Preparation method and application of grapefruit essential oil nanoemulsion

Abstract

The invention discloses a preparation method and application of grapefruit essential oil nanoemulsion, and belongs to the technical field of daily chemical products. The invention provides a preparation method of a grapefruit essential oil nanoemulsion. The preparation method comprises the following steps: S1, heating and mixing a surfactant and a cosurfactant to obtain a mixed solution A; the cosurfactant comprises at least one of absolute ethyl alcohol, glycerol and 1, 2-propylene glycol; s2, mixing grapefruit essential oil with the mixed solution A to obtain a mixed solution B; s3, mixing and homogenizing water and the mixed solution B to obtain the grapefruit essential oil nanoemulsion. The grapefruit essential oil nano-emulsion prepared through optimization has uniform particle size distribution, low polydispersity index and high negative zeta potential, the grapefruit essential oil nano-emulsion with the concentration of 1.5 mL / dL is used for carrying out film coating treatment on strawberries, the rotting rate can be reduced, and the weight loss is controlled within 8.65%. The appearance quality of the strawberries can be effectively maintained, and the change of the pH value and titratable acidity is slowed down.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of daily chemical products, and in particular to a preparation method and application of grapefruit essential oil nanoemulsion. BACKGROUND

[0002] Fruit preservation, especially postharvest preservation of perishable fruits, has always been a challenge. As a representative of berry fruits, strawberries are prone to spoilage due to their high water content and high respiration intensity. They have a relatively short shelf life at room temperature. Although traditional chemical preservatives (such as sulfites) have certain effects, they pose a risk of drug resistance and health hazards. In contrast, natural plant essential oils are ideal alternatives due to their excellent antibacterial and antioxidant properties. In existing strawberry preservation technologies, the application of ordinary essential oils faces two major challenges: first, their water insolubility and high volatility make it difficult to form a uniform and effective antibacterial film; second, the conventional nanoemulsion system is thermally unstable, and the particle size may uncontrollably increase during storage, leading to a sharp decrease in specific surface area, which in turn reduces the release rate of essential oils and their antibacterial efficiency. In severe cases, the nanoemulsion system may be damaged and the essential oils may leak. Therefore, there is an urgent need to develop a method that can effectively extend the shelf life of perishable fruits. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a preparation method of grapefruit essential oil nanoemulsion. The nanoemulsion prepared by optimization has a uniform particle size distribution, a low polydispersity index, and a high negative zeta potential, and exhibits excellent stability at 4°C. The nanoemulsion formulation significantly improves the free radical scavenging capacity of grapefruit essential oil (p<0.05) and has a significant inhibitory effect on Candida albicans and Staphylococcus aureus. Application tests show that using 1.5 mL / dL concentration of grapefruit essential oil nanoemulsion for coating treatment of strawberries can reduce the rot rate and control the weight loss within 8.65%. After 6 days of storage at room temperature, the appearance quality of strawberries can be effectively maintained, and the changes in pH and titratable acidity can be slowed down.

[0004] According to a first aspect of the present application, a preparation method of grapefruit essential oil nanoemulsion is provided, comprising the following preparation steps:

[0005] S1: heating and mixing a surfactant and a co-surfactant to obtain a mixed solution A;

[0006] The surfactant includes Span 80 and Tween 20, and the co-surfactant includes at least one of anhydrous ethanol, glycerol, and 1,2-propanediol;

[0007] S2: mixing grapefruit essential oil and the mixed solution A to obtain a mixed solution B;

[0008] S3: Mix and homogenize water and mixed solution B to obtain grapefruit essential oil nanoemulsion.

[0009] This invention successfully developed a grapefruit essential oil nanoemulsion system with ultrafine particle size (38.20 nm), narrow particle size distribution (PDI 0.15), and high stability (zeta potential -60.88 mV), effectively solving the technical problems of uneven particle size and poor stability in traditional nanoemulsions. After nanoemulsion encapsulation, the antioxidant properties of grapefruit essential oil were significantly improved (p<0.05), and the antibacterial effect against Candida albicans and Staphylococcus aureus was significantly enhanced, laying a solid foundation for its application in the field of food preservation. Strawberry preservation experiments confirmed that a 1.5 mL / dL concentration of nanoemulsion coating can reduce the strawberry rot rate by 62.5%, control the weight loss rate to within 8.65%, and effectively maintain the quality indicators of strawberries such as pH value and titratable acidity, demonstrating excellent preservation effect.

