E. coli and S. aureus were cultured in Luria-Bertani (LB) liquid medium (1% of tryptone, 0.5% of yeast extract, 1% of sodium chloride, pH 7.2–7.4) at 37°C with shaking of 100 r/min. L. monocytogenes was cultured in tryptic soy broth (TSB) medium (1.7% of tryptone, 0.3% of soy peptone, 0.5% of sodium chloride, 0.25% of dipotassium phosphate, 0.25% of glucose, pH = 7.1–7.5) at 37°C with shaking of 100 r/min. All the strains were cultured to the log phase, and the bacterial dilutions were obtained by adjusting the final cell density to 10⁷ CFU/mL according to OD₆₀₀-cell count standard curve, and then the bacterial dilutions were used for subsequent experiments.

The MIC and MBC of LEO was determined according to previous study (Yazgan, 2020) with slightly modification: 1.5% DMSO was added to the LB liquid medium or TSB liquid medium. Then, different amounts of LEO were added to achieve final concentrations of 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00, 20.00, 40.00, 60.00, and 80.00 μL/mL. Only the tubes containing DMSO were used as the control group, while only the tubes containing culture medium were used as the blank group. One hundred microliters of the bacterial dilutions was added, and the mixture was cultured at 37°C for 24 h. The MIC values represent the lowest concentration of the sample that inhibited visible growth of the tested microorganism. To determine the MBC, the contents of the tubes with the MIC were subcultured onto solid medium and incubated at 37°C. The minimal concentration of the samples that showed no growth after 24 h of incubation was considered as the MBC.

The agar diffusion test was slightly modified (Luo et al., 2023) and used to determine the antibacterial activity of LEO in vitro. The Petri plate medium (liquid medium supplemented with 1.5% agar) surface was inoculated by spreading 10 μL of the bacterial dilutions over the entire surface of the agar. After air-drying, three filter paper discs with a diameter of 8 mm were placed on each culture medium, ensuring a certain distance between the discs. Ten microliters of LEO was pipetted onto the filter paper discs at 37°C for 24 h. The essential oil diffused from the center to the outer edge of the agar plate, forming transparent inhibitory zones. The blank group was not treated with LEO. The diameter of the inhibitory zones was measured. The standards for determining the bacteriostatic effect were as follows: “highly sensitive”: diameter ≥ 20 mm; “medium sensitive”: 20 mm ≥ diameter ≥ 12 mm; and “slightly sensitive”: diameter ≤ 12 mm.

According to previous methods with minor modifications (Costa et al., 2022), E. coli, S. aureus, and L. monocytogenes culture mediums were mixed with 1.5% of DMSO, and then the culture mediums were mixed with LEO at concentrations of 2 MIC, MIC, 0.5 MIC, respectively. For the blank group: only E. coli culture medium was used, while for the S. aureus culture medium, L. monocytogenes culture medium was used. In the control group, E. coli, S. aureus, and L. monocytogenes culture mediums were mixed with 1.5% of DMSO. Then the cells were cultured at 37°C with shaking, and the OD600 values of each group were measured at 0, 2, 4, 6, 8, 10, 12, and 24 h, respectively, by using a 1,550 microplate reader (Thermo Scientific, MA, United States).

Kiwifruits were preserved at 4°C until they reached the edible stage (soluble solids content ≥10%), after which they were subjected to fresh-cut processing (Hu et al., 2022). Undamaged samples (90 ± 5 g) of a uniform size, at the edible stage, were rinsed. Kiwifruit tissue was cut into 1 cm thick thin slices using a stainless steel knife after peeling. The slices were randomly divided into 6 groups: the blank group was soaked in ultrapure water. The control group was soaked in an equal concentration of nanoemulsion without embedding essential oils. 0.5, 1.0, 1.5, and 2.0% LEO nanoemulsion groups were soaked in 0.5, 1.0, 1.5, and 2.0%(v/v) LEO nanoemulsion, respectively. Both samples were air-dried for 10 min after soaking in different concentrations of nanoemulsion for 3 min. Afterward, both samples were stored in plastic containers at 4°C for 8 days. During storage, the samples were sampled at 2-d intervals for analysis.

The weight loss rate was determined by the weighing method, and the samples were weighed once every 2 days during storage. The weight loss rate was calculated by Formula (3):

where Pi is the weight of the sample before storage (g) and Pf is the weight of the sample after storage (g).

The firmness of kiwifruit was measured with a TA-XT plusC texture instrument (StableMicro System, Godalming, United Kingdom). The parameter settings were as follows: 2 mm probe with a puncture speed of 1 mm⁻¹ and a puncture distance of 5 mm. The firmness was the maximum force (N) required to puncture the slices.

The color values (a* and L*) of the samples were measured from 3 random points on each sample using an NH300 colorimeter (Threenh Technology Co., Ltd., China) during the storage period. Color values were determined by taking the average of these points.

The total chlorophyll content was analyzed using commercial assay kits according to the manufacturer’s instructions (Nanjing Jiancheng Biological Engineering Research Institute, China).

The decay rate was assessed by visually inspecting the presence of spoilage on the surface of kiwifruit slices at the beginning and end of each storage interval. Visually spoiled slices, characterized by changes in color, visible structural integrity, and appearance, were removed when approximately 70% of the fruit surface area decayed. The decay rate was calculated as the number of decayed slices divided by the initial number of slices, multiplied by 100.

The SCC of the kiwifruit was determined using a PAL-1 Akabe refractometer (ATAGO CO., Ltd., Guangzhou, China), and the results are expressed as percentages.

TA was determined by the titration method. Ten grams of sample was homogenized and subsequently diluted to 100 mL with distilled water. Then the samples were filtered for 30 min to collect the filtrate. Three to five drops of 1% phenolphthalein indicator were added to 25 mL of filtrate. The mixture was stirred and titrated with 0.1 M NaOH solution until the solution turned pink and did not fade within 30 s. The conversion Factor f (lemon acid) is based on the consumption of NaOH titrant and the results are expressed as the mass fraction (%) of the titratable content of the sample (Su et al., 2023). The TA content was calculated by Formula (4)

where V is the total volume of extraction solution (mL); C is the molar concentration of NaOH (mol/L); V₁ is the volume of NaOH consumed by the filtrate (mL); V₀ is the volume of NaOH consumed by distilled water (mL); V_S is the volume of extraction solution (mL); W is the weight of the samples (g); the f is the conversion factor (g/mmol).

After the kiwifruit slices were homogenized, use a PHSJ-4F pH meter (INASE Scientific Instrument Co., Ltd., Shanghai, China) to measure the pH of the sample.

VC was analyzed using commercial assay kits according to the manufacturer’s instructions (Nanjing Jiancheng Biological Engineering Research Institute, China).

Five grams of kiwifruit were homogenized in an ice bath, then it was centrifuged at 4°C, and 10,000 × g for 10 min and collected as a test sample. The DPPH radical scavenging ability of the kiwifruit was determined according to the methods of section 2.3.

Peroxidase (POD), catalase (CAT), phenylalaninammo-nialyase (PAL), and polyphenol oxidase (PPL) were analyzed using commercial assay kits according to the manufacturer’s instructions (Nanjing Jiancheng Biological Engineering Research Institute, China).

According to the Chinese national standard GB4789.2—2016, the total colony count of freshly cut kiwifruit on the 8th day of storage was determined and expressed as log10 (CFU/g).

As shown in Figure 1A and Table 1, the studied LEOs act differently on the tested bacteria, L. monocytogenes has the best antibacterial effect, with an antibacterial circle diameter of 14.23 ± 0.61 mm, followed by S. aureus (12.20 ± 0.67 mm), both of which are highly sensitive; however, LEO has the worst antibacterial effect on E. coli (9.50 ± 1.08 mm), which is slightly sensitive and significantly less than the antibacterial effect of LEO on the other two kinds of bacteria. One possible reason is that limonene, a main volatile component of LEO, can damage the cell membranes of S. aureus and L. monocytogenes, leading to the leakage of biomolecules from the cells (Han et al., 2021), while the outer membranes of gram-negative bacteria are rich in lipopolysaccharides and are almost unable to penetrate lipophilic compounds, thus preventing the penetration of various antibacterial components of the essential oil. The presence of hydrolases in the periplasmic space may also help hydrolyze antibacterial substances. However, LEO can still act on gram-negative bacteria because other volatile substances, such as γ-terpinene and β-pinene in LEO still have antibacterial effects on E. coli (Tang et al., 2023), these two small molecular compounds can act on gram-negative bacteria through a sufficiently large enough channel generated by porins in the outer membrane (Seow et al., 2014).

