Eugenol liposome / chitosan / polyoxyethylene hydrophobic electrospun fibrous membrane and preparation method thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of bio-based food preservative materials, and in particular to a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane and a preparation method thereof. Background Art

[0002] Eugenol and other plant essential oils are considered ideal natural food preservatives due to their broad-spectrum antimicrobial properties and strong antioxidant capacity. However, the high volatility, water insolubility, and photo-thermal instability of these essential oils severely restrict their application in food preservation. Liposome encapsulation technology, which encapsulates active ingredients within a phospholipid bilayer, can significantly reduce volatility losses and delay oxidative degradation. The ethanol injection method for preparing eugenol liposomes is a relatively mature technology, achieving a high eugenol encapsulation efficiency. However, single liposome encapsulation techniques suffer from issues such as uneven particle size, poor storage stability, and weak interfacial bonding with polymer substrates. Electrospinning technology is simple to operate and requires no heating during the encapsulation process, making it suitable for encapsulating non-bioactive substances. The negatively charged eugenol liposomes in the spinning solution stabilize the encapsulation of eugenol through electrostatic interactions with the cations on the chitosan backbone, minimizing eugenol loss during the electrospinning process and improving the encapsulation efficiency of eugenol in nanofibers. However, due to the large specific surface area of the nanofibers prepared by electrospinning technology, the initial burst release effect of eugenol is severe, which is not conducive to the slow and long-term release of plant essential oils such as eugenol.

[0003] Chitosan and polyethylene oxide are non-toxic, environmentally friendly, and are both common electrospun fiber substrates. Pure chitosan solutions have high viscosity, and the repulsion between cations on the chitosan backbone hinders the formation of electrospun fibers. Adding polyethylene oxide and eugenol liposomes to the chitosan solution not only reduces the viscosity of the solution, but also significantly improves the spinnability of the solution by utilizing the electrostatic interaction between eugenol liposomes and chitosan. On the other hand, the hydrophilicity of polyethylene oxide and eugenol liposomes further deteriorates 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] Cross-linking is an effective method for enhancing the hydrophobicity of chitosan membranes. However, studies on the hydrophobic modification of chitosan-based composite hydrophilic systems have often used non-food additive cross-linkers such as glutaraldehyde, or toxic organic reagents during the preparation process. The resulting modified membranes are unsuitable for use as packaging materials for direct food contact. Currently reported results show that the extent of hydrophobic modification achieved with chitosan / polyethylene oxide composite hydrophilic systems using food additives as cross-linkers is limited. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane and a preparation method, aiming to solve the problem in the prior art that the fiber membrane has a low loading rate and poor encapsulation of eugenol, resulting in poor preservation effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, a method for preparing a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane comprises: (1) Phospholipid compounds and cholesterol were dissolved in anhydrous ethanol at a mass ratio of 5.5:1. The concentrations of phospholipid compounds and cholesterol were 13.75 mg / mL and 2.5 mg / mL, respectively. The mixture was stirred in a 60°C water bath for 30 min. After cooling to room temperature, eugenol and Tween 80 were added to a beaker in sequence. The mixture was stirred at room temperature for 30 min to obtain a lipid solution. The lipid solution was injected into water or phosphate buffer solution. A eugenol liposome suspension was prepared under reduced pressure at a temperature not exceeding 35°C. The entrapment rate of eugenol in the liposome suspension was greater than 88%. (2) Chitosan with a deacetylation degree of ≥95% was dissolved in a 0.5 mol / L acetic acid solution, and dissolved under magnetic stirring in a 60°C water bath to prepare a 5% chitosan acetic acid solution. A crosslinking agent was added to the 5% chitosan acetic acid solution, and dissolved in the chitosan acetic acid solution under stirring in a 60°C water bath, and 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 a 4% polyethylene oxide acetic acid solution. Mix the cross-linked chitosan acetic acid solution and the polyethylene oxide acetic acid solution to prepare a substrate spinning solution. (4) The eugenol liposome suspension and the substrate spinning solution were mixed to prepare the final spinning solution, and the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane was 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 the phosphate buffer is 7.2-7.4, and the concentration is 50 mmol / L. The lipid solution is injected into water or phosphate buffer and stirred to mix. The resulting liquid is rotary evaporated at 35°C to remove ethanol, and then ultrasonicated at 350 W for 20 min to form a uniform eugenol liposome suspension.

