REPLACEMENT OF CONVENTIONAL CHEESE COATINGS BY NATURAL WHEY PROTEIN EDIBLE COATINGS WITH ANTIMICROBIAL ACTIVITY

Journl of Hygienic Engineering nd Design Originl scientific pper UDC 637.3.045.055 REPLACEMENT OF CONVENTIONAL CHEESE COATINGS BY NATURAL WHEY PROTEIN EDIBLE COATINGS WITH ANTIMICROBIAL ACTIVITY Mrt Henriques 1, 2*, Gustvo Sntos 1, An Rodrigues 1, Dvid Gomes 1, Crlos Pereir 1, Mri Gil 2 1 Deprtment of Food Science nd Technology, Escol Superior Agrári de Coimr - Polytechnic Institute of Coimr, Bencnt, 3040-316 Coimr, Portugl. 2 Chemicl Engineering Deprtment, CIEPQPF, Fculty of Science nd Technology - University of Coimr, Ru Sílvio Lim Pólo II, 3030-790 Coimr, Portugl. * e-mil: mhenriques@esc.pt Astrct The present work ssesses the efficcy of whey protein edile cotings with ntimicroil properties pplied to ripened cheese s lterntives to commercil cheese cotings. Whey protein edile cotings were produced from ovine whey protein concentrte (WPC) with lctic cid nd ntmycin s ntimicroils. Two methods of coting polymeriztion were performed seprtely nd in comintion; the het denturtion method (HD) nd the innovtive polymeriztion method (). Their effectiveness ws evluted y mesuring the physico-chemicl, microiologicl nd sensoril properties of coted cheeses throughout 45 dys. Cotings produced only y HD did not significntly improve the coting efficiency; however, the polymeriztion in comintion with therml tretment () originted cotings tht showed good performnce. With regrd to physico-chemicl evlution, no significnt differences were found etween cheeses ering commercil cotings or edile cotings ( nd ) in terms of weight loss, ft, protein nd slt contents, s well s w, ph nd hrdness, reveling tht the ntimicroil edile cotings could e used s n lterntive to their commercil counterprt(s). Microiologicl nlysis proved tht edile cotings prevented growth of Stphylococcus spp., Pseudomons spp., Enteroctericee, yests nd molds wht demonstrtes their ility to ssure the sfety of cheese. In fct cotings produced y showed etter inhiition or reduction in microil growth s result of the synergistic effect of the ntimicroils nd light. The commercil coting hd the est performnce ginst yests nd molds due to its higher mount of ntmycin. With regrd to sensoril nlysis, cheeses with ntimicroil edile cotings did not show significnt differences with the commercil coted ones. Key words: Whey protein edile cotings, Antimicroil coting, -polymeriztion. 1. Introduction Food sfety depends on hygienic chrcteristics of foodstuffs during production, storge nd commerciliztion. Severl physicl nd chemicl methods to preserve food qulity, such s high pressure, steriliztion, irrdition, ultrsounds nd cidifiction were developed iming tht gol. However, none of these methods ws self sufficient without the use of n pproprite pckge s the finl step of the preservtion process. The use of plstic films is pprised due to their efficiency in protecting nd reducing the mss trnsfer etween food nd its surroundings. Nevertheless, their incresing use nd non iodegrdility chrcteristics led to disposl prolems. Edile films nd cotings re nturl nd iodegrdle products tht contriute to the environment protection while reducing residues from gro-industry. Chrcteristics fcilitting hndling nd crrige of foods cn e improved y the use of edile films nd cotings, enhncing their sensoril ttriutes like color, trnsprency, roughness or stickiness. Though mny functions of edile pckging re identicl to the synthetic ones, such s gses, vpor nd solute permeility, they pper to e complementry prmeter for the qulity of fresh or treted products ccording to their non toxic chrcter nd crrier cpcity [1, 2, 3, 4 nd 5]. A novel function ttriuted to edile cotings is their ction s crriers of ctive ingredients nd dditives (e.g. flvors, pigments, ntioxidnts nd ntimicroil gents) protecting 34

Journl of Hygienic Engineering nd Design nd improving food qulity. Whey protein films nd cotings hve shown to e poor moisture rriers ecuse of their hydrophilic nture, ut present very interesting oxygen rrier properties, comprle to synthetic polymers [6, 7, nd 8], nd ccording Miller nd Krocht [9] even etter mechnicl chrcteristics thn polyscchride-sed edile films. The improvement of whey protein edile films nd cotings with ntioxidnts nd ntimicroil ingredients enhncing food sfety nd shelf life is their most promising ppliction [ nd 11]. Antimicroil performnce of whey protein cotings ws tested in some pplictions: fresh or processed met (Min et l. [2], Cgri et l. [12], nd Zinovidou et l. [4]), fish (Stuchell nd Krocht [13], Min et l. [14], Neetoo et l. [15]) nd cheese (Frnssen [16] nd Rmos [17]). The presence of ntimicroil gents in the coting pplied to the surfce of these food products my reduce or even prevent growth of pthogenic nd spoilge microorgnisms llowing the use of ntimicroils t lower initil levels nd ssuring constnt ckground of these compounds during storge [18]. Antimicroil selection is primrily dependent of the food ppliction nd their trget pthogenic microorgnisms tht re intended to eliminte or control growth. Hence, wide spectrum ntimicroils or very specific ones cn e selected. The ddition of lctic cid into edile films nd cotings hs proven to hve high efficiency [17, 19 nd 20]. It is frequently dded to food for preservtion purposes reducing or eliminting pthogenic grmpositive cteri. However, ccording Ry [21], it is not considered wide spectrum ntimicroil due to its inefficiency ginst yests nd molds. Ntmicyn is successful ntimycotic polyene tht prevents yests nd molds growth t cheese surfces or slices thereof [22 nd 23] nd susges [12]. It is considered s GRAS (Generlly Recognized s Sfe) y the U.S. Food nd Drug Administrtion nd ssigned to e the numer E-235-nturl preservtive in Europen Union. Formtion of protein cotings on food products my involve dipping, sprying, enroing or pnning the food with the coting formultion requiring in ll cses drying of the solvent from the protein coting formultion fter its