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International Journal of Food Micro

Potential antimicrobials to control Listeria monocytogenes invacuum-packaged cold-smoked salmon pâté and fillets

Hudaa Neetoo, Mu Ye, Haiqiang Chen ⁎

Department of Animal & Food Sciences, University of Delaware, Newark, DE 19716-2150, United States

Received 28 December 2007; received in revised form 31 January 2008; accepted 3 February 2008

Abstract

In the wake of recent outbreaks associated with Listeria monocytogenes in ready-to-eat foods and an increasing desire for minimally processedfoods, there has been a burgeoning interest in the use of natural antimicrobials by the food industry to control this pathogen. The minimum inhibitoryconcentrations (MICs) of nisin and salts of organic acids (sodium lactate (SL), sodium diacetate (SD), sodium benzoate (SB), and potassium sorbate(PS)) against twelve strains of L. monocytogenes in a TSBYE broth medium at 35 °C were determined. The MICs were strain-dependent and fell inthe range of 0.00048–0.00190% for nisin, 4.60–5.60% for SL, 0.11–0.22% for SD, 0.25–0.50% for SB and 0.38–0.75% for PS, respectively. Thetwo most antimicrobial-resistant strains were used as a cocktail in the following experiments to represent a worst case scenario. The fiveantimicrobials alone and in binary combinations were screened for their efficacy against the two-strain cocktail in TSBYE at sub-MIC and sub-legallevels at 35 °C. Seven effective antimicrobial treatments were then selected and evaluated for their long-term antilisterial effectiveness in cold-smoked salmon pâté and fillets during refrigerated storage (4 °C) of 3 and 6 weeks, respectively. The two most effective antimicrobial formulationsfor smoked salmon pâté, 0.25% SD and 2.4% SL/0.125% SD, were able to inhibit the growth of L. monocytogenes during the 3 weeks of storage.Surface application of 2.4% SL/0.125% SD was the most effective treatment for smoked salmon fillets which inhibited the growth ofL. monocytogenes for 4 weeks. These antimicrobial treatments could be used by the smoked salmon industry in the U.S. and Europe in their efforts tocontrol L. monocytogenes as they are effective against even the most antimicrobial-resistant strains tested in this study.© 2008 Elsevier B.V. All rights reserved.

Keywords: Listeria monocytogenes; Smoked salmon; Antimicrobials; Storage; Pâté

1. Introduction

Listeria monocytogenes is a public health concern in manycountries including the United States and European countries.Several ready-to-eat (RTE) foods including smoked fish andpâté (also known as “spread or dip”) have been implicated inthe outbreaks of human listeriosis. In recent years, occurrencesof listeriosis associated with the consumption of these foods(Gilbert et al., 1993; Ericsson et al., 1997; Miettinen et al., 1999)have sparked interest in studying the prevalence of this causative

⁎ Corresponding author. Department of Animal & Food Sciences, 020Townsend Hall, University of Delaware, Newark, DE 19716-2150, UnitedStates. Tel.: +1 302 831 1045; fax: +1 302 831 2822.

E-mail address: haiqiang@udel.edu (H. Chen).

0168-1605/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2008.02.001

agent in smoked fish (Heinitz and Johnson, 1998; Jorgensen andHuss, 1998; Lyhs et al., 1998; Hansen et al., 2006) and pâté(Farber and Daley, 1994; Wilson, 1995; Nichols et al., 1998).

Despite considerable efforts to improve process hygiene andsanitation procedures, the complete elimination of L. mono-cytogenes from processing environments in which RTE foodsare produced is currently considered impossible. This is critical,as typical product characteristics, including pH, water activity(aw), salt, and smoke components, are insufficient to prevent thegrowth of L. monocytogenes in chilled and vacuum-packagedsmoked salmon products (Mejlholm and Dalgaard, 2007).Products with a long chilled shelf-life which are consumedwithout further cooking especially constitute a risk since it hasbeen demonstrated that L. monocytogenes could grow to highlevels during long-term refrigerated storage (Rocourt et al.,

221H. Neetoo et al. / International Journal of Food Microbiology 123 (2008) 220–227

2003; Gimenez and Dalgaard, 2004; Neetoo et al., 2008).Buchanan et al. (1997) inferred that the “initial focus for risk-management decisions should be the prevention of the growthof this pathogen in foods to high levels”. We thus evaluated thegrowth-inhibitory effect of appropriate concentrations andbinary combinations of nisin and salts of organic acids (sodiumlactate (SL), sodium diacetate (SD), sodium benzoate (SB) andpotassium sorbate (PS)). These antimicrobials are GenerallyRecognized as Safe (GRAS).

Nisin is known to inhibit the growth of gram-positivebacteria and its antilisterial effect has been widely reported invarious food products including milk (Bhatti et al., 2004),frankfurters (Luchansky and Call, 2004), cheese (Samelis et al.,2003), pork (Murray and Richard, 1997), salmon (Nilsson et al.,1997) and beef (Avery and Buncic, 1997). SL (2–4%) has beenshown to retard bacterial growth in beef (Maca et al., 1997),pork (Brewer et al., 1991; Lamkey et al., 1991), turkey (Maaset al., 1989) and salmon (Pelroy et al., 1994). Its antilisterialactivity (2–3%) has also been demonstrated in meat withminimal effects on the pH and sensory characteristics of theproduct (Shelef and Yang, 1991; Chen and Shelef, 1992; Pelroyet al., 1994; Houtsma et al., 1996a, b). SD is a flavoring andantimicrobial agent that has also been used in various foods forcontrolling L. monocytogenes (Lungu and Johnson, 2005). Atconcentrations of 0.1 to 0.3%, SD has been shown to inhibit thegrowth of L. monocytogenes in meat (Shelef and Addala, 1994;Islam et al., 2002). SB and PS have also been shown to inhibitthe growth of L. monocytogenes (El-Shenawy and Marth, 1988;Glass et al., 2007; González-Fandos and Dominguez, 2007).The maximum legal limits for use in foods in the U.S. are10,000 IU/g (0.025%) for nisin in RTE foods (Code of FederalRegulations, 2003), 4.8% for SL in meats, 0.25% for SD inmeats (Code of Federal Regulations, 2000), 0.1% for SB infoods (Code of Federal Regulations, 2007), and 0.3% for PS inmeat and fish products (Sofos, 1989). The maximum limitsallowed by the European Commission for these antimicrobialsin foods are: nisin (0.00125% or 500 IU/g for ripened cheeseand processed cheese), PS (0.2%), SB (0.2%), while no upperlimits are imposed for SL and SD (Nordic Council of Ministers,2002).

Typically, it was common to use only one antimicrobial in afood product for preservation purpose. However, in recent years,the use of combined antimicrobials in a food system has becomemore frequent (Schlyter et al., 1993). Combining antimicrobialagents theoretically provides a broader spectrum of activity, withenhanced antimicrobial action against pathogenic and/or spoilageorganisms. It is believed that the combined agents would targetdifferent species of a mixed microbiota, or act on differentmetabolic elements within similar species or strains, which wouldtheoretically result in improved microbial control over the use ofone antimicrobial agent alone (Schlyter et al., 1993).

The goal of this project was to determine the effect of thesefive antimicrobials, when employed alone or in binary combina-tions, on inhibiting the growth of L. monocytogenes. Theefficacy of the antimicrobials was initially screened in a brothmedium and the effective antimicrobial treatments weresubsequently tested in smoked salmon pâté and fillets.

2. Materials and methods

2.1. Bacterial strains

Twelve strains of L. monocytogenes consisting of PSU1,PSU2, PSU9, PSU21, PSU23, ATCC 19115, ATCC 19113,F5069, CA, CCR8, V7, and Scott A were tested. The cultureswere prepared by transferring a single colony of each strainfrom Tryptic Soy Agar supplemented with 0.6% Yeast Extract(TSAYE) into 10 ml of Tryptic Soy Broth plus 0.6% YeastExtract (TSBYE). The cultures were incubated at 35 °C for 24 hand 0.1 ml of the overnight cultures was then transferred into10 ml of fresh TSBYE and incubated at 35 °C for another 24 h.

2.2. Antimicrobial compounds

Antimicrobials evaluated included a commercial product ofnisin (contains 2.5% pure nisin) (Sigma-Aldrich, St. Louis,MO), SL (Fisher Scientific, Hampton, NH), SD (Sigma), SB(Fisher) and PS (Fisher). Stock solutions (0.125% nisin(concentrations of nisin mentioned here and elsewhere representpure nisin concentrations), 30% SL, 12% SD, 10% SB and 12%PS (w/v) were prepared by dissolving appropriate amounts ofthe compounds in sterile 0.02 M acetic acid which weresubsequently filter-sterilized using 0.22 μm filters (MilliporeExpress™ PLUS, Fisher) and stored at 4 °C.

2.3. Minimum inhibitory concentrations (MICs) assay

Culture of each of the twelve strains of L. monocytogenesmentioned above was diluted to approximately 5 log10 CFU/mlin sterile TSBYE and 50 μl of each diluted culture wasdispensed into sterile wells (370 μl capacity) of a 96-well platewith a flat bottom. Serial dilutions of the antimicrobials in0.02 M sterile acetic acid were prepared to cover the range:Nisin (0–0.025%), SL (0–15%), SD (0–0.6%), SB (0–0.5%)and PS (0–1.2%). Aliquots of the antimicrobial dilutions (50 μl)were then added to the wells containing TSBYE. The pH ofTSBYE broths with antimicrobials were determined by mixingbroth and antimicrobial solutions in culture tubes in proportionto the amounts that were used in wells. The pH values of thesolutions were then measured using an Oakton pH meter(Fisher). pH of TSBYE broth prior to antimicrobial additionwas 6.8 and final pH of broth mixtures incorporating nisin, SL,SB, SD and PS were 5.5, 6.0, 5.9, 5.1, 6.2 respectively. Cultureplates were covered with lids to avoid evaporation of waterduring incubation at 37 °C and to prevent contamination of theplates. The MICs were recorded as the lowest concentration thatresulted in no visible turbidity after 24 h incubation (Ukuku andShelef, 1997; Castellano et al., 2001).

2.4. Binary antimicrobial combinations at sub-MIC level

The strains V7 and Scott A, showing the higher resistancetowards all the antimicrobials, were selected for the subsequentexperiments. Binary combinations of the five antimicrobialstogether with individual antimicrobials at sub-MIC levels were

Table 1MIC values of five GRAS antimicrobials for twelve strains of L. monocytogenesin TSBYE broth after 24 h incubation at 35 °C

Strains Antimicrobials (% w/v)

SL SD SB PS Nisin

PSU 1 5.60 0.15 0.37 0.60 0.00095PSU 2 5.60 0.18 0.37 0.60 0.00190PSU 9 5.60 0.18 0.37 0.60 0.00095PSU 21 4.60 0.18 0.37 0.45 0.00190PSU 23 5.60 0.18 0.37 0.60 0.00190ATCC 19115 4.60 0.18 0.37 0.60 0.00048V7 5.60 0.18 0.50 0.75 0.00190CA 5.60 0.18 0.37 0.60 0.00048CCR8 4.60 0.13 0.25 0.38 0.00095F5069 5.60 0.18 0.37 0.60 0.00048ATCC 19113 4.60 0.11 0.37 0.45 0.00048Scott A 5.60 0.22 0.50 0.75 0.00095

SL = Sodium Lactate, SD = Sodium Diacetate, SB = Sodium Benzoate, PS =Potassium Sorbate.

Table 2Growth/No Growth response in the presence of binary combinations ofantimicrobials at sub-MIC levels in TSBYE broth after 24 h incubation at 35 °C

Antimicrobials(% w/v)

Nisin(0.00095)

SL(2.80)

SD(0.11)

SB(0.10)

PS(0.30)

Individual G G G G GNisin (0.00095) ⁎ G G G GSL (2.80) ⁎ ⁎ NG G NGSD (0.11) ⁎ ⁎ ⁎ G NGSB (0.10) ⁎ ⁎ ⁎ ⁎ G

Note: G = growth, NG = no growth,⁎ = not applicable, SL = Sodium Lactate,SD = Sodium Diacetate, SB = Sodium Benzoate, PS = Potassium Sorbate.

222 H. Neetoo et al. / International Journal of Food Microbiology 123 (2008) 220–227

used to evaluate their efficacy against this two-strain cocktail ofL. monocytogenes. Nisin, SL and SD were used at concentrationscorresponding to the half of higher MIC recorded, respectively(0.00095% nisin, 2.8% SL and 0.11% SD). These half-MICconcentrations were lower than their respective legal limits. Sincethe half-MIC concentrations of PS and SB were greater thantheir legal limits, PS and SB were used at 0.3% and 0.1%, theconcentrations corresponding to their legal limits allowed infoods, respectively so that results obtained could be used by theindustry. One ml of each culture of V7 and Scott A strains ofL. monocytogenes prepared as described in Section 2.1 werepooled to form a two-strain cocktail and diluted to approxi-mately 5 log10 CFU/ml in sterile TSBYE. Individual or bi-nary combinations of antimicrobials that were inhibitory toL. monocytogenes were then identified using the well-assaymethod described in Section 2.3. It was found that SB was noteffective at its maximum legally allowed concentration in the invitro assay. Therefore it was not included in the following study.

