This research aimed to develop, optimize, and evaluate a new antifungal nanoemulsion system based on the crude reuterin-synergistic essential oils (EOs) hybrid to overcome the EOs application limits. At first, the anti- fungal effects of the Lactobacillus plantarum and Lactobacillus reuteri cell-free extracts (CFE) were tested against the Botrytis cinerea, Penicillium expansum, and Alternaria alternata as indicator fungus using broth microdilution method. The L. reuteri CFE with the MIC of 125 μL/mL for B. cinerea and 250 μL/mL for P. expansum and A. alternata showed more inhibitory effects than L. plantarum. Next, reuterin as a significant antibacterial compound in the L. reuteri CFE was induced in glycerol-containing culture media. To reach a nanoemulsion with maximum antifungal activity and stability, the reuterin concentration, Tween 80 %, and ultrasound time were optimized using response surface methodology (RSM) with a volumetric constant ratio of 5 % v/v oil phase including triple synergistic EOs (thyme, cinnamon, and rosemary) at MIC concentrations. Based on the Box- Behnken Design, the maximum antifungal effect was observed in the treatment with 40 mM reuterin, 1 % Tween 80, and 3 min of ultrasound. The growth inhibitory diameter zones of B. cinerea, P. expansum, and A. alternata were estimated 6.15, 4.25, and 4.35 cm in optimum nanoemulsion, respectively. Also, the minimum average particle size diameter (16.3 nm) was observed in nanoemulsion with reuterin 40 mM, Tween 80 5 %, and 3 min of ultrasound treatment. Zeta potential was relatively high within 30 mV range in all designed nano- emulsions which indicates the nanoemulsion\\\\\\\'s stability. Also, the prepared nanoemulsions, despite initial particle size showed good stability in a 90-d storage period at 25 ◦C. In vivo assay, showed a significant improvement in the protection of apple fruit treated with reuterin-EOs nanoemulsions against fungal spoilage compared to free reuterin nanoemulsion. Treatment of apples with nanoemulsion containing 40 mM reuterin showed a maximum inhibitory effect on B. cinerea (5.1 mm lesion diameter compared to 29.2 mm for control fruit) within 7 d at 25 ◦C. In summary, the present study demonstrated that reuterin-synergistic EOs hybrid with boosted antifungal activities can be considered as a biopreservative for food applications.

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