A preparation method of p-coumaric acid nanoliposomes and its antibacterial and fresh-keeping application

Abstract

The present invention provides a preparation method and antibacterial preservation application of p-coumaric acid nanoliposomes. Soybean lecithin and cholesterol are used as membrane materials, p-coumaric acid is used as an encapsulation, and p-coumaric acid nanoliposomes are prepared by thin film dispersion combined with ultrasonic method. Soybean lecithin, cholesterol and p-coumaric acid are dissolved in an organic solvent in the following proportions, and a liquid mixture is obtained after full dissolution: the mass ratio of soybean lecithin to cholesterol is 2:1-10:1, and the mass ratio of soybean lecithin to p-coumaric acid is 5:1-50:1; the organic solvent is obtained by mixing chloroform and methanol; then, p-coumaric acid nanoliposomes are obtained by evaporation, hydration and ultrasonic treatment, and the final concentration of soybean lecithin is 2-10 mg / mL. The encapsulation of the nanoliposomes obtained by the present invention improves the inhibitory effect of p-coumaric acid on Botrytis cinerea, reduces the weight loss rate and decay rate of fruits and vegetables, and prolongs the shelf life of fruits and vegetables.

Description

Technical Field

[0001] The invention belongs to the field of food material technology and antibacterial and fresh-keeping, and specifically relates to a preparation method of p-coumaric acid nanoliposomes and their antibacterial and fresh-keeping applications. Background Art

[0002] p-Coumaric acid, also known as p-hydroxycinnamic acid, is a natural phenolic acid compound that can be found in various fruits, vegetables, grains and fungi in free or combined forms. The biosynthesis of p-coumaric acid uses phenylalanine and tyrosine as precursors and is synthesized through the shikimic acid pathway through deamination and hydroxylation reactions. p-Coumaric acid is an important precursor of secondary metabolites in plants, such as chlorogenic acid, resveratrol, lignin, anthocyanins, etc. Studies have shown that p-coumaric acid has a variety of biological activities, such as antibacterial, antioxidant, hypolipidemic, neuroprotective, etc., and has broad application prospects in the fields of medicine, cosmetics and food. However, p-coumaric acid has poor water solubility and is sensitive to the environment, which limits its scope of application. In the Chinese patent application with application number 201910516849.7, a 'nano drug delivery system based on p-coumaric acid polymer and its preparation method and application' is disclosed, which involves the anti-tumor activity of p-coumaric acid polymer. The Chinese patent application number 201711237205.1 discloses "an application of p-coumaric acid ester in fruit and vegetable sterilization and preservative agents", but does not mention the antibacterial and preservative effects of p-coumaric acid. How to use existing technologies to improve the biological activity of p-coumaric acid requires further research.

[0003] Liposomes are vesicles with a bilayer structure similar to biological membranes formed by self-assembly of phospholipids and cholesterol. The hydrophilic head and hydrophobic tail of phospholipid molecules form a bilayer structure of phospholipids through hydrophobic interactions. Lipid-soluble substances can be encapsulated between the bilayers, and water-soluble substances can be encapsulated in the inner aqueous phase. In addition, compared with other delivery systems, the biomimetic structure of liposomes shows unique advantages, such as biocompatibility, high safety, targeting and sustained release, which can improve the physical and chemical properties of active ingredients and enhance their stability and efficacy.

[0004] As an efficient delivery system, liposomes have attracted extensive attention due to their nano-size and amphiphilic properties. Liposomes have been successfully used in the fields of medicine, cosmetics and food. They can encapsulate antibacterial drugs and improve their stability and solubility in aqueous media, thereby increasing their bioavailability and biological functions. There are no reports on the encapsulation of p-coumaric acid and its application in the antibacterial and fresh-keeping of fruits and vegetables in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method of p-coumaric acid nanoliposomes and their antibacterial and fresh-keeping applications, so as to solve the problems existing in the above-mentioned prior art.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A method for preparing p-coumaric acid nanoliposomes, using soybean lecithin and cholesterol as membrane materials, using p-coumaric acid as an encapsulation, and adopting thin film dispersion combined with ultrasonic method to prepare p-coumaric acid nanoliposomes, specifically comprising the following steps:

