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obiology 122 (2008) 8–15www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micr

Use of nisin-coated plastic films to control Listeria monocytogenes onvacuum-packaged cold-smoked salmon

Hudaa Neetoo a, Mu Ye a, Haiqiang Chen a,⁎, Rolf D. Joerger a,Doris T. Hicks b, Dallas G. Hoover a

a Department of Animal & Food Sciences, University of Delaware, Newark, DE 19716-2150, United Statesb Department of Marine & Earth Studies, University of Delaware, DE 19958, United States

Received 1 September 2006; received in revised form 8 August 2007; accepted 12 November 2007

Abstract

Cold-smoked (Salmo salar) salmon samples were surface-inoculated with a cocktail of three nisin-resistant strains of L. monocytogenes (PSU1,PSU2 and PSU21) to a level of approximately 5×102 or 5×105 CFU/cm2 of salmon surface. The inoculated smoked salmon samples were vacuum-packaged with control film (no nisin) or nisin-coated plastic films and stored at either 4 or 10 °C. When the inoculated smoked salmon samples werepackaged with film coated with 2000 IU/cm2 of nisin, a reduction of 3.9 log CFU/cm2 (compared with control) was achieved at either temperature forsamples inoculated with 5×102 CFU/cm2 of L. monocytogenes after 56 (4 °C) and 49 (10 °C) days of storage while reductions of 2.4 and 0.7 logCFU/cm2 were achieved for samples inoculated with a high level of L. monocytogenes (5×105 CFU/cm2) after 58 (4 °C) and 43 (10 °C) days,respectively. For samples packaged in film coated with 500 IU/cm2 of nisin, reductions of 0.5 and 1.7 log CFU/cm2 were achieved for samplesinoculated with a low level of L. monocytogenes (5×102 CFU/cm2) after 56 (4 °C) and 49 (10 °C) days of storage while reductions of 1.8 and 0.8 logCFU/cm2 were achieved for samples inoculated with high level of L. monocytogenes after 58(4 °C) and 43 (10 °C) days, respectively. In addition,nisin inhibited the proliferation of background microbiota on smoked salmon in a concentration-dependent manner at both storage temperaturesalthough the bacteriostatic effect was more pronounced at refrigeration temperature. This work highlights the potential for incorporating nisin intoplastic films for enhancing the microbial safety of smoked salmon as well as controlling its microbial spoilage.© 2007 Elsevier B.V. All rights reserved.

Keywords: Nisin; Listeria monocytogenes; Smoked salmon; Antimicrobial packaging; Storage

1. Introduction

Listeria monocytogenes has been isolated from a variety ofseafoods, such as fresh and cold-smoked salmon, on aregular basis (Jinneman et al., 1999; Rorvik, 2000). BenEmbarek (1994) reported a contamination rate of 6–36% inready-to-eat cold-smoked salmon and cooked fish products.Jørgensen and Huss (1998) reported a contamination rate of34–60% in cold-smoked fish and 4–12% in heat-treated andcured seafoods. The organism was isolated from 3.3% of

⁎ 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 © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2007.11.043

ready-to-eat (RTE) raw seafood samples in Japan and 54% offresh rainbow trout purchased on the retail markets in theUSA (Draughon et al., 1999; Inoue et al., 2000). In a surveyconducted by Gombas et al. (2003), L. monocytogenes wasfound in 4.31% of smoked seafood in Maryland andCalifornia in the years of 2000 and 2001.

L. monocytogenes is a Gram-positive, food-borne pathogenthat is widely distributed in the environment and occursnaturally in many raw foods. It is psychrotrophic, halotolerant,and can grow in the temperature range of 1 to 45 °C and NaClrange of 0 to 10% (Seelinger and Jones, 1986). As aconsequence it may grow in many refrigerated food productswith extended shelf-lives (Barakat and Harris, 1999; Rorviket al., 1991). RTE products such as cheeses, meat and fish, andother delicatessen products, may contain L. monocytogenes,

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and many of these foods have been linked to listeriosis(McLauchlin, 1997).

