Calcium for Extending the Shelf Life of Fresh Whole and Minimally Processed Fruits and Vegetables

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1 Dulin Institute of Tehnology Artiles Shool of Foo Siene n Environmentl Helth Clium for Extening the Shelf Life of Fresh Whole n Minimlly Proesse Fruits n Vegetles An Belen Mrtin-Din Dulin Institute of Tehnology, nelen.mrtinin@it.ie Dniel Rio Dulin Institute of Tehnology, niel.rio@it.ie Jesus Mri Fris Dulin Institute of Tehnology, Jesus.Fris@it.ie J. Brt Universi Politeni e Cmino e Ver Gry Henehn Dulin Institute of Tehnology, gry.henehn@it.ie Follow this n itionl works t: Prt of the Foo Miroiology Commons Reommene Cittion Mrtin-Din, A. et l. (27). Clium for extening the shelf life of fresh whole n minimlly proesse fruits n vegetles: review.trens in Foo Siene & Tehnology,18 (4),pp oi:1.116/j.tifs This Artile is rought to you for free n open ess y the Shool of Foo Siene n Environmentl Helth t ARROW@DIT. It hs een epte for inlusion in Artiles y n uthorize ministrtor of ARROW@DIT. For more informtion, plese ontt yvonne.esmon@it.ie, rrow.min@it.ie, rin.wiis@it.ie. This work is liense uner Cretive Commons Attriution- Nonommeril-Shre Alike 3. Liense

2 Effet of ozone n lium ltte tretments on rowning n texture properties of fresh-ut lettue D. Rio, A. B. Mrtin-Din, J.M Fris, GTM Henehn n Ctherine Brry-Ryn Shool of Foo Siene n Environmentl Helth, Dulin Institute of Tehnology (DIT), Cthl Brugh Street, Dulin 1, Ireln Astrt: The effets of three tretments, 1 mg L 1 ozone t 18 2 C, 15 g L 1 lium ltte (CL) t 5 C n omintion thereof, were ompre on fresh-ut lettue over 1 ys of refrigerte storge. Respirtion rte, rowning n texture were exmine s min qulity initors. The use of ozone proue signifintly (P <.5) higher oxygen eline thn the use of CL (from y 3 to y 1). At the en of storge, CL (lone or omine with ozone) smples h higher oxygen ontent ( 9%) thn ozone smples ( 6%). Enzymti tivit y erese signifintly (P <.5) in ozone smples. Polyphenol oxise tivity in fresh-ut lettue trete with ozone (lone or omine with CL) showe lower vlues on y 1 (<25 units g 1 ) n t the en of storge (<3 units g 1 ) thn CL smples (4 48 units g 1 ). Ozone lso reue peroxise tivity to 3 units g 1 fter tretment. Finlly, petin methylesterse tivity ws lso reue with ozone, showing negtive effet on texturl properties. Dt suggeste tht CL mintine qulity mrkers etter thn tretments with ozone n ozone/cl omintion over 1 ys of storge. Journl of the Siene of Foo n Agriulture, 86, Keywors: ozone; lium ltte; texture; rowning; Ieerg lettue INTRODUCTION Consumers inresingly require foo prouts tht preserve their nutritionl vlue, retin nturl n fresh olour, flvour n texture n ontin fewer itives suh s preservtives.1 Reent reserh hs unerline the importne of fruit n vegetle onsumption to helth n hs reporte new tehniques to preserve the nutritionl n sensory qulities emne y onsumers. Even though fresh-ut proue hs een sol sine the 194s, the qulity is unpreitle n the shelf life limite. The extension of qulity retention for fresh-ut prouts is relevnt for the inustry owing to its eonomi impt. It is importnt tht the wshing tretments pplie to fresh vegetles help mintin their qulity, sine onsumers emn fresh prout s well s onveniene n long shelf life.2 Tissue rowning is one of the mjor uses of loss of qulity of fresh-ut vegetles. Browning is influene y the onentrtion of phenoli ompouns n y other ftors suh s the tivity of polyphenol oxise n peroxise enzymes. Woun-inue loss of ellulr omprtmentlistion