ppliction to food [7]. Therml protein denturtion is the most commonly used polymeriztion method for whey protein coting production [24, 25, nd 26], while protein crosslinking cn lso e induced chemiclly [27 nd 28], enzymticlly [29] or y mens of irrdition. The used of /g-irrdition, presents some dvntges: it is clen nd well-known process for the steriliztion of goods [30] nd less expensive thn the use of enzymes. g-irrdition ws used y Mezgheni et l. [31], Vchon et l. [32] nd Lcroix et l. [33] to produce edile films from cseintes nd whey proteins. The proposed mechnism ws rdicl polymeriztion through tyrosine with the formtion of ityrosine linkges etween protein chins. The lck of tyrosine residues in whey proteins cn e overtken y the use of chemicls like photoinititors tht under irrdition crete rdicls tht strt the polymeriztion rection [33 nd 34]. Our ssumption for this reserch ws tht the comintion of oth ctive compounds (lctic cid nd ntmycin) nd -irrdition would generte whey protein edile cotings with n improved cohesion nd ntimicroil properties, mking them suitle for coting trditionl cheeses nd e n lterntive to the existing commercilly ville cheese cotings (PVA-sed). The effect of the protein polymeriztion method in the production of WPCsed edile cotings nd its ntimicroil ctivity could e ssessed vi physico-chemicl, microil nd sensoril evlution of coted cheeses, throughout 45 dys of ripening, nd y comprison with cheeses with commercil coting or uncoted t ll. 2. Mterils nd Methods 2.1 Mterils Ovine freeze dried WPC otined y ultrfiltrtion s descried y Henriques et l. [35] ws used s protein source for whey protein sed coting formultions (61.53 g protein 0 g -1 nd 28.28 g lctose 0 g -1 ). Glycerol (99% purity) supplied y JM Vz Pereir Ld. (Portugl) ws the plsticizer used, gur gum ws dded s nturl thickener nd emulsifier (Formul Aditivos Alimentres, LDA, Portugl), sunflower oil (Olimmo) ws provided y Cidcel S.A. (Portugl) nd the surfctnt tween 20 ws supplied y Fluk Chemik (Spin). The photoinititor 2-hydroxy-1-[4- (2-hydroxyethoxy)phenyl]-2-methyl-1-propnone (Irgcure 2959) ws used for cotings produced y polymeriztion nd ws supplied y Ci Specilty Chemicls. The ctive ntimicroil compounds were: lctic cid (90% purity, JM Vz Pereir Ld. Portugl) nd ntmycin (50% purity, Enzil Ld., Portugl). The commercil coting (Redom D, Enzil Ld., Portugl) ws composed of polyvinyl cette (s se mteril) nd c. 0.25 g 0 ml -1 ntmycin s ctive component (indicted y the supplier). Microil nlysis of Stphylococcus spp. ws performed in Bird-Prker Agr Bse, BPA (HIMEDIA) supplemented with egg yolk nd telurite emulsion (Fluk Chemik); Pseudomons spp. in Pseudomons Agr F, PAF (DIFCO Lortories); Enteroctericee in Violet Red Bile Glucose Agr, VRBGA (HIMEDIA) with Tripton Soy Agr, TSA (HIMEDIA) nd yest nd molds in Rose-Bengl Chlormphenicol Agr, RBC 35

Journl of Hygienic Engineering nd Design (Merck). Peptone wter (Merck, Germny) ws used for the smple deciml dilutions. All other chemicls were regent-grde or etter, nd were used without further purifiction. 2.2 Cheese Cylindricl nd semi-hrd ovine cheeses of pproximtely 120 g were produced in Escol Superior Agrári (ESAC) fcilities (Coimr, Portugl) nd used s trditionl Portuguese rw milk cheese food model mteril for whey protein-sed coting ppliction. Stndrdized (3.5 ± 0.1 g ft 0 g -1 ) ovine milk ws heted nd mintined t (30 ± 0.5 C) during cogultion (45 min) in the presence of 0.04 g 0 ml -1 CCl 2 solution (36 g 0 g -1, Betegeux), mg L -1 of Mesófilo Plus Strter (Ais, Spin), 25 mg L -1 of Lysozyme nd 20 mg L -1 of rennet (> 92 g 0 g -1 chimosin, Tecnilc-Portugl) previously diluted in tp wter. The curd ws molded in plstic molds nd stored t 5 ± 1 C nd 80 ± 3% reltive humidity during one dy, efore coting ppliction. 2.3 Coting production nd ppliction The coting formultion developed y Rmos [17] ws the sis for the coting formultion used in this reserch with some specific dpttions. The protein se mteril, WPI in the former, ws replced y ovine WPC t the sme protein content ( g 0 g -1 ) nd the mount of ntmycin s ntimicroil ws reduced from 0.025 g 0 ml -1 to 0.0125 g 0 ml -1, in order to etter evlute the ntimicroil effect of -irrdition. All the remining coting ingredients were mintined t the optimized concentrtion chieved y Rmos [17]: glycerol ws dded t 50 g 0 g -1 (protein sis), gur gum (0.7 g 0 g -1 ), sunflower oil nd tween 20 ( g 0 g -1 nd 0.2 g 0 g -1, respectively), nd the ntimicroil gent lctic cid t 0.6 g 0 ml -1. Three different types of whey protein-sed ntimicroil cotings were mde for cheese ppliction ccording to ech polymeriztion method: het denturtion (HD), polymeriztion () nd oth methods comined (). Cheeses coted with the whey protein cotings were compred to the negtive control (uncoted cheeses) nd positive control (cheeses with commercil coting). The production of WPC cotings y the HD technique implies tht the therml tretment nd consequently protein denturtion occurs in the coting solution efore the coting ppliction to the cheese surfce. Glycerol nd WPC were homogenized in deionized wter until complete dissolution. Susequently the solution ws heted in wter th t 80 ± 2 ºC for 20 min., under continuous gittion, nd cooled down to pproximtely 30 ºC. Gur gum, sunflower oil nd tween 20 were dded under stirring for c. 20 min. t room temperture. Afterwrds the ntimicroil gents; lctic cid nd ntmycin were dded nd ph ws djusted to 7.0 using NOH (40 g L -1 ). The coting solution ws homogenized using T25 Ultr-Turrx (IKA from Stufen, Germny) t 000-13000 rpm for c. 2 min efore ppliction into cheeses. In the polymeriztion method, protein polymeriztion y irrdition only occurs fter the coting solution ppliction to the cheese surfce. The coting solution ws prepred s descried in the forementioned method, without ny het tretment. During coting solution preprtion the photoinititor t 3.5 g 0 g -1 (protein sis) ws lso dded under continuous stirring. Antimicroil compounds were incorported into the edile coting mtrix followed