Dual-response antibacterial nanofiber membrane and preparation method thereof

Abstract

The invention provides a dual-response antibacterial nanofiber membrane and a preparation method thereof, an antibacterial aminated gelatin nano inclusion compound is loaded into a polyvinyl alcohol-chitosan matrix through an electrostatic spinning technology to prepare a nanofiber membrane, citral is further loaded in a closed adsorption mode, and the dual-response antibacterial nanofiber membrane is prepared. The antibacterial nanofiber membrane with the humidity and gelatinase dual responsiveness is obtained. The antibacterial nanofiber membrane disclosed by the invention can directly wrap the periphery of a meat product, and citral is adaptively released by increasing the environmental humidity so as to inhibit the proliferation of microorganisms; besides, when endogenous or exogenous microorganisms infect the surfaces of the meat products, gelatinase secreted by the microorganisms is used as a sensor to further release antibacterial substances, the antibacterial substances can be released according to actual fresh-keeping requirements in the storage, transportation and circulation process of the meat products, the problems that antibacterial components are released too early and the timeliness is short are solved, the storage time of the meat products can be remarkably prolonged, and the safety of the meat products is improved. And the spoilage is delayed.

Description

Technical Field

[0001] The present invention belongs to the field of food preservation, and particularly relates to a dual-responsive antibacterial nanofiber membrane and a preparation method thereof. Background Art

[0002] During the storage, transportation and circulation of meat products, their spoilage forms mainly include microbial spoilage, enzymatic spoilage, lipid oxidation and protein oxidation. Among them, microbial spoilage is an important reason for the deterioration of the quality of meat products. Proteins and other nitrogen-containing compounds contained in meat products are easily degraded by microorganisms, and the resulting spoilage substances such as amines, organic acids, and ketones combine with microbial biofilms to form mucus, further accelerating the spoilage and deterioration of meat. The use of active packaging loaded with natural antibacterial essential oils can effectively extend the shelf life of perishable meat products. However, natural antibacterial essential oils are volatile, have a strong pungent odor, are easily decomposed by light and heat, and have poor acid and alkali resistance. In addition, the release of antibacterial essential oils in antibacterial active packaging is mostly achieved through passive diffusion or shell rupture, and the delivery efficiency is low, making its application effect in meat food preservation unsatisfactory. Therefore, developing a stimulus-responsive intelligent antibacterial packaging material to deliver antibacterial substances to specific target positions and release them precisely based on actual preservation needs is an effective strategy to enhance the preservation effect of antibacterial active packaging. The response system of intelligent responsive antibacterial materials depends on the type of stimulating factors. Groups with different response functions can be incorporated into responsive materials to achieve antibacterial effects in different response systems. According to the source of stimulation, intelligent responsive antibacterial materials can be classified into those responsive to physical stimuli (temperature, humidity, salt, etc.) and those responsive to bacterial metabolites (pH, enzymes, etc.). So far, there have been some patents for the preservation of fresh food. Chinese Patent CN202310384907.1 discloses the preparation of a polylactic acid / citral electrospun nanofiber membrane and its refrigerated application in turbot fish meat. Although the preparation materials of this fresh-keeping film are similar to those of the present invention, it only targets the preservation of turbot fish meat and does not have the function of targeted release of antibacterial agents. Chinese Patent CN201910988026.4 discloses a method for preparing a nano fresh-keeping film by electrospinning technology, with thymol as the core and poly(lactic-co-glycolic acid) and ethylene-vinyl alcohol copolymer as the shell. This fresh-keeping film can only respond to humidity and intelligently release thymol essential oil, targeting the preservation of fruits and vegetables. Chinese Patent CN202311015251.2 discloses a method for preserving meat food with dual responses to cellulase and pH. This fresh-keeping film uses cellulase secreted by bacteria to respond to carboxymethyl cellulose in the coating, and uses the increase in pH inside the film to respond to the change in the structure of anthocyanins in the coating. Although this fresh-keeping film has the functions of targeted antibacterial and pH indicating freshness, the pH response causes a change in the color of the film when the meat spoils to a certain extent. In addition, the single-stimulus responsive release of antibacterial agents may not be able to adapt to various environmental changes in food storage, transportation, and circulation in a timely manner. Research shows that packaging systems for storing foods such as meat usually have a relative humidity (RH) of more than 65%, but the growth of microorganisms also increases with the increase in humidity. The invasion of microorganisms into meat first involves attachment and colonization on the surface of the meat, followed by the secretion of a series of enzymes to decompose the surrounding organic substances, promoting the growth of microorganisms, the increase in humidity, and the formation of biofilms. Mature biofilms enable microorganisms to multiply rapidly and continuously secrete enzymes to invade the tissue components of the meat, causing meat spoilage. In the middle and late stages of spoilage, the action of microorganisms accelerates the oxidative degradation of amino acids and fatty acids, producing substances such as organic acids and amines, which cause a significant change in the pH of the meat. Therefore, high humidity conditions and the response to specific enzymes can be used to design intelligent active fresh-keeping films that precisely and release functional factors on demand, enabling the fresh-keeping film to exert antibacterial performance when invaded by microorganisms and extending the shelf life of meat. Summary of the Invention

[0003] Technical problem to be solved: The object of the present invention is to provide a dual-responsive antibacterial nanofiber membrane and its preparation method. By means of electrospinning technology, an antibacterial amino-gelatin nano-inclusion complex is loaded into a polyvinyl alcohol-chitosan matrix to prepare a nanofiber membrane, and further loaded with citral (Cit) by means of sealed adsorption to obtain a humidity and gelatinase dual-responsive antibacterial nanofiber membrane. The antibacterial nanofiber membrane of the present invention can be directly wrapped around the periphery of meat products, and adaptively release citral to inhibit the proliferation of microorganisms as the environmental humidity increases; in addition, when endogenous or exogenous microorganisms infect the surface of meat products, the gelatinase secreted by the microorganisms is used as a sensor to further release antibacterial substances, which can release antibacterial substances according to the actual fresh-keeping needs during the storage and circulation of meat products, overcome the problems of premature release and short timeliness of antibacterial components, can significantly extend the storage time of meat products, and delay their spoilage.

[0004] Technical solution: A dual-responsive antibacterial nanofiber membrane, wherein the dual response is humidity and gelatinase dual responsiveness. A dual-responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of amino-gelatin: Dissolve gelatin in phosphate buffer, add 1,2-ethylenediamine while stirring, adjust the pH to 5.0 with 5M hydrochloric acid, add water-soluble carbodiimide hydrochloride (EDC), and make up the volume to 250 mL with phosphate buffer. After incubating at 30-40 °C for 2-3 h, dialyze in pure water and freeze-dry to obtain amino-gelatin (AGNPS); S2. Preparation of amino-gelatin dispersion: Dissolve 1.0-1.5 g of freeze-dried AGNPS in 25 mL of distilled water, fully dissolve it, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence, adjust the pH to 4.0 with 1M hydrochloric acid, slowly stir and dropwise add acetone until the solution becomes milky white, then add 160-240 μL of glutaraldehyde solution, incubate AGNPS at 45-55 °C for 3-5 h, then rotary evaporate and make up the volume to a 250 mL volumetric flask to obtain an AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of antibacterial amino-gelatin nano-inclusion complex: Mix the AGNPS dispersion with 20% thymol essential oil, shear at 12000 rpm for 1 min, and homogenize at 500 MPa for 3 min to obtain an antibacterial amino-gelatin nano-inclusion complex (AG-Thy); S4. Preparation of Polyvinyl Alcohol-Chitosan PVA / CS / AG-Thy Nanofiber Membrane Loaded with Antibacterial Amino Gelatin Nanocapsules: Using a mixed solution of water and acetic acid as the solvent, prepare an electrospinning solution containing 0.15 - 0.25 g / mL PVA, 0.05 - 0.15 g / mL CS, and 0.03 - 0.13 g / mL antibacterial amino gelatin nanocapsules. After stirring at 35 - 45 °C for 3 - 5 h, perform electrospinning at a voltage of 20 - 25 kV for 3 - 5 h to obtain a nanofiber membrane (PVA / CS / AG-Thy) loaded with antibacterial amino gelatin nanocapsules; S5. Preparation of Dual-Responsive Nanoantibacterial Fiber Membrane: Place the PVA / CS / AG-Thy nanofiber membrane and citral together in a sealed space and leave it at room temperature for 3 - 5 days to obtain the Cit@PVA / CS / AG-Thy dual-responsive antibacterial nanofiber membrane. Further, in step S1, the mass-to-volume ratio of gelatin to phosphate buffer solution is 1:(20 - 25); the concentration of the phosphate buffer solution is 0.1 M and the pH is 5.0. Further, in step S1, the mass ratio of 1,2-ethylenediamine to gelatin is (3.0 - 3.5):1; the mass ratio of water-soluble carbodiimide hydrochloride to gelatin is (0.5 - 0.8):1. Further, in step S3, the volume ratio of AGNPS dispersion to thyme essential oil is (4.0 - 5.0):1. Further, in step S4, the volume ratio of water to acetic acid is (2.5 - 3.5):1. Further, in step S4, the electrospinning parameters are that the distance between the electrospinning nozzle and the substrate is set to 16 - 18 cm, and the relative humidity is 50 - 60%. Further, in step S5, the mass-to-volume ratio of the PVA / CS / AG-Thy nanofiber membrane to citral is 1:(2 - 3). Further, application of the dual-responsive antibacterial nanofiber membrane prepared by the preparation method in meat preservation. Beneficial effects: 1. In the present invention, an antibacterial nanofiber membrane is prepared by electrospinning technology using antibacterial nanocapsules stabilized by amino gelatin, polyvinyl alcohol, and chitosan as the matrix, and the attachment of citral is achieved by utilizing the space existing between polymer chains to allow small molecules to pass through. During this process, citral molecules penetrate into the voids inside the PVA / CS polymer chains. Part of them react with the amino groups of chitosan through chemical reactions to form Schiff base bonds and graft into the nanofibers; the other part is physically adsorbed and loaded into the voids of the nanofibers. 