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he use, and sometimes misuse, of antimicrobials in both human and veterinary medicine during the years has resulted in the emergence of bacterial strains that no longer respond to antimicrobial therapy. So far, resistance has developed to almost all antimicrobial drugs (Gold and Moellering 1996). Moreover, bacterial strains resistant to all available antimicrobial agents have been identified among clinical isolates of different bacterial species (Tomasz 1994). A study aiming at evaluating the resistance of 5 major foodborne pathogens (Campylobacter, Salmonella, Yersinia enterocolitica, pathogenic Escherichia coli, and Listeria monocytogenes) isolated from 922 meat products showed that enteric bacteria are highly resistant against a wide range of antimicrobial agents and also possess multiresistance phenotypes of distinct advantages (Mayrhofer and others 2004). The alarming emergence of resistance among bacteria has led the World Health Organization (WHO) to announce antimicrobial drug resistance as a main public health concern (WHO 1995). Over the years, massive use of common food safety barriers led to the development of resistance by various food microorganisms (Samson and others 1995; Holyoak and others 1996; Lin and others 1996; Park and others 1996; Henriques and others 1997; Kathariou 2002). The difficulty in the food industry in preventing outbreaks of foodborne pathogens such as Salmonella, E. coli O157:H7, Staphylococcus, L. monocytogenes, and Clostridium perfringens resulted in
thousands deaths and million cases of foodborne illnesses each year (Mead and others 1999). In addition to well-known pathogens that may cause global pandemics, new unrecognized and uncontrolled pathogens are emerging as a result of changing ecology and technology or by the transfer of mobile virulence factors such as bacteriophage, thus dramatically changing the spectrum of foodborne illnesses (Tauxe 2002). Enteric bacteria are especially tolerant to adverse environmental conditions such as low pH and high salt concentrations (Small and others 1994; Cheville and others 1996; Brown and others 1997; Mayrhofer and others 2004). Virulent strains of E. coli are increasingly recognized as foodborne pathogens. Among the 6 virotypes, enterohemorrhagic E . coli (EHEC) are considered to be highly significant due to their low infectious dose and the severe consequences of infection (Buchanan and Doyle 1997). Escherichia coli O157:H7 may cause hemorrhagic colitis and hemolytic uremic syndrome (Riley and others 1983). Outbreaks of foodborne illness due to E. coli O157:H7 have been reported from various parts of the world (Doyle 1991; Griffin and Tauxe 1991; Besser and others 1993; Anonymous 1995). Acid foods such as apple juice were reported to be associated with these outbreaks (Steele and others 1982). Therefore, contamination and proliferation of food pathogens are a great concern for food safety and public health. Consumer awareness and demands as well as food legislation have made the task of providing high-quality products even more challenging to the food industry. Consumers demand higher quality, preservative-free, safe yet mildly processed foods with an exMS 20060146 Submitted 3/2/2006, Accepted 8/26/2006. Authors are with tended shelf life. Since acidity and sterilization are the most comDept. of Biotechnology & Food Engineering, TechnionIsrael Inst. of mon preservation techniques that control outgrowth of pathogenic Technology, Haifa, 32000, Israel. Direct inquiries to author Mor (E-mail: spore-forming bacteria, addressing this consumers need calls for amor@tx.technion.ac.il). innovative approaches to ensure product preservation.