y ph djustment. The coting solution homogeniztion ws performed in the sme conditions mentioned previously nd pplied to the cheese surfce tht ws exposed for min. on ech side t direct irrdition using lmps (G8 T5-8W, PHILIPS ) t c. 23 cm from the cheeses. The comintion of oth polymeriztion techniques implies the protein denturtion in the solution y het tretment, efore coting ppliction, nd the protein polymeriztion, fter the coting ppliction to the cheese surfce. The coting composition nd preprtion steps were similr to the forementioned methods. The ltter nd the commercil coting (Redom D) were directly pplied on the cheese surfce one dy fter cheese mnufcture. Cotings were mnully pplied y rushing until ll cheese surfces were covered with the residul coting eing llowed to drip off. Cheeses were then stored in n pproprite chmer for 45 dys, t 11 ºC nd 85% RH y turning them from time to time. The coted cheeses were compred with their uncoted counterprts. 2.4 Coting solutions rheologicl nlysis Before ppliction to cheese, the rheologicl ehvior of the coting solutions ws evluted sed on their pprent viscosity determined on controlled stress rheometer (Rheostress 1 - RS1, Hke, Thermo Fisher Scientific, Germny) using prllel plte sensor (TP20 Ti, 20 mm dimeter, 115 mm gp). Flow tests were crried out t 20 ± 0.1 ºC with upwrd nd downwrd liner sher rte rmps etween 0.1 nd s -1. For ech thixotropic cycle, the sher rte ws incresed over period of 0 s, held t the upper limit for 20 s nd then decresed gin over period of 0 s. Thixotropy ws recorded s the resultnt re etween the upwrd nd 36

Journl of Hygienic Engineering nd Design downwrd sher stress (s) curves s function of sher rte (g). For ech coting solution three mesurements were performed. The upwrd sher rte rmp ws used to determine the consistency index (k) nd the power lw fctor (n) ccording to the Power Lw model eqution: ( g) n 1 h = k (1) where h is the pprent viscosity (P.s) nd g is the sher rte (1/s). 2.5 Physico-chemicl nlyses Physico-chemicl nlyses of cheeses were ssessed in triplicte, y 1, 15, 30 nd 45 dys fter coting ppliction. Weight loss ws determined y individully cheese weighing with Mettler Toledo PB2 (Switzerlnd) nlyticl lnce t the eginning nd during the storge period. The percentge of the reltive weight loss ( W) ws clculted sed on eqution 2. Iwo Fwi % W = 0 (2) I wo where I wo is the initil cheese weight nd F wi is the finl cheese weight t time i. Cheese moisture content ws grvimetriclly determined ccording to the Portuguese stndrd method (NP 3544 [36]). Protein evlution ws performed y the Kjeldhl method (ISO 8968-1 [37]) t the first dy of storge nd fter 45 dys. The ft content ws determined using the Vn Gulik method (NP 25 [38]). Totl chloride in cheese ws determined using the officil AOAC method 935.43 [39]. The ph of cheeses ws mesured directly using ph meter (PHM61 Lortory ph Meter, Denmrk) equipped with proe for reding solids nd the titrtle cidity expressed s g lctic cid 0 g -1 ccording to the method AOAC [40]. Wter ctivity ( w ) of cheese smples (representtive from the ulk nd cheese surfce) ws mesured fter temperture stiliztion (20ºC) using n hygrometer (Rotronic Hygroskop BT, Zurich, Switzerlnd) coupled with DMS 0H device nd equipped with WA- 14TH proe connected to thermosttic th. 2.6 Texture Cheese hrdness ws determined in Stle Micro Systems Texture Anlyzer, model TA.XT Express Enhnced used to perform texturl nlysis, fter dt tretment y the Specific Expression PC Softwre. Cheese texture profile nlysis (TPA) ws performed with penetrtion distnce of 15 mm t 2 mm/s test speed, using n crylic cylindricl proe with dimeter of 5 mm nd 38.1 mm of height. Three penetrtions were performed per cheese t distinct loctions. 2.7 Color Cheese color ws determined y portle colorimeter HP-2132, Zheijng Top Instruments Co, Ltd., previously clirted with stndrd white plte of known prmeters (L* stndrd = 97.03; *stndrd = -0.67; * stndrd = 5.57), using C illuminnt in the color spce CIEL***. The color of cheeses ws expressed y the individul three coordintes of CIEL*** nd y the totl color difference (ΔE). For ech type of coting nd ripening time, three cheese smples were mesured nd three redings per cheese were mde. 2.8 Microiologicl nlysis Microiologicl development on the cheese surfce ws evluted vi enumertion of vile cells, y 1, 15, 30 nd 45 dys fter ppliction of sid cotings. g of cheese were septiclly removed from the upper surfce re of ech cheese into stomcher g, nd ccordingly diluted to 1: (w/v) in sterile 1 g 0 ml -1 sodium citrte (Merck) nd lended in stomcher (Mstictor IUL Instruments) for 1.5 min t 260 rpm. Susequently, deciml dilutions were prepred with 0.1 g 0 ml -1 peptone wter nd plted, in triplicte, on the corresponding medi. Stphylococcus spp. were enumerted on Bird-Prker Agr Bse, BPA supplemented with egg yolk nd telurite emulsion, s originlly proposed y Bird-Prker [41]. Pseudomons spp. were counted on Pseudomons Agr F, PAF. Both medi were incuted eroiclly t 37 ºC for 48 h. Enteroctericee were counted on Violet Red Bile Glucose Agr, VRBGA with Tripton Soy Agr, TSA fter incution t 30 ºC for 48 h. Yests nd molds were determined fter 5 dys of incution t 25 ºC on Rose-Bengl Chlormphenicol Agr, RBC. Except for the enumertion of Enteroctericee on VRBGA (for which the pour plte technique ws used) the surfce plting technique descried y Miles et l. [42] ws followed for ll other smples nd growth medi. 2.9 Sensoril nlysis Sensoril tests were crried out t the end of the ripening period (45 dys), in the sensory room of ESAC y trined pnel of 12 memers, from oth genders - nd fmilir with trditionl Portuguese cheeses. Two tests were performed y ech pnelist; the first for the evlution of the glol cheese ppernce, where 37

