Abstract
The generation of a zone of inhibition on a solid substrate indicates the bioactivity of antimicrobial peptides such as bacteriocin and enterocin. The indicator strain plays a significant role in bacteriocin assays. Other characteristics of bacteriocins, such as their dispersal ability and the different zymogram components, also affect bacteriocin assays. However, universal well diffusion assays for antimicrobials, irrespective of their ability to diffuse (bacteriocin and enterocin), do not exist. The ability of different zymography components to generate non-specific activities have rarely been explored in the literature. The purpose of the present work was to evaluate the impact of major factors (diffusion and rate of diffusion) in a solid substrate bioassay, and to document the adverse effects of sodium dodecyl sulfate in zymograms used to estimate the approximate molecular weight of bacteriocins.
Highlights
Bacteriocins/enterocins are produced by Gram-positive bacteria, and they exhibit narrow to broad spectrum antimicrobial activities that are heat stable, and bacteriocin/enterocin-producing strains are immune to the effects of these antibacterial peptides
We show that zymograms are not sufficiently specific because they can result in the formation of a non-specific zone of growth inhibition, irrespective of the presence or absence of bacteriocins and enterocins
We showed that the conventional agarose well diffusion assay is not suitable and applicable for enterocins, but are well suited for assaying bacteriocins
Summary
Bacteriocins/enterocins are produced by Gram-positive bacteria, and they exhibit narrow to broad spectrum antimicrobial activities that are heat stable, and bacteriocin/enterocin-producing strains are immune to the effects of these antibacterial peptides. Certain standard and control strains are extensively used to detect the antimicrobial activities of bacteriocins. Micrococcus luteus is the microbe used to assay bacteriocins antimicrobial activity in a plate assay. Many more indicator strains, such as Lactobacillus, Leuconostoc, and Pediococcus strains, have been used in bacteriocin assays, they are not widely available. The plate assay does not provide any information pertaining to the approximate molecular weights of bacteriocins. A standard zymography procedure has been used to estimate the approximate molecular weights of bacteriocins for many years. Recent observations in our laboratory demonstrate that zymograms are not suitable for determining the approximate molecular weights of antimicrobial proteins because of the lack of specificity due to a non-specific zone of growth suppression. The present manuscript reports various kinds of a nonspecific zone of growth inhibition observed by following a zymogram approach
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