Listeria monocytogenes Inhibition by Whey Protein Films and Coatings Incorporating the Lactoperoxidase System EACHEOL

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1 JFS M: Food Microbiology nd Sfety Listeri monocytogenes Inhibition by Whey Protein Films nd Cotings Incorporting the Lctoperoxidse System SEA EACHEOL MIN IN,, LINDA J. HARRIS ARRIS, AND JOHN M. KROCHT OCHTA ABSTRACT CT: : Antimicrobil effects of whey protein isolte (WP WPI) films nd cotings incorporting the lctoperoxidse system (LPOS) ginst Listeri monocytogenes wer ere e studied by turbidity,, plte counting, disc-cover ering, nd disc-surfce-spreding tests using vrious growth medi. Survivl of L. monocytogenes pplied to smoked slmon before or fter the coting ws monitored immeditely fter ppliction nd during storge t 4 C nd 10 C for up to 35 d. Tensile proper operties (elstic modulus [EM], tensile strength [TS], elongtion [E]), oxygen perme- bility (OP), nd color (Hunter L,, b) ) of WPI I films,, with nd without LPOS, wer ere e lso compred. LPOS inhibited L. monocytogenes in broth nd on gr medi. WPI I films incorporting 29 mg of LPOS per grm of film (dry bsis) inhibited 4.2 log colony-for -forming units (CFU)/cm 2 of L. monocytogenes inoculted on gr medi. WPI cotings prepred with LPOS t 0.7% (w/w) in coting solution (40 mg LPOS/g coting [dry bsis]) initilly reduced 3 3 nd 1 log CFU/g of L. monocytogenes nd totl erobic microorgnisms in smoked slmon, respec- tively ely. The WPI I cotings incorporting LPOS prev evented ented the growth of L. monocytogenes in smoked slmon t 4 C nd 10 C for 35 d nd 14 d, respectiv espectively ely. The tensile proper operties ties,, oxygen permebility mebility,, nd color of WPI I films wer ere not significntly chnged by incorportion of LPOS (P > 0.05). Keywords: Listeri monocytogenes,, ntimicrobil film nd coting, whey protein film nd coting, lctoperoxi- dse system, smoked slmon Introduction Listeri monocytogenes is recognized s n importnt foodborne pthogen due to its wide distribution in the environment, bility to survive for long periods of time under dverse conditions, nd bility to grow t tempertures s low s 0.4 C (IFT 2004). Listeriosis, cused by L. monocytogenes, ffects n estimted 1600 individuls nd cuses n estimted 415 deths nnully in the United Sttes (IFT 2004). Listeriosis hs been ssocited with the consumption of rnge of food products, including cole slw, diry products, poultry products, nd sefoods (Kennedy nd others 2000). Smoked slmon is redy-to-et (RTE) food with shelf life of severl weeks when stored t refrigertion tempertures in vcuum pckges (Rorvik 2000). L. monocytogenes hve been found spordiclly in smoked slmon (Rorvik nd Yndestd 1991; Cortesi nd others 1997), indicting tht the processed slmon product is vulnerble to contmintion (Zuckermn nd Avrhm 2002). The slt content, ph, nd wter ctivity of smoked slmon re normlly within rnge permitting the growth of L. monocytogenes under refrigerted nd vcuum pckging conditions (Rorvik 2000; Gimenez nd Dlgrd 2004). Novel mens to control post-processing contmintion of L. monocytogenes in RTE foods hve been sought (Wkbyshi nd others 1992). Among potentil pproches, ntimicrobil edible films nd cotings, which cn minimize or prevent growth of undesirble microorgnisms during storge, hve received ttention (Outtr nd others 2000; Cgri nd others 2004). Mjor potentil food pplictions MS Submitted 1/7/05, Revised 2/23/05, Accepted 5/24/05. The uthors re with Dept. of Food Science nd Technology, Univ. of Cliforni, Dvis, One Shields Ave., Dvis, CA Direct inquiries to uthor Krocht (E-mil: jmkrocht@ucdvis.edu). of ntimicrobil cotings include fish, poultry, bkery goods, cheese, fruits, nd vegetbles (Lbuz nd Breene 1988). Efforts hve focused on the incorportion of nturl ntimicrobil gents in the edible films nd cotings (Den nd Zottol 1996; Delves-Broughton nd others 1998). Exmples of gents being studied include bcteriocins (for exmple, nisin, pediocin, lcticin), enzymes (for exmple, lysozyme), nd extrcts from nturl sources (Hn 2000). Lctoperoxidse system (LPOS) is nturl ntimicrobil system in milk nd in humn secretions such s sliv nd ter fluid (Kussendrger nd vn Hooijdonk 2000). The use of LPOS hs been suggested s preservtive in foods nd phrmceuticls (Bosch nd others 2000). Lctoperoxidse (LPO) in LPOS ctlyzes the oxidtion of thiocynte ion (SCN ), generting oxidizing products such s hypothiocynite (OSCN ) nd hypothiocynous cid (HOSCN), which inhibit microorgnisms by the oxidtion of sulphydryl (SH) groups of microbil enzymes nd other proteins (Kussendrger nd vn Hooijdonk 2000). The structurl dmge of microbil cytoplsmic membrnes by the oxidtion of SH groups results in lekge of potssium ions, mino cids, nd peptides from microbil cells nd eventul deth of the cells (Reiter nd Hrnulv 1984; Kussendrger nd vn Hooijdonk 2000). Antimicrobil edible films nd cotings must lso provide dequte physicl nd brrier protection, s well s mintin cceptble color, for foods during production nd storge. Tensile (elstic modulus, tensile strength, elongtion), brrier (for exmple, oxygen permebility), nd color properties of edible films re importnt becuse they ffect the usefulness of the films for food coting nd pckging (Debeufort nd others 1998). Thus, combined nlyses of ntimicrobil, tensile, nd physicl properties re crucil for predicting the behvior of ntimicrobil edible films (McHugh nd Krocht 1994; Cgri nd others 2001). M: Food Microbiology & Sfety 2005 Institute of Food Technologists Further reproduction without permission is prohibited Vol. 70, Nr. 7, 2005 JOURNAL OF FOOD SCIENCE M317 Published on Web 8/19/2005

