A eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane and a preparation method thereof

Abstract

The application discloses a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane and a preparation method thereof. The method comprises the following steps: based on soybean lecithin and cholesterol, adding eugenol and Tween 80 to prepare a lipid solution, injecting the lipid solution into water or a phosphate buffer to prepare eugenol liposome suspension with an eugenol embedding rate greater than 88%; based on chitosan and a crosslinking agent, preparing a crosslinked chitosan acetic acid solution; preparing a polyethylene oxide acetic acid solution, mixing the crosslinked chitosan acetic acid solution and the polyethylene oxide acetic acid solution to obtain a base spinning solution; mixing the eugenol liposome suspension with the base spinning solution to prepare a final spinning solution, and adopting an electrospinning method to prepare the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane. The eugenol embedding rate is high, the water contact angle of the fiber membrane can reach more than 90 degrees, the slow-release effect of the eugenol is good, and the antibacterial and fresh-keeping effects are improved.

Description

Technical Field

[0001] This invention relates to the field of bio-based food preservation materials technology, and in particular to an eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane and its preparation method. Background Technology

[0002] Eugenol and other plant essential oils are considered ideal natural food preservatives due to their broad-spectrum antibacterial properties and strong antioxidant capabilities. However, their high volatility, water insolubility, and photothermal instability severely limit their application in food preservation. Liposome encapsulation technology, by embedding active ingredients in a phospholipid bilayer, can significantly reduce volatilization loss and delay oxidative degradation. Ethanol injection is a relatively mature technology for preparing eugenol liposomes, achieving high encapsulation rates. However, single-liposome encapsulation technology suffers from problems such as uneven particle size, poor storage stability, and weak interfacial bonding with polymer substrates. Electrospinning technology is simple to operate, requiring no heating during encapsulation, and is suitable for encapsulating non-bioactive substances. The negatively charged eugenol liposomes in the spinning solution, through electrostatic interaction with cations on the chitosan backbone, further stabilize the encapsulation of eugenol, resulting in minimal eugenol loss during electrospinning and thus improving the encapsulation efficiency of eugenol by nanofibers. However, nanofibers prepared by electrospinning technology have a large specific surface area, resulting in a severe burst release effect of eugenol at the beginning, which is not conducive to the slow and long-term release of eugenol and other plant essential oils.

[0003] Chitosan and polyethylene oxide are both non-toxic and environmentally friendly, and are common substrates for electrospun fibers. Pure chitosan solutions have high viscosity, and the repulsive forces between cations in the chitosan backbone hinder the formation of electrospun fibers. Adding polyethylene oxide and eugenol liposomes to the chitosan solution not only reduces the viscosity but also significantly improves the spinnability of the solution by utilizing the electrostatic interaction between the eugenol liposomes and chitosan. However, on the other hand, the hydrophilicity of polyethylene oxide and eugenol liposomes further degrades the water resistance and strength of the chitosan / polyethylene oxide electrospun fiber membrane. In the preservation of high-moisture foods, the burst release effect of eugenol after fiber swelling is more severe, making it difficult to achieve long-term preservation of high-moisture foods.

[0004] Crosslinking is an effective method to improve the hydrophobicity of chitosan films. In studies on the hydrophobic modification of chitosan-based composite hydrophilic systems, non-food additive crosslinking agents such as glutaraldehyde are often used, or toxic organic reagents are used in the preparation process. The resulting modified films are unsuitable for use as packaging materials in direct contact with food. Current reports indicate that the use of food additives as crosslinking agents has limited effect on the hydrophobic modification of chitosan / polyvinyl oxide composite hydrophilic systems. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane and its preparation method, aiming to solve the problems of low loading rate and poor encapsulation of eugenol in the existing fiber membrane, resulting in poor preservation effect.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a method for preparing an eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane includes: (1) Phospholipids and cholesterol were dissolved in anhydrous ethanol at a mass ratio of 5.5:1. The concentrations of phospholipids and cholesterol were 13.75 mg / mL and 2.5 mg / mL, respectively. The mixture was stirred in a water bath at 60°C for 30 min. After cooling to room temperature, eugenol and Tween 80 were added to the beaker in sequence and stirred at room temperature for 30 min to obtain a lipid solution. The lipid solution was injected into water or phosphate buffer and eugenol liposome suspension was prepared under reduced pressure at a temperature not exceeding 35°C. The encapsulation rate of eugenol in the liposome suspension was greater than 88%. (2) Chitosan with a degree of deacetylation ≥ 95% was dissolved in 0.5 mol / L acetic acid solution and magnetically stirred under a water bath at 60°C to prepare a 5% chitosan acetic acid solution. A crosslinking agent was added to the 5% chitosan acetic acid solution and dissolved in the above chitosan acetic acid solution under stirring at a water bath at 60°C. The mixture was stirred evenly to obtain a crosslinked chitosan acetic acid solution. (3) Dissolve polyethylene oxide in 0.5 mol / L acetic acid solution and stir until dissolved to prepare 4% polyethylene oxide acetic acid solution. Mix the above cross-linked chitosan acetic acid solution and polyethylene oxide acetic acid solution to prepare substrate spinning solution. (4) The final spinning solution is prepared by mixing the eugenol liposome suspension with the substrate spinning solution, and the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane is prepared by electrospinning.

