Limonin Enhances the Antifungal Activity of Eugenol Nanoemulsion against Penicillium Italicum In Vitro and In Vivo Tests

Abstract

Penicillium italicum, the cause of citrus blue mold, is a pathogenic fungus that seriously affects the postharvest quality of citrus fruit and causes serious economic loss. In this study, a eugenol nanoemulsion containing limonin, an antimicrobial component from citrus seeds, was prepared using a high-pressure microfluidizer and the antifungal activity of the nanoemulsions against P. italicum was evaluated based on the conidial germination rate, mycelial growth, and scanning electron microscopy analysis. The results showed that the minimum inhibitory concentration and the inhibition rate of limonin-loaded eugenol nanoemulsion was 160 μg/mL and 59.21%, respectively, which was more potent than that of the limonin-free eugenol emulsion. After treatment with the nanoemulsions, the integrity of the P. italicum cell membrane was disrupted, the cell morphology was abnormal, and the leakage of nucleic acid and protein was observed. In addition, the challenge test on citrus fruits revealed that the limonin-loaded eugenol emulsion inhibited citrus infection for longer periods, with an infection rate of 29.2% after 5 days. The current research shows that nanoemulsions containing limonin and eugenol have effective antifungal activity against P. italicum, and may be used as a substitute for inhibiting blue mold in citrus fruits.

Eugenol and Aloe vera blended natural wax-based coating for preserving postharvest quality of Kaji lemon (Citrus jambhiri)

“…The diameter of the zones of inhibition of EuNE-30 against Saccharomyces cerevisiae and Rhizopus stolonifera were 35.56 mm and 37.59 mm, respectively, while EuNE-5 induced zones of inhibition against the same fungi were 28.95 mm and 29.71 mm, respectively. A similar fungal inhibition pattern was observed by Basumatary, Kalita, et al, 2022 , Basumatary, Mukherjee, et al, 2022 with isolated fungi ( Basumatary, Mukherjee, et al, 2022 ) and Li, Zhao, Li, & Zhou, 2021 against Penicillium Italicum when exposed to eugenol nanoemulsions ( Li et al, 2021 ).

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Preparation and Characterization of Eugenol Incorporated Pullulan-Gelatin Based Edible Film of Pickering Emulsion and Its Application in Chilled Beef Preservation

“…The mean particle size of the Pickering emulsion added to Eu was 296.0 ± 11.2 nm, the PDI value was 0.457 ± 0.039, and the absolute value of zeta potential was 20.1 ± 0.9 mV. In a study, Li et al prepared an Eu nanoemulsion containing limonin (an antimicrobial component from citrus seeds), which showed that the mean particle size of Eu nanoemulsions was 213.7 nm, and presented a larger particle size of 245.7 nm with the addition of limonin [25]. In another study, a sub-micron injectable emulsion of Eu was developed with an average particle size of 176.1 ± 10.3 nm and a potential of −40.2 ± 1.8 mV, which showed excellent stability and safety compared to Eu solutions [26].…”

Preparation and potential of nanoparticles containing curcuminoids to control fungal diseases in tropical fruits

“…Concerning antifungal and protection properties of nanoformulations of phytochemicals, eugenol (4-allyl-2-methoxyphenol) and limonin were formulated into a nanoemulsion and showed high inhibition against blue mold caused by P. italicum in citrus fruits [6]. Cinnamaldehyde, eugenol and carvacrol nanoemulsions strongly inhibited P. digitatum and were applied to the postharvest preservation of citrus fruit [21].…”