2 L. monocytogenes inhibition by LPOS-WPI films/cotings... M: Food Microbiology & Sfety Although ntimicrobil ctivity of LPOS ginst L. monocytogenes hs been demonstrted (Denis nd Rmet 1989; Kmu nd others 1990; Gy nd others 1991; Rvishnkr nd Hrrison 1999; Kennedy nd others 2000), no reserch hs been reported on the ntimicrobil effect ginst L. monocytogenes when the LPOS is incorported in whey protein edible films nd cotings. In ddition, the ntimicrobil effect of LPOS ginst psychrotrophic pthogen hs not been studied in ny food other thn milk. The objectives of this reserch were to (1) evlute the effect of LPOS on the inhibition of L. monocytogenes in nd on recovery medi; (2) study the nti L. monocytogenes effect of LPOS whey protein isolte (WPI) films/cotings; (3) compre tensile, brrier, nd color properties of LPOS-WPI films with those of plin WPI films; nd (4) evlute the effect of LPOS-WPI cotings on the microbil stbility of cold-smoked slmon, inoculted with L. monocytogenes, during storge t 4 C nd 10 C for 35 d. Mterils nd Methods Bcteri strins Dr. Lrry Beucht, Univ. of Georgi, provided the bcteril strins used in this study. A cocktil ws prepred with the following 5 L. monocytogenes isoltes: V7 (serotype 4b, milk), LCDC (serotype 4b, rw cbbge), Scott A (serotype 4b, humn isolte ssocited with milk), 101 M (unknown serotype, beef ), nd 108M (unknown serotype, beef). All strins were dpted to grow in the presence of 50 g/ml nlidixic cid, used to inhibit growth of other microorgnisms (Lng nd others 2004). Medi Tryptic soy gr nd brin hert infusion gr (Difco, Becton Dickinson, Sprks, Md., U.S.A.) supplemented with 50 g/ml of nlidixic cid (Sigm-Aldrich, St. Louis, Mo., U.S.A.), TSAN nd BHIAN, respectively, tryptic soy broth (TSB), nd brin hert infusion broth (BHIB) were used s recovery medi. Modified Oxford gr (Difco, Becton Dickinson) supplemented with 50 g/ml of nlidixic cid (MOXN) ws used s selective medium. Nlidixic cid ws filtered (0.2 m, Gelmn Sciences, Ann Arbor, Mich., U.S.A.) nd dded to bsl medi fter utoclving nd cooling to pproximtely 50 C. Nlidixic cid ws dded to minimize colonies of bcteri present on cold-smoked slmon, thus fcilitting detection of L. monocytogenes. Plte count gr (PCA; Difco, Becton Dickinson) nd dichlorn rose-bengl chlormphenicol gr (DRBC; Merck, Drmstdt, Germny) were used to enumerte totl erobic microorgnisms nd yests/molds, respectively. Inoculum preprtion The inoculum ws prepred following the method of Lng nd others (2004). Frozen stock cultures of L. monocytogenes were streked weekly on BHIAN. Cultures were incubted t 37 C for 24 h, nd n isolted colony of ech strin ws trnsferred to BHIB (10 ml). At 2 consecutive 24-h intervls, further trnsfers were mde in BHIB using sterile loop (pproximtely 10 L). Cells of ech overnight (18 h) culture were collected by centrifugtion (4000 g, 15 min, 22 C) (Centrifuge 5417, Eppendorf, Hmburg, Germny) nd suspended in 0.1% (w/v) peptone. The cocktil ws prepred by combining equl portions of ech strin to produce n inoculum of pproximtely 10 9 colonyforming units (CFU)/mL. The cocktil ws diluted in 0.1% (w/v) peptone to produce the desired inoculum concentrtion. (KSCN) (Sigm-Aldrich), nd hydrogen peroxide (H 2 O 2 ) (EM Science, Gibbstown, N.J., U.S.A.). The rtio of the components in the system ws 1.00:0.35:108.70:1.09:2.17 in the order of LPO, GO, Glu, KSCN, nd H 2 O 2. The concentrtions of the components were selected on the bsis of those used by other reserchers (Thoms nd Aune 1978; Denis nd Rmet 1989; Ernshw nd others 1989; Atmer nd others 1999; Boussouel nd others 1999; Rvishnkr nd Hrrison 1999; Bosch nd others 2000). The components were dissolved seprtely in Butterfield s buffer (Weber Scientific, Hmilton, N.J., U.S.A.). The dissolved system ws filter-sterilized (0.2 m, Gelmn Sciences). The filter sterilized system ws incubted t 23 ± 2 C for 24 h with shking t 160 rev/min using wter bth shker (Model G76, New Brunswick Scientific, Edison, N.J., U.S.A.) to increse ntimicrobil ctivity of LPOS (Bosch nd others 2000). The incubted LPOS solution ws used without storge. As preliminry test, the ntimicrobil ctivity of LPOS ginst L. monocytogenes ws exmined t 4 C, 10 C, nd 22 C for up to 42 d, 28 d, nd 14 d, respectively, using the turbidity test, described in the next section. Results from the test indicted tht the LPOS ntimicrobil ctivity ws mintined during the test period t ech temperture. Turbidity test LPOS ws prepred t 0%, 0.1%, 0.25%, nd 0.5% (w/v) in 1% (w/v) peptone, 0%, 0.25%, 0.5%, 0.75%, nd 1.0% (w/v) in TSB, nd 0%, 0.25%, 0.5%, 0.75%, 1.0%, nd 1.25% (w/v) in BHIB. L. monocytogenes (50 L of CFU/mL) ws inoculted into 10 ml of LPOS-contining broth nd incubted t 37 C. The extent of growth of L. monocytogenes ws determined by mesuring opticl density (OD) of the inoculted broth t 600 nm on 0 d, 1 d, nd 2 d (Spectrophotometer, DU 640, Beckmn Coulter, Inc., Fullerton, Clif., U.S.A.). Deionized wter ws used s the blnk for the OD mesurement. For confirmtion of the bsence of cells in L. monocytogenes inoculted nd LPOS-treted liquid medium for which bsorbnce did not chnge during the incubtion, 1 ml (250 L 4) of the liquid medium ws plted onto TSAN. The TSAN pltes were incubted t 37 C for 48 h before ssessing the presence of ny colonies. Plte counting test with LPOS solution Molten TSAN nd BHIAN gr (1.5 ml/well) ws introduced into pre-sterilized tissue culture pltes (24 wells/plte, di of ech well: 1.6 cm, Nunc, Denmrk). Inoculum t different concentrtions ws spotted (100 L) nd spred on the surfce of ech gr well using pre-sterilized disposble hockey stick (Fisher Scientific Inc., Fir Lwn, N.J., U.S.A.) to obtin levels of pproximtely 10, 10 2, nd 10 3 CFU/well. After holding t room temperture for 1 h, 100 L of LPOS solution (0%, 0.01%, 0.025%, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4% [w/w]) in sterile deionized wter ws spred on the surfce of the inoculted gr of ech well using the hockey stick. A control ws prepred by spreding 100 L sterile deionized wter, insted of LPOS solution. All pltes were incubted t 37 C for 48 h. Film-forming gents Whey protein isolte ws supplied by Dvisco Foods Intl. (Le Sueur, Minn., U.S.A.). Glycerol (Gly), used s plsticizer to improve film nd coting flexibility, ws purchsed from Fisher Scientific Inc. Product informtion for WPI from the supplier indicted tht the WPI contined n undetectble number of molds (pproximtely <10 cells/g) nd 2.0% ± 0.2% (w/w) sh (Dvisco Foods 2003). Lctoperoxidse system The LPOS ws composed of LPO (102 U/mg), glucose oxidse (GO) (235 U/mg), -D-glucose (Glu), potssium thiocynte Film preprtion WPI edible films were prepred following the method of McHugh nd Krocht (1994). Briefly, 10% (w/w) WPI solution ws M318 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 7, 2005 URLs nd E-mil ddresses re ctive links t