of phenoli ompouns (minly in the vuole) n polyphenol oxise (in the ytoplsm) results in tissue rowning t rte tht inreses with temperture n wter loss.3 The other use of qulity loss is erese in firmness. Tissue softening n ssoite loss of integrity n lekge of juie from some fresh-ut prouts n e the primry use of poor qulity n unmrketility. Chlorine solutions hve een wiely use to snitise fruit n vegetles in the fresh-ut inustry. However, the ssoition of hlorine with the possile formtion of rinogeni hlorinte ompouns in wter hs lle into question the use of hlorine in foo proessing.4,5 For tht reson, there is rel nee to fin lterntives for preservtion of fresh-ut vegetles in orer to improve the effiy of wshing tretments. This will le to inrese miroil sfety n exten the qulity retention of prouts. Alterntives or moifie methos hve een propose, e.g. ntioxints, irrition, ozone, orgni is, moifie tmosphere pkging, whey permete, et.,6 11 ut none hve yet gine wiespre eptne y the inustry. Clium ltte (5 3 g L 1 ) hs een use s firming gent for fruits suh s ntloupe, strwerry, lettue n others.1 14 It hs een reporte to e goo lterntive to lium hlorie euse it vois the itterness or off-flvours ssoite with tht slt15 n the ntiteril properties.1,16 Het shok omine with lium ltte hs een use to prevent rowning retions in vegetles n fruits Firming effets otine from het

3 tretments lone or omine with lium tretments hve een ttriute to the tion of het-tivte petin methylesterse n/or to inrese lium iffusion into tissues t higher tempertures.19,22 The min ojetive of this work ws to etermine the effet of lium ltte, n effetive n sfe iopreservtive, omine with het shok on freshut lettue n ompre it with tht of ozone, novel preservtive wshing solution. Ozone is strong ntimiroil gent with high retivity n penetrility n spontneous eomposition to non-toxi prout.23,24 Severl reserhers hve shown tht tretment with ozone ppers to hve enefiil effet in extening the storge life of rooli n seeless uumer.23,25 Although the ntimiroil pity of ozone hs een wiely reporte, few stuies on qulity hve een rrie out,26,27 n none hve evlute the potentil for synergisti effets of ozone omine with other wshing tretments. This stuy is fouse on the respirtion rte, pperne of rown isolourtion n loss of firmness thorough monitoring of suitle qulity mrkers. Experimentl esign Experiments were onute from Deemer 23 to My 24. All proeures were performe in speil foo-proessing room t 18 2 C. The tretmen ts were onute in prllel n prepre fro m the sme th of prout. Eh th ws 2 kg ( 5 hes of lettue of 4 g eh). The first wshing tretment onsiste of 1 mg L 1 ozone in istille wter, the seon tretment ws solution of 15 g L 1 lium ltte (CL) t 5 C n the thir tretment onsiste of first wshing the smples in 1 mg L 1 ozone n then treting them with 15 g L 1 CL t 5 C. Three inepenent trils (165 gs per th, 55 gs per tretment) were onute (Fig. 1). For eh tretment: three gs were use for hespe monitoring throughout the entire storge perio; sensory nlysis require two gs per pnellist (14), i.e. 28 in totl; lso, on eh of ys 1, 3, 7 n 1, two gs for texture, two gs for olour n two gs for enzymes were use fter eing opene n poole. For texture n olour, more thn 25 smples were nlyse per tretment n y. For enzymes, mesurements were uplite. MATERIALS AND METHODS Rw mteril Ieerg lettue (Ltu stiv sp.) ws grown in Ireln. The prout ws purhse from lol supermrket n store t 4 C for 2 4 h until use. Seletion of wshing tretments Previous work showe tht CL ws promising lterntive to the inustril