Nisin has a highly selective spectrum of activity suppressing gram-positive and gram-negative bacteria. Applications of this bacteriocin include conservation of food, dental care products, pharmaceutical products such as stomach ulcers, colon infection treatment and potential birth control. This work aims to optimize large-scale nisin production utilizing diluted skimmed milk (2.27g_{total solids}) and milk whey (generously provided by a commercial dairy industry) as growth media, aiming, at low-costs process, to stimulate its use. L. lactis ATCC 11454 pre culture was poured into 1.5L of the milk whey (pH 6.30) in a 2L bench–scale bioreactor. The initial cell concentration in the bioreactor was 0.39 and 0.36g.L^-1 (SD=0.10). The incubation conditions were 36 hours, 200 rpm at 30ºC with and without air injection (up to 1.5mL.min^-1). Nisin activity was quantified by the agar diffusion assay utilizing L. sake as a sensitive indicator microorganism. Without air injection, nisin activity was observed only in the milk whey (3.8x10⁶AU.mL^-1), but no nisin activity was detected in the diluted skim milk. However, for 1.5mL.min^-1 condition, nisin activity in the diluted skim milk (3.31 x 10³AU.mL^-1) was 1 log₁₀ higher than in the milk whey (4.8 x 10²AU.mL^-1). In this study, the air absence enhanced the nisin activity in the milk whey media, reproducing the disposal conditions of the milk whey by dairy industries. Nisin production can be attained with a low cost growth media (milk whey).