The ingestion of high numbers of L. monocytogenes is asignificant threat to health for people in risk groups such asthe immuno-compromised, elderly, and pregnant women.Ingestion of L. monocytogenes by pregnant women presentsa danger to fetuses and newborns. In these groups, mortalityfrom listeriosis is high, typically 20 to 30% (McLauchlin,1997). Because L. monocytogenes can be readily isolatedfrom smoked fish and because inoculation trials havedemonstrated significant growth in such products, the riskof listeriosis from consuming these types of products must betaken seriously. Hence the challenge for the smoked salmonindustry is not only to limit contamination of the end productwith L. monocytogenes as much as practically possible, but alsoto provide a control strategy that suppresses the pathogen'sgrowth. This goal is difficult to achieve with the current levels ofsmoke and salt commonly used in salmon processing, as they aretoo low to exert a major inhibitory effect on the growth ofL. monocytogenes (Phillips, 1996).

A growing consumer demand for natural and minimallyprocessed foods that mandate alternative preservationtechnologies to assure microbial food safety has sparkedheightened interest in the use of nisin. The application ofnisin in the preservation of fish and shellfish products hasbeen investigated. Nilsson et al. (1997) studied the effect ofnisin on the growth of L. monocytogenes both in an in vitromodel, as well as in trials with cold-smoked salmon, anddemonstrated that growth of L. monocytogenes can beprevented by a combination of carbon dioxide, nisin, NaCl,and low temperature. Szabo and Cahill (1999) assessed theeffect of nisin on the growth of L. monocytogenes on smokedsalmon packaged under vacuum or 100% CO2. Smokedsalmon slices were inoculated with L. monocytogenes andsurface-treated with nisin. Nisin reduced the growth ofL. monocytogenes on vacuum-packaged smoked salmon. Thegrowth of L. monocytogenes was completely inhibited bypackaging the nisin-treated smoked salmon in 100% CO2.

Additionally, nisin has been incorporated into packagingfilms to control the growth of L. monocytogenes. Forexample, Bower et al. (1995) demonstrated that nisinadsorbed onto silanized silica surfaces inhibited the growthof L. monocytogenes. In contrast, surfaces contacted withfilms of heat-inactivated nisin allowed L. monocytogenes togrow. Franklin et al. (2004) coated nisin onto a commercialbarrier plastic film and used it to vacuum-package hot dogsinoculated with L. monocytogenes. L. monocytogenes countson hot dogs packaged in films coated with 7500 IU/ml nisindecreased by greater than 2 log CFU per package throughout60 days of storage at 4 °C, while the counts ofL. monocytogenes in the control package increased from 5 to9 log CFU per package.

The objective of this study was to use a plastic film coatedwith nisin to inhibit the growth of L. monocytogenes andbackground microbiota on the surface of cold-smokedsalmon stored at refrigeration (4 °C) and abuse temperatures(10 °C).

2. Materials and methods

2.1. Selection of most nisin-resistant strains of L. monocytogenes

2.1.1. CulturesVariation in resistance to nisin among strains of

L. monocytogenes has been demonstrated (Benkerroum andSandine, 1988; Castellano et al., 2001) and it is logical to usethe most nisin-resistant strains as a worst case scenario.Twelve strains of L. monocytogenes were tested for theirsensitivity to nisin. The strains were ATCC 19115, ATCC19113, CCR8, CA, F5069, V7, PSU1, PSU2, PSU9, PSU21,PSU23, and Scott A. All strains were maintained on trypticsoy agar plus 0.6% yeast extract (TSAYE) (Difco Labora-tories, Sparks, MD) plates and stored at 4 °C. Before eachuse, they were transferred to 10 ml of tryptic soy broth plus0.6% yeast extract (TSBYE) (Difco Laboratories) andincubated 16 h at 35 °C.

2.1.2. Nisin standardsTo prepare a nisin concentration of 10000 IU/ml, 1 g of

nisin (Sigma-Aldrich, St. Louis, MO) was placed in a 100-mlvolumetric flask and 0.02 M hydrochloric acid was added tothe 100-ml mark. The nisin solution was then diluted two-fold with 0.02 M hydrochloric acid to obtain nisinconcentrations of 5000, 2500, 1250, and 625 IU/ml.Hydrochloric acid (0.02 M) was used as a control.

2.1.3. Sensitivity testingThe agar well diffusion assay technique previously

described by Tramer and Fowler (1964) was used to measurethe susceptibility of the various strains to nisin. Briefly,20 ml of TSAYE was poured onto each petri dish andallowed to solidify. Each agar plate was then overlaid with8 ml of semi-soft TSBYE agar (0.5% agar) containingapproximately 106 CFU/ml of L. monocytogenes. Plates wererefrigerated for 3 h and allowed to solidify. Six wells werebored on each plate using Pasteur pipettes. Fifty microliter ofthe nisin standard solutions prepared in the previous step(0, 625, 1250, 2500, 5000, 10000 IU/ml) were thendispensed into the wells. The plates were refrigerated for24 h to allow diffusion of nisin and subsequently incubatedat 35 °C for 16 h or until inhibition zones were evident. Theinhibition zone diameters were measured using a digitalcaliper (VWR International, Bridgeport, NJ). Based on thediameters of the zones of inhibition measured, three mostnisin-resistant strains, PSU1, PSU2, and PSU21, wereselected for the storage study.

2.2. Evaluation of the effect of nisin-coated plastic film oncontrolling growth of L. monocytogenes and natural microbiotaon the surface of cold-smoked salmon

2.2.1. Experimental designA 3×3×2 factorial design was used for collecting data

totaling 18 treatments. Three nisin concentrations (0, 500, and2000 IU/ml), three inoculum levels (0, 5 × 102 and

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5×105 CFU/cm2 of salmon surface) and two storagetemperatures (4 and 10 °C) were investigated during eachtrial. The experiment was replicated three different timesand, for each replicate, single samples were analyzed induplicates on each sampling day.

2.2.2. Preparation of nisin-coated plastic (LDPE) filmPreliminary study indicated that it was impossible to coat

nisin directly onto plastic films of LDPE, and therefore acoating solution was needed as a carrier for nisin. Thecoating solution was prepared by mixing 1.4 g ofMethylcellulose (MC) (Sigma-Aldrich, St. Louis, MO) and0.6 g of Hydroxypropyl Methylcellulose (HPMC) (Sigma-Aldrich) in 40 ml of 95% ethanol (Fisher Scientific,Hampton, NH) until completely dissolved, followed by theaddition of 40 ml of sterile distilled water. Subsequently,1.2 ml of polyethylene glycol 400 (Fisher Scientific), aplasticizer, was added to the mixture. Once a homogeneousmixture was obtained, 1 g (for film containing 500 IU ofnisin per cm2 film surface) or 4 g (for film containing2000 IU of nisin per cm2 film surface) nisin (Sigma-Aldrich)was dissolved in 20 ml of 0.02 M acetic acid (FisherScientific) and the resulting nisin solution was well mixedwith the coating liquid mixture (Franklin et al., 2004;Cooksey; 2005). Air bubbles in the coating solution wereremoved using a vacuum-packaging machine (Model Ultra-vac 225 with digital control panel, Koch Equipment, KansasCity, MO).

A low-density polyethylene (LDPE) plastic film (DuPontCompany, Wilmington, DE) was taped to 20×20 cm glassplates, and the coating solution was cast onto the film using athin-layer chromatography plate coater (TLC, CAMAG,Muttenz, Switzerland). The volume of the coating solutionwas enough to coat five film-lined glass plates. The coatedfilms were then air-dried at room temperature overnight.

2.2.3. Inoculation of smoked salmon samplesA composite consisting of the three most nisin-resistant

strains, L. monocytogenes PSU1, PSU2, and PSU21 wasprepared. Each strain was grown separately in TSBYE for24 h at 35 °C and 100 μl of each overnight culture wastransferred to fresh TSBYE broths for another 24-hincubation. On the day of the experiment, a 1-ml volumeof each culture was pooled to provide a cocktail of the threestrains. Dilutions in 0.1% peptone water (Difco Laboratories)were made to obtain inocula of ca. 108 CFU/ml and105 CFU/ml. Dilutions were plated on TSAYE plates andincubated at 35 °C for 24 h to determine cell numbers.