2. The prepared PVA / CS / AG-Thy nanofibers in the present invention have humidity and gelatinase-responsive properties. The nanofibers absorb moisture in a high-humidity environment, inducing an increase in the gas permeability of the polymer and an enlargement of the internal pore structure, causing the swelling of polyvinyl alcohol, thereby triggering the responsive release of citral. At the same time, the amidated gelatin in the fibers acts as a "gatekeeper" for gelatinase response. When microorganisms secrete gelatinase, the thyme essential oil encapsulated in the amidated gelatin will be released in a targeted manner to kill bacteria, achieving dual-stimulus-responsive release at the levels of humidity and microorganisms, which is of great significance for the long-term preservation of meat products. 3. Compared with other food preservation materials, the advantages of the prepared fresh-keeping nanofiber membrane of the present invention are as follows: (1) The method of the present invention uses polyvinyl alcohol and chitosan to prepare a fiber matrix, loads an antibacterial amidated gelatin nano-inclusion in the matrix, and grafts citral into the fiber pores, significantly increasing the loading amount of the antibacterial agent; (2) The present invention selects the amidated gelatin nano-inclusion as the carrier of thyme essential oil, which has gelatinase responsiveness and can respond to the enzymes secreted by the growth of microorganisms inside and outside the packaging environment to release the antibacterial agent. Amidated gelatin, as the modified gelatin, has the characteristics of good biocompatibility, improving the embedding rate and stability of the antibacterial agent; (3) The present invention selects a humidity and gelatinase response system, which has selectivity and specificity in meat preservation, and the humidity response is targeted for the storage of meat in a high-humidity environment; (4) The present invention realizes the controllable and precise release of multiple antibacterial agents under dual responses, can release antibacterial substances according to the actual fresh-keeping needs during the storage, transportation and circulation of meat products, overcomes the problems of premature release and short timeliness of antibacterial components, and can significantly extend the storage time of meat products and delay their spoilage. Description of the Drawings Figure 1 It is the preparation flow chart of the dual-response antibacterial nanofiber membrane; Figure 2 It is the scanning electron microscope image of the dual-response antibacterial nanofiber membrane, where a is PVA / CS1 / A1 (Comparative Example 1); b is Cit1@PVA / A1 (Comparative Example 3); c is Cit1@PVA / CS1 (Comparative Example 2); d is Cit1@PVA / CS1 / A1 (Example 1); Figure 3 It is the change graph of the pH of fresh pork during the storage of the dual-response antibacterial nanofiber membranes prepared in Examples 1-7 and Comparative Examples 1-3; Figure 4 It is the change graph of the total number of colonies of fresh pork during the storage of the dual-response antibacterial nanofiber membranes prepared in Examples 1-7 and Comparative Examples 1-3; Figure 5 It is the gelatinase response behavior graph of the Cit1@PVA / CS1 / A1 dual-response antibacterial nanofiber membrane; Figure 6 It is a humidity response behavior diagram of the Cit1@PVA / CS1 / A1 dual-responsive antibacterial nanofiber membrane. Specific implementation manners The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Embodiment 1 A preparation method of a humidity- and gelatinase-dual-responsive antibacterial nanofiber membrane includes the following steps: S1. Preparation of amino-gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution, add 16 g of 1,2-ethylenediamine while stirring, adjust the pH to 5.0 with 5 M hydrochloric acid, add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, make up the volume to 250 mL with phosphate buffer solution, incubate at 37 °C for 2 h, dialyze in pure water for 3 days, and freeze-dry to obtain amino-gelatin AGNPS; S2. Preparation of amino-gelatin dispersion: Dissolve 1.25 g of freeze-dried amino-gelatin in 25 mL of distilled water, fully dissolve it, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence, adjust the pH to 4.0 with 1 M hydrochloric acid, slowly stir and dropwise add acetone until the solution turns milky white, then add 200 μL of glutaraldehyde solution, incubate AGNPS at 50 °C for 3 h, then perform rotary evaporation and make up the volume to a 250 mL volumetric flask to obtain an AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of antibacterial amino-gelatin nanocapsule AG-Thy1: Mix the AGNPS dispersion and thyme essential oil in a volume ratio of 4:1, shear at 12000 rpm for 1 min, and homogenize at 500 MPa for 3 min to obtain AG-Thy1, denoted as A1; S4. Preparation of a polyvinyl alcohol-chitosan PVA / CS1 / A1 nanofiber membrane loaded with antibacterial amino-gelatin nanocapsule: Use 0.15 g / mL PVA, 0.05 g / mL CS (CS1), and 0.03 g / ml A1 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio of 3:1) as the solvent. After stirring at 40 °C for 4 h, perform electrospinning at a voltage of 22 kV for 4 h to obtain a nanofiber membrane; S5. Preparation of a dual-responsive nanoantibacterial fiber membrane: Place the PVA / CS1 / A1 nanofiber membrane and citral in a closed space with a mass-volume ratio of 1:2, and leave it at room temperature for 3 days to obtain a Cit1@PVA / CS1 / A1 dual-responsive antibacterial nanofiber membrane. Embodiment 2 A preparation method of a humidity- and gelatinase-dual-responsive antibacterial nanofiber membrane includes the following steps: S1. Preparation of amino-gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution. While stirring, add 16 g of 1,2-ethylenediamine. Adjust the pH to 5.0 with 5 M hydrochloric