Carvyl ketone nanoemulsion, preparation method and application thereof

Abstract

The application discloses carvone nanoemulsion, a preparation method and application thereof. The preparation method comprises the following steps: dissolving pea protein isolate in water, adjusting the pH to be greater than or equal to 10, adjusting the pH to be less than or equal to 8.5 after heating and stirring, and obtaining a pea protein isolate solution; dissolving octenyl succinic acid starch in water, heating and gelatinizing to obtain an octenyl succinic acid starch dispersion; mixing the pea protein isolate solution and the octenyl succinic acid starch dispersion in equal volumes to obtain a pea protein isolate-octenyl succinic acid starch composite dispersion; adding non-volatile vegetable oil and carvone into the composite dispersion, performing homogenization treatment to obtain carvone nano coarse emulsion, and performing ice bath ultrasonic refining treatment to obtain carvone nanoemulsion. The carvone nanoemulsion has excellent stability and antibacterial activity, the preparation process is simple and easy to operate, safe, non-toxic and green, raw materials are all food grade, and the carvone nanoemulsion has strong compatibility and wide application prospect in the field of fruit and vegetable preservation.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product storage and preservation technology, and relates to a carvone nanoemulsion, its preparation method and application. Background Technology

[0002] Kiwifruit, a specialty fruit rich in Vitamin C and dietary fiber, is favored by consumers worldwide for its unique flavor and high nutritional value, and has become an important export fruit for my country. However, kiwifruit is highly susceptible to soft rot caused by *Staphylococcus aureus* after harvest, leading to rapid softening and decay of the fruit. This not only severely reduces its commercial value but also causes significant post-harvest losses and economic damage. Therefore, controlling post-harvest soft rot in kiwifruit, extending its shelf life, and achieving post-harvest value enhancement have become the focus of industry attention, and exploring new preservation technologies for controlling soft rot has become a current research focus. Meanwhile, although traditional chemical preservatives are widely used in post-harvest preservation of fruits and vegetables, they pose a risk of chemical residues and may affect the original flavor of the fruit, potentially threatening food safety and product quality. Based on this, bio-based preservation materials with natural active substances at their core, with their core advantages of safety, non-toxicity, and environmental friendliness, are considered a highly promising green alternative and have become a research hotspot in both academia and industry, receiving continuous and in-depth exploration.

[0003] Plant essential oils, with their natural, highly effective, and safe antibacterial activity, have great potential in the postharvest preservation of fruits and vegetables. However, plant essential oils generally suffer from drawbacks such as volatility, poor chemical stability, and strong hydrophobicity. These problems accelerate the loss of active ingredients, further reducing their effective utilization rate. Furthermore, their hydrophobic nature makes them difficult to spread evenly when applied directly to the surface of fruits and vegetables, easily leading to problems such as excessively high local concentrations that burn the fruit and vegetable tissue, or insufficient local concentrations that fail to inhibit bacteria, severely limiting the preservation effect. Carvone, as a plant essential oil, has a better inhibitory effect on various postharvest fungi than some chemical preservatives and can fundamentally avoid the risk of chemical reagent residues. However, it also suffers from volatility, instability, and strong hydrophobicity. Therefore, there is an urgent need to develop a safe and natural preservative that can simultaneously solve the above problems, improving the hydrophobicity, volatility, and biocompatibility of carvone while achieving long-lasting antibacterial effects, thereby effectively controlling postharvest soft rot in kiwifruit and increasing the practical application value of carvone in the postharvest preservation of kiwifruit. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a carvone nanoemulsion with excellent stability, long-lasting antibacterial properties and fruit and vegetable preservation properties, as well as its preparation method and application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0006] A method for preparing a carvone nanoemulsion includes the following steps: (1) Dissolve pea protein isolate in water, adjust the pH to ≥10, heat and stir, and then adjust the pH to ≤8.5 to obtain pea protein isolate solution; (2) Dissolve octenyl succinic acid starch in water, heat and stir to gelatinize, and obtain octenyl succinic acid starch dispersion; (3) Mix the pea protein isolate solution and the octenyl succinic acid starch dispersion in equal volumes, and stir to obtain a pea protein isolate-octenyl succinic acid starch composite dispersion; (4) Add non-volatile vegetable oil and carvone to the pea protein isolate-octenyl succinic acid starch composite dispersion and homogenize to obtain carvone nano-crude emulsion; (5) The carvone nano-emulsion was subjected to ice bath ultrasonic refining treatment to obtain carvone nano-emulsion; In step (1), the mass ratio of the pea protein isolate to the volume of water is 0.2g~0.4g∶10mL; In step (2), the mass ratio of the octenyl succinic acid starch to the volume ratio of water is 0.1g~0.2g∶10mL; In step (4), the ratio of the mass of soybean oil, the mass of carvone, and the volume of pea protein isolate-octenyl succinic acid starch composite dispersion is 0.1g~0.3g∶0.2g~0.6g∶10mL.

[0007] In the preferred method for preparing the above-mentioned carvone nanoemulsion, in step (1), the pH is adjusted to 10-12, the heating is water bath heating, the heating temperature is 45 ℃-48 ℃, and the pH is further adjusted to 7.5-8.5.

[0008] In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (2), the heating is water bath heating, and the heating temperature is 85 ℃~95 ℃.