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

[0010] Furthermore, in step (3), 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.

[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 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 °C, and relative humidity 60%.

[0013] In the second aspect, a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane has a fiber diameter of 60~200 nm, and irregular pores with approximately circular shapes are formed between the nanofibers, with a pore diameter of 100 nm~2 μm.

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

[0015] The technical solution adopted by the present invention has the following beneficial effects: Cinnamaldehyde and citric acid are permitted additives in food. In this invention, they are used to physically crosslink chitosan. The addition of cinnamaldehyde and citric acid also modulates the phase separation of PEO, resulting in a hydrophobic eugenol liposome / chitosan / polyethylene oxide fiber membrane structure. In applications involving the preservation of high-moisture foods, this novel electrospun fiber structure facilitates the slow and prolonged release of the bioactive substance eugenol, enhancing the preservation of high-moisture foods.

[0016] (1) The present invention adopts liposome combined with electrospinning technology to prepare eugenol liposome / chitosan / polyethylene oxide 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 preservative film.

[0017] (2) The present invention uses liposome and electrospinning technologies to embed the biological preservative eugenol. During the preparation process, eugenol is not heated, the loss is small, and the eugenol embedding rate is high. This method is not only conducive to improving the utilization rate of eugenol, but also can effectively reduce the impact of eugenol on the flavor of food during the preservation process.

[0018] (3) The present invention adopts the ethanol injection method to prepare eugenol liposomes, with an embedding rate of more than 88%. The particle size of the eugenol liposomes is between 80 and 180 nm, and it is a unilamellar vesicle structure.

[0019] (4) When preparing the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane using the method of the present invention, the crosslinking agents cinnamaldehyde and citric acid are both food additives permitted in GB 2760. The amount of cinnamaldehyde used is 1 / 50 of that used in eugenol, and the amount of citric acid used is 1 / 5 of that used in eugenol. The amount of crosslinker added during the preparation process is small, and the membrane can be used in direct contact food packaging.

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

[0021] (6) The present invention uses hydrophilic swellable chitosan and hydrophilic polyethylene oxide as substrates, water-soluble liposomes as carriers of the biological preservative eugenol, cross-links the chitosan with cinnamaldehyde and citric acid, and regulates the phase separation of polyethylene oxide, thereby achieving a transition from hydrophilic to hydrophobic properties of the electrospun fiber membrane. Without the addition of the cross-linking agents cinnamaldehyde and citric acid, the water contact angle of the electrospun fiber membrane is 40.6°. After cross-linking modification using the present invention, the water contact angle of the electrospun fiber membrane can reach over 90°.

[0022] (7) The eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the present method has water-absorbing 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 a slow and long-lasting release of eugenol in a simulated food preservation solution.