Journl of Hygienic Engineering nd Design the whole cheese ws first nlyzed; nd the second for the evlution of cheese chrcteristics, using cheese slices of c. 1 cm thickness tht were plced on individul plstic Petri dishes coded using rndom tree-digit-code. A 5-point scle ws used y pnelists to evlute ll the ttriutes used to clssify whole cheeses smples nd sliced cheese smples. For whole cheese evlution, the sensoril ttriutes ssessed were: shpe (1 = not chrcteristic nd 5 = idel); rind color (1 = white nd 5 = drk yellow); color homogeneity (1 = heterogenic nd 5 = homogeneous); hrdness (1 = very soft nd 5 = very hrd). For cheese slices evlution the sensoril ttriutes were: differences etween pste nd ring color (1= imperceptile nd 5= intense); odor (1 = imperceptile nd 5 = intense); consistency (1 = very soft nd 5 = very hrd); flvor (1 = imperceptile nd 5 = intense) nd overll cceptility (1 = less ccepted nd 5 = most ccepted). For etter definition of coting ttriutes, pnelists were sked to include useful informtion in the oservtions section included on ech evlution crd. 2. Sttisticl nlysis Sttisticl nlysis of the dt ws crried out employing nlysis of vrince (ANOVA) pckge included in SttSoft Sttistic 8.0 (Hill nd Lewicki [43]). Tukey-HSD post-hoc test, with 95% confidence level, ws pplied to ssessed differences etween physicochemicl, microiologicl nd sensory properties of cheeses coted with WPC-sed cotings, commercil coting nd uncoted cheeses. One-Wy ANOVA tests were performed to compre the mens of the coting solutions rheologicl properties (thixotropy nd pprent viscosity) nd the cheese smples ttriutes used for the sensoril evlution of the whole cheese nd sliced cheese. Two-wy ANOVA with interction ws employed to determine the effects of oth storge time nd coting type on physico-chemicl properties (moisture, ft nd slt contents; weight loss, wter ctivity, titrtle cidity, ph; hrdness nd color) nd microiologicl properties (Stphylococcus spp.; Pseudomons spp.; Enteroctericee nd yest nd molds) of ripened cheeses. 3. Results nd Discussion 3.1 Cotings rheologicl properties The pprent viscosity of the WPC-sed cotings produced y the different polymeriztion methods (HD, nd ) ws nlyzed nd compred with the commercil coting mtrix viscosity, efore its ppliction into the cheese surfces in order to etter understnd in wht extent this property would influenced the coting phenomenon. These results re shown in Figure 1. For the entire rnge of sher rtes it ws oserved similr flow ehvior for ll the produced coting solutions; eing however clerly visile the distinction etween individul tch viscosities over the tested sher rte rnge. The shpe of coting solutions flow curves exhiited sherthinning ehvior, chrcteristic of pseudoplstic fluids, nd showed decresing pprent viscosity with incresing sher rte which is typicl of wekly ggregting dispersion systems [46]. Coting mtrixes sujected to het denturtion ( nd HD) hd the highest pprent viscosity vlues, while the commercil nd coting solutions displyed the lower viscosity vlues. These differences cn e relted to the mount nd nture of polymeric se mteril (e.g. whey proteins or PVA), coting dditives nd the method used to perform coting polymeriztion. It is importnt to notice tht in coting solution, the polymeriztion process is induced only fter its ppliction to the cheese surfce nd irrdition exposure. The smller viscosity otined for this cse corroortes tht, until this stge only colloidl prticles re present in the coting solution, without ny indiction tht polymeriztion hs strted. On the other hnd, the ppliction of therml process to the whey protein coting solutions promotes protein denturtion nd the eginning of polymeriztion efore coting ppliction leding to higher viscosity vlues. h(p.s) 00 0 1 0,1 0 1 2 3 4 5 6 7 8 9 g (1/s) HD Commercil Figure 1. Apprent viscosity (h) s function of sher rte of the ntimicroil WPC coting solutions produced y ( ) HD; ( ) nd ( ) polymeriztion compred with ( ) commercil coting solution, efore its ppliction to the cheese surfce Thixotropic or hysteresis loops were generted for the tested coting smples s shown in Figure 2 nd the thixotropy (P/s) plotted in Tle 1. It ws oserved tht HD coting solution showed the higher thixotropic 38

Journl of Hygienic Engineering nd Design ehvior. The presence of the photoinititor in formultion my proly contriute to more effective nd ccurte moleculr rerrngement of dentured proteins fter het tretment, explining the distinct rheologicl ehvior etween oth smples. similr thickness it ws decided to pply the coting solutions mnully with rush until ll the cheese surfces were covered. 3.2 Physico-chemicl profile s (P) 0 80 60 40 HD s (P) 5 4 3 2 Commercil Physico-chemicl properties of cheese were ssyed y 1, 15, 30 nd 45 dys of storge nd re displyed in Figure 3 (weight loss, wter ctivity ( w ), moisture, ft, protein nd slt (NCl) contents), Tle 2 (titrtle cidity nd ph) nd Figure 4 (hrdness). 20 0 0 1 2 3 4 5 6 7 8 9 g (1/s) 1 0 0 1 2 3 4 5 6 7 8 9 g (1/s) Figure 2. Thixotropic loops of the ntimicroil WPC coting solutions produced y ( ) HD, ( ) nd ( ) polymeriztion compred with the ( ) commercil coting solution, efore its ppliction to the cheese surfce The power lw model (eqution 1) ws pplied in order to descrie the flow ehvior of coting solutions t the cheese surfces y determining the consistency index (k) nd the power lw fctor (n). The consistency index (Tle 1) ws in greement with the forementioned oservtions. Significnt higher vlues (P < 0.05) were otined for nd HD coting solutions (36.68 nd 29.30 P.s n, respectively) ginst 0.81 P.s n for the nd 0.65 P.s n for commercil coting solution. According to the power lw fctor no significnt differences were oserved etween smples nd the pseudoplstic flow ehvior ws confirmed (n < 1) for ll the coting solutions. According to these findings it ws possile to predict tht during coting ppliction more viscous solutions would exhiit higher dherence to cheese surfces thn the less viscous ones, which could result in very distinct coting thickness mong cheeses if dipping process is used. In order to produce cotings with Weight loss (%) Moisture (g 0 g -1 ) Protein (g 0 g -1 ) 0 80 60 40 20 Uncoted HD Commercil 0 70 time (dys) c 60 50 40 30 20 35 