[0009] In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (4), the rotation speed of the homogenization treatment is 8000 rpm to 12000 rpm, and the homogenization treatment time is 3 min to 5 min.

[0010] In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (5), the power of the ultrasonic refining treatment is 300 W to 350 W, and the time of the ultrasonic refining treatment is 15 min to 20 min.

[0011] In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (4), the non-volatile vegetable oil is a plant-derived triglyceride, which is one or more of soybean oil, corn oil, palm oil and medium-chain triglycerides.

[0012] In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (1), the stirring speed is 300 rpm to 600 rpm and the stirring time is 60 min to 90 min.

[0013] In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (2), the stirring speed is 300 rpm to 600 rpm and the stirring time is 30 min to 60 min; In the above-mentioned method for preparing carvone nanoemulsion, preferably, in step (3), the stirring speed is 300 rpm to 500 rpm and the stirring time is 10 min to 15 min.

[0014] As a general technical concept, the present invention also provides a carvone nanoemulsion prepared by the above-mentioned method.

[0015] Preferably, the carvone nanoemulsion has a particle size of 100 nm to 300 nm and a polydispersity index of 0.1 to 0.2.

[0016] As a general technical concept, the present invention also provides an application of the above-mentioned carvone nanoemulsion in postharvest preservation of fruits and vegetables.

[0017] The preferred application described above is the use of carvone nanoemulsion in the prevention and preservation of postharvest soft rot in kiwifruit.

[0018] In this invention, the carvone nanoemulsion contains nanoparticles (mainly carvone nanoparticles) and belongs to a dispersion system.

[0019] Compared with the prior art, the advantages of the present invention are as follows: (1) The method for preparing carvone nanoemulsion of the present invention involves coating with a proteoglycan complex system and ultrasonic emulsification technology to prepare carvone nanoemulsion. On the one hand, the nanoemulsion has a nanoscale particle size and excellent polymer dispersibility index (0.1-0.2), and exhibits excellent stability, remaining stable at 4 °C for more than one month. Furthermore, compared with traditional Tween 80 emulsion, the encapsulation rate of carvone in this nanoemulsion is as high as 94% or more. On the other hand, pea protein isolate and octenyl succinate starch are combined into a proteoglycan complex system through hydrogen bonding, electrostatic interaction, and hydrophobic interaction, which can act as an effective stabilizer. The present invention effectively improves the volatility, hydrophobicity, and biocompatibility of carvone, achieving slow release of carvone.

[0020] (2) Compared with commercially available compound preservatives, the carvone nanoemulsion prepared in this invention has stronger antibacterial activity. In vivo verification experiments on kiwifruit show that it can significantly reduce the diameter of lesions and effectively control fruit rot caused by soft rot bacteria. Adding this nanoemulsion to pectin solution can endow the solution with both antioxidant and antibacterial properties; the resulting coating solution can form a dense protective film on the fruit surface, reducing the weight loss rate and rot rate of kiwifruit, and has significant effects in controlling kiwifruit soft rot and extending its shelf life.

[0021] (3) The present invention successfully prepared a natural and safe fruit and vegetable preservative. The raw materials of the composite carvone nanoemulsion are all natural compounds, which are green and environmentally friendly. The preparation process is simple and has excellent slow release, antioxidant and antibacterial properties. It can effectively preserve the freshness of harvested fruits and vegetables, especially the prevention and control of soft rot disease of kiwifruit and the preservation effect of kiwifruit. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy (TEM) images of carvone nanoemulsions from Examples 1, 1, and 2 of this invention.

[0023] Figure 2 The particle size distribution, PDI diagram, and zeta potential diagrams of the carvone nanoemulsions of Examples 1, 1, and 2 of this invention are shown.

[0024] Figure 3 The Fourier transform infrared spectra of carvone nanoemulsions from Examples 1, 1, and 2 of this invention are shown.

[0025] Figure 4 The graph shows the changes in particle size and PDI of carvone nanoemulsions from Examples 1, 1, and 2 of this invention during storage at 4 °C.

[0026] Figure 5 The graph shows the particle size variation of carvone nanoemulsions in Examples 1, 1, and 2 of this invention at different pH values.

[0027] Figure 6 The graph shows the particle size variation of carvone nanoemulsions in Examples 1, 1, and 2 of this invention under different concentrations of salt ions.

[0028] Figure 7 The diagram shows the carvone encapsulation rate of the carvone nanoemulsions of Example 1, Comparative Example 1, and Comparative Example 2 of this invention.

[0029] Figure 8 The graphs show the DPPH and ABTS free radical scavenging capabilities of the carvone nanoemulsions of Examples 1, 1, and 2 of this invention.

[0030] Figure 9 The images show the appearance of the carvone nanoemulsions of Example 1, Comparative Example 1 and Comparative Example 2 of the present invention, as well as the control group 1 without any treatment, inhibiting the growth of *Botrytis cinerea* hyphae.

[0031] Figure 10 The images show a comparison of colony diameters and inhibition rates of *Vitis vinifera* hyphae grown using carvone nanoemulsions in Examples 1, 1, and 2 of this invention.