[0023] (8) The eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the method of the present invention has a high eugenol loading rate. In food preservation applications, the cross-linking agents cinnamaldehyde and citric acid and the biological preservative eugenol have a combined antioxidant and antibacterial effect. It shows an excellent preservation effect in the preservation process of sea bass fillets and has potential application value in the field of preservation materials such as food active packaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Graph showing the water contact angle 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 prepared under the process conditions of Example 1 of the present invention; Figure 3 1 is the XRD pattern of the electrospun fiber membrane prepared under the process conditions of Example 1 of the present invention; Figure 4 This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane in Example 2 of the present invention; Figure 5 This is a scanning electron micrograph of the surface of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared under the process conditions of Example 2 of the present invention; Figure 6 Graph showing the cumulative release curve of eugenol in a simulated release solution (volume ratio of phosphate buffer to anhydrous ethanol of 90:10) from the electrospun fiber membrane prepared under the process conditions of Examples 1 and 2 of the present invention; Figure 7 Graph showing changes in total bacterial count (A), TVB-N (B), TBA (C), and color difference values (D-F) during storage of sea bass fillets preserved using the electrospun fiber membranes prepared by the processes of Examples 1 and 2 of the present invention; Figure 8 : This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3 of the present invention; Figure 9 is a scanning electron micrograph of the surface of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3; Figure 10 : This is the XRD pattern of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 3 of the present invention; Figure 11 This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 4 of the present invention; Figure 12 : This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 5 of the present invention; Figure 13 : This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 6 of the present invention; Figure 14: This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 7 of the present invention; Figure 15 This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared according to the process of Example 8 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] In the present invention, a method for preparing a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane comprises the following specific steps: (1) Preparation of liposome solution Soy lecithin and cholesterol were dissolved in a volume of anhydrous ethanol at a mass ratio of 5.5:1 to concentrations of 13.75 mg / mL and 2.5 mg / mL, respectively. The mixture was stirred in a 60°C water bath for 30 minutes. After cooling to room temperature, a predetermined amount of eugenol and Tween 80 were added to a beaker and stirred at room temperature for 30 minutes to obtain a lipid solution. The lipid solution in the beaker was poured into water or phosphate buffer (pH = 7.2-7.4, 50 mmol / L) containing twice the volume of the anhydrous ethanol and stirred to mix thoroughly. The resulting solution was rotary evaporated at 35°C to remove the ethanol. The solution was then sonicated at 350 W for 20 minutes to form a uniform 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 entrapment rate of eugenol must reach more than 88%; the rotary evaporation process is carried out under certain reduced pressure conditions, and the temperature does not exceed 35°C. The reduced pressure conditions only need to ensure the removal of ethanol, and the negative pressure can be set according to actual needs.

[0028] (2) Cross-linked chitosan acetate solution Weigh a certain amount of chitosan (degree of deacetylation ≥ 95%) and dissolve it in a certain volume of 0.5 mol / L acetic acid solution. Dissolve in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetic acid solution. Weigh a certain amount of citric acid and cinnamaldehyde and dissolve them in the chitosan acetic acid solution in a 60°C water bath with stirring. Stir thoroughly to obtain a cross-linked chitosan acetic acid solution.

[0029] Among them, only one crosslinking agent, citric acid or cinnamaldehyde, or both citric acid and cinnamaldehyde can be added. When only one crosslinking agent, citric acid or cinnamaldehyde, is added, the amount of cinnamaldehyde added is 0.8% to 1% of the chitosan mass; the amount of citric acid added is 5% to 9% of the chitosan mass; when both crosslinking agents are added, the amount of cinnamaldehyde added is 0.8% to 1.2% of the chitosan mass, and the amount of citric acid added is 1.5% to 2% of the chitosan mass.

[0030] (3) Preparation of substrate spinning solution Weigh a certain amount of polyethylene oxide (average molecular weight ~600,000) and dissolve it in a certain volume of 0.5 mol / L acetic acid solution. Stir until dissolved to prepare a 4% (m / v) polyethylene oxide acetate solution. Add the 4% polyethylene oxide acetate solution to the cross-linked chitosan acetate solution at a specific mass ratio and mix thoroughly 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 electrospinning membrane The final spinning solution was prepared by mixing the eugenol liposome suspension with the substrate spinning solution at a mass ratio of 0.8:2 to 1:2. Electrospinning membranes were prepared using a uniaxial electrospinning method. The electrospinning process parameters were optimized to form a well-defined Taylor cone. Reference process parameters were: positive voltage 19 kV, negative voltage -2.86 kV, flow rate 0.84 mL / h, spinning distance 14 cm, drum speed 25 r / min, translation speed 100 mm / min, temperature 25°C, 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the 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 minutes to obtain eugenol liposomes for later use.

[0034] When the amount of eugenol added was zero, blank liposomes were prepared, and the rest of the preparation process was 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. Dissolve it in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 25 mg of cinnamaldehyde into the chitosan acetic acid solution and stir it in a 60°C water bath 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 the cross-linked chitosan acetate solution and 10 g of the polyethylene oxide acetate solution to prepare the chitosan / polyethylene oxide spinning solution.