[0008] 1) dissolving soybean lecithin, cholesterol and p-coumaric acid in an organic solvent in the following ratios to obtain a liquid mixture after sufficient dissolution: the mass ratio of soybean lecithin to cholesterol is 2:1-10:1, and the mass ratio of soybean lecithin to p-coumaric acid is 5:1-50:1; the organic solvent is obtained by mixing chloroform and methanol, and the volume ratio of chloroform to methanol is 0.5:1-1.5:1;

[0009] 2) the liquid mixture obtained in step 1) is subjected to rotary evaporation at 45-55° C. under vacuum or reduced pressure conditions to remove the organic solvent, thereby forming a lipid film at the bottom of the evaporation container;

[0010] 3) under normal pressure, adding a phosphate buffer containing a surfactant to the obtained lipid film, rotating and hydrating at 50° C. under normal pressure, and obtaining a liposome suspension after the film falls off; the final concentration of soybean lecithin is 2 to 10 mg / mL;

[0011] 4) Finally, the liposome suspension obtained in step 3) is subjected to ultrasonic treatment at a power of 100 to 500 W in an ice bath for 4 to 12 minutes to obtain p-coumaric acid nanoliposomes.

[0012] The soybean lecithin concentration is 6±1 mg / mL.

[0013] The mass ratio of the soybean lecithin to cholesterol is 7:1.

[0014] The mass ratio of soybean lecithin to p-coumaric acid is 20:1.

[0015] In the step 1), the volume ratio of chloroform to methanol is 1:1.

[0016] In the step 2), the rotation temperature is 50° C. and the rotation time is 1 h.

[0017] In the step 3), the concentration of the surfactant Tween 80 is 0-0.8wt%, and the volume of the phosphate buffer is 50mL.

[0018] In the step 4), the ultrasonic power is 100-500 W, preferably 300 W, and the ultrasonic time is 4-12 min, preferably 8 min.

[0019] The p-coumaric acid nanoliposomes obtained by the method have the following product properties: an encapsulation rate of 40-60%, a drug loading rate of 1-5%, and a particle size of 70-120 nm.

[0020] The p-coumaric acid nanoliposomes are used for inhibiting the growth of Botrytis cinerea in pepper fruits and reducing the incidence rate thereof for antibacterial and fresh-keeping purposes.

[0021] The beneficial effects of the present invention are:

[0022] The invention adopts a thin film dispersion combined with an ultrasonic method to prepare p-coumaric acid nanoliposomes, takes the encapsulation efficiency and particle size of the p-coumaric acid nanoliposomes as main evaluation indicators, investigates the influence of different factors on the preparation of the p-coumaric acid nanoliposomes, and obtains the optimal preparation process parameters as follows: the mass ratio of soybean lecithin to cholesterol is 7:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, the concentration of Tween 80 is 0.3wt%, the particle size is 83.55nm, and the encapsulation efficiency is 55.70%.

[0023] The p-coumaric acid nanoliposomes prepared by the invention have a microscopic morphology of a nearly spherical vesicle structure, a small particle size and uniform distribution, and can improve the water solubility and stability of p-coumaric acid through encapsulation of the nanoliposomes.

