Preparation method and application of acetic acid-2-(1H-indole-3-yl)-ethyl ester nano-emulsion

Abstract

The invention discloses a preparation method and application of acetic acid-2-(1H-indole-3-yl)-ethyl ester nanoemulsion, the preparation method comprises the following steps: dissolving sucrose fatty acid ester in ultrapure water, stirring and dissolving in a water bath at 60-80 DEG C to obtain a water phase; the preparation method comprises the following steps: dissolving acetic acid-2-(1H-indole-3-yl)-ethyl ester in corn oil, and stirring to obtain an oil phase; mixing the oil phase with the water phase, and then carrying out homogeneous emulsification to obtain a crude emulsion; and carrying out ultrasonic disruption treatment on the crude emulsion, so as to obtain the acetic acid-2-(1H-indole-3-yl)-ethyl ester nano-emulsion. According to the invention, the problems of poor water solubility and low bioavailability of acetic acid-2-(1H-indole-3-yl)-ethyl ester are solved; the obtained acetic acid-2-(1H-indol-3-yl)-ethyl ester nano-emulsion can inhibit the growth of penicillium expansum and can be compounded with a film-forming substrate for fresh keeping of apples.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation and application of oil-in-water emulsions, and specifically relates to a preparation method and application of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion. Background Art

[0002] Apple postharvest mold caused by Penicillium expansum (Pe) causes huge economic losses to the industry. Traditional chemical methods for controlling Pe pollution no longer meet the requirements of sustainable development in my country at this stage. Therefore, it is of great significance to find new bio-source antibacterial substances that are safe, efficient, low-toxic and low-residue.

[0003] 2-(1H-indol-3-yl)-ethyl acetate is a new indole ester isolated from the metabolites of Kluyveromyces marx and has high efficiency in inhibiting Penicillium expansum. It has a good antibacterial effect on Pe. However, 2-(1H-indol-3-yl)-ethyl acetate has poor water solubility and low bioavailability, which greatly affects its physiological efficacy. It is necessary to solve this limitation and develop a new anti-mold preservative. Summary of the invention

[0004] Technical problem to be solved: In view of the above technical problem, the present invention provides a method for preparing 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion and its application, using hydrophilic sucrose fatty acid ester as an emulsifier, dissolving 2-(1H-indol-3-yl)-ethyl acetate in corn oil as an oil phase, preparing 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion by ultrasonic crushing technology, and evaluating the quality of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion. The prepared 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is compounded with a film-forming matrix for apple coating preservation, and the quality of the apples is evaluated.

[0005] Technical solution: In the first aspect, the present invention provides a method for preparing a nanoemulsion of 2-(1H-indol-3-yl)-ethyl acetate, comprising the following steps:

[0006] S1. Dissolve sucrose fatty acid ester in ultrapure water, and stir to dissolve in a 60-80° C. water bath to obtain an aqueous phase;

[0007] S2, dissolving 2-(1H-indol-3-yl)-ethyl acetate in corn oil, and stirring to obtain an oil phase;

[0008] S3, mixing the oil phase obtained in step S2 with the water phase obtained in step S1, and then homogenizing and emulsifying to obtain a crude emulsion;

[0009] S4, subjecting the crude emulsion to ultrasonic crushing treatment to obtain 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion.

[0010] Preferably, the w / v concentration of sucrose fatty acid ester in the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is 0.5-2.5%.

[0011] Preferably, the w / v concentration of 2-(1H-indol-3-yl)-ethyl acetate in the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is 1-2%, and the volume ratio of the oil phase to the water phase is 7:93-19:81.

[0012] Preferably, the homogenization emulsification conditions in step S3 are: 12000 r / min, 5 min.

[0013] Preferably, the power of the ultrasonic crushing treatment in step S4 is 180-420 W, the ultrasonic time is 5-25 min, and it is turned on and off every 5 seconds.

[0014] In a second aspect, the present invention provides an acetic acid-2-(1H-indol-3-yl)-ethyl ester nanoemulsion, which is prepared by the preparation method described in the first aspect.

[0015] In a third aspect, the present invention provides use of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion described in the second aspect in controlling Penicillium expansum.