[0010] According to some embodiments of the present invention, in step S1, the mass ratio of the surfactant to the co-surfactant is 1:(2-6).

[0011] According to some embodiments of the present invention, the mass ratio of Span 80 to Tween 20 is (0.2 to 1):1.

[0012] According to some embodiments of the present invention, the mass ratio of the mixed solution A to grapefruit essential oil is (4-9):1.

[0013] According to some embodiments of the present invention, in step S1, the heating temperature is 60-90°C.

[0014] According to some embodiments of the present invention, in step S3, the homogenization time is 4 to 8 minutes, and the homogenization power is 70 to 280 W.

[0015] According to some embodiments of the present invention, in step S3, the homogenization time is 4 to 8 minutes and the homogenization power is 70 to 90 W.

[0016] According to some embodiments of the present invention, the grapefruit essential oil nanoemulsion has a particle size of 37-39 nm, a narrow distribution index of 0.13-0.17, and a zeta potential of -58 to -66 mV.

[0017] According to a second aspect of the present invention, the application of grapefruit essential oil nanoemulsion prepared by the aforementioned preparation method in fruit preservation is provided.

[0018] According to some embodiments of the present invention, the fruit includes strawberries.

[0019] According to some embodiments of the present invention, the method for preserving fruit involves coating the grapefruit essential oil nanoemulsion onto the surface of the fruit.

[0020] According to some embodiments of the present invention, the concentration of the grapefruit essential oil nanoemulsion is 0.5 to 1.5 mL / dL.

[0021] The grapefruit essential oil nanoemulsion of this invention effectively inhibits microbial growth through a slow-release mechanism, significantly reducing strawberry spoilage and maintaining a bright appearance. The nanoemulsion coating forms a physical barrier, slowing down moisture evaporation and microbial metabolism, controlling weight loss to approximately 31% after 8 days. Simultaneously, by slowly releasing and regulating the strawberry microenvironment, it slows pH increases and titratable acidity decreases (maintaining 0.60% on day 6), ensuring stable quality. Thanks to its small particle size and high zeta potential, the nanoemulsion maintains excellent stability at 4°C for 30 days, continuously releasing essential oils to achieve long-lasting antibacterial and antioxidant effects, significantly extending the shelf life of strawberries.

[0022] Unless otherwise specified, the term "about" in this invention actually means that the error is allowed to be within ±2%, for example, about 100 is actually 100 ± 2% × 100.

[0023] Unless otherwise specified, "between" in this invention includes the number itself, for example, "between 2 and 3" includes the endpoint values ​​2 and 3.

[0024] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0026] Figure 1 The perturbation diagram (A) and the actual value vs. predicted value (B) of the grapefruit nanoemulsion particle size of the present invention are shown.

[0027] Figure 2 This is a visualization of the 3D and 2D models of response surface optimization in this invention.

[0028] Figure 3 These are the characterization results of grapefruit essential oil nanoemulsion in the embodiments of the present invention.

[0029] Figure 4 This is an appearance diagram showing the environmental stability of grapefruit essential oil nanoemulsion according to an embodiment of the present invention.

[0030] Figure 5 This relates to the environmental stability of grapefruit essential oil nanoemulsion in this embodiment of the invention.

[0031] Figure 6The antioxidant activity of grapefruit essential oil nanoemulsion in this embodiment of the invention is shown in terms of DPPH and ABTS.

[0032] Figure 7 The MBC assay was performed on grapefruit essential oil and grapefruit essential oil nanoemulsion in the examples to measure the growth of Candida albicans and Streptococcus aureus after subculturing.

[0033] Figure 8 This refers to the changes in the appearance of strawberries during storage.

[0034] Figure 9 It refers to the rate of spoilage during the storage of strawberries.

[0035] Figure 10 It is the weight loss rate of strawberries during storage.

[0036] Figure 11 It refers to the pH changes of strawberries during storage.

