Antibacterial and antioxidant intelligent indicating double-layer preservative film and preparation method and application thereof

Abstract

The application discloses an antibacterial and antioxidative intelligent indication double-layer preservative film and a preparation method and application thereof. The beet red is modified to generate a beet red-acylated product, so that the beet red-acylated product has better thermal stability and stronger DPPH free radical scavenging rate. The beet red-acylated product is prepared into a first layer of preservative film together with a film-forming material. The plant essential oil is prepared into a Pickering emulsion, and then prepared into a second layer of preservative film together with the film-forming material, so that the antibacterial, antioxidative and intelligent indication double-layer preservative film is prepared.

Description

Technical Field

[0001] This invention relates to the fields of chemical structure modification and food packaging technology, and in particular to an antibacterial and antioxidant intelligent indicator double-layer preservation film, its preparation method and application. Background Technology

[0002] As consumers place increasing demands on food safety and quality, food is more susceptible to microbial contamination and oxidative spoilage during storage and transportation, leading to shortened shelf life and economic losses. Traditional food packaging primarily relies on physical barriers, lacking active antibacterial and antioxidant functions, and cannot monitor food freshness and safety in real time, failing to meet the demands of the modern food industry for active packaging. Furthermore, microbial metabolites (such as volatile basic nitrogen) produced during food spoilage may pose food safety risks, necessitating the development of intelligent packaging materials that combine antibacterial, antioxidant, and real-time monitoring functions.

[0003] Beetroot red is a water-soluble nitrogenous pigment, mainly extracted and purified from plants such as beetroot and dragon fruit. The powder is purplish-red and possesses excellent coloring ability, making it widely used in food, cosmetics, and pharmaceuticals. Studies show that beetroot red maintains good stability at pH 3–7, with the highest stability at pH 4–6. When beetroot red is in a system with pH < 3, it readily undergoes isomerization or dehydrogenation reactions to form iso-beetroot red or neo-beetroot red, changing its color from red to purplish-red. Due to its high pH sensitivity, beetroot red is often used as an effective indicator for detecting food freshness in active or smart packaging films. Under alkaline conditions, the color change of beetroot red is beneficial for detecting the freshness of various protein-rich foods, including shrimp, fish, pork, chicken, and milk. Chinese patent application CN202410720840.9 discloses a *Auricularia auricula-judae* polysaccharide-curcumin-betaine red indicator film, its preparation method, and its application. Curcumin and betaine red are used as indicators, and chitosan and *Auricularia auricula-judae* polysaccharide are used as film-forming matrices. The film is prepared by casting and drying. The color change of the indicator film reflects the freshness of salmon. A linear fit is performed between the change in volatile basic nitrogen (TVB-N) content during salmon storage and the change in the color difference of the indicator film, which can be used to monitor the freshness of salmon. Chinese patent application CN202111182867.X discloses a method for preparing an anthocyanin-betaine-k-carrageenan freshness indicator film. Anthocyanins, betaine, and k-carrageenan are mixed, poured, and then formed into a film. This invention utilizes anthocyanins to monitor pH changes due to the presence of phenols or conjugated substances, and betaine to detect changes in alkaline environments. By combining both with k-carrageenan polymers, the pH-responsive color change sensitivity of natural pigments is enhanced and their chemical stability is improved, thereby monitoring the freshness and spoilage of packaged products.

[0004] The aforementioned patent obtained a freshness indicator film by combining the indicator material with a polymer film-forming material. However, the sensitivity of beetroot red to heat, light, and pH limits its practical application. Furthermore, the prepared indicator film only has an indicator function and cannot achieve effective antibacterial function. Therefore, constructing a preservation film with antibacterial, antioxidant, and intelligent indicator capabilities is of great significance to the field of food packaging technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an antibacterial and antioxidant intelligent indicator double-layer preservation film, its preparation method, and its applications. By acylation modification of betaine, phenolic acid compounds are associated with betaine glycoside molecules to form acyl glycosides, reducing the breaking of aldehyde-imine bonds and thus improving the stability of betaine. Simultaneously, antibacterial plant essential oils are loaded onto polysaccharide colloidal particles to prepare a Pickering emulsion. This system not only improves the compatibility between essential oils and film-forming materials but also protects the essential oils from external environmental influences and slows down the release rate of the essential oils to prolong the antibacterial duration. Furthermore, an intelligent indicator double-layer preservation film based on natural ingredients is designed to achieve the functions of indicating the freshness of protein-based foods, antibacterial properties, and preservation.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] One objective of this invention is to disclose a method for preparing a beetroot red-phenolic acid acylated derivative, comprising the following steps:

[0008] (1) Dissolve beetroot in a solvent to make a solution with a concentration of 2.0~5.0 mg / mL;

[0009] (2) Dissolve the activator, phenolic acid and condensing agent in a solvent and activate for 0.5-3 h. Then add them dropwise to the beet red solution and stir at 300-800 r / min for 12-36 h at 25℃-30℃.

[0010] (3) After the reaction is complete, the water and / or organic solvent are removed by rotary evaporation, and then the solvent is added and the solid is precipitated by ultrasonic vibration.

[0011] (4) Centrifuge to remove the supernatant, and finally freeze-dry the bottom precipitate to obtain the beet red-phenolic acid acylated product.

[0012] Further, the solvent is one or more selected from water, methanol, ethanol, acetone, isopropanol, chloroform, ethyl acetate, tetrahydrofuran, and N,N-dimethylformamide;

[0013] The activator is at least one of 1-hydroxy-benzotriazole, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, and N-hydroxysuccinimide.

[0014] The condensing agent is at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, and diisopropylcarbodiimide (DIC);

[0015] The phenolic acid is at least one of chlorogenic acid, ferulic acid, gentic acid, vanillic acid, cinnamic acid, syringic acid, sinapic acid, p-coumaric acid, caffeic acid, protocatechuic acid, and gallic acid.

[0016] Furthermore, the molar ratio of the activator to phenolic acid is 1:1 to 5:1, the molar ratio of the condensing agent to phenolic acid is 1:1 to 5:1, and the molar ratio of phenolic acid to betaine is 0.1:1 to 3:1.

[0017] The second objective of this invention is to provide beetroot red-phenolic acid acylated derivatives prepared by any of the above preparation methods.

[0018] The third objective of this invention is to provide a method for preparing an antibacterial, antioxidant, intelligent indicator double-layer food preservation film, comprising the following steps:

[0019] S1. Disperse polysaccharides in deionized water to obtain a polysaccharide dispersion, and then simultaneously drip plant essential oils into the aqueous phase and perform shear emulsification to obtain a plant essential oil Pickering emulsion.

[0020] S2. Dissolve the film-forming material in deionized water, stir magnetically to dissolve, add plasticizer, and then obtain film-forming solution A. Add the beet red-phenolic acid acylated derivative mentioned above, pour into a mold, and dry at room temperature to obtain an indicator layer.

[0021] S2. Dissolve the film-forming material in deionized water, stir magnetically to dissolve, add plasticizer to obtain film-forming liquid B; then mix the emulsion prepared in step S1 with film-forming liquid B in a volume ratio of 1:9 to 9:1, pour into the indicator layer obtained in step S2, and dry at room temperature to obtain a double-layer plastic wrap.

[0022] Furthermore, in step S1,

[0023] The polysaccharide is one or more of the following: carrageenan, gellan gum, konjac gum, gum arabic, pectin, xanthan gum, agar, guar gum, ebony gum, tamarind gum, and sophora bean gum;

[0024] The rotation speed of the shear emulsification is 10000~12000 r / min, and the shear emulsification time is 3~8 min;

[0025] The plant essential oils include at least one of the following: thyme essential oil, galangal essential oil, tea tree essential oil, lemon essential oil, lemongrass essential oil, oily skin essential oil, mustard essential oil, peppermint essential oil, cinnamon essential oil, clove essential oil, garlic essential oil, almond essential oil, eucalyptus essential oil, senna essential oil, and lavender essential oil.

[0026] The concentration of the polysaccharide is 0.1-3.0%, and the concentration of the plant essential oil is 1.0-50%.

[0027] Furthermore, the film-forming material is at least one of chitosan, carboxymethyl chitosan, quaternary ammonium salt chitosan, quaternary phosphorus salt chitosan, soluble starch, cellulose, polyvinyl alcohol, zein, cyclodextrin, gelatin, sodium alginate, carboxymethyl cellulose, konjac glucomannan, and pullulan.

[0028] The plasticizer is at least one of ethylene glycol, glycerol, sorbitol, polyethylene glycol with a molecular weight of less than 4000, ethylenediamine, and triethanolamine.

[0029] Further, in step S2, the amount of plasticizer added is 20-30 wt% of the mass of the film-forming material; the amount of beetroot red-phenolic acid acylated derivative added is 5-40 wt% of the mass of the film-forming material.

[0030] In step S3, the amount of plasticizer added is 20-30 wt% of the mass of the film-forming material.

[0031] In film-forming solution A and film-forming solution B, the mass concentration of the film-forming material is 0.5%-10%.

[0032] The fourth objective of this invention is to provide an antibacterial and antioxidant intelligent indicator double-layer preservation film prepared by any of the above preparation methods.

[0033] The present invention also aims to provide the application of the above-mentioned antibacterial and antioxidant intelligent indicator double-layer preservation film in food packaging or food freshness detection.

[0034] The beneficial effects of this invention include at least the following:

[0035] (1) This invention provides a method to reduce the chromophore group (aldeimine bond breakage) of beet red by structurally modifying the unstable group of beet red molecule. By grafting bioactive phenolic acid onto beet red molecule, not only is the stability of beet red improved, but its antioxidant activity is also increased.

[0036] (2) The acylation reaction conditions involved in this invention are mild, simple to process, and do not require harsh conditions such as high temperature and high pressure, thus avoiding the degradation of beet red due to external environment such as high temperature, strong acid or strong alkali during the structural modification process, which reduces its stability.

[0037] (3) This invention utilizes polysaccharide colloids to construct a Pickering emulsion of plant essential oils. The polysaccharide used is a high-viscosity colloid containing multiple carboxyl groups. The spatial network structure enhances the strength of the interfacial film, prevents droplet aggregation, and maintains the stability of the emulsion. The preparation method is simple and suitable for large-scale synthesis.