[0016] In a fourth aspect, the present invention provides use of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion described in the second aspect in apple preservation.

[0017] Preferably, the coating solution prepared by compounding 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion with chitosan is used in the preservation of post-harvest apples.

[0018] Furthermore, the application comprises the following steps:

[0019] (1) Weigh 1 g of chitosan powder, add 10 mL of 1.5% 2-(1H-indol-3-yl)ethyl acetate nanoemulsion, dilute to 90 mL with distilled water, and stir overnight to obtain a coating solution of 2-(1H-indol-3-yl)ethyl acetate nanoemulsion chitosan;

[0020] (2) Surface cleaning of apples;

[0021] (3) Using the coating solution prepared in step (1) and prepared by mixing 2-(1H-indol-3-yl)ethyl acetate nanoemulsion with chitosan to perform a coating treatment on apples.

[0022] Beneficial effects: 1) The present invention uses sucrose fatty acid ester as an emulsifier, and the prepared 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is of O / W type, the droplets are spherical, the average particle size is less than 200nm, the particle size distribution is uniform and concentrated, and the stability is good; 2) The present invention solves the problem that 2-(1H-indol-3-yl)-ethyl acetate has poor water solubility and low bioavailability; 3) The present invention solves the problem that in practical applications, when the nanoemulsion is sprayed on the smooth fruit surface, the high fluidity and low adhesion of the emulsion cause it to gather at the bottom of the fruit, thereby reducing the overall protectiveness; 4) The present invention uses chitosan combined with 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion to coat apples, which significantly improves the quality of the fruit, reduces the occurrence of mold, improves the fresh-keeping effect, and can avoid the harm of pesticide preparations to people and the pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart for preparing the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion;

[0024] Figure 2 The effects of different prescriptions and different preparation process conditions on 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, wherein A is the result of emulsifier dosage optimization, B is the result of oil phase dosage optimization, C is the result of ultrasound time optimization, and D is the result of ultrasound power optimization;

[0025] Figure 3 The physicochemical properties of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion of the present invention, wherein A is a Tyndall effect diagram of a ten-fold diluted nanoemulsion, B is the diffusion of Sudan Red III and methylene blue in the nanoemulsion, C is a particle size diagram of the AIE-NE nanoemulsion, and D is a potential diagram of the AIE-NE nanoemulsion;

[0026] Figure 4 The transmission electron microscope morphology of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion of the present invention;

[0027] Figure 5 Fourier infrared analysis of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, blank nanoemulsion, corn oil, sucrose fatty acid ester and 2-(1H-indol-3-yl)-ethyl acetate of the present invention;

[0028] Figure 6The present invention is a stability test result of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion stored at 4°C, 25°C and 37°C for 28 days, wherein A, B and C are the appearance diagrams of the emulsion stored at 4°C, 25°C and 37°C, respectively, D, E and F are the particle size changes of the emulsion stored at 4°C, 25°C and 37°C, respectively, and G, H and I are the PDI changes of the emulsion stored at 4°C, 25°C and 37°C, respectively;

[0029] Figure 7 The ion, heating, freeze-thaw and pH stability test results of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion of the present invention, wherein A, B and C are respectively the particle size, PDI and Zeta potential results of the nanoemulsion at different ion concentrations, D, E and F are respectively the particle size, PDI and Zeta potential results of the nanoemulsion at different heating temperatures, G, H and I are respectively the particle size, PDI and Zeta potential results of the nanoemulsion after freeze-thaw, and J, K and L are respectively the particle size, PDI and Zeta potential results of the nanoemulsion under different pH conditions;

[0030] Figure 8 The results of the investigation of the antibacterial activity of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion on Penicillium expansum, wherein A is the inhibitory effect of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion on Penicillium expansum in a liquid culture medium, and B is the inhibitory effect of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion on Penicillium expansum in a solid culture medium;

[0031] Fig. 9 The results of the investigation of the artificial inoculation of extended Penicillium lesions on apples after treatment with the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion of the present invention, wherein A is the appearance of the apple lesion wound, B, C and D are the lesion diameters of the apples on the 3rd, 5th and 7th days and the lesion inhibition rate of the nanoemulsion on the apple wound, respectively;