As shown in Figures 1B–D and Table 1, the MIC and MBC of LEO against S. aureus were determined to be 0.63 μL/mL and 1.25 μL/mL, respectively. For E. coli, the MIC and MBC of LEO were found to be 60 μL/mL and 80 μL/mL, respectively. LEO exhibited an MIC and an MBC of 2.50 μL/mL against L. monocytogenes. In this study, compared to the gram-negative bacteria (E. coli), the gram-positive bacteria (S. aureus and L. monocytogenes) exhibited greater sensitivity and lower concentrations required for inhibition by LEO. The findings are consistent with the results of the agar diffusion test and the conclusions of He et al. (2022).

Although the MIC and MBC are important for evaluating the antibacterial activity of LEO, the results of these analyses are static. Therefore, it is necessary to dynamically assess the activity over time. As shown in Figure 2A, the bacterial counts in the blank and control groups rapidly increased logarithmically with extended cultivation time, and the bacteria entered a stable phase after 12 h. LEO exhibited bactericidal activity against S. aureus at 2 MIC (1.25 μL/mL) and significant growth inhibition at the MIC (0.63 μL/mL). The lag phase of the growth curve of S. aureus treated with LEO was observed, with a lag phase of 6 h for MIC-treated S. aureus, and 4 h for the 1/2 MIC, 1/4 MIC, control, and blank groups.

As shown in Figure 2B, LEO exhibited bactericidal activity against E. coli at 2 MIC (120 μL/mL) and significant growth inhibition at the MIC (60 μL/mL) and 1/2 MIC (30 μL/mL). The lag phase of the growth curve of E. coli treated with LEO was observed, with a lag phase of 8 h for MIC-treated Escherichia coli, 4 h for the 1/2 MIC, and 2 h for the 1/4 MIC, control, and blank groups.

As shown in Figure 2C, LEO completely inhibited the growth of L. monocytogenes at 2 MIC (5.00 μL/mL), MIC (2.50 μL/mL), and 1/2 MIC (1.25 μL/mL), and exhibited significant growth inhibition at 1/4 MIC (0.63 μL/mL). The lag phase of the growth curve of L. monocytogenes treated with LEO was observed, with a lag phase of 4 h for 1/4 MIC-treated L. monocytogenes, and 2 h for the control and blank groups.

After 24 h of treatment, compared to those in the blank group, the inhibition rates of LEO at the MIC, 1/2 MIC, and 1/4 MIC against S. aureus were 54.19, 17.47, and 6.53%, respectively. The inhibition rates of LEO at the MIC, 1/2 MIC, and 1/4 MIC against E. coli were 72.26, 41.27, and 22.84%, respectively. The inhibition rate of 1/4 MIC LEO against L. monocytogenes was 43.72%.

Weight loss is a major factor affecting fruit quality and storage life and is primarily caused by water evaporation from the fruit surface, which leads to shrinkage and deterioration (Guerreiro et al., 2015). As shown in Figure 5A, with increasing storage time, the percentage of weight loss gradually increased in all the groups. Kiwifruit slices treated with LEO nanoemulsions exhibited less weight loss as the concentration of LEO increased. Beginning on the 6th day, there was a significant difference (p < 0.05) in weight loss between the kiwifruit slices treated with the nanoemulsion and those without the nanoemulsion. Additionally, there were significant differences (p < 0.05) in weight loss between kiwifruit slices without added LEO and those treated with 1.5 and 2% LEO nanoemulsions. On the 8th day, the weight loss rate of the 2% LEO nanoemulsion group was only 1.16%. This result is consistent with the application of nanoemulsions containing 1.0 and 2.0% oregano essential oil for papaya preservation (Tabassum et al., 2023). This difference may be attributed to the formation of nanoemulsions on the surface of the kiwifruit, which reduce water evaporation. Essential oils can also reduce the permeability of water vapor (Pardeike et al., 2009) and can be further utilized to improve the water resistance properties of edible coatings (Atarés and Chiralt, 2016).

