Bilid: 1821-4487 (2015) 19; 5; p 219-223 DK: 577.151.3 ENZYMATIC ACTIVITY AND METABOLISM OF FESH-CT FIT AS A FNCTION OF IPENING DEGEE ENZIMSKA AKTIVNOST I METABOLIZAM SVEŽE EZANOG VOĆA KAO FNKCIJA STEPENA ZELOSTI Originl Scientific Pper Originlni nučni rd Silvi TAPPI *, Cinzi MANNOZZI *, rszul TYLEWICZ */**, Sntin OMANI */**, Mrco DALLA OSA */**, Pietro OCCLI */** * Deprtment of Agriculturl nd Food Sciences - DISTAL, niversity of Bologn, Cmpus of Food Science, Cesen, Itly ** Interdeprtmentl Centre for Agri-Food Industril eserch, niversity of Bologn, Cmpus of Food Science, Cesen, Itly e-mil: silvi.tppi2@unio.it ABSTACT The im of this work ws to evlute the influence of the ripening stge on three fresh-cut fruit (pple, kiwifruit nd melon) in terms of qulity nd tissue metolism, during ccelerted storge (AS) in controlled conditions. For this purpose, two different ripening stges were selected for ech fruit. At ech ripening stge the fruits were peeled nd cut into 10 mm thick slices nd stored t 10 C, H 90 %, for 24 hour for pples nd 4 dys for kiwifruit nd melon. During storge, smples were monitored for the min qulity indexes nd the ctivity of the min degrdtive enzymes (polyphenoloxidse, peroxidse nd pectinesterse). Production of the endogenous metolic het ws lso monitored using isotherml clorimetry. esults showed tht, whether for melon nd kiwifruit mrked differences were found in the evolution of the prmeters considered during storge nd in reltion to their ripening degree, pple qulity nd metolic chrcteristics proved to e less ffected y the ripening degree during storge. Key words: fresh-cut fruit, wounding response, ripening degree, enzymtic ctivity, tissue metolism. EZIME Cilj ovog rd je ocen uticj stepen zrelosti tri sveže rezne voćne vrste (juke, kivij i dinje) u odnosu n kvlitet i metolizm tkiv, tokom postupk urznog skldištenj u kontrolisnim uslovim. tu svrhu, dv rzličit stdijum zrelosti su odrn z svku voćnu vrstu. Svki uzork rzličitih stepen zrelosti je oljušten i izrezn n kolutove deljine 10 mm i potom skldišten n 10 C pri reltivnoj vlžnosti vzduh 90%, u trjnju od 24 st z juku i četiri dn z kivi i dinju. Tokom skldištenj, prćeni su glvni indeksi kvlitet uzork i ktivnosti glvnih degrdtivnim enzim (poliphenolokidze, peroksidze i pektinsterse). Proizvodnj endogene metoličke toplote je tkođe prćen korišćenjem metode izotermne klorimetrije. ezultti su pokzli uočljive rzlike u ocenjivnju prmetr tokom skldištenj kd je reč o dinji i kiviju i njihovom stepenu zrenj, ispostvilo se d n kvlitet juke i metoličke krkteristike, stepen zrenj im mnji uticj tokom proces skldištenj. Ključne reči: sveže rezno voće, uticj sečenj, stepen zrenj, enzimsk ktivnost, metolizm tkiv. INTODCTION Fresh-cut fruit re products tht undergo miniml processing opertion such s peeling, cutting, dicing, shredding etc. nd mintin their qulity chrcteristics similr to those of fresh products. However, their qulity degrde fster compred to tht of the intct product due to the physiologicl response to the mechnicl stress clled wounding response (Brecht, 1995). The loss of cellulr comprtmentlistion, promoted y processing opertions, leds to the contct etween enzymes nd their sustrtes nd generl increse of enzymtic nd metolic ctivity tht ccelerte the qulity degrdtion. Appernce nd texture chnges re the min fctor determining consumer cceptility of fresh-cut fruit nd, eing strictly relted to tissue deteriortion, re often used s mesures of product freshness nd qulity. The selection of rw mteril is fundmentl fctor for product qulity nd its shelf-life. Degree of