[0037] When the amount of cinnamaldehyde added was zero, a chitosan / polyethylene oxide spinning solution without adding a cross-linking agent was prepared, and the rest of the preparation process was the same as above.

[0038] (4) After the substrate spinning solution has cooled, 13 g of the eugenol liposome suspension is dissolved in 26 g of the above-mentioned substrate spinning solution and mixed evenly under magnetic stirring to prepare the final spinning solution. Eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber 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.

[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 a eugenol liposome / chitosan / polyethylene oxide solution without adding a 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 membrane, chitosan / polyethylene oxide fiber membrane, blank liposome / chitosan / polyethylene oxide fiber membrane, and eugenol liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent. The electrospinning preparation process parameters were the same as above.

[0042] Figure 1The water contact angle diagram of the electrospun fiber membrane prepared under the process conditions of Example 1 of the present invention. Among them, (a) chitosan / polyethylene oxide fiber membrane without crosslinking agent; (b) blank liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; (c) eugenol liposome / chitosan / polyethylene oxide fiber membrane without crosslinking agent; (d) eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane. As can be seen from the figure, 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 by the substrate has good hydrophilicity, and the hydrophilicity is even better after adding liposomes. However, after adding cinnamaldehyde cross-linking agent, the electrospun fiber membrane becomes hydrophobic.

[0043] Figure 2 The following are scanning electron micrographs of the surfaces of electrospun fiber membranes prepared under the process conditions of Example 1. (a) shows a chitosan / polyethylene oxide fiber membrane without a crosslinker; (b) shows a blank liposome / chitosan / polyethylene oxide fiber membrane without a crosslinker; (c) shows a eugenol lipid / chitosan / polyethylene oxide fiber membrane without a crosslinker; and (d) shows a hydrophobic electrospun fiber membrane composed of cinnamaldehyde / eugenol liposomes / chitosan / polyethylene oxide. Figure 2 The diameters of most fibers in (d) are between 60 and 140 nm, with an average diameter of about 99 nm. Figure 2 Compared to (a-c), some nanofibers show entanglement, and a few fibers have beads. The nanofiber network structure with the addition of cinnamaldehyde modifier is denser, forming irregular, nearly circular pores with diameters ranging from 100 nm to 5 μm.

[0044] Figure 3 The following are XRD patterns of electrospun fiber membranes produced under the process conditions of Example 1. (a) shows a polyethylene oxide fiber membrane; (b) shows a chitosan / polyethylene oxide fiber membrane without a crosslinker; (c) shows a blank liposome / chitosan / polyethylene oxide fiber membrane without a crosslinker; (d) shows a eugenol lipid / chitosan / polyethylene oxide fiber membrane without a crosslinker; and (e) shows a hydrophobic electrospun fiber membrane containing cinnamaldehyde / eugenol liposomes / chitosan / polyethylene oxide. Comparing Figure (e) with Figures (b-d), the relative intensities of the diffraction peaks at 19.1° and 23.6°, corresponding to PEO crystals, have increased significantly, indicating that the addition of cinnamaldehyde alters the phase separation process of PEO during membrane 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the 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. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0046] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of 0.5 mol / L acetic acid solution in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 125 mg of citric acid into the chitosan acetic acid solution and stir evenly in a 60°C 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 cross-linked chitosan acetic acid solution and 10.25 g of the polyethylene oxide acetic acid solution (mass ratio of the two solutions is 1.6:1) and stir evenly in a 60°C water bath to prepare the substrate spinning solution.

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

[0049] Figure 4 This is a water contact angle diagram of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane in Example 2 of the present invention, and the water contact angle is 95.0°.

[0050] Figure 5 This is a scanning electron micrograph of the surface of a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane produced under the process conditions of Example 2. The nanofibers intertwine and adhere to each other, forming a dense network structure with fiber diameters ranging from 50 to 200 nm. The fibers are larger in diameter at the points where the nanofibers intertwine, with a small amount of beading formed. Irregular, nearly circular pores are formed between the nanofibers, with pore diameters ranging from 100 nm to 1.5 μm.