3 L. monocytogenes inhibition by LPOS-WPI films/cotings... prepred in distilled wter. A weight of Gly equl to tht of WPI ws dded to the WPI solution, nd the WPI-Gly solution ws degssed under vcuum. The solution ws mintined t 90 C for 30 min in wter bth nd cooled on ice. Once cooled, the solution ws degssed gin. LPOS ws dded into the degssed solution to prepre n LPOS-WPI film-forming solution. Films were cst by pipetting the LPOS-WPI film-forming solution onto utoclved high-density polyethylene (HDPE) pltes (8.5- or 15.5-cm inner di), resting on leveled grnite surfce. The HDPE pltes were prepred to specifiction in the Dept. of Biologicl nd Agriculturl Engineering, Univ. of Cliforni, Dvis. The mount of the filmforming solution pipetted ws selected to produce 0.1-mm-thick trnsprent film. For exmple, the weights to produce LPOS films on the 8.5-cm-di pltes with the concentrtions of 17 nd 29 mg LPOS/g film were 4.49 g nd 4.53 g, respectively. The film-forming solutions were dried for 18 h t 23 ± 2 C/35% ± 5% reltive humidity (RH). Dried films were peeled intct from the csting surfce nd stored in chmber controlled t 23 ± 2 C/50% ± 2% RH until used. Plte counting test with LPOS-WPI films WPI films incorporting LPOS (LPOS-WPI films) were prepred t 0, 11, 17, 23, nd 29 mg LPOS/g film on dry bsis, which correspond to 0%, 0.2%, 0.3%, 0.4%, nd 0.5% (w/v) in the WPI film solution. TSAN (1.5 ml/well) ws septiclly distributed into ech well of 24-well tissue culture plte. The LPOS-WPI films were cut into 1.6-cm-di discs using sterile cheese borer. Two tretments were evluted by plcing the films on top of the gr well either before (F + I) or fter (I + F) inoculting with L. monocytogenes. L. monocytogenes t level of 10 3 CFU/mL ws prepred s the inoculum nd 20 L of the inoculum ws spotted nd spred with pre-sterilized disposble hockey stick either directly on the TSAN or on the film discs covering the TSAN, to obtin n pproximte concentrtion of 70 CFU/well. The survivl of L. monocytogenes ws mesured by the bility of the orgnism to form colonies on the surfce of gr or on the film disc fter incubtion t 37 C for 48 h. Disc-covering test TSAN Petri dishes (8.5 cm-di) were prepred with 15 ml of molten TSAN. The inoculum (100 L) ws spred on the surfce of the TSAN plte to produce lwn estimted to be pproximtely 4.2 log CFU/cm 2. Discs (1.6-cm-di) of LPOS-WPI films with LPOS concentrtion of 29 mg/g film (dry bsis) were plced on the surfce of the TSAN pltes inoculted with L. monocytogenes. Pltes were refrigerted t 4 C for 3 h to llow diffusion of ntimicrobil gents from the films into the gr nd then incubted t 37 C for 48 h. After 48 h of incubtion, pltes were observed for growth below the film discs. Disc-surfce-spreding test Discs (1.6-cm di) of LPOS-WPI films (29 mg LPOS/g film [dry bsis]) were plced on the surfce of uninoculted TSAN pltes. After holding t room temperture for 1 h, 100 L of inoculum ws spred on the plte to produce lwn of pproximtely 4.2 log CFU/cm 2. The pltes were incubted t 35 C for 48 h fter refrigertion t 4 C for 3 h. Inhibition of growth on the disc surfce ws exmined fter incubtion. Coting on cold-smoked slmon Cold-smoked slmon ws chosen to test the effect of the LPOS- WPI coting ginst L. monocytogenes in food system. Pre-sliced commercilly produced cold-smoked slmon ws purchsed t locl supermrket nd stored t 4 C for mximum of 1 d before use in experiments. The ingredients of the smoked slmon, listed on the pckge, were Atlntic frm-rised slmon, slt, brown sugr, nturl hrdwood smoke, nd color. The thickness of the slice ws 3 mm. Ech piece ws trimmed to mke 10 ± 0.4 g (pproximtely 6 cm 6 cm) smple. The coting solution ws prepred using the methods described previously to prepre the LPOS-WPI film-forming solution in the film preprtion. A 1.5-mL coting solution volume ws either pplied to the surfce of ech slmon smple using disposble hockey stick before (C + I) or fter (I + C) inoculting with L. monocytogenes. Preliminry tests tht incorported blue dye (FD&C blue nr 1, SENSIENT, St. Louis, Mo., U.S.A.) into the coting confirmed tht uniform surfce spreding ws chieved with this technique. The weight gin on the slmon smple fter coting ws pproximtely 0.7 g. Inhibitory effects of LPOS-WPI coting ginst L. monocytogenes in cold-smoked slmon Initil inhibition. Slmon smples were plce in single lyer on wire screen in lminr flow biohzrd hood (SterilGARD Hood, the BAKER COMPANY, Inc. Snford, Mine, U.S.A.) t 28 ± 2 C t 30% RH. The slmon smples were coted in 2 wys, I + C nd C + I, s previously described. For I + C, inoculted slmon smples were dried for 0.5 h nd then coted. For C + I, coted smples were dried for 1 h nd then inoculted. Inoculum (100 L) ws spotted in 25 to 30 pproximtely equl volumes either directly onto the slmon or onto the top of the coting previously pplied to the slmon nd spred with hockey stick. The levels of inoculum were pproximtely 2.0, 3.0, nd 4.0 log CFU/g. The concentrtions of LPOS in the coting solution were 0%, 0.1%, 0.3%, 0.5%, nd 0.7% (w/w). After drying, smples were plced in stomcher bgs (19 30 cm, Nsco WHIRL-PAK, Fort Atkinson, Wis., U.S.A.). The smples were diluted 10-fold with 0.1% (w/v) peptone nd then pummeled by stomcher blender (STOMACHER 400, Sewrd, Norfolk, U.K.) for 1 min t norml speed. The homogente ws diluted serilly nd plted (100 L or 1 ml) on MOXN. The MOXN pltes were incubted for 48 h t 37 C before colonies of presumptive L. monocytogenes were counted. When the counts fell below the level of detection (1.0 log CFU/g), 100 ml of Listeri Enrichment Broth (LEB; Oxoid, Hmpshire, U.K.) ws dded to ech stomcher bg contining homogenized smple. The stomcher bgs were then incubted for 48 h t 37 C. The incubted smple ws streked onto MOXN pltes nd incubted for 48 h t 37 C before being exmined for presumptive colonies of L. monocytogenes. If presumptive positive colonies were present, 5 of those colonies were selected rndomly for confirmtion using API Listeri (biomerieux s, Durhm, N.C., U.S.A.). Uninoculted slmon smples were lso enriched for L. monocytogenes detection using the sme method. Inhibition during storge. Slmon smples were prepred s described previously with n inoculum level of 4.0 log CFU/g of L. monocytogenes. Treted smples (I + C nd C + I) nd control smples (without coting) were individully pcked erobiclly in sterile bgs (18 oz, Nsco WHIRL-PAK, Fort Atkinson, Wis., U.S.A.) nd stored t 4 C or 10 C. Smples were held for up to 35 d. At ech time point, smples were diluted 10-fold with 0.1% (w/v) peptone nd homogenized for 1 min t norml speed in the stomcher blender. The homogente ws serilly diluted nd plted on MOXN, PCA, nd DRBC to enumerte L. monocytogenes, totl erobes, nd yests/molds, respectively. MOXN nd PCA pltes were incubted for 48 h t 37 C nd 35 C, respectively, wheres DRBC pltes were incubted for 5 d t 22 C before counting colonies. When L. monocytogenes ws not detected on MOXN (detection limit, 1.0 log CFU/g), smples were enriched for L. monocytogenes s described previously. Wter ctivity ( w ) ws mesured fter the slmon ws removed from commercil pckges, before being stored, nd fter 21 d nd 28 d of storge using wter M: Food Microbiology & Sfety URLs nd E-mil ddresses re ctive links t Vol. 70, Nr. 7, 2005 JOURNAL OF FOOD SCIENCE M319

4 L. monocytogenes inhibition by LPOS-WPI films/cotings... ctivity meter (AquLb CX-2, Decgon Devices, Inc., Pullmn, Wsh., U.S.A.). The ph of smples ws mesured before storge nd fter 21 d nd 28 d of storge. Solutions obtined from stomching the slmon smples with 1% (w/v) peptone (1:10) in stomcher bgs were collected for ph mesurement t 22 C using ph meter (370, Orion, Beverly, Mss., U.S.A.). Tensile proper operties of films The Americn Society of Testing nd Mterils (ASTM) stndrd method D (ASTM 1997) ws used to mesure tensile properties of films. The LPOS concentrtion in LPOS-WPI films ws 40 mg/g (dry bsis). Control films were prepred without LPOS ddition. All films were conditioned for 48 h t 23 ± 2 C/50% ± 2% RH before testing. A sturted slt solution of mgnesium nitrte (Fisher Scientific Inc.) ws used to equilibrte films to n environment t 50% ± 2% RH. Films were cut into strips with test dimension of 50 mm 8 mm. Dt from n Instron (Model 1122, Instron, Cnton, Mss., U.S.A.) ws used to determine elstic modulus (EM), tensile strength (TS), nd elongtion t brek (E) (Sothornvit nd Krocht 2001). The vlues for sttic lod cell nd cross hed speed of the Instron were 4.9 kn nd 50 mm/min, respectively. Film thickness A micrometer (nr , Mitutoyo, Kwski, Jpn) ws used to determine film thickness to the nerest mm ( in). Mesurements were rndomly tken t 5 different loctions, nd the men vlue ws used in clcultions for tensile properties nd oxygen permebility. Oxygen permebility An Ox-Trn 2/20 ML modulr system (MOCON, Minnepolis, Minn., U.S.A.) ws used to determine oxygen permebility (OP) of films t 23 C nd 50% ± 1% RH, ccording to ASTM stndrd method D 3985 (ASTM 1995). Tested films were identicl to those used for the test of tensile properties. Tble 1 Inhibition of Listeri monocytogenes with inoculum levels of 10 1, 10 2, 10 3, nd 10 4 colony-forming units [CFU]/cm 2 by the lctoperoxidse system t concentrtions of 0%, 0.01%, 0.025%, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, nd 0.4% (w/v) on tryptic soy gr nd brin hert infusion gr () Popultion (CFU/well),b Concentrtion of lctoperoxidse Inoculum level system %(w/v) ± 4 c 359 ± 12 b TNTC c ± 2 c 358 ± 10 b TNTC ± 4b c 355 ± 10 b TNTC ± 3b c 268 ± 11b b TNTC ± 3c c 186 ± 8c b TNTC ± 2d c 46 ± 5d b TNTC ± 0e c 3 ± 1e b 52 ± 5b ± 0e 0 ± 0f 0 ± 0c ± 0e 0 ± 0f 0 ± 0c (b) Popultion (CFU/well),b Concentrtion of lctoperoxidse Inoculum level system %(w/v) ± 5 c 361 ± 16 b TNTC ± 4 c 358 ± 19 b TNTC ± 3b c 358 ± 8 b TNTC ± 3b c 298 ± 10b b TNTC ± 3c c 247 ± 12c b TNTC ± 2c c 216 ± 6d b TNTC ± 2d c 130 ± 7e b 326 ± 12b ± 0e b 0 ± 0f b 12 ± 4c ± 0e 0 ± 0f 0 ± 0d Vlues re men ± stndrd devitions, n = 4. b Men vlues (CFU/well) in columns were nlyzed for significnt differences ( = 0.05). First letter = within inoculum level, vlues not followed by the sme letter re significntly different; 2nd letter = within concentrtion of lctoperoxidse system, vlues not followed by the sme letter re significntly different. ctntc = too numerous to count. M: Food Microbiology & Sfety Color A Hunter Lb colorimeter (Lbscn II, Hunter Assocites Lbortory Inc., Reston, V., U.S.A.) ws used for film color mesurement nd Hunter L,, b vlues were reported. The colorimeter ws clibrted using white nd blck stndrd tiles, Illuminte D 65, nd 2 stndrd observer. Eight redings were tken per prmeter. Tested films were identicl to those used for the tests of tensile properties nd oxygen permebility. Sttisticl nlysis The order of experimentl tretment ws determined by rndom numbers. All experiments except the storge study, which ws done once with 4 smples for ech tretment, were duplicted. Ech observtion within ech repliction ws determined in duplicte except tht of tensile properties. Eight repeted mesurements in ech repliction were performed to determine tensile properties. Significnt differences nd the number of repliction were determined t 5% significnce level. Dt were nlyzed by 1-wy nlysis of vrince (ANOVA). All the sttisticl