[0032] Fig.10 The present invention is a result of investigating the natural storage quality of apples after compounding the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion with chitosan, wherein A is an appearance diagram of apple storage, B is a change in weight loss rate of apples during storage, C is a change in rot rate of apples during storage, D is a change in hardness of apples during storage, E is a change in soluble solids content of apples during storage, F is a change in titratable acid content of apples during storage, G is a change in ascorbic acid content of apples during storage, and H is a browning degree of apples during storage. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0034] like Figure 1As shown, a method for preparing 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, the raw materials used are: sucrose fatty acid ester (emulsifier), 2-(1H-indol-3-yl)-ethyl acetate, corn oil and ultrapure water;

[0035] Preparation process: a. Weigh the raw materials according to the formula; b. Place the formulated amount of sucrose fatty acid ester in a beaker, add the formulated amount of ultrapure water, and stir magnetically at 60° C. as the aqueous phase; c. Mix 1.5% of 2-(1H-indol-3-yl)-ethyl acetate and corn oil as the oil phase, and then mix with the aqueous phase, homogenize at a speed of 12000r / min for 5 minutes, and treat the crude emulsion with an ultrasonic cell disruptor for 15 minutes (300W, 5s on, 5s off), and finally obtain 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion.

[0036] Example 1: Investigation of emulsifier dosage

[0037] The oil phase content was fixed at 13%. Different emulsifier dosages (0.5%, 1.0%, 1.5%, 2.0% and 2.5%) were weighed and mixed with a certain amount of ultrapure water. Figure 1 The preparation method shown is used to prepare 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, and the effect of different emulsifier amounts on the particle size and PDI of the nanoemulsion is examined.

[0038] Example 2: Investigation of the amount of oil phase used

[0039] The concentration of sucrose fatty acid ester screened in Example 1 was added with a corresponding amount of ultrapure water for mixing. Figure 1 The preparation method shown was used to prepare 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, and the effects of different oil phase amounts (7%, 10%, 13%, 16% and 19%) on the particle size and PDI of the nanoemulsion were screened.

[0040] Example 3: Screening of Ultrasonic Time

[0041] The best formula composition selected from Examples 1 and 2 is as follows: Figure 1 The preparation method shown is used to prepare 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, and the particle size and PDI of the nanoemulsion are used as evaluation indicators to investigate the effects of ultrasonic time of 5, 10, 15, 20 and 25 min on the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion.

[0042] Example 4: Screening of Ultrasonic Power

[0043] The best formula composition selected from Examples 1-3 is as follows: Figure 1The preparation method shown is used to prepare 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, and the particle size and PDI of the nanoemulsion are used as evaluation indicators to investigate the effects of ultrasonic powers of 180, 240, 300, 360, and 420 W on the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion.

[0044] The results of Examples 1-4 are as follows: Figure 2 As shown in AD:

[0045] (1) Effect of different dosages of sucrose fatty acid ester emulsifier on 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion: Emulsifier can effectively reduce the interfacial tension of the oil-water interface at a certain concentration. When the emulsifier is used at a dosage of 2.0%, the particle size and PDI of the emulsion are the smallest, while too much emulsifier will cause the droplets to break and the emulsion particle size to increase.

[0046] (2) Effect of the total amount of oil phase on 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion: Corn oil, as a dispersant, helps to evenly disperse the droplets in the liquid, but as its concentration increases, the emulsifier is insufficient to maintain stability, resulting in droplet aggregation. When the total amount of oil phase is 10%, the particle size and PDI of the emulsion are the smallest;

[0047] (3) Effect of ultrasonic time on 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion: When the ultrasonic time is 5-15 min, the ultrasonic action leads to an enhanced dispersion effect, thereby reducing the particle size of the emulsion. However, as the ultrasonic time increases, the dispersant loses its activity and the emulsion begins to coagulate, resulting in an increase in particle size. When the ultrasonic treatment time is 15 min, the particle size and PDI of the emulsion are relatively minimum;

[0048] (4) Effect of ultrasonic power on 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion Ultrasound can effectively reduce the average particle size of the emulsion. However, when the power is too high, it may cause adverse effects and lead to an increase in particle size. When the ultrasonic power is low, the dispersion effect of ultrasound is not enough to further reduce the droplets, while excessive ultrasonic power will also cause some negative effects, leading to an increase in particle size. With the increase of ultrasonic power, the particle size and PDI of the emulsion first decrease and then increase, but when the ultrasonic power is 300W, the average particle size and PDI of the emulsion are the smallest.