[0032] Figure 11 Visual representation of the changes over time in kiwifruit inoculated with Fibrocystic soft rot during storage of the carvone nanoemulsion prepared in Example 1 of this invention, the pea protein isolate-octenyl succinic acid starch composite dispersion in Comparative Example 3, the commercial preservative in Comparative Example 4, and the untreated control group 1.

[0033] Figure 12 The graph shows the changes in the diameter of soft rot lesions in kiwifruit during storage, including the carvone nanoemulsion prepared in Example 1 of this invention, the pea protein isolate-octenyl succinic acid starch composite dispersion in Comparative Example 3, the commercial preservative in Comparative Example 4, and the untreated control group 1.

[0034] Figure 13 The graph shows the changes in conductivity of kiwifruit during storage for the carvone nanoemulsion prepared in Example 1 of this invention, the pea protein isolate-octenyl succinic acid starch composite dispersion in Comparative Example 3, the commercial preservative in Comparative Example 4, and the untreated control group 1.

[0035] Figure 14 The images show the apparent quality of kiwifruit during storage for pectin coating treatment containing the carvone nanoemulsion prepared in Example 1 of this invention, pectin coating treatment containing the commercial preservative of Comparative Example 4, control group 1 without any treatment, and control group 2 treated only with pectin coating.

[0036] Figure 15Comparison charts of weight loss, rot rate, and hardness during storage of kiwifruit containing the carvone nanoemulsion prepared in Example 1 of this invention, the pectin coating treatment containing the commercial preservative of Comparative Example 4, the control group 1 without any treatment, and the control group 2 treated only with pectin coating. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available. Pea protein isolate and octenyl succinate starch were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and carvone was purchased from Shandong Wanhua Chemical Technology Co., Ltd.

[0038] Example 1 A method for preparing the carvone nanoemulsion of the present invention, as follows: Figure 1 As shown, it includes the following steps: (1) Add pea protein isolate to deionized water, adjust the pH to 11, stir continuously at 500 rpm in a 45 ℃ hot water bath for 60 min, and then adjust the pH to 8 to obtain pea protein isolate solution (homogeneous solution system); wherein, the mass ratio of pea protein isolate to deionized water is 0.2 g: 10 mL.

[0039] (2) Add octenyl succinic acid starch to deionized water, with the mass ratio of octenyl succinic acid starch to the volume ratio of deionized water being 0.1 g: 10 mL. Dissolve the starch by stirring at 500 rpm for 30 min in a 90 ℃ hot water bath to obtain an octenyl succinic acid starch dispersion.

[0040] (3) Mix the pea protein isolate solution obtained in step (1) with the octenyl succinic acid starch dispersion obtained in step (2) in equal volumes, stir at 400 rpm for 10 min to obtain pea protein isolate-octenyl succinic acid starch composite dispersion.

[0041] (4) Soybean oil and carvone were added dropwise to the pea protein isolate-octenyl succinic acid starch composite dispersion. The ratio of the mass of soybean oil, the mass of carvone and the volume of pea protein isolate-octenyl succinic acid starch composite dispersion was 0.2 g: 0.4 g: 10 mL. The mixture was homogenized at 9000 rpm for 3 min to obtain a crude emulsion.

[0042] Soybean oil can be replaced by other non-volatile vegetable oils, such as corn oil, palm oil, medium-chain triglycerides (MCT oil) or mixtures thereof; soybean oil is used mainly because it is readily available, economically priced, safe and non-toxic, and meets the requirements of the food industry.

[0043] (5) The crude emulsion was placed in an ice bath (0 °C) for ultrasonic treatment. The ultrasonic power was 300 W and the ultrasonic time was 15 min to obtain carvone nanoemulsion with a particle size of 190 nm to 200 nm.

[0044] The carvone nanoemulsion prepared in this embodiment is used for the prevention and control of soft rot in kiwifruit and for preservation.

[0045] Comparative Example 1 A method for preparing carvone nanoemulsion, the preparation process is basically the same as in Example 1, the difference being that in step (4), the ratio of the mass of carvone, the mass of soybean oil and the volume of pea protein isolate-octenyl succinic acid starch composite dispersion is 0.1 g∶0.2 g∶10 mL.

[0046] Comparative Example 2 A method for preparing carvone nanoemulsion, the preparation process is basically the same as in Example 1, the difference being that in step (4), the ratio of the mass of carvone, the mass of soybean oil and the volume of pea protein isolate-octenyl succinic acid starch composite dispersion is 0.8 g∶0.2 g∶10 mL.

[0047] Comparative Example 3 A method for preparing a pea protein isolate-octenyl succinic acid starch composite dispersion, the preparation process is the same as steps (1), (2) and (3) in Example 1.

[0048] Comparative Example 4 A commercially available fruit preservative was purchased from Henan Yinsheng Health Technology Co., Ltd. The concentrate was diluted 500 times with deionized water according to the instructions.

[0049] The embodiments and comparative examples were tested and characterized in the following manner: (1) Transmission electron microscopy: First, ensure the emulsion is homogeneous by ultrasonic dispersion. Then, drop 10 μL of the emulsion onto a copper mesh support and let it stand for 15 min. Next, stain with 2% phosphotungstic acid for 1–3 min. After removing excess staining solution and allowing it to air dry for 15 min, take images using a transmission electron microscope at 100 kV to ensure the droplets are clearly visible. During the operation, avoid excessive drying and contamination of the sample to ensure that the sample structure is not damaged.

