To determine the antimicrobial activity of nisin and ncAA-modified nisin(BocK) variants synthesized by L. lactis, solid medium was inoculated with 100 μl of nisin-sensitive L. lactis NZ9000 pNZnisPT pIL253 overnight culture. The first plasmid (pNZnisPT) leads to expression of NisP, which can activate the nisin variants later added to the agar by proteolytic cleavage of the leader peptide. Plasmid pIL253 is an “empty” control plasmid conferring erythromycin resistance, thus avoiding growth inhibition from potential carryover of the antibiotic from the L. lactis production strain supernatant. Fifty microliters of either filtered culture supernatant or HPLC-purified peptides was applied to the testing wells (diameter: 7.5 mm) and incubated at 30°C overnight, until inhibition halos were visible.