2.5. Binary antimicrobial combinations at legal and half-legallimits

The cocktail of V7 and Scott A was used and the sameexperimental procedure was followed as described in Sections 2.3and 2.4. Binary combinations of the four antimicrobials togetherwith individual antimicrobials were studied. SL, SD, and PSwereused at their U.S. legal limits (4.8% for SL, 0.25% for SD, and0.30% for PS) and at half of those concentrations. Nisin was usedat 0.00125%and at 0.00250% to represent a sub-MIC and a super-MIC level, respectively. Antimicrobial treatments resulting in nogrowth (NG) and incorporating the lowest overall concentrationswere selected for the subsequent experiment.

2.6. Effect of selected antimicrobial treatments on the growth ofL. monocytogenes in smoked salmon pâté and fillets duringlong-term refrigerated storage

Cold-smoked salmon fillets were thawed at 2±2 °C (b4 °C)for 1 day immediately before use as described by Besse et al.

(2004). Fillets of smoked salmon (pH 6.0, aw 0.908, salt 1.4%,and fat 4.4%) of similar thicknesses (∼4.2 mm) were punchedaseptically into 5.7-cm diameter round discs weighing 10±1 g.Appropriate dilutions of a two-strain L. monocytogenes cocktailof V7-Scott A (100 μl) were then surface-inoculated on one sideof the slices, spread evenly using a hockey stick, and left to dry for5 min before the slices were flipped and inoculated on the otherside to a final level of approximately 103 CFU/g. Smoked salmonpâté was prepared by mixing 50% smoked salmon, 18.75%butter, 27.5% heavy cream, 3% lemon juice, and 0.75% salt (w/w)in a sterile blender. The pâté had pH 5.6, aw 0.906, salt 1.3%, andfat 33%. The pâté samples were inoculated with the V7-Scott Acocktail to a final level of approximately 103 CFU/g and dividedequally into 100 g portions. Various antimicrobial treatments thathad been pre-screened and found effective in Section 2.5 werethen tested in smoked salmon pâté and fillets. Antimicrobialsolutions were spread on each side of the salmon discs (50 μl) ormixed into the 100 g pâté portions by stomaching (200 μl).Aliquots of pâté (10 g) and salmon discs with an approximateweight of 10 g (the exact weights were determined and recorded)were then placed into vacuum pouches and vacuum-sealed(Model Ultravac 225 vacuummachine, Koch Equipment, KansasCity,MO). The pâté and salmon discswere stored at 4 °C for 3 and6 weeks, respectively. The typical shelf-lives for cold-smokedsalmon pâté and fillets are 3 and 3–6 weeks, respectively. At eachweekly sampling time, the contents of each product wastransferred into a sterile stomacher bag, 40 ml of 0.1% peptonewater was added, and the contents were stomached for 2 min.Serial dilutions were made in 0.1% peptone water and spread-plated in duplicate onto TSAYE plates. The plates were incubatedat 35 °C for 3 h to recover injured cells and overlaidwithModifiedOxford agar (Difco) (Kang and Fung, 1999). The plates wereincubated at 35 °C for 48 h. All colonies with black haloes werecounted. Presence of L. monocytogenes in the un-inoculatedsalmon samples was determined by a primary enrichment inUVM broth (Difco Laboratories) and a secondary enrichment inFraser broth (Difco Laboratories) according to the USDAMicrobiology Laboratory Guidebook (USDA/FSIS, 1999) atthe beginning of the experiment.

2.7. Data analysis

Three independent trials were conducted for all experiments.Data were analyzed using Microsoft® Excel® 2004 for Mac

Table 3Growth/no growth response in the presence of binary combinations of antimicrobials in TSBYE broth after 24 h incubation at 35 °C

Antimicrobials (% w/v) Nisin SL SD PS

0.00125 0.00250 2.40 4.80 0.125 0.250 0.15 0.30

Individual 0 G NG G G G NG G GNisin 0.00125 ⁎ ⁎ G NG NG NG NG NG

0.00250 ⁎ ⁎ NG NG NG NG NG NGSL 2.40 ⁎ ⁎ ⁎ ⁎ NG NG G G

4.80 ⁎ ⁎ ⁎ ⁎ NG NG G NGSD 0.125 ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ NG NG

0.250 ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ NG NG

SL, SD, and PS were used at their legal and half-legal limits. Nisin was used at sub- and super-MIC levels. Note: G = growth, NG = no growth,⁎ = not applicable,SL = Sodium Lactate, SD = Sodium Diacetate, SB = Sodium Benzoate, PS = Potassium Sorbate.

223H. Neetoo et al. / International Journal of Food Microbiology 123 (2008) 220–227

(Version 11.3.6.). One way ANOVA was used to comparesignificant differences between treatments.

3. Results

3.1. MIC assay

Table 1 shows the antilisterial activity of the five antimicro-bials represented by MIC values. MIC values spanned the rangeof 0.00048–0.00190% for nisin, 4.6–5.6% for SL, 0.11–0.22%for SD, 0.25–0.50% for SB and 0.38–0.75% for PS. The MICvalues for all the salts of organic acids did not differ significantlyacross strains of L. monocytogenes varying by a maximum of atwo-fold dilution factor while the antilisterial activity of nisinwas clearly more strain-dependent. Despite some variations,strains ATCC 19113 and CCR8 appeared to be most sensitive,and L. monocytogenes strains V7 and Scott A appeared to bemost resistant overall to the antimicrobials.

3.2. Binary antimicrobial combinations at sub-MIC level

Binary combinations of all antimicrobials at sub-MIC levelswere tested against the two-strain composite of L. monocyto-genes of V7-Scott A (Table 2). Several combinations achieved“No Growth” outcomes in the well assay and these effectivebinary mixtures were: 2.8% SL/0.11% SD, 0.11% SD/0.3% PSand 2.8% SL/0.3% PS. These findings showed that althoughrelatively high concentrations of the preservatives were requiredto achieve no growth when employed individually, lowerconcentrations of antimicrobials could be equally effective

Table 4Counts of L. monocytogenes in smoked salmon pâté (initial level 3.1 log10 CFU/g)

Week control SD Nisin/SL SL/SD

(0.25) (0.0025/2.4) (2.4/0.125)

1 5.5±0.5 2.8±0.6 ⁎ 4.4±0.7 3.7±1.12 5.9±0.4 3.4±0.5 ⁎ 3.0±0.5 ⁎ 3.0±0.9 ⁎

3 6.1±0.6 3.0±0.2 ⁎ 5.2±0.7 2.6±0.6 ⁎

The antimicrobial concentrations used were % (w/w). Data are the means of log (C⁎ means statistically significant differences (Pb0.05) between particular treatmen

when used in combinations. Nisin and SB were the only twocompounds that did not produce a “No Growth” response whenthey were combined with the other antimicrobials at their sub-MIC levels. Since SB used at 0.1% was already at its maximumlegal limit, it was considered not effective in the in vitro assayand was removed from the following study. To increase theefficacy of nisin, it was used in the following study at sub- andsuper-MIC levels (0.00125 and 0.0025%) which were stillsubstantially lower than its legal limit of 0.025%.

3.3. Binary antimicrobial combinations at legal and half-legallimits

Nisin, SL, SD, and PS were then combined to form variousbinary combinations (Table 3). The concentrations of SL, SD,and PS were set at their legal limits (high) or half-legal limits(low) while nisin was used at levels of 0.0025% (high) and0.00125% (low). Nisin (0.0025%) and SD (0.25%) were theonly preservatives which were found to inhibit the growth of L.monocytogenes at concentrations ≤ their legal limits whenemployed alone. SL and PS achieved “No-Growth” outcomesonly when present in binary combinations. Although “No-Growth” was achieved with various binary combinationsincorporating the same two antimicrobials at low and highconcentrations, we were most interested in binary combinationsthat produced a “No-Growth” response at their lowest overallconcentrations. The seven combinations highlighted in bold inTable 3 represented concentrations that would hold mostrelevance to food processors. The efficacy of these sevencombinations was then tested in two real food systems.

subjected to different antimicrobial treatments during storage at 4 °C

Nisin/PS Nisin/SD Nisin SD/PS

(0.00125/0.15) (0.00125/0.125) (0.0025) (0.125/0.15)

2.6±0.5 ⁎ 2.8±0.2 ⁎ 4.4±0.4 3.0±0.6 ⁎

3.1±0.3 ⁎ 4.0±0.6 ⁎ 5.1±0.4 3.1±0.2 ⁎

4.1±1.3 4.4±0.8 6.4±0.1 4.0±1.2

FU/g)±one standard deviation.ts and the control.

