International Dairy Journal 15 (2005) 133–143 Isolation and characterisation of antibacterial peptides derived from the f(164–207) region of bovine a S2 -casein K.B. McCann a , B.J. Shiell b , W.P. Michalski b , A. Lee c , J. Wan c , H. Roginski a , M.J. Coventry c,Ã a Institute of Land and Food Resources, Gilbert Chandler College, The University of Melbourne, Sneydes Road, Werribee, Vic. 3030, Australia b Australian Animal Health Laboratory, CSIRO Livestock Industries, Private Bag 24, Geelong, Vic. 3220, Australia c Food Science Australia, Private Bag 16, Sneydes Road, Werribee, Vic. 3030, Australia Received 23 May 2003; accepted 24 June 2004 Abstract A chymosin digest of sodium caseinate, which showed antibacterial activity against Listeria innocua, was fractionated using reverse phase high performance liquid chromatography and the purified antibacterial peptides were characterised by mass spectrometry, N-terminal amino acid sequencing and comparison to peptide masses of theoretical enzymic digests of milk proteins. Five antibacterial peptides, Cr1, Cr3, Cr4, Cr5 and Cr7 corresponding to amino acid residues 181–207, 180–207, 175–207, 164–207 and 172–207 of bovine a S2 -casein, respectively, were isolated. The minimal inhibitory concentration of peptides Cr1, Cr4 and Cr5 was determined against a range of Gram-positive and Gram-negative bacteria and showed similar activities to those of the bacteriocin peptide, nisin and the antibacterial peptide, lactoferricin B against certain Gram-positive bacteria. A partially purified chymosin digest of sodium caseinate (Cr MIX ) was prepared and observed to be heat stable for up to 15 min on exposure to 121 1C. Although Cr MIX showed bactericidal activity against Salmonella Typhimurium in 0.1% (w/v) peptone medium, no antibacterial activity was observed when tested in skim milk at the same concentration. r 2004 Elsevier Ltd. All rights reserved. Keywords: Antibacterial; Peptide; a S2 -Casein 1. Introduction There has been a continuing interest in naturally occurring bioactive molecules and in their potential use as aids in food and health-care product preservation and human and veterinary therapeutic treatment (Bellamy, Takase, Wakabayashi, Kawase, & Tomita, 1992a; Gould, 1996). Jones and Simms (1930) reported the antibacterial activity of milk in vitro as far back as 1930, and it has long been recognised that breast-feeding of infants reduces the rate of enteric and respiratory infections (Welsh, Skurrie, & May, 1978; Portelli, Gordon, & May, 1998). The antibacterial activity of milk is attributed mainly to the immunoglobulins, but the non-immune proteins, lactoferrin, lactoperoxidase and lysozyme also exhibit distinct antibacterial activities (Pakkanen & Aalto, 1997). In addition to the naturally occurring antimicrobial proteins present in milk, there are also a variety of antibacterial peptides encrypted within the sequence of milk proteins that are released upon suitable hydrolysis of the precursor protein. To date, antibacterial peptides have been derived from the bovine milk proteins, lactoferrin (Bellamy et al., 1992b), a S1 -casein (Lahov & Regelson, 1996), a S2 -casein (Zucht, Forssmann, Raida, & Ader- mann, 1996; Recio & Visser, 1999), a-lactalbumin (Pellegrini, Thomas, Bramaz, Hunziker, & von Fellenberg, 1999), b-lactoglobulin (Pellegrini, Dettling, Thomas, & Hunziker, 2001) and k-casein (Malkoski et al., 2001). ARTICLE IN PRESS www.elsevier.com/locate/idairyj 0958-6946/$-see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.06.008 Ã Corresponding author. Tel.: +61-3-9731-3447; fax: +61-3-9731- 3250. E-mail address: john.coventry@foodscience.afisc.csiro.au (M.J. Coventry).