[0007] Furthermore, in step (1), the phospholipid compound is soybean lecithin or other phospholipid compounds with a structure similar to soybean lecithin, the concentration of eugenol in the lipid solution is 30~60 μL / mL, and the concentration of Tween 80 is 13~16 μL / mL.

[0008] Furthermore, in step (1), the volume ratio of anhydrous ethanol to phosphate buffer is 1:2, the pH value of phosphate buffer is 7.2-7.4, and the concentration is 50 mmol / L. The lipid solution is injected into water or phosphate buffer, stirred and mixed, and the resulting liquid is evaporated at 35°C to remove ethanol. Then, it is sonicated at 350 W for 20 min to form a uniform eugenol liposome suspension.

[0009] Furthermore, in step (2), the crosslinking agent is cinnamaldehyde or citric acid or a combination thereof. When the crosslinking agent is cinnamaldehyde, the amount of cinnamaldehyde added is 0.8 to 1% of the mass of chitosan; when the crosslinking agent is citric acid, the amount of citric acid added is 5 to 9% of the mass of chitosan; when the crosslinking agent is cinnamaldehyde and citric acid, the amount of cinnamaldehyde added is 0.8 to 1.2% of the mass of chitosan, and the amount of citric acid added is 1.5 to 2% of the mass of chitosan.

[0010] Furthermore, in step (3), the substrate spinning solution is a cross-linked chitosan acetate solution and a polyethylene oxide acetate solution mixed at a mass ratio of 1.4:1 to 1.6:1.

[0011] Furthermore, in step (4), the eugenol liposome suspension and the substrate spinning solution are mixed at a mass ratio of 0.8:2 to 1:2 to prepare the final spinning solution.

[0012] Furthermore, in step (4), the electrospinning process parameters are: positive voltage 19 kV, negative voltage -2.86 kV, flow rate 0.84 mL / h, spinning distance 14 cm, roller speed 25 r / min, translation speed 100 mm / min, temperature 25℃, and relative humidity 60%.

[0013] The second aspect is a hydrophobic electrospun fiber membrane of eugenol liposome / chitosan / polyoxyethylene, wherein the fiber diameter of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane is 60~200 nm, and the nanofibers form approximately circular irregular pores with a pore diameter of 100 nm~2 μm.

[0014] Furthermore, the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane is greater than 90°.

[0015] The technical solution adopted in this invention has the following beneficial effects: Cinnamaldehyde and citric acid are permitted additives in food. In this invention, cinnamaldehyde and citric acid are used to physically crosslink with chitosan. The addition of cinnamaldehyde and citric acid also regulates the phase separation process of PEO, resulting in an eugenol liposome / chitosan / polyethylene oxide fiber membrane structure with certain hydrophobic properties. In the preservation of high-moisture foods, the novel electrospun fiber structure facilitates the slow and long-lasting release of the bioactive substance eugenol, improving the preservation effect in high-moisture foods.

[0016] (1) The present invention uses liposome combined with electrospinning technology to prepare eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane. The method is simple and the raw materials are non-toxic. It is a green, environmentally friendly and safe method for preparing food preservation film.

[0017] (2) The present invention uses two technologies, liposomes and electrospinning, to encapsulate the biological preservative eugenol. During the preparation process, eugenol is not heated, resulting in less loss and a high eugenol encapsulation rate. This method not only helps to improve the utilization rate of eugenol, but also effectively reduces the influence of eugenol on the flavor of food during the preservation process.

[0018] (3) The present invention uses the ethanol injection method to prepare eugenol liposomes with an embedding rate of over 88% and a particle size of 80~180 nm, which is a single-layer vesicle structure.

[0019] (4) In the preparation of eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane by the method of the present invention, the crosslinking agents cinnamaldehyde and citric acid used are both food additives permitted in GB 2760. The amount of cinnamaldehyde used is less than 1 / 50 of the amount of eugenol used, and the amount of citric acid used is less than 1 / 5 of the amount of eugenol used. The amount of crosslinking agent added during the preparation process is small, which can be applied to food packaging that comes into direct contact with the product.

[0020] (5) The average diameter of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane nanofibers prepared by the method of the present invention is between 60 and 200 nm. The nanofibers interweave to form a dense network structure, and irregular pores of approximately circular shape are formed between the nanofibers, with pore diameters between 100 nm and 2 μm.

[0021] (6) The method of the present invention uses hydrophilic swelling chitosan and hydrophilic polyethylene oxide as substrates, water-soluble liposomes as carriers for the biopreservative eugenol, crosslinking chitosan with cinnamaldehyde and citric acid, and adjusting the phase separation process of polyethylene oxide to achieve the transformation of electrospun fiber membrane from hydrophilic to hydrophobic. Without the addition of crosslinking agents cinnamaldehyde and citric acid, the water contact angle of the electrospun fiber membrane is 40.6°. After crosslinking modification by the method of the present invention, the water contact angle of the electrospun fiber membrane can reach more than 90°.

[0022] (7) The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the method of the present invention has water absorption and swelling properties, and is suitable as an active packaging material for absorbing food juices. The hydrophobic electrospun fiber membrane has reduced water solubility and water swelling rate, enhanced structural stability, and exhibits slow and long-lasting release of eugenol in simulated food preservation solutions.