[0024] The p-coumaric acid nanoliposomes prepared by the invention can improve the antibacterial activity of p-coumaric acid, inhibit the growth of Botrytis cinerea in chili pepper fruits, reduce the incidence rate thereof, and can also reduce the weight loss rate and decay rate of chili pepper fruits, thereby maintaining the storage quality thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of the soybean lecithin concentration on the preparation of p-coumaric acid nanoliposomes of the present invention;

[0026] Figure 2 The effect of the mass ratio of soybean lecithin to cholesterol on the preparation of p-coumaric acid nanoliposomes of the present invention;

[0027] Figure 3 The effect of the mass ratio of soybean lecithin to p-coumaric acid on the preparation of p-coumaric acid nanoliposomes of the present invention;

[0028] Figure 4 The effect of Tween 80 concentration on the preparation of p-coumaric acid nanoliposomes of the present invention;

[0029] Figure 5 The effect of the ultrasonic power on the preparation of p-coumaric acid nanoliposomes of the present invention;

[0030] Figure 6 The effect of ultrasound time on the preparation of p-coumaric acid nanoliposomes of the present invention;

[0031] Figure 7 This is an appearance diagram of the p-coumaric acid nanoliposome of the present invention;

[0032] Figure 8 The microscopic morphology of the p-coumaric acid nanoliposomes of the present invention under transmission electron microscopy (A) and atomic force microscopy (B);

[0033] Fig. 9 The figure shows the effect of the p-coumaric acid nanoliposomes of the present invention on the lesion area (A) and the incidence rate (B) of pepper fruit.

[0034] Fig.10 The figure shows the effect of the p-coumaric acid nanoliposomes of the present invention on the weight loss rate (A) and decay rate (B) of chili pepper fruit. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0036] The method for determining the encapsulation efficiency and drug loading efficiency of p-coumaric acid nanoliposomes in the present invention is as follows:

[0037] Prepare 0.5, 1, 2.5, 5, 10 μg / mL p-coumaric acid solution with ethyl acetate, measure absorbance at 290nm, and draw p-coumaric acid concentration-absorbance standard curve. Adopt organic solvent extraction method to determine the free p-coumaric acid content in nanoliposomes. Pipette 1mL p-coumaric acid nanoliposome solution in a centrifuge tube, add 4mL ethyl acetate, vortex mix for 2min, centrifuge at 3000r / min for 15min, take the supernatant, and measure absorbance at 290nm. Calculate the encapsulation efficiency and drug loading rate of p-coumaric acid in nanoliposomes according to the standard curve equation. The formula is as follows:

[0038]

[0039]

[0040] The particle size, PDI and Zeta potential of p-coumaric acid nanoliposomes in the present invention are determined as follows:

[0041] After the p-coumaric acid nanoliposome sample to be tested is diluted to a certain concentration, an appropriate amount of the sample is taken and loaded into a polystyrene cuvette. The refractive coefficients of phospholipids and dispersed phase are set to 1.49 and 1.33, respectively. The sample is kept in equilibrium at 25°C for 10 minutes. The scattering angle is 90°. In order to reduce the measurement error caused by multiple light scattering, each sample is measured three times, and the particle size, PDI and Zeta potential of the sample are recorded.

[0042] Example 1 Effect of soybean lecithin concentration on the preparation of p-coumaric acid nanoliposomes

[0043] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 4:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0.2 wt% Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the final concentration of phospholipids is 2-10 mg / mL; an ultrasonic disruptor is used to perform ultrasound under ice bath conditions, with an ultrasound power of 300 W and an ultrasound time of 8 min to obtain a p-coumaric acid nanoliposome solution, which is stored at 4° C.

[0044] Depend on Figure 1 It can be seen that when the concentration of soybean lecithin is 6 mg / mL, the encapsulation efficiency of p-coumaric acid nanoliposomes reaches the maximum value of 51.04%, the drug loading rate decreases with the increase of soybean lecithin concentration, the particle size increases with the increase of soybean lecithin concentration, and the PDI is less than 0.3, indicating that the p-coumaric acid nanoliposomes are evenly dispersed.

[0045] Example 2 Effect of the mass ratio of soybean lecithin to cholesterol on the preparation of coumaric acid nanoliposomes

[0046] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 2:1-10:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0.2 wt% Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the final phospholipid concentration is 6 mg / mL; an ultrasonic disruptor is used to perform ultrasound under ice bath conditions, with an ultrasound power of 300 W and an ultrasound time of 8 min to obtain a p-coumaric acid nanoliposome solution, which is stored at 4° C.