[0037] Figure 12 Changes in titratable acidity of strawberries during storage. Detailed Implementation

[0038] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0039] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0040] Example 1

[0041] The components, their amounts, and reaction conditions in Example 1 are shown in Table 1 below. The preparation method is as follows:

[0042] S1: Heat and mix the surfactant and co-surfactant to obtain mixed solution A;

[0043] S2: Mix grapefruit essential oil and mixed solution A to obtain mixed solution B;

[0044] S3: Add water to mixed solution B, mix well, stir for 15 minutes, homogenize, and filter through a 0.22μm microporous membrane to obtain the grapefruit essential oil nanoemulsion.

[0045] Comparative Example 1

[0046] The difference between this comparative example and the embodiment is that tea tree oil is used instead of grapefruit oil, while all other conditions are the same.

[0047] Table 1. Grapefruit essential oil nanoemulsions of Examples 1-13 and Comparative Examples 1-12 of the present invention

[0048]

[0049]

[0050] Response surface model optimization:

[0051] This invention optimizes grapefruit essential oil nanoemulsion formulations using a central composite design (CCD) method, selecting the following three key independent variables for investigation:

[0052] (1) Mass ratio of emulsifier / co-emulsifier to grapefruit essential oil (grapefruit essential oil) (D / O);

[0053] (2) The mixing ratio of Span 80 and Tween 20;

[0054] (3) Mass ratio of co-emulsifier to emulsifier (Km).

[0055] The experimental ranges for each variable were determined through previous single-factor experiments, with specific coding levels shown in Table 2. This experimental design systematically evaluates the independent effects and interaction effects of each variable on nanoemulsion particle size (response variable) at five levels. A total of 20 randomized tests were conducted to determine the optimal preparation parameters and analyze the correlation patterns among the variables. Table 2 lists the three independent variables and their actual values ​​at different coding levels (-α, -1, 0, +1, +α). These values ​​are derived from the ranges of the single-factor experiments and extended using a star-point design to capture nonlinear effects. The three parameters are:

[0056] The significance of coding level and actual value:

[0057] -α and +α: Axis points in star point design, representing the upper and lower limits of the parameter range, used to detect curvature effects.

[0058] -1 and +1: Factor levels, representing the middle range in the experimental design.

[0059] 0: Center point, repeated multiple times to assess experimental error and model stability.

[0060] Table 2: Coding and Actual Level of Each Parameter in Design-Expert Star Design

[0061]

[0062] Response surface optimization:

[0063] Based on the preliminary screening results, three key process variables for the preparation of grapefruit essential oil nanoemulsions were identified: the mass ratio of co-emulsifier to emulsifier, the mixing ratio of 80 / Tween 20, and the mass ratio of emulsifier / co-emulsifier to grapefruit essential oil. The RSM study was conducted using a central composite design, incorporating three factors at five levels, and included 20 experimental runs. As shown in Table 2, the particle size range of the obtained nanoemulsions was 33.14±1.16 nm (F10, minimum) to 88.16±3.67 nm (F1, maximum). Design-Expert software was used for quadratic model analysis of the data. Detailed information on the ANOVA data is shown in Tables 1 and S3. The results showed excellent model fitness, with an F-value of 56.62 (p<0.0001), indicating no significant fit (F=2.57, p=0.1613). 2 The adjusted R² value (R² = 0.9634; predicted R² = 0.8798) and sufficient precision (28.1052) confirm the predictive ability of the model. The coefficient of variation (CV < 10%) further validates the reliability of the model. The three process variables—the mass ratio of co-emulsifier to emulsifier, the Span 80 / Tween 20 mixing ratio, and the mass ratio of emulsifier / co-emulsifier to grapefruit oil—all significantly affect particle size (p < 0.05). The derived quadratic equation for particle size prediction is as follows: Y = 55.25 – 12.45A + 3.81B + 9.05C + 2.61AB – 2.2AC + 0.925BC + 5.02A 2 +0.1753B 2 –0.3338C 2 .

[0064] Where Y represents particle size, A represents the mass ratio of emulsifier / co-emulsifier to grapefruit essential oil, B represents the mixing ratio of Span 80 / Tween 20, and the C ratio of co-emulsifier to emulsifier (Table 3, Table 4).