Table 5Counts of L. monocytogenes in cold-smoked salmon fillets (initial level of 3.2 log10 CFU/g) subjected to different antimicrobial treatments during storage at 4 °C

Week Control SD Nisin/SL SL/SD Nisin/PS Nisin/SD Nisin SD/PS

(0.250) (0.00250/2.40) (2.40/0.125) (0.00125/0.150) (0.00125/0.125) (0.0025) (0.125/0.150)

1 5.2±1.2 3.3±1.3 2.6±1.4 3.0±0.4 2.5±0.5 3.0±1.2 5.2±1.2 4.0±0.92 6.0±1.3 3.0±0.2 2.6±0.4 3.7±1.1 3.4±1.2 3.5±1.9 3.7±1.5 4.9±0.83 7.2±0.6 3.8±1.1 ⁎ 4.7±0.6 ⁎ 3.8±0.7 ⁎ 3.8±1.3 ⁎ 5.3±0.2 6.1±0.8 4.5±0.5 ⁎

4 5.7±1.2 5.2±1.5 4.1±0.5 3.3±1.4 5.7±0.5 3.7±1.9 5.5±1.7 3.0±0.75 6.8±0.4 4.6±1.7 4.2±0.8 5.6±0.5 4.3±1.2 4.8±1.1 6.0±0.2 5.7±0.86 6.7±0.5 3.9±1.0 ⁎ 5.1±1.1 4.8±0.8 4.3±1.1 4.9±0.4 5.7±1.0 5.8±1.0

The antimicrobial concentrations used were % (w/w). Data are the means of log (CFU/g)±one standard deviation.⁎ means statistically significant differences (Pb0.05) between particular treatments and the control.

224 H. Neetoo et al. / International Journal of Food Microbiology 123 (2008) 220–227

3.4. Effect of selected antimicrobial treatments on the growth ofL. monocytogenes in smoked salmon pâté and fillets duringlong-term refrigerated storages

Counts of L. monocytogenes in pâté and fillet samplestreated with various antimicrobials are shown in Tables 4 and 5,respectively. Representative samples of both products had nodetectable L. monocytogenes before inoculation. The meanpopulation of L. monocytogenes on inoculated samples asrecovered just after inoculation was 3.1 log10 CFU/g in pâté and3.2 log10 CFU/g in fillets. L. monocytogenes populations inuntreated (without antimicrobials added) smoked salmon pâtéand fillets grew quickly. After only one week storage, the countsof L. monocytogenes in both products increased by ≥2.0 log10.Application of antimicrobials on these two products inhibited orslowed down the growth of L. monocytogenes. The two mosteffective treatments, 0.25% SD and 2.4% SL/0.125% SD, wereable to inhibit the growth of L. monocytogenes in pâté duringthe 3–week storage. The counts of L. monocytogenes at the endof the storage in these two treatments were more than 3.1 log10CFU/g lower than that in the untreated sample (Pb0.05).For smoked salmon fillets, the most effective treatment was2.4% SL/0.125% SD which was able to inhibit the growth ofL. monocytogenes in smoked salmon fillets for 4 weeks (countswere consistentlyb4 log10 CFU/g). The other effective treat-ments were 0.25% SD and 0.00125% Nisin/0.15% PS whichwere able to inhibit the growth of L. monocytogenes for 3 weeks(counts were consistentlyb4 log10 CFU/g).

4. Discussion

Although the legal limits for application of nisin, SL, SD, SBand PS in foods have already been defined, the MICs andoptimal combinations of these compounds have not been wellestablished and potential variations in resistance among strainsof L. monocytogenes need to be evaluated. This study thereforepurported to determine the MICs of the five antimicrobialsagainst twelve strains of L. monocytogenes and the efficacy ofthese antimicrobials (individually and in binary combinations)against antimicrobial-resistant strains of L. monocytogenes infoods.