[0023] (8) The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the method of the present invention has a high eugenol loading rate. In food preservation applications, the crosslinking agents cinnamaldehyde and citric acid have a combined antioxidant and antibacterial effect with the biological preservative eugenol. It has shown excellent preservation effect in the preservation process of sea bass fillets and has potential application value in the field of food active packaging and other preservation materials. Attached Figure Description

[0024] Figure 1 This is a water contact angle diagram of the electrospun fiber membrane prepared under the process conditions of Example 1 of the present invention; Figure 2 This is a scanning electron microscope image of the surface of the electrospun fiber membrane obtained under the process conditions of Example 1 of the present invention; Figure 3 The image shows the XRD pattern of the electrospun fiber membrane prepared under the process conditions of Example 1 of this invention. Figure 4 This is a water contact angle diagram of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane in Example 2 of the present invention; Figure 5 This is a scanning electron microscope image of the surface of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process conditions of Example 2 of the present invention. Figure 6 The cumulative release curves of eugenol in a simulated release solution (phosphate buffer and anhydrous ethanol volume ratio of 90:10) are shown for the electrospun fiber membranes prepared under the process conditions of Examples 1 and 2 of this invention. Figure 7 The graph shows the changes in total bacterial count (A), TVB-N (B), TBA (C), and color difference (D~F) of sea bass fillets preserved by electrospun fiber membranes prepared according to the process of Example 1 and Example 2 during storage. Figure 8 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3 of this invention. Figure 9 The image shows a scanning electron microscope (SEM) image of the surface of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3. Figure 10 The image shows the XRD pattern of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the process in Example 3 of this invention. Figure 11 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared according to the process in Example 4 of this invention. Figure 12 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared according to the process in Example 5 of this invention. Figure 13 This is a water contact angle diagram of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the process in Example 6 of the present invention. Figure 14The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared according to the process in Example 7 of this invention. Figure 15 This is a water contact angle diagram of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the process in Example 8 of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] In this invention, a method for preparing an eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane is described, with the following specific steps: (1) Preparation of liposome solution Soybean lecithin and cholesterol were dissolved in anhydrous ethanol at a mass ratio of 5.5:1, with concentrations of 13.75 mg / mL and 2.5 mg / mL, respectively. The solution was stirred in a 60°C water bath for 30 min. After cooling to room temperature, eugenol and Tween 80 were added sequentially to a beaker, and the mixture was stirred at room temperature for 30 min to obtain a lipid solution. The lipid solution in the beaker was then added to twice the volume of anhydrous ethanol in water or phosphate buffer (pH=7.2-7.4, 50 mmol / L), and stirred until homogeneous. The resulting liquid was then subjected to rotary evaporation at 35°C to remove the ethanol, followed by sonication at 350 W for 20 min to form a homogeneous eugenol liposome suspension for later use.

[0027] Among them, soybean lecithin can be replaced by other phospholipids with similar structures; eugenol liposome suspension can be prepared by other methods, but the encapsulation rate of eugenol needs to reach more than 88%; the rotary evaporation process is carried out under certain reduced pressure conditions, the temperature does not exceed 35℃, and the reduced pressure conditions are sufficient to remove ethanol, and the negative pressure can be set according to actual needs.

[0028] (2) Cross-linked chitosan acetate solution A certain mass of chitosan (degree of deacetylation ≥ 95%) was weighed and dissolved in a certain volume of 0.5 mol / L acetic acid solution. The solution was magnetically stirred in a water bath at 60℃ to prepare a 5% (m / v) chitosan acetic acid solution. A certain mass of citric acid and cinnamaldehyde were weighed and dissolved in the above chitosan acetic acid solution under stirring in a water bath at 60℃. The solution was stirred evenly to obtain a cross-linked chitosan acetic acid solution.

[0029] The process can involve adding only one cross-linking agent, either citric acid or cinnamaldehyde, or both. When only citric acid or cinnamaldehyde is added, the amount of cinnamaldehyde is 0.8% to 1% of the chitosan mass, and the amount of citric acid is 5% to 9% of the chitosan mass. When both cross-linking agents are added, the amount of cinnamaldehyde is 0.8% to 1.2% of the chitosan mass, and the amount of citric acid is 1.5% to 2% of the chitosan mass.

[0030] (3) Preparation of substrate spinning solution A certain mass of polyethylene oxide (average molecular weight ~600,000) was weighed and dissolved in a certain volume of 0.5 mol / L acetic acid solution, and stirred until dissolved to prepare a 4% (m / v) polyethylene oxide acetic acid solution. The 4% polyethylene oxide acetic acid solution was added to the cross-linked chitosan acetic acid solution at a certain mass ratio and mixed evenly to obtain the substrate spinning solution.

[0031] The substrate spinning solution is a mixture of cross-linked chitosan acetate solution and polyethylene oxide acetate solution in a mass ratio of 1.4:1 to 1.6:1.

[0032] (4) Preparation of electrospun membrane The final spinning solution was prepared by mixing eugenol liposome suspension and substrate spinning solution at a mass ratio of 0.8:2 to 1:2. Electrospun membranes were prepared using uniaxial electrospinning. The electrospinning process parameters were set to achieve a good Taylor cone. The reference process parameters were: positive voltage 19 kV, negative voltage -2.86 kV, flow rate 0.84 mL / h, spinning distance 14 cm, roller speed 25 r / min, translation speed 100 mm / min, temperature 25℃, and relative humidity 60%.

