Original article The effect of calcium chloride and calcium lactate pretreatment concentration on peach cell integrity after high-pressure processing

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1 Interntionl Journl of Food Science nd Technology 2017, 52, Originl rticle The effect of clcium chloride nd clcium lctte pretretment concentrtion on pech cell integrity fter high-pressure processing Chukwn Techknon 1,2 & Dine M. rrett 1 * 1 Deprtment of Food Science nd Technology, University of Cliforni, One Shields venue, Dvis, C 95616, US 2 Fculty of Science nd Industril Technology, Prince of Songkl University, Surt Thni Cmpus, 31 Mkhm Ti, Mung Surt Thni, Surtthni 84000, Thilnd (Received 8 October 2016; ccepted in revised form 4 November 2016) Summry Keywords The objective of this study ws to determine the effect of clcium chloride nd clcium lctte pretretments t different concentrtions (1, 1.5 nd 2% w/v) on enzymtic browning of clingstone nd freestone peches following high-pressure processing () t 200 MP for 10 min. Proton nucler mgnetic resonnce ( 1 H-NMR) relxometry indicted tht following clcium pretretment nd, both pech types hd lower percentges of wter in the vcuole comprtment nd simultneous increse in the percentge of wter in the cytoplsm comprtment. Clcium pretretment of freestone peches did not ffect the development of browning, regrdless of the form of clcium or the concentrtion, wheres clcium lctte pretretment undesirly enhnced clingstone pech discolortion. Clcium pretretments hve the potentil to reduce the loss of cell integrity tht results from ; however, in this study, the effect ws not sufficient to inhibit the interction between the polyphenol oxidse enzyme tht ctlyses browning nd its substrtes. rowning, peches, physicl preservtion methods, polyphenol oxidse. Introduction Peches (Prunus persic) hve become populr fruit consumed worldwide. s they re only ville for short seson, in ddition to fresh consumption, mny peches re preserved in the form of cnned or frozen products. lthough there is n dvntge to long-term preservtion using these methods, fruit chrcteristics such s texture nd flvour s well s vitmin content my drmticlly decrese during the preservtion process. High-pressure processing () yields higher qulity product with better retention of texture, flvour, nutrients nd helth ttributes (rb et l., 2012, 2015); therefore, it hs strong potentil to be used s n lterntive choice for pech preservtion. However, during pressuristion nd pressure relese, loss of membrne permeility nd subcellulr comprtmentlistion my occur, resulting in texturl chnges nd triggering the enzymtic browning rection. Discolortion of fruits nd vegetles is therefore mjor concern fter. Colour chnges during storge of -processed fruits hve been reported, *Correspondent: E-mil: dmbrett@ucdvis.edu for instnce in bnn puree (Plou et l., 1999), mngo (Guerrero-eltrn et l., 2005) nd nvel ornge juice (Polyder et l., 2005). This limittion hs motivted food scientists to explore enzymtic browning t the cellulr level in n ttempt to limit these browning rections. Clcium chloride nd clcium lctte hve been employed s firming gents in wide rnge of fruits. These compounds function by binding to the negtively chrged crboxylic cid groups of glcturonic cid residues in the pectin chin nd forming n eggbox gel model resulting in the strengthening of the cell wll (Grnt et l., 1973). There is lso possibility tht positively chrged clcium ions cn bind the negtively chrged hed groups in the lipids tht mke up cellulr membrnes (Pedersen et l., 2006). Clcium pretretment is common not only becuse of its ility to retin firmness in plnt-bsed mterils, but lso becuse there were severl reports indicting its use in reducing browning (Drke & Spyd, 1983; Hopfinger et l., 1984; olin & Huxsoll, 1989). primry objective of the pretretment with clcium chloride or clcium lctte in this study is the desire to hve C 2+ doi: /ijfs.13316

2 636 Clcium effects on treted peches C. Techknon nd D. M. rrett penetrte into the plsm membrne, which surrounds ech plnt cell nd the tonoplst, which is the membrne surrounding the lrge wter-filled vcuole, nd stilise these membrnes prior to. Nucler mgnetic resonnce (NMR), which is nondestructive method, hs been used to detect physiologicl nd biochemicl chnges in the vrious wter comprtments in plnt tissues. The ppliction of NMR hs been used recently for the determintion of the effect of pectin methyl esterse (PME) nd CCl 2 infusion on cell integrity in fresh-cut nd frozenthwed mngoes (Kirtil et l., 2014). In this study, NMR relxometry is used for evlution of the permeility of ech wter comprtment in smples pretreted with clcium, following. Observtion of pech cells using light microscopy is lso included in this study s nother tool for determintion of the tonoplst integrity. The technique developed by dmon et l. (1980) uses neutrl red (NR), lipophilic phenzine dye which diffuses into nd ionises in the cidic environment of the vcuole. The intct vcuole ppers s n intense red colour, but in dmged vcuoles, the dye spreds throughout the cell nd the colour is therefore less intense. The number nd colour intensity of intct vcuoles correlte with the ility of the membrnes to resist dmge from. Thus, the evlution of microgrphs from smples treted with different pretretments cn be correlted with the degree of de-comprtmentlistion. 1 H-NMR relxometry nd the observtion of pech cells under light microscopy were used to determine whether pretretment with clcium t different concentrtions results in n improvement in cell integrity in peches following. Prmeters involved in the browning rection, which include degree of browning, polyphenol oxidse (PPO) ctivity nd totl phenol content, were lso quntified. Mterils nd methods Rw mterils The clingstone pech cultivr Extr Lte 1 nd the freestone type cultivr Summerset were hrvested by hnd from Foundtion Plnt Services on the University of Cliforni Dvis cmpus. Peches were nondestructively sorted with T-XT2 Texture nlyzer (Stle Micro Systems Ltd., Surrey, UK) using compression test for 3 mm distnce nd 5.0-cm-dimeter probe, nd those with firmness of N were stored t 4 C for pproximtely 2 dys until processing. The fruit were hnd-peeled, nd ech of the fruit ws segmented into different smple sizes bsed on their further nlysis. flt circulr disc of 1 cm thickness ws used for colour determintion, cylinder with 15 mm dimeter nd 15 mm height ws cut using cork borer for NMR nlysis, nd cm cube ws used for nlysis of PPO ctivity, totl phenols nd microscopic evlution of cell viility. There were two control tretments, represented by (i) untreted peches t pproximtely 0 MP pckged in vcuum pckge, polyethylene bg (4 mil vcuum pouch; Ultrsource, North Knss City, MO, US) with n oxygen trnsmission of 1.2 cc sq m 1 per dy, nd (ii) untreted peches which were vcuum-pckged in the sme pouch nd then treted with t 200 MP to compre the effect of pressure without pretretment. The sme fruit ws nlysed for ll prmeters, for exmple difference in lightness, NMR relxtion, PPO ctivity, totl phenols nd determintion of vile cells using light microscopy. Clcium pretretment The smples were soked in either clcium chloride or clcium lctte t concentrtion of 1, 1.5 nd 2% w/ v for 5 min before vcuum pckging in polyethylene bgs (4 mil vcuum pouches; Ultrsource, Knss City, MI, US). Ech bg contined pech smples receiving the sme pretretment, one from ech of the three replicte fruit, nd seprte bgs were creted for ech nlysis method. ll of the smples were kept t mbient temperture (22 2 C) for 30 min fter pckging prior to tretment. High-pressure processing () Following pretretment, the pckged smples were processed t 200 MP for 10 min in unit (vure Technologies Inc, Kent, W, US). The pressure level used in this experiment, t 200 MP, ws