nlysis were mde using Minitb (Minitb, Inc., Stte College, P., U.S.A.). defined s the lowest concentrtion tht inhibits growth of microorgnism, with no resulting significnt increse in bsorbnce (Murdock nd Mtthews 2002). In the current study, the MIC ( = 0.05) of LPOS inhibiting 4.0 log CFU/mL of L. monocytogenes ws 0.5%, 1.0%, nd 1.3% (w/v) fter 2 d of incubtion t 37 C in 1% (w/v) peptone, TSB, nd BHIB, respectively. No cells of L. monocytogenes were detected in the inoculted medium tht did not increse in bsorbnce during incubtion (detection limit, 1 CFU/mL inoculted medium), indicting no growth of the cells t the observed MIC vlues. Plte counting test with LPOS solution The number of cells enumerted decresed s the inocultion level decresed nd the concentrtion of LPOS incresed (Tble 1). No colonies were observed in the wells treted with 0.3% nd 0.4% (w/v) of LPOS with inocultion levels of 10 to 10 3 CFU/well. The MICs on TSAN nd BHIAN, which resulted in no growth of L. monocytogenes of 10 3 CFU/well, were 0.3% nd 0.4% (w/v), respectively. Inhibition effects depended on the medium used. The rnge of the LPOS concentrtion suitble for use in the film tests ws bsed on the results from the broth nd gr ssys. Results nd Discussion Turbidity test Chnges in the bsorbnce of broth medi inoculted with L. monocytogenes fter the ddition of LPOS t different concentrtions were mesured. A minimum inhibition concentrtion (MIC) is Effects of WPI I films incorporting LPOS ginst L. monocytogenes WPI films incorporting LPOS (LPOS-WPI films) were evluted for their bility to inhibit L. monocytogenes using the plte counting test, the disc-covering test, nd the disc-surfce-spreding test. Plte counting test. An imge from the plte counting test with M320 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 7, 2005 URLs nd E-mil ddresses re ctive links t

5 L. monocytogenes inhibition by LPOS-WPI films/cotings... Tble 2 Inhibition of Listeri monocytogenes with different inoculum levels of 2.0, 3.0, nd 4.0 log colony-forming units (CFU)/g on smoked slmon by whey protein isolte cotings incorporting the lctoperoxidse system in the concentrtions of 0%, 0.1%, 0.3%, 0.5%, nd 0.7% (w/w) in the coting solution Popultion (log CFU/g)b,c Concentrtion of lctoperoxidse system (% [w/w]) Estimted inoculum level (log CFU/g) Tretment Inocultion without coting I + Cd C + Ie I+C C+I I+C C+I I+C C+I I+C C+I ± 0.3 c 1.6 ± 0.3 c 1.6 ± 0.2 c 1.1 ± 0.3 b <1.0 (+) fb c <1.0 (+)b c <1.0 (+)b c <1.0 (+)b c <1.0 (+)b b <1.0 (+)b b <1.0 (+)b ± 0.2 b 2.8 ± 0.2 b 2.7 ± 0.2 b 2.0 ± 0.3b 1.8 ± 0.2bc b 1.4 ± 0.2c b 1.3 ± 0.1c b 1.3 ± 0.2c b <1.0 (+)d b <1.0 (+)d b <1.0 (+)d ± ± ± ± 0.3b 2.4 ± 0.2b 2.2 ± 0.3b 2.2 ± 0.2b 2.0 ± 0.2b 2.1 ± 0.1b 1.4 ± 0.2c <1.0 (+)d The concentrtion of lctoperoxidse only in the coting solution contining 0.7% lctoperoxidse system ws 6 ppm (353 ppm on dry bsis). b Vlues re men ± stndrd devitions, n = 4. c Men vlues (log CFU/g) in columns were nlyzed for significnt differences ( = 0.05). First letter = within inoculum level, vlues not followed by the sme letter re significntly different; 2nd letter = within concentrtion of lctoperoxidse system, vlues not followed by the sme letter re significntly different. d Inocultion before coting. e Coting before inocultion. f Positive on the confirmtion test (API Listeri) for the presence of L. monocytogenes. LPOS-WPI films (29 mg/g film [dry bsis]) inhibited L. monocytogenes t level of 4.2 log CFU/cm2, regrdless of whether the film ws bove or below the inoculum. Post-processing surfce-contmintion with L. monocytogenes is mjor issue for the food industry (Cgri nd others 2004). The results from the disc-covering test nd the I + F tretment (plte counting test) simulte wht would hppen if LPOS-WPI films were wrpped or LPOS-WPI cotings were formed over L. monocytogenes contminted surfce of food (Appendini nd Hotchkiss 2002). Results from the disc-surfce-spreding test nd the F + I tretment (plte counting test) simulte wht would hppen if L. monocytogenes post-contmintion occurred on LPOS-WPI film-wrpped or coted foods. M: Food Microbiology & Sfety LPOS-WPI films incorporting vrious concentrtions of LPOS is shown in Figure 1. L. monocytogenes ws inoculted into ech well t level of pproximtely 70 CFU/well directly onto the gr surfce followed by plcement of the film (I + F). Identicl results were obtined when inocultion occurred fter the film hd been plced on top of the gr (F + I). Thus, L. monocytogenes ws inhibited eqully well when inoculted over or under the film. LPOS t concentrtion of 29 mg/g in the films prevented growth of 70 CFU of L. monocytogenes. Disc-covering nd disc-surfce-spreding tests. The disc-covering nd disc-surfce-spreding tests were conducted to determine if LPOS-WPI films could inhibit higher concentrtion (4.2 log CFU/cm2) of L. monocytogenes. These tests showed tht Figure 1 An imge from the plte counting test with whey protein isolte (WPI) films incorporting the lctoperoxidse system (LPOS-WPI films), showing the effects of the films on the inhibition of Listeri monocytogenes on tryptic soy gr. Film discs were plced fter inocultion (I + F). The inocultion level ws pproximtely 70 colony-forming units (CFU)/well. Ech pir of rows ws treted with LPOS-WPI films t different concentrtions of the lctoperoxidse system. The concentrtions were 0, 11, 17, 23, nd 29 mg/g film (dry bsis) for ech pir from the 2nd left column to the right, respectively. The 1st left column ws inoculted with Listeri monocytogenes but not treted with the films. The concentrtion of lctoperoxidse in the 29 mg/g LPOS-WPI film ws 256 ppm on dry bsis. URLs nd E-mil ddresses re ctive links t Vol. 70, Nr. 7, 2005 JOURNAL OF FOOD SCIENCE M321