[0033] Example 1: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Inject this lipid suspension into 20.0 mL of phosphate buffer (pH 7.2-7.4, 50 mmol / L) and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min to obtain eugenol liposomes for later use.

[0034] When the amount of eugenol added is zero, blank liposomes are prepared, and the rest of the preparation process is the same as above.

[0035] (2) Weigh 2.5 g of chitosan (degree of deacetylation ≥ 95%) and dissolve it in 50 mL of 0.5 mol / L acetic acid solution. Stir the solution magnetically in a water bath at 60 °C to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 25 mg of cinnamaldehyde into the above chitosan acetic acid solution and stir evenly in a water bath at 60 °C to obtain a cross-linked chitosan acetic acid solution.

[0036] (3) Weigh 1.5625 g of polyethylene oxide (average molecular weight ~600,000) and dissolve it in 39 mL of 0.5 mol / L acetic acid solution. Stir until dissolved to prepare a 4% (m / v) polyethylene oxide solution. Mix 16 g of crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution to prepare a chitosan / polyethylene oxide spinning solution.

[0037] When the amount of cinnamaldehyde added is zero, a chitosan / polyvinyl oxide spinning solution without crosslinking agent is prepared, and the rest of the preparation process is the same as above.

[0038] (4) After the substrate spinning solution has cooled, dissolve 13g of eugenol liposome suspension in 26g of the above substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane is prepared by uniaxial electrospinning. The electrospinning process should be carried out to form a good Taylor cone. The reference process parameters given above can be slightly adjusted.

[0039] According to the above method, 13 g of blank liposomes were dissolved in 26 g of chitosan / polyethylene oxide spinning solution without crosslinking agent to prepare blank liposome / chitosan / polyethylene oxide spinning solution without crosslinking agent.

[0040] According to the above method, 13 g of eugenol liposomes were dissolved in 26 g of uncrosslinked chitosan / polyethylene oxide spinning solution to prepare an eugenol liposome / chitosan / polyethylene oxide solution without the addition of crosslinking agent.

[0041] Polyethylene oxide solution, chitosan / polyethylene oxide spinning solution without crosslinking agent, blank liposome / chitosan / polyethylene oxide spinning solution without crosslinking agent, and eugenol liposome / chitosan / polyethylene oxide solution without crosslinking agent were electrospun to prepare polyethylene oxide fiber membranes, chitosan / polyethylene oxide fiber membranes, blank liposome / chitosan / polyethylene oxide fiber membranes, and eugenol liposome / chitosan / polyethylene oxide fiber membranes without crosslinking agent. The electrospinning process parameters were the same as above.

[0042] Figure 1The figures show the water contact angles of the electrospun fiber membranes prepared under the process conditions of Example 1 of this invention. (a) is a chitosan / polyethylene oxide fiber membrane without crosslinking agent; (b) is a blank liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; (c) is an eugenol liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; and (d) is an eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane. As can be seen from the figures, the water contact angle of the chitosan / polyethylene oxide fiber membrane is 75.2°; the water contact angle of the blank liposome / chitosan / polyethylene oxide fiber membrane is 41.5°; the water contact angle of the eugenol liposome / chitosan / polyethylene oxide electrospun fiber membrane prepared without crosslinking agent is 40.6°; and the water contact angle of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared with cinnamaldehyde crosslinking agent is 104.1°. The electrospun fiber membrane prepared from the substrate has good hydrophilicity, and the hydrophilicity is even better after the addition of liposomes. However, the electrospun fiber membrane becomes hydrophobic after the addition of cinnamaldehyde crosslinking agent.

[0043] Figure 2 The images show scanning electron microscope (SEM) images of the surface of the electrospun fiber membranes prepared under the process conditions of Example 1 of this invention. (a) is a chitosan / polyethylene oxide fiber membrane without crosslinking agent; (b) is a blank liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; (c) is an eugenol liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; and (d) is a cinnamaldehyde / eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane. Figure 2 In (d), most fibers have a diameter between 60 and 140 nm, with an average diameter of approximately 99 nm. (Compared to...) Figure 2 Compared to (a~c), some nanofibers exhibit entanglement, and a small number of fibers have bead-like structures. The nanofibers modified with cinnamaldehyde have a denser network structure, forming irregular, nearly circular pores with diameters ranging from 100 nm to 5 μm.

[0044] Figure 3 The XRD patterns of the electrospun fiber membranes prepared under the process conditions of Example 1 of this invention are shown below. (a) is a polyethylene oxide fiber membrane; (b) is a chitosan / polyethylene oxide fiber membrane without crosslinking agent; (c) is a blank liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; (d) is an eugenol liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; and (e) is a cinnamaldehyde / eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane. Comparing Figure (e) with Figures (b-d), it can be seen that the relative intensities of the diffraction peaks corresponding to PEO crystals at approximately 19.1° and 23.6° are significantly increased, indicating that the addition of cinnamaldehyde alters the phase separation process of PEO during film formation.

[0045] Example 2: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Inject this lipid suspension into 20.0 mL of phosphate buffer (pH 7.2-7.4, 50 mmol / L) and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0046] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of 0.5 mol / L acetic acid solution under magnetic stirring in a 60℃ water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 125 mg of citric acid into the above chitosan acetic acid solution and stir evenly in a 60℃ water bath to obtain a cross-linked chitosan acetic acid solution.