[0065] Table 3. Experimental formulation and results for response surface model optimization

[0066]

[0067]

[0068] Table 4 Regression coefficients of the final simplified model

[0069]

[0070] The established quadratic polynomial model accurately characterizes the quantitative relationship between the independent variable (AC) and the particle size response. The interaction terms (AB, AC, BC) and the quadratic term (A...) are also included. 2 B 2 C 2 The coefficients reflect the interactions and nonlinear effects between variables, with positive coefficients indicating synergistic effects and negative coefficients indicating antagonistic effects. Model analysis shows that the emulsifier / co-emulsifier to grapefruit oil ratio has a negative coefficient, indicating a negative correlation with particle size; while the Span80 / Tween20 ratio and the co-emulsifier to emulsifier mass ratio both have positive coefficients, confirming a positive correlation with particle size. These results are consistent with the perturbation plot ( Figure 1 The observations in section A) are consistent: increased emulsifier concentration leads to decreased particle size, consistent with the mechanism of high-concentration emulsifier enhancing interfacial coverage; while increasing the Span80 / Tween20 ratio and the proportion of co-emulsifier increases particle size. Model validation results show that the predicted particle size is in high agreement with the measured value (R0). 2 =0.9808), scatter plot ( Figure 1 Data B) in the table confirms that the predicted values ​​and observed values ​​have a significant linear correlation, which fully verifies the accuracy and reliability of the model (Table 5).

[0071] Table 5 Regression coefficients of the quadratic model fitting the particle size of grapefruit essential oil nanoemulsion

[0072]

[0073]

[0074] This study uses 3D response surfaces and contour plots ( Figure 2 The system analyzed the control law of key process parameters on the particle size of nanoemulsions, fully demonstrating the accurate predictive ability of the established model. The changes in surface elevation visually show that increasing the mass ratio of emulsifier / co-emulsifier to grapefruit oil (A) significantly reduces the particle size. Figure 2 In the A1 and A2 groups, the increase in the co-emulsifier / emulsifier ratio (C) leads to an increase in particle size. Figure 2The coefficients (B1, B2) in the model equation are completely consistent with the coefficient relationships quantified in the model equation. Notably, the interaction between the Span / Tween ratio (B) and parameter C did not show a significant effect (p = 0.3536), a finding that corroborates previous statistical analyses and further verifies the reliability of the model. The optimization model established based on the central composite design successfully locked the optimal combination of process parameters (A = 7.63, B = 0.40, C = 2.95), with a predicted particle size (37.83 nm) having an error of only 0.97% compared to the measured value (Example 1: 38.20 nm). This near-perfect match not only confirms the accuracy of the response surface methodology but also highlights the excellent applicability of this model in the optimization of nanoemulsion formulations. The research results fully demonstrate that the constructed response surface model can accurately capture the complex interactions between multiple variables, providing a scientifically reliable mathematical tool for optimizing nanoemulsion preparation processes.

[0075] This study uses 3D response surfaces and contour plots ( Figure 2 The system analyzed the control law of key process parameters on the particle size of nanoemulsions, fully demonstrating the accurate predictive ability of the established model. The changes in surface elevation visually show that increasing the mass ratio of emulsifier / co-emulsifier to grapefruit oil (A) significantly reduces particle size, while increasing the co-emulsifier / emulsifier ratio (C) leads to an increase in particle size. Figure 2 The coefficients (B1, B2) in the model equation are completely consistent with the coefficient relationships quantified in the model equation. Notably, the interaction between the Span / Tween ratio (B) and parameter C did not show a significant effect (p = 0.3536), a finding that corroborates previous statistical analyses and further verifies the reliability of the model. The optimization model established based on the central composite design successfully locked the optimal combination of process parameters (A = 7.63, B = 0.40, C = 2.95), with a predicted particle size (37.83 nm) having an error of only 0.97% compared to the measured value (38.20 nm). This near-perfect match not only confirms the accuracy of the response surface methodology but also highlights the excellent applicability of this model in the optimization of nanoemulsion formulations. The research results fully demonstrate that the constructed response surface model can accurately capture the complex interactions between multiple variables, providing a scientifically reliable mathematical tool for optimizing nanoemulsion preparation processes.