[0038] (4) This invention uses acylated beetroot red derivatives as indicator materials and polysaccharide-stabilized essential oil Pickering emulsions as antibacterial materials to prepare an antibacterial indicator preservation film. Aromatic acid modification is used to enhance the antioxidant activity of beetroot red; polysaccharide stabilization of essential oils is used to achieve solubilization, enhancement and efficient transport of essential oils, while improving the antibacterial properties of packaging materials, and applying the packaging film to food freshness detection. Attached Figure Description

[0039] Figure 1 The infrared spectrum of the prepared betaine red-caffeic acid acylated derivative, which includes betaine red (BT), caffeic acid (CA), and betaine red-caffeic acid acylated derivative (BT-g-CA).

[0040] Figure 2 The UV-Vis spectrum of the prepared betaine red-caffeic acid acylated derivative is shown, which includes betaine red (BT), caffeic acid (CA), and betaine red-caffeic acid acylated derivative (BT-g-CA).

[0041] Figure 3 Retention curves of beetroot red (BT) and beetroot red-caffeic acid acylated derivative (BT-g-CA) at different temperatures.

[0042] Figure 4 The retention curves of betaine red (BT) and betaine red-caffeic acid acylated derivative (BT-g-CA) under ultraviolet light (500W) irradiation are shown.

[0043] Figure 5 The DPPH free radical scavenging ability of vitamin C (VC), betaine (BT), and betaine-caffeic acid acylated derivative (BT-g-CA) was evaluated.

[0044] Figure 6 Optical microscope image of the prepared cinnamon essential oil (CEO) Pickerin emulsion.

[0045] Figure 7 Particle size distribution of the prepared cinnamon essential oil (CEO) Pickerin emulsion.

[0046] Figure 8 To investigate the antibacterial properties of the prepared cinnamon essential oil (CEO) Pickerin emulsion.

[0047] Figure 9To assess the light-blocking properties of the prepared intelligent indicator double-layer food preservation film.

[0048] Figure 10 The application of the prepared intelligent indicator double-layer preservation film in the detection of fresh shrimp freshness.

[0049] Figure 11 The graph shows the growth trend of volatile basic nitrogen (TVB-N) in the prepared intelligent indicator double-layer preservation film during the storage of fresh shrimp. Detailed Implementation

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0051] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0052] One objective of this invention is to disclose a method for preparing a beetroot red-phenolic acid acylated derivative, comprising the following steps:

[0053] Beetroot red was dissolved in a solvent to prepare a solution with a concentration of 2.0–5.0 mg / mL. The activator, phenolic acid, and condensing agent were dissolved in the solvent and activated for 0.5–3 h. These were then added dropwise to the beetroot red solution. The reaction was carried out at room temperature (25–30 °C) with stirring at 300–800 r / min for 12–36 h. After the reaction was complete, water and organic solvent were removed by rotary evaporation. Then, an appropriate amount of solvent was added (to remove unreacted phenolic acid), and the solid was precipitated by ultrasonic oscillation. The supernatant was then removed by centrifugation, and the above operation was repeated three times. Finally, the bottom precipitate was freeze-dried to obtain the beetroot red-phenolic acid acylated product.

[0054] In some embodiments, the activator is preferably at least one of 1-hydroxy-benzotriazole (HOBT), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU), and N-hydroxysuccinimide (NHS). Thus, the activator is used to activate carboxylic acids, which can enhance the reaction rate and suppress side reactions, thereby increasing the yield of the target product. HBTU, TBTU, HOBt, and NHS are urea-ion peptide condensing agents, multifunctional reagents for activating carboxylic acids. They can react beet red with phenolic acids in one step without prior acid activation, and their advantages include rapid in-situ activation, fewer side reactions, good stability, and good solubility.

[0055] In some embodiments, the condensing agent is preferably at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC). Thus, a condensing agent refers to a reaction facilitator added in a condensation reaction, generally acting as a catalyst. EDC, DCC, and DIC are inexpensive, solid, convenient to use, and their byproducts are relatively easy to remove. Furthermore, EDC, DCC, and DIC are suitable for amide condensation reactions of carboxylic acids and amines.

[0056] In some embodiments, the molar ratio of the activator to phenolic acid is 1:1 to 5:1, the molar ratio of the condensing agent to phenolic acid is 1:1 to 5:1, and the molar ratio of phenolic acid to betaine is 0.1:1 to 3:1.

[0057] In some embodiments, the polyphenol is at least one selected from chlorogenic acid, ferulic acid, gentic acid, vanillic acid, cinnamic acid, syringic acid, sinapic acid, p-coumaric acid, caffeic acid, protocatechuic acid, and gallic acid.

[0058] In some embodiments, the solvent includes one or more of water, methanol, ethanol, acetone, isopropanol, chloroform, ethyl acetate, tetrahydrofuran, and N,N-dimethylformamide.

[0059] The second objective of this invention is to disclose a method for preparing a Pickering emulsion of plant essential oils, comprising the following steps:

[0060] Polysaccharides were dispersed in deionized water to obtain a polysaccharide dispersion. Then, plant essential oils were added dropwise to the aqueous phase while undergoing shear emulsification to obtain a plant essential oil Pickering emulsion.