[0047] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 16.4 g of the above crosslinked chitosan acetic acid solution and 10.25 g of polyethylene oxide acetic acid solution (mass ratio of the two solutions 1.6:1) and stir evenly in a water bath at 60℃ to prepare the substrate spinning solution.

[0048] (4) After the substrate spinning solution has cooled, dissolve 13g of eugenol liposome suspension in 26g of substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared using a uniaxial electrospinning method. The electrospinning process should be sufficient to form a good Taylor cone; the reference process parameters given above can be slightly adjusted.

[0049] Figure 4 The diagram shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane in Example 2 of this invention, with a water contact angle of 95.0°.

[0050] Figure 5 This is a scanning electron microscope (SEM) image of the surface of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process conditions of Example 2 of this invention. The nanofibers are interwoven and adhered to form a dense network structure, with fiber diameters ranging from 50 to 200 nm. The fiber diameters are larger at the entanglement points, and a small number of bead-like structures are formed. Approximately circular irregular pores are formed between the nanofibers, with pore diameters ranging from 100 nm to 1.5 μm.

[0051] Table 1 shows the encapsulation rate data of eugenol in some electrospun fiber membranes prepared in Examples 1 and 2. The encapsulation rate of eugenol in the electrospun fiber membrane is the percentage of eugenol content in the fiber membrane relative to the total eugenol content in the eugenol liposome suspension added to the spinning solution. The encapsulation rate of eugenol in the eugenol liposome suspension is the percentage of eugenol in the liposome structure relative to the total eugenol content in the eugenol liposome suspension (the sum of eugenol in the liposomes and free eugenol). The eugenol concentration was determined by ultraviolet spectrophotometry (282 nm). The encapsulation rate of eugenol in the eugenol liposome suspension was 88.06%. In Example 1, the eugenol encapsulation rate in the eugenol liposome / chitosan / polyethylene oxide electrospun fiber membrane prepared without crosslinking agents was 59.68%. In the hydrophobic electrospun fiber membranes prepared in Example 1 and Example 2, the eugenol encapsulation rates were increased to 85.74% and 89.04%, respectively, due to the addition of crosslinking agents cinnamaldehyde and citric acid. Therefore, the addition of crosslinking agents cinnamaldehyde and citric acid significantly reduced the loss of eugenol during the electrospinning process.

[0052] Table 1 shows the encapsulation rate data of eugenol in some electrospun fiber membranes prepared in Examples 1 and 2 of the present invention. Fiber membrane Eugenol encapsulation rate (%) Eugenol liposome / chitosan / polyoxyethylene fiber membrane prepared in Example 1 without cross-linking agent <![CDATA[59.68±0.02 c ]]> Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared in Example 1 <![CDATA[85.74±0.01 b ]]> Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared in Example 2 <![CDATA[89.04±0.02 a ]]>

[0053] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0054] Figure 6 The cumulative release curves of eugenol in a simulated release solution (phosphate buffer and anhydrous ethanol volume ratio of 90:10) for the electrospun fiber membranes prepared under the process conditions of Examples 1 and 2 of this invention. (a) Eugenol liposome / chitosan / polyoxyethylene fiber membrane without crosslinking agent prepared in Example 1; (b) Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared in Example 1; (c) Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared in Example 2.

[0055] Eugenol concentration was determined using ultraviolet spectrophotometry (282 nm). In the figure, the black control curve represents the eugenol liposome / chitosan / polyoxyethylene fiber membrane prepared in Example 1 without crosslinking agent. At 4°C, the total release of each fiber membrane was similar in the first 12 hours. After reaching release equilibrium at 120 h, the final cumulative release of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared with cinnamaldehyde in Example 1 and the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared with citric acid in Example 2 was significantly higher than that of the control curve, which is consistent with the eugenol encapsulation rate data in the fiber membranes in Table 1. It can be inferred that the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared with cinnamaldehyde in Example 1 and the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared with citric acid in Example 2 slowed down the release of eugenol in the first 12 hours. This may be related to the hydrophobicity of the hydrophobic electrospun fiber membrane and the structural changes of the nanofiber membrane after the addition of a crosslinking agent.

[0056] Figure 7 The graphs show the changes in total bacterial count (A), TVB-N (B), TBA (C), and color difference (D~F) of sea bass fillets preserved using the electrospun fiber membranes prepared according to the processes of Examples 1 and 2 of this invention during storage; (a) control group without fiber membrane; (b) eugenol liposome / chitosan / polyoxyethylene fiber membrane group prepared in Example 1 without crosslinking agent; (c) eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane group prepared in Example 1; (d) eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane group prepared in Example 2.

[0057] The storage temperature was 4℃. The control group was not treated with the fiber membrane and was placed directly in a covered plastic box. Compared with the control group, the preservation indicators of other groups were better. The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane groups prepared in Examples 1 and 2 had the best preservation indicators. Under refrigeration conditions of 4℃, the shelf life of sea bass fillets prepared in Examples 1 and 2 with the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane groups could reach 9 days. The extended shelf life of sea bass fillets is due to the slow release of eugenol in the hydrophobic fiber membrane and the addition of cross-linking agents cinnamaldehyde and citric acid, which have a synergistic antibacterial effect with eugenol.