[0076] Figure 3 The particle size distribution characteristics of the grapefruit essential oil nanoemulsion in the optimized embodiment are shown, and its corresponding macroscopic appearance is as follows. Figure 6As shown in B in the figure. The particle size of the nanoemulsion prepared by response surface methodology was measured to be 37.9-38.7 nm, which is highly consistent with the theoretical prediction of 37.83 nm (p>0.05). The nanoemulsion system has excellent uniformity, which is manifested in: (1) the polydispersity index (PDI) is maintained in the range of 0.139-0.168 (<0.2); (2) the zeta potential is stable between -58.20 and -65.77 mV. These characteristics together ensure the long-term stability of the nanoemulsion. TEM analysis results ( Figure 3 The results from D) further confirm that the optimized nanoemulsion exhibits a regular spherical morphology and a uniform particle size distribution, which is consistent with the test results of dynamic light scattering (DLS) (Table 6).

[0077] Table 6. Average particle size, PDI, and Zeta potential of nanoemulsions after response surface optimization.

[0078]

[0079] Experimental example:

[0080] Single-factor optimization of grapefruit essential oil nanoemulsion:

[0081] In this invention, the particle size of Example 1 was 37.9 nm, the Zeta potential was -60.88 mV, and the PDI was 0.14; the particle size of Example 2 was 61.5 nm, and the PDI was 0.19; the particle size of Example 3 was 95.3 nm, and the PDI was 0.28; the particle size of Example 4 was 66.9 nm, and the PDI was 0.1; the particle size of Example 5 was 76.5 nm, and the PDI was 0.06; and the particle size of Example 6 was 51.4 nm, and the PDI was 0.21.

[0082] As the proportion of essential oils increases, the particle size first decreases and then increases. When the ratio reaches 8:1, the particle size reaches its minimum value, and the PDI is also relatively small.

[0083] Temperature has little effect on the particle size of grapefruit essential oil nanoemulsion. When the temperature is 80℃, the PDI reaches its minimum value and the particle size is suitable.

[0084] As the homogenization power increases, the particle size remains stable. When the homogenization power is 90W, the PDI reaches its minimum value and the particle size is suitable.

[0085] As the homogenization time increases, there is almost no effect on particle size and PDI, so a homogenization time of 4 minutes is sufficient.

[0086] Test example:

[0087] 1. Characterization of grapefruit essential oil nanoemulsion:

[0088] Particle size and potential analysis:

[0089] The average particle size, polydispersity index (PDI), and zeta potential of nanoemulsions were determined using dynamic light scattering. Before testing, the samples were diluted 10-fold with deionized water to reduce multiple scattering effects. All measurements were performed at 25°C with a refractive index of 1.53. The diluted samples were injected into the sample cell and equilibrated for 120 seconds before detection. Each sample was measured in triplicate, and the results were averaged.

[0090] Morphological characterization: The microstructure of the nanoemulsion was observed using transmission electron microscopy (TEM). The specific procedures are as follows:

[0091] Take 10 μL of diluted sample and add it to a carbon film copper grid; negatively stain with 2% phosphotungstic acid solution for 60 seconds; carefully remove excess stain with filter paper; after drying at room temperature, perform TEM observation and acquire images at an accelerating voltage of 80 kV.

[0092] Storage stability of the optimized grapefruit essential oil nanoemulsion:

[0093] like Figure 4 As shown, the sample stored at 4℃ remained translucent blue for 30 days without phase separation or flocculation; while the group stored at 25℃ gradually became turbid. Particle size analysis ( Figure 5 The results showed that at 4℃, the nanoemulsion particle size only increased to 46.38 nm, and the PDI value remained stable, confirming the system's excellent storage stability. In contrast, the 25℃ storage group showed a significant increase in particle size after 5 days (reaching 243.17 nm on day 30), accompanied by significant fluctuations in the PDI value. These results indicate that grapefruit essential oil nanoemulsions are highly stable at 4℃.