[0061] In some embodiments, the polysaccharide is preferably carrageenan, gellan gum, konjac gum, gum arabic, pectin, xanthan gum, agar, guar gum, ebony gum, tamarind gum, sophora bean gum, etc.

[0062] In some embodiments, the rotation speed of the shear emulsification is 10,000 to 12,000 r / min, and the shear emulsification time is 3 to 8 min.

[0063] In some embodiments, the plant essential oil includes at least one selected from thyme essential oil, galangal essential oil, tea tree essential oil, lemon essential oil, lemongrass essential oil, oily skin essential oil, mustard essential oil, peppermint essential oil, cinnamon essential oil, clove essential oil, garlic essential oil, almond essential oil, eucalyptus essential oil, senna essential oil, and lavender essential oil.

[0064] In some embodiments, the concentration of the polysaccharide is 0.1-3.0% (w / v), where w / v refers to the percentage concentration of the polysaccharide in the aqueous solution as a mass / volume ratio, expressed in g / mL; and the volume concentration of the plant essential oil (in mL / mL) is 1.0-50% (v / v).

[0065] The present invention also aims to disclose a method for preparing a smart indicator double-layer preservation film based on the above-mentioned natural ingredients, which realizes the functions of indicating freshness, antibacterial properties, and preservation of protein-based foods. The method includes the following steps:

[0066] A two-step casting method was used to prepare a smart indicator double-layer food preservation film. First, the film-forming material was dissolved in deionized water under magnetic stirring. A plasticizer (20-30 wt%, based on the mass of the film-forming matrix) was added, followed by the addition of the indicator material, beetroot red-phenolic acid acylated product (5-40 wt%, based on the mass of the film-forming matrix). The mixture was poured into a mold and dried at room temperature to obtain the indicator layer. Next, the film-forming material was dissolved in deionized water under magnetic stirring. A plasticizer (20-30 wt%, based on the mass of the film-forming matrix) was added. The prepared emulsion and film-forming solution were mixed at a volume ratio of 1:9 to 9:1, poured into a mold, and dried at room temperature to obtain the double-layer food preservation film.

[0067] In some embodiments, the film-forming material is at least one selected from chitosan, carboxymethyl chitosan, quaternary ammonium salt chitosan, quaternary phosphorus salt chitosan, soluble starch, cellulose, polyvinyl alcohol, zein, cyclodextrin, gelatin, sodium alginate, carboxymethyl cellulose bundles, konjac glucomannan, and pullulan; the plasticizer is at least one selected from ethylene glycol, glycerol, sorbitol, polyethylene glycol with a molecular weight of less than 4000, ethylenediamine, and triethanolamine; and the mass concentration of the film-forming material in the film-forming base solution is 0.5%-10%.

[0068] This invention also provides an application of intelligent indicator double-layer preservation film in detecting the freshness of protein-based foods; including the following steps:

[0069] Fresh shrimp, beef, pork, fish, and other protein-rich foods are placed in a mold (9cm in diameter), sealed with a double-layer film (indicator layer facing out), and then stored at 25°C or 4°C. The volatile basic nitrogen (TVB-N) and pH value of the food are measured, and the color change of the film is recorded. The freshness of the food is determined by colorimetry.

[0070] The following specific embodiments illustrate the solution proposed in this invention:

[0071] Example 1: Beetroot Red-Caffeic Acid Acylation Derivative

[0072] At 25°C, 0.275 g (0.5 mmol) of beetroot red was weighed and dissolved in 5 mL of water and 2.5 mL of tetrahydrofuran (THF) until fully dissolved. Then, 0.5 mmol of caffeic acid (0.09 g) was weighed and added to 7.5 mL of THF. 0.5 mmol (0.09585 g) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.5 mmol (0.0575 g) of N-hydroxysuccinimide (NHS) were dissolved in 7.5 mL of water and mixed thoroughly with the caffeic acid solution. The mixture was stirred at room temperature in the dark for 2 hours to fully activate the solution. Subsequently, the activated solution was slowly added dropwise to the beetroot red solution while stirring continuously. The reaction was carried out at room temperature in the dark for 24 hours. After the reaction was complete, the solution was rotary evaporated at 55°C to remove water and THF. 25 mL of anhydrous ethanol was added to remove unreacted caffeic acid, and the solid was precipitated by ultrasonic oscillation. Subsequently, the mixture was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was removed, and the above operation was repeated 3 times (adding 25 mL of anhydrous ethanol, precipitating solid, and centrifuging for 10 min). Finally, the bottom precipitate was freeze-dried to obtain the beet red-caffeic acid acylated product (BT-g-CA).