[0051] Table 1 shows the eugenol entrapment efficiency data for some electrospun fiber membranes prepared in Examples 1 and 2. The eugenol entrapment efficiency in the electrospun fiber membranes is the percentage of eugenol in the fiber membranes to the total amount of eugenol in the eugenol liposome suspension added to the spinning solution. The eugenol entrapment efficiency in the eugenol liposome suspension is the percentage of eugenol in the liposome structure to the total amount of eugenol in the eugenol liposome suspension (the sum of the eugenol in the liposomes and the free eugenol). Eugenol concentration was determined by UV spectrophotometry (282 nm). The eugenol entrapment efficiency in the eugenol liposome suspension was 88.06%. The eugenol entrapment efficiency in the eugenol liposome / chitosan / polyethylene oxide electrospun fiber membrane prepared in Example 1 without a crosslinker was 59.68%. However, the entrapment efficiency in the hydrophobic electrospun fiber membranes prepared in Example 1 and Example 2 increased to 85.74% and 89.04% respectively due to the addition of the crosslinkers cinnamaldehyde and citric acid. Therefore, the addition of the crosslinkers cinnamaldehyde and citric acid significantly reduced the loss of eugenol during the electrospinning process.

[0052] Table 1 shows the embedding rate data of eugenol in some electrospun fiber membranes prepared in Example 1 and Example 2 of the present invention. fiber membrane Eugenol embedding rate (%) Eugenol liposome / chitosan / polyethylene oxide fiber membrane prepared in Example 1 without adding a crosslinking agent <![CDATA[59.68±0.02 c ]]> Eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared in Example 1 <![CDATA[85.74±0.01 b ]]> Eugenol liposome / chitosan / polyethylene oxide 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 Cumulative release curves of eugenol from electrospun fiber membranes prepared under the process conditions of Examples 1 and 2 of the present invention in a simulated release solution (phosphate buffer to anhydrous ethanol, volume ratio: 90:10). (a) Eugenol liposome / chitosan / polyethylene oxide fiber membrane prepared in Example 1 without the addition of a crosslinker; (b) Eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared in Example 1; (c) Eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared in Example 2.

[0055] Eugenol concentration was determined by UV spectrophotometry (282 nm). In the figure, the black control curve represents the eugenol liposome / chitosan / polyethylene oxide fiber membrane prepared in Example 1 without a crosslinker. At 4°C, the total release from each fiber membrane was similar within the first 12 hours. After reaching release equilibrium after 120 hours, the final cumulative release from the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared with cinnamaldehyde in Example 1 and the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared with citric acid in Example 2 was significantly higher than that from the control curve. This is consistent with the eugenol entrapment efficiency data in Table 1. It can be inferred that the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared with cinnamaldehyde in Example 1 and the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared with citric acid in Example 2 slowed the release of eugenol within the first 12 hours. This may be related to the hydrophobicity of the hydrophobic electrospun fiber membrane and the change in the structure of the nanofiber membrane after the addition of the cross-linking agent.

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

[0057] The storage temperature is 4°C. The control group was not treated with a fiber membrane and was directly placed in a plastic box with a lid. Compared with the control group, the preservation indicators of the other groups were better than those of the control group. The preservation indicators of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane group prepared in Example 1 and Example 2 were the best. Under refrigerated conditions of 4°C, the shelf life of sea bass fillets in the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane group prepared in Example 1 and Example 2 can reach 9 days. The extension of the shelf life of sea bass fillets is due to the sustained release of eugenol in the hydrophobic fiber membrane and the addition of cross-linking agents cinnamaldehyde and citric acid, which have a combined 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the 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. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0059] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of 0.5 mol / L acetic acid solution in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 225 mg of citric acid into the chitosan acetic acid solution and stir evenly in a 60°C 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 acetate solution. Mix 16 g of the cross-linked chitosan acetate solution and 10 g of the polyethylene oxide acetate solution (mass ratio of the two solutions is 1.6:1) and stir evenly in a 60°C water bath to prepare the substrate spinning solution.