[0094] Antioxidant activity assay of grapefruit essential oil nanoemulsion:

[0095] To determine the antioxidant activity of the grapefruit essential oil nanoemulsion in the examples, concentration gradient samples ranging from 313 to 5000 μg / mL were first prepared. In the ABTS assay, a 7 mmol / L ABTS aqueous solution was reacted with 2.45 mmol / L potassium persulfate to generate ABTS· + Free radicals were incubated at 25°C in the dark for 16 hours, and then the absorbance at 734 nm was adjusted to 0.70±0.02 with deionized water. 40 μL of samples of different concentrations were mixed with 160 μL of ABTS· +After mixing the working solutions and reacting at room temperature for 10 min, the absorbance was measured at 734 nm. In the DPPH assay, freshly prepared 0.1 mmol / L DPPH ethanol solution was used. 40 μL of sample was mixed with 160 μL of DPPH solution and reacted at 37 ± 1 °C in the dark for 30 min. The absorbance was measured at 517 nm using a BioTek Synergy Neo2 microplate reader (BioTek Instruments, USA). All experiments were performed in triplicate. The free radical scavenging rate was calculated using formula (1):

[0096]

[0097] Among them, A control It is a free radical solution without the sample (ABTS· + The absorbance of (or DPPH), A sample It contains ABTS. + Or the absorbance of grapefruit essential oil nanoemulsion samples in DPPH solution, A blank It does not contain ABTS. + Or the absorbance of the blank group of DPPH solution.

[0098] The antioxidant properties of grapefruit essential oil nanoemulsion were systematically evaluated using DPPH and ABTS free radical scavenging assays. The results showed that the grapefruit essential oil nanoemulsion of Example 1 exhibited significant concentration-dependent antioxidant activity (p<0.05). At the highest tested concentration (5000 μg / mL), the DPPH scavenging rate of the nanoemulsion (40.33%) was 2.1 times higher than that of free grapefruit essential oil (19.00%); the ABTS scavenging rate increased from 37.98% at 313 μg / mL to 69.12% at 5000 μg / mL. The mechanism by which the nanoemulsion significantly enhances the antioxidant efficacy of grapefruit essential oil may include improving the dispersibility of hydrophobic active ingredients, enhancing the contact efficiency with free radicals, and protecting active ingredients from degradation. The ABTS experiment showed that the IC50 value of the nanoemulsion (1152 μg / mL) was 48.7% lower than that of free grapefruit essential oil (2365 μg / mL), confirming its significantly enhanced antioxidant activity. The research results indicate that nanoemulsification technology can effectively enhance the antioxidant capacity of grapefruit essential oil, making it of significant application value in fields requiring antioxidant protection, such as food preservation. The results are as follows... Figure 6 As shown.

[0099] Antibacterial activity of grapefruit essential oil nanoemulsion:

[0100] The antibacterial activity of grapefruit peel essential oil (GPEO) and its nanoemulsion (Example 1) was determined using the micro-broth dilution method. First, a stock solution of 4.25 g / 100 mL of GPEO and its nanoemulsion was prepared. Suspensions of Staphylococcus aureus or Candida albicans were diluted to approximately 10...6 The concentration of GPEO and its nanoemulsion was serially diluted with sterile water containing 0.1 mL / dL Tween 80 to achieve a final concentration range of 0.85-4.25 g / 100 mL. During the experiment, 100 μL of bacterial suspension and 100 μL of different concentrations of GPEO or nanoemulsion were added to each well. A positive control (liquid culture medium + bacterial strain) and a negative control (0.1 mL / dL Tween 80) were also included. After incubation at 37°C for 16-18 hours, the lowest concentration at which no obvious growth was observed was determined as the minimum inhibitory concentration (MIC). Subsequently, the broth from the wells without growth was transferred to agar plates for further incubation, and the lowest concentration at which no colonies grew was determined as the minimum bactericidal concentration (MBC).

[0101] Although grapefruit essential oil itself is highly effective against Candida albicans (1.0% inhibition, 1.5% complete bactericidal), its nanoemulsion formulation (Example 1) successfully maintained the key complete bactericidal efficacy (MBC remained at 1.5%). Notably, the MIC (2.5%) of the nanoemulsion was slightly higher than that of free grapefruit essential oil, reflecting its sustained-release characteristics—a more gradual release of the essential oil in the initial stage, which is more conducive to a long-lasting effect. Although the Candida albicans biofilm may pose a challenge to the initial penetration of the oil-in-water nanoemulsion, the nanoemulsion demonstrated efficacy equivalent to that of the free essential oil at the key bactericidal concentration. In contrast, Staphylococcus aureus showed low sensitivity to both (1.5% only inhibition, 2.5% still no bactericidal effect), failing to demonstrate a long-lasting effect. The tea tree oil nanoemulsion system in Comparative Example 1 exhibited poor stability and sustained-release failure (Table 7).

