Bovine lactoferrin and lactoferricin on plasma-deposited coating against spoilage Pseudomonas spp

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 optimal 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 natural 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. 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 buffered saline; PP, polypropylene; PCB, Plate Count Broth; PCA, Plate Count Agar; II, Inhibition 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 Keywords Antimicrobial peptides; Plasma processing; Active packaging; Food spoilage; HM Mozzarella cheese -------------------------------------------------------------------------------- 1. Introduction Antimicrobial peptides (< 10 kDa; 3–50 amino acid residues) have been extensively investigated for promising applications in food preservation (Meng, Huanli, & Fengshan, 2010). Among milk proteins, bovine 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 antimicrobial activity against fungi, yeasts and Gram-negative and positive 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 and 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 releasing systems, most researches were addressed to control pathogens in foods such as Listeria monocytogenes ( Cha et al., 2003, dos Santos Pires et al., 2008, Limjaroen et al., 2005, Nguyen et al., 2008, Santiago-Silva et al., 2009 and Trinetta et al., 2010), whereas fewer studies have concerned with antimicrobial packaging for the control of spoilage bacteria. Appendini and Hotchkiss (2001) found that a 14-amino-acid residue peptide, covalently immobilized on polystyrene by solid phase peptide synthesis, was microcidal in a concentration and time dependent 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 combination of both lysozyme and BLF, incorporated into a paper containing carboxymethyl cellulose, was the most effective against two Gram-positive and Gram-negative bacteria target strains; the lysozyme/BLF active packaging also performed the best control of mesophilic aerobic population in carpaccio, a ready-to-eat uncooked thin-cut veal meat. The release of organic acids and essential oils included into a chitosan matrix did not affect lactic acid bacteria, whereas they delayed the growth of Enterobacteriaceae, including S. liquefaciens, during 21 days of refrigerated period of Bologna type sausage (Quattara, Simard, Piette, Begin, & Holley, 2000). Similar results were also obtained by Conte, Scrocco, Sinigaglia, and Del Nobile (2007), who demonstrated the inhibitory effect of lemon extract against coliforms and pseudomonads, when the governing liquid of Mozzarella cheese was replaced with active gels. Note to users: Corrected proofs are Articles in Press that contain the authors' corrections. Final citation details, e.g., volume/issue number, publication year and page numbers, still need to be added and the text might change before final publication. Although corrected proofs do not have all bibliographic details available yet, they can already be cited using the year of online publication and the DOI , as follows: author(s), article title, journal (year), DOI. Please consult the journal's reference style for the exact appearance of these elements, abbreviation of journal names and use of punctuation. When the final article is assigned to an issue of the journal, the Article in Press version will be removed and the final version will appear in the associated published issue of the journal. The date the article was first made available online will be carried over. Elsevier homepage (opens in a new window) About ScienceDirect About Elsevier Contact and support Information for advertisers Terms and conditions Privacy policy -------------------------------------------------------------------------------- Copyright © 2013 Elsevier B.V. All rights reserved. SciVerse® is a registered trademark of Elsevier Properties S.A., used under license. ScienceDirect® is a registered trademark of Elsevier B.V. Cookies are used by this site. To decline or learn more, visit our Cookies page Bibliographic information Citing and related articles Related articles Antimicrobial efficacy of pepsin-digested bovine lactoferrin on spoilage bacteria contaminating traditional Mozzarella cheese 2012, Food Microbiology Show more information . Innovative Active Packaging Systems to Prolong the Shelf Life of Mozzarella Cheese 2007, Journal of Dairy Science Show more information . Agar hydrogel with silver nanoparticles to prolong the shelf life of Fior di Latte cheese 2011, Journal of Dairy Science Show more information . Related reference work articles No articles found. ADVERTISEMENT Applications and tools Workspace • Close ScienceDirect 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 optimal 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 natural 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. 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 buffered saline; PP, polypropylene; PCB, Plate Count Broth; PCA, Plate Count Agar; II, Inhibition 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 Keywords Antimicrobial peptides; Plasma processing; Active packaging; Food spoilage; HM Mozzarella cheese -------------------------------------------------------------------------------- 1. Introduction Antimicrobial peptides (< 10 kDa; 3–50 amino acid residues) have been extensively investigated for promising applications in food preservation (Meng, Huanli, & Fengshan, 2010). Among milk proteins, bovine 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 antimicrobial activity against fungi, yeasts and Gram-negative and positive 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 and 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 releasing systems, most researches were addressed to control pathogens in foods such as Listeria monocytogenes ( Cha et al., 2003, dos Santos Pires et al., 2008, Limjaroen et al., 2005, Nguyen et al., 2008, Santiago-Silva et al., 2009 and Trinetta et al., 2010), whereas fewer studies have concerned with antimicrobial packaging for the control of spoilage bacteria. Appendini and Hotchkiss (2001) found that a 14-amino-acid residue peptide, covalently immobilized on polystyrene by solid phase peptide synthesis, was microcidal in a concentration and time dependent 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 combination of both lysozyme and BLF, incorporated into a paper containing carboxymethyl cellulose, was the most effective against two Gram-positive and Gram-negative bacteria target strains; the lysozyme/BLF active packaging also performed the best control of mesophilic aerobic population in carpaccio, a ready-to-eat uncooked thin-cut veal meat. The release of organic acids and essential oils included into a chitosan matrix did not affect lactic acid bacteria, whereas they delayed the growth of Enterobacteriaceae, including S. liquefaciens, during 21 days of refrigerated period of Bologna type sausage (Quattara, Simard, Piette, Begin, & Holley, 2000). Similar results were also obtained by Conte, Scrocco, Sinigaglia, and Del Nobile (2007), who demonstrated the inhibitory effect of lemon