Bovine lactoferrin and lactoferricin on plasma-deposited coating against spoilage Pseudomonas spp. L. Quintieri a , B.R. Pistillo b , L. Caputo a , P. Favia b, c , F. Baruzzi a, ⁎ a Institute of Sciences of Food Production, National Research Council of Italy, Via Amendola 122/O, 70126 Bari, Italy b University of Bari “Aldo Moro”, Department of Chemistry, Via Orabona 4, 70126 Bari, Italy c Institute of Inorganic Methodologies and Plasmas, National Research Council of Italy, Via Orabona 4, 70125 Bari, Italy abstract article info Article history: Received 3 December 2012 Accepted 23 April 2013 Editor Proof Receive Date 24 May 2013 Keywords: Antimicrobial peptides Plasma processing Active packaging Food spoilage HM Mozzarella cheese Bovine lactoferrin and lactoferricin B, well-known for their antimicrobial properties, were individually immobilized on two different coatings functionalized with -COOH groups deposited in the inner part of polyethylene micro tubes by means of a plasma deposition (PE-CVD) process fed with ethylene and acrylic acid vapors. The resulting functionalized tubes were tested for antimicrobial activity against three Pseudomonas strains responsible for casein hydrolysis and cheese pigmentation. The cell counts of these spoilage bacteria, incubated for 30 h under their op- timal growth conditions, were found to be significantly reduced after 24 h in micro tubes functionalized with lactoferricin B, whereas a very low antimicrobial activity against the same strains, often undistinguishable from that of control samples, was observed in tubes functionalized with lactoferrin. This is the first work in which a plasma coating functionalized by lactoferricin B was studied to make an active packaging useful to control cheese spoilage by Pseudomonas. Industrial relevance: The current study describes a new method to immobilize two food grade proteinaceous nat- ural compounds. The resulting plasma-functionalized lactoferricin B-immobilized coating is a promising tool for the control of spoilage microorganisms and shelf-life extension of cheeses. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Antimicrobial peptides (b 10 kDa; 3–50 amino acid residues) have been extensively investigated for promising applications in food pres- ervation (Meng, Huanli, & Fengshan, 2010). Among milk proteins, bo- vine lactoferrin (BLF) has gained much interest as functional bioactive ingredient for applications in food, personal care, pharmaceutical products (Wakabayashi, Yamauchi, & Takase, 2006) and for its anti- microbial activity against fungi, yeasts and Gram-negative and posi- tive bacteria (Naidu, 2000). Some authors have demonstrated that the digestion of BLF with pepsin releases the peptide lactoferricin B (LfcinB), largely responsible for BLF antibacterial activity (Bellamy et al., 1992; Tomita et al., 1991). Active packaging systems can be categorized into adsorbing (e.g. oxygen and ethylene scavengers) and releasing systems (e.g. flavor and odor releasers and antimicrobials), as recently reviewed (Pereira de Abreu, Cruz, & Paseiro Losada, 2012). Concerning antimicrobial releas- ing systems, most researches were addressed to control pathogens in foods such as Listeria monocytogenes (Cha, Chen, Park, & Chinnan, 2003; dos Santos Pires et al., 2008; Limjaroen, Ryser, Lockhart, & Harte, 2005; Nguyen, Gidley, & Dykes, 2008; Santiago-Silva et al., 2009; Trinetta, Floros, & Cutter, 2010), whereas fewer studies have concerned with anti- microbial packaging for the control of spoilage bacteria. Appendini and Hotchkiss (2001) found that a 14-amino-acid resi- due peptide, covalently immobilized on polystyrene by solid phase peptide synthesis, was microcidal in a concentration and time depen- dent manner against several microorganisms re-suspended in buffer, including the food spoilers Bacillus subtilis, Kluyveromyces marxianus, Pseudomonas fluorescens and Serratia liquefaciens. Other in vitro assays demonstrated that partially purified lysozyme (in combination with chickpea albumin extract, bovine serum albumin and disodium EDTA) or thymol in zein films could control the growth of B. subtilis and Escherichia coli (Güçbilmez, Yemenicioğlu, & Arslanoğlu, 2007) or Bacillus cereus, Candida lusitaniae and Pseudomonas spp. (Del Nobile, Conte, Incoronato, & Panza, 2008). Other authors (Barbiroli et al., 2012) found that the combina- tion of both lysozyme and BLF, incorporated into a paper containing carboxymethyl cellulose, was the most effective against two Innovative Food Science and Emerging Technologies 20 (2013) 215–222 Abbreviations: PE-CVD, plasma enhanced-chemical vapor deposition; BLF, bovine Lactoferrin; LfcinB, Lactoferricin B; HM, Mozzarella cheese High Moisture Mozzarella cheese; pdEthAA, plasma deposited Ethylene/Acrylic Acid; XPS, X-ray Photoelectron Spectroscopy; IEX, ion-exchange chromatography; LOQ, limit of quantification; EDC, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; PBS, phosphate buff- ered saline; PP, polypropylene; PCB, Plate Count Broth; PCA, Plate Count Agar; II, Inhi- bition Index; GLM, General Linear Model; ANOVA, Analysis of variance; LSD, Fisher's least significant difference; P, statistical probability; LC/MS, Liquid chromatography– mass spectrometry; HPLC, High-performance liquid chromatography. ⁎ Corresponding author at: Institute of Sciences of Food Production, National Research Council of Italy, Via G. Amendola 122/O, 70126 Bari, Italy. Tel.: +39 080 5929319; fax: +39 080 5929374. E-mail address: federico.baruzzi@ispa.cnr.it (F. Baruzzi). 1466-8564/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ifset.2013.04.013 Contents lists available at ScienceDirect Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset