CHALON Miriam Carolina
congresos y reuniones científicas
Nisin purification and evaluation of its antimicrobial activity in combination with EDTA against Gram-negative pathogens
LANZA, L; CHALON M.C.; BELLOMIO, A
Congreso; Congreso Nacional de la Sociedad de Investigaciones en Bioquímica y Biología Molecular; 2019
Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular
Nisin is an antimicrobial peptide, produced by Lactococcus lactis, as a defense system against competing microorganisms. Nisin exerts a dual mode of action to kill Gram-positive bacteria. Firstly, it inhibits cell wall synthesis by binding toward lipid II. Secondly, the peptide permeabilizes the cell membrane by forming pores that disrupt vital ion gradients. This peptide is widely used as a food preservative. Nevertheless, it is difficult for nisin to penetrate the outer membrane of Gram-negative bacteria, and thus, it cannot reach its target in the inner membrane. This leads to the inactivity against Gram-negative bacteria. Notably, nisin can inhibit the growth of these bacteria when chelating agents, like EDTA, are used to destabilize the outer membrane. Thus, the bottleneck for nisin to be active against Gram-negative bacteria appears to be its ability to pass the outer membrane. The aim of this work was to improve the purification of nisin from commercial preparations and evaluate its activity against Gram-negative foodborne pathogens. Methods: TECNIS® (commercial preparation of nisin 2.5%) was suspended in water and stirred at room temperature (rt) before dichloromethane was added. Instantaneously, a white precipitate was formed at the interface of the yellow aqueous layer and the colorless organic layer. The obtained emulsion was centrifuged, after which the desired nisin fraction formed a brown solid pellet at the interface of both layers. The pellet was dried to remove any residual solvent. The nisin extract was subsequently dissolved in water, followed by filtration. This solution was further purified by RP-HPLC in a C18 preparative column. The eluted fractions were dried in vacuo and stored at -20°C. Purity was checked with an analytical RP-HPLC system. Nisin activity (MIC) was evaluated against Gram-negative foodborne pathogens in liquid and solid media, using EDTA as a chelator (0.625 and 2.5 mM for Escherichia coli O157 and Salmonella enterica serovar Typhimurium 14028, respectively). Listeria monocytogenes FBUNT was employed as a positive activity control. The MIC was defined as the lowest nisin concentration which produces no visible microbial growth.Results: pure nisin was obtained as a brown powder and its MIC in liquid media was 1.16, 4.66 and 9.33 μM against L. monocytogenes FBUNT, S. Typhimurium 14028 and E. coli O157, respectively. In solid media, the MIC was 0.58 μM against L. monocytogenes FBUNT, and 149 μM against S. Typhimurium 14028 and E. coli O157. Conclusions: here we describe an efficient protocol for the scalable purification of nisin that allows us to obtain the enriched peptide from commercial preparations that contains only 2.5% of the desired peptide, with high antimicrobial activity against foodborne pathogens.