stnr of 12 mg L 1 free hlorine in wshing tretments for fresh-ut lettue.1 CL tretment prove to e s effetive s hlorine tretment n enhne the nutritionl vlue of the finl prout. A lter extensive stuy of the effet of temperture n CL onentrtion Ieerg lettue. Eh th ws ~2 kg (~5 hes of lettue of ~4 g). Three inepenent trils were rrie out from De 23 to My 24 Miniml proessing (utting lettue) Wshing tretment (1 min) Ozone (1 mg L-1) Clium ltte (15 g L-1) t 5 Χ Ozone (1 mg L-1) Remove wter Clium ltte (15 g L-1) t 5 Χ Drie, poole, mixe n pkge in gs (2 mm 32 mm) 35 μ OPP Storge 1 ys t 4 Χ Figure 1. Flow igrm showing the proeure for the proution of minimlly proesse fresh-ut lettue. 2

4 on fresh-ut lettue ws investigte.11 The results showe tht 15 g L 1 CL t 5 C ws the optiml onition to reue rowning n mintin firmness (rispness).11,21,28 The seon tretment selete ws ozone s novel tretment to preserve fresh-ut vegetles, sine ozone hs een reporte to e n effiient reuer of rowning23,29 owing to its high retivity n penetrility n its spontneous eomposition to non-toxi prout. Following guielines n levels reporte in the literture, 1 mg L 1 ozone tretment ws use s optiml for mintining the shelf life in optiml n sfe onitions.3 Proessing (wshing n pkging proeure) Lettue smples were trete oring to miniml proessing proeures. Eh lettue ws prepre oring to epte prties. The two outer leves were remove n the ore ws exise with stinless steel knife. The lettue ws ut in hlf n eh hlf ws further ut into four piees. Different snitise knifes were use uring the proess in orer to voi ross-ontmintion. Wshing tretments were performe y immersion of the fresh-ut lettue in the ozone n CL tretment solutions. Ozonte wter ws generte y uling gseous ozone into istille wter. Ozone gs mixture ws proue using n tive oxygen genertor mhine (moel HV-13, Anlytil Tehnology In., Hmpshire, UK) onnete to issolve ozone monitor (moel C15-64, Anlytil Tehnology In.). The gs ws then pumpe into the system using n quti ir pump t flow rte of 2.5 L min 1. Ozonte wter ws hel in ontiner until it rehe onentrtion of 1 mg L 1 t 4 C. The ozone onentrtion ws mesure in the rnge 1 mg L 1 with sensitivity of.1 mg L 1 ove.5 mg L 1. Repetility ws ±.1 mg L 1 n linerity ws.5%. Clium ltte (Sigm, St Louis, MO, USA) ws ilute to 15 g L 1 (ph 6.5) in istille wter t 5 C. Eh tretment ws rrie out in ifferent sket ( 2 g vegetle prout L 1 ) y immersion in the orresponing wshing solution for 1 min with gittion n susequently rying for 5 min in n utomti sl spinner. To minimise prout heterogeneity, proesse vegetles were poole, mixe n susequently pkge in gs (2 mm 32 mm) of 35 m oriente polypropylene (OPP, Amor Flexiles Europe, Bristol, UK). The permeility of the film ws efine y oxygen (O2 ) n ron ioxie (CO2 ) trnsmission rtes of 12 n 13 ml m 2 y 1 tm 1 respetively t 5 C. Eh pkge ontine 1 g of prout. The pkges were hille in lst freezer t C for 2 min efore eing het sele uner tmospheri onitions. Qulity mrkers Vrious qulity mrkers were nlyse s initors of respirtion, rowning n texture: hespe, olour mesurement, rowning-relte enzymes, texturerelte enzymes, Instron n sensory nlysis. Hespe nlysis A Gspe nlyser (Systeh Instruments, Oxon, UK) ws use to monitor levels of CO2 n O2 uring storge. Gs extrtions were performe with hypoermi neele, inserte through n hesive septum previously fixe to the gs, t flow rte of 3 ml min 1 for 1 s, monitoring with sensitivity of.1 (O2 ) n.1 (CO2 ). Aury of reings ws: ±.5% elow 1% n ±.1% ove 1% for O2 ; ±.1% elow 1%, ±.2% etween 1 n 1% n ±2% ove 1% for