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.