ripening t hrvesting nd t processing is n importnt fctor tht cn influence the intensity of the wounding response (Hodges nd Toivonen, 2008). Generlly, processing fruits tht re unripe or slightly unripe, led to etter preservtion of qulity during storge ut it could e detrimentl for the orgnoleptic nd flvour profile (Beulieu et l., 2004). However, few studies hve een performed out the influence of ripening degree on the metolic response to processing stress in fresh-cut fruit. Therefore, the im of this work ws to evlute the influence of the ripening stge on fresh-cut pple, kiwifruit nd melon in terms of qulity nd tissue metolism, during ccelerted storge (AS) in controlled conditions. MATEIAL AND METHOD w mterils s (Actinidi delicios vr delicios cv Hywrd) nd pples (Mlus domestic vr. Pink Ldy) were rought on the locl mrket. They were prtilly ripened t 4 ± 1 C nd 90-95 % of reltive humidity in ir for out month, selecting two different ripening stges defined s nripe () nd ipe (). (Cucumis melo vr. eticultus) ws hrvested t different degrees of development of the scission lyer, lso clled slip, in prticulr t ½ slip s unripe () nd full slip s ripe (). For ech ripening stge the fruits were hnd peeled nd cut into 10 mm thick slices with shrp knife nd stored in climtic chmers t 10 C, 90 % H, for different time period depending on the kinetics of qulity degrdtion tht were defined in preliminry tests s 24 hours for pples nd 4 dys for kiwifruit nd melon. Journl on Processing nd Energy in Agriculture 19 (2015) 5 219
Tppi, Silvi et l. / Enzymtic Activity nd Metolism of Fresh-Cut s Function of ipening Degree Dry mtter content, solule solid content nd titrle cidity were determined to chrcterize the rw mteril. Dry mtter ws determined grvimetriclly fter drying in vcuum oven (AOAC Interntionl, 2002). Solule solids content (SSC) ws determined t 20 C y mesuring the refrctive index with digitl refrctometer (P1, Atgo, Jpn) clirted with distilled wter. Titrle cidity (TA) ws determined y titrtion with NOH 0.1 N until ph 8.1 (AOAC Officil Method 942.15) nd expressed s mg of citric cid/100 g FW. Triplicte mesurements were conducted for ech smple. The rtio etween SSC nd TA hs een used s ripening index. esults re presented in tle 1. Tle 1. Physico-chemicl prmeters of pple, kiwifruit nd melon t the two ripening stges selected Mturity ipening SSC TA (mg citric Dry mtter index Stge ( Brix) cid/100 g) (g/100g) ( Brix/TA) 13.0 ± 0.2 0.36 ± 0.02 35.9 ± 0.4 13.4 ± 0.2 13.4 ± 0.2 0.27 ± 0.01 50.6 ± 1.1 14.5 ± 0.7 11.6 ± 0.9 1.45 ± 0.02 7.9 ± 0.2 15.9 ± 0.5 14.9 ± 0.6 1.42 ± 0.01 10.0 ± 0.4 16.3 ± 0.5 11.8 ± 0.1 0.7 ± 0.1 16.8 ± 0.3 13.1 ± 0.9 14.3 ± 0.1 0.8 ± 0.1 17.9 ± 0.3 16.2 ± 0.5 Qulittive determintions Firmness (N) ws evluted y performing penetrtion test on fruit pulp using TA-HDi500 texture nlyzer (Stle Micro Systems, Surrey, K) with 5 kg lod cell. Experiment ws run with metl proe of 6 mm dimeter, nd rte nd depth of penetrtion of 1 mm s 1 nd 6 mm, respectively (Beirão-d-Cost et l., 2006). Firmness (N) ws evluted s the first pek force vlue. Surfce colour ws mesured with colourspectrophotometer mod. Colorflex (Hunterl, SA) using the CIEL scle nd Illuminnt D65. A white tile (L* 93.47, * 0.83, * 1.33) ws used for clirtion. esults were expressed s L* (luminosity) nd h (hue ngle). Enzymtic ctivity The ctivity of the enzyme minly responsile for qulity degrdtion for ech fruit ws evluted. Polyphenoloxidse (PPO) ctivity ws ssyed spectrophotometriclly ccording to the methods proposed y Britux et l. (1991) on fresh-cut pples nd fter 2, 4, 12 nd 24 