[0049] Therefore, the optimal formula composition is: sucrose fatty acid ester 2% (w / v), corn oil 10% (v / v), and the balance is ultrapure water; the optimal process conditions are: ultrasonic time 15 min, ultrasonic power 300 W (5 s on, 5 s off).

[0050] Example 5: Average particle size, PDI and Zeta potential of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsions of different concentrations

[0051] Determination of particle size, PDI, and Zeta potential: The attraction between droplets usually decreases as the droplet diameter decreases. The small particle size of nanoemulsions balances the Brownian motion of droplets with the attraction between droplets, thereby reducing the aggregation of droplets and keeping the emulsion stable. Related studies have shown that after nano-sizing, the particle size of the emulsion decreases and the stability improves. The polydispersity index (PDI) can represent the particle size distribution of the nanoemulsion. The larger the PDI, the more dispersed the particle size distribution of the emulsion, and the more likely the nanoemulsion is to undergo stratification or precipitation. The smaller the PDI, the more uniform the distribution of the nanoemulsion. Zeta potential represents the surface charge properties of the nanoemulsion. The larger the absolute value, the greater the charge repulsion between the nanoemulsion molecules, which can overcome the intermolecular attraction (i.e., van der Waals force and hydrophobic attraction), and the more stable the emulsion system. It is generally believed that when the absolute value of the potential is higher than 30mV, the droplets have a sufficiently large electrostatic repulsion to overcome the natural aggregation tendency, thereby maintaining the stability of the emulsion. To reduce the multiple light scattering effect, the nanoemulsion was diluted 100 times with ultrapure water. The particle size, PDI and Zeta potential were measured by Malvern nanoparticle sizer. The average value was taken after three repetitions. Each measurement contained 15 independent runs, each run lasted 10 s, and the equilibrium waiting time between two measurements was 120 s.

[0052] 0.1 g, 0.15 g and 0.2 g of 2-(1H-indol-3-yl)-ethyl acetate were weighed and dissolved in 1 mL of corn oil as the oil phase; 3 portions of 0.2 g of sucrose fatty acid ester were weighed and added to 9 mL of ultrapure water respectively, and magnetically stirred at 60° C. as the water phase; the three portions of oil phase were mixed with the water phase respectively, homogenized at a speed of 12000 r / min for 5 min, and the crude emulsion was treated with an ultrasonic cell disruptor for 15 min (300 W, 5 s on, 5 s off) to obtain 2-(1H-indol-3-yl)-ethyl acetate nanoemulsions with concentrations of 1%, 1.5% and 2%, and the average particle size, PDI and Zeta potential were measured. The results are shown in Table 1 below:

[0053] Table 1 Average particle size, PDI and Zeta potential of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsions with different concentrations

[0054]

[0055] Note: Different lowercase letters in the same column indicate significant differences among the samples in each group (p﹤0.05).

[0056] like Figure 3As shown in C and D: After ultrasonic treatment, the mixed solutions containing 2-(1H-indol-3-yl)-ethyl acetate can form nanoemulsions with droplet diameters below 200 nm, PDI values ​​are all less than 0.3, and the three emulsions are all negatively charged, with Zeta potential absolute values ​​greater than 30, indicating that the prepared nanoemulsions have high stability, small particle size and uniform distribution.