Uncoted e 30 HD 25 20 Commercil 15 5 1 15 30 45 time (dys) w Ft (g 0 g -1 ) NCl (g 0 g -1 ) 1,00 0,95 0,90 0,85 0,80 50 time (dys) d 40 30 20 time (dys) f 8 6 4 2 0 time (dys) Figure 3. Weight loss (n = 3), wter ctivity ( w ) (n = 3), moisture (n = 9), ft (n = 3), protein (n = 9) nd slt (NCl) (n = 6) contents of cheese smples coted with ntimicroil whey protein edile cotings produced y ( ) HD, ( ) nd ( ) polymeriztion compred with ( ) uncoted cheese nd ( ) cheese with commercil coting, during 45 dys of ripening t 11 ºC nd 85% RH Tle 1. Thixotropy, consistency index (k) nd power lw fctor (n) of the power lw model of the ntimicroil WPC coting solutions produced y HD, nd polymeriztion compred with the commercil coting solution Coting type Thixotropy (P/s) k (P.s n ) Power lw model n HD 43.30 ± 0.35 c 29.30 ± 13.39 0.35 ± 0.13 3.35 ± 0.70 0.81 ± 0.08 0.37 ± 0.02 24.08 ± 12.7 36.68 ± 0.32 0.44 ± 0.01 Commercil 4.40 ± 0.84 0.65 ± 0.13 0.41 ± 0.08,,c mens (n = 3) ± stndrd devition with different letters within column re significntly different (P < 0.05) 39

Journl of Hygienic Engineering nd Design Tle 2. Titrle cidity (g lctic cid) nd ph of cheese smples coted with ntimicroil whey protein edile cotings produced y HD, nd polymeriztion compred with uncoted cheese nd cheese with commercil coting, during 45 dys of ripening t 11 ºC nd 85% RH Coting type Ripening time (dys) 1 15 30 45 Uncoted 0.42 ± 0.09 A 0.97 ± 0.03 B 0.95 ± 0.02 B 1.08 ± 0.15 B Titrtle cidity (g lctic cid) HD 0.42 ± 0.09 A 0.96 ± 0.07 B 0.98 ± 0.16 B 1.13 ± 0.40 B 0.42 ± 0.09 A 1.04 ± 0.05 B 1.05 ± 0.02 B 1.16 ± 0.16 B 0.42 ± 0.09 A 1.29 ± 0.06 B 1.54 ± 0.05 cc 1.40 ± 0.09 BC Commercil 0.42 ± 0.09 A 1.06 ± 0.07 B 1.17 ± 0.03 B 1. ± 0.14 B Uncoted 5.19 ± 0.23 B 4.62 ± 0.04 A 4.69 ± 0. A 4.76 ± 0.19 A HD 5.19 ± 0.23 B 4.56 ± 0.04 A 4.37 ± 0.06 A 4.69 ± 0.07 A ph 5.19 ± 0.23 B 4.49 ± 0.02 A 5.03 ± 0.05 cb 4.81 ± 0.06 AB 5.19 ± 0.23 C 4.49 ± 0.01 A 4.93 ± 0.03 cb 4.70 ± 0.05 AB Commercil 5.19 ± 0.23 B 4.73 ± 0.03 A 4.62 ± 0.03 A 4.57 ± 0.02 A mens (n = 6) ± stndrd devition. mens (n = 9) ± stndrd devition. A,B mens ± stndrd devition with different cpitl letters re significntly different (P < 0.05) during ripening time for the sme coting type (line).,, mens ± stndrd devition with different smll letters re significntly different (P < 0.05) etween coting types t the sme ripening dy (column). Hrdness (g) 8000 6000 4000 2000 Uncoted HD Commercil 0 time (dys) nd > 6000 g Figure 4. Hrdness of cheese smples coted with ntimicroil whey protein edile cotings produced y ( ) HD, ( ) nd ( ) polymeriztion compred with ( ) uncoted cheese nd ( ) cheese with commercil coting, during 45 dys of ripening t 11 ºC nd 85% RH Our nlyses provided informtion on how cheese weight loss throughout 45 dys ws ffected y the presence of coting, its type nd the influence of the polymeriztion method used. In Figure 3, one finds n increse in weight loss for ll cses throughout storge; such increse is sttisticlly higher (P < 0.05) during the first 30 dys, exception mde for the commercil coting whose weigh loss is less pronounced fter 15 dys. No differences in weight loss (P > 0.05) were oserved mong coted cheeses. However, WPC coting produced y method nd the commercil coting hd the est performnce. Cheeses covered with WPC edile cotings produced y polymeriztion showed sttisticlly (P < 0.05) lower vlues thn uncoted cheeses, which presented the higher weight loss. Wter ctivity ( w ) is the min fctor ffecting cheese stility during ripening displying reltively high vlues (0.87-0.92) in some wy expected since this type of cheese generlly hs w close to unity [45]. This property remins prcticlly constnt during the entire storge period for ll tested cheeses, with no significnt differences (P > 0.05) during the first 30 dys. Only for uncoted cheeses nd cheeses coted with WPC produced y HD nd polymeriztion (Figure 3) significntly decrese (P < 0.05) etween the 30 th nd 45 th dy of study ws oserved. The wter loss (Figure 3c) is the min fctor pointed s responsile for tht decrese though protein degrdtion y relese of croxyl nd mino groups my lso contriute to wter ctivity decrese [46]. Cheese moisture significntly decreses (P < 0.05) during storge, displying moisture losses from c. 33.4 to 40.0% for coted cheeses. The moisture loss profile (Figure 3c) ws distinct etween cheeses during the ripening period, with significnt differences (P < 0.05) etween them t the 15 th nd 45 th dy. For uncoted cheese 40

Journl of Hygienic Engineering nd Design nd cheese with HD coting, the moisture loss ws less pronounced during the first 15 dys, while the remining smples showed the opposite trend. At the end of the ripening period cheeses coted with WPC cotings produced y the nd method presented the highest levels of humidity, followed y the commercil coted cheese nd finlly y HD coted nd uncoted cheese. The fster decrese in moisture oserved t the eginning of the storge for cheeses coted with WPC when the polymeriztion ws used, nd its similrity to the moisture profile of commercil coted cheese my suggest nlogous wter vilility conditions to microil prolifertion which is crucil t the first dys of ripening. These results prove tht the presence of coting, its nture nd polymeriztion method influence the wter trnsfer phenomenon etween cheeses nd their surroundings during ripening. Moreover, fter 45 dys, the presence of whey protein ntimicroil edile cotings decresed the weight loss nd moisture loss of cheese in 5.8% nd 4.4% respectively, if the coting ws prepred y HD; 8.0% nd.9% when only polymeriztion ws used; 11.2% nd.2% using cotings prepred y HD + ; while in the cse of commercil coting those vlues decresed y.4% nd 6.4% respectively. Thus, whey protein edile cotings hd etter or similr performnces compred to the commercil one, exception mde to the HD coting. Ft (Figure 3d) nd protein content (Figure 3e) incresed (P < 0.05) during the ripening period for ll cheese smples independently of the presence of coting. However, this