The MIC data for the five antimicrobials garnered in ourstudy were in agreement with the findings reported by otherresearchers. L. monocytogenes Scott A and V7 were two highly

nisin tolerant strains and displayed the highest resistance to allantimicrobials overall. Similarly, Benkerroum and Sandine(1988); Ukuku and Shelef (1997) and Castellano et al. (2001)reported Scott A to be one of their most nisin-resistant strains intheir study. In addition, Benkerroum and Sandine (1988)and Ennahar et al. (2000) found L. monocytogenes V7 to beparticularly resistant to nisin. The MIC range of SL obtained(4.6–5.6%) was consistent with the value of 446 mM (∼5.0%)obtained by Houtsma et al. (1996a,b) for L. monocytogenesWAU and DSM 20600. The MIC of SB determined byEl-Shenawy and Marth (1988) at 4 and 21 °C were foundto be 0.2% and 0.3%, respectively. Although they did notdetermine the MIC of SB at 35 °C, it would be logical toassume that at 35 °C the MIC would be higher than 0.3%and comparable to our findings for the MIC range of SB(0.25–0.50%). El-Shenawy and Marth (1988) showed thatthe legal limit of 0.3% PS was not able to inhibit thegrowth of L. monocytogenes in TSB incubated at 35 °C(pH 5.6). The MIC of PS determined by Moir and Eyles(1992) was found to be N0.5% (pH 6). The MIC of PSobtained in our study was strain-dependent and had anaverage value of ∼0.6% (pH 6.2). For SD, a lower MIC(≤0.22%) (TSBYE with pH 5.1) was observed comparedwith the MIC value of approximately 0.5% (BHI brothwith pH of 5.3–6.3) reported by Shelef and Addala(1994). This could be due to the use of different brothswith different pH values. It is known that the antimicro-bial activity of SD increases with decreasing pH. The factthat all compounds studied could inhibit the growth ofthe various L. monocytogenes strains to various extentsdemonstrate that these antimicrobials have a broad spec-trum of antilisterial activity with the ability to target amultitude of strains. The MIC assay also enabled us to pre-liminarily select two highly antimicrobial-resistant strains forsubsequent tests in a broth medium and in foods.

Combining two antimicrobials at their respective sub-MIClevels allowed us to narrow down all possible binary combina-tions to select few which exhibited favorable additive or syn-ergistic interactions between each other. The combinations of2.8% SL/0.11% SD, 2.8% SL/0.3% PS, and 0.11% SD/0.3%PS which produced “No-Growth” outcomes are thought to workadditively or synergistically since 2.8% SL, 0.11% SD, and0.3% PS could not achieve a “No-Growth” response on theirown (Table 2). Therefore, the efficacy of SL, SD, and PS at

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concentrations conforming to the legal limits was subsequentlytested. Nisin was also included in this experiment at a sub-MIC(0.00125%) and a super-MIC level (0.0025%). The treatmentsthat resulted in “No-Growth” incorporating the lowest antimicro-bial concentrations were 0.0025%Nisin, 0.00125%Nisin/0.125%SD, 0.00125% Nisin/0.15% PS, 0.0025% Nisin/2.4% SL, 2.4%SL/0.125% SD, 0.125% SD/0.15% PS, and 0.25% SD (Table 3).These treatments were then tested in smoked fish pâté and smokedsalmon fillets which have different intrinsic food characteristicssuch as pH, aw, fat and salt content. Moreover, pâté has a shortershelf-life (≤3 weeks at 4 °C) compared to cold-smoked salmon(3–6 weeks at 4 °C). Therefore, certain antimicrobials may bemore effective in certain products than others depending on theirmatrices, pH, composition and aw. Final selection ofmost effectiveantimicrobial treatments specific to particular products wastherefore considered important.