Firmness, which is related to water content and metabolic changes (Saha et al., 2023), is an important quality parameter for consumer acceptance of fresh fruits and vegetables. As shown in Figure 5B, the increase in the storage period and the cell damage caused by cutting resulted in the release of cell wall-degrading enzymes (Ragaert et al., 2007). Under the catalysis of these enzymes, the polysaccharides in the cell wall are degraded, ultimately leading to a decrease in firmness. The blank group samples exhibited the maximum decrease in firmness, with a firmness value of only 20.459 g on the 8th day. Although the samples with nanoemulsion coatings showed a slight decrease in firmness, this decrease was relatively lower than that in the blank group at the end of storage, which is consistent with the results of previous research (Hu et al., 2022). This difference may be attributed to the addition of KGM to the nanoemulsion, which acts as a polysaccharide gas barrier, hindering the exchange of water vapor between the fruit and the external environment (Miranda et al., 2022).

The kiwifruit slices treated with the LEO nanoemulsion had greater firmness values than that did those in the emulsion-coated group, and decrease in firmness was also slighter than that in the blank group. Starting on the 2nd day, significant differences (p < 0.05) were observed between the kiwifruit slices treated with 2.0% LEO nanoemulsion and the untreated kiwifruit slices, indicating that the LEO nanoemulsion was beneficial for maintaining the firmness of the kiwifruit slices. This difference may be due to the continuous release of some active ingredients (including limonene) from the nanoemulsion by LEO during storage. These ingredients play a role in inhibiting bacteria, thus reducing the firmness decrease in firmness of kiwifruit slices caused by microbial deterioration (Ben Ghnaya et al., 2016). On the other hand, it is possible that the nanoemulsion coating disrupts the balance of O₂/CO₂ in the kiwifruit flesh, reducing the activity of pectin-related glycosidases involved in the hydrolysis of glycosidic bonds in kiwifruit (Huang et al., 2023), thereby slowing down cell wall degradation and alleviating the decrease in kiwifruit firmness.

The color of fruits and vegetables is closely related to their quality during storage and directly influences consumer choices. The L* value represents the brightness of the sample color. As shown in Figure 5C, the L* values of all the kiwifruit slices decreased throughout the entire storage period, possibly due to browning on the surface of kiwifruit, resulting in a gradual decrease in brightness during storage (Yu et al., 2023). By the 8th day of storage, the L* value of the blank group decreased from 44.38 on the 1st day to 28.02, indicating a darker color. In contrast, the L* value of kiwifruit slices immersed in LEO nanoemulsions decreased at a slower rate, indicating better maintenance of color brightness. Among them, the L* value of the 2% LEO nanoemulsion group was 33.07, which was significantly different from that of the blank group and the control group (p < 0.05). This may be attributed to the smaller particle size and antioxidant properties of the nanoemulsion containing LEO, which can slowly release into the kiwifruit pulp, reduce gas exchange between the pulp and the external environment, inhibit the activity of enzymes that cause browning, and decrease the oxidation of phenolic substances catalyzed by enzymes, thus reducing the formation of brown pigments (Toivonen and Brummell, 2008).

The a* value represents the color from green to red (Passafiume et al., 2020) As shown in Figure 5D, the a* values of kiwifruit slices in all groups increased during the entire storage period, which was attributed to the degradation of chlorophyll. The a* values of the blank group and the control group on the 8th day were-1.15 and-1.25, respectively, with chlorophyll contents of 9.26 and 9.41 mg/kg, respectively (Figure 5E). In contrast, the a* value of the 2% LEO nanoemulsion sample group was −2.74, with a chlorophyll content of 13.64 mg/kg, indicating the highest green color retention ability. This finding suggested that the 2% LEO nanoemulsion had the greatest effect on preserving green color, which was similar to what was observed in fresh-cut kiwifruit treated with shortwave ultraviolet radiation on the 7th day (Hu et al., 2022). The changes in L* and a* values are consistent with the color of kiwifruit in Figure 6. These results demonstrated that immersion treatment with LEO nanoemulsions can maintain the typical bright green color of kiwifruit slices by preventing the oxidation and degradation of chlorophyll.