evolution ws coting type dependent nd vries in n opposite wy to the cheese moisture content. Dried cheeses hve higher percentge of ft. It ws lso oserved tht the protein nture of the WPC edile cotings did not ffect the protein composition of cheese, since no differences (P > 0.05) were oserved etween cheeses fter coting. The slt content (Figure 3f) ws not ffected y the presence of coting (P > 0.05); however during ripening it ws oserved significntly (P < 0.05) increse. Cheese with WPC coting produced y hd the lower increse (0.43%) nd very similr vlues to commercil coted cheese. WPC coted cheese produced y HD showed the more pronounced vrition (1.36%). Tle 2 shows results for cheese titrtle cidity nd ph vrition throughout ripening. During the first 15 dys, titrtle cidity significntly incresed (P < 0.05) oth for coted nd uncoted cheese smples. Cheese owing WPC coting produced y presented significntly higher vlues (1.40 g lctic cid) thn its counterprts. The ctivity of indigenous cultures of lctic cid cteri tht metolize lctose to lctte is responsile for the production of cids which results in cidity increse nd consequent ph reduction. The oserved ph decrese etween the 1 st nd the 45 th dy of storge (Tle 2) showed some vritions nd did not follow the opposite ehvior of titrtle cidity. However, t the 30 th dy of storge significnt differences were otined (P < 0.05) etween ph mong smples. Figure 4 presents the hrdness vlues for cheese smples throughout storge showing significnt increse (P < 0.05) in ll cses. It ws oserved tht the presence of coting, its nture nd the method of polymeriztion considerly influenced this prmeter. Some uthors (Cerqueir et l. [47], Rmos [17]) pointed tht cheeses with lower moisture content re hrder. This reson could justify our results during the first 15 dys of ripening when cheeses with higher moisture content (uncoted, HD nd ) (Figure 3c) hd lower hrdness vlues (Figure 4). The moisture content for the different cheese smples ecome similr t the 30 th dy of storge ut hrdness vlues were significntly different t this point nd t the end of the ripening period (45 th dy). The most dehydrted cheeses (uncoted nd HD) hd the lower hrdness vlues, while cheeses tht hd significntly higher humidity (, nd commercil) showed higher hrdness vlues. It is importnt to notice tht cheese hrdness determintion does not depend exclusively of the cheese ulk consistency ut it is lso influenced y the rind consistency; where the polymeric mteril used in coting formultion nd the type of chemicl interctions tht occurred during the coting formtion ply n importnt role. The similrity in hrdness profiles otined for cheeses coted with commercil coting nd coted y WPC coting produced method (Figure 4) my indicte tht the moleculr nd chemicl interctions hve the sme nture, despite the distinct polymeric se mteril (PVA for the commercil coting). The fster drying nd rind formtion during the first dys of ripening tht occurred in the forementioned cheeses, led to hrder crust which prevented further dehydrtion from the cheese ulk. 3.3 Cheese ppernce The ppernce of cheeses coted with ntimicroil WPC-sed cotings produced y the three different polymeriztion methods (HD, nd ) ws compred with uncoted cheese nd commercil coted cheese during the 45 dys of ripening (Tle 3). 41

Journl of Hygienic Engineering nd Design Tle 3. Appernce of cheeses of c. 120 g, coted with ntimicroil whey protein edile cotings produced y HD, nd polymeriztion compred with uncoted cheese nd cheese with commercil coting, during 45 dys of storge t 11 ºC nd 85% RH. Arrows presence of molds Coting Ripenning time (dys) 1 15 30 45 Uncoted HD Commercil During coting ppliction it ws oserved tht coting solutions with lower viscosity, especilly the coting solution, resulted in lower dherence to the cheese surfce originting significnt dringe from it. Het denturtion (HD nd ) ws responsile for incresing coting solutions viscosity contriuting to good dherence. The dopted coting procedure (spreding the coting solution with rush) is resonly efficient for the cotings with higher viscosities, since it llowed for the production of thinner cotings, less sticky nd with higher drying rtes during storge. However, the use of this method in low viscosity cotings my proly compromise the coting efficiency s result of the extremely thinner cotings produced. For this reson the use of n lterntive dipping procedure could e etter lterntive to improve coting thickness in these cses. Identicl nd homogeneous ppernce of the vrious cheese surfces (top, ottom nd lterl surfces) ws oserved one dy fter coting ppliction with no visul differences etween coted nd uncoted cheese, neither mong the different types of coted cheeses (commercil nd WPC-sed cotings). The most relevnt visul chnges in cheese throughout ripening occurred during the first 15 dys (Tle 3). 42

Journl of Hygienic Engineering nd Design The initil white color of cheese nd wet ppernce gve rise to dry ppernce nd light yellow color. By visul inspection it ws not esy to detected color differences etween uncoted nd coted cheeses, ut with respect to microil growth, uncoted cheese presented significntly mount of molds t its surfce fter 30 dys of storge, which incresed until the 45 th dy. The presence of some molds on WPC cotings produced y HD or polymeriztion ws lso oserved ut in much smller extension (Tle 3). Possile resons include the lesser ntimicroil efficiency of these cotings or proly the insufficient mount of coting deposited on the cheese surfce. It ws not possile to visully detect the presence of molds in commercil coted cheese nd in cheese with ntimicroil WPC coting produced y polymeriztion during the 45 dys. In oth cses similr good ppernce ws chieved. Color nlysis sed on L*, *, * coordintes nd color difference ( E*) (Figure 5) confirmed tht ll cheese smples chnged their color throughout storge; with sttisticlly significnt differences (P < 0.05) recorded etween them. The most pronounced color chnge occurs during