[0047] Depend on Figure 2 It can be seen that when the mass ratio of soybean lecithin to cholesterol is 6:1, the encapsulation efficiency and drug loading rate of p-coumaric acid nanoliposomes reach the highest values. The particle size first decreases and then increases with the increase of the mass ratio of soybean lecithin to cholesterol, and is the smallest when the mass ratio is 6:1. PDI does not change much.

[0048] Example 3 Effect of the mass ratio of soybean lecithin to p-coumaric acid on the preparation of p-coumaric acid nanoliposomes

[0049] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 4:1, the mass ratio of soybean lecithin to p-coumaric acid is 5:1-50:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0.2 wt% Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the final phospholipid concentration is 6 mg / mL; an ultrasonic disruptor is used to perform ultrasound under ice bath conditions, with an ultrasound power of 300 W and an ultrasound time of 8 min to obtain a p-coumaric acid nanoliposome solution, which is stored at 4° C.

[0050] Depend on Figure 3 It can be seen that the encapsulation efficiency of p-coumaric acid nanoliposomes increases with the increase of the mass ratio of soybean lecithin to p-coumaric acid, while the drug loading rate is the opposite. The particle size decreases with the increase of the mass ratio of soybean lecithin to p-coumaric acid, and the PDI remains at about 0.26.

[0051] Example 4 Effect of Tween 80 concentration on the preparation of p-coumaric acid nanoliposomes

[0052] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 4:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0-0.8 wt % Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the final phospholipid concentration is 6 mg / mL; an ultrasonic disruptor is used to perform ultrasound under ice bath conditions, with an ultrasound power of 300 W and an ultrasound time of 8 min to obtain a p-coumaric acid nanoliposome solution, which is stored at 4° C.

[0053] Depend on Figure 4 It can be seen that the encapsulation efficiency and drug loading rate of p-coumaric acid nanoliposomes first increase and then decrease with the increase of Tween 80 concentration, and are the highest when the Tween 80 concentration is 0.2wt%. The particle size decreases with the increase of Tween 80 concentration, and the PDI is less than 0.3.

[0054] Example 5 Effect of Ultrasonic Power on Preparation of Coumaric Acid Nanoliposomes

[0055] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 4:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0.2 wt% Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the final phospholipid concentration is 6 mg / mL; an ultrasonic disruptor is used to perform ultrasound under ice bath conditions, the ultrasound power is 100-500 W, and the ultrasound time is 8 min to obtain a p-coumaric acid nanoliposome solution, and the solution is stored at 4° C.

[0056] Depend on Figure 5 It can be seen that the encapsulation efficiency and drug loading rate of p-coumaric acid nanoliposomes decrease with the increase of ultrasonic power, the particle size decreases with the increase of ultrasonic power, and the PDI is less than 0.3.

[0057] Example 6 Effect of Ultrasonic Time on Preparation of Coumaric Acid Nanoliposomes

[0058] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 4:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0.2 wt% Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the phospholipid concentration is 6 mg / mL; an ultrasonic crusher is used to perform ultrasound under ice bath conditions, the ultrasound power is 300 W, and the ultrasound time is 4 to 12 minutes to obtain a p-coumaric acid nanoliposome solution, and the solution is stored at 4° C.

[0059] Depend on Figure 6 It can be seen that the encapsulation efficiency and drug loading rate of p-coumaric acid nanoliposomes reached the maximum value when the ultrasonic time was 10 min, and the particle size and PDI showed a decreasing trend with the increase of ultrasonic time.