As shown in the Figure 5F, all the samples exhibited an increase in decay rate during storage. However, the samples in the nanoemulsion group exhibited a significantly lower decay rate than did those in the blank group. On the 2nd day, the decay rate of the blank samples was 10.57%, and after 8 days of storage, 80.42% of the kiwifruit slices were infected and decayed. With an increase in LEO concentration, the decay rate of the kiwifruit slices gradually decreased. On the 8th day of storage, when the LEO concentration increased from 0 to 2.0%, the decay rate of the kiwifruit slices significantly decreased from 73.24 to 41.55%. This indicates that the nanoemulsion coating with LEO can effectively reduce the decay of kiwifruit slices during storage, and that a higher concentration of LEO results in a better inhibitory effect on decay. LEO has antimicrobial activity, inhibits the growth of foodborne pathogens by forming a uniform surface on kiwifruit plants. By being uniformly dispersed in the nanoemulsion, the volatilization of the effective antimicrobial components of LEO can be minimized, thereby enhancing the antimicrobial properties of the LEO nanoemulsion.

The soluble solid content (SSC) is an important indicator for evaluating the quality of kiwifruit, and it is positively correlated with the sugar content. Additionally, SSC is closely related to metabolism, where stronger respiratory activity leads to greater consumption of its own substances and lower SSC. As shown in Figure 7A, the blank group, control group, and 0.5% group exhibited an initial increase followed by a decrease in SSC. This trend may be attributed to the respiratory activity of kiwifruit, where polysaccharides are hydrolyzed into sugars and other soluble compounds during metabolism, leading to an increase in soluble solids content. As the kiwifruit deteriorates, the converted sugars continue to hydrolyze into reducing sugars (Tian et al., 2023), and simple sugars further degrade and transform into acidic substances (Tirkey et al., 2014), resulting in a decrease in soluble solids content. In addition, the remaining treatment groups demonstrated a gradual increase in soluble solids content, possibly due to the ability of LEO concentrations exceeding 0.5% to inhibit the reproduction of microorganisms and reduce their consumption of carbohydrates. In addition, LEO reduced the respiration intensity of kiwifruit, thereby slowing its physiological metabolic activity and facilitating the accumulating of soluble sugars, thus better preserving the flavor of freshly cut kiwifruit.

TA is an important factor that affects product flavor and directly influences consumer preference. As shown in Figure 7B, all treatment groups exhibited a decreasing trend in TA content during storage, possibly due to the respiratory activity of fresh-cut kiwifruit, which consumes some of its organic acids as substrates during respiration, resulting in a decrease in titratable acidity (Yu et al., 2021). On the 2nd day of storage, the TA content of all the treatment groups decreased rapidly, which may be attributed to external damage to the fresh fruit and respiratory consumption. Compared to those of the control group and the blank group, the soaking treatment with the LEO nanoemulsion reduced the decline in TA. The sustained-release effect of LEO helps maintain TA at a relatively high level, possibly due to the antimicrobial activity of LEO, which inhibits microbial growth and reduces the oxygen content on the surface of kiwifruit, thereby reducing the consumption of organic acids (Yan et al., 2020).

As shown in Figure 8A, on the 0th day, kiwifruit slices treated with the LEO nanoemulsion exhibited greater DPPH radical scavenging ability than did those in the blank and control groups. During storage, this activity gradually decreased, beginning on the 6th day and then significantly decreased compared to that on 4th day (p < 0.05). Compared to those of the blank and control groups, the kiwifruit slices treated with the LEO nanoemulsion demonstrated improved DPPH scavenging ability, indicating the ability of the LEO nanoemulsion to enhance the antioxidant capacity of freshly cut kiwifruit. As shown in Figure 8B, during storage, the VC content in the blank group gradually decreased. In comparison to those in the blank and control groups, kiwifruit slices treated with LEO nanoemulsions exhibited a slower degradation rate of VC. Beginning on the 2nd day, the VC content in the blank group samples significantly differed (p < 0.05) from that on Day 0, while VC content did not significantly differ between the samples treated with the different concentrations of LEO nanoemulsion compared to those on Day 0. This difference may be due to the superior oxygen barrier ability of the LEO nanoemulsion, which reduces the oxidation of ascorbic acid by oxygen compared to that of the nanoemulsion without lemon essential oil. Considering the trend of DDPH scavenging ability changes, this may also be attributed to the superior oxygen isolation capability of the nanoemulsion containing LEO. Moreover, some terpenes in essential oils, for instance, γ-terpinene in LEO (Nunes-Damaceno et al., 2013) have synergistic antioxidant effects (Guo et al., 2021) with phenolic substances in kiwifruit and are gradually released from the nanoemulsions during storage, contributing to the maintenance of VC content and DPPH radical scavenging ability.