[0050] (2) Particle size, polydispersity index (PDI) and zeta potential: The particle size, PDI and zeta potential of the nanoemulsion were determined by a nanoparticle size analyzer. The emulsion was diluted 100 times before measurement to eliminate the effect of dispersion.

[0051] (3) Fourier transform infrared spectroscopy: The carvone nanoemulsion was measured at 4000–400 cm⁻¹ using a Fourier transform infrared spectrophotometer. -1Infrared spectroscopy in the wavenumber range was used to analyze the interaction between the composite dispersion and carvone.

[0052] (4) Storage stability: The carvone nanoemulsion was stored at 4 °C for 40 days, and the particle size and PDI were measured on days 0, 10, 20, 30 and 40.

[0053] (5) pH stability: The particle size was measured after adjusting the pH of the carvone nanoemulsion to 3, 5, 7, 9 and 11 with 1 M NaOH or HCl.

[0054] (6) Salt ion stability: The particle size was measured after adding sodium chloride solutions of different concentrations (0, 50, 100, 150 and 200 mM) to the carvone nanoemulsion.

[0055] (7) Encapsulation efficiency: The absorbance of carvone dissolved in n-hexane at 235 nm was measured using a UV-Vis spectrophotometer and a standard curve was plotted. Then, carvone nanoemulsion (1 mL) was dissolved in n-hexane (9 mL) and centrifuged for 5 minutes. Subsequently, the absorbance of the supernatant at 235 nm was measured, and the encapsulation efficiency was calculated based on the standard curve.

[0056] (1) Where M a and M t These represent the total carvone content and the unencapsulated carvone content, respectively.

[0057] (8) DPPH and ABTS free radical scavenging ability: Dissolve 100 μL of carvone nanoemulsion in 1 mL of deionized water, and then mix the sample with 3 mL of DPPH methanol solution or ABTS working solution. Keep in a 37 ℃ water bath for 10 minutes to ensure sufficient reaction. The antioxidant capacity of carvone nanoemulsion is reflected by its ability to scavenge DPPH and ABTS free radicals (ABTS working solution preparation: 7 mM ABTS and 2.45 mM potassium persulfate solution are mixed at a ratio of 1:1 (v / v) for 12 h, and then diluted with ethanol to an absorbance of 0.70±0.02 at 734 nm). Finally, the free radical scavenging rate is calculated according to the following formula.

[0058] (2) A0 and A1 are the absorbance of the blank control and the sample, respectively.

[0059] (9) Inhibition of mycelial growth of *Vitis vinifera* by carvone nanoemulsion: 6 mm fungal cakes were placed in the center of a PDA medium plate containing 200 μL of carvone nanoemulsion. The plate was then placed in a constant temperature and humidity incubator at 28 ℃ and 90% relative humidity for 7 days, and mycelial growth was observed.

[0060] (10) Inhibition of *Botrytis cinerea* hyphae growth by carvone nanoemulsion: A 6 mm fungal cake was placed in the center of a PDA medium plate containing 200 μL of carvone nanoemulsion. The plate was then placed in a constant temperature and humidity incubator at 28 ℃ and 90% relative humidity for 7 days. The hyphae diameter was measured and the inhibition rate was calculated. The inhibition rate was calculated according to formula (3).

[0061] (3) Where D0 and D1 represent the hyphal diameter of the blank control and the hyphal diameter of the sample group, respectively. (11) In vivo verification experiment of carvone nanoemulsion inhibiting *Staphylococcus aureus* in kiwifruit: Kiwifruit with similar maturity, uniform size, good color, and no mechanical damage were selected for in vivo experiments. A sterile needle was used to puncture a hole at the equator of the kiwifruit, with a wound size of 5 mm deep and 3 mm wide. *Staphylococcus aureus* was then inoculated at the wound site, and 10 μL of carvone nanoemulsion prepared in Example 1, the composite dispersion of Comparative Example 3, and the commercial preservative of Comparative Example 4 were added respectively. Finally, all fruits were placed in transparent and breathable boxes and stored in a constant temperature and humidity incubator at 28 ℃ and 90% RH. The diameter of kiwifruit lesions was measured on days 0, 2, 4, 6, and 8, and the average value was calculated.

[0062] (12) In vivo verification experiment of kiwifruit: Changes in conductivity during storage: On days 0, 2, 4, 6, and 8, pulp near the lesions of kiwifruit was collected, washed with distilled water, and dried. 2 g of sample was weighed and placed in an Erlenmeyer flask, 40 mL of distilled water was added, and its conductivity E0 was measured immediately. After 10 min, the conductivity E1 was measured again. Subsequently, it was transferred to a flask and heated to boiling on an electric stove for 10 min. After cooling, it was weighed and distilled water was added to the original weight. After equilibration at room temperature, the conductivity E2 after boiling was measured. The relative conductivity was calculated according to formula (4).