justified on the bsis of previous studies (Techknon, 2015), which found tht rupture of the plnt cellulr membrnes occurred t this pressure level. The initil high-pressure unit temperture (T i ) ws pproximtely 23 C. The mximum temperture in the high-pressure chmber ws dependent on the set pressure, which for 200 MP ws 28 C. The high-pressure unit hd 2.0-L vessel nd mximum pressure of 600 MP. The pressurising medium ws wter. Nucler mgnetic resonnce (NMR) relxometry Following high-pressure processing, the smples were removed from the plstic bg nd blotted dry before being plced into covered NMR tube. NMR relxometry mesurements were performed using n NMR spectrometer (spect I; Industril re Hvel Modi in, Shohm, Isrel) equipped with 1.02 T mgnetic field, 43.5 MHz frequency nd 42.6 rd s 1 T -1 gyromgnetic rtio. T 2 ws mesured using the Crr Purcell Meiboom Gill (CPMG) sequence echoes with n Interntionl Journl of Food Science nd Technology 2017

3 Clcium effects on treted peches C. Techknon nd D. M. rrett 637 echo time of 0.5 ms. T 2 spectrum inversion ws performed on the rw dt using non-negtive lest squre lgorithm using Prosp (Mgritek, Wellington, New Zelnd). Light Microscopy study Section preprtion fter the process, the cube destined for microscopy evlution ws further cut into smll rectngulr shpes pproximtely cm 3 before being plced in smple holder. Then, Vibrtome 1000 Plus (The Vibrtome Co., St. Louis, Mo, US) ws used for sectioning specimens with pproximtely 200 lm thickness. stin ws prepred using 0.5% neutrl red in cetone nd filtered twice with Whtmn pper # 1 before being diluted to 0.04% in 0.55 M mnnitol 0.01 M N-(2-hydroxyethyl) piperzine-n -2-ethne-sulphonic cid (HEPES) buffer djusted to ph 7.8. The specimens were submerged in the solution for 2 h nd then rinsed for 0.5 h in the 0.55 M mnnitol 0.01 M HEPES buffer solution. n observtion under light microscope (Olympus System Microscope, Model HS, Shinjuku-Ku, Tokyo, Jpn) ws done t 409 mgnifiction, nd colour photomicrogrphs were cptured using digitl colour cmer (Olympus MicroFire; Olympus, Tokyo, Jpn). Imge processing nd nlysis Fifteen microgrphs were rndomly selected from the three replictes of control smple (0 MP), Control (untreted with clcium, only 200 MP pplied) nd ech clcium pretretment for imge nlysis using softwre IMGEJ (NIH, ethesd, MD, US). Cell counter, PLUGIN softwre developed by De Vos (2008), ws used for semiquntifiction of the number of vile cells. The cells with smooth red to pink stin were distinguished s vile cells, wheres invile cells hd visully rough membrne with no red dye retined. Degree of browning The browning of pech smples ws reported s the difference in lightness (DL*), which is the difference between initil lightness nd the lightness of the sme fruit smples fter 2 weeks storge t 4 C. Colour ws determined using Minolt CR-410 colorimeter (Minolt cmer Co, Ltd., Rmsey, NJ, US). white tile ws used for clibrtion (L* =96.88, * =0.02, b* =2.05). The vlues were expressed by the CIE L**b* system. Polyphenol oxidse (PPO) ssy The enzyme ws ssyed using the spectrophotometric method described by Espin et l. (1995) with some modifictions. ssys were performed t room temperture in 1.0 ml of medium contining 5.0 mm dihydroxyhydrocinnmic cid (Sigm ldrich, St. Louis, MO, US), 0.2 m M 3-methyl-2-benzothizolinone (MTH) (Sigm ldrich), 0.1% (w/v), nd 100 m M cette buffer (ph 5.5). Incubtions were strted with the ddition of 10 ll of pech extrct. n increse in sorbnce t 500 nm ws monitored for up to 120 s using UV spectrophotometry (UV2101PC; Shimdzu Scientific Instruments Inc., Columbi, MD, US). PPO ctivity ws clculted in units using the eqution below, where bs(0) is the initil sorbnce nd bs(1) is sorbnce t the end of linerity. PPO ctivity ðunits ml 1 Þ¼bsð1Þ bsð0þ=min ml of juice nlysis of totl phenols nlysis of totl phenols ws performed by mens of the Folin Cioclteu nlysis of