Introduction
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Table 1 --- Amino acid sequence of representative gene-encoded antimicrobial peptides Designation Sequencea Reference Rogers 1928 Nielsen and others 1990 Hastings and others 1991 Tichaczek and others 1994 Tomita and others 1996 Aymerich and others 1996 Cintas and others 1997 Zasloff 1987 Ge and others 1999a Shi and others 1996 Thouzeau and others 2003 Cole and others 1997 Mor and Nicolas 1994a Rydlo and others 2006 Lee and others 1997 Wilcox and Eisenberg 1992 Johansson and others 1998 Lee and others 1997 Ganzle and others 1999
AMPs produced by microbial cells Nisin ITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK Pediocin PA1 KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC Leucocin A KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW Sakacin P KYYGNGVHCGKhSGCTVDWGTAIGNIGNNAAANWATGGNAGWNK Bacteriocin 31 ATYYGNGLYCNKQKCWVDWNKASREIGKIIVNGWVQHGPWAPR Enterocin A TTHSGKYYGNGVYCTKNKCTVDWAKATTCIAGMSIGGFLGGAIPGQC Enterocin P ATRSYGNGVYCNNSKCWVNWGEAKENIAGIVISGWASGLAGMGH AMPs produced by animal cells Magainin GIGKFLHSAKKFGKAFVGEIMNS MSI-78 GIGKFLKKAKKFGKAFVKILKK CONH2 PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP Spheniscin SFGLCRLRRGFCAHGRCRFPSIPIGRCSRFVQCCRRVW Pleurocidin GWGSFFKKAAHVGKHVGKAALHTYL Dermaseptin S4 ALWMTLLKKVLKAAAKALNAVLVGANA K 4 S4(1-14) ALWKTLLKKVLKAA CONH2 Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGPR Melittin GIGAVLKVLTTGLPALISWIKRKRQQ LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Clavanin A VFQFLGKIIHHVGNFVHGFSHVF Curvacin A ARSYGNGVYCNNKKCNVNRGEATQSIIGGMISGWASGLAGM
a
Amino acid sequence in the 1 letter code. Positively charged residues are highlighted in bold and underlined.
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their biological activity (Figure 1). Accordingly, cytotoxicity results from nonspecific interactions involving either membrane perturbations (Matsuzaki 1998; Wu and others 1999; Huang 2000) and/or cytoplasmic translocation followed by interaction with anionic elements such as nucleic acids (Friedrich and others 2000; Brogden 2005). In the case of cell-wall containing microorganisms (for example, Gram-negative bacteria), the external membranes may act as additional barriers. It has been proposed that AMPs first target the external membrane and undergo a self-promoted uptake (Hancock 1997), a process that involves displacement of divalent cations from their binding site on lipopolysaccharides (LPS) in the external membrane by competitor peptides. This leads to increased permeability and access to the cytoplasmic membrane. Hydrophobic peptides, which often aggregate in aqueous media, fail in this competition, resulting in poor bactericidal potency. Due to their nonspecific target and mode of action, the generation of resistance toward these antimicrobial agents was shown to be less likely to occur (Navon and others 2002; Yeaman and Yount 2003). Although most AMPs act by nonspecific mechanisms, they often display some selectivity between different microorganisms, for example, Gram-negative compared with Gram-positive bacteria (Boman and others 1991; Meister and others 1997), fungi preference over other eukaryotic cells (Tailor and others 1997), as well as normal compared with cancerous mammalian cells (Utsugi and others
1991; Perez-Paya and others 1994). The basis for this discrimination appears to be linked to a number of parameters, including lipid composition, membrane fluidity, extent of trans-membrane electric potential (Andreu and Rivas 1998; Matsuzaki 1999; Tossi and others 2000), and peptides self-assembly in solution (Pouny and others 1992; Ghosh and others 1997; Feder and others 2000; Kustanovitch and others 2002; Radzishevski and others 2005; Rydlo and others 2006).
Figure 1 --- Proposed antibacterial mechanism of action of linear AMPs: Unfolded cationic peptides associate with the negatively charged surface of the outer membrane (OM) and neutralize the charge over a patch of the membrane or competitively displace divalent cations from their binding sites on lipoplysacharides (LPS), creating cracks through which peptides can cross the outer membrane. In the periplasmic space (PS), the peptides adhere to negatively charged phospholipids in the cytoplasmic membrane (CM), which induces their amphipathic fold. Insertion of multiple monomers within the membrane lipid core ultimately leads to disruption of the membrane structure and function (Hancock 2001; Rydlo and others 2006).