[0073] The changes in the molecular structure of the prepared beet red-caffeic acid acylated derivative were analyzed using a German Tensor 27 infrared spectroscopy (FT-IR) instrument. The results are as follows: Figure 1 As shown. In the infrared spectrum of beet red (BT), in the range of 3500~3000 cm⁻¹ -1 The broad absorption band is due to the superimposed vibrational absorption peaks of -COOH, -OH, =CH, -NH- and pyridine heteroaromatic rings in beetroot red, at 2933 cm⁻¹. -1 2886cm -1 It is the asymmetric and symmetric stretching vibration of the -CH2 group of beetroot glucose, at 1640 cm⁻¹. -1 It is the characteristic absorption peak of the benzene ring skeleton structure of beetroot red, at 1016 cm⁻¹. -1 This is the vibrational absorption peak of the -CH2-OH group of betaine glucose. In the infrared spectrum of caffeic acid (CA), the peak is at 3398 cm⁻¹.-1 It is the absorption peak of the phenolic hydroxyl group -OH vibration of the benzene ring in caffeic acid, at 3219 cm⁻¹. -1 The absorption peaks are due to the stretching vibration of the free -OH group in the carboxyl group of caffeic acid, at 1632 and 1450 cm⁻¹. -1 The characteristic absorption peak of the caffeic acid benzene ring skeleton structure. Compared with the infrared spectrum of betaine (BT), the 3292 cm⁻¹ peak is more prominent in the infrared spectrum of the betaine red-caffeic acid acylated derivative (BT-g-CA). -1 The increased intensity of the stretching vibration absorption peak of -OH at 2933 cm⁻¹ indicates the introduction of -OH into the beetroot red structure. Additionally, the absorption peak at 2933 cm⁻¹... -1 The increased intensity of the CH vibration absorption peak at 1707 cm⁻¹ indicates the introduction of a methyl or methylene group into the beetroot red structure. A characteristic C=O absorption peak for the ester group (COO) appears at 1707 cm⁻¹, and similar peaks are observed at 1640 and 1562 cm⁻¹. -1 The presence of a characteristic absorption peak indicating a benzene ring backbone structure suggests the introduction of a benzene ring into the betaine glucoside structure. These results demonstrate the successful preparation of a betaine-caffeic acid acylated derivative by grafting caffeic acid onto the betaine structure.

[0074] The functional group structure changes of the prepared beetroot red-caffeic acid acylated derivatives were analyzed using a Shimadzu UV spectrophotometer (U765S). The results are as follows: Figure 2 As shown in the UV-Vis spectrum of betaine (BT), a maximum absorption wavelength appears in the visible light region at 523 nm, and a lower absorption peak appears in the UV region at 265 nm. In the UV-Vis spectrum of caffeic acid (CA), a maximum absorption wavelength appears at 328 nm, which is a characteristic peak of the benzene ring. In the UV-Vis spectrum of the betaine-caffeic acid acylated derivative (BT-g-CA), a lower absorption peak appears at 523 nm, which is a characteristic peak of betaine aldehydes, and a strong absorption peak appears at 281 nm, mainly due to the grafting of caffeic acid into the betaine molecule. These results also prove that caffeic acid was successfully grafted onto the structure of betaine.

[0075] Time-temperature stability test: 10 mg / mL beetroot red solutions before and after modification were heated in a constant temperature water bath at 60℃ and 80℃ for different times in the dark. The absorbance values ​​were measured, the retention rate was calculated, and curves were plotted. The results are shown below. Figure 3As shown in the figure, under high temperature conditions (60℃ and 80℃), the retention rate of acylated betaine red (BT-g-CA) is significantly higher than that of betaine red at the same concentration. When heated at 60℃ for 4 hours, the retention rate of betaine red BT was 32.78%, while the retention rate of betaine red-caffeic acid acylated derivative (BT-g-CA) was 44.64%. When heated at 80℃ for 2 hours, the retention rate of betaine red BT was 8.87%, while the retention rate of betaine red-caffeic acid acylated derivative (BT-g-CA) was 19.79%. This indicates that caffeic acid acylation of betaine red is beneficial for its thermal stability.

[0076] UV stability test: 10 mg / mL beetroot red solutions before and after modification were irradiated with UV light (500 W) for different times, absorbance values ​​were measured, retention rates were calculated, and curves were plotted. The results are shown below. Figure 4 As shown in the figure, the retention rate of betaine-caffeic acid acylated derivative (BT-g-CA) under ultraviolet light irradiation is significantly higher than that of betaine at the same concentration. After 2 hours (120 minutes) of irradiation, the retention rate of betaine is only 2.27%, while the retention rate of betaine-caffeic acid acylated derivative (BT-g-CA) is as high as 38.69%. This is mainly because the molecular structure of betaine-caffeic acid acylated derivative (BT-g-CA) introduces phenolic hydroxyl groups with anti-ultraviolet properties. During ultraviolet irradiation, the phenolic hydroxyl groups help protect the chromophore (betaine aldehyde) of betaine from degradation.

[0077] The in vitro antioxidant activity of betaine-caffeic acid acylated derivatives was determined by measuring their DPPH free radical scavenging ability.

[0078] DPPH free radical scavenging capacity determination: Vitamin C solutions of different concentrations were obtained by diluting with anhydrous ethanol, and sample solutions of different concentrations were obtained by diluting with ultrapure water. A certain amount of DPPH powder was weighed and dissolved in anhydrous ethanol to obtain a 0.2 mM DPPH working solution. 1 mL of sample or Vitamin C solution of different concentrations was taken and mixed thoroughly with an equal volume of DPPH working solution. The mixture was reacted at 25°C in the dark for 30 min, and the absorbance of the mixture was measured at 517 nm. Vitamin C was used as a positive control. The zeroing was performed using an equal volume of anhydrous ethanol and ultrapure water mixture. The absorbance of the sample and DPPH mixture was designated as As, and the absorbance of the equal volume of anhydrous ethanol and DPPH mixture was designated as the blank control Ac. The DPPH free radical scavenging rate was calculated using the following formula:

[0079] ;

[0080] The DPPH free radical scavenging rate of Vc was calculated, and the DPPH value of the sample was expressed as μmol Vcequiv. / g.