CO2. Three gs per tretment were monitore for eh experiment, n the gs for other nlyses were mesure seprtely. The trils were lso triplite. Browning-relte enzymes: peroxise (POD, EC ) n polyphenol oxise (PPO, EC ) Both enzymes were ssye in homogentes tht were prepre s follows. Lettue (1 g) ws ple in homogeniser (Polytron moel PT 3, Ontrio, Cn) in 1:2 (w/v) rtio with.5 mol L 1 phosphte uffer, ph 6.5, ontining 5 g L 1 polyvinylpyrrolione. Homogenistion ws rrie out twie t 4 C n 55 rpm for 1 min eh time, with rek of 3 min etween homogenistions in orer to voi exess heting of the smple. The homogente ws then entrifuge t g for 3 min t 4 C n filtere through one lyer of repe nge. The resulting rue extrt ws use without further purifition. All extrts were store t 4 C in the rk n use immeitely. POD tivity ws ssye spetrophotometrilly. The retion mixture onsiste of.2 ml of extrt n 2.7 ml of.5 mol L 1 phosphte uffer, ph 6.5, ontining 1 L of hyrogen peroxie (1 ml L 1 ) s oxint n 2 L of p-phenylenimine s hyrogen onor. The oxition of p-phenylenimine ws monitore t 485 nm n 25 C. One unit of enzyme tivity ws efine s n inrement of.1 in sorne per minute. PPO tivity ws ssye spetrophotometrilly y moifie metho se on Refs 31 n 32. The retion mixture ontine.1 ml of rue extrt n 2.9 ml of sustrte solution (.2 mol L 1 tehol s sustrte in.5 mol L 1 phosphte uffer, ph 6.5). The rte of tehol oxition ws followe t 4 nm for 2 min t 25 C. One unit of enzyme tivity ws efine s n inrement of.1 in sorne per minute. Three inepenent trils were rrie out. All enzymti mesurements were me in uplite. Petin methylesterse (PME, EC ) PME tivity ws mesure using the metho esrie y Kimll.33 Briefly, 1 g of smple ws 3

5 ilute in n extrtion solution (.2 mol L 1 soium phosphte uffer, ph 7.5, ontining 1 mol L 1 soium hlorie n 1 mmol L 1 ithiothreitol) n homogenise t 4 C for 2 min t 55 rpm. The merte ws inute t 4 C for 3 min with gittion n then entrifuge t 12 5 g for 3 min t 4 C. A 1 ml liquot of this extrt ws mixe with 4 ml of sustrte solution (1 g L 1 petin). The solution ws juste to ph 7 with 1 mol L 1 NOH n then rejuste to ph 7.5 with.5 mol L 1 NOH. After the ph h rehe 7.5,.2 ml of 25 mmol L 1 NOH ws e. The time require to return to ph 7.5 ws reore. Ativity ws quntifie s roxyl groups forme y the hyrolysis of methyl esters of petin n ws mesure y titrtion using ph eletroe to monitor the proution of H+. PME tivity units were lulte using the formul33 PME = (25 mmol L 1 NOH) (X ml extrte) (.2 ml NOH) 16 /(1 ml smple) (prllel, olique or perpeniulr orienttion) with respet to the sher ell les iretly ffets the mesurement.34 For this reson, photosyntheti tissue with perpeniulr orienttion ws selete for the experiments. Vlues were expresse s (mximum lo - minimum lo)/mximum lo, whih ws efine s the rispiness oeffiient (CC).21 Three inepenent trils were rrie out. More thn 25 smples were nlyse per tretment n y. Sensory nlysis Sensory nlysis of lettue ws rrie out over 1 ys of storge y pnel of 14 untrine memers with n ge rnge of 25 4 yers. Fresh pperne of smples ws sore on heoni sle from (poor fresh pperne) to 5 (exellent fresh pperne). The sensory pnel ws selete from memers of the eprtment n the nlysis ws rrie out in the sensory evlution lortory. Dt nlysis ws performe using Compusense Five softwre (relese 4.4, Ontrio, Cn). Three inepenent trils were rrie out. (1 g smple) time (min) (1) Three inepenent trils were rrie out. All enzymti mesurements were me in uplite. Colour mesurement Colour ws quntifie using olorimeter (Hunter- L, Reston, UK). A