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Food Preservation
onsumers concern about possible adverse health effects of some food additives has stimulated research of new effective alternatives. The bacteriocins produced by Gram-positive LAB bacteria have been found to be appropriate candidates to fulfill these requirements, mostly due to their natural origin in foods such as fermented dairy and meat products and due to their safety aspects (Cleveland and others 2001; Chen and Hoover 2003; Papagianni 2003). Nisin, a bacteriocin produced by Lactococcus lactis bacteria, was found to be of a particular interest. Discovered in 1928 (Rogers 1928), nisin was initially evaluated as a clinical antibiotic in the 1940s (Hirsch and Mattick 1949). Later, it was found to be suitable for food preservation due to its lethal activity against foodborne pathogens and spoilage microorganisms, inhibition of cell wall biosynthesis and spore outgrowth, and, most of all, its safe use and consumption with no apparent adverse effects (Hurst 1981; Montville and others 1995). So far, nisin is the only purified antimicrobial peptide approved by the U.S Food and Drug Administration for use in particular food products (FDA Federal Register 1988). It is used in a crude extract containing up to 5% of nisin of the solid. Of the different variants of nisin, the most used commercial form is NisaplinTM , which contains 2.5% of the active ingredient (Nisin A), 77.5% NaCl, and 12% nonfat dry milk (Chen and Hoover 2003). Currently it is permitted for use mostly in dairy products (especially cheese) and canned goods (vegetables, soups). In European countries it is also used in baby foods, baked goods, mayonnaise, and milk shakes. Nisin however, displays several shortcomings: low solubility at physiologic pH reduces its activity and limits its use in most cured meat products (Rayman and others 1983), and it is inactive against yeast, molds, and Gram-negative bacteria, unless other processing technologies are used in combination. These technologies include adding chelator agents like EDTA (Cutter and Siragusa 1995, Branen and Davidson 2004), preheating the product (Boziaris and others 1998), or pH reduction (Rayman and others 1983). The partial success of nisin as a food preservative has prompted examination of other bacteriocins. Pediocin PA-1 showed the most promising results (Nielsen and others 1990). But it is not yet an approved food additive in the United States. The use of bacteriocins in food preservation presents serious limitations because of their relatively narrow activity spectra and moderate antibacterial effects. Synthetic peptides have been tested in food products such as apple juice. SAR studies of synthetic model AMPs aiming at understanding their mechanism of action resulted in a 14-residue, long peptide, 8K6L, composed of 8 lysine and 6 leucine residues (Blondelle and Houghten 1992; Haynie and others 1995). The peptide showed bactericidal effect against E. coli O157, reducing its population by 6 log units after 1 h of incubation at the concentration of 50 g/mL in a buffer medium (Appendini and Hotchkiss 1999). A more recent study aimed at using this synthetic peptide as a preservative in food packaging materials demonstrated the ability of the peptide to reduce E.coli O157 population by 3.5 log units after 10-min incubation in citrate buffer (pH 3.5) at a concentration of 5 g/mL (Appendini and Hotchkiss 2000). Testing the bactericidal effect of the peptide in apple juice at 25 C (pH 3.7) revealed only 3.5 log unit reduction after a long incubation period of 8 h at the high peptide concentration of 100 g/mL. As pointed out above, unlike lactobacteria AMPs that inhibit growth of mainly Gram-positive organisms, AMPs that are produced by animal cells often display activity against a much larger specR128 JOURNAL OF FOOD SCIENCEVol. 71, Nr. 9, 2006
Mammalian AMPs
A 26 amino acid peptide derived from the sequence of PR39, a proline-arginine rich polypeptide isolated from porcine neutrophils, was found to be effective in killing E. coli, Salmonella Typhimurium, and Streptococcus suis, at 37 C (Shi and others 1996). The peptide was proposed to penetrate the outer membrane of E. coli, gain entry into the cytoplasm, and thus affect bacterial viability by interfering with DNA and/or protein synthesis (Boman and others 1993; Cabiaux and others 1994). Scanning electron microscopy studies of bacterial cells exposed to PR-26 indicated that the peptide does not lyse cells by pore-forming mechanisms (Shi and others 1996). Its effectiveness against pathogenic strains E. coli O157:H7 and L. monocytogenes was tested at different temperatures (Annamalai and others 2001). Although after 24-h incubation the peptide decreased bacterial populations by 4 and 5 log units at 24 and 37 C, respectively, it had poor activity at the lower temperatures representing normal refrigeration.