acid. Add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Make up the volume to 250 mL with phosphate buffer solution. Incubate at 37 °C for 2 h, then dialyze in pure water for 3 days and freeze-dry to obtain amino-gelatin AGNPS; S2. Preparation of amino-gelatin dispersion: Dissolve 1.25 g of freeze-dried amino-gelatin in 25 mL of distilled water and dissolve it completely. Add 25 mL of acetone and 25 mL of distilled water to the solution in sequence. Adjust the pH to 4.0 with 1 M hydrochloric acid. Stir slowly and dropwise add acetone until the solution turns milky white. Then add 200 μL of glutaraldehyde solution. Incubate AGNPS at 50 °C for 3 h, then rotary evaporate and make up the volume to a 250 mL volumetric flask to obtain AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of antibacterial amino-gelatin nanocapsule AG-Thy2: Mix the AGNPS dispersion and thymol essential oil at a volume ratio of 4.5:1. Shear at 12000 rpm for 1 min, then homogenize at 500 MPa for 3 min to obtain AG-Thy2, denoted as A2; S4. Preparation of polyvinyl alcohol-chitosan PVA / CS1 / A2 nanofiber membrane loaded with antibacterial amino-gelatin nanocapsule: Use 0.15 g / mL PVA, 0.05 g / mL CS (CS1) and 0.03 g / ml A2 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. Stir at 40 °C for 4 h, then perform electrospinning at 22 kV voltage for 4 h to prepare the nanofiber membrane; S5. Preparation of dual-responsive nano antibacterial fiber membrane: Place the PVA / CS1 / A2 nanofiber membrane and citral in a closed space at a mass-volume ratio of 1:2 and let it stand at room temperature for 3 days to prepare the Cit1@PVA / CS1 / A2 dual-responsive antibacterial nanofiber membrane. Example 3 A preparation method of a humidity and gelatinase dual-responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of amino-gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution. While stirring, add 16 g of 1,2-ethylenediamine. Adjust the pH to 5.0 with 5 M hydrochloric acid. Add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Make up the volume to 250 mL with phosphate buffer solution. Incubate at 37 °C for 2 h, then dialyze in pure water for 3 days and freeze-dry to obtain amino-gelatin AGNPS; S2. Preparation of amino - gelatin dispersion: Dissolve 1.25 g of freeze - dried amino - gelatin in 25 mL of distilled water, and fully dissolve it. Then, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence. Adjust the pH to 4.0 with 1 M hydrochloric acid, slowly stir and dropwise add acetone until the solution turns milky white. Subsequently, add 200 μL of glutaraldehyde solution. After incubating AGNPS at 50 °C for 3 h, perform rotary evaporation and make up the volume to a 250 - mL volumetric flask to obtain the AGNPS dispersion, and store it refrigerated at 4 °C for later use; S3. Preparation of antibacterial amino - gelatin nano - inclusion complex AG - Thy3: Mix the AGNPS dispersion and thymol essential oil with a volume ratio of 5:1. After shearing at 12000 rpm for 1 min, homogenize at 500 MPa for 3 min to obtain AG - Thy3, denoted as A3; S4. Preparation of polyvinyl alcohol - chitosan PVA / CS1 / A3 nanofiber membrane loaded with antibacterial amino - gelatin nano - inclusion complex: Use 0.15 g / mL PVA, 0.05 g / mL CS (CS1), and 0.03 g / ml A3 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. After stirring at 40 °C for 4 h, perform electrospinning at a voltage of 22 kV for 4 h to prepare the nanofiber membrane; S5. Preparation of dual - responsive nano - antibacterial fiber membrane: Place the PVA / CS1 / A3 nanofiber membrane and citral in a closed space with a mass - to - volume ratio of 1:2, and leave it at room temperature for 3 days to prepare the Cit1@PVA / CS1 / A3 dual - responsive antibacterial nanofiber membrane. Example 4 A preparation method of a humidity - and gelatinase - dual - responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of amino - gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution. While stirring, add 16 g of 1,2 - ethylenediamine, adjust the pH to 5.0 with 5 M hydrochloric acid, add 3.055 g of 1 - ethyl - 3 - (3 - dimethylaminopropyl) - carbodiimide hydrochloride, make up the volume to 250 mL with phosphate buffer solution. After incubating at 37 °C for 2 h, dialyze in pure water for 3 days and then freeze - dry to obtain amino - gelatin AGNPS; S2. Preparation of amino - gelatin dispersion: Dissolve 1.25 g of freeze - dried amino - gelatin in 25 mL of distilled water, and fully dissolve it. Then, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence. Adjust the pH to 4.0 with 1 M hydrochloric acid, slowly stir and dropwise add acetone until the solution turns milky white. Subsequently, add 200 μL of glutaraldehyde solution. After incubating AGNPS at 50 °C for 3 h, perform rotary evaporation and make up the volume to a 250 - mL volumetric flask to obtain the AGNPS dispersion, and store it refrigerated at 4 °C for later use; S3. Preparation of antibacterial amino - gelatin nanocapsule AG - Thy1: Mix the AGNPS dispersion and thyme essential oil with a volume ratio of 4:1. After shearing at 12000 rpm for 1 min, homogenize at 500 MPa for 3 min to obtain AG - Thy1, denoted as A1; S4. Preparation of polyvinyl alcohol - chitosan PVA / CS2 / A1 nanofiber membrane loaded with antibacterial amino - gelatin nanocapsule: Use 0.15 g / mL PVA, 0.08 g / mL CS (CS2), and 0.03 g / ml A1 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. Stir at 40 °C for 4 h, and then perform electrospinning at 22 kV for 4 h to obtain the nanofiber