[0063] (4) (13) Application experiment of carvone nanoemulsion in the in vitro preservation of kiwifruit: Kiwifruit with similar maturity, uniform size, good color, no mechanical damage, and no pathogen infection were selected for preservation experiments. After simply removing surface dust from all kiwifruit, a 2% pectin coating solution was prepared. First, 20g of pectin powder was dissolved in 1L of deionized water and stirred for 12 h. After dissolution, 5% (5% of the volume of the pectin solution without glycerol) of the carvone nanoemulsion prepared in Example 1 was added and stirred continuously for 30 min. Then, glycerol was added at 30% of the weight of the pectin powder, and finally, the mixture was ultrasonically treated for 30 min to remove air bubbles. A pectin coating solution containing the commercial preservative of Comparative Example 4 was prepared in the same manner. Kiwifruit coated with the pectin coating solution served as control group 2. The pectin coating solution containing the commercial preservative of Comparative Example 4 and the pectin coating solution containing the carvone nanoemulsion prepared in Example 1 were used as blank control. After all samples were air-dried, they were packed into PE plastic boxes in groups of six kiwifruit and stored at room temperature with 75% relative humidity. Samples were taken and observed on days 0, 3, 6, 9, 12, and 15.

[0064] (14) Appearance quality: Observe the appearance quality of kiwifruit by taking photos of its appearance.

[0065] (15) Weight loss rate: The weight change of kiwifruit during storage was measured by weighing with an electronic balance using the gravimetric method. The weight loss rate was calculated according to formula (5).

[0066] (5) Where W0 and W1 are the initial weight of the fruit and the weight of the fruit at different storage times, respectively.

[0067] (16) Rot rate: 100 kiwifruits were selected from each group to count the number of rotten kiwifruits during storage. The rot rate was calculated according to formula (6).

[0068] (6) Where N0 and N1 are the number of rotten fruits and the total number of fruits, respectively. (17) Hardness: The hardness change of kiwifruit during storage was determined using a texture analyzer. A TA-41 probe was used to puncture the equatorial region of the kiwifruit. The test speed was 1 mm / s and the probe puncture depth was 8 mm. The results are expressed as N.

[0069] The above detection and characterization results are analyzed as follows: (1) Morphology of nanoemulsions Figure 1These are transmission electron microscopy (TEM) images of the carvone nanoemulsions of Example 1, Comparative Example 1, and Comparative Example 2 of this invention. TEM observations show that all three nanoemulsions exhibit regular, spherical droplet morphology, confirming the successful construction of the oil-in-water (O / W) emulsion system. Specifically, the nanoemulsion of Example 1 has the most uniform droplet size and excellent dispersibility, with no obvious agglomeration or large particles. While the overall droplet size of Comparative Example 1 is relatively small, it contains a small number of larger particles, resulting in slightly less uniform dispersion than Example 1. The droplets of Comparative Example 2 exhibit uneven particle size, a high proportion of large particles, and localized agglomeration.

[0070] (2) Particle size, PDI and ζ potential of nanoemulsion Figure 2 The particle size, PDI diagram, and zeta potential diagrams of the carvone nanoemulsions of Example 1, Comparative Example 1, and Comparative Example 2 of this invention are shown. Figure 2 As shown in the particle size distribution diagram (1), the sizes of the carvone nanoemulsions in Examples 1, 1, and 2 all decreased to below 300 nm under ultrasonic treatment. Among them, the carvone nanoemulsion in Example 1 had the smallest particle size, at 234.8 nm. The smaller particle size laid the foundation for the uniform dispersion and stability of the emulsion. Figure 2 As shown in (2) the PDI plot, the PDI (polydispersity index) of all samples is below 0.3, which meets the requirements for uniformity of nanoemulsions. Among them, the PDI value of Example 1 is the lowest (0.1019), which is significantly lower than that of Comparative Example 1 (0.2539) and Comparative Example 2 (0.2238), indicating that the nanoemulsion of Example 1 has the most uniform droplet size distribution and the best dispersibility. Figure 2 As shown in the ζ-potential diagram (3), the absolute potential values ​​of the carvone nanoemulsions of Example 1 and Comparative Example 1 are greater than 30 mV, indicating stronger stability.

[0071] (3) Viscosity diagram and Fourier transform infrared spectrum of nanoemulsion Figure 3 The images show the Fourier transform infrared spectra of the carvone nanoemulsions of Examples 1, 1, and 2 of this invention. Figure 3 As shown, overall, no new absorption peaks appeared in any of the nanoemulsions, indicating that there were no significant chemical interactions between them. (1700–1600 cm⁻¹) -1 The characteristic C=O peak of the aldehyde group of carvone was completely preserved, confirming the effective encapsulation of carvone by the composite material; in the range of 3000–2800 cm⁻¹ -1 The obvious CH stretching vibration peaks indicate the presence of hydrogen bonding in the system, which enhances emulsion stability. This is corroborated by the characterization results of particle size and morphology. In summary, the infrared spectroscopy results further confirm the structural stability of the carvone nanoemulsion, providing support for the long-term efficacy of its active functions.

[0072] (4) Storage stability of nanoemulsions Figure 4 This diagram shows the changes in particle size and PDI of carvone nanoemulsions from Examples 1, 1, and 2 of the present invention during storage at 4 °C. A 40-day storage test was conducted on the carvone nanoemulsions, and their storage stability was evaluated by the changes in droplet size and PDI over storage time. Figure 4 As shown in (1), throughout the entire storage period, compared to Comparative Example 2, the particle size of the carvone nanoemulsions in Example 1 and Comparative Example 1 remained around 250 nm, and the growth rate was significantly smaller than that in Comparative Example 2. Figure 4 (2) As shown, although the PDI of Example 1 fluctuated slightly, it remained below 0.25 throughout the process, and the droplet dispersion remained good. This excellent storage stability is mainly due to the strong affinity and permeability of the hydrophobic groups of the composite material to the oil phase, which can form a dense and stable oil-water interface with both steric hindrance and electrostatic repulsion, thereby providing a long-term stable encapsulation and sustained release environment for carvone.