Avian AMPs
Two beta-defensins, termed spheniscins, were recently isolated from the stomach content of the king penguin Aptenodytes patagonicus (Thouzeau and others 2003). It has been proposed that in combination with other AMPs, spheniscins may be involved in a longterm preservation of food in the male birds stomach. This 38-residue AMP displayed mainly bacteriostatic effect against Gram-negative bacteria but showed bactericidal effect against most Gram-positive bacteria tested at a concentration range of 0.4 to 15 M and was active against yeast and filamentous fungi at concentrations ranging from 1.5 to >100 M. The peptide showed stable activity at a range of moderate acidity (pH 4 to 6), suggesting that it is not affected by conserving conditions in the stomach. No studies aiming to characterize these peptides in food systems as preservatives have been published to date.
Fish AMPs
Protamine, extracted from fish milt, demonstrated antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, yeasts, and molds (Islam and others 1984; Uyttendaele and Debevere 1994; Johansen and others 1995). This 30 amino acid AMP (of which 66% are arginine) is believed to disrupt the cytoplasmatic membrane by inducing leakage of K+ , ATP , and intracellular enzymes of sensitive cells (Islam and others 1987; Johansen and others 1997; Stumpe and Bakker 1997). In recent studies this AMP was evaluated for its efficacy against L. monocytogenes and E. coli at pH levels ranging from 5.5 to 8 (Hansen and Gill 2000) and at temperatures ranging from 5 to 30 C (Hansen and others 2001). The peptide showed better activity against Gram-negative bacteria, showing similar MIC and MBC values, but was mainly bacteriostatic toward Gram-positive bacteria. In both cases the peptides activity increased at alkaline pH in a medium containing positively charged protein (gelatin A) where the competitive electrostatic interactions between protamine and culture media and bacterial surface were in favor for the latter. Addition of 0.9 mM EDTA was found to be either synergistic to protamine (reduced MIC) or bactericidal, depending on the bacterial strain tested. The effect of temperature on protamine MICs varied greatly among strains, species, and genera. Therefore, no definite conclusion regarding the effect of temperature on bactericidal potency could be made.
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Another fish AMP , pleurocidin, isolated from the edible winter flounder, was active against Gram-positive and Gram-negative bacteria and was heat and salt tolerant (Cole and others 1997, 2000). Pleurocidin was active against foodborne microorganisms at levels well below the legal limit for nisin (10,000IU/g) without significant effect on human red blood cells (Burrowes and others 2004), indicating its potential as a food preservative and a natural alternative to conventional chemicals. It is noteworthy, however, that pleurocidin was inhibited by magnesium and calcium (Cole and others 2000), which may limit the use of this AMP in environments laden with these cations.