[0058] Example 3: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Inject this lipid suspension into 20.0 mL of phosphate buffer (pH 7.2-7.4, 50 mmol / L) and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0059] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of 0.5 mol / L acetic acid solution under magnetic stirring in a 60℃ water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 225 mg of citric acid into the above chitosan acetic acid solution and stir evenly in a 60℃ water bath to obtain a cross-linked chitosan acetic acid solution.

[0060] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 16 g of the above crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution (mass ratio of the two solutions 1.6:1) and stir evenly in a water bath at 60℃ to prepare the substrate spinning solution.

[0061] (4) After the substrate spinning solution has cooled, dissolve 13 g of eugenol liposome suspension in 26 g of substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared using a uniaxial electrospinning method. The electrospinning process should be sufficient to form a good Taylor cone; the reference process parameters given above can be slightly adjusted.

[0062] Figure 8 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3 of this invention. The water contact angle is 101.2°.

[0063] Figure 9 This is a scanning electron microscope (SEM) image of the surface of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3. The nanofibers are interwoven and adhered to form a dense network structure, with fiber diameters ranging from 80 to 200 nm. Irregular, approximately circular pores are formed between the nanofibers, with pore diameters ranging from 100 nm to 1.0 μm.

[0064] Figure 10The XRD patterns of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membranes prepared according to the process of Example 3 of this invention are shown below: (a) Polyoxyethylene fiber membrane prepared in Example 1; (b) Chitosan / polyoxyethylene fiber membrane without crosslinking agent prepared in Example 1; (c) Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared in Example 1; (d) Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared in Example 3. The figures also include control XRD curves. Figure 10 (a~c) represent the polyethylene oxide fiber membrane, the chitosan / polyethylene oxide fiber membrane without crosslinking agent, and the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared in Example 1 of the present invention, respectively. Figure 10 (d) and Figure 10 (c) Similarly, the relative intensities of the diffraction peaks corresponding to PEO crystals at 19.1° and 23.6° are significantly increased, indicating that the crosslinking of cinnamaldehyde, citric acid and chitosan alters the phase separation process of PEO during film formation.

[0065] Example 4: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Inject this lipid suspension into 20.0 mL of phosphate buffer (pH 7.2-7.4, 50 mmol / L) and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0066] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) under magnetic stirring in a 60 °C water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 20 mg of cinnamaldehyde and 50 mg of citric acid into the above chitosan acetic acid solution and stir evenly in a 60 °C water bath to obtain a cross-linked chitosan acetic acid solution.

[0067] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 16 g of the above crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution, dissolve them in the above chitosan acetic acid solution under stirring in a 60°C water bath, and stir evenly to prepare the substrate spinning solution.

[0068] (4) After the substrate spinning solution has cooled, dissolve 13 g of eugenol liposome suspension in 26 g of substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared using a uniaxial electrospinning method. The electrospinning process should be sufficient to form a good Taylor cone; the reference process parameters given above can be slightly adjusted.

[0069] Figure 11 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process of Example 4 of this invention, wherein the water contact angle is 92.0°.

[0070] Example 5: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Inject this lipid suspension into 20.0 mL of phosphate buffer (pH 7.2-7.4, 50 mmol / L) and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0071] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) under magnetic stirring in a 60 °C water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 25 mg of cinnamaldehyde and 37.5 mg of citric acid into the above chitosan acetic acid solution and stir evenly in a 60 °C water bath to obtain a cross-linked chitosan acetic acid solution.

[0072] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 14 g of the above crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution, dissolve them in the above chitosan acetic acid solution under stirring in a 60°C water bath, and stir evenly to prepare the substrate spinning solution.

[0073] (4) After the substrate spinning solution has cooled, 10.8 g of eugenol liposome suspension is dissolved in 24 g of substrate spinning solution and mixed evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared by uniaxial electrospinning. The electrospinning process is sufficient to form a good Taylor cone. The reference process parameters given above can be slightly adjusted.

[0074] Figure 12 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process of Example 5 of this invention, wherein the water contact angle is 91.2°.

[0075] Example 6: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 130 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Inject this lipid suspension into 20.0 mL of phosphate buffer (pH 7.2-7.4, 50 mmol / L) and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0076] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) under magnetic stirring in a 60 °C water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 20 mg of cinnamaldehyde into the above chitosan acetic acid solution and stir evenly in a 60 °C water bath to obtain a cross-linked chitosan acetic acid solution.

[0077] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 14 g of the above crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution, dissolve them in the above chitosan acetic acid solution under stirring in a 60°C water bath, and stir evenly to prepare the substrate spinning solution.

[0078] (4) After the substrate spinning solution has cooled, dissolve 9.6 g of eugenol liposome suspension in 24 g of substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared using a uniaxial electrospinning method. The electrospinning process should aim to form a good Taylor cone; the reference process parameters given above can be slightly adjusted.

[0079] Figure 13 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process described in Example 6 of this invention, wherein the water contact angle is 95.9°.

[0080] Example 7: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 130 μL of Tween 80 and 300 μL of eugenol to the beaker while stirring magnetically. Pour this lipid suspension into 20.0 mL of water and transfer it to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0081] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) under magnetic stirring in a 60 °C water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 20 mg of cinnamaldehyde and 20 mg of citric acid into the above chitosan acetic acid solution and stir evenly in a 60 °C water bath to obtain a cross-linked chitosan acetic acid solution.

[0082] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 14 g of the above crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution to prepare the substrate spinning solution.