lettue piee ws ple iretly on the olorimeter sensor (3.5 m imeter) n mesure; 2 3 mesurements were tken per tretment. The instrument ws lirte with white tile (L = 93.97, =.88, = 1.21) n green tile (L = 56.23, = 21.85, = 8.31) stnrs uner D65 luminosity onitions. Totl olour hnge E = [(Lf Li )2 + (f i )2 + (f i )2 ]1/2 ws nlyse, where Li = initil luminosity, Lf = finl luminosity, f = vlue t finl time, i = vlue t initil time, f = vlue t finl time n i = vlue t initil time. Three inepenent trils were rrie out. More thn 25 smples were nlyse per tretment n y. Texture nlysis Texturl properties of lettue were ssesse using texture nlyser (Instron 432 universl testing mhine, Cnton, MA, USA) with 5 N lo ell tthe. A Krmer sher ell with n eightle proe tthe to the instrument ws use. The spee setting for the experiment ws 1 mm min 1. Tissue segregtion n orienttion were one for the nlysis. All tests were performe with photosyntheti tissue. Retngulr piees (3 m 6.5 m) were ut n ple in the Krmer ell. Dt were nlyse with the Instron series IX softwre for Winows. The instrumentl mesurement of lettue texture is iffiult, minly owing to the high vriility of the prout. Lettue ontins two ifferent types of tissue (vsulr n photosyntheti) tht re not lwys esy to ifferentite n hve n irregulr istriution, n the reltive position of vsulr pkges Sttistil nlysis Anlysis of vrine (multiftor n one-wy ANOVA) ws use to fin ifferenes etween tretments, storge n their intertion for eh of the vriles stuie. Mens were ompre y the lest signifint ifferene (LSD) test t signifine level of P =.5 using Sttgrphis softwre (version 2.1, Sttistil Grphis Co., Rokville, MD, USA). Three inepenent trils were rrie out. RESULTS AND DISCUSSION Hespe nlysis Chnges in hespe gs omposition (oxygen n ron ioxie) of fresh-ut lettue were mesure over 1 ys. The oxygen ontent in the pkges erese uring storge, while the ron ioxie ontent inrese, s expete (Fig. 2). Chnges in oxygen n ron ioxie onentrtion were more rmti from y 1 to y 3 thn from y 3 to y 1 for ll tretments. Oxygen erese from its initil tmospheri onentrtion (21%) to vlues roun 15% fter 1 y of storge (Fig. 2()). This rpi erese reflete high respirtion rte, presumly use y the stress proue y miniml proessing.35 On the other hn, ron ioxie inrese signifintly (P <.5) in ll smples over the entire storge perio (Fig. 2()), with the highest inreses eing oserve from y to y 2. Wshing tretments signifintly ffete (P <.5) the oxygen n ron ioxie levels (Fig. 2). The use of ozone in the wshing tretment proue more rpi oxygen eline thn the use of CL in the storge perio from y 3 to y 1 (Fig. 2()). This ws evient t the en of storge, when smples trete with CL h higher oxygen ontents thn 2182

6 ozone-trete smples. The fresh-ut lettue wshe with the omine tretment h intermeite vlues, whih inites tht the stress proue y the use of ozone ws prtilly reue y the use of CL. The results inite tht CL use reution in the respirtion of lettue (Fig. 2()), whih is in greement with the finings of other uthors who suggeste tht CL reues the respirtion of minimlly proesse fresh vegetles.36,37 Sine the rte of respirtion of vegetle is relte to its senesene stte,38 lower onsumption of oxygen might e ue to lower stress response in lettue. The lower respirtion rte in CL-trete smples oul e expline y possile post-wouning protetive effet of CL t 5 C.21,28 Clium seems to mintin ell wll struture y interting with the peti i present in ell wlls to form lium pette, therey mintining wter tivity n thus reuing the stress usully ssoite with