[0060] Example 7 Preparation of p-coumaric acid nanoliposomes

[0061] Soybean lecithin, cholesterol and p-coumaric acid are dissolved in a mixed solution of chloroform and methanol, wherein the mass ratio of soybean lecithin to cholesterol is 7:1, the mass ratio of soybean lecithin to p-coumaric acid is 20:1, and the volume ratio of chloroform to methanol is 1:1; the mixed solution is rotary evaporated under vacuum conditions at 50° C. for 1 h to remove chloroform and methanol; 50 mL of phosphate buffer containing 0.3 wt% Tween 80 is added to the obtained lipid film, and rotary hydration is performed under normal pressure conditions at 50° C. to obtain a liposome suspension, wherein the final phospholipid concentration is 6 mg / mL; an ultrasonic disruptor is used to perform ultrasound under ice bath conditions, with an ultrasound power of 300 W and an ultrasound time of 8 min to obtain a p-coumaric acid nanoliposome solution, which is stored at 4° C.

[0062] Table 1 Encapsulation efficiency, drug loading rate, particle size, PDI and Zeta potential of p-coumaric acid nanoliposomes

[0063]

[0064] Encapsulation efficiency and drug loading rate are important indicators for evaluating the encapsulation effect of liposomes. PDI and Zeta potential can reflect the uniformity and stability of liposome dispersion. As shown in Table 1, the optimal p-coumaric acid nanoliposomes have an encapsulation efficiency of 55.70%, a drug loading rate of 2.44%, a particle size of 83.55 nm, a PDI of 0.25, and a Zeta potential of -8.83 mV, indicating that the liposomes are evenly dispersed and have a negative charge on the surface. Figure 7 It can be seen that the p-coumaric acid nanoliposomes exhibit light blue opalescence.

[0065] Example 8 Microscopic morphology of p-coumaric acid nanoliposomes

[0066] Example 7 was used to prepare p-coumaric acid nanoliposomes, and their microscopic morphology was observed using a transmission electron microscope (TEM) and an atomic force microscope (AFM). A certain volume of p-coumaric acid nanoliposome suspension was dropped on a copper mesh with a carbon film and allowed to stand for 10 minutes, excess liquid was removed with filter paper, 3% uranyl acetate solution was added for negative staining, and the copper mesh was observed at 100 kV with a TEM after air-drying at room temperature. For AFM, the pre-diluted p-coumaric acid nanoliposome sample was added dropwise to the surface of a mica sheet and air-dried at room temperature for 30 minutes. After the sample was fully dried, it was placed under an AFM scanning probe and scanned in a tapping mode with a frequency of 300 kHz and an elastic modulus of 0.4 N / m. The silicon nitride microcantilever with a probe moved in a wave direction perpendicular to the sample surface to obtain surface morphology information of the sample.

[0067] Depend on Figure 8 It can be seen that the p-coumaric acid nanoliposomes are spherical or nearly spherical with a smooth surface and a typical vesicle-like structure. AFM shows that the liposomes are uniformly dispersed.

[0068] Example 9 Antibacterial activity of p-coumaric acid nanoliposomes

[0069] Example 7 was used to prepare p-coumaric acid nanoliposomes, and its in vivo antibacterial activity was verified using chili pepper fruit as an example. Fresh and disease-free chili pepper fruits were selected and divided into three groups. After surface disinfection with sodium hypochlorite solution, p-coumaric acid, p-coumaric acid nanoliposomes and sterile water (control) were punctured on the surface of chili pepper fruits and p-coumaric acid, p-coumaric acid nanoliposomes and sterile water (control) were injected respectively. After being placed at room temperature for 2 hours, a suspension of Botrytis cinerea spores was injected into the small holes of chili pepper fruits, and stored at 20°C and 85-90% RH after natural drying. The incidence of chili pepper fruits was regularly counted, the diameter of the lesions was measured and the area of ​​the lesions was calculated, and the inhibitory effect of p-coumaric acid nanoliposomes on gray mold after harvest of chili pepper fruits was evaluated.

[0070] Depend on Fig. 9 It can be seen that liposome encapsulation improved the inhibitory effect of p-coumaric acid on Botrytis cinerea and reduced the incidence of gray mold in postharvest pepper fruit. On the 8th day, the lesion area of ​​the p-coumaric acid nanoliposome group was 43.0% lower than that of the control group, and on the 2nd day, the incidence was 6.8% lower than that of the control group.