Freshly processed cold-smoked salmon (Salmo salar)samples were obtained from a producer. They were keptfrozen at −20 °C and thawed at 2±2 °C (b4 °C) for 1 dayimmediately before use as described by Besse, Audinet,Beaufort, Colin, Cornu and Lombard (2004). Slices ofsmoked salmon were punched aseptically into 5.72-cmdiameter round pieces weighing 10±1 g. The samples weresurface-inoculated with 125 μl of a 105 or a 108 CFU/mldilution of the three-strain cocktail of L. monocytogenes to

achieve final concentrations of 5 × 102 CFU/cm2 or5×105 CFU/cm2 of salmon surface. Control and nisin-coated (500 and 2000 IU/cm2) LDPE films were wrappedaround the inoculated and un-inoculated smoked salmonsamples. The wrapped samples were then inserted into 3-mmthick high barrier pouches (nylon/polyethylene, KochSupplies, Kansas City, MO) and subsequently sealed usinga vacuum-packaging machine (Model Ultravac 225 withdigital control panel, Koch Equipment, Kansas City, MO).The samples were stored at either 4 or 10 °C.

2.2.4. Microbial enumeration of inoculated samplesInitially, 10 °C samples were analyzed every 2–3 days

while 4 °C samples were analyzed every 5 days, and laterduring the study, samples were analyzed according toinoculation level. Low inoculation samples (5×102 CFU/cm2) were analyzed every 7 days while high inoculationsamples (5×105 CFU/cm2) were analyzed every 5 days. Formicrobial analysis, smoked salmon samples were individuallyplaced in stomacher bags that contained 40 ml of 0.1% sterilepeptone water and stomached for 2 min. Serial dilutions weremade in 0.1% peptone water, and counts of L. monocytogeneswere determined by an overlay method (Kang and Fung,1999). Briefly, the serial dilutions were spread plated onsolidified TSAYE agar plates and the plates were incubated at35 °C for 3 h. Approximately 7 ml of modified Oxfordmedium (Difco Laboratories) at 45 °C was overlaid on theTSAYE plates. The plates were incubated at 35 °C for 48 hand black colonies on the plates were counted. Occasionally,colonies were confirmed to be L. monocytogenes using aBAX™ for Screening/L. monocytogenes PCR assay (Quali-con-DuPont, Wilmington, DE).

The numbers of L. monocytogenes per cm2 of salmon werecalculated by dividing the total count of L. monocytogenes persalmon disc by the total surface area (51.4 cm2). The absenceof L. monocytogenes in the smoked salmon samples wasconfirmed by a primary enrichment in UVM broth (DifcoLaboratories) and a secondary enrichment in Fraser broth(Difco Laboratories) according to the USDA MicrobiologyLaboratory Guidebook (USDA, 2005).

2.2.5. Microbial enumeration of un-inoculated samplesUn-inoculated samples were analyzed for L. monocyto-

genes, aerobic, anaerobic, lactic acid bacteria (LAB), andyeast and mold counts. Counts of L. monocytogenes weredetermined using the overlay method as described above.Black colonies were confirmed as L. monocytogenes by thePCR method. Anaerobic bacteria counts were determinedon Liver Veal Agar (Difco Laboratories) plates incubated inanaerobic jars with Gas Paks (BBL) at 35 °C for 2 days.Aerobic bacteria counts were determined by plating ontoTSAYE plates while LAB counts were performed in pour-plates (double layer) in MRS agar (Difco Laboratories). TheTSAYE and MRS plates were incubated aerobically at35 °C for 2 days. Yeast and mold counts were performed onPotato Dextrose Agar (Difco Laboratories) after aerobicincubation at 35 °C for 3 days.

Table 1Sensitivity variations of twelve strains of L. monocytogenes to nisin

Strains Nisin concentrations (IU/ml) y-coordinate

Slope

625 1250 2500 5000 10000

PSU 1 8.00±0.41 9.58±1.05 10.73±0.98 12.52±0.56 14.10±0.23 8.0 5.0PSU 2 7.94±0.85 9.88±1.55 11.42±1.50 12.64±1.67 13.88±1.76 8.2 4.9PSU 21 6.60±1.05 9.14±1.09 11.51±1.06 13.80±1.09 15.35±0.83 6.8 7.4PSU 23 8.63±0.69 11.83±0.65 14.30±0.50 16.05±0.80 17.57±0.96 9.3 7.3PSU 9 9.39±1.61 9.59±1.97 11.11±1.84 13.16±1.62 14.32±1.49 8.8 4.5V7 9.26±1.01 10.70±1.46 12.81±1.19 14.44±1.23 15.60±1.28 9.3 5.5CA 8.62±0.54 11.06±1.47 13.27±1.38 14.81±0.97 16.09±0.66 9.0 6.2CCR8 8.77±2.08 10.43±1.47 11.69±1.81 12.30±2.55 14.11±2.54 9.0 4.2F5069 8.29±0.71 9.80±0.71 10.93±0.60 12.63±0.62 14.22±0.66 8.2 4.9ATCC 19113 20.76±0.26 22.07±1.33 23.63±1.05 25.36±1.22 27.09±0.68 20.6 5.3ATCC 19115 9.28±1.83 10.97±2.11 12.76±1.81 13.98±1.67 15.35±1.53 9.4 5.0Scott A 10.09±1.07 12.61±0.91 14.50±0.47 15.54±0.55 16.83±0.48 10.6 5.5

Data are the means of diameter of inhibition zone±one standard deviation (mm). The y-coordinates were calculated with nisin concentration at 625 IU/ml.

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2.3. Statistical analysis

The counts of L. monocytogenes were converted to logCFU/cm2 and analyzed by a two-way ANOVA procedure usingthe Graphpad Software Prism 4.0 (GraphPad Software, Inc.,

Fig. 1. Effect of nisin concentration on LDPE film, inoculation level, and storage tempera(PSU 1, PSU 2 and PSU 21) on cold-smoked salmon. Samples were inoculated with lowat 4 or 10 °C. (A) low inoculation level and 4 °C; (B) low inoculation level and 10 °C; (C)represent±1 standard deviation.

San Diego, CA). Nisin concentrations, storage temperatures,and storage times were considered principal factors. Differ-ences in mean log CFU/cm2 among treatments were deter-mined using Bonferroni's test (α=0.05) (GraphPad Software,Inc., San Diego, CA).

ture on the growth of a composite of three nisin-resistant strains ofL. monocytogenes(5×102 CFU/cm2) or high (5×105 CFU/cm2) levels of L. monocytogenes and storedhigh inoculation level and 4 °C; and (D) high inoculation level and 10 °C. Error bars

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3. Results

The nisin resistance of the twelve strains was assessed using amodified method of Wolf and Gibbons (1996). The inhibitionzone sizes and the corresponding nisin concentrations used in thewell assay are shown in Table 1. The zone sizes were plottedversus the log of nisin concentrations (figure not shown). Thediameter of inhibition zones had an almost linear relationshipwith log (nisin concentration). Therefore, the data points werefitted with linear regression equations and the y-coordinates atthe smallest nisin concentration of 625 IU/ml and slopesobtained from these equations are shown in Table 1. A larger

Fig. 2. Effect of nisin concentration on LDPE film and storage temperature on the gaerobic, anaerobic, and LAB counts at 4 °C while B, D, and F show the counts for

slope indicated that a given increase in nisin concentrationresulted in a larger change in the inhibition zones. The size ofthe y-coordinate at 625 IU/ml of nisin of the regression lines isalso indicative of the sensitivity of the strains to nisin (Wolf andGibbons, 1996). Hence, the most nisin-resistant strains werethe ones with the smallest y-coordinate at 625 IU/ml of nisinand slope. Taking both the y-coordinate at 625 IU/ml of nisinand slope into consideration, the PSU1, PSU2, and PSU21were the most nisin-resistant strains.

Samples of smoked salmon obtained from the producer hadno detectable L. monocytogenes before inoculation. Counts ofL. monocytogenes on smoked salmon samples packaged in

rowth of background microbiota on cold-smoked salmon. A, C, and E show thesamples stored at 10 °C. Error bars represent±1 standard deviation.

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plain and nisin-coated LDPE films and stored at 4 or 10 °C areshown in Fig. 1. LDPE film (no nisin) allowed L. monocytogeneson smoked salmon to grow rapidly, especially at higher storagetemperatures. The populations of L. monocytogenes reached N7.0log CFU/cm2 after 16 days storage at 4 °C and 6 days of storage at10 °C, respectively, for the low inoculation level. For the highinoculation level, counts reached N7.0 log CFU/cm2 after 6 daysstorage at 4 °C and 3 days storage at 10 °C. Nisin-coated filmsslowed down or inhibited the growth of L. monocytogenes. Theoverall trend for both inoculation levels and storage tempera-tures showed that counts for samples packaged in film coatedwith 2000 IU/cm2 of nisin were consistently lower than thosefor samples packaged in film coated with 500 IU/cm2 of nisinwhich in turn were lower than those for samples packaged incontrol film.