higher respirtion rtes.38 The higher respirtion rte in smples trete with ozone oul e ssoite with tissue n photosyntheti pprtus eing mge, whih might lter the metoli stte. Browning-relte enzymes (polyphenol oxise n peroxise) After tissue wouning, mny enzymes re relese t the woun site. Two of them, PPO n POD, re involve in tissue isolourtion (e.g. russet spotting, rusty rown isolourtion, et.), ontriuting to reution in qulity. Previous results showe signifint ifferenes in PPO n POD tivity in fresh-ut lettue epening on the type of tissue exmine (photosyntheti or vsulr).28 For this reson, the present stuy ws rrie out entirely with photosyntheti tissue to minimise vriility etween experiments. () Ozone() Ozone Ozone + CL Ozone + CL f CL CL Hespe gs (ron Hespe ioxie %) gs (oxy 18 gen 18 %) e B 9 9 AB B AB 6 A A Figure 2. Chnges in hespe gs omposition (() oxygen n () ron ioxie) in pkges of fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL ). Points esignte on ny urve y the sme letter re not signifintly ifferent (P >.5). Lowerse letters re use for omprisons uring storge n upperse letters for tretment omprisons. Three inepenent trils were rrie out in triplite. 5 Polyphenol B oxise (units 46 g-1) 42 AB 38 A 34 Ozone Ozone + CL CL Figure 3. Chnges in polyphenol oxise tivity in fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL). Points esignte on ny urve y the sme letter re not signifintly ifferent (P >.5). Lowerse letters re use for omprisons uring storge n upperse letters for tretment omprisons. Three inepenent trils were rrie out in uplite. 5

7 Figure 3 shows the PPO tivity profile uring storge of fresh-ut lettue. Smples trete with ozone n ozone omine with CL showe signifintly lower (P <.5) PPO enzymti tivities thn smples trete with CL lone. This erese in PPO tivity oul e use y the high oxition potentil of ozone ompre with CL. Although CL hs een esrie s n inhiitor of PPO synthesis28 uring storge, ozone showe higher effiy in the reution of these enzymes. Sftner et l.39 onsiere ozone to e n effiient orgni mtter oxint tht oul reue the miroil ontent, giving rise to lower respirtion rte, n erese enzyme synthesis.4 Intermeite PPO vlues in the smples trete with the omine metho n e ttriute to the opposing effets of the inrese in stress (oule wshing proess), whih might inrese enzyme tivity, n the ozone, whih ereses it. The oxitive effet of ozone might lso hve proue lower POD tivity (Fig. 4). Although signifintly (P <.5) lower vlues were oserve with the use of ozone tretments, either lone or omine, ompre with CL lone, these signifint ifferenes were only oserve t the eginning of storge (ys 1 3). From y 7, no effet of the tretment ws oserve. Texture-relte enzymes (petin methylesterse) PME tivity inrese signifintly uring storge for ll tretments, lthough showing flututions within the storge perio (Fig. 5). This irregulr ehviour my e ue to wouning response n/or to hnges in the soluility of the enzyme uring storge.41 Suh ehviour hs een oserve for ertin rowningrelte enzymes.42 Other uthors hve ttriute this vriility to intrinsi ftors n to pre- n 1 e Peroxise (units 8 g-1) A AB 6 B 4 2 Ozone Ozone + CL CL 1 Figure 4. Chnges in peroxise tivity in fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL). Points esignte on ny urve y the sme letter re not signifintly ifferent (P >.5). Lowerse letters re use for omprisons uring storge n upperse letters for tretment omprisons. Three inepenent trils were rrie out in uplite. Petin methylesters 68 e (mol COO- min-1 g-1) 58 B A 48 A Ozone 8 Ozone + CL CL Figure 5. Chnges in petin methylesterse tivity in fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL). Points esignte on ny urve y the sme letter re not signifintly ifferent (P >.5). Lowerse letters re use for omprisons uring storge n upperse letters for tretment omprisons. Three inepenent trils were rrie out in uplite. 6