[0057] Example 6: Investigation of the physicochemical properties of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion

[0058] 1) Appearance of emulsion: observe its appearance with naked eyes;

[0059] 2) Identification of emulsion type: Take a freshly prepared nanoemulsion, add Sudan III and methylene blue dyes to it respectively, and observe the diffusion speed of the two dyes. If the diffusion speed of Sudan III dye is faster, it means that the nanoemulsion is a W / O type nanoemulsion; if the diffusion speed of methylene blue dye is faster, it means that the nanoemulsion is an O / W type nanoemulsion;

[0060] 3) pH determination: Take an appropriate amount of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion and place it in a small beaker, and measure it with a pH meter at room temperature;

[0061] 4) Morphological observation of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion: The nanoemulsion morphology was observed using a transmission electron microscope. An appropriate amount of nanoemulsion was diluted to an appropriate concentration and dropped on the surface of a copper mesh. After 5-6 minutes, excess liquid was removed with filter paper. A 2% tungsto-molybdic acid solution was added for staining for 1-2 minutes. Excess liquid was removed with filter paper. After drying, the nanoemulsion was observed using a Hitachi HT7800 transmission electron microscope.

[0062] 5) Fourier transform infrared spectroscopy study of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion: At 25°C, 2-(1H-indol-3-yl)-ethyl acetate (AIE), sucrose fatty acid ester sample (SE) and dried potassium bromide were mixed and pressed into tablets; after the potassium bromide was pressed into tablets, corn oil (Corn Oil) and 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion sample (AIE-NE) were smeared on it and the resolution was 4 cm -1 , scanning times 64 times, scanning range 4000-400cm -1 The test was carried out under the conditions of

[0063] The results are as follows:

[0064] 1) The three groups of nanoemulsions prepared under the optimal conditions were milky white uniform emulsions without stratification or flocculent precipitation. After being diluted 10 times, the emulsions glowed blue and had an obvious Tyndall effect ( Figure 3 A);

[0065] 2) The dye method was used to identify the type of nanoemulsion as O / W type ( Figure 3 B);

[0066] 3) The pH values ​​of the three groups of emulsions were 7.86±0.02, 7.56±0.1 and 7.1±0.05, respectively, which were weak alkaline solutions;

[0067] 4) The nanoemulsion was analyzed by transmission electron microscopy. The nanoemulsion was spherical, with uniform particle size distribution, uniform size and good formability ( Figure 4 );

[0068] 5) Infrared analysis of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion: Figure 5 As shown, the five samples have a wavelength of 1450-1750 cm -1 The stretching vibration of C=O bond and C=C bond on aromatic ring appears in the range of 800-680cm -1 The bending vibration of the CH bond on the aromatic ring appeared in all samples, and the four samples except AIE had a peak at 2800-3000 cm -1 The stretching vibration of saturated CH bonds appeared in both blank emulsion and AIE emulsion. -1 A broad peak in the wavenumber range is the stretching vibration of the intermolecular hydrogen bond OH, which is usually related to the strong intramolecular / intermolecular hydrogen bonds formed between water and emulsifiers. In addition, the characteristic peak of AIE is 3386cm -1 、2964cm -1 、1721cm -1 、1557cm -1 、1245cm -1 、1226cm -1 、740cm -1 It disappeared or weakened in the AIE nanoemulsion, and the peak intensity and shape also changed significantly, indicating that AIE was successfully encapsulated.

[0069] Example 7: Study on the stability of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion

[0070] 1) Storage stability at different temperatures: The 2-(1H-indol-3-yl)-ethyl acetate nanoemulsions of different concentrations in Example 2 were placed at 4° C., 25° C., and 37° C. for 28 days, and the appearance of the emulsions was observed on the 0th, 7th, 14th, 21st, and 28th days, respectively, to investigate the changes in the average particle size and PDI of the nanoemulsions;

[0071] 2) Stability under different ion concentrations: 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion was mixed with an equal volume of NaCl solution (0.1, 0.2, 0.3, 0.4 and 0.5 mmol / L) and stored at room temperature for 12 h to investigate the average particle size, PDI and Zeta potential of the nanoemulsion;

[0072] 3) Stability at different heating temperatures: The 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion was treated in a hot water bath at 60°C, 80°C and 100°C for 30 min, and the average particle size, PDI and Zeta potential of the nanoemulsion were investigated;

[0073] 4) Freeze-thaw stability: The 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion was frozen at -20°C for 22 h and then thawed at 40°C for 2 h for three cycles to investigate the average particle size, PDI and Zeta potential of the nanoemulsion.