the first 15 dys of storge. Lightness (L*) decresed significntly from ner 95 (white smples) to pproximtely 70; * vlues chnge from negtive (green) to positive vlues (red) nd * incresed in the positive xis direction from c. to 20 (yellow direction) (dt not shown). After tht period nd during the following 30 dys, color chnges were not so pronounced; however the presence of coting nd the coting polymeriztion method plys n importnt influence on L* nd E* vlues (Figure 5). It ws oserved tht the use of het denturtion to produce cotings (HD nd ) leds to drker cheeses thn uncoted ones. A possile reson is tht the high lctose mounts (28.28 g 0 g -1 ) present in WPC, when exposed to therml tretments induce chemicl Millrd rowning rections. On the other hnd, cheeses coted with commercil coting nd WPC cotings produced only y polymeriztion exhiited lower color differences thn uncoted cheeses. Sid color chnge for uncoted cheese cn e ttriuted in prt, to oxygen nd light oxidtion, which is lower in coted cheeses due to the reduction in oxygen permeility nd higher opcity [47]. Cheese dehydrtion rte during ripening, tht ws lower for cheeses with commercil nd coting, my lso e ssocited to less dry nd therefore less drk cheese rind t the end of the study. It ws lso mentioned y Cgri et l. [48]) tht cotings with lctic cid incorportion nd its cidulnt feture hve the cpcity to reduce color chnge. However, our results did not show tht ehvior y compring WPC sed cotings (which contined lctic cid s ntimicroil gent) with the commercil counterprts, proly due to the use of WPC insted WPI. The presence of higher lctose concentrtions in WPC nd its tendency to ecome drker during time proly msked the lctic cid effect [49]. L* E* 0 90 80 70 60 50 50 40 30 20 Uncoted HD Commercil 1 15 30 45 time (dys) Figure 5. Color coordinte L* nd color difference E* of cheese coted with ntimicroil whey protein edile cotings produced y HD, nd polymeriztion compred with uncoted cheese nd cheese with commercil coting, during 45 dys of storge t 11 ºC nd 85% RH., mens with different letters differ significntly (P < 0.05) etween coting types t the sme ripening dy 3.4 Microiologicl profile The ntimicroil performnce of the vrious WPCsed cotings, with lctic cid nd ntmycin s ioctive gents, ws scertined using set of spoilge/pthogenic microflor frequently found on the cheese surfce, especilly in rw milk cheeses. Stphylococcus spp. working s grmpositive cterium model; Pseudomons spp. nd Enteroctericee s grm-negtive cterium model nd finlly yests nd molds. The microiologicl results of the cheese smples during ripening re presented in Figure 6. These dt shows tht ll the evluted types of pthogenic or contminnt microorgnisms were detected t the cheese surfce. Grm-positive cteri (Stphylococcus spp.) were found in lower levels (< 6.5 log (cfu g -1 )) thn grm-negtive cteri (Pseudomons spp. nd Enteroctericee) or even yests nd molds. Microiologicl nlysis lso indicted tht y 45 dys of storge, there re sttisticl differences (P < 0.05) etween uncoted cheese nd cheeses with the tested ntimicroil cotings. 43

Journl of Hygienic Engineering nd Design Log (cfu g -1 ) Log (cfu g -1 ) 12 11 9 8 7 6 5 4 3 12 Enteroctericee 11 9 8 7 6 5 4 Stphylococcus spp. Uncoted HD Commercil 3 time (dys) Pseudomons spp. Yests & Molds time (dys) Figure 6. Vile cell counts (log(cfu g -1 )) (n =3) of Stphylococcus spp., Pseudomons spp., Enteroctericee nd yests nd molds in cheese smples coted with ntimicroil whey protein edile cotings produced y ( ) HD, ( ) nd ( ) polymeriztion compred with ( ) uncoted cheese nd ( ) cheese with commercil coting, during 45 dys of storge t 11 ºC nd 85% RH The est results for microil growth control or inhiition were found for cheese coted with ntimicroil WPC-sed edile coting produced y the polymeriztion method, independently of the evluted microorgnism. It ws very cler the microil inhiition of Stphylococcus spp. nd the growth control of Enteroctericee (Figure 6) in this coting type in contrst to the performnce of the remining cotings ginst grm-positive nd grm-negtive cteri. The results otined for yests nd molds showed similr performnce (P > 0.05) for nd commercil coting. In oth cses it ws not displyed growth of yests nd molds y comprison with uncoted cheeses or cheeses with nd HD cotings. Furthermore, significnt reduction (P < 0.05) of these types of microorgnisms ws recorded fter 30 dys of storge. This outcome ws somehow expected for the commercil coting, since it includes ntmycin s ctive compound tht hs well-estlished success in preventing growth of yests nd molds on cheese surfces [23]. However the effectiveness ginst this type of microorgnisms y the ppliction of the coting which hd reltive lower mount of ntmycin (0.0125 g 0 ml -1 ginst 0.25 g 0 ml -1 in the cse of the commercil coting) turns it extremely ttrctive s n efficient sustitute. According to these results the first postulted hypothesis for tht ehvior ws the possile germicidl effect of -irrdition promoted during the polymeriztion process. The irrdition hs een studied s n efficient method of cteril growth inctivtion. The germicidl effects re minly due to DNA muttions induced through sorption of light y DNA molecules [50 nd 51]. It ws lso mentioned tht grm-positive cteri shows higher resistnce thn grm-negtive cteri to irrdition exposure [52 nd 53]. However it ws not cler if this difference in susceptiility to -rdition is cused y the difference in the cell-wll structure etween the two types of cteri, ecuse grmpositive cteri hve mny lyers of peptidoglycn, forming thick nd rigid cell wlls, while grm-negtive cteri hve only single lyer or few lyers of peptidoglycn [54]. From the oservtion of the microiologicl results (Figure 6) the germicidl effect of irrdition is not cler since the ntimicroil efficiency in coting, in which only polymeriztion ws pplied, ws extremely poor, concerning to the growth prevention of Stphylococcus spp., Enteroctericee nd especilly for yests nd molds. A possile explntion for tht cn e the very short time of exposure to -irrdition (only min t the eginning of the ripening period) tht my e not sufficient for the germicidl effect, ut essentil for the polymeriztion process strts to occur. Another possiility tht cn e pointed ws the extremely low thickness of this coting type s result of the reduced dhesion to the cheese surfce during coting, which limited the coting effectiveness. 