Amphibian AMPs
In addition to mammalian-like neuropeptides and hormones, the skin of amphibians also contains a rich arsenal of broad-spectrum, cytolytic AMPs (Nicolas and Mor 1995; Simmaco and others 1998). Prepro-dermaseptins are processed and then stored in the large granules of dermal glands (Nicolas and others 2003) and released onto the skin surface upon stimulation to provide an effective and fast-acting defense against noxious microorganisms (Bevins and Zasloff 1990; Simmaco and 1998). All known amphibian AMPs are linear and able to form amphipathic helical structures in hydrophobic environments (Papagianni 2003). As wide-spectrum microbicides, amphibian AMPs stimulate increasing interest because of their unique characteristics and potential therapeutic usefulness. Two of the most investigated representative frog AMPs, magainins and dermaseptins, are discussed below. Magainins discovery was triggered by the observation that wounded frogs manage to thrive in waters dense with bacteria (Zasloff 1987). Initially, the magainins were isolated from the skin of the African clawed frog Xenopus laevis, but have also been found in their stomach (Moore and others 1991). These 21 to 27 residue AMPs form an amphipathic -helix upon binding to membranes (Marion and others 1988) and displayed a broad spectrum of antimicrobial activity. At micromolar concentrations, they induced lysis of bacteria, fungi, protozoa, and tumor cells (Zasloff and others 1988; Soballe and others 1995). SAR studies yielded a 22-residue peptide termed MSI-78 showing improved potency and broader spectrum of activity, including against multiresistant bacterial species (Ge and others 1999a), and was found to be suitable for development as human therapeutic agent. A formulation based on this peptide has been taken through all phases of clinical trials for topical treatment of infected diabetic foot ulcers (Ge and others 1999b). The bactericidal effect of magainin and its synthetic analogs was investigated against 13 food-related pathogens (Abler and others 1995) displaying MICs ranging from <3 to 50 g/mL. When tested at various temperatures, Mag2 showed reduced potency at 4 and 25 C. Bovine serum albumin, which was selected as a representative food constituent that may interfere with the peptides inhibitory effect, indeed reduced its activity, suggesting that magainin peptides might be employed, during food processing as a final spray or dip for products such as meat and poultry or integrated into primary food packaging materials. The South American hylid frogs of the phyllomedusinae subfamily produce a rich array of AMPs (Vanhoye and others 2003). Several intriguing peptides have been isolated from the skin of these arboreal frogs, most of them derived from a group of precursors collectively known as preprodermorphin/dermaseptin (Amiche and others 1994; 1999; Auvynet and others 2006). Eight members of the dermaseptin S family (Mor and others 1991; Mor and Nicolas 1994a; Chen and others 2003) and 6 dermaseptin B members (Mor and others 1994b; Charpentier 1998) have been isolated and characterized. These linear AMPs often display rapid cytolytic ac-
tivity against a wide range of microorganisms, including viruses (Belaid and others 2002; Lorin and others 2005), Gram-negative and Gram-positive bacteria and filamentous fungi (Mor and others 1991; Mor and Nicolas 1994a; DeLucca and others 1998), protozoa (Hernandez and others 1992), and yeasts (Coote and others 1998). They were also found to possess intracellular antiparasitic activities (Ghosh and others 1997; Krugliac and others 2000; Dagan and others 2002; Efron and others 2002). As shown by circular dichoism, FTIR, and NMR analysis, these peptides are medium sensitive and tend to adopt helical conformations in membrane-mimicking environments (Mor and others 1991; Mor and others 1994b; Kustanovich and others 2002; Shalev and others 2002, 2005; Lequin and others 2003). Their cytolytic activity is typically mediated by interaction of the Nterminal sequence with plasma membrane phospholipids (Hernandez and others 1992; Mor and Nicolas 1994b; Gaidukov and others 2003; Radzishevski and others 2005; Shalev and others 