6 L. monocytogenes inhibition by LPOS-WPI films/cotings... M: Food Microbiology & Sfety Inhibitory effects of LPOS-WPI coting ginst L. monocytogenes in cold-smoked slmon Initil inhibition. Coting before (C + I) or fter (I +C) inocultion ws to simulte contmintion of smoked slmon with L. monocytogenes before or fter pplying the film, respectively. Levels of L. monocytogenes on smoked slmon were not significntly different for smples with or without films tht did not contin LPOS, indicting tht the coting itself did not impct survivl of L. monocytogenes (P > 0.05). LPOS-WPI cotings (0.7% [w/w]) inhibited L. monocytogenes of pproximtely 3 log CFU/g in smoked slmon smples. No colonies were detected (detection limit, 1.0 log CFU/g) in some smples (Tble 2). However, ll smples were positive upon enrichment of () (b) (c) Figure 2 Effects of whey protein isolte (WPI) cotings incorporting the lctoperoxidse system (0.7% [w/w] in the coting solution) on the microbil counts of Listeri monocytogenes inoculted cold-smoked slmon determined on modified oxford gr () to count L. monocytogenes, plte count gr (b) to count totl erobic microorgnisms, nd dichlorn rose-bengl chlormphenicol gr (c) to count yests/molds during storge t 4 C nd 10 C for 35 d. The detection limit (1.0 log colonyforming units [CFU]/g) ws indicted with dotted line nd zero counts on the pltes were plotted s 0 on the verticl xis in Figure g of slmon, indicting tht L. monocytogenes ws not eliminted by the LPOS-WPI coting. In most cses, there ws no significnt difference (P > 0.05) between I + C nd C + I. When significnt difference (P < 0.05) ws observed (0.1% nd 2 log CFU/g; 0.5% nd 3 log CFU/g; 0.7% nd 4 log CFU/g), the counts were lwys lower in C + I. Antimicrobil films or cotings re often more effective in inhibiting trget microorgnisms when pplied to nutrient medi thn on rel food systems (Dwson nd others 2002). To compre inhibition efficiency of the coting on smoked slmon to tht of the film on TSAN, the expression of the inhibition efficiency of the coting ws chnged by using the pproximte surfce re of slmon smples of 36 cm 2 nd by considering the fct tht the concentrtion of 0.5% (w/w) of LPOS ws 29 mg/g film on dry bsis. Approximtely 1.5 log CFU/cm 2 of L. monocytogenes ws inhibited by the coting on the surfce of slmon, wheres pproximtely 4.2 log CFU/cm 2 ws inhibited on TSAN (results from disc-covering nd disc-surfce-spreding tests) t the sme concentrtion of LPOS. This considerble difference might be due to higher diffusion of oxidizing products (for exmple, OSCN, HOSCN) through the gr. Diffusion is fcilitted t higher w (Nesens nd others 1982). The w of the slmon ws 0.95, wheres tht of TSAN is close to 1.0. Fcilitted movement of oxidizing products from the film mtrix to TSAN might improve inhibition of the cells. In ddition, more sulfhydryl (SH) compounds my be present in smoked slmon. The oxidizing products inhibit microorgnisms by the oxidtion of SH groups of microbil enzymes nd other proteins (Kussendrger nd vn Hooijdonk 2000). The oxidizing products cn rect with SH compounds in foods (Kmu nd others 1990). The possibly higher presence of SH compounds in smoked slmon thn TSAN could reduce LPOS effectiveness nd thus, higher concentrtion of LPOS might be required to mtch effectiveness observed on TSAN. Inhibition during storge. Presumptive L. monocytogenes ws not detected in uninoculted slmon. The microbil counts of the commercil slmon product were 4.8 log CFU/g nd 2.9 log CFU/g for totl erobic microorgnisms nd yests/molds, respectively. The LPOS concentrtion (0.7% [w/w]) in the coting solution nd the inoculum size (4.0 log CFU/g) for the storge study were bsed on the results from the study of inhibitory effects of LPOS- WPI coting ginst L. monocytogenes in smoked slmon. A storge temperture of 10 C ws chosen to simulte temperture-buse refrigertion. Storge of met nd sefood products frequently occurs bove 4 C s the result of overloding or poor mintennce of refrigertion systems, nd this cn llow L. monocytogenes to grow more rpidly in products supporting its growth (Kennedy nd others 2000). Considering the weight of the slmon smple (10 g), the concentrtion of LPO in the smple ws estimted s 35 ppm (dry bsis). This mount is in the rnge of the concentrtions nturlly present in bovine milk (30 ppm) (Bjorck 1978) nd those suggested for food pplictions (100 ppm) (Kennedy nd others 2000). The LPOS-WPI cotings initilly reduced L. monocytogenes to below the limit of detection (Figure 2). L. monocytogenes in control smples reched 4 log CFU/g t 4 C fter 21 to 28 d, wheres the numbers in the smples coted by the LPOS-WPI were consistently <1.0 log CFU/g t 4 C for 35 d (both I + C nd C + I tretments). At 10 C, the number of L. monocytogenes in control smples incresed grdully nd reched 6.5 log CFU/g t 21 d. Growth of L. monocytogenes in I + C smples t 10 C ws detected t 21 d of storge, but not from C + I smples throughout 35 d of storge t 10 C. This might be prtly due to more efficient contct of the coting with the cells. Cells my be protected from the ntimicro- M322 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 7, 2005 URLs nd E-mil ddresses re ctive links t

7 L. monocytogenes inhibition by LPOS-WPI films/cotings... Tble 3 Tensile, oxygen, nd color properties of whey protein isolte films, with nd without the lctoperoxidse system,b Tensile properties c Oxygen permebility d Hunter vlue e Film EM (MP) TS (MP) E (%) (cm 3 m/m 2 d kp) L b Control ± ± ± ± ± ± ± LPOS-WPI ± ± ± ± ± ± ± Concentrtion of LPOS in the WPI films on dry bsis ws 40 mg/g film (lctoperoxidse only: 353 ppm [dry bsis]). b No significnt difference on ech property t 5% significnce level. c,d,e Vlues re men ± stndrd devition, n = 16, 4, nd 4 for c, d, nd e, respectively. bil by the rough geometry of smoked slmon (I + C). Another possibility is tht nutrients required for the cells to recover from the LPOS tretment might be more vilble on slmon thn the WPI cotings. The inhibited cells in I + C nd C + I smples filed to grow during storge t 4 C for 35 d. The filure of L. monocytogenes to recover to the sme levels on coted smples compred