extract against coliforms and pseudomonads, when the governing liquid of Mozzarella cheese was replaced with active gels. Note to users: Corrected proofs are Articles in Press that contain the authors' corrections. Final citation details, e.g., volume/issue number, publication year and page numbers, still need to be added and the text might change before final publication. Although corrected proofs do not have all bibliographic details available yet, they can already be cited using the year of online publication and the DOI , as follows: author(s), article title, journal (year), DOI. Please consult the journal's reference style for the exact appearance of these elements, abbreviation of journal names and use of punctuation. When the final article is assigned to an issue of the journal, the Article in Press version will be removed and the final version will appear in the associated published issue of the journal. The date the article was first made available online will be carried over. Elsevier homepage (opens in a new window) About ScienceDirect About Elsevier Contact and support Information for advertisers Terms and conditions Privacy policy -------------------------------------------------------------------------------- Copyright © 2013 Elsevier B.V. All rights reserved. SciVerse® is a registered trademark of Elsevier Properties S.A., used under license. ScienceDirect® is a registered trademark of Elsevier B.V. Cookies are used by this site. To decline or learn more, visit our Cookies page Bibliographic information Citing and related articles Related articles Antimicrobial efficacy of pepsin-digested bovine lactoferrin on spoilage bacteria contaminating traditional Mozzarella cheese 2012, Food Microbiology Show more information . Innovative Active Packaging Systems to Prolong the Shelf Life of Mozzarella Cheese 2007, Journal of Dairy Science Show more information . Agar hydrogel with silver nanoparticles to prolong the shelf life of Fior di Latte cheese 2011, Journal of Dairy Science Show more information . Related reference work articles No articles found. ADVERTISEMENT Applications and tools Workspace • Close ScienceDirect 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 optimal 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 natural 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. 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 buffered saline; PP, polypropylene; PCB, Plate Count Broth; PCA, Plate Count Agar; II, Inhibition 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 Keywords Antimicrobial peptides; Plasma processing; Active packaging; Food spoilage; HM Mozzarella cheese -------------------------------------------------------------------------------- 1. Introduction Antimicrobial peptides (< 10 kDa; 3–50 amino acid residues) have been extensively investigated for promising applications in food preservation (Meng, Huanli, & Fengshan, 2010). Among milk proteins, bovine 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 antimicrobial activity against fungi, yeasts and Gram-negative and positive 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 and 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 releasing systems, most researches were addressed to control pathogens in foods such as Listeria monocytogenes ( Cha et al., 2003, dos Santos Pires et al., 2008, Limjaroen et al., 2005, Nguyen et al., 2008, Santiago-Silva et al., 2009 and Trinetta et al., 2010), whereas fewer studies have concerned with antimicrobial packaging for the control of spoilage bacteria. Appendini and Hotchkiss (2001) found that a 14-amino-acid residue peptide, covalently immobilized on polystyrene by solid phase peptide synthesis, was microcidal in a concentration and time dependent 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 combination of both lysozyme and BLF, incorporated into a paper containing carboxymethyl cellulose, was the most effective against two Gram-positive and Gram-negative bacteria target strains; the lysozyme/BLF active packaging also performed the best control of mesophilic aerobic population in carpaccio, a ready-to-eat uncooked thin-cut veal meat. The release of organic acids and essential oils included into a chitosan matrix did not affect lactic acid bacteria, whereas they delayed the growth of Enterobacteriaceae, including S. liquefaciens, during 21 days of refrigerated period of Bologna type sausage (Quattara, Simard, Piette, Begin, & Holley, 2000). Similar results were also obtained by Conte, Scrocco, Sinigaglia, and Del Nobile (2007), who demonstrated the inhibitory effect of lemon extract against coliforms and pseudomonads, when the governing liquid of Mozzarella cheese was replaced with active gels. Note to users: Corrected proofs are Articles in Press that contain the authors' corrections. Final citation details, e.g., volume/issue number, publication year and page numbers, still need to be added and the text might change before final publication. Although corrected proofs do not have all bibliographic details available yet, they can already be cited using the year of online publication and the DOI , as follows: author(s), article title, journal (year), DOI. Please consult the journal's reference style for the exact appearance of these elements, abbreviation of journal names and use of punctuation. When the final article is assigned to an issue of the journal, the Article in Press version will be removed and the final version will appear in the associated published issue of the journal. The date the article was first made available online will be carried over. Elsevier homepage (opens in a new window) About ScienceDirect About Elsevier Contact and support Information for advertisers Terms and conditions Privacy policy -------------------------------------------------------------------------------- Copyright © 2013 Elsevier B.V. All rights reserved. SciVerse® is a registered trademark of Elsevier Properties S.A., used under license. ScienceDirect® is a registered trademark of Elsevier B.V. Cookies are used by this site. To decline or learn more, visit our Cookies page Bibliographic information Citing and related articles Related articles Antimicrobial efficacy of pepsin-digested bovine lactoferrin on spoilage bacteria contaminating traditional Mozzarella cheese 2012, Food Microbiology Show more information . Innovative Active Packaging Systems to Prolong the Shelf Life of Mozzarella Cheese 2007, Journal of Dairy Science Show more information . Agar hydrogel with silver nanoparticles to prolong the shelf life of Fior di Latte cheese 2011, Journal of Dairy Science Show more information . Related reference work articles No articles found. ADVERTISEMENT Applications and tools Workspace • Close ScienceDirect Antimicrobial peptides (b10 kDa; 3–50 amino acid residues) have been extensively investigated for promising applications in food preservation (Meng, Huanli, & Fengshan, 2010). Among milk proteins, bovine 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 antimicrobial activity against fungi, yeasts and Gram-negative and positive 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).