Identification of food-grade photosensitizers using 2',7'-dichlorodihydrofluorescein for photodynamic inactivation of spoilage microorganisms on baby spinach.

Antimicrobial photodynamic inactivation (aPDI) is a promising non-thermal approach for microbial control in food systems, but efficient screening and validation approaches for food-grade photosensitizers remain limited. In this study, an integrated workflow combining DCFH2-based fluorescence screening, antimicrobial validation, and fresh-produce evaluation was established to identify and assess food-grade photosensitizers for aPDI applications. DCFH2 was synthesized by alkaline hydrolysis and characterized using a Fenton system as a reproducible ROS-generating fluorescence reference. Nine food colorants were screened under visible LED illumination, with erythrosin B, curcumin, and chlorophyllin showing the strongest photoactivity. Antimicrobial assays confirmed their effectiveness against the food-spoilage bacteria Bacillus cereus and Pseudomonas fluorescens and excluded tartrazine and allura red from the final candidate pool because of insufficient measurable antibacterial activity. Erythrosin B at 20 μM achieved >5-log reductions of both bacteria within 30 min of green LED illumination, while curcumin and chlorophyllin showed greater activity against B. cereus than against P. fluorescens. Practical performance was further evaluated on baby spinach during 14 days of refrigerated storage. Colorant misting combined with LED illumination reduced indigenous bacterial populations by 70.14-81.32% while largely maintaining texture, color, and visual quality. The lower antimicrobial efficacy observed on spinach compared with in vitro assays highlights the influence of food-surface and matrix effects on treatment performance. Overall, the results support food colorant-mediated aPDI as a promising non-thermal preservation strategy for reducing microbial spoilage of fresh produce.