[0081] Figure 5The graph shows the DPPH radical scavenging abilities of vitamin C (VC), betaine (BT), and its betaine-caffeic acid acylated derivative (BT-g-CA). As can be seen from the graph, the DPPH radical scavenging ability of BT-g-CA is significantly stronger than that of betaine (BT). At a concentration of 125 μg / mL, BT-g-CA exhibits a DPPH radical scavenging rate of 94.98%, while BT's DPPH radical scavenging rate is only 3.34%. This indicates that modification significantly improves the antioxidant capacity of betaine. This is because BT-g-CA introduces the catechol hydroxyl group from caffeic acid into BT through esterification, endowing betaine with phenolic antioxidant activity. BT's own antioxidant capacity mainly relies on the isolated hydroxyl and glycosyl groups in betaine, which have weak hydrogen donation capacity and low reaction rates, resulting in an overall low scavenging rate.

[0082] Example 2: Carrageenan-stabilized cinnamon oil Pickering emulsion

[0083] 1 g of carrageenan was dissolved in 100 mL of deionized water at 80 °C by heating, preparing a 1% (w / v, g / mL) carrageenan aqueous solution. Then, cinnamon essential oil at different volume fractions of 1%, 3%, 5%, and 7% was added to the carrageenan aqueous solution. The mixture was emulsified using a high-speed disperser at 10,000 rpm for 3 minutes to obtain a carrageenan-stabilized cinnamon essential oil Pickering emulsion.

[0084] The microstructure of the carrageenan-stabilized cinnamon oil Pickering emulsion was observed using an EVOS XL Core inverted optical microscope manufactured by Thermo Fisher Scientific, USA. The results are as follows: Figure 6 As shown in the figure, the emulsion droplets are all regularly shaped and have clear boundaries between them. The figure also shows that the particle size of the emulsion gradually increases with the increase of the proportion of cinnamon essential oil (CEO).

[0085] The particle size of the emulsion was measured using a Mastersizer 3000 laser scattering particle size analyzer. The pump stirring speed of the dispersant was 1800 rpm. The refractive indices of the dispersant and cinnamon essential oil were 1.333 and 1.592, respectively. The emulsion particle size is expressed as a volume-weighted average diameter (D4,3). The results are as follows: Figure 7As shown, the particle size of the cinnamon essential oil Pickering emulsion exhibits a normal distribution, and the particle size gradually increases with increasing essential oil content. When the essential oil content is 3%, the emulsion distribution is relatively concentrated, with an average particle size of 6.019 ± 0.816 μm. The rheological property curves reveal that the apparent viscosity of the carrageenan-stabilized cinnamon essential oil Pickering emulsion decreases with increasing shear rate, exhibiting shear thinning. This indicates that the carrageenan-stabilized cinnamon essential oil Pickering emulsion is a pseudoplastic fluid. This is because carrageenan, as a polysaccharide solid particle, can form a three-dimensional network structure or particle aggregation state through hydrogen bonds or van der Waals forces under static or low shear conditions, increasing flow resistance. When the shear rate increases, the shear force disrupts this temporary structure of the three-dimensional network or particle aggregation state, causing the particles or droplets to rearrange, thereby reducing the apparent viscosity. Furthermore, the graph shows that the viscosity of the emulsion gradually decreases with increasing essential oil concentration. This is because the low viscosity of cinnamon essential oil dilutes the carrageenan network structure of the continuous phase (aqueous phase), weakening its thickening ability.

[0086] The rheological properties of the carrageenan-stabilized cinnamon oil Pickering emulsion in this embodiment were studied at 25°C using a Thermofisher / MARS Hack rheometer. The apparent viscosity of the sample was measured in a dynamic oscillation experiment using parallel steel plates (60 mm in diameter, 0.5 mm gap) within a shear rate range of 0.1–300 s⁻¹. The results are as follows: Figure 8 As shown in the figure, the antibacterial effects and magnitudes of emulsions prepared with 1.0% carrageenan and different volume fractions of cinnamon essential oil (0%, 1%, 3%, 5%, 7%) against *Escherichia coli* and *Staphylococcus aureus* are as follows. The figure shows that the antibacterial ability of the emulsion is positively correlated with the essential oil content. When the essential oil content is 1%, a clear inhibition zone is visible, significantly inhibiting the growth of *E. coli* and *Staphylococcus aureus*. The diameter of the inhibition zone against these two bacteria was measured, and the results are shown in Table 1. The larger the diameter of the inhibition zone, the better the effect of the substance in inhibiting microbial growth. An inhibition zone diameter less than 7 mm indicates weak antibacterial ability; an inhibition zone diameter greater than 20 mm indicates extremely strong antibacterial ability. When the essential oil content in the emulsion was 3%, the diameters of the inhibition zones against Escherichia coli and Staphylococcus aureus were 35.2±0.22 mm and 30.6±0.13 mm, respectively, demonstrating extremely strong antibacterial activity.