totl phenols ws performed by mens of the Folin Cioclteu method s described by Wterhouse (2002). Pech smples (20 g) were homogenised with 30 ml deionised wter, nd then 6.4 g of the blended smples ws homogenised with 27.6 ml of 76% (v/v) queous cetone for 2 min. fter 10-min shking, the cell wll prticles were seprted using room temperture centrifugtion (Centr CL2 tletop centrifuge, IEC; Needhm, M, US) t g for 10 min mL liquot of 2N Folin regent (Sigm ldrich, uchs, Switzerlnd) ws dded to 1 ml of superntnt. The solution ws then vortexed nd llowed to stnd for 5 min, fter which 6 ml of sodium crbonte ws dded nd mixed well before dding 2.64 ml of deionised wter. The solution ws vortexed gin nd incubted t 50 C for 5 min. fterwrds, the solution ws cooled to room temperture for 1 h, nd then, the sorbnce ws red t 760 nm. Gllic cid (rcos Orgnics, Geel, elgium) concentrtions of mg L 1 were used for clibrtion of the stndrd curve. The results were expressed s gllic cid equivlents (GE)/fresh weight of peches (g). Sttisticl nlysis This experiment ws crried out in three replicte processing runs on three seprte dys. Smples were obtined from three seprte peches, which formed one replicte mesurement of ech prmeter. nlysis of vrince ws used for determining the effects of ech clcium pretretment on the T 2 relxtion time, % reltive re, % vile cells, difference in lightness, PPO ctivity nd totl phenols. Tukey s test ws used to compre mens of ech tretment t P < 0.05 (SS version 9.4, Cry, NC, US). Interntionl Journl of Food Science nd Technology 2017

4 638 Clcium effects on treted peches C. Techknon nd D. M. rrett Results nd discussion T 2 relxtion nd reltive percentge of wter in different comprtments of clingstone nd freestone pech cells Following ppliction in untreted control peches, there re three distinct comprtments, ech generted from different proton environment (Fig. 1). Snr & Vn s (1992) pioneered the ppliction of 1 H-NMR in plnt cell studies nd ssigned ech of these three peks to the seprte cellulr wter comprtment, for exmple the vcuole, cytoplsm nd cell wll/extrcellulr spce. In the present study, the highest pek with the longest T 2 relxtion time nd men vlue of 790 ms (pek 1) is ssigned to the vcuole, comprtment tht contins s much s 50 80% of the totl cellulr wter (Krmer & oyer, 1995). Plnt cell wlls re very rigid, being comprised of cellulose nd hemicellulose mtrix, which re cemented together with pectin. With its restricted re, the cell wll fcilittes proton exchnge between wter nd neighbouring molecules. s result, the T 2 signl of the wter in the cell wll comprtment is the shortest, ppering s pek 3 with men vlue of 37 ms. The cytoplsm hs the second highest mount of wter fter the vcuole nd hs previously been reported with n intermedite T 2 relxtion time (Zhng & McCrthy, 2013; Kirtil et l., 2014). Hence, the cytoplsm wter pek is displyed s pek 2 with men T 2 vlue of 143 ms. T 2 distributions nd reltive re of ech wter comprtment in ll smples evluted in this study re summrised in Tle 1. ccording to these results, tretment t 200 MP lone (Control ) did not ffect T 2 relxtion time or % reltive re of the min vcuolr comprtment (pek 1) compred to the untreted control (Control) in either clingstone or freestone peches. However, there ws significnt decrese in the T 2 time of both the cytoplsm comprtment (pek 2) nd the cell wll comprtment (pek 3) in clingstone peches. This my hve resulted from high-pressure-induced increse in membrne permeility; however, it is surprising tht the vcuolr comprtment (pek 1) ws not ffected. Pek 3 (cell wll) showed reltively smll but significnt chnge, s the cell wll contins only smll mount of wter nd its structure is more resistnt to dmge. There ws no significnt high-pressure effect in freestone peches, possibly becuse the middle lmell of this pech type tends to undergo greter degree of degrdtion during ripening, which in fct