[0083] (4) After the substrate spinning solution has cooled, dissolve 12 g of eugenol liposome suspension in 24 g of substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared using a uniaxial electrospinning method. The electrospinning process should be sufficient to form a good Taylor cone; the reference process parameters given above can be slightly adjusted.

[0084] Figure 14 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process of Example 7 of this invention, wherein the water contact angle is 92.4°.

[0085] Example 8: (1) Dissolve 0.1375 g of soybean lecithin and 0.025 g of cholesterol in a beaker containing 10.0 mL of anhydrous ethanol and stir magnetically for 30 minutes in a water bath at 60 °C. After cooling to room temperature, add 130 μL of Tween 80 and 600 μL of eugenol to the beaker while stirring magnetically. Pour this lipid suspension into 20.0 mL of water and transfer it to a round-bottom flask. Remove the ethanol by rotary evaporation at 35 °C, and sonicate the resulting suspension at 300 W for 20 min for later use.

[0086] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) under magnetic stirring in a 60 °C water bath to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 20 mg of cinnamaldehyde and 40 mg of citric acid into the above chitosan acetic acid solution and stir evenly in a 60 °C water bath to obtain a cross-linked chitosan acetic acid solution.

[0087] (3) Weigh 1.5625 g of polyethylene oxide and dissolve it in 39 mL of 0.5 mol / L acetic acid solution under magnetic stirring to prepare a 4% (m / v) polyethylene oxide acetic acid solution. Mix 15 g of the above crosslinked chitosan acetic acid solution and 10 g of polyethylene oxide acetic acid solution to prepare the substrate spinning solution.

[0088] (4) After the substrate spinning solution has cooled, dissolve 10 g of eugenol liposome suspension in 25 g of substrate spinning solution and mix evenly under magnetic stirring to prepare the final spinning solution. The electrospun membrane is prepared using a uniaxial electrospinning method. The electrospinning process should be designed to form a good Taylor cone. The reference process parameters given above can be slightly adjusted.

[0089] Figure 15 The image shows the water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared under the process of Example 8 of this invention, wherein the water contact angle is 97.7°.

[0090] As verified by the above embodiments, the natural polymer chitosan in this invention has good biocompatibility and biodegradability, and has wide applications in food, medicine, agriculture, and other fields. However, pure chitosan solution has high viscosity and exhibits cationic electrolyte characteristics. The interionic repulsion on the chitosan backbone hinders the formation of electrospun fibers. Many researchers have introduced polyethylene oxide into chitosan solution, which has greatly improved the spinnability of the solution. However, due to the hydrophilicity of chitosan and polyethylene oxide, the resulting composite fiber membrane has poor structural stability and water resistance during the preservation of foods with high water content. Adding eugenol in the form of liposomes to the chitosan / polyethylene oxide spinning solution can improve the stability of liposomes and the encapsulation rate of bioactive substances due to the electrostatic interaction between the cations on the chitosan chains and the negatively charged surface of the liposomes. However, eugenol liposomes are water-soluble, which further increases the hydrophilicity of the fiber membrane. The highly hydrophilic composite fiber membrane will swell and rupture rapidly, accelerating the release of the encapsulated eugenol.

[0091] The Schiff base crosslinking of cinnamaldehyde and chitosan, and the esterification of chitosan with citric acid, are both green crosslinking modification strategies to enhance the hydrophobic and film-forming properties of chitosan membranes. Utilizing the dynamic covalent bonds formed by the Schiff bases and the hydrolysis of the esterification reaction, the release of eugenol bioactive components can be regulated in food preservation applications. Simultaneously, because the crosslinking agents cinnamaldehyde and citric acid possess certain antibacterial and antioxidant capabilities, although used in small quantities, they also exert a synergistic antibacterial effect with eugenol in the preservation application of composite fiber membranes.

[0092] The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the method of this invention has the following beneficial effects: (1) The present invention uses liposome combined with electrospinning technology to prepare eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane. The method is simple and the raw materials are non-toxic. It is a green, environmentally friendly and safe method for preparing food preservation film.

[0093] (2) The present invention uses two technologies, liposomes and electrospinning, to encapsulate the biological preservative eugenol. During the preparation process, eugenol is not heated, resulting in less loss and a high eugenol encapsulation rate. This method not only helps to improve the utilization rate of eugenol, but also effectively reduces the influence of eugenol on the flavor of food during the preservation process.

[0094] (3) The present invention uses the ethanol injection method to prepare eugenol liposomes with an embedding rate of over 88% and a particle size of 80~180 nm, which is a single-layer vesicle structure.

[0095] (4) In the preparation of eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane by the method of the present invention, the crosslinking agents cinnamaldehyde and citric acid used are both food additives permitted in GB 2760. The amount of cinnamaldehyde used is less than 1 / 50 of the amount of eugenol used, and the amount of citric acid used is less than 1 / 5 of the amount of eugenol used. The amount of crosslinking agent added during the preparation process is small, which can be applied to food packaging that comes into direct contact with the product.

[0096] (5) The average diameter of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane nanofibers prepared by the method of the present invention is between 60 and 200 nm. The nanofibers interweave to form a dense network structure, and irregular pores of approximately circular shape are formed between the nanofibers, with pore diameters between 100 nm and 2 μm.