[0087] Table 1. Antibacterial diameters at different essential oil contents

[0088]

[0089] Example 3: Intelligent Indicator Double-Layer Food Preservation Film

[0090] A two-step casting method was used to prepare a smart indicator double-layer food preservation film. First, carboxymethyl cellulose (CMC) was dissolved in deionized water at 60°C with magnetic stirring. The solution was then cooled to 25°C to prepare a 2% (w / v, g / mL) aqueous solution, with 20 wt% (based on solute mass) of glycerol added as a plasticizer. 0.2 g of betaine red-caffeic acid acylated derivative (MBT, 20 wt% of the CMC matrix mass) was added to 50 mL of the 2% CMC solution, poured into a mold, and dried at room temperature to obtain the indicator layer. Then, sodium alginate (SA) was added to deionized water and dissolved with stirring at 60°C. After cooling to 25°C, 30 wt% of glycerol (based on the mass of SA solute) was added to obtain a 2% (w / v, g / mL) SA solution. 17.5 mL of SA solution was mixed with 7.5 mL of carrageenan-stabilized cinnamon oil Pickering emulsion (PE) prepared above at a ratio of 7:3 (v / v). 25 mL of the mixture was poured into a dry indicator layer and dried at 25 °C to obtain a double-layer plastic wrap CMC-SA-MBT-PE. Three control groups were set up: CMC-SA, CMC-SA-MBT, and CMC-SA-PE.

[0091] Figure 9 The light-blocking performance of the prepared intelligent indicator double-layer food preservation film was studied. The UV-Vis light barrier is crucial for food packaging because light can cause oxidative spoilage. As shown in the figure, the double-layer food preservation film CMC-SA-MBT-PE, with added acylated beetroot red and essential oil emulsion, exhibits significant light-blocking performance in the 200-800 nm range. Specifically, at 350 nm, the optical transmittance of the CMC-SA composite film was 58.73%, while that of the CMC-SA-MBT composite film was only 0.38%, indicating that the addition of acylated beetroot red improved the light-blocking performance of the composite film. The light transmittance of the CMC-SA-PE and CMC-SA-MBT-PE composite films with added essential oil emulsion was significantly lower than that of the CMC-SA and CMC-SA-MBT composite films. At 550 nm, the optical transmittance of CMC-SA was 71.05%, CMC-SA-MBT was 46.32%, CMC-SA-PE was 1.44%, and CMC-SA-MBT-PE was 0.83%, indicating that the addition of emulsions improved the light-blocking properties of the materials. This is mainly because MBT and essential oil emulsions contain unsaturated bonds that can absorb ultraviolet-visible radiation, such as benzene rings, phenolic hydroxyl groups, and double bonds. In conclusion, the addition of MBT and essential oil Pickering emulsions can significantly improve the optical blocking performance of the film.

[0092] Example 4: Application of intelligent indicator double-layer plastic wrap in detecting the freshness of fresh shrimp

[0093] Fresh shrimp (80g) were placed in a plastic petri dish (9cm in diameter), sealed with a double-layer film (indicator layer facing out), and then stored at 25°C or 4°C for 48 hours. The volatile basic nitrogen (TVB-N) value of the shrimp was measured every 8 hours, and the color change of the film was recorded. The freshness of the food was determined by colorimetry.

[0094] Figure 10 This study investigates the application of the prepared intelligent indicator double-layer preservation film in detecting the freshness of shrimp. As shown in the figures, in the KB (blank) and CMCS-SA groups, the shrimp spoiled by day 6, and by day 8, the shrimp heads showed a noticeable yellow tinge, with the surface flesh beginning to rot. In contrast, the composite film CMC-SA-MBT-PE gradually turned yellow over time, indicating that the shrimp began to spoil by day 8.

[0095] Figure 11 The prepared intelligent indicator double-layer preservation film was used to measure the volatile basic nitrogen (TVB-N) during the storage of fresh shrimp. Shrimp have high water activity and their muscle components (protein, amino acids) are rich in nitrogen, making them prone to rapid spoilage under refrigeration conditions (4℃). Endogenous enzymes and microbial activity produce TVB-N and biogenic amines. The initial TVB-N level (7.23±1.02 mg / 100g) classified the sample as Grade 1 freshness. On day 4, the TVB-N values ​​of KB (blank), CMCS-SA group, and CMC-SA-MBT-PE were 28.5±1.19, 23.1±1.71, and 17.43±1.75 mg / 100g, respectively. The KB (blank) and CMCS-SA groups exceeded the Chinese regulatory limit (GB2707-2016: ≤20 mg / 100g). In comparison, CMC-SA-MBT-PE exhibited better performance, maintaining TVB-N at 17.43±1.75 mg / 100g. This is attributed to the synergistic effect of the antioxidant MBT and antibacterial PE in the CMC-SA-MBT-PE bilayer membrane. By day 8, TVB-N accumulation in KB (blank) (39.82±2.14 mg / 100g) and the CMCS-SA group (30.33±1.87 mg / 100g) exceeded the international threshold (≤30 mg / 100g), while the CMC-SA-MBT-PE bilayer membrane maintained it at 21.27±1.19 mg / 100g. These results indicate that bilayer membranes containing antioxidants and antibacterial materials can extend the shelf life of shrimp by 2-4 days.