mkes the cells more flexible nd therefore resistnt to (Techknon, 2015). This finding is lso supported by the greter number of vile cells (43% vs. 30%) in freestone smples following lone (Tle 2). Following pretretment with clcium chloride nd clcium lctte t different concentrtions nd then tretment, pech smples still hd three comprtments. The T 2 relxtion time of the vcuolr comprtment (pek 1) ws unffected by clcium pretretment in most cses, which implies tht C 2+ my not hve reched this comprtment. The study by Kirtil et l. (2014) observed drmtic chnge in the T 2 of vcuolr comprtment of mngo smples (from 930 to 736 ms) following infusion of U ml 1 PME + 1% (w/w) CCl 2 t 50 kp. n impregntion technique such s this is therefore recommended, rther thn the dipping nd pssive diffusion tht were used in this study, to deliver C 2+ to the trget re. There were some significnt chnges in the T 2 relxtion times nd % reltive re of wter in peks 2 nd 3 in clingstone peches. significnt increse in the T 2 of the cytoplsm comprtment (pek 2) fter pretretment indictes less interction of wter protons; therefore, clcium tretment my hve reched the cytoplsmic (plsm) membrne, improving membrne integrity nd decresing permeility of wter in the cytoplsm comprtment. Kirtil et l. (2014) reported similr observtion of n incresed T 2 relxtion time in the cytoplsm comprtment of fresh-cut mngo pretreted with 1% CCl 2. There ws no significnt difference mong the vrious mplitude (u) Time (s) Figure 1 representtive T 2 relxtion spectrum of control (untreted) pech smple following t 200 MP. Interntionl Journl of Food Science nd Technology 2017

5 Clcium effects on treted peches C. Techknon nd D. M. rrett 639 Tle 1 verge T 2 relxtion times nd per cent reltive res (R) of the three comprtments of pretreted clingstone pech cultivr Extr Lte 1 nd freestone pech cultivr Summerset processed using t 200 MP for 10 min T 2 (ms) Reltive re (%) Tretment Concentrtion Pek 1 Pek 2 Pek 3 Pek 1 Pek 2 Pek 3 Clingstone pech Control d 90 8 b 2 d Control c 37 bc b 4 bc Clcium lctte 1.0% b 15 4 c 1.5% b % c 40 c 81 b 15 4 bc Clcium chloride 1.0% c 80 b 16 4 c 1.5% c 79 b % 717 b 149 bc 37 c 81 b 16 3 cd Freestone pech Control b b 2 cd Control 699 b 136 b b 2 d Clcium lctte 1.0% b 18 3 bcd 1.5% b % b 17 4 Clcium chloride 1.0% b 18 4 c 1.5% b % 693 b b 18 4 Vlues within columns followed by the sme letter re not significntly different to ech other t P < Tle 2 Per cent vile cells in clingstone nd freestone peches treted with clcium lctte nd clcium chloride t 1, 1.5 nd 2% nd processed with t 200 MP for 10 min Concentrtion Clingstone (EL1) (%) Freestone (Summerset) (%) Control Control 30 7 bc 43 6 b Clcium lctte Clcium chloride 1.0% 28 4 bc 23 4 e 1.5% 31 5 bc 37 7 cb 2.0% 26 5 c 22 6 e 1.0% 27 2 c 24 5 e 1.5% 25 5 c 32 5 cd 2.0% 34 3 b 29 5 de Vlues represent the men with its stndrd devition for ech determintion. Vlues with the sme letter re not significntly different cross tretment vriles t significnce level of P < concentrtions of either clcium lctte or clcium chloride used for pretretment of either pech type; therefore, the lowest concentrtion (1.0%) of either clcium species ws sufficient to imprt this chnge. In contrst to clingstone peches, clcium tretments did not significntly ffect the T 2 relxtion of ny of the freestone pech wter comprtments. The chnge in reltive re, which refers to chnge in the mount of wter in ech comprtment, hs been widely used to evlute the chnge in cell integrity of fruit nd vegetle smples, for instnce in strwberry (Mrigheto et l., 2004), per (Hernndez-Snchez et l., 2007), onion (Gonzlez et l., 2010) nd pomegrnte (Zhng & McCrthy, 2012). High-pressure tretment lone (Control ) resulted in no significnt chnge in reltive re in most cses, except in one of the six clcium pretretments for pek 3 (cell wll) of clingstone peches, which showed higher per cent re fter. This chnge, while