Two methods of antimicrobial application on smokedsalmon pâté and fillets were studied: (1) direct addition intothe product formulation for pâté and (2) surface application forsmoked salmon fillets. L. monocytogenes populations in un-treated (without antimicrobials added) smoked salmon pâté andfillets grew quickly while application of antimicrobials onthese two products inhibited or slowed down the growthof L. monocytogenes (Tables 4 and 5). Growth of L. mono-cytogenes during refrigerated storage was not unexpected.The bacterium is known to grow at refrigeration temperature(Tienungoon et al., 2000;Neetoo et al., 2008;Ye et al., 2008). Pâtésamples receiving organic acid salt treatments (0.25% SD, 2.4%SL/0.125%SD or 0.125%SD/0.15% PS) had lower counts by theend of three weeks compared to those incorporating nisin or nisinwith other antimicrobials (Table 4). It is possiblethat the ingredients in pâté provided nisin protection forL. monocytogenes. It is known that presence of fat in food mayhinder the uniform distribution of nisin and render it unavailablefor activity (Jung et al., 1992). Indeed, the high fat content(∼33%) characteristic of pâté could be the main factor adverselyaffecting nisin's antilisterial activity.

Consistent with observations made by Glass et al. (2002), thelevel of antimicrobial agent SD used individually (0.25%) canbe reduced when applied in combination with 2.4% SL (2.4%SL/0.125% SD, Tables 4 and 5). The authors showed that theantimicrobial activity of SL plus SD was enhanced in smokedproducts due to the possible interactions of the antimicrobialswith components provided by the smoking process. Ourfindings also corroborate other studies which have demon-strated effective antilisterial activity of SL or potassium lactatein combination with SD in a dipping solution or as part of theformulation (Barmpalia et al., 2004; Yoon et al., 2004; Schultzeet al., 2006; Geornaras et al., 2006; Vogel et al., 2006; Knightet al., 2007). When used in the formulation either alone ortogether, 2 to 3% SL and 0.125 to 0.25% SD were shown tocontrol the growth of L. monocytogenes in cooked vacuum-packed meats for storage periods ranging from 21–120 days attemperatures ranging from 3–10 °C (Samelis et al., 2001;Mbandi and Shelef, 2002; Stekelenburg, 2003; Barmpalia et al.,2004; Serdengecti et al., 2006). Our results demonstrated thatthese antimicrobials could also be used in smoked salmon pâté

and fillets which have product characteristics different fromRTE meats.

Various investigations have shown that PS has an excep-tional ability for preventing foodborne listeriosis. Sorbates havebeen shown to inhibit or kill L. monocytogenes (El-Shenawyand Marth, 1988; Wederquist et al., 1994; Sofos, 2000) and theantilisterial effect of PS can be enhanced by the addition of nisinsuch that nisin/PS combination has been shown to producemarked listericidal effect (Buncic et al., 1995). Avery andBuncic (1997) demonstrated the listeriostatic and listericidalability of 0.1% PS/0.00025% nisin combination in vitro as wellas on packaged beef at refrigeration temperatures for 4 weeks.We were also able to show that 0.00125% Nisin/0.15% PScould suppress the growth of L. monocytogenes in smokedsalmon fillets over three weeks storage at 4 °C (Table 5).

Based on the results of this study, it can be concluded that directaddition of either 0.25% SD or 2.4% SL/0.125% SD in smokedsalmon pâté formulation can provide an effective control ofL. monocytogeneswithin the commercial shelf-life of the productat 4 °C. Post-process surface application of 2.4% SL/0.125% SDmay also constitute a measure to enhance the safety of refrigeratedvacuum-packaged cold-smoked salmon fillets with a recom-mended shelf-life of four weeks at 4 °C. These two antimicrobialformulations can be utilized by the smoked salmon industry in theU.S. and EuropeanUnion (EU) since the concentrations of SL andSD are within the legal limits of the U.S. and EU.

Natural antimicrobials have great potential usefulness to thefood industry. Effective application however entails preliminaryresearch of the efficacy of these antimicrobials againstappropriate strains of the target organism, appropriate assaysto determine right concentrations for use as well as testing inrelevant foods having specific intrinsic food characteristics(Vigil et al., 2005). The results of this study would assist thesmoked salmon industry in its efforts to meet the regulatoryrequirements for control of L. monocytogenes. From a practicalstandpoint, the levels and conditions of use of the antimicrobialstested in this study must be treated with caution as theseconcentrations need to be validated in the relevant productshaving their specific formulations or process conditions. Inaddition, the effect of extrinsic factors such as storage tem-perature must not be disregarded.

Acknowledgments

This publication is the result of research sponsored, in part,by NOAA Office of Sea Grant, Department of Commerce,under Grant No. NA050AR4171041 (Project No. R/CT-1)University of Delaware Sea Grant and a grant from the NationalFisheries Institute Fisheries Scholarship Foundation.

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