanol eluent was collected, concentrated under reduced pressure, frozen under vacuum, and dried to obtain the lyophilized powder of each berry anthocyanin. S3: Weigh KGM powder and dissolve it in distilled water with added glycerin, and continuously stir in a water bath to prepare KGM emulsion; S4: The prepared KGM emulsion is stirred for 0.5 h in a water bath at 60 °C to serve as a membrane matrix; the lyophilized anthocyanin powders of each berry from step S2 are weighed and prepared into a 6 mg / mL anthocyanin solution, which is then added to the membrane matrix at a ratio of 1 ml: 100 g and stirred thoroughly to obtain a KGM-anthocyanin mixed emulsion. S5: The KGM-anthocyanin mixed emulsion is inverted in a petri dish to spread it evenly and then dried. The total KGM content in the resulting sample is kept at 1.0g to obtain a KGM-anthocyanin membrane.

2. The method for preparing a KGM-anthocyanin membrane according to claim 1, characterized in that, The step of harvesting ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants, drying them in a vacuum freeze dryer to obtain dried fruit, and then pulverizing the dried fruit to obtain berry powder includes: Ripe blueberries, black goji berries, honeysuckle berries, and blackcurrants are harvested and dried in a vacuum freeze dryer to obtain dried fruit. The dried fruit is then pulverized and passed through an 80-mesh sieve to collect the residue, resulting in berry powder. The berry powder is then stored in a -18°C refrigerator.

3. The method for preparing a KGM-anthocyanin membrane according to claim 1, characterized in that: The concentration-to-volume ratio of the methanol-formic acid solution is 98:

2.

4. The method for preparing a KGM-anthocyanin membrane according to claim 1, characterized in that, The steps of weighing the dried berry powder, placing it in an Erlenmeyer flask, adding methanol-formic acid solution, sealing the flask opening with plastic wrap, and repeatedly extracting it at room temperature and in the dark include: Weigh the dried berry powder and place it in an Erlenmeyer flask. Add a methanol-formic acid solution with a volume ratio of 98:

2. Seal the flask with plastic wrap and extract at room temperature and in the dark for 8-10 hours. Repeat the extraction 3 times and combine the resulting extracts.

5. The method for preparing a KGM-anthocyanin membrane according to claim 4, characterized in that, The step of combining the obtained extracts and then centrifuging them includes: combining the obtained extracts and centrifuging them at 3000g for 10min at 4℃.

6. The method for preparing a KGM-anthocyanin membrane according to claim 1, characterized in that, The step of dissolving KGM powder in distilled water with added glycerin and continuously stirring in a water bath to prepare a KGM emulsion includes: KGM powder was weighed and dissolved in distilled water with added glycerol (1% glycerol content), and the mixture was stirred continuously in a water bath at 60°C for 2 hours to prepare a KGM emulsion.

7. The method for preparing a KGM-anthocyanin membrane according to claim 1, characterized in that: The mass ratio of the lyophilized berry anthocyanin powder to the membrane matrix is ​​6:

100.

8. The method for preparing a KGM-anthocyanin membrane according to claim 1, characterized in that, The step of inverting the KGM-anthocyanin mixed emulsion in a petri dish to spread it evenly and drying it, maintaining the total KGM content in the resulting sample at 1.0g, to obtain the KGM-anthocyanin membrane, includes: The KGM-anthocyanin mixed emulsion was inverted in a petri dish and dried at 35°C for 48 hours. The total KGM content in the resulting sample was kept at 1.0 g to obtain a KGM-anthocyanin membrane. The KGM-anthocyanin membrane was stored at 25°C and 4.0% relative humidity for later use.

9. A KGM-anthocyanin membrane prepared according to the method for preparing a KGM-anthocyanin membrane according to claims 1-8.

10. The application of the KGM-anthocyanin membrane according to claim 9 as an active packaging film for extending the shelf life of food and as a smart packaging film for real-time monitoring of food freshness.

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