significnt, ws quite smll in comprison with the mount of wter in the whole fruit. In the present study, both clingstone nd freestone peches demonstrted the sme trend of significnt decrese in per cent reltive re of the vcuolr comprtment (pek 1) simultneous with n increse in the % R belonging to the cytoplsmic comprtment (pek 2) fter pretretment with either form of clcium nd t ny concentrtions. This scenrio demonstrtes migrtion of wter from the vcuole to the cytoplsm. It is possible tht the clcium pretretments ct s hypertonic solution, nd plsmolysis tkes plce with the resulting osmotic differences cusing loss of vcuolr membrne integrity nd movement of wter. Sftner et l. (1998) suggested tht the immersion of fruits in high clcium concentrtion slts enhnces the risk for fruit injury, which is possibly result of osmotic effects. Interntionl Journl of Food Science nd Technology 2017

6 640 Clcium effects on treted peches C. Techknon nd D. M. rrett Evlution of vile cells using light microscopy Clingstone nd freestone peches both showed significnt decrese in the number of vile cells fter high-pressure tretment, from pproximtely 61 63% to 30 43%, respectively (Tle 2). This finding is supported by previous study of -treted onions, which suggested tht pressures ove 100 MP cuse membrne dmge, which induces relese of PPO s well s its phenolic substrtes, nd the subsequent enzymtic browning rection (utz et l., 1994). sk & Rmswmy (1998) suggested tht the cellulr membrnes of fruits nd vegetles rupture t pressures of 200 MP nd ove. previous study by our group lso found significnt decrese in vile cell number in onion smples following t 200 MP (Gonzlez et l., 2010b). In clingstone peches, none of the clcium pretretments used prior to resulted in significnt improvements in the mintennce of cell viility. However, in freestone peches, which hd higher number of vile cells following (43 vs. 30%), there ws in fct significnt undesirle decrese in the percentge of vile cells in most of the smples pretreted with either clcium solution t ny percentge prior to. sed on our previous experiments, -treted freestone peches without clcium pretretment retined their cell integrity better thn clingstone types nd showed higher percentges of cell viility (Techknon, 2015). This could be explined by the looser cell mtrix of freestone peches, which undergo more extensive degrdtion of the middle lmell s the fruit mtures. s consequence, the cells re more flexible nd potentilly less dmged during nd subsequent sectioning for the microscopic study. The clcium tretment, on the other hnd, imprts less cell flexibility due to the interction of clcium with pectin to form bridges. Therefore, following most of the pretretments nd, freestone peches showed significnt decrese in the percentge of vile cells. This finding is in greement with the degree of browning results tht freestone smples showed greter difference in lightness fter pretretment nd, compred to clingstone peches. Degree of browning Enzymtic browning in fruit is direct consequence of the interction between the enzyme polyphenol oxidse, originlly locted in the plstid nd its phenolic substrtes, which re initilly in the vcuole. fter cells lose comprtmentlistion, the membrne brrier seprting the two components from ech other is ruptured. In the present study, pech smples were highpressure-treted t 200 MP, nd t this level, the cell membrnes were ruptured, leding to relese of vcuolr contents to the cytoplsm. significnt increse in the difference in lightness following tretment lone (Control vs. Control) ws observed in both pech types fter 2 weeks of storge (Fig. 2). previous study by our group reported similr finding in onion cells (Gonzlez et l., 2010,b). These uthors found tht loss in membrne integrity ws observed using light microscopy t this pressure level. sk & Rmswmy (1998) reported significnt chnges in pple flesh colour fter