SYNTHESIS, CHARACTERIZATION, AND ANTIBACTERIAL ACTIVITY OF ANTIMICROBIAL PEPTIDE Ctx(Ile21)-Ha ANALOG AGAINST Salmonella Enteritidis

Brazil is a global reference in broiler chicken exports. However, trade barriers imposed by demanding markets, such as the European Union, which restrict the use of antibiotics in animal production due to the threat of antimicrobial resistance, drive the search for biotechnological alternatives. In this scenario, Salmonella enterica subsp. enterica serovar Enteritidis emerges as a highly dangerous zoonotic pathogen. Operating as a silent threat in poultry flocks, it easily contaminates the food chain and is responsible for severe foodborne outbreaks worldwide, causing gastroenteritis that can escalate to life-threatening systemic infections in humans. Antimicrobial peptides (AMPs) have gained attention for their broad spectrum of biological activities and arise as promising substitutes with disruptive mechanisms. Specifically, the AMP Ctx(Ile²¹)-Ha has demonstrated promising activity in combating pathogenic and multidrug-resistant bacteria (MDR) in previous studies. Therefore, the objective of this study was to synthesize, purify, and characterize an antimicrobial peptide analog to Ctx(Ile²¹)-Ha, and to evaluate its in vitro antibacterial activity against S. Enteritidis. The peptide synthesis was performed by Solid-Phase Peptide Synthesis (SPPS) using the Fmoc strategy. Following synthesis, purification was carried out by Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC), and the peptide's identity was confirmed by Mass Spectrometry. For the antibacterial evaluation, the Minimum Inhibitory Concentration (MIC) was assessed through the serial microdilution methodology in Mueller-Hinton broth. The SPPS methodology was highly feasible for obtaining the molecule, yielding a final peptide with a purity greater than 95%. In the in vitro assays, the synthesized AMP demonstrated a potent antibacterial effect, with a MIC of 0.0625 mg/mL (62.5 µg/mL; ~ 26.8 µmol L^-1) against S. Enteritidis. These findings represent a promising result, demonstrating that the successfully synthesized peptide possesses significant inhibitory activity against a highly virulent pathogen. This validates the potential of AMPs as viable alternatives to traditional antibiotics, aligning chemical synthesis with the sanitary safety requirements of the international animal protein market.