[0097] (6) The method of the present invention uses hydrophilic swelling chitosan and hydrophilic polyethylene oxide as substrates, water-soluble liposomes as carriers for the biopreservative eugenol, crosslinking chitosan with cinnamaldehyde and citric acid, and adjusting the phase separation process of polyethylene oxide to achieve the transformation of electrospun fiber membrane from hydrophilic to hydrophobic. Without the addition of crosslinking agents cinnamaldehyde and citric acid, the water contact angle of the electrospun fiber membrane is 40.6°. After crosslinking modification by the method of the present invention, the water contact angle of the electrospun fiber membrane can reach more than 90°.

[0098] (7) The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the method of the present invention has water absorption and swelling properties, and is suitable as an active packaging material for absorbing food juices. The hydrophobic electrospun fiber membrane has reduced water solubility and water swelling rate, enhanced structural stability, and exhibits slow and long-lasting release of eugenol in simulated food preservation solutions.

[0099] (8) The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the method of the present invention has a high eugenol loading rate. In food preservation applications, the crosslinking agents cinnamaldehyde and citric acid have a combined antioxidant and antibacterial effect with the biological preservative eugenol. It has shown excellent preservation effect in the preservation process of sea bass fillets and has potential application value in the field of food active packaging and other preservation materials.

[0100] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the solutions disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

Claims

1. A method for preparing an eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane, characterized in that, include: (1) Phospholipids and cholesterol were dissolved in anhydrous ethanol at a mass ratio of 5.5:

1. The concentrations of phospholipids and cholesterol were 13.75 mg / mL and 2.5 mg / mL, respectively. The mixture was stirred in a water bath at 60°C for 30 min. After cooling to room temperature, eugenol and Tween 80 were added to the beaker in sequence and stirred at room temperature for 30 min to obtain a lipid solution. The lipid solution was injected into water or phosphate buffer and eugenol liposome suspension was prepared under reduced pressure at a temperature not exceeding 35°C. The encapsulation rate of eugenol in the liposome suspension was greater than 88%. (2) Chitosan with a degree of deacetylation ≥ 95% was dissolved in 0.5 mol / L acetic acid solution and magnetically stirred under a water bath at 60°C to prepare a 5% chitosan acetic acid solution. A crosslinking agent was added to the 5% chitosan acetic acid solution and dissolved in the above chitosan acetic acid solution under stirring at a water bath at 60°C. The mixture was stirred evenly to obtain a crosslinked chitosan acetic acid solution. (3) Dissolve polyethylene oxide in 0.5 mol / L acetic acid solution and stir until dissolved to prepare 4% polyethylene oxide acetic acid solution. Mix the above cross-linked chitosan acetic acid solution and polyethylene oxide acetic acid solution to prepare substrate spinning solution. (4) The final spinning solution was prepared by mixing the eugenol liposome suspension with the substrate spinning solution, and the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane was prepared by electrospinning. In step (2), the crosslinking agent is cinnamaldehyde or citric acid or a combination thereof. When the crosslinking agent is cinnamaldehyde, the amount of cinnamaldehyde added is 0.8 to 1% of the mass of chitosan; when the crosslinking agent is citric acid, the amount of citric acid added is 5 to 9% of the mass of chitosan; when the crosslinking agent is cinnamaldehyde and citric acid, the amount of cinnamaldehyde added is 0.8 to 1.2% of the mass of chitosan, and the amount of citric acid added is 1.5 to 2% of the mass of chitosan.

2. The method for preparing the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane according to claim 1, characterized in that, In step (1), the phospholipid compound is soybean lecithin or other phospholipid compounds with a structure similar to soybean lecithin, the concentration of eugenol in the lipid solution is 30~60 μL / mL, and the concentration of Tween 80 is 13~16 μL / mL.

3. The method for preparing the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane according to claim 1, characterized in that, In step (1), the volume ratio of anhydrous ethanol to phosphate buffer is 1:2; the phosphate buffer is prepared by dissolving Na2HPO4 and KH2PO4 in distilled water, with a pH of 7.2-7.4 and a phosphate concentration of 50 mmol / L; the lipid solution is injected into water or phosphate buffer, stirred and mixed, and the resulting liquid is evaporated at 35°C to remove ethanol, and then sonicated at 350 W for 20 min to form a uniform eugenol liposome suspension.

4. The method for preparing the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane according to claim 1, characterized in that, In step (3), the substrate spinning solution is a cross-linked chitosan acetate solution and a polyethylene oxide acetate solution mixed at a mass ratio of 1.4:1 to 1.6:

1.

5. The method for preparing the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane according to claim 1, characterized in that, In step (4), the eugenol liposome suspension and the substrate spinning solution are mixed at a mass ratio of 0.8:2 to 1:2 to prepare the final spinning solution.

6. The method for preparing the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane according to claim 1, characterized in that, In step (4), the electrospinning process parameters are: positive voltage 19 kV, negative voltage -2.86 kV, flow rate 0.84 mL / h, spinning distance 14 cm, roller speed 25 r / min, translation speed 100 mm / min, temperature 25℃, and relative humidity 60%.

7. An eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane prepared by the method described in any one of claims 1-6, characterized in that, The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane has a fiber diameter of 60~200 nm, and forms approximately circular irregular pores between the nanofibers with a pore diameter of 100 nm~2 μm.

8. The eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane according to claim 7, characterized in that, The water contact angle of the eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fiber membrane is greater than 90°.

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