8 posthrvest ftors, whih n ffet enzyme tivity, vitmin ontent, et.43 Fresh-ut lettue trete with CL h signifintly (P <.5) higher PME tivity vlues thn smples trete with ozone or the omine metho (Fig. 5). Previous stuies hve shown tht CL omine with het shok proues n tivtion of PME.11,19,22 The enefiil effets on texture of het tretments n CL solution hve een expline y the tivtion of PME.19 PME is responsile for leving the methoxyl groups from methylte peti sustnes, generting free peti is44 tht ontin newly ville roxyl groups. This tivtion of PME use y CL n het shok ws inhiite y the tretment with ozone. Smples trete with ozone in omintion with CL showe the sme levels of PME tivity s smples trete with ozone lone. This suggests stronger inhiitory effet on this enzyme (lso on PPO n POD) use y ozone tretment thn the tivtion proue y CL tretment. orreltion. Fresh-ut lettue trete with ozone n CL omine h similr reution in PPO (throughout the entire storge perio) n POD (uring the first y of storge) tivities to smples trete with ozone. Instron nlysis (rispiness oeffiient) The vrition in CC ws nlyse uring storge (1 ys) for ll smples (Fig. 7). During storge signifint (P <.5) reution in CC ws oserve in ll ses, whih ws ssoite with loss of turgor n fresh-like texturl properties. Signifint ifferenes etween tretments were foun. Smples trete with ozone showe the lowest vlues of CC, while smples trete with CL h the highest vlues. This fining might e prtly ssoite with PME tivtion (Fig. 6). The use of het shok (5 C) n the ition of CL n mke tissues firmer y ining to the petin roxyl groups tht re generte through the tion of PME.45 Smples sujete to the oule tretment h similr vlues to smples trete with ozone lone. Colour nlysis Totl olour ifferenes ( E) were nlyse using CIE L prmeters for fresh-ut lettue. Chnges in olour uring storge (P <.5) were oserve (Fig. 6). Smples sujete to the oule tretment showe greter hnges in olour thn smples sujete to iniviul tretments. However, no ifferenes were foun etween smples trete with CL n smples trete with ozone. This greter hnge in olour with the oule tretment oul e ssoite with the mge n stress proue y the oule mnipultion (physil rowning) rther thn eing use y enzymti rowning (Fig. 3). Although olour vrition n e ssoite with enzymti rowning y phenoli oxition over time,45 49 our results i not show Sensory nlysis Fresh pperne is the min ttriute tht onsumers use to evlute the qulity of vegetles n fruits, sine people uy with their eyes.5,51 Apperne, rowning n texture re key spets use in sensory nlysis to evlute the generl qulity of prout. This is espeilly true for lettue, where rowning n lk of firmness re ritil ftors in pereive loss of qulity.52 Sensory nlysis ws use to ssess the qulity of fresh-ut Ieerg lettue over 1 ys. Men sores for sensory fresh pperne re given in Tle 1. Fresh-ut lettue showe signifintly lower sores for pperne t the en (1 ys) of storge thn from y 1 to y 7. The verge vlues on y 7 e 12 B Totl olour iffe1 rene ( E) A 8 A Ozone Ozone + CL CL Figure 6. Totl olour hnge (CIE L prmeters) in fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL). Points esignte on ny urve y the sme letter re not signifintly ifferent (P >.5). Lowerse letters re use for omprisons uring storge n upperse letters for tretment omprisons. Three inepenent trils were rrie out; >25 smples per tril were nlyse. 7