ppliction of pressures of 200 MP nd higher nd stted tht this ws due to the interction of PPOs nd their substrtes. Difference in lightness C c Clingstone Freestone b 0 Control Control 1.0% 1.5% 2.0% Tretment CCl2 1.0% CCl2 1.5% CCl2 2.0% Figure 2 Difference in lightness in clingstone nd freestone peches treted with clcium lctte nd clcium chloride t 1.0, 1.5 nd 2.0% nd then -treted t 200 MP for 10 min. Vlues with the sme letter re not significntly different cross tretment vriles t significnce level of P < Cpitl letters (,, C) refer to clingstone peches, nd smll letters (, b, c) refer to freestone peches. Interntionl Journl of Food Science nd Technology 2017

7 Clcium effects on treted peches C. Techknon nd D. M. rrett 641 In the current study, it ws hoped tht this problem would be solved using pretretments with clcium solutions, which theoreticlly my id in retention of cell integrity during nd prevent loss of membrne integrity. However, results in Fig. 2 illustrte tht there ws no significnt effect of pretreting with clcium chloride on reducing the discolortion of -treted nd cold-stored peches. This is supported by previous findings of strwberry pretretment with CCl 2 t 1% nd 4%, which lso found tht there ws no significnt effect of clcium pretretment on strwberry firmness (Mrigheto et l., 2004). Clcium lctte, in contrst, hd significnt negtive effect on clingstone pech browning, with significnt increse in the difference in lightness in the smples pretreted with ll concentrtions prior to processing. There ws no significnt difference in freestone peches pretreted with ny level of clcium lctte, s compred to the Control smple. In previous study by Gorny et l. (2002) in fresh-cut pers, these uthors lso found tht tretment with clcium lctte did not prevent cut surfce drkening. In fct, the pretreted smple developed lower hue ngle (higher flesh drkening) thn the control smple (wter dip) during 8 dys of storge t 0 C in continuously flowing (100 ml min 1 ) humidified ir. nother study on clcium tretment of pech fruit (cv. ndross) confirmed the sme finding, s significnt discolortion compred to the control ws found in smples during 4 weeks of cold storge following clcium lctte tretment t high concentrtion of 187 mm C (Mngnris et l., 2007). Effect of pretretment on PPO ctivity Figure 3 illustrtes tht high-pressure tretment lone hd no effect on PPO ctivity of freestone peches, but cused significnt increse in tht of clingstone peches. similr observtion ws reported in our previous study, in which clingstone pech cultivrs Crson nd Ross hd significntly higher PPO level following t 100 MP for 10 min (Techknon, 2015). PPO ctivtion induced by hs been reported in other fruits nd vegetles, for instnce, per extrcts following t 400 MP or higher (sk et l., 1994), strwberries treted t 400 MP (Cno et l., 1997) nd onions treted t MP (utz et l., 1994). This study further compred the PPO ctivity fter use of different pretretments in the two pech types. Freestone peches re generlly higher in PPO ctivity, with men vlue of units ml 1 in the control group, while the untreted clingstone peches hd men PPO ctivity of 9776 units ml 1. These PPO ctivity levels re in greement with our previous study (Techknon, 2015). There ws no significnt difference (P < 0.05) in PPO ctivity following pretretment with clcium lctte or clcium chloride in either clingstone or freestone types. lthough other studies hve suggested n effect of CCl 2 on PPO inhibition, through the interction between the chloride ion nd copper t the ctive site of the enzyme (Grci & rrett, 2002), this experiment did not observe ny significnt chnges in PPO ctivity. One possible reson could be the low concentrtion of clcium chloride used in the current experiment, s chloride is wek inhibitor. Some uthors hve suggested tht n PPO ctivity (x100 units ml 1 ) Clingstone Freestone b b Tretment Figure 3 PPO ctivity in clingstone nd freestone peches treted with clcium lctte nd clcium chloride t 1.0, 1.5 nd 2.0% followed by tretment t 200 MP for 10 min. Vlues with the sme letter re not significntly different cross tretment vriles t significnce level of P < Cpitl letters (,, C) refer to clingstone peches, nd smll letters (, b, c) refer to freestone peches. Interntionl Journl of Food Science nd Technology 2017