2005). Various dermaseptin derivatives display selective activity (Mor and Nicolas 1994a; Mor and others 1994a; Feder and others 2000, 2001; Krugliac and others 2000; Navon and others 2002). Members from the dermaseptin S family exhibited dramatic synergy in combination, resulting in some cases in a 100-fold increase in activity compared to the activity of the peptides separately (Mor and others 1994a). Dermaseptins efficiently kill slowly growing and nongrowing bacteria, suggesting a potential use in the eradication of bacteria placed in a dormant state and/or subjected to low oxygen tension (Jouenne and others 1998). Dermaseptins are also effective killers of spoilage yeasts and pathogenic food-related bacteria, implying a possible application as food preservatives (Coote and others 1998; Yaron and others 2003; Rydlo and others 2006). Due to its unique molecular structure, dermaseptin S4 was used to unravel structure-function relationships that eventually led to potent derivatives (Mor and Nicolas 1994b; Feder and others 2000; Kustanovich and others 2002; Gaidukov and others 2003; Radzishevski and others 2005; Shalev and others 2005; Rydlo and others 2006). Dermaseptin S4 and it derivatives were subjected to CD and NMR studies correlating the peptides 3-D amphipathic organization with its cytotoxic profile (Mor and Nicolas 1994b; Kustanovich and others 2002; Shalev and others 2005). C-terminus truncated derivatives were investigated as model AMPs for investigating the effect of N-terminal acyl conjugation which resulted in potent and selective derivatives (Radzishevsky and others 2005; Shalev and others 2005). Binding parameter studies of the peptides to model membranes were used to investigate the mode of action and membrane permeating properties of dermaseptins. Assuming that peptide binding to the target membrane proceeds by a 2-stage mechanism (that is, an initial surface adsorption stage, due to electrostatic interaction, followed by insertion into the membrane bilayer, due to hydrophobic interaction), it was shown that the 2nd step is the crucial step leading to cell death (Gaidukov and others 2003; Shalev and others 2005; Rydlo and others 2006). Two recent studies investigated the molecular elements in dermaseptin S4 that are necessary for maintaining antimicrobial potency against E. coli O157:H7, where full length, truncated, and acylconjugated derivatives were examined for their potency, secondary structure, self-assembly state, and binding parameters (Yaron and others 2003; Rydlo and others 2006). The studies emphasize the effect of net charge and nonaggregative hydrophobicity on the peptides activity and point to 3 elements that affect peptide activity under such incubation conditions: peptide-membrane interaction, bacterial stress response, and peptide availability. The outcome of these and similar studies is discussed below in terms of how peptide performance might be affected by incubation conditions.
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Effects of salt
Electrostatic interactions between peptide and negatively charged targets maybe masked at high salt concentrations. Salt might also affect peptide organization in solution in terms of either secondary structure, salting out or induce aggregation, thus limiting its activity. The -helical content of a synthetic bactericidal peptide [RLLR] 5 was reduced from 72% to 13% in the presence of 200 mM NaCl, leading to an 8- to 32-fold decrease in its antimicrobial activity against bacteria and fungi (Park and others 2004). This study also indicated that conjugation of -helix-capping motives at the peptides termini helped maintain its helical structure under salty conditions, resulting in improved activity. In different studies, however, salt supplement actually improved bactericidal activity: Adding up to 300 mM NaCl did not impair cecropin P1 activity against E. coli but progressively led to activity loss for magainin (Lee and others 1997). Activity of nisin against E.coli O157 was achieved at high salt concentrations of 5% irrespective of whether bacteria were precultured in the presence or absence of salt in the incubation medium (Ganzle and others 