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

[0062] Figure 8 This is a water contact angle graph of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared under the process conditions of Example 3 of the present invention, and its water contact angle is 101.2°.

[0063] Figure 9 This is a scanning electron micrograph of the surface of a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane produced under the process conditions of Example 3. The nanofibers intertwine and adhere to each other, forming a dense network structure with fiber diameters ranging from 80 to 200 nm. Irregular, nearly circular pores with diameters ranging from 100 nm to 1.0 μm are formed between the nanofibers.

[0064] Figure 10The XRD patterns of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membranes prepared in Example 3 of the present invention are as follows: (a) the polyethylene oxide fiber membrane prepared in Example 1; (b) the chitosan / polyethylene oxide fiber membrane prepared in Example 1 without adding a crosslinking agent; (c) the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared in Example 1; (d) the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared in Example 3. Figure 10 (a~c) are respectively the polyethylene oxide fiber membrane prepared in Example 1 of the present invention, the chitosan / polyethylene oxide fiber membrane without adding a cross-linking agent, and the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane. Figure 10 (d) with Figure 10 (c) is similar. The relative intensities of the diffraction peaks at 19.1° and 23.6° corresponding to PEO crystals are significantly increased, indicating that cross-linking of cinnamaldehyde, citric acid and chitosan changes 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the 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. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0066] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetate solution. Weigh 20 mg of cinnamaldehyde and 50 mg of citric acid into the chitosan acetate solution and stir evenly in a 60°C water bath to obtain a cross-linked chitosan acetate 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 cross-linked chitosan acetic acid solution and 10 g of the polyethylene oxide acetic acid solution, dissolve them in the 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, 13 g of the eugenol liposome suspension is dissolved in 26 g of the substrate spinning solution and mixed evenly under magnetic stirring to prepare the final spinning solution. The electrospinning membrane is prepared by a uniaxial electrospinning method. The electrospinning process is sufficient to form a good Taylor cone. The reference process parameters given above can be slightly adjusted.

[0069] Figure 11 This is a water contact angle graph of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 4 of the present 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 160 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the 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. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0071] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetate solution. Weigh 25 mg of cinnamaldehyde and 37.5 mg of citric acid into the chitosan acetate solution and stir evenly in a 60°C water bath to obtain a cross-linked chitosan acetate 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 cross-linked chitosan acetic acid solution and 10 g of the polyethylene oxide acetic acid solution, dissolve them in the 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 the eugenol liposome suspension is dissolved in 24 g of the substrate spinning solution and mixed evenly under magnetic stirring to prepare the final spinning solution. The electrospinning membrane is prepared by a uniaxial electrospinning method. The electrospinning process is sufficient to form a good Taylor cone. The reference process parameters given above can be slightly adjusted.

[0074] Figure 12 This is a water contact angle graph of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 5 of the present 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 in a water bath at 60°C for 30 minutes. After cooling to room temperature, add 130 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the 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. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0076] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetic acid solution. Weigh 20 mg of cinnamaldehyde into the 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 cross-linked chitosan acetic acid solution and 10 g of the polyethylene oxide acetic acid solution, dissolve them in the 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, 9.6 g of the eugenol liposome suspension is dissolved in 24 g of the substrate spinning solution and mixed evenly under magnetic stirring to prepare the final spinning solution. The electrospinning membrane is prepared by a uniaxial electrospinning method. The electrospinning process is sufficient to form a good Taylor cone. The reference process parameters given above can be slightly adjusted.

[0079] Figure 13 This is a water contact angle graph of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 6 of the present 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 130 μL of Tween 80 and 300 μL of eugenol to the beaker under magnetic stirring. Pour the lipid suspension into 20.0 mL of water and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35°C. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0081] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetate solution. Weigh 20 mg of cinnamaldehyde and 20 mg of citric acid into the chitosan acetate solution and stir evenly in a 60°C water bath to obtain a cross-linked chitosan acetate 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 acetate solution. Mix 14 g of the cross-linked chitosan acetate solution and 10 g of the polyethylene oxide acetate solution to prepare the substrate spinning solution.

