Measuring Nisin Yield by Agar Diffusion

Nisin yield was determined by the agar well diffusion method [12 (link)] with minor modifications. Briefly, the broth of the tested strains after fermentation was boiled for 10 min and cells were removed by centrifugation at 8000 rpm for 3 min. Then, the supernatant was appropriately diluted with 0.02 M HCl. M. luteus (10⁷ CFU/mL) was used as indicator and inoculated at a concentration of 1% (v/v) into 30 mL melted/cooled LB agar. To enhance nisin diffusion, 1.5% (v/v) Tween 80 (JiangTian, Tianjin, China) was added to the medium and mixed well. Then, the medium was quickly poured into sterile petri dish. After solidification and pre-cultivation, a 7 mm diameter sterile cork borer (MRS Scientific Ltd., Wickford, UK) was used to generate agar well for loading samples. Standard nisin solutions (concentrations of 20, 40, 80, 100, 200 and 400 IU/mL) were prepared using nisin powder (Sigma, St. Louis, MO, USA). Subsequently, standard nisin solutions and sample solutions were, respectively, loaded into the wells (80 μL per well), and the plates were incubated at 37 °C for 24 h. The diameter of inhibition zone was measured with a calliper. A regression equation was derived from the nisin standard data.