1999). Salt resistance to -helical cationic AMPs was also reported against P . aeruginosa PAO1 at salt concentrations of 300 mM (Freidrich and others 1999). Unfortunately, none of these studies addressed changes in secondary structure in the presence of high salt concentrations. The bee venom melittin was reported to convert from a monomeric random coil to a helical tetramer as ionic strength was increased from 0 to 500 mM NaCl (Wilcox and Eisenberg 1992). Furthermore, 2 -helix forming peptides produced by gene engineering methods were able to maintain the secondary structure at extremely high salt concentrations up to 1.5 M (Kojima and others 1996). High salt concentration was explained to mask electrostatic repulsion between similarly charged side chains of lysine (in acidic conditions) or glutamic acid (in alkali conditions) on the hydrophilic
Figure 2 --- Effects of incubation conditions on the antimicrobial properties of dermaseptin derivatives on E. coli 0157:H7. Plotted in column A are data obtained after 2 h of incubation with a 28 residue dermaseptin. Columns B and C present data obtained with a 14-residue truncated derivative and its acylated version, respectively, at same peptide concentration (8 M). CFU values represent the mean standard deviations (SD) obtained from two independent experiments performed in duplicates. Bars represent the SD from the mean. Absence of a bar indicates that the SD value is smaller than the symbol size. Zero CFU indicate negative cultures (Rydlo and others 2006). R130 JOURNAL OF FOOD SCIENCEVol. 71, Nr. 9, 2006
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surface of the helix bundle, thus stabilizing the peptide structure. In addition, hydrophobic interaction was strengthened when increasing salt concentration, thus facilitating peptide association. Another study showed that the human cathelicidin, LL-37, converted from random coil in water to -helix in the presence of various salts, including 160 mM NaCl, and that the -helix content correlated the observed antibacterial activity (Johansson and others 1998). The salting out effect explained in this study causes peptide oligomerization observed with increasing helical content. Reducing repulsive forces between positively charged residues along the peptide chain was also explained as an outcome of salt addition, resulting in stabilization of the helix structure. Changes in antibacterial activity of dermaseptin derivatives did not correlate with structural changes as function of salt concentration since the peptides exhibited the highest helical structure at 6% NaCl where activity was significantly reduced (Rydlo and others 2006). Acylation remarkably improved efficacy, enabling the peptide to eliminate large bacterial populations (107 CFU) at high salt concentrations of 1.5% (approximately 0.3 M NaCl) as shown in Figure 2. At 3%, on the other hand, the peptide did not display improved activity. At such high salt concentrations, growth of the control group was also hampered.
tion (Richard and Foster 2004). The pH effect is bound to influence the peptides interaction with bacterial membrane components in a variety of manners. In support of this view, surface plasmon resonance (SPR) experiments performed with dermaseptin derivatives revealed high binding affinity at low pH but a low tendency to penetrate into the membrane inner core (Rydlo and others 2006). A solidstate NMR spectroscopy study also reported histidine-rich peptides to alter their orientation in a model membrane as a function of the surrounding pH, changing from parallel to transmembrane orientations, respectively, at acidic and elevated pH conditions (Bechinger 1996). The pH differences also affected the mode of action by which clavanin A permeabilized Lactobacillus sake membrane (van Kan and others 2002). Whereas the peptide efficiently released fluorophores from unilamellar vesicles at neutral pH according to a nonspecific permeabilization mechanism, it did not permeabilize model bilayers at low pH levels although displaying antimicrobial activity at those conditions. It was therefore suggested that this peptide uses distinct modes of action at acidic and neutral pH.