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

[0084] Figure 14 This is a water contact angle graph of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 7 of the present 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 in a 60°C water bath for 30 minutes. After cooling to room temperature, add 130 μL of Tween 80 and 600 μL of eugenol to the beaker under magnetic stirring. Pour the lipid suspension into 20.0 mL of water and transfer to a round-bottom flask. Remove the ethanol by rotary evaporation at 35°C. Ultrasonicate the resulting suspension at 300 W for 20 minutes and set aside.

[0086] (2) Weigh 2.5 g of chitosan and dissolve it in 50.0 mL of acetic acid solution (0.5 mol / L) in a 60°C water bath with magnetic stirring to prepare a 5% (m / v) chitosan acetate solution. Weigh 20 mg of cinnamaldehyde and 40 mg of citric acid into the chitosan acetate solution and stir evenly in a 60°C water bath to obtain a cross-linked chitosan acetate 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 acetate solution. Mix 15 g of the cross-linked chitosan acetate solution and 10 g of the polyethylene oxide acetate solution to prepare the substrate spinning solution.

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

[0089] Figure 15 This is a water contact angle graph of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the process of Example 8 of the present invention, wherein the water contact angle is 97.7°.

[0090] Verification by the above-described embodiments shows that, in the present invention, natural polymer chitosan has good biocompatibility and biodegradability, and is widely used in fields such as food, medicine and health, and agriculture. However, pure chitosan solution has a higher viscosity and exhibits cationic electrolyte properties, and the interionic repulsion on the chitosan main chain hinders the formation of electrospun fibers. Many researchers have introduced polyethylene oxide into chitosan solutions, which has improved the spinnability of the solution to a great extent. However, due to the hydrophilicity of chitosan and polyethylene oxide, the composite fiber membrane obtained has poor structural stability and water resistance during the preservation of high-water-content foods. Eugenol is added to the chitosan / polyethylene oxide spinning solution in the form of a liposome structure. Due to the electrostatic interaction between the cations on the chitosan chain and the negatively charged surface of the liposome, the stability of the liposome and the embedding efficiency of the bioactive substances can be improved. However, eugenol liposomes are water-soluble, which further increases the hydrophilicity of the fiber membrane. The highly hydrophilic composite fiber membrane can rapidly swell and rupture, accelerating the release of the embedded eugenol.

[0091] The Schiff base crosslinking of cinnamaldehyde and chitosan, and the esterification of citric acid and chitosan, are both eco-friendly crosslinking modification strategies designed to enhance the hydrophobicity and film-forming properties of chitosan membranes. The dynamic covalent Schiff base bond formed and the hydrolysis of the esterification reaction are utilized to control the release of the bioactive component eugenol in food preservation applications. Furthermore, because the crosslinkers cinnamaldehyde and citric acid possess certain antimicrobial and antioxidant properties, they, even at relatively low dosages, exhibit a combined antimicrobial effect with eugenol in the preservation of composite fiber membranes.

[0092] The eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the method of the present invention has the following beneficial effects: (1) The present invention adopts liposome combined with electrospinning technology to prepare eugenol liposome / chitosan / polyethylene oxide 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 preservative film.

[0093] (2) The present invention uses liposome and electrospinning technologies to embed the biological preservative eugenol. During the preparation process, eugenol is not heated, the loss is small, and the eugenol embedding rate is high. This method is not only conducive to improving the utilization rate of eugenol, but also can effectively reduce the impact of eugenol on the flavor of food during the preservation process.

[0094] (3) The present invention adopts the ethanol injection method to prepare eugenol liposomes, with an embedding rate of more than 88%. The particle size of the eugenol liposomes is between 80 and 180 nm, and it is a unilamellar vesicle structure.