8 642 Clcium effects on treted peches C. Techknon nd D. M. rrett 12 Clingstone Freestone Totl Phenols (mg g 1 smple) b 0 Control Control 1.0% 1.5% Tretment 2.0% CCl2 1.0% CCl2 1.5% CCl2 2.0% Figure 4 Totl phenol content in clingstone nd freestone peches treted with clcium lctte nd clcium chloride t 1, 1.5 nd 2% following tretment t 200 MP. Vlues with the sme letter re not significntly different cross tretment vriles t significnce level of P < Cpitl letters (,, C) refer to clingstone peches, nd smll letters (, b, c) refer to freestone peches. elevted level of chloride is required to cuse PPO inhibition (Myer & Hrel, 1991). Effect on totl phenols The selected cultivrs of clingstone nd freestone peches hd similr levels of phenolic compounds, for exmple in the rnge of 6 12 mg g 1 smple (Figure 4). These compounds ct s substrtes for PPO in enzymtic browning rections. oth pech types showed stle level of totl phenols following s compred to the control smples. No significnt differences in totl phenols in other -treted fruits were lso reported by other investigtors, for exmple strwberry nd blckberry purees (Ptrs et l., 2009) nd Grnny Smith pple puree (Lndl et l., 2010). High-pressure tretment is therefore excellent for retining these nutrients. Summry nd conclusions Enzymtic browning in fruit following directly involves the loss of cell integrity, which leds to the interction between the enzyme in the plstid nd the substrtes stored in the vcuole. Clcium pretretments my help prevent enzymtic browning rections during if the penetrtion of C 2+ reches the trget re, which is the tonoplst or vcuolr membrne of pech cells. However, the results of this study showed no significnt chnges in T 2 relxtion time of the vcuolr comprtment nd no improvement in the number of vile cells in most of the pretreted smples, in either clingstone nd freestone peches. These findings indicte high probility tht C 2+ could not penetrte ll the wy to the tonoplst membrne. This result is lso in greement with the observtion of the difference in lightness, in which clcium chloride nd clcium lctte pretretments t 1 2% could not improve pech colour following 2 weeks of refrigerted storge. For future investigtions, higher clcium concentrtion nd/or improved impregntion (potentilly using vcuum infusion) is suggested for efficient delivery of C 2+ inwrd to the tonoplst membrne. cknowledgment This reserch ws finncilly supported by Prince of Songkl University, Surt Thni Cmpus, 2016, nd the Royl Thi government scholrships (Thilnd). The uthors would like to thnk Lu Zhng, Thoms Grdziel, Judy Jernstedt nd our student ssistnt, Ptchrin Sefrung, for their support. References dmon,., Jcoby,. & Goldschmidt, E.E. (1980). ssessment of cytoplsmic contmintions in isolted vcuole preprtions. Plnt Physiology, 65, sk, M., oym, Y., Nknishi, R. & Hyshi, R. (1994). Purifiction of ltent form of polyphenoloxidse from L Frnce per fruit nd its pressure-ctivtion. ioscience, iotechnology, nd iochemistry, 58, rb, F.J., Esteve, M.J. & Frıgol,. (2012). High pressure tretment effect on physicochemicl nd nutritionl properties of fluid foods during storge: review. Comprehensive Reviews in Food Science nd Food Sfety, 11, rb, F.J., Terefe, N.S., uckow, R., Knorr, D. & Orlien, V. (2015). New opportunities nd perspectives of high pressure tretment to improve helth nd sfety ttributes of foods. review. Food Reserch Interntionl, 77, Interntionl Journl of Food Science nd Technology 2017

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