[0096] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0097] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0098] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing an antibacterial, antioxidant, intelligent indicator double-layer food preservation film, characterized in that, Includes the following steps: S1. Disperse polysaccharides in deionized water to obtain a polysaccharide dispersion, and then simultaneously drip plant essential oils into the aqueous phase and perform shear emulsification to obtain a plant essential oil Pickering emulsion. S2. Dissolve the film-forming material in deionized water, stir magnetically to dissolve, add plasticizer, and then obtain film-forming solution A. Add beet red-phenolic acid acylated derivative, pour into a mold, and dry at room temperature to obtain an indicator layer. S3. Dissolve the film-forming material in deionized water, stir magnetically to dissolve, add plasticizer to obtain film-forming liquid B; then mix the emulsion prepared in step S1 with film-forming liquid B in a volume ratio of 1:9 to 9:1, pour into the indicator layer obtained in step S2, dry at room temperature to obtain double-layer plastic wrap. In step S2, the preparation of the beetroot red-phenolic acid acylated derivative includes the following steps: (1) Dissolve beetroot in a solvent to prepare a solution with a concentration of 2.0~5.0 mg / mL; (2) Dissolve the activator, phenolic acid and condensing agent in a solvent and activate for 0.5-3 h. Then add them dropwise to the beet red solution and stir at 300-800 r / min for 12-36 h at 25℃-30℃. (3) After the reaction is complete, the water and / or organic solvent are removed by rotary evaporation, and then the solvent is added and the solid is precipitated by ultrasonic vibration. (4) Centrifuge to remove the supernatant, and finally freeze-dry the bottom precipitate to obtain the beet red-phenolic acid acylated product.

2. The method for preparing the antibacterial and antioxidant intelligent indicator double-layer food preservation film according to claim 1, characterized in that, In the preparation of beetroot red-phenolic acid acylated derivatives, The solvent is one or more selected from water, methanol, ethanol, acetone, isopropanol, chloroform, ethyl acetate, tetrahydrofuran, and N,N-dimethylformamide; The activator is at least one of 1-hydroxy-benzotriazole, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid, and N-hydroxysuccinimide; The condensing agent is at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, and diisopropylcarbodiimide (DIC); The phenolic acid is at least one of chlorogenic acid, ferulic acid, gentic acid, vanillic acid, cinnamic acid, syringic acid, sinapic acid, p-coumaric acid, caffeic acid, protocatechuic acid, and gallic acid.

3. The method for preparing the antibacterial and antioxidant intelligent indicator double-layer food preservation film according to claim 1, characterized in that, In the preparation of beetroot red-phenolic acid acylated derivatives, The molar ratio of the activator to phenolic acid is 1:1 to 5:1, the molar ratio of the condensing agent to phenolic acid is 1:1 to 5:1, and the molar ratio of phenolic acid to betaine is 0.1:1 to 3:

1.

4. The method for preparing the antibacterial and antioxidant intelligent indicator double-layer food preservation film according to claim 1, characterized in that, In step S1, The polysaccharide is one or more of the following: carrageenan, gellan gum, konjac gum, gum arabic, pectin, xanthan gum, agar, guar gum, ebony gum, tamarind gum, and sophora bean gum; The rotation speed of the shear emulsification is 10000~12000 r / min, and the shear emulsification time is 3~8 min; The plant essential oils include at least one of the following: thyme essential oil, galangal essential oil, tea tree essential oil, lemon essential oil, lemongrass essential oil, oily skin essential oil, mustard essential oil, peppermint essential oil, cinnamon essential oil, clove essential oil, garlic essential oil, almond essential oil, eucalyptus essential oil, senna essential oil, and lavender essential oil. The concentration of the polysaccharide is 0.1-3.0%, and the concentration of the plant essential oil is 1.0-50%.

5. The method for preparing the antibacterial and antioxidant intelligent indicator double-layer food preservation film according to claim 1, characterized in that, The film-forming material is at least one of chitosan, carboxymethyl chitosan, quaternary ammonium salt chitosan, quaternary phosphorus salt chitosan, soluble starch, cellulose, polyvinyl alcohol, zein, cyclodextrin, gelatin, sodium alginate, carboxymethyl cellulose, konjac glucomannan, and pullulan. The plasticizer is at least one of ethylene glycol, glycerol, sorbitol, polyethylene glycol with a molecular weight of less than 4000, ethylenediamine, and triethanolamine.

6. The method for preparing the antibacterial and antioxidant intelligent indicator double-layer food preservation film according to claim 1, characterized in that, In step S2, the amount of plasticizer added is 20-30 wt% of the mass of the film-forming material; the amount of beet red-phenolic acid acylated derivative added is 5-40 wt% of the mass of the film-forming material. In step S3, the amount of plasticizer added is 20-30 wt% of the mass of the film-forming material. In film-forming solution A and film-forming solution B, the mass concentration of the film-forming material is 0.5%-10%.

7. The antibacterial and antioxidant intelligent indicator double-layer preservation film prepared by any of the preparation methods of claims 1-6.

8. The application of the antibacterial and antioxidant intelligent indicator double-layer preservation film according to claim 7 in food packaging or food freshness detection.

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