with control (no coting) smples suggests tht the ntimicrobil effect of the LPOS-WPI cotings ws sustined during the 35 d storge period t the storge conditions used in this study. One of the possible cuses of the sustined inhibition could be continued vilbility of the ntimicrobils by their relese from the coting interior. Denis nd Rmet (1989) nd Ernshw nd Bnks (1989) reported tht LPOS contining glucose nd glucose oxidse possessed extended bctericidl effects ginst L. monocytogenes. The LPOS, continuously generting oxidizing products, would hve strong nd prolonged ntimicrobil effects (Kmu nd others 1990). The LPOS used in this reserch contined glucose nd glucose oxidse. Thus, there is possibility tht oxidizing products were continuously generted in the coting during storge. The LPOS-WPI coting inhibited not only L. monocytogenes, but lso erobic microorgnisms present in smoked slmon (Figure 2b). The LPOS-WPI coting initilly reduced number of totl erobic microorgnisms excluding tht of L. monocytogenes by pproximtely 1 log CFU/g. The growth of totl erobic microorgnisms ws retrded by the LPOS-WPI coting t both 4 C nd 10 C. Yests/molds were not significntly reduced initilly by the LPOS-WPI cotings (P > 0.05) (Figure 2c). However, the growth of yests/molds ws retrded by the LPOS-WPI coting during storge t both 4 C nd 10 C (P > 0.05). At 4 C, 7 d, 21 d, nd 21 d lpsed for the number of yests/molds in control, I + C, nd C + I smples to rech 5.4 to 5.6 log CFU/g, respectively. At 10 C, the number of yests/molds in control, I + C, nd C + I smples reched 6.2 to 6.5 log CFU/g on dys 7, 14, nd 14, respectively. The number of yests/molds in I + C smples t 4 C reched tht of the control t 35 d storge. The growth rte of yests/molds ws slower in coted smples t 10 C during the erly stge of storge, but the number of yests/molds in the coted smples reched tht of control t the end of storge t 10 C (21 d). The w of cold-smoked slmon out of the pckge ws 0.95, nd the w vlues for I + C, C + I, nd control smples were initilly 0.95 ± No significnt chnges were observed in w between 0-d nd 21-d or 28-d smples for ech tretment or mong tretments (P > 0.05). L. monocytogenes is cpble of multiplying between ph 4.39 nd 9.4 (IFT 2004). Initil ph vlues of coting-treted (I + C nd C + I) slmon smples nd untreted slmon smples (controls) rnged from 6.3 to 6.7. Thus, coting with LPOS-WPI solution (ph 6.7) did not ffect initil ph of slmon smples. The ph of the control smples t 10 C, smpled t 21 d, ws 6.3 ± 0.2. The ph of treted slmon smples t 10 C ws 6.1 ± 0.2 t 28 d (lst dy of smpling). The ph vlues of treted smples nd controls stored t 4 C for 35 d rnged from 6.2 to 6.7 nd 6.1 to 6.7, respectively. There were no significnt differences in ph vlues mong smples of different tretments nd different storge tempertures during the storge study (P > 0.05). Thus, L. monocytogenes growth or survivl ws not influenced by ph in the present study. Tensile proper operties Vlues of EM, TS, nd E of WPI films, with nd without LPOS, re shown in Tble 3. The concentrtion of LPOS in LPOS-WPI films ws 40 mg/g film (dry bsis), which corresponds to 0.7% (w/w) LPOS in the coting solution used for the storge study. There were no significnt differences in EM, TS, nd E between WPI films with nd without LPOS (P > 0.05). EM, TS, nd E cn be used to describe how the tensile properties of film mterils relte to their structures (Ninnemnn 1968). Our results suggest tht the incorportion of the LPOS did not significntly chnge the structure of WPI films. Insignificnt chnge in the tensile properties of WPI film fter incorportion of LPOS t 59 mg/g film (dry bsis) ws previously reported (Min nd Krocht 2005). Brrier nd color properties Oxygen permebility nd Hunter L,, nd b vlues of WPI films, with nd without LPOS, re listed in Tble 3. The incorportion of the LPOS (40 mg/g film [dry bsis]) in the WPI film did not significntly ffect oxygen permebility of the film (P > 0.05). No significnt differences were observed in Hunter L,, nd b vlues between the films with nd without LPOS (P > 0.05). Systems with SCN were reported fintly yellow (Bosch nd others 2000). However, colors of both films were not significntly different (P > 0.05). This my be due the smll quntities of KSCN (pproximtely 384 ppm in dry bsis) in the LPOS-WPI films. Insignificnt chnges in oxygen permebility nd color of WPI film fter incorportion of LPOS t 59 mg/g film (dry bsis) were reported by Min nd Krocht (2005). Conclusions WPI films nd cotings incorporting LPOS effectively reduced the number of L. monocytogenes nd inhibited the growth of the cells, inoculted before or fter ppliction of films nd cotings, on microbil medium (TSAN) nd smoked slmon. Aerobic microorgnisms nd yests nd molds on smoked slmon were lso inhibited. Thus, LPOS-WPI films nd cotings show potentil for inhibiting microorgnisms present on RTE fish nd other RTE met products before or fter ppliction of cotings, with tensile, brrier, nd color properties identicl to those of WPI films without LPOS. Acknowledgments This reserch ws supported by the Univ. of Cliforni through UC Discovery Grnt bio , which included mtching funds from the Cliforni Diry Reserch Foundtion (CDRF). We thnk group members of the UC Dvis Biopolymer Film nd Food Sfety Lbortories for their support. We cknowledge nd thnk Dvisco Foods Intl. for supplying us with whey protein isolte for the reserch. M: Food Microbiology & Sfety URLs nd E-mil ddresses re ctive links t Vol. 70, Nr. 7, 2005 JOURNAL OF FOOD SCIENCE M323