[0071] Example 10: Preservation effect of coumaric acid nanoliposomes

[0072] Use Example 7 to prepare p-coumaric acid nanoliposomes, and use Chinese prickly ash fruit as an example to verify its effect on fruit and vegetable preservation. Fresh and disease-free Chinese prickly ash fruits were selected and divided into three groups. After surface disinfection with sodium hypochlorite solution, the Chinese prickly ash fruits were immersed in p-coumaric acid, p-coumaric acid nanoliposomes and sterile water (control). After natural drying, they were stored at 20°C and 85-90% RH. The weight loss rate and rot rate of Chinese prickly ash fruits were regularly counted to evaluate the preservation effect of p-coumaric acid nanoliposomes on post-harvest Chinese prickly ash fruits. Fig.10 It can be seen that the encapsulation of nanoliposomes improved the preservation effect of p-coumaric acid on chili pepper fruit, and reduced the weight loss rate and decay rate of chili pepper fruit. After 25 days of storage, the weight loss rate of the p-coumaric acid nanoliposome group was 26.6% lower than that of the control group, and the decay rate was 38.7% lower than that of the control group, effectively extending the shelf life of chili pepper fruit.

Claims

1. A method for preparing p-coumaric acid nanoliposomes, characterized in that: Soybean lecithin and cholesterol are used as membrane materials, p-coumaric acid is used as an encapsulation material, and p-coumaric acid nanoliposomes are prepared by thin film dispersion combined with ultrasonic method, which specifically includes the following steps: 1) dissolving soybean lecithin, cholesterol and p-coumaric acid in an organic solvent in the following proportions to obtain a liquid mixture after sufficient dissolution: the mass ratio of soybean lecithin to cholesterol is 2:1-10:1, and the mass ratio of soybean lecithin to p-coumaric acid is 5:1-50:1; the organic solvent is obtained by mixing chloroform and methanol, and the volume ratio of chloroform to methanol is 0.5:1-1.5:1; 2) the liquid mixture obtained in step 1) is subjected to rotary evaporation at 45-55° C. under vacuum or reduced pressure conditions to remove the organic solvent, thereby forming a lipid film at the bottom of the evaporation container; 3) Under normal pressure, a phosphate buffer solution containing a surfactant is added to the obtained lipid film, and the film is rotated and hydrated at 50°C and normal pressure, and a liposome suspension is obtained after the film is detached; the final concentration of soybean lecithin is 2-10 mg / mL; 4) Finally, the liposome suspension obtained in step 3) is subjected to ultrasonic treatment at a power of 100 to 500 W in an ice bath for 4 to 12 minutes to obtain p-coumaric acid nanoliposomes; The p-coumaric acid nanoliposomes obtained by the method have the following product properties: encapsulation efficiency is 40-60%, drug loading efficiency is 1-5%, and particle size is 70-120nm.

2. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: The soybean lecithin concentration is 6±1 mg / mL.

3. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: The mass ratio of the soybean lecithin to cholesterol is 7:

1.

4. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: The mass ratio of soybean lecithin to p-coumaric acid is 20:

1.

5. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: In the step 1), the volume ratio of chloroform to methanol is 1:

1.

6. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: In the step 2), the rotation temperature is 50° C. and the rotation time is 1 h.

7. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: In the step 3), the concentration of the surfactant Tween 80 is 0-0.8wt%, and the volume of the phosphate buffer is 50mL.

8. The method for preparing p-coumaric acid nanoliposomes according to claim 1, characterized in that: In the step 4), the ultrasonic power is 300 W and the ultrasonic time is 8 min.

9. The antibacterial and fresh-keeping application of the p-coumaric acid nanoliposomes according to any one of claims 1 to 8 in inhibiting the growth of Botrytis cinerea in pepper fruits and reducing their incidence rate.

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