Rational design of hyperstable antibacterial peptides for food preservation.

We describe the design of peptides with properties like thermostability, pH stability, and antibacterial activity against a few
bacterial food pathogens. Insights obtained from classical structure-function analysis of natural peptides and their mutants through
antimicrobial and enzymatic assays are used to rationally develop a set of peptides. pH and thermostability assays were performed
to demonstrate robust antimicrobial activity post-treatment with high temperatures and at wide pH ranges. We have also
investigated the mode of action of these hyperstable peptides using membrane permeability assays, electron microscopy, and
molecular dynamics simulations. Notably, through mutational studies, we show that these peptides elicit their antibacterial action
via both membrane destabilization and inhibition of intracellular trypsin—the two functions attributable to separate peptide
segments. Finally, toxicity studies and food preservation assays demonstrate the safety and efficacy of the designed peptides for
food preservation. Overall, the study provides a general ‘blueprint’ for the development of stable antimicrobial peptides (AMPs).
Insights obtained from this work may also be combined with combinatorial methods in high-throughput studies for future
development of antimicrobials for various applications.