[0074] 5) pH stability: The pH of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion was adjusted to 2, 3, 4, 5, 6, 7, 8 and 9 with 1 mol / L hydrochloric acid and 1 mol / L sodium chloride. The average particle size, PDI and Zeta potential of the nanoemulsion were measured after the pH adjusted nanoemulsion was left for 12 h, because pH induced ionization may be a slow process.

[0075] The results are as follows Figure 6-Figure 7 As shown:

[0076] 1) The long-term storage stability results show that the nanoemulsion is a thermodynamically unstable system. As the temperature rises, the molecular motion rate increases, the particles collide and aggregate with each other, resulting in a larger particle size. Sucrose fatty acid esters as emulsifiers can effectively overcome the particle aggregation caused by molecular thermal motion. The nanoemulsion has good physical stability at 4°C and 25°C, and the Zeta potential value is negative, indicating that the droplets are negatively charged, which can provide electrostatic repulsion to inhibit the occurrence of droplet flocculation during storage. On the 21st day of storage at 37°C, it was found that the average particle size of the 2% acetic acid-2-(1H-indol-3-yl)-ethyl ester nanoemulsion increased significantly to 206.3±2.36nm. On the 28th day, the average particle size increased to 219.63±1.78nm, and droplet agglomeration occurred in all three concentrations of nanoemulsions. Comprehensive analysis shows that 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion should be stored at 4℃ or 25℃, when the system is most stable and is conducive to storage;

[0077] 2) Effect of ionic strength: The particle size and PDI of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion change little at an ionic strength of 0.1-0.5mmol / L, but the absolute value of the potential tends to decrease with the increase of ion concentration. This may be because 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion attracts positive ions, causing the charge of the nanoemulsion to move toward neutrality faster. In general, 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is stable at an ionic strength of 0.1-0.5mmol / L;

[0078] 3) Effect of heating temperature: After heating at 60℃, 80℃ and 100℃ for 30min, compared with the initial state, with the increase of heating temperature, the particle size and PDI of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion slowly increased, but the average particle size was still below 200nm, which indicates that 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion has good stability at high temperature;

[0079] 4) Freeze-thaw stability: After three freeze-thaw cycles, the average particle size of the nanoemulsion increased from 163.9 ± 0.62 nm to 848.7 ± 16.71 nm, and the particle size distribution was not concentrated, which may be due to crystallization during the thawing process, resulting in interfacial film rupture, droplet aggregation and oil-water separation;

[0080] 5) pH stability: In the pH range of 6-9, the particle size of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion did not change significantly, but when the pH was 2-5, the particle size increased significantly and the absolute value of the potential decreased. This may be because as the pH value decreases, the hydrogen ions in the emulsion continue to increase, gradually neutralizing the negative charge of the droplets. This shows that due to the relatively strong charge repulsion between the droplets, 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion (pH 4-13) can still maintain a stable system. However, 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion (pH 2-3) is no longer stable, the droplets aggregate and demulsification or peeling occurs because the weaker charge repulsion is not enough to overcome the effect of gravity separation.

[0081] Example 8: Inhibitory effect of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion on Penicillium expansum

[0082] 1) Effect of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion on spore germination of Penicillium expansum in liquid culture medium: Penicillium expansum was inoculated on potato dextrose agar medium and placed in a constant temperature incubator at 25°C for 7 days. The Penicillium expansum spores were washed with saline and the spore concentration was adjusted to 1×10 6spores / mL, 1 mL of spore suspension was added to 9 mL of potato glucose medium and mixed to prepare 1×10 5 Spore suspension of spores / mL. The spore suspension and nanoemulsion were added to the microplate by the microbroth dilution method, respectively, so that the concentrations of acetic acid-2-(1H-indol-3-yl)-ethyl ester nanoemulsion in the wells reached 0.047, 0.094, 0.188, 0.375, 0.75, 1.5 and 3 g / L, respectively. The blank culture medium without nanoemulsion and spore solution was used as a blank control, and the spore solution without nanoemulsion was used as a growth control. The culture medium was placed in a constant temperature incubator at 25°C for 48 hours to investigate the effects of different concentrations of acetic acid-2-(1H-indol-3-yl)-ethyl ester nanoemulsion on Penicillium expansum.

