| Summary: | The objective of this work was to examine the effect of controlled doses of O<sub>3</sub> (0, 5 µL L<sup>−1</sup> of O<sub>3</sub> for 60 min, and 5 µL L<sup>−1</sup> of O<sub>3</sub> for 720 min) on the quality and phytochemical content of broccoli florets during postharvest storage. The optimal dose was found at 5 µL L<sup>−1</sup> of O<sub>3</sub> for 60 min, from the color retention of broccoli florets exposed to the gas treatment. Overall, the antioxidant capacity of the florets was significantly affected by both doses of O<sub>3</sub> compared to the non-exposed florets. The profile of glucosinolates was determined for up to 14 days in broccoli florets stored at 4 °C by LC-MS. The amount of total glucobrassicins and total hydroxy-cinnamates in florets significantly (<i>p</i> ≤ 0.05) improved by the application of 5 µL L<sup>−1</sup> of O<sub>3</sub> for 60 min compared to non-treated florets. The up-regulation of genes of the tryptophan-derived glucosinolate pathway was observed immediately after both treatments. The gene expression of CYP79A2 and CYP79B3 in broccoli was significantly higher in broccoli florets exposed to 5 µL L<sup>−1</sup> of O<sub>3</sub> for 720 min compared to non-exposed florets. Although enhancement of secondary metabolites can be achieved by the fumigation of broccoli florets with low doses of ozone, quality parameters, particularly weight loss, can be compromised.
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