[0102] The results are as follows Figure 7 As shown.

[0103] Table 7. Effective antibacterial concentrations and minimum bactericidal concentrations of grapefruit essential oil and essential oil nanoemulsions against Candida albicans and Staphylococcus aureus.

[0104]

[0105] Grapefruit essential oil nanoemulsion for strawberry preservation: reducing spoilage and weight loss.

[0106] Fresh strawberries with uniform ripeness, size, good color, and no mechanical damage were selected as experimental materials. Three treatment groups were set up: Blank NE and grapefruit essential oil nanoemulsion solutions containing 0.5 mL / dL, 1.0 mL / dL, and 1.5 mL / dL of essential oil, respectively, were applied; the control group was treated with sterile distilled water. All strawberry samples were soaked for 3 minutes, followed by air drying at 25±1℃ for 2.5±0.5 hours. The treated strawberries were stored in a constant temperature and humidity environment (25℃, 75% RH) using food-grade plastic containers with polypropylene (PP) ventilation holes for 8 days. During storage, a professional sensory evaluation team regularly monitored physiological indicators such as appearance changes, decay rate, and weight loss rate of the strawberries. Formulas (2, 3) used to determine the decay rate and weight loss rate are calculated as follows.

[0107]

[0108] Where Nd represents the number of rotten strawberries, Nt represents the total number of strawberries, and W0 and W1 are the initial and final weights of the strawberries, respectively.

[0109] Figure 8 The changes in appearance of strawberries during 8 days of storage were demonstrated. The results showed that both the blank nanoemulsion and the grapefruit oil nanoemulsion coatings of different concentrations (0.5-1.5 mL / dL) from Example 1 adhered well to the strawberry surface. During storage, strawberries in the control group and the blank nanoemulsion treatment group gradually showed obvious shrinkage, dehydration, and mold growth. In contrast, the strawberry samples treated with grapefruit oil nanoemulsion maintained their initial fresh appearance better. Quantitative analysis data ( Figure 9 The results showed that the spoilage rate of strawberries in all groups increased with prolonged storage time, but the spoilage rate of the grapefruit essential oil nanoemulsion treatment group was significantly lower than that of the control group (p<0.05), demonstrating a good preservation effect. The role of grapefruit essential oil nanoemulsion in the weight loss rate of fresh strawberries:

[0110] Figure 10Data showed that strawberries in all experimental groups experienced continuous weight loss during the 8-day storage period. Notably, the samples treated with 1.0 mL / dL and 1.5 mL / dL grapefruit oil nanoemulsions exhibited significantly lower weight loss rates than the control group (p<0.05). This protective effect is mainly attributed to the grapefruit oil nanoemulsion coating effectively inhibiting dehydration caused by moisture evaporation and microbial activity. The 1.5 mL / dL (Example 1) treatment group showed the best preservation effect: after 8 days of storage, the weight loss rate was only 31.46%, comparable to the control group's weight loss level (32.27%) at 5-6 days. The experimental results indicate that the grapefruit oil nanoemulsion coating can significantly delay the quality deterioration of strawberries under room temperature storage conditions, demonstrating outstanding potential for preservation applications.

[0111] The role of grapefruit essential oil nanoemulsion in pH:

[0112] This study used a pH meter to measure the pH changes of strawberry samples. Measurements were taken on the day of harvest (day 0) and on the 3rd and 6th days of storage. Simultaneously, the titratable acidity of the strawberries was measured at the same time points: 10g of strawberry sample was homogenized in 100mL of ultrapure water, filtered, and 20mL of the filtrate was accurately transferred. Two drops of 1% (w / v) phenolphthalein indicator were added, and finally titrated to the endpoint with 0.1mol / L NaOH standard solution. The titratable acidity was expressed as citric acid and calculated using formula (4):

[0113]

[0114] Where V NaOH K is the volume (mL) of NaOH solution consumed during the titration, c is the molar concentration (mol / L) of the NaOH solution, and K is the volume of NaOH solution consumed during the titration. c It is the lemon equivalent conversion factor (0.064 g / mmol), V C Vs is the total volume of the prepared strawberry extract (mL), Vs is the volume of the strawberry extract used for titration (mL), and m is the mass of the strawberry sample used (g).