membrane; S5. Preparation of dual - responsive nano - antibacterial fiber membrane: Place the PVA / CS2 / A1 nanofiber membrane and citral in a closed space with a mass - to - volume ratio of 1:2, and leave it at room temperature for 3 days to obtain the Cit1@PVA / CS2 / A1 dual - responsive antibacterial nanofiber membrane. Example 5 A preparation method of a humidity - and gelatinase - dual - responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of amino - gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer. While stirring, add 16 g of 1,2 - ethylenediamine, adjust the pH to 5.0 with 5M hydrochloric acid, add 3.055 g of 1 - ethyl - 3 - (3 - dimethylaminopropyl) - carbodiimide hydrochloride, and make up the volume to 250 mL with phosphate buffer. After incubating at 37 °C for 2 h, dialyze in pure water for 3 days and freeze - dry to obtain amino - gelatin AGNPS; S2. Preparation of amino - gelatin dispersion: Dissolve 1.25 g of freeze - dried amino - gelatin in 25 mL of distilled water, and fully dissolve it. Then, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence. Adjust the pH to 4.0 with 1M hydrochloric acid, slowly stir and dropwise add acetone until the solution turns milky white. Subsequently, add 200 μL of glutaraldehyde solution. Incubate AGNPS at 50 °C for 3 h, then perform rotary evaporation and make up the volume to a 250 - mL volumetric flask to obtain the AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of antibacterial amino - gelatin nanocapsule AG - Thy1: Mix the AGNPS dispersion and thyme essential oil with a volume ratio of 4:1. After shearing at 12000 rpm for 1 min, homogenize at 500 MPa for 3 min to obtain AG - Thy1, denoted as A1; S4. Preparation of Polyvinyl Alcohol-Chitosan PVA / CS3 / A1 Nanofiber Membrane Loaded with Antibacterial Amino-Gelatin Nanocapsules: Using 0.15 g / mL PVA, 0.10 g / mL CS (CS3), and 0.03 g / ml A1 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. After stirring at 40 °C for 4 h, electrospinning was carried out at 22 kV for 4 h to obtain the nanofiber membrane; S5. Preparation of Dual-Responsive Nano-Antibacterial Fiber Membrane: Placing the PVA / CS3 / A1 nanofiber membrane and citral in a closed space with a mass-to-volume ratio of 1:2 and leaving it at room temperature for 3 days to obtain the Cit1@PVA / CS3 / A1 dual-responsive antibacterial nanofiber membrane. Example 6 A method for preparing a humidity and gelatinase dual-responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of Amino-Gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution, add 16 g of 1,2-ethylenediamine while stirring, adjust the pH to 5.0 with 5M hydrochloric acid, add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and make up the volume to 250 mL with phosphate buffer solution. After incubating at 37 °C for 2 h, dialyze in pure water for 3 days and freeze-dry to obtain amino-gelatin AGNPS; S2. Preparation of Amino-Gelatin Dispersion: Dissolve 1.25 g of freeze-dried amino-gelatin in 25 mL of distilled water, fully dissolve it, successively add 25 mL of acetone and 25 mL of distilled water to the solution, adjust the pH to 4.0 with 1M hydrochloric acid, slowly stir and drip acetone until the solution turns milky white, then add 200 μL of glutaraldehyde solution. After incubating AGNPS at 50 °C for 3 h, rotary evaporate and make up the volume to a 250 mL volumetric flask to obtain the AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of Antibacterial Amino-Gelatin Nanocapsules AG-Thy1: Mix the AGNPS dispersion and thyme essential oil with a volume ratio of 4:1, shear at 12000 rpm for 1 min, and homogenize at 500 MPa for 3 min to obtain AG-Thy1, denoted as A1; S4. Preparation of Polyvinyl Alcohol-Chitosan PVA / CS1 / A1 Nanofiber Membrane Loaded with Antibacterial Amino-Gelatin Nanocapsules: Using 0.15 g / mL PVA, 0.05 g / mL CS1, and 0.03 g / ml A1 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. After stirring at 40 °C for 4 h, electrospinning was carried out at 22 kV for 4 h to obtain the nanofiber membrane; S5. Preparation of dual-responsive nano-antibacterial fiber membrane: Place the PVA / CS1 / A1 nanofiber membrane and citral in a closed space at a mass-to-volume ratio of 1:2.5 and leave it at room temperature for 3 days to obtain the Cit2@PVA / CS1 / A1 dual-responsive antibacterial nanofiber membrane. Example 7 A method for preparing a humidity- and gelatinase-dual-responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of amino-gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution, add 16 g of 1,2-ethylenediamine while stirring, adjust the pH to 5.0 with 5 M hydrochloric acid, add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, make up the volume to 250 mL with phosphate buffer solution, incubate at 37 °C for 2 h, dialyze in pure water for 3 days, and freeze-dry to obtain amino-gelatin AGNPS; S2. Preparation of amino-gelatin dispersion: Dissolve 1.25 g of freeze-dried amino-gelatin in 25 mL of distilled water, fully dissolve it, successively add 25 mL of acetone and 25 mL of distilled water to the solution, adjust the pH to 4.0 with 1 M hydrochloric acid, slowly stir and dropwise add acetone until the solution turns milky white, then add 200 μL of glutaraldehyde solution, incubate AGNPS at 50 °C for 3 h, rotary evaporate, and make up the volume to a 250 mL volumetric flask to obtain the AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of antibacterial amino-gelatin nano-inclusion complex AG-Thy1: Mix the AGNPS dispersion and thyme essential oil at a