Applied Biochemistry and Biotechnology 619 Vol. 121–124, 2005 Copyright © 2005 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/05/01–09/619–638/$30.00 Production of Nisin by Lactococcus lactis in Media with Skimmed Milk THEREZA CHRISTINA VESSONI PENNA, *,1 ANGELA FAUSTINO JOZALA, 1 LETÍCIA CÉLIA DE LENCASTRE NOVAES, 1 ADALBERTO PESSOA JR., 1 AND OLIVIA CHOLEWA 2 1 Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Science, University of São Paulo, Rua Antonio de Macedo Soares, 452, São Paulo, SP, Brazil 04607-000, E-mail: tcvpenna@usp.br 2 Molecular Probes, Eugene, OR, 97402 Abstract Nisin is a bacteriocin that inhibits the germination and growth of Gram- positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media com- ponents, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30ºC for 36 h) in both M17 and MRS stan- dard broth media (pH 6.0–7.0) supplemented with sucrose (1.0–12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30ºC for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of “standard nisin” (Nisaplin ® , g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other sup- plements, and the activity increased to 142.5 AU/L in M17 diluted with liq- uid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed. Index Entries: Nisin; Lactococcus lactis; growth conditions; skimmed milk; Lactobacillus sake. Introduction Lantibiotics are small peptides characterized by the presence of an unusual amino acid, lanthionine. Jung (1) defines lantibiotics as bacteriocins produced by lactic acid bacteria that are divided into two subgroups, type A and type B. Nisin, a type A bacteriocin, is a 34-residue monomeric penta- *Author to whom all correspondence and reprint requests should be addressed.