[0073] (5) pH stability and salt ion stability of nanoemulsions Figure 5 The graph shows the particle size variation of carvone nanoemulsions in Examples 1, 1, and 2 of this invention at different pH values. Figure 6 This diagram shows the particle size variation of carvone nanoemulsions from Examples 1, 1, and 2 of the present invention under different salt ion concentrations. To evaluate the pH and salt ion stability of the carvone nanoemulsions, the particle size was tested at pH values ​​from 3 to 11 and salt ion concentrations from 0 mM to 200 mM. Figure 5 It is known that all emulsions exhibit very small particle size variations and remain essentially stable at pH values ​​between 7 and 11; however, they become highly unstable at pH values ​​between 3 and 5, with a sharp increase in particle size. This is because acidic conditions disrupt the structure or charge balance of the composite material, leading to droplet aggregation. Furthermore, from... Figure 6 It can be seen that as the treatment concentration increased from 0 mM to 200 mM, the particle size of the nanoemulsions in Example 1 and Comparative Example 1 remained stable compared to Comparative Example 2. This indicates that the carvone nanoemulsions in Example 1 and Comparative Example 1 are more tolerant to changes in salt ion concentration and can better maintain the small particle size and dispersibility of the emulsion. The results show that Example 1 and Comparative Example 1 exhibit good pH and salt ion stability.

[0074] (6) Encapsulation efficiency of nanoemulsions Figure 7 The encapsulation efficiency of the carvone nanoemulsions in Examples 1, 1, and 2 of this invention is shown. Figure 7As shown, the encapsulation efficiency of the three emulsions for carvone was at a high level (93.13%-96.53%), with Comparative Example 1 having the highest encapsulation efficiency (96.53%), followed by Example 1 (94.63%), and Comparative Example 2 having the lowest (93.13%). Overall, the efficiency slightly decreased with increasing carvone concentration, a phenomenon attributed to gravity-induced oil-water separation caused by the increase in oil phase fraction. The carvone nanoemulsion of Example 1 maintained a high encapsulation efficiency of over 94%, effectively inhibiting the volatilization and decomposition of carvone and providing an ideal sustained-release environment, thereby enhancing the stability and sustainable antibacterial properties of carvone.

[0075] (7) Antioxidant capacity of nanoemulsions Figure 8 This diagram shows the antioxidant capacity of the carvone nanoemulsions from Examples 1, 1, and 2 of this invention. The excellent antioxidant function of the carvone nanoemulsion is key to its application value. The scavenging activity of DPPH and ABTS free radicals can reflect whether the nanoemulsion retains the antioxidant properties of carvone. Figure 8 As shown in (1) and (2), the scavenging activities of DPPH and ABTS are related to the content of carvone. The antioxidant capacity of the carvone nanoemulsion shows an increasing trend with the increase of carvone content. Among them, the scavenging activities of DPPH (54.5%) and ABTS (73.2%) free radicals of the carvone nanoemulsion in Example 1 fully demonstrate that carvone endows the nanoemulsion with good antioxidant capacity. The results show that the above-mentioned carvone nanoemulsion can effectively retain the antioxidant activity of carvone while ensuring the stability of the emulsion.

[0076] (8) Antibacterial properties of nanoemulsions against Staphylococcus aureus Figure 9 The images show the appearance of the carvone nanoemulsions of Example 1, Comparative Example 1 and Comparative Example 2 of the present invention, as well as the growth inhibition of *Botrytis cinerea* hyphae in the untreated control group 1. Figure 10 The figures show the hyphal diameter and inhibition rate of *Botrytis cinerea* mycelial growth on the carvone nanoemulsions prepared in Examples 1, 1, and 2 of this invention. The inhibition rate in control group 1 was 0%. Kiwifruit is highly susceptible to soft rot caused by *Botrytis cinerea* infection during storage; therefore, kiwifruit preservatives must possess excellent antifungal activity. The inhibitory effect of carvone nanoemulsions on *Botrytis cinerea* is as follows: Figure 9 As shown, the *Botrytis cinerea* hyphae in control group 1 completely covered the plate, comparative example 1 showed only a weak antibacterial effect, while no obvious hyphal growth was observed on the plates of example 1 and comparative example 2, and the inhibition zones were clear. Figure 10 As can be seen from (1) in the text, the colony diameter of Comparative Example 1 is the largest (about 56.5 mm), while the hyphae on the plates of Example 1 and Comparative Example 2 hardly grow. Figure 10As shown in (2), the antibacterial rates of Example 1 and Comparative Example 2 were both close to 100%, significantly higher than that of Comparative Example 1 (31.33%). This indicates that the carvone nanoemulsion has good antibacterial activity against Staphylococcus aureus, and the effect is positively correlated with the carvone content. It also confirms that the composite material is an ideal carrier for maintaining the antibacterial activity of carvone. Carvone exerts its effects through mechanisms such as disrupting cell membranes and inducing the accumulation of reactive oxygen species. The high encapsulation rate of Example 1 further ensures the long-term release of carvone, thereby effectively inhibiting the growth of pathogens. In summary, considering the comprehensive results of stability, encapsulation rate, and antibacterial activity, the carvone nanoemulsion of Example 1 of this invention effectively solves the problems of high volatility and high hydrophobicity of carvone, and achieves excellent antifungal activity through high encapsulation rate.