h of storge for oth ripening degrees. Pectinmethylesterse (PME) ws extrcted nd ssyed spectrophotometriclly ccording to the methods proposed y Hgermn nd Austin (1986) on fresh-cut kiwifruit fter 0, 1, 2 nd 4 dys of storge for oth ripening degrees. Peroxidse (POD) ws extrcted nd ssyed spectrophotometriclly ccording to the methods proposed y Morles Blncs et l. (2002) on fresh-cut melon fter 0, 1, 2 nd 4 dys of storge for oth ripening degrees. Metolic determintions A TAM ir isotherml clorimeter (Thermometric AB, Järfäll, Sweden), descried in detils y Wdsö nd Gόmez- Glindo (2009), ws used to ssess metolic het production. Six cylinders were smpled using core orer from the fruit tissue nd plced in seled 20 ml glss mpoule. Four replictes for ech smple were performed. The rte of het production ws continuously mesured t 10 C for 24 h. Immeditely fter the end of the nlysis, the O 2 nd CO 2 percentges were mesured in the hedspces y gs nlyzer (MFA III S/L gs nlyzer, Witt-Gsetechnik, Witten, Germny). ESLTS AND DISCSSION Qulittive determintions The vlues of hrdness mesured in the fruit smples t different ripening stges during storge re reported in tle 2. As it cn e oserved, hrdness incresed upon ripening in pple smples, oth etween ripening stges nd during 24 h storge. On the contrry, melon nd kiwifruit slices showed notle loss of hrdness etween the two ripening degrees selected, lmost tenfold for kiwifruit. After 4 d of storge, it cn e oserved loss of hrdness up to 23 nd 60 % compred to the initil vlues for respectively unripe melon nd unripe kiwifruit, while ripe fruit showed lwys low nd similr vlues. Texturl chrcteristics re relted to ripening degree nd vritions of such properties re often due to enzymtic hydrolysis of cell wll components. nd kiwifruit re prticulrly sujected to softening during storge (Silveir et l., 2011; Gil et l., 2006) nd, in greement with results found y Tle 2. Hrdness (N) of pple, kiwifruit nd melon t the two ripening stges selected during ccelerted storge in controlled conditions. Different letters indicte significnt differences (p-level < 0.05) etween ripe nd unripe fruit for the sme storge time ipe. Storge time (dys) Stge 0 0.5 1 2 4 15.22 ± 3.41 20.12 ± 3.71 19.99 ± 2.84 - - 19.79 ± 7.80 22.67 ± 4.37 25.35 ± 4.00 - - 18.42 ± 3.77-16.63 ± 3.31 16.08 ± 3.91 14.14 ± 4.12 9.43 ± 4.61-10.81 ± 4.40 10.16 ± 4.78 8.98 ± 2.87 11.28 ± 5.22-12.21 ± 3.49 17.23 ± 5.26 4.43 ± 0.79 Kiwi. 1.66 ± 0.32-1.48 ± 0.37 1.26 ± 0.31 1.03 ± 0.18 Beulieu et l. (2004), texture ws significntly ffected y the mturity stge ut the effect of storge time decresed s mturity incresed. As it cn e oserved in tle 3, in the first 12 h of storge pple slices underwent relevnt vrition of colour prmeters in terms of luminosity nd hue ngle decrese, tht indictes generl surfce rowning tht cn e ttriuted to PPO ctivity. In the second prt of the storge, oth prmeters further decresed ut no significnt differences were found etween ripening stges. Colour chnges in fresh-cut melon nd kiwifruit re minly due to vrition in the internl structure of the tissue nd the induction of trnslucent wter-soked tissue while enzymtic rowning is scrcely influent. Whether in oth fruits nd in oth ripening stges surfce drkening ws oserved during 4 d of storge, degree of ripening significntly ffected lightness in kiwifruit ut not in melon. Conversely, hue ngle lthough decresing during storge, ws consistently higher in riper melon slices indicting vrition from yellow to red tonlity possily due to higher concentrtion in β-crotene upon ripening (Simndjuntk et l., 1996). 220 Journl on Processing nd Energy in Agriculture 19 (2015) 5