For salmon samples inoculated at 500 CFU/cm2 level andstored at 4 °C (Fig. 1A), LDPE film coated with 2000 IU/cm2 ofnisin significantly inhibited the growth of L. monocytogenes onthe surface of smoked salmon (Pb0.05) through 56 days ofstorage compared with the control film without nisin. Filmscoated with 500 IU/cm2 of nisin allowed L. monocytogenes togrow although the growth was slower than that for controlfilm. Similarly, for smoked salmon samples inoculated at500 CFU/cm2 level and stored at 10 °C (Fig. 1B), films con-taining 2000 IU/cm2 of nisin significantly suppressed growthof L. monocytogenes throughout the 49 day-storage comparedwith the control film. Although the counts for the samplespackaged in film coated with 500 IU/cm2 of nisin were con-sistently lower than the control samples, the degree of inhi-bition was marginal. These results indicated the importance ofnisin concentration for inhibiting L. monocytogenes on thesurface of smoked salmon.

For smoked salmon samples inoculated at 5×105 CFU/cm2

and stored at 4°C (Fig. 1C), film incorporating 2000 IU/cm2 ofnisin significantly inhibited growth of L. monocytogenes whencompared to the control samples (Pb0.05) and achieved areduction of 2.4 log CFU/cm2 (compared to the control) by theend of the trial. Film incorporating 500 IU/cm2 nisin alsohindered the growth of L. monocytogenes on salmon althoughthis film was not as effective as films coated with 2000 IU/cm2.For samples inoculated at a level of 5×105 CFU/cm2 and stored at10°C (Fig. 1D), nisin (500 or 2000 IU/cm2) did not significantlyinhibit any growth of L. monocytogenes on salmon (PN0.05)although counts were consistently lower for nisin-treated samplescompared to control samples throughout the 43-day study.

A two-way ANOVA analysis of the growth data forL. monocytogenes under the various combinations of inoculaand nisin levels at each storage temperature showed that storagetemperature was not a significant factor affecting bacterialgrowth (PN0.05); however, in all instances, counts for smokedsalmon samples stored at 4 °C were lower than those of theircounterparts stored at 10 °C. This contradiction was probablydue to the large variations associated with this kind of growthstudies in foods. The growth of L. monocytogenes at 4 °C wasnot unexpected since the bacterium is known to grow atrefrigeration temperatures (Berrang et al., 1989; Beuchat andBrackett 1990; Barbosa et al., 1994).

Un-inoculated samples always tested negative forL. monocytogenes after plating on TSAYEwithModified OxfordOverlay. Occasional black colonies detected onMOX plates weretested negative when confirmed by PCR. Primary and secondaryenrichments of un-inoculated smoked salmon samples in UVMand Fraser Broths respectively did not reveal the presence ofL. monocytogenes. There was also no detectable growth ofyeasts and molds on the PDA plates (b1 CFU/cm2). The changesin aerobic, anaerobic and presumptive LAB counts in smokedsalmon stored at 4 and 10 °C are summarized in Fig. 2A–E. Thepopulations of aerobes in control (no nisin) samples grew steadilyover timewhen stored at either 4 or 10 °C (Fig. 2A and B). Countsreached N7.0 log CFU/cm2 after 28 days of storage at 4 °C or after11 days of storage at 10 °C. At both storage temperatures, nisinsuppressed the growth of aerobic bacteria to a slightly higherdegree at 2000 than at 500 IU/cm2. At 4 °C, counts for samplespackaged in film containing 500 IU/cm2 of nisin reached ≥7.0log CFU/cm2 after 49 days of storage at 4 °C, while counts forsalmon samples packaged in film containing 2000 IU/cm2 of nisinnever reached 7.0 log CFU/cm2 during 56 days of storage at 4 °C.At 10 °C, counts reached N7.0 log CFU/cm2 after 18 and 33 daysof storage for 500 and 2000 IU/cm2 treated samples, respectively.