volume ratio of 4:1, shear at 12000 rpm for 1 min, and homogenize at 500 MPa for 3 min to obtain AG-Thy1, denoted as A1; S4. Preparation of polyvinyl alcohol-chitosan PVA / CS1 / A1 nanofiber membrane loaded with antibacterial amino-gelatin nano-inclusion complex: Use 0.15 g / mL PVA, 0.05 g / mL CS1, and 0.03 g / ml A1 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio of 3:1) as the solvent. After stirring at 40 °C for 4 h, perform electrospinning at a voltage of 22 kV for 4 h to obtain the nanofiber membrane; S5. Preparation of dual-responsive nano-antibacterial fiber membrane: Place the PVA / CS1 / A1 nanofiber membrane and citral in a closed space at a mass-to-volume ratio of 1:3 and leave it at room temperature for 3 days to obtain the Cit3@PVA / CS1 / A1 dual-responsive antibacterial nanofiber membrane. Comparative Example 1 The difference between this comparative example and Example 1 is that citral is not added, specifically as follows: A method for preparing a humidity- and gelatinase-dual-responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of Amino Gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution. While stirring, add 16 g of 1,2-ethylenediamine. Adjust the pH to 5.0 with 5M hydrochloric acid. Add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Make up the volume to 250 mL with phosphate buffer solution. Incubate at 37 °C for 2 h, then dialyze in pure water for 3 days and freeze-dry to obtain amino gelatin AGNPS; S2. Preparation of Amino Gelatin Dispersion: Dissolve 1.25 g of freeze-dried amino gelatin in 25 mL of distilled water. After complete dissolution, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence. Adjust the pH to 4.0 with 1M hydrochloric acid. Slowly stir and dropwise add acetone until the solution turns milky white. Then add 200 μL of glutaraldehyde solution. Incubate AGNPS at 50 °C for 3 h, then rotary evaporate and make up the volume to a 250 mL volumetric flask to obtain AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of Antibacterial Amino Gelatin Nanocapsule AG-Thy1: Mix the AGNPS dispersion and thymol essential oil at a volume ratio of 4:1. After shearing at 12000 rpm for 1 min, homogenize at 500 MPa for 3 min to obtain AG-Thy1, denoted as A1; S4. Preparation of Polyvinyl Alcohol-Chitosan PVA / CS1 / A1 Nanofiber Membrane Loaded with Antibacterial Amino Gelatin Nanocapsule: Use 0.15 g / mL PVA, 0.05 g / mL CS1, and 0.03 g / ml A1 as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. Stir at 40 °C for 4 h, then perform electrospinning at 22 kV voltage for 4 h to obtain the PVA / CS1 / A1 nanofiber membrane. Comparative Example 2 The difference between this comparative example and Example 1 is that the antibacterial amino gelatin nanocapsule AG-Thy is not added. Specifically as follows: A preparation method of a humidity and gelatinase dual-responsive antibacterial nanofiber membrane includes the following steps: S1. Preparation of Polyvinyl Alcohol-Chitosan PVA / CS1 Nanofiber Membrane: Use 0.15 g / mL PVA and 0.05 g / mL CS (CS1) as the electrospinning solution, and a mixed solution of water and acetic acid (volume ratio 3:1) as the solvent. Stir at 40 °C for 4 h, then perform electrospinning at 22 kV voltage for 4 h to obtain the nanofiber membrane; S2. Preparation of Dual-Responsive Nanobacterial Fiber Membrane: Place the PVA / CS1 nanofiber membrane and citral in a closed space at a mass-volume ratio of 1:2 and leave it at room temperature for 3 days to obtain the Cit1@PVA / CS1 dual-responsive antibacterial nanofiber membrane. Comparative Example 3 The difference between this comparative example and Example 1 is that chitosan was not added, and the details are as follows: A method for preparing a humidity and gelatinase dual-responsive antibacterial nanofiber membrane, comprising the following steps: S1. Preparation of amino-gelatin: Dissolve 5.0 g of gelatin in 125 mL of phosphate buffer solution, add 16 g of 1,2-ethylenediamine while stirring, adjust the pH to 5.0 with 5 M hydrochloric acid, add 3.055 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, and make up the volume to 250 mL with phosphate buffer solution. After incubating at 37 °C for 2 h, dialyze in pure water for 3 days and freeze-dry to obtain amino-gelatin AGNPS; S2. Preparation of amino-gelatin dispersion: Dissolve 1.25 g of freeze-dried amino-gelatin in 25 mL of distilled water, dissolve it completely, add 25 mL of acetone and 25 mL of distilled water to the solution in sequence, adjust the pH to 4.0 with 1 M hydrochloric acid, stir slowly and dropwise add acetone until the solution turns milky white, then add 200 μL of glutaraldehyde solution. After incubating AGNPS at 50 °C for 3 h, perform rotary evaporation and make up the volume to a 250 mL volumetric flask to obtain AGNPS dispersion, and store it at 4 °C for later use; S3. Preparation of antibacterial amino-gelatin nano-inclusion A1: Mix the AGNPS dispersion and thymol essential oil in a volume ratio of 4:1, shear at 12000 rpm for 1 min, and homogenize at 500 MPa for 3 min to obtain AG-Thy1, denoted as A1; S4. Preparation of polyvinyl alcohol PVA / A1 nanofiber membrane loaded with antibacterial amino-gelatin nano-inclusion: Using 0.15 g / mL PVA and 0.03 g / ml A1 as the electrospinning solution, a mixed solution of water and acetic acid (volume ratio of 3:1) as the solvent, stir at 40 °C for 4 h, and then perform electrospinning at 22 kV for 4 h to prepare a nanofiber membrane; S5. Preparation of dual-responsive nano-antibacterial fiber