Effects of pH
Variations in pH can affect the peptides net charge, which in turn affects its secondary structure, binding properties, and cytotoxic activity. Histidine-containing amphipathic helical peptides were reported to exhibit random coil structure at acidic pH levels and typical -helix structure at basic conditions (Vogt and Bechinger 1999). Another histidine-based peptide, clavanin A, showed enhanced activity at acidic conditions while at neutral pH, the uncharged peptide became inactive. Its substituted analog, clavanin AK, having a higher pKa value, exhibited potency at all conditions (Lee and others 1997). The human cathelicidin LL-37 displayed random coil structure at acidic conditions (pH 2 and 3.8) and -helix at neutral and basic pH levels (Johansson and others 1998). The acidic conditions reportedly destabilized ion pairs between acidic and basic side chains due to the protonated state of acidic residues, whereas at basic conditions, repulsive forces between basic side chains were limited due to deprotonation of positively charged residues, leading to helix stabilization. Thus, a peptides pKa value can determine loss or gain of activity as a function of the environmental pH conditions. Dermaseptin S4 derivatives also displayed reduced bactericidal potency against E. coli O157:H7 at acidic conditions of pH 3.6 (Figure 2). A few possible reasons might account for differences in peptide efficacy: Bacterial stress responses may reduce bacterial susceptibility to AMPs either by modifications of the membrane composition, SOS gene expression, or modification of the trans-membrane potential. Several studies revealed that rpoS, a gene involved in regulating the expression of a variety of stress response genes, could be induced in response to a number of inimical processes used in the food industry, including changes in water activity, osmotic shock, and highly acidic conditions (Foster and Spector 1995; Fang and others 1996; Turner and others 2000; Dodd and Aldsworth 2002). Preculture of E. coli in acidic conditions led to resistance to curvacin A (Ganzle and others 1999). A similar outcome was obtained with dermaseptins where bacteria that had been pre-incubated in pH 3.6 and assayed in neutral pH became less susceptible (Rydlo and others 2006). However, the fact that bacterial viability was hampered in absence of peptide may point to stress response adaptation of bacteria being the cause for reduced potency. As part of the stress response, it has been reported that E. coli transmembrane potential decreases significantly at acidic condiURLs and E-mail addresses are active links at www.ift.org
Food Packaging
ntimicrobial packaging has become one of the most interesting and challenging topics in the area of active packaging (AP). In AP , the product, the package, and the environment interact during food preparation and storage, resulting either in an improved product quality and safety and an extended shelf life or in the attainment of some product characteristics that cannot be obtained by any other means (Miltz and others 1995; Yam and others 2005). The most developed AP technology is oxygen scavenging. A reduction of oxygen in a package can inhibit oxidative reactions as well as the growth of microorganisms. However, a reduction in the oxygen concentration in a package to very low levels may encourage the growth of pathogenic anaerobic microorganisms. Antimicrobial packaging overcomes this problem. Several antimicrobial packaging systems have been proposed and reviewed in detail (Appendini and Hotchkiss 2002) where most of them use synthetic additives. The subject of antimicrobial packaging has been reviewed recently in 2 additional articles by Suppakul and others (2003a, 2003b). In recent years, however, the public perception is that synthetic agents may cause side effects and therefore consumers prefer natural over synthetic additives. Miltz and others (2004) have developed an antimicrobial film containing the natural components of basil: linalool and methyl chavicol. This film has shown a significant inhibition of E. coli and Listeria as well as other microorganisms and extended the shelf life of cheddar cheese. Miltz and others (2006) have studied very recently the properties of an antimicrobial coating based
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Concluding Remarks
hile the data collected in this review highlight part of the massive research that is currently being conducted, many signs designate the potential of natural AMPs in food preservation. Future challenges lie in our ability to adapt these extraordinary compounds to perform tasks specifically related to food safety. Better understanding of the mode(s) by which AMPs rapidly eliminate microorganisms should provide solid grounds for engineering new and upgraded derivatives with optimized potency and stability under the range of incubation conditions typical to food. This endeavor is indeed stimulating an increasing number of multidisciplinary studies that tackle these issues by a large variety of strategies: So far, success in significantly reducing the molecular size while increasing resistance to proteases was achieved through SAR studies aiming at optimizing known native peptides or model peptide sequences through conventional and/or combinatorial methods. In the future, the use of biotechnology techniques should further enable mass production of AMPs to minimize their costs. The recent isolation of plectasin, a defensin-like AMP expressed in fungi, clearly represents a breakthrough toward achieving several of these goals. There is therefore little doubt that as progress is made, new derivatives will be designed which will enhance the suitability of these simple yet phenomenal molecules for various antimicrobial applications, including food safety, for their demonstrable ability to escape most of the known resistance mechanisms.
Acknowledgments
The authors gratefully acknowledge the financial support by THE ISRAEL SCIENCE FOUNDATION (grant nr 387/03) and by funds from the Technions Vice President for Research.
References
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