[0077] (10) In vivo verification of nanoemulsion in kiwifruit Figure 11 Visual representation of the changes over time in kiwifruit inoculated with Fibrocystic soft rot during storage of the carvone nanoemulsion prepared in Example 1 of this invention, the composite dispersion of Comparative Example 3, the commercial preservative of Comparative Example 4, and the untreated control group 1. Figure 12 The graph shows the changes in the diameter of soft rot lesions in kiwifruit during storage for the carvone nanoemulsion prepared in Example 1 of this invention, the composite dispersion of Comparative Example 3, the commercial preservative of Comparative Example 4, and the untreated control group 1. Figure 13 The graph shows the changes in conductivity of kiwifruit during storage for the carvone nanoemulsion prepared in Example 1 of this invention, the composite dispersion of Comparative Example 3, the commercial preservative of Comparative Example 4, and the untreated control group 1. Kiwifruit soft rot is caused by *Botrytis cinerea* infection. This experiment verified the in vivo antibacterial effect of the carvone nanoemulsion by inoculating the pathogen. Figure 11 It can be seen that the kiwifruit in control group 1 showed obvious soft rot lesions on the 4th day after inoculation, and by the 8th day the lesions had expanded over a large area and the fruit was severely rotten; the fruits in comparison groups 3 and 4 also showed lesion expansion to varying degrees; while the kiwifruit in example 1 only showed very small lesions on the 8th day and the appearance remained relatively intact. Figure 12 The lesion diameter data further confirmed that the lesion diameter of control group 1 was the largest (33.47 mm), followed by control groups 3 and 4 (27.223 mm and 27.63 mm respectively), and the lesion diameter of example 1 was the smallest (11.18 mm). Figure 13 The relative conductivity results showed that the control group 1 had the highest conductivity and the fastest increase, reflecting the most severe cell membrane damage; the conductivity of Example 1 increased the least, indicating that it could better maintain the integrity of the fruit cell membrane and delay cell damage caused by the disease. In summary, the carvone nanoemulsion of Example 1 can significantly inhibit kiwifruit soft rot caused by *Vitis vinifera*, with significantly lower lesion expansion rate and conductivity than the control group and the comparative group, demonstrating excellent antibacterial and preservation effects.

[0078] (11) The effect of carvone nanoemulsion coating on the appearance preservation of kiwifruit Figure 14 Images show the apparent quality of kiwifruit during storage for pectin coating treatment containing the carvone nanoemulsion prepared in Example 1 of this invention, pectin coating treatment containing the commercial preservative of Comparative Example 4, control group 1 without any treatment, and control group 2 treated only with pectin coating. Figure 14 As shown, control groups 1 and 2 showed signs of spoilage on day 6 of storage, and by day 15, the fruit skin was wrinkled and severely rotten. While the commercial preservative group in Comparative Example 4 delayed spoilage, significant water loss and wrinkling appeared on the fruit surface by day 9, accompanied by localized rot by day 15. In contrast, the carvone nanoemulsion treatment group in Example 1 maintained a relatively intact fruit appearance throughout the 15-day storage period, showing only slight wrinkling and color changes, without significant rot. This is due to the synergistic effect of the carvone nanoemulsion and pectin in Example 1: the two form a dense composite protective film on the fruit surface. Carvone in the nanoemulsion can inhibit the infection of pathogens such as *Botrytis cinerea* and reduce oxidative stress in the fruit through its antioxidant effect. Simultaneously, the dense film structure effectively reduces water loss and slows down fruit wrinkling, thus maintaining a good fruit appearance. In summary, the carvone nanoemulsion in Example 1 has a significantly better preservation effect on the appearance of kiwifruit than the control group and the commercial preservative in Comparative Example 4.

[0079] (12) The preservation effect of nanoemulsion coating on kiwifruit during storage Figure 15 Figure 15(1) shows a comparison of weight loss, rot rate, and firmness of kiwifruit during storage for pectin coating treatments containing the carvone nanoemulsion prepared in Example 1 of this invention, pectin coating treatments containing the commercial preservative from Comparative Example 4, control group 1 without any treatment, and control group 2 treated only with pectin coating. These three indicators quantify the differences in preservation effects of different treatments. As can be seen from Figure 15(1), the weight loss rate of all groups increased with the extension of storage time, but the weight loss rate of Example 1 was always the lowest, significantly lower than that of control group 1 and comparative example 4, indicating that the treatment of Example 1 can effectively slow down the transpiration and respiration of the fruit and reduce water loss. Figure 15 (2) The rot rate results showed that Example 1 had the smallest increase in rot rate (15 days: 6%), while the rot rate of Control Group 1 was 18%, and Comparative Example 4 also reached 12%, confirming that the nanoemulsion of Example 1 can inhibit the infection of pathogens such as Gracilaria spp. and significantly reduce the risk of fruit rot. Figure 15(3) Regarding the change in hardness, the hardness of the fruit in all groups decreased with storage time, but the rate of decrease in hardness in Example 1 was the slowest. At 15 days, it remained at a relatively high level of 10.22 N, which was higher than that of Control Group 1 (7.33 N) and Comparative Example 4 (7.29 N). This result is directly related to the weight loss rate. Reducing the loss of water and nutrients can effectively maintain the integrity of the fruit cell structure, thereby maintaining a high hardness. In summary, throughout the entire storage period, the carvone nanoemulsion pectin coating treatment group in Example 1 showed significant advantages of low weight loss rate, low rot rate, and high hardness. By inhibiting respiration, reducing water loss and pathogen infection, it effectively delayed the deterioration of fruit quality, thereby significantly extending the shelf life of kiwifruit.

