Purification and Quantification of Nisin

The peptides used in this work were synthesized by Pepscan (Lelystad, The Netherland) (Table 1). For the purification and quantification of nisin, commercial nisin was suspended in 0.05% acetic acid and further purified by HPLC (Agilent 1260 Infinity LC) equipped with a semi-preparative C12 column (Phenomenex 250 × 10 mm). The fractions were collected, tested for activity against L. lactis, and analyzed with MALDI-TOF. The active, fully dehydrated and pure fractions were freeze-dried. The freeze-dried nisin was weighted and dissolved with 0.05% acetic acid^{54 (link)}. Vancomycin, polymyxin B, MgCl₂, lipopolysaccharide (LPS) from Escherichia coli O55:B5, HEPES buffer, and chloral hydrate were purchased from Sigma-Aldrich. Sodium phosphate tablets, DMEM, non-essential amino acids (NEAA) were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA). CellTiter-glo reagent was purchased from Promega (Leiden, the Netherlands). Human plasma EDTA K2 Mixed-Gender was purchased from Sera Laboratories International, Ltd (West Sussex, United Kingdom).