[0083] 2) Effect of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion on hyphal growth of Penicillium expansum in solid culture medium: Mix 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion of different concentrations with heated and melted potato dextrose agar medium to prepare plate culture medium of different concentrations for later use. Take a 7.5 mm diameter bacterial cake from the Penicillium expansum colony cultured on potato dextrose agar medium for 1 day, inoculate the bacterial cake in the center of the plate culture medium, inoculate one bacterial cake on each plate, with the hyphae facing down, and culture in a 25°C incubator for 7 days. Repeat 3 times for each group. Mycelial growth inhibition rate = [(control group colony diameter - treatment group colony diameter) / control group colony growth diameter] × 100. SPSS software was used to analyze the toxicity regression equation and half maximal effective concentration (EC 50 ).

[0084] The results are as follows Figure 8 As shown:

[0085] 1) After 48 hours of action of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, the inhibitory effect of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion gradually increased with the increase of concentration, and the inhibitory effect showed a certain dose effect. It can be clearly seen that the mycelium in the growth control group grew vigorously. When the concentration was 0.375g / L, the liquid in the well was clear and there was no mycelium growth, and there was no mycelium growth in the microwells with higher concentrations.

[0086] 2) With the increase of the mass concentration of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, the inhibitory effect on the mycelial growth of Penicillium expansum gradually increased. The independent regression equation obtained by analysis was y=2.4897x-6.7770, and the half-maximum effect concentration was 0.527g / L.

[0087] Example 9: Effect of 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion treatment on artificially inoculated mold in apples

[0088] Use a sterile hole puncher to evenly punch three holes (5 mm in diameter, 5 mm in depth) at the equatorial position on the surface of each apple fruit, and then randomly divide the fruits into four groups, namely, a blank nanoemulsion control group (NE), and acetic acid-2-(1H-indol-3-yl)-ethyl ester nanoemulsion treatment groups (1.5 g / L AIE-NE, 3 g / L AIE-NE, and 6 g / L AIE-NE). 20 μL of nanoemulsion was added to the holes of the four groups of fruits, and 3 fruits were treated in each group. After the liquid in the wells dried for 2 hours, 20 μL of Penicillium expansum spore suspension (10 5 The spores / mL) were inoculated into the wells, and the fruits were placed in plastic boxes for storage at room temperature. The lesion diameter of each group of fruits during the test was measured and recorded on the 3rd, 5th and 7th days. Lesion inhibition rate (%) = [(lesion diameter of control group - lesion diameter of treatment group) / lesion diameter of control group] × 100.

[0089] The results are as follows Fig. 9 As shown: on the 3rd day of storage, the diameter of the lesions in the treatment group was significantly lower than that in the control group, which were 28.57%, 30.36% and 32.14% lower than those in the control group, respectively; on the 5th day of storage, the diameter of the lesions in the treatment group were 25.53%, 26.95% and 28.37% lower than those in the control group, respectively; on the 7th day of storage, the diameter of the lesions in the treatment group were 10.71%, 15.30% and 22.45% lower than those in the control group, respectively, and showed a concentration-dose effect.

[0090] Example 10: Application of a coating liquid prepared by mixing 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion with chitosan in postharvest preservation of apples

[0091] 1) Preparation of coating liquid:

[0092] Chitosan coating (CS): 1 g of chitosan powder was dissolved in 100 mL of distilled water and stirred overnight;

[0093] Blank nanoemulsion chitosan composite coating solution (NE / CS): weigh 1 g chitosan, add 10 mL blank nanoemulsion, dilute to 90 mL with distilled water, and stir overnight;

[0094] 2-(1H-indol-3-yl)ethyl acetate nanoemulsion chitosan composite coating liquid (AIE-NE / CS): Weigh 1 g of chitosan, add 10 mL of 1.5% 2-(1H-indol-3-yl)ethyl acetate nanoemulsion, dilute to 90 mL with distilled water, and stir overnight.

