Pepsin-digested bovine lactoferrin prevents Mozzarella cheese blue discoloration caused by Pseudomonas fluorescens Leonardo Caputo a, * , Laura Quintieri a , Daniela Manila Bianchi b , Lucia Decastelli b , Linda Monaci a , Angelo Visconti a , Federico Baruzzi a a Institute of Sciences of Food Production, National Research Council of Italy, Via G. Amendola 122/O, 70126 Bari, Italy b S.C. Controllo Alimenti e Igiene delle Produzioni, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna, 148, 10154 Torino, Italy article info Article history: Received 7 February 2014 Received in revised form 6 June 2014 Accepted 20 June 2014 Available online 28 June 2014 Keywords: Cheese spoilage Blue Mozzarella Indigoidine ESI-Orbitrap-MS Antimicrobial peptides Cheese shelf life abstract The aim of this work was to check the efficacy of bovine lactoferrin hydrolyzed by pepsin (LFH) to prevent blue discoloration of Mozzarella cheese delaying the growth of the related spoilage bacteria. Among 64 Pseudomonas fluorescens strains, isolated from 105 Mozzarella samples, only ten developed blue discoloration in cold-stored Mozzarella cheese slices. When Mozzarella cheese samples from dairy were treated with LFH and inoculated with a selected P. fluorescens strain, no pigmentation and changes in casein profiles were found up to 14 days of cold storage. In addition, starting from day 5, the count of P. fluorescens spoiling strain was steadily ca. one log cycle lower than that of LFH-free samples. ESI- Orbitrap-based mass spectrometry analyses allowed to reveal the pigment leucoindigoidine only in the blue LFH-free cheese samples indicating that this compound could be considered a chemical marker of this alteration. For the first time, an innovative mild approach, based on the antimicrobial activity of milk protein hydrolysates, for counteracting blue Mozzarella event and controlling psychrotrophic pig- menting pseudomonads, is here reported. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Italian traditional Mozzarella is a fresh table pasta filata cheese with a high moisture (HM) content (50e60%), usually dipped into a governing liquid (GL), mainly made up of tap water, brine and whey that preserve the soft-springy texture and high amounts of expressible serum throughout 10e12 days of cold storage. A combination of longer storage times and refrigeration tem- peratures causes an advantage particularly to psychrotrophic pseudomonads that can become the dominant non-lactic bacteria population in milk and in fresh cheeses such as Mozzarella (Cantoni et al., 2003; De Jonghe et al., 2011; Franciosi et al., 2011; Martin et al., 2011; Morales et al., 2005). Recently, the occurrence of very high loads of non-lactic acid bacteria populations, mainly composed of Pseudomonas, Acineto- bacter and Rhanella strains, was found to be responsible for casein hydrolysis and exfoliation of the outer surface of Mozzarella (Baruzzi et al., 2012). In addition, several cases of anomalous discoloration were reported in HM Mozzarella cheese and referred to the contamination by Pseudomonas putida (reddish discolor- ation; Soncini et al., 1998), Pseudomonas fluorescens biovar IV and Pseudomonas libanensis (bluish discoloration; Cantoni et al., 2003), Pseudomonas gessardii (yellowepurple spots; Cantoni et al., 2006) and P. fluorescens (greenish and fluorescent discoloration; Franzetti and Scarpellini, 2007) thanks to the production of different pig- ments (pyoverdin, pyocianin, pyorubin and pyomelanin; Palleroni, 2005). In June 2010, the Rapid Alert System for Food and Feed (RASFF) reported many cases referred to as “blue Mozzarella cheese”. At first, it was developed on high moisture (HM) Mozzarella cheese manufactured in Germany, and latter in other European countries. These cheeses, properly kept in cold storage conditions, became blue after opening the packs. German authorities demonstrated that tap water, containing Pseudomonas spp., was the source of cheese contamination (RASFF, 2010). Many approaches have been undertaken to control the micro- biota responsible for HM Mozzarella cheese spoilage such as the use of lysozyme and Na 2 eEDTA (Sinigaglia et al., 2008), essential oil (Gammariello et al., 2008) or the use of silver nanoparticles in bio- based nanocomposite coatings (Gammariello et al., 2011). The * Corresponding author. Tel.: þ39 080 5929323. E-mail address: leonardo.caputo@ispa.cnr.it (L. Caputo). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm http://dx.doi.org/10.1016/j.fm.2014.06.021 0740-0020/© 2014 Elsevier Ltd. All rights reserved. Food Microbiology 46 (2015) 15e24