9 16 Crispiness oeffiient (CC)12 B A A Ozone Ozone + CL CL 1 Figure 7. Effet of wshing tretment on rispiness oeffiient (CC). Mesurement of CC of fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL). Points esignte on ny urve y the sme letter re not signifintly ifferent (P >.5). Lowerse letters re use for omprisons uring storge n upperse letters for tretment omprisons. Three inepenent trils were rrie out; >25 smples per tril were nlyse. Tle 1. Sensory evlution of fresh pperne rting (sle 1 5) of fresh-ut lettue store t 4 C for 1 ys n trete with (1) ozone (1 mg L 1 ), (2) lium ltte (CL, 15 g L 1 t 5 C) or (3) ozone omine with lium ltte (ozone + CL) Tretment Dy 1 Dy 3 Dy 7 Dy 1 Ozone Ozon e + CL CL 3.45 ± ± ± ± ± ± ± ± ± ± ± ±.2 Eh vlue represents men ± stnr evition for fresh pperne. Vlues followe y ifferent letters in the sme row inite signifint ifferenes uring storge. No signifint ifferenes were oserve etween tretments. Three inepenent trils were rrie out; 14 pnellists in eh tril sore the smples. Rtings: = poor fresh pperne; 3 = eptle fresh pperne; 5 = exellent fresh pperne. were onsiere eptle (sore ove 3), showing tht ll tretments mintine goo qulity until the en of storge. Differenes etween tretments were not oserve. At the en of storge, lower fresh pperne vlues were oserve in smples wshe with ozone n ozone omine with CL. However, the ifferenes were not signifint. The results otine in the sensory nlysis i not orrelte with the instrumentl results, whih might e ue to the high vriility of the prout n/or the limite isrimintive ility of humn pereption. CONCLUSIONS The use of n ozone wshing tretment to exten the qulity of fresh-ut lettue ws ompre with tretment using CL t 5 C n omintion of oth, with unequl results. A reution in enzyme tivity ws proue y ozone tretments, resulting in potentilly enefiil effet of reue enzymti rowning, lthough this potentil reution in rowning ws not revele y olour nlysis. However, this enzyme intivtion showe negtive effet, s the reution in tivity of the texture-relte enzyme PME ws orrelte with lower rispiness oeffiient. Depening on further stuies to evlute the effet on the miroil lo of these two tretments (n the omintion of oth), some vntges of CL ompre with ozone mke the former more suitle tretment, i.e. sfety of use, no y-prout proution, iry inustry wste reutilistion, lium enrihment of the prout n possily more lsting effet, s ozone rpily isppers. For these resons the uthors onsier CL (use t 5 C) more suitle tretment thn ozone in orer to exten the qulity of fresh-ut lettue. ACKNOWLEDGEMENTS The uthors woul like to knowlege the finnil support of Tehnologil Setor Reserh (22 26). Thnks re lso extene to Amor Flexiles Europe (Bristol, UK) for kinly proviing the film gs. REFERENCES 1 Alzmor SM, Lopez-Mlo A n Tpi MS, Overview, in Minimlly Proesse Fruits n Vegetles: Funmentl Aspets n Applitions, e. y Alzor SM, Tpi MS n Lopez- Mlo A. Aspen, Githersurg, MD, pp. 1 9 (2). 2 Ohlsson T, Miniml proessing preservtion methos of the future n overview. Trens Foo Si Tehnol 5: (1994). 3 Ker AA, Posthrvest iology n tehnology: n overview, in Posthrvest Tehnology of Hortiulturl Crops, e. y Ker AA. University of Cliforni Press, Okln, CA, pp (22). 4 Wei CI, Hung TS, Kim JM, Lin WF, Tmplin ML n Brtz JA, Growth n survivl of Slmonell montevieo on 8

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