[0080] This invention employs ultrasonic emulsification technology, using a pea protein isolate-octenyl succinic acid starch composite dispersion as the emulsifying carrier to stably encapsulate hydrophobic carvone within a nanoemulsion system. This effectively solves the technical challenges of carvone's high volatility, high hydrophobicity, and difficulty in stable dispersion, thus constructing a natural and safe antibacterial and preservative material. This carvone nanoemulsion possesses excellent antioxidant and antifungal properties, effectively inhibiting the growth of *Staphylococcus aureus*, the pathogen causing soft rot in kiwifruit, and blocking disease spread. When combined with pectin to prepare a composite coating solution, it forms a dense protective film on the fruit surface, reducing moisture loss and respiration while inhibiting pathogen infection, thereby significantly maintaining the appearance quality and physicochemical properties of kiwifruit during storage and greatly extending its shelf life. The preservative provided by this invention combines safety and high efficiency, offering a green and feasible technical solution for post-harvest preservation of kiwifruit.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method of preparing a nanemulsion of carvyl ketone, characterized in that, Includes the following steps: (1) Dissolve pea protein isolate in water, adjust the pH to ≥10, heat and stir, and then adjust the pH to ≤8.5 to obtain pea protein isolate solution; (2) Dissolve octenyl succinic acid starch in water, heat and stir to gelatinize, and obtain octenyl succinic acid starch dispersion; (3) Mix the pea protein isolate solution and the octenyl succinic acid starch dispersion in equal volumes, and stir to obtain a pea protein isolate-octenyl succinic acid starch composite dispersion; (4) Add non-volatile vegetable oil and carvone to the pea protein isolate-octenyl succinic acid starch composite dispersion and homogenize to obtain carvone nano-crude emulsion; (5) The carvone nano-emulsion was subjected to ice bath ultrasonic refining treatment to obtain carvone nano-emulsion; In step (1), the mass ratio of the pea protein isolate to the volume of water is 0.2g~0.4g∶10mL; In step (2), the mass ratio of the octenyl succinic acid starch to the volume ratio of water is 0.1g~0.2g∶10mL; In step (4), the ratio of the mass of soybean oil, the mass of carvone, and the volume of pea protein isolate-octenyl succinic acid starch composite dispersion is 0.1g~0.3g∶0.2g~0.6g∶10mL.

2. The method of claim 1, wherein the preparation of the nanemulsion of carvyl ketone is characterized in that, In step (1), the pH is adjusted to 10-12, the heating is water bath heating, the heating temperature is 45 ℃-48 ℃, and the pH is then adjusted to 7.5-8.

5.

3. The method of claim 1, wherein the preparation of the nanemulsion of carvyl ketone is characterized by, In step (2), the heating is water bath heating, and the heating temperature is 85 ℃~95 ℃.

4. The method for preparing carvone nanoemulsion according to claim 1, characterized in that, In step (4), the rotation speed of the homogenization process is 8000 rpm to 12000 rpm, and the homogenization time is 3 min to 5 min; In step (5), the power of the ultrasonic refining process is 300 W to 350 W, and the time of the ultrasonic refining process is 15 min to 20 min.

5. The method for preparing carvone nanoemulsion according to any one of claims 1 to 4, characterized in that, In step (4), the non-volatile vegetable oil is a plant-derived triglyceride, which is one or more of soybean oil, corn oil, palm oil and medium-chain triglycerides.

6. The method for preparing the carvone nanoemulsion according to any one of claims 1 to 4, characterized in that, In step (1), the stirring speed is 300 rpm to 600 rpm, and the stirring time is 60 min to 90 min; In step (2), the stirring speed is 300 rpm to 600 rpm, and the stirring time is 30 min to 60 min; In step (3), the stirring speed is 300 rpm to 500 rpm, and the stirring time is 10 min to 15 min.

7. A carvone nanoemulsion prepared by a method according to any one of claims 1 to 6.

8. The carvone nanoemulsion according to claim 7, characterized in that, The obtained carvone nanoemulsion has a particle size of 100 nm to 300 nm and a polydispersity index of 0.1 to 0.

2.

9. The application of the carvone nanoemulsion as described in claim 7 or 8 in postharvest preservation of fruits and vegetables.

10. The application according to claim 9, characterized in that, The application described is the use of carvone nanoemulsion in the prevention and preservation of postharvest soft rot in kiwifruit.

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