membrane: Place the PVA / A1 nanofiber membrane and citral in a closed space with a mass-volume ratio of 1:2, and let it stand at room temperature for 3 days to prepare the Cit1@PVA / A1 dual-responsive antibacterial nanofiber membrane. Performance test: (1) Mechanical properties of the nanofiber membrane Measure the mechanical properties of the nanofiber membranes prepared in Examples 1-7 and Comparative Examples 1-3. The tensile strength and elongation at break of the plastic wrap are tested according to the GB / T 1040-2006 standard. Use a strip sampler to cut strip samples with a specification of 100 mm × 10 mm on coated paper, and fix the samples between the tensile clamps of an electronic universal tensile machine. The initial distance between the clamps is 50 mm. Table 1 Mechanical properties of the nanofiber membranes prepared in Examples 1-7 and Comparative Examples 1-3 As can be seen from Table 1, the tensile strength of the dual-responsive antibacterial nanofiber membranes prepared in Examples 1-7 was 28.56-39.52 MPa, and the elongation at break was 87.92-115.87%. The tensile strength of the nanofiber membrane prepared in Comparative Example 1 without adding citral decreased significantly, and the elongation at break increased significantly; for the nanofiber membranes prepared in Comparative Example 2 without adding antibacterial amino-gelatin nanocomposites and Comparative Example 3 without adding chitosan, the mechanical strength changed little. (2) Microstructure of the nanofiber membrane The electrospun nanofiber membranes with different shell materials prepared by the methods described in Example 1 and Comparative Examples 1-3 above were observed by scanning electron microscopy. The electrospun nanofiber membranes with different shell materials were cut into 1 cm × 1 cm pieces. After sputtering gold on the surface, the surface structure of the fibers was observed with a scanning electron microscope. From Figure 2 it can be seen that after adding chitosan and antibacterial amino-gelatin nanocomposites, nodules appeared on the fibers and the thickness was uneven, indicating the successful loading of antibacterial amino-gelatin nanocomposites in the fibers. Among them, the nodules of the fibers added with chitosan were particularly obvious; Figure 2 (b) and (c) prove that the successful loading of citral in the fibers requires the addition of chitosan; Figure 2 (d) Compared with Figure 2 (c), the citral loading amount is significantly increased, and the fiber distribution is more dense and uniform, indicating that the addition of antibacterial amino-gelatin nanocomposites and chitosan improves the citral loading in the fibers. (3) Fresh-keeping performance of the nanofiber membrane for meat products After applying the nanofiber membranes prepared in Examples 1-7 and Comparative Examples 1-3 to store fresh pork for 20 days, the pH and total colony count were measured. The results are as Figure 3 、 4 shown. As Figure 3 can be seen, with the prolongation of the storage time, the pH values of each group increased to varying degrees. Among them, the pH values of the groups of Comparative Examples 1-3 increased relatively rapidly, while the growth rates of the groups of Examples 1-7 were relatively slow. The growth rate of Example 1 group was the slowest. At the 20th day of storage, its pH value was still lower than 6.3, belonging to the first-class freshness range; the groups of Examples 3 and 6-7 had initial spoilage after 16 days of storage; however, the pH values of the groups of Comparative Examples 1-3 were greater than 6.7 after 16 days of storage, belonging to spoiled meat. As Figure 4 can be seen, the total colony count in fresh meat showed an upward trend during storage. Among them, the growth rate of Example 1 group was the slowest and always remained at the lowest level. (4) Study the release behavior of thyme essential oil in the Cit1@PVA / CS1 / A1 nanofiber membrane at different concentrations of gelatinase 100 mg of the fiber membranes prepared in Example 1 were respectively placed into 50 mL of PBS solutions containing gelatinase (30,000 U / mL) at concentrations of 0, 0.5, 1.0, and 1.5 mg / mL, oscillated at a rotation speed of 150 rpm. 1 mL of the release solution was taken at set time intervals, and fresh PBS solution was replenished. The experiment was repeated 3 times. The absorbance of the release solution was measured using a UV-visible spectrophotometer. The release concentration and mass of thymol essential oil were calculated according to the standard curve, and the cumulative release curve of thymol essential oil at different gelatinase concentrations was plotted. As Figure 5 shown, as the gelatinase concentration increased, the release rate of thymol essential oil accelerated. (5) Study the release behavior of Cit in the Cit1@PVA / CS1 / A1 nanofiber membrane at different relative humidities (35%, 55%, 75%, 95%) The results are as Figure 6 shown. When the humidity of the Cit1@PVA / CS1 / A1 nanofiber membrane prepared in Example 1 was 35%, only about 10% of citral was released on the 12th day; when the humidity was 55%, about 31% of citral was released on the 12th day; when the humidity was 75%, 80% of citral was released; when the humidity was 95%, citral was basically completely released on the 10th day. Therefore, the release results indicate that the release of citral from the Cit1@PVA / CS1 / A1 nanofiber membrane can be triggered by high humidity, and the fresh-keeping film can be stored at low humidity.

