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1 Dipartimento di Protezione delle Piante e Microbiologia Applicata,
2 Dipartimento di Chimica, and
3 Laboratorio di Spettrometria di Massa per la Proteomica, Università degli Studi di Bari, 70126 Bari, Italy
Corresponding author: M. Gobetti; e-mail: gobbetti{at}agr.uniba.it.
Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: 
S1-casokinin (e.g., sheep S1-casein (CN) f22–30 of Pecorino Romano and cow S1-CN f24–33 of Canestrato Pugliese); isracidin (e.g., sheep S1-CN f10–21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow 
-CN f106–115 of Canestrato Pugliese and Crescenza); and ß-casomorphin-11 (e.g., goat ß-CN f60–68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 µg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.
Key Words: antibacterial peptide • Italian cheese
Abbreviation key: ACE = angiotensin-I converting enzyme, ESI-IT = electrospray ionization-ion trap, MS = mass spectrometry, OPA = o-phthaldialdehyde, RP-FPLC = reversed phase fast protein liquid chromatography, TFA = trifluoroacetic acid.
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