[0095] (4) When preparing the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane using the method of the present invention, the crosslinking agents cinnamaldehyde and citric acid are both food additives permitted in GB 2760. The amount of cinnamaldehyde used is 1 / 50 of that used in eugenol, and the amount of citric acid used is 1 / 5 of that used in eugenol. The amount of crosslinker added during the preparation process is small, and the membrane can be used in direct contact food packaging.

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

[0097] (6) The present invention uses hydrophilic swellable chitosan and hydrophilic polyethylene oxide as substrates, water-soluble liposomes as carriers of the biological preservative eugenol, cross-links the chitosan with cinnamaldehyde and citric acid, and regulates the phase separation of polyethylene oxide, thereby achieving a transition from hydrophilic to hydrophobic properties of the electrospun fiber membrane. Without the addition of the cross-linking agents cinnamaldehyde and citric acid, the water contact angle of the electrospun fiber membrane is 40.6°. After cross-linking modification using the present invention, the water contact angle of the electrospun fiber membrane can reach over 90°.

[0098] (7) The eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the present method has water-absorbing 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 a slow and long-lasting release of eugenol in a simulated food preservation solution.

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

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

Claims

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

1. The concentrations of phospholipid compounds and cholesterol were 13.75 mg / mL and 2.5 mg / mL, respectively. The mixture was stirred in a 60°C water bath for 30 min. After cooling to room temperature, eugenol and Tween 80 were added to a beaker in sequence. The mixture was stirred at room temperature for 30 min to obtain a lipid solution. The lipid solution was injected into water or phosphate buffer solution. A eugenol liposome suspension was prepared under reduced pressure at a temperature not exceeding 35°C. The entrapment rate of eugenol in the liposome suspension was greater than 88%. (2) Chitosan with a deacetylation degree of ≥95% was dissolved in a 0.5 mol / L acetic acid solution, and dissolved under magnetic stirring in a 60°C water bath to prepare a 5% chitosan acetic acid solution. A crosslinking agent was added to the 5% chitosan acetic acid solution, and dissolved in the chitosan acetic acid solution under stirring in a 60°C water bath, and 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 a 4% polyethylene oxide acetic acid solution. Mix the cross-linked chitosan acetic acid solution and the polyethylene oxide acetic acid solution to prepare a substrate spinning solution. (4) The eugenol liposome suspension and the substrate spinning solution were mixed to prepare the final spinning solution, and the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane was prepared by electrospinning.

2. The method for preparing the eugenol liposome / chitosan / polyethylene oxide 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 / polyethylene oxide 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 value of 7.2-7.4 and a phosphate concentration of 50 mmol / L; the lipid solution is injected into water or phosphate buffer and stirred to mix. The resulting liquid is rotary evaporated at 35°C to remove ethanol, and then ultrasonicated at 350 W for 20 min to form a uniform eugenol liposome suspension.

4. The method for preparing the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane according to claim 1, characterized in that: In step (2), the cross-linking agent is cinnamaldehyde or citric acid or a combination thereof. When the cross-linking agent is cinnamaldehyde, the amount of cinnamaldehyde added is 0.8-1% of the mass of chitosan; when the cross-linking agent is citric acid, the amount of citric acid added is 5-9% of the mass of chitosan; when the cross-linking agent is cinnamaldehyde and citric acid, the amount of cinnamaldehyde added is 0.8-1.2% of the mass of chitosan, and the amount of citric acid added is 1.5-2% of the mass of chitosan.

5. The method for preparing the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane according to claim 1, characterized in that: In step (3), 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.

6. The method for preparing the eugenol liposome / chitosan / polyethylene oxide 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.

7. The method for preparing the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane according to claim 1, characterized in that: In step (4), the electrospinning 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 °C, and relative humidity 60%.

8. A eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane prepared by the method for preparing a eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane according to any one of claims 1 to 7, characterized in that: The fiber diameter of the eugenol liposome / chitosan / polyethylene oxide hydrophobic electrospun fiber membrane is 60~200 nm, and irregular pores with approximately circular shapes and pore diameters of 100 nm~2 μm are formed between the nanofibers.

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

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