Claims

1. A dual-response antibacterial nanofiber membrane, characterized in that: The dual response is dual responsiveness to humidity and gelatinase.

2. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 1, characterized in that: The following steps are involved: S1. Preparation of amino gelatin: dissolve gelatin in phosphate buffer, add 1,2-ethylenediamine while stirring, adjust the pH to 5.0 with hydrochloric acid, add water-soluble carbodiimide hydrochloride, incubate at 30-40°C for 2-3h, dialyze, and freeze-dry to obtain amino gelatin; S2. Preparation of amino gelatin dispersion: Dissolve freeze-dried amino gelatin in distilled water until fully dissolved, add acetone and distilled water to the solution in sequence, adjust the pH to 4.0 with hydrochloric acid, slowly stir and drop acetone until the solution turns milky white, then add glutaraldehyde solution, incubate the amino gelatin at 45-55°C for 3-5h, and then rotary evaporate to obtain an amino gelatin dispersion, refrigerate at 4°C for later use; S3. Preparation of antibacterial amino gelatin nano-inclusion complex: The amino gelatin dispersion was mixed with 20% thyme essential oil, sheared at 12000rpm for 1min, and homogenized at 500MPa for 3min to obtain an antibacterial amino gelatin nano-inclusion complex; S4. Preparation of nanofiber membrane loaded with antibacterial amino-gelatin nano-inclusion complex: using a mixed solution of water and acetic acid as a solvent, preparing an electrospinning solution containing 0.15-0.25 g / mL PVA, 0.05-0.15 g / mL CS and 0.03-0.13 g / mL antibacterial amino-gelatin nano-inclusion complex, and electrospinning for 3-5 h at a voltage of 20-25 kV to obtain a nanofiber membrane loaded with antibacterial amino-gelatin nano-inclusion complex; S5. Preparation of dual-responsive antibacterial nanofiber membrane: Place the nanofiber membrane loaded with antibacterial amino-gelatin nanoinclusion complex together with citral in a closed space and leave it at room temperature for 3-5 days to obtain a dual-responsive antibacterial nanofiber membrane.

3. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 2, characterized in that: In step S1, the mass volume ratio of gelatin to phosphate buffer is 1:(20-25); the concentration of the phosphate buffer is 0.1 M and the pH is 5.

0.

4. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 2, characterized in that: In the step S1, the mass ratio of 1,2-ethylenediamine to gelatin is (3.0-3.5):1; the mass ratio of the water-soluble carbodiimide hydrochloride to gelatin is (0.5-0.8):

1.

5. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 2, characterized in that: In step S3, the volume ratio of the amination gelatin dispersion to the thyme essential oil is (4.0-5.0):

1.

6. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 2, characterized in that: The volume ratio of water to acetic acid in step S4 is (2.5-3.5):

1.

7. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 2, characterized in that: The electrospinning parameters in step S4 are: the distance between the electrospinning nozzle and the substrate is 16-18 cm, and the relative humidity is 50-60%.

8. The method for preparing a dual-responsive antibacterial nanofiber membrane according to claim 2, characterized in that: In the step S5, the mass volume ratio of the nanofiber membrane loaded with the antibacterial amino-gelatin nano-inclusion complex to citral is 1:(2-3).

9. Use of the dual-responsive antibacterial nanofiber membrane according to claim 1 in meat preservation.

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