DETECTION AND DEMONSTRATION OF INHIBITORY ACTIVITIES OF BACTERIOCINS BY ISOELECTRIC FOCUSING

Abstract

AbstractA technique utilizing isoelectric focusing (IEF) was developed and compared with a modified Bhunia's sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) method for directly detecting antimicrobial activities of inhibitory peptides or proteins (bacteriocins). In IEF, the gel containing separated peptide or protein bands was directly overlaid with indicator bacteria without any fixation or rinsing as done in the SDS‐PAGE method. The IEF gave clear zones of inhibition surrounding inhibitory bands. Bhunia's method was modified to reduce the time for fixation and rinsing, and a 15‐min fixation followed by 2‐h rinsing with dd H2O was determined to be necessary for a detection similar to the IEF. In addition, results of this study showed that denaturation of bacteriocins and, subsequently, failure to detect the presence of a bacteriocin could occur in the SDS‐PAGE analysis while little denaturation was observed in the IEF assay. Therefore, the IEF assay developed in this study was more rapid and less destructive as compared with the SDS‐PAGE method.