Nisin

The activity levels of nisin and new lanthipeptides were determined by a previously described agar diffusion method, with minor modifications.^[37] Briefly, a stock nisin solution 1000 IU mL⁻¹) was prepared by adding 50 mg of commercial nisin (10⁶ IU g⁻¹; Sigma‐Aldrich, St. Louis, MO, USA) into 50 mL of sterile 0.02 mol L⁻¹ HCl. Standard nisin solutions of 1000, 500, 250, 200, 100, 20, and 5 IU mL⁻¹ were prepared using the stock solution and diluted with 0.02 mol L⁻¹ HCl to construct a standard curve. The bioassay medium contained 1.2% tryptone, 0.75% yeast extract, 0.75% NaCl, 0.3% NaH₂PO₄, and 2% agar. After the addition of sterile 0.75% glucose and 0.5% Tween 20, the agar medium was cooled to 50°C and inoculated with 1.5% overnight culture of the indicator strain M. luteus NCIB 8166. The bioassay agar (25 mL) was aseptically poured into sterile Petri dishes, and several holes were made on each plate after solidification. Then, 2 µL of the CFPS mixture supernatant and an equal volume of the nisin Z standard solution were separately added to the holes. After incubation at 30 °C for 18 h, a standard curve of the nisin zone of inhibition versus units of the nisin standard solution was created by measuring the zone diameter using digital calipers (TAJIMA Tool Co., Ltd., Shanghai, China) horizontally and vertically. nisin concentrations for each CFPS mixture were estimated.

A stock solution of nisin was prepared at 1000 µg/mL by dissolving nisin (1000 UI/mg, 2.5% purity) (Sigma-Aldrich, USA) in 0.02 M HCl (Merck, Germany). A pexiganan stock solution was prepared at 2048 µg/mL by dissolving pexiganan (>95% purity, Innovagen, Sweden) in deionized sterile water. Both nisin and pexiganan stock solutions were filtered using a 0.22 µm filter (Millipore Corporation, Billerica, MA, US) and stored at 4 °C. Working solutions of pexiganan were diluted in water or incorporated within the biogel at concentrations ranging from 1 to 256 µg/mL and incorporated in the collagen model at 256 µg/mL. The dual-AMPs solutions were prepared by supplementing pexiganan solutions with nisin at MIC values, as determined in a previous study by Santos et al. [30 (link)]. Briefly, nisin was added at 12.5 µg/ mL for the dual-AMP suspension in water and at 22.5 µg/mL for the dual-AMP incorporated within the biogel. Working solutions of nisin were diluted in water at a concentration of 12.5 µg/mL, incorporated within the biogel at 22.5 µg/mL and used in the collagen model at 125 µg/mL.

The standard nisin (Sigma-Aldrich, St. Louis, MO, USA—containing 2.5% nisin, with 1,000,000 IU/g in its composition) solution was prepared by dissolving 1 g of nisin in 10 mL of phosphate buffer solution (PBS-pH 7.0), as in Table 1. The solution was centrifuged at 13,201 g for 10 min at 10 °C and the supernatant was collected and filtered in a 0.22 µm membrane (Millipore, Burlington, MA, USA). The nisin standard curve was evaluated by an agar diffusion assay. The nisin bioindicator Lactobacillus sakei ATCC 15521 was used for the agar diffusion assay [6 (link),23 (link)].
The concentrations of standard nisin were related by the diameter of the inhibition halo (H, mm), and the activity of nisin was determined and expressed in arbitrary units per mL (AU/mL). The activity of nisin was based on the dilution of the standard nisin calibration curves. The correlation between AU/mL and international units per mL (IU/mL) was 1.09 ± 0.17 AU to 1.0 IU (40 IU = 1 µg of pure nisin A) [14 (link),26 (link)].