A One-Step Dual Chromogenic Plate Assay for Reliable Screening of Naringinase-Producing Microorganisms

Naringinase is an industrially important enzyme system with applications in citrus debittering, aroma enhancement, and the production of bioactive flavonoid aglycones. Accurate identification of true naringinase-producing microorganisms remains challenging due to the dual-enzyme nature of naringinase, which requires coordinated α-L-rhamnosidase and β-D-glucosidase activities. Conventional screening methods based on naringin agar or single chromogenic substrates often generate false-positive results, as they fail to distinguish partial enzyme producers from microorganisms capable of complete naringin hydrolysis. In this study, a single-plate, one-step dual chromogenic screening assay was developed for reliable identification of true naringinase producers. The method employs p-nitrophenyl-α-L-rhamnopyranoside and X-gal to simultaneously detect α-L-rhamnosidase and β-glycosidase-like activities, producing distinct yellow and blue signals, respectively. The spatial separation of these chromogenic products enables unambiguous differentiation of microbial enzymatic profiles on a single agar plate. The assay was validated using bacterial strains with known enzymatic characteristics and further confirmed by spectrophotometric enzyme assays and thin-layer chromatography. Only isolates exhibiting both chromogenic responses demonstrated complete biotransformation of naringin to naringenin. A positive correlation between chromogenic halo diameter and measured enzyme activity was also observed. The proposed assay provides a simple, reliable, and scalable platform for high-throughput screening of naringinase-producing microorganisms in applied microbiology and biotechnology.