[0115] A systematic analysis was conducted on pH changes during strawberry storage. Figure 11Experimental results showed that all samples exhibited an increasing pH trend during storage, but there was a significant difference between the grapefruit essential oil nanoemulsion treatment group and the control group. Specifically, the pH of the control group strawberries increased sharply from the initial 2.76 to 3.35, while the pH of the samples treated with grapefruit essential oil nanoemulsion changed more gradually. Among them, the 1.5 mL / dL treatment group in Example 1 showed the best pH stability, with its pH only increasing slightly from 2.74 to 2.82. This stabilizing effect may be due to the following mechanisms: (1) the physical barrier formed by the nanoemulsion coating effectively inhibited microbial growth and reduced the accumulation of alkaline metabolites; (2) the antibacterial and antioxidant properties of grapefruit essential oil were enhanced through the slow-release mechanism of the nanoemulsion, further reducing the metabolic activity of microorganisms; (3) the two together regulated the strawberry microenvironment, significantly reducing the respiration intensity and delaying the decomposition and metabolism of sugars and organic acids. The results confirmed that the synergistic effect of grapefruit essential oil and nanoemulsion is significantly better than that of a single component, which can effectively regulate the physiological metabolic activities of strawberries, delay the process of quality deterioration, and thus significantly extend the shelf life.

[0116] The role of grapefruit essential oil nanoemulsion in titratable acidity:

[0117] Figure 12 The changes in titratable acidity of strawberries under different treatments during storage were shown. The titratable acidity of all treatment groups decreased with prolonged storage time, with the control group and the blank nanoemulsion group showing the most significant decreases, dropping to 0.55% and 0.46%, respectively. In contrast, the grapefruit essential oil nanoemulsion coating treatment group maintained a titratable acidity of 0.60% on day 6, significantly higher than the control group. In particular, the 1.5 mL / dL grapefruit essential oil nanoemulsion treatment group in Example 1 showed the best acidity retention effect. This result confirms the effectiveness of grapefruit essential oil nanoemulsion coating in maintaining the quality of strawberries. (1) The physical barrier formed by the nanoemulsion coating effectively slows down water loss and acid degradation caused by microbial metabolism; (2) The antibacterial and antioxidant properties of grapefruit essential oil are optimized through the slow-release mechanism of nanoemulsion, significantly inhibiting microbial activity and organic acid decomposition; (3) Both regulate the physiological metabolism of strawberries, delaying the metabolic consumption of sugars and organic acids. Experimental results confirm that the synergistic effect of grapefruit essential oil and nanoemulsion is significantly better than that of a single component, effectively maintaining the titratable acidity of strawberries, thereby ensuring their quality and extending their shelf life.

[0118] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A method for preparing grapefruit essential oil nanoemulsion, characterized in that, The preparation steps include the following: S1: Heat and mix the surfactant and co-surfactant to obtain mixed solution A; The surfactants include Span 80 and Tween 20, and the co-surfactant includes at least one of anhydrous ethanol, glycerol and 1,2-propanediol; S2: Mix grapefruit essential oil and mixed solution A to obtain mixed solution B; S3: Mix and homogenize water and mixed solution B to obtain grapefruit essential oil nanoemulsion.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of the surfactant to the co-surfactant is 1:(2-6).

3. The preparation method according to claim 1, characterized in that, The mass ratio of Span 80 to Tween 20 is (0.2~1):

1.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the mixed solution A to grapefruit essential oil is (2-14):

1.

5. The preparation method according to claim 1, characterized in that, In step S1, the heating temperature is 60–90°C.

6. The preparation method according to claim 1, characterized in that, In step S3, the homogenization time is 4 to 8 minutes, and the homogenization power is 70 to 280 W.

7. The preparation method according to claim 1, characterized in that, The grapefruit essential oil nanoemulsion has a particle size of 37-39 nm, a narrow distribution index of 0.13-0.17, and a zeta potential of -58-66 mV.

8. The application of grapefruit essential oil nanoemulsion prepared by any one of claims 1 to 7 in fruit preservation.

9. The application according to claim 8, characterized in that, The method for preserving fruit involves coating the surface of the fruit with grapefruit essential oil nanoemulsion.

10. The application according to claim 9, characterized in that, The concentration of the grapefruit essential oil nanoemulsion is 0.5–1.5 mL / dL.

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