Anaerobic counts for untreated samples also proliferatedrapidly and reached N7.0 log CFU/cm2 after 28 days of storage at4 °C and after 11 days of storage at 10 °C (Fig. 2C and D). At bothtemperatures, anaerobic counts for 2000 IU/cm2 samples wereconsistently lower than those of 500 IU/cm2 samples which inturn were lower than the control samples although the differenceswere not found to be significant at most of the time points(PN0.05). At 4 °C, counts reached 7.2 and 6.7 log CFU/cm2 after56 days of storage for 500 and 2000 IU/cm2 nisin-treated sampleswhile at 10 °C, anaerobic counts reached≥7.0 log CFU/cm2 after28 days of storage for 500 IU/cm2 nisin-treated samples and after33 days for 2000 IU/cm2 nisin-treated samples, respectively.

Presumptive LAB counts in the control samples without nisinreached N7.0 log CFU/cm2 after 28 days of storage at 4 °C and11 days of storage at 10 °C, respectively (Fig. 2E and F). Nisin-coated films inhibited the growth of LAB. This inhibition effectwas higher at lower storage temperature. At 4 °C, nisin achievedgreater inhibition at 2000 IU/cm2 than at 500 IU/cm2 during thefirst 42 days of storage. Beyond 42 days, counts for samplesincorporating low and high levels of nisin were almost identical.Counts for nisin-treated samples (both levels) reached above 6.0log CFU/cm2 after 56 days of storage at 4 °C. For samplestreated at either nisin concentration and stored at 10 °C, thecounts reached N7.0 log CFU/cm2 after 16–18 days of storage.

4. Discussion

In this study, the effect of storage temperature, nisin concen-tration on LDPE film, and inoculation level on the growth andsurvival of L. monocytogenes was investigated. At 4 °C (lowand high inoculum levels) and 10 °C (low inoculum level), itwas found that the degree of inactivation or growth inhibitionof L. monocytogenes was directly related to the concentrationof nisin; however, for samples inoculated with high levels ofL. monocytogenes and stored at the abusive temperature of

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10 °C, neither nisin concentration was adequate in controllingproliferation of the pathogen. Moreover, storage at 4 °C al-lowed slower growth of L. monocytogenes than at 10 °C re-gardless of the inoculation level or the nisin concentration onthe films. Therefore combination of refrigeration temperature(4 °C) storage in the presence of packaging film incorporating2000 IU/cm2 nisin was found to be the most effective of thetreatment conditions for limiting the growth of L. mono-cytogenes on smoked salmon. The fact that nisin delayed thegrowth of L. monocytogenes populations in smoked salmon atboth low and high inoculum levels show that nisin might beused to control post-processing contamination of L. mono-cytogenes in cold-smoked salmon.

Incorporation of nisin into packaging films to preserve themicrobial quality of smoked salmon was also studied. Coating ofLDPE film with nisin led to varying degrees of inhibition of thevarious spoilage microorganisms (aerobes, anaerobes and LAB).For all three categories of spoilage organisms, it was found thatnisin inhibited their growth in a concentration-dependent fashion.The nisin-coated films (500 and 2000 IU/cm2) suppressed thegrowth of spoilage microflora during storage at both temperatures;however, the extent ofmicrobial suppressionwas higher in samplesincorporating nisin at a level of 2000 compared to those at 500 IU/cm2. Growth inhibition was also more prominent at 4 °C than at10 °C at either nisin concentration. Therefore the treatmentcombination that resulted in highest degree of inhibition wasvacuum-packaging of smoked salmon in 2000 IU/cm2 andsubsequent storage at 4 °C.

The survival of L. monocytogenes on smoked salmon wasdependent on the concentration of nisin and film incorporating theantimicrobial at a level of 2000 IU/cm2 significantly suppressed itsgrowth at 4 °C. Hence this study demonstrates the potential for theapplication of nisin-coated films onto vacuum-packaged cold-smoked salmon in order to overcome the problems associated withpost-process contamination of L. monocytogenes. Nisin alone didnot achieve a satisfactory degree of inhibition of spoilageorganisms, although there were initial reductions in the numbersof aerobes, anaerobes and lactic acid bacteria, counts eventuallyreached levels comparable to those of untreated samples afterextended storage. It should be pointed out that the limitations oflaboratory inoculation studies should be kept in mind and resultsobtained from this study need to be interpreted with caution.

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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