Fresh-cut kiwifruit undergo mechanical damage, such as peeling and cutting, leading to the production of a large amount of reactive oxygen species (ROS), which disrupts the balance of free radical metabolism in kiwifruit and accelerates membrane peroxidation (Zeng et al., 2019), severely affecting product quality. CAT and POD are important antioxidant enzymes in fruits that can scavenge ROS and reduce oxidative damage. As shown in Figures 9A,B the CAT and POD activities exhibited an increasing trend followed by a decrease in all treatment groups. The CAT and POD activities of the blank group and the control group peaked on the 4th day of storage, while those of the LEO nanoemulsion-treated group peaked on the 6th day and were greater than those of the blank group and the control group, indicating that immersion in LEO nanoemulsions can enhance the CAT and POD activities of fresh-cut kiwifruit.

As shown in Figure 9C, PAL activity increased followed by a decrease in all treatment groups. The PAL activity in the blank group and the control group remained relatively low throughout the storage period, with a slight increase on the 2nd day. In contrast, the PAL activity in the LEO nanoemulsion group was consistently greater than that in the blank group and the control group, peaking on the 6th day and slightly decreasing on the 8th day. Kiwifruit plants are susceptible to chilling injury and sensitive to temperature. Storage at 4°C can lead to chilling injury in kiwifruit, resulting in browning and lignification, which affects the storage quality and edibility of kiwifruit. PAL is a key enzyme in phenylpropane metabolism and is involved in the synthesis of phenolic substances (Yu et al., 2021), which can enhance the antioxidant capacity of fresh-cut kiwifruit. Through the treatment with LEO nanoemulsions, the active components in LEO are continuously released, inhibiting microbial growth, enhancing PAL activity, and delaying the senescence of fresh-cut kiwifruit.

PPO is a key enzyme involved in the browning of fruits and vegetables. This process oxidizes phenolic substrates to quinones, resulting in the formation of brown pigments. Therefore, inhibiting PPO activity is an important measure for fruit and vegetable preservation. As shown in Figure 9D, the PPO activity of the samples treated with the LEO nanoemulsion was lower than that of the control group and the blank group throughout the entire storage period. This may be because VC has an inhibitory effect on PPO activity, and LEO inhibits PPO activity by reducing the loss of VC, thereby alleviating browning caused by the oxidation of phenolic substances. This finding is consistent with the previous results for kiwifruit color.

After peeling and cutting, fresh-cut kiwifruit undergoes mechanical damage, losing the protection of the peel and increasing the exposed surface area, increasing susceptibility to microbial contamination. Additionally, during storage, the leakage of fruit juice provides an excellent environment for microbial growth and reproduction. As shown in Figure 10, there were significant differences in the total colony count between the group treated with the LEO nanoemulsion and the blank group and control group (p < 0.05). The values for both the blank group and the control group exceeded the edible safety standard [6 log10 (CFU/g)]. In contrast, the samples treated with the LEO nanoemulsion had total colony count within 6 log10 (CFU/g), and the 1.0, 1.5, and 2.0% of the LEO nanoemulsion groups had total colony count within 4 log10 (CFU/g). This indicates that the LEO nanoemulsion effectively inhibits microbial activity during storage. On the one hand, the immersion treatment with LEO nanoemulsions results in the formation of a protective film on the surface of fresh-cut kiwifruit, which helps to prevent microbial contamination. Moreover, LEO has certain antibacterial properties that induce bacterial cell membrane lysis, resulting in reduced total colony counts (Ghosh et al., 2024), and is gradually released from nanoemulsions, where it can achieve sustained antibacterial effects. These findings indicate that the coating fresh-cut kiwifruit with LEO nanoemulsion can extend their shelf life.