3.5 Sensoril profile The results oserved in sensory nlysis of cheese smples coted with ntimicroil whey protein edile cotings nd with commercil coting (or none) re presented in Tle 4. Sensory ssessment ws performed for the externl ttriutes (whole cheese evlution) nd lso for the internl cheese ttriutes (sliced cheese). After the externl evlution, ll the cheese smples were mnully wshed nd dried t mient temperture in order to eliminte ny contminnt from the cheese surfce to use them for internl sensory evlution. 44

Journl of Hygienic Engineering nd Design Tle 4. Whole cheese nd sliced cheese sensoril evlution in 5 point scle (mens ± stndrd devition) etween cheese coted with ntimicroil whey protein edile cotings produced y HD, nd polymeriztion compred with uncoted cheese nd cheese with commercil coting, fter 45 dys of storge Sensoril test Attriutes Coting type Uncoted HD Commercil Whole cheese shpe 3.21±0.78 3.58±0.72 3.46±0.96 2.90±0.87 2.77±0.89 rind color 2.94±0.59 3.43±0.81 3.46±0.68 2.85±0.40 3.82±0.69 color homogeneity 3.30±0.85 4.02±0.64 3.07±0.92 2.91±0.75 2.56±0.91 hrdness 4.33±0.34 4.13±0.43 4.59±0.27 4.03±0.59 4.43±0.52 Sliced cheese differences etween pste nd ring color 3.75±0.83 3.44±0.70 3.47±0.66 3.30±0.80 2.96±0.82 odor 3.06±0.83 3.19±0.82 3.19±1.13 3.23±1.11 2.69±0.78 consistency 3.90±0.67 3.93±0.47 3.49±0.56 3.65±0.64 3.43±0.66 flvor 3.17±0.83 3.23±0.90 3.19±0.63 3.43±0.87 3.22±0.39 overll cceptility 2.60±0.88 2.88±0.93 2.72±1.02 3.30±0.89 3.73±0.72,, mens (n = 12) ± stndrd devition with different smll letters in the sme line re significntly different (P < 0.05) etween coting types. With respect to externl cheese evlution (Tle 4) no differences were oserved (P > 0.05) etween cheeses concerning shpe nd rind color y visul inspection. Only, for color homogeneity nd hrdness, sensoril differences were found (P < 0.05). It ws oserved tht commercil coted cheese hd the lowest score in color homogeneity wheres the cheese with the coting produced y HD polymeriztion ws clssified s the most uniform. These results re consistent with the E* vlues otined ove (Figure 5) regrding color mesurement indicting tht commercil coting ws the lighter nd HD coting ws the drker t the end of the ripening period. In some pplictions, cheeses ering drker cotings could enefit in terms of homogeneity ecuse smller defects t the cheese surfce my e msked, mking them more ttrctive to consumers. Tested cheeses were clssified s hrd y pnelists scoring this ttriute with vlues higher then 4 in 5-point scle (very hrd). These results corroorte the ehvior of those cheeses during hrdness mesurements with vlues higher thn 3000 g (Figure 4). 45 dys of ripening, induced higher levels of dehydrtion on cheeses (out 40%), indicting tht the ripening period ws too long for the smll cheeses (120 g) used in this study. During ripening it ws oserved tht fter 30 dys cheeses displyed the idel texture. Concerning the internl cheese evlution, no sttisticlly significnt differences (P > 0.05) were otined mong cheeses ccording to the color difference etween pste nd rind, odor, consistency nd flvor. Nevertheless, it ws oserved tht for the first three ttriutes the cheese ering the commercil coting hd the lower clssifiction. In fct, the lighter rind color otined for the commercil cheese (Figure 5) led to lower color difference etween the rind nd pste. Cheese coted with WPC edile cotings hd more intense odor, proly due to the presence of lctose in the coting. However, this difference does not influence significntly the cheese flvor (P > 0.05). Rmos [17] reported tht cheeses coted with whey protein edile coting solutions exhiited itter flvor nd high stringency, ut this ws not oserved in our study. Finlly, in terms of overll cceptility, the uncoted cheese ws the less ccepted y the pnelists nd cheeses coted with edile cotings showed sttisticlly similr (P > 0.05) cceptility to the commercil coted cheese. 4. Conclusions - The distinct rheologicl ehvior of the whey protein coting solutions, s result of the pplied protein polymeriztion method is determinnt for the coting dhesion to the cheese surfce. More viscous coting solutions were produced when het denturtion ws pplied leding to etter dhesion. - The polymeriztion method significntly influences the performnce of WPC-edile cotings in the physico-chemicl, microiologicl nd sensoril chrcteristics of cheeses. The ppliction of -irrdition enhnced whey protein coting properties, especilly in terms of protein crosslinking during the coting formtion. - Antimicroil edile cotings sed on WPC showed tht cn e used s less expensive nd 45

Journl of Hygienic Engineering nd Design suitle lterntive to edile cotings sed on WPI or commercil cotings since cheese smples coted with either cotings displyed similr (P > 0.05) vlues in terms of physicochemicl, microiologicl nd sensoril properties, prticulrly in the cse of the whey protein edile coting produced y het denturtion nd polymeriztion (). -tretment of WPCsed cotings my disply improved functionlity nd provide opportunities for incresed utiliztion of this technology in the food industry, deserving therefore reserch ttention. 5. References [1] Olivs G. I. nd Bros-Cánovs G. V. (2005). Edile cotings for fresh-cut fruits. CRC Criticl Reviews Food Science, 45, pp. 657-670. [2] Min S., Hrris L. J. nd Krocht J. M. (2006). Inhiition of Slmonell enteric nd Escherichi coli O157:H7 on rosted turkey y edile whey protein cotings incorporting lctoperoxidse system. Journl of Food Protection, 69, pp. 784-793. [3] Min S. nd Krocht J. M. (2007). 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