Comparative Analysis of Inhibition-Based and Indicator-Independent Colorimetric Assay for Screening of...

Screening of bacteriocin-producing lactic acid bacteria (LAB) is an important aspect for the search of new/novel probiotic strains. Here, a vesicle-based colorimetric assay was compared with conventional inhibition-based antimicrobial assays using 54 isolates of LAB. All isolates demonstrated zone of growth inhibition ranging from 2.5 to 7.5 mm against indicator strain, Micrococcus luteus MTCC106 using point inoculation method. Cell-free supernatant of the isolates showed zone of growth inhibition varying from 14.5 to 25 mm using agar well diffusion assay. These isolates inhibited the growth of indicator strain by 89.56-98.65%. The antimicrobial activity present in cell-free supernatant of different isolates was found to be in the range of 10-160 AU ml^- 1. The treatment of polydiacetylene (PDA) vesicles with cell-free supernatant of selected isolates led to blue-red color transition, and presence of protein band on tricine SDS-PAGE confirmed the presence of membrane-acting peptides, bacteriocins. The colorimetric responses (CR%) varied between 0 and 59%, and the assay was found to be more sensitive, faster, and reliable as compared to the other conventional indicator-based methods used. Therefore, the colorimetric assay may be specifically applied for screening of bacteriocin-producing LAB.