[0095] 2) Application of coating liquid on apple surface:

[0096] Select ripe fruits with no pests and diseases, smooth surface and uniform size, and disinfect them with 0.1% sodium hypochlorite solution for 3-5 minutes, then wash with clean water and dry them naturally for later use. Divide the apples into 3 groups, 36 in each group, soak them in different coating solutions for 2 minutes, dry them naturally and store them alternately at 25℃ day and night, respectively, and record them as CS group, NE / CS group and AIE-NE / CS group. The weight, hardness, rot rate, browning degree, titratable acid content, ascorbic acid content and soluble solid content of each group of apples were measured on the 0th, 7th, 14th, 21st and 28th day.

[0097] like Fig.10 As shown in the figure, the weight loss rate, decay rate and browning degree of the fruit during storage showed an overall increasing trend, while the hardness and acid content showed a decreasing trend. Compared with the control group, the coating treatment containing 2-(1H-indol-3-yl)ethyl acetate nanoemulsion can effectively maintain the quality of apples. The soluble solid content fluctuated during storage, which may be due to the consumption of sugar by the fruit during the initial respiration or external infection, the metabolic conversion of soluble carbohydrates in the ripening state, and the consumption of carbohydrates by fruit aging or infection. Some fruits in the CS group and NE / CS group were infected by mold, and the AIE-NE / CS coating treatment could effectively prevent the infection of external mold.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion, characterized in that: The following steps are involved: S1. Dissolve sucrose fatty acid ester in ultrapure water, and stir to dissolve in a 60-80° C. water bath to obtain an aqueous phase; S2, dissolving 2-(1H-indol-3-yl)-ethyl acetate in corn oil, and stirring to obtain an oil phase; S3, mixing the oil phase obtained in step S2 with the water phase obtained in step S1, and then homogenizing and emulsifying to obtain a crude emulsion; S4. The crude emulsion is subjected to ultrasonic crushing treatment to obtain 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion.

2. The method for preparing a nanoemulsion of 2-(1H-indol-3-yl)-ethyl acetate according to claim 1, characterized in that: The w / v concentration of sucrose fatty acid ester in the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is 0.5-2.5%.

3. The method for preparing a nanoemulsion of 2-(1H-indol-3-yl)-ethyl acetate according to claim 1, characterized in that: The w / v concentration of 2-(1H-indol-3-yl)-ethyl acetate in the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion is 1-2%, and the volume ratio of the oil phase to the water phase is 7:93-19:

81.

4. The method for preparing a nanoemulsion of 2-(1H-indol-3-yl)-ethyl acetate according to claim 1, characterized in that: The conditions for homogenization and emulsification in step S3 are: 12000 r / min, 5 min.

5. The method for preparing a nanoemulsion of 2-(1H-indol-3-yl)-ethyl acetate according to claim 1, characterized in that: The power of the ultrasonic crushing treatment in step S4 is 180-420 W, the ultrasonic time is 5-25 min, and it is turned on and off every 5 seconds.

6. A nanoemulsion of 2-(1H-indol-3-yl)-ethyl acetate, characterized in that: The invention is prepared by the preparation method described in any one of claims 1 to 5.

7. Use of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion according to claim 6 in controlling Penicillium expansum.

8. Use of the 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion according to claim 6 in apple preservation.

9. The use according to claim 8, characterized in that: Application of the coating solution prepared by compounding 2-(1H-indol-3-yl)-ethyl acetate nanoemulsion with chitosan in the postharvest preservation of apples.

10. The use according to claim 9, characterized in that: The following steps are involved: (1) Weigh 1 g of chitosan powder, add 10 mL of 1.5% 2-(1H-indol-3-yl) ethyl acetate nanoemulsion, dilute to 90 mL with distilled water, and stir overnight to obtain a coating solution of 2-(1H-indol-3-yl) ethyl acetate nanoemulsion and chitosan; (2) Cleaning the surface of the apple; (3) The apples are immersed in the coating solution prepared by mixing the 2-(1H-indol-3-yl) ethyl acetate nanoemulsion and chitosan prepared in step (1) to form a coating.

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