8 L. monocytogenes inhibition by LPOS-WPI films/cotings... References Appendini P, Hotchkiss JH Review of ntimicrobil food pckging. Innov Food Sci Emerg Technol 3: [ASTM] Americn Society for Testing nd Mterils Stndrd test method for oxygen gs trnsmission rte through plstic film nd sheeting using coulometric sensor. D Phildelphi, P.: ASTM. [ASTM] Americn Society for Testing nd Mterils Stndrd test method for tensile properties of thin plstic sheeting. D Phildelphi, P.: ASTM. Atmer M, Kock C, Cimer A, Odbsi S, Tmucy B, Ymner N Some qulity chrcteristics of Ksr cheese mnufctured from milk preserved by ctivtion of lctoperoxidse/thiocynte/hydrogen peroxide (LP) system. Milchwissenschft. 54: Bjorck L Antibcteril effect of the lctoperoxidse system on psychotrophic bcteri in milk. J Diry Res 45: Bosch EH, Vn Doorne H, De Vries S The lctoperoxidse system: the influence of iodide nd the chemicl nd ntimicrobil stbility over the period of bout 18 months. J Appl Microbiol 89: Boussouel N, Mthieu F, Benoit V, Linder M, Revol-Junelles AM, Milliere JB Response surfce methodology, n pproch to predict the effects of lctoperoxidse system, nisin, lone or in combintion, on Listeri monocytogenes in skim milk. J Appl Microbiol 86: Cgri A, Ustunol Z, Ryser ET Antimicrobil, mechnicl, nd moisture brrier properties of low ph whey protein-bsed edible films contining p- minobenzoic or sorbic cids. J Food Sci 66: Cgri A, Ustunol Z, Ryser ET Antimicrobil edible films nd cotings. J Food Prot 67: Cortesi ML, Srli T, Sntoro A, Murru N, Pepe T Distribution nd behvior of Listeri monocytogenes in three lots of nturlly-contminted vcuumpcked slmon stored t 2 nd 10 C. Int J Food Microbiol 37: Dvisco Foods Product informtion of whey protein isolte. Le Sueur, Minn., Dvisco Foods Intl. Dwson PL, Crl GD, Acton JC, Hn IY Effect of luric cid nd nisin-impregnted soy-bsed films on the growth of Listeri monocytogenes on turkey bologn. Poultry Sci 81: Den M, Zottol EA Use of nisin in ice crem nd effect on the survivl of Listeri monocytogenes. J Food Prot 59: Debeufort F, Quezd-Gllo JA, Voilley A Edible films nd cotings: tomorrow s pckgings: review. Crit Rev Food Sci 38: Delves-Broughton J, Blckburn RJ, Evns P, Hugenholtz J Use of bcteriocinogenic lctic cid bcteri to inhibit spontneous nisin-resistnt mutnts of Listeri monocytogenes Scott A. J Appl Microbiol 85: Denis F, Rmet JP Antibcteril ctivity of the lctoperoxidse system on Listeri monocytogenes in trypticse soy broth, UHT milk nd French soft cheese. J Food Prot 52: Ernshw RG, Bnks JG A note on the inhibition of Listeri monocytogenes NCTC in milk by n ctivted lctoperoxidse system. Lett Appl Microbiol 8: Ernshw RG, Bnks JG, Defrise D, Frncotte C The preservtion of cottge cheese by n ctivted lctoperoxidse system. Food Microbiol 6: Gy P, Medin M, Nunez M Effect of the lctoperoxidse system on Listeri monocytogenes behvior in rw milk t refrigertion tempertures. Appl Environ Microbiol 57: Gimenez B, Dlgrd P Modeling nd predicting the simultneous growth of Listeri monocytogenes nd spoilge micro-orgnisms in cold-smoked slmon. J Appl Microbiol 96: Hn JH Antimicrobil food pckging. Food Technol 54: [IFT] Inst. Food Technologists Bcteri ssocited with foodborne diseses. IFT Sci Sttus Summ 2004(Aug):1 25. Kmu DN, Doores S, Pruitt KM Antibcteril ctivity of the lctoperoxidse system ginst Listeri monocytogenes nd Stphylococcus ureus in milk. J Food Prot 53: Kennedy M, O Rourke AL, McLy J, Simmonds R Use of ground beef model to ssess the effect of the lctoperoxidse system on the growth of Escherichi coli O157:H7, Listeri monocytogenes nd Stphylococcus ureus in red met. Int J Food Microbiol 57: Kussendrger KD, vn Hooijdonk ACM Lctoperoxidse: physico-chemicl properties, occurrence, mechnism of ction nd pplictions. Br J Nutr 84:S19 S25. Lbuz TP, Breene WM Applictions of ctive pckging for improvement of shelf-life nd nutritionl qulity of fresh nd extended shelf-life foods. J Food Proc Preserv 13:1 69. Lng MM, Hrris LJ, Beucht LR Survivl nd recovery of Escherichi coli O157:H7, Slmonell, nd Listeri monocytogenes on lettuce nd prsley s ffected by method of inocultion, time between inocultion nd nlysis, nd tretment with chlorinted wter. J Food Prot 67: McHugh TH, Krocht JM Sorbitol- vs glycerol-plsticized whey protein edible films: Integrted oxygen permebility nd tensile property evlution. J Agric Food Chem 42: Min S, Krocht JM Inhibition of Penicillium commune by edible whey protein films incorporting lctoferrin, lctoferrin hydrolyste, nd lctoperoxidse systems. J Food Sci 70(2):M Murdock CA, Mtthews KR Antibcteril ctivity of pepsin-digested lctoferrin on foodborne pthogens in buffered broth systems nd ultr-high temperture milk with EDTA. J Appl Microbiol 93: Nesens W, Bresseleers G, Tobbck P Diffusionl behvior of triplmitin in freeze-dried model system t different wter ctivities. J Food Sci 47: Ninnemnn KW Mesurement of physicl properties of flexible films. In: Sweeting OJ, editor. The science nd technology of polymer films. New York, N.Y.: Interscience. p Outtr B, Simrd RE, Piette G, Begin A, Holley RA Inhibition of surfce spoilge bcteri in processed mets by ppliction of ntimicrobil films prepred with chitosn. Int J Food Microbiol 62: Rvishnkr S, Hrrison MA Acid dpttion of Listeri monocytogenes strins does not offer cross-protection ginst n ctivted lctoperoxidse system. J Food Prot 62: Reiter B, Hrnulv G Lctoperoxidse ntibcteril system: nturl occurrence, biologicl functions nd prcticl pplictions. J Food Prot 47: Rorvik LM Listeri monocytogenes in the smoked slmon industry. Int J Food Microbiol 62: Rorvik LM, Yndestd M Listeri monocytogenes in foods in Norwy. Int J Food Microbiol 13: Sothornvit R, Krocht JM Plsticizer effect on mechnicl properties of lctoglobulin films. J Food Eng 50: Thoms EL, Aune TM Susceptibility of Escherichi coli to bctericidl ction of lctoperoxidse, peroxide, nd iodide or thiocynte. Antimicrob Agents Chemother 13: Wkbyshi H, Bellmy W, Tkse M, Tomit M Inctivtion of Listeri monocytogenes by lctoferricin, potent ntimicrobil peptide derived from cow s milk. J Food Prot 55: Zuckermn H, Avrhm RB Control of growth of L. monocytogenes in fresh slmon using Microgrd TM nd Nisin. Lebensm Wiss Technol 35: M: Food Microbiology & Sfety M324 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 7, 2005 URLs nd E-mil ddresses re ctive links t