Tppi, Silvi et l. / Enzymtic Activity nd Metolism of Fresh-Cut s Function of ipening Degree Tle 3. Colour of pple, kiwifruit nd melon t the two ripening stges selected during ccelerted storge in controlled conditions. Different letters indicte significnt differences (p-level < 0.05) etween ripe nd unripe fruit for the sme storge time ipening Storge time (dys) Stge 0 0.5 1 2 4 81.45 ± 2.05 69.33 ± 2.45 65.57 ± 1.38 - - 82.33 ± 3.05 71.55 ± 2.56 67.67 ± 2.05 - - L* 48.45 ± 2.11-47.98 ± 3.07 47.2 1 ± 3.16 44.08 ± 3.77 38.11 ± 2.45-31.93 ± 3.61 31.88 ± 3.78 31.75 ± 1.81 57.24 ± 2.90-45.25 ± 1.72 45.26 ± 2.12 40.43 ± 4.97 57.85 ± 4.50-42.92 ± 4.04 44.11 ± 2.48 42.38 ± 4.97 97.57 ± 4.55 93.25 ± 2.56 88.15 ± 2.07 - - 98.67 ± 3.18 90.03 ± 3.56 84.66 ± 2.28 - - h 103.93 ± 2.11-103.72 ± 0.76 102.19 ± 2.16 101.17 ± 3.11 103.83 ± 2.70-104.70 ± 2.80 104.44 ± 2.05 106.32 ± 4.39 61.81 ± 0.98-60.89 ± 1.05 59.83 ± 1.76 60.08 ± 1.03 64.57 ± 0.89-62.91 ± 1.16 62.85 ± 1.37 62.11 ± 1.75 Enzymtic ctivity Figure 1 reports PPO ctivity mesured in fresh cut pples during 24 h of storge. Stright fter cutting, riper fruit showed higher ctivity compred to unripe one ut differences were not sttisticlly significnt. After 2 h, it incresed shrply until douling its vlues, ut then decresed gin. PPO is rpidly ctivted y peeling nd cutting opertions tht led to the contct with its sustrte. After 12 nd 24 h the ctivity ws very low nd did not show ny significnt differences etween smples. Texture loss is one of the min fctor limiting fresh-cut kiwifruit shelf-life (Gil et l., 2006) Softening nd texturl chnges re rought out y the ctions of multitude of cell-wll-loclized enzymes cting on specific, potentilly highly loclized sustrtes. According to Vroquux et l. (1990) texture rekdown in kiwifruit slices is minly due to the enzymtic rekdown of pectic compounds, due to demethoxyltion nd depolymeriztion y pectinolytic enzymes such s PME nd polyglcturonse (PG). In prticulr, PME cuses the removl of methylester groups from pectins in the cell wll tht re further depolymerised y PG. Figure 2 shows Pectinmethylesterse (PME) ctivity in fresh-cut kiwifruit slices during 4 d of storge. nripe fruits showed n ctivity tenfold compred to ripe fruit. PME ctivity is generlly higher erly in ripening (Toivonen nd Brummel, 2008) incresing ccessiility of PG to its sustrte. Peroxidse (POD) is n uiquitous enzyme found in most vegetle tissues nd comprises mny isozymes (Lmiknr nd Wtson, 2000). Its ctivity cuses the oxidtion of mono nd diphenols when even smll quntities of hydrogen peroxide ct s oxidising gent. It is involved in mny degrdtive rections tht ffect colour, rom, texture nd nutritionl chrcteristics of fruit nd vegetles. According to Lmiknr et l. (2005), high POD ctivity is relted to the PPO ctivity (/min) 3,0 2,5 2,0 1,5 1,0 0,5 0,0 0 4 8 12 16 20 24 Time of storge (h) Fig. 1. Polyphenoloxidse (PPO) ctivity (/ml) of pple t different ripening degrees during 24 h of storge in controlled conditions. Different letters indicte significnt differences (p-level < 0.05) etween ripe nd unripe fruit for the sme storge time PME ctivity (/ml) 2,0 1,5 1,0 0,5 0,0 Time of storge (dys) Fig. 2. Pectinmethylesterse (PME) ctivity (/ml) of kiwifruit t different ripening degrees during 4 d of storge in controlled conditions. Different letters indicte significnt differences (p-level < 0.05) etween ripe nd unripe fruit for the sme storge time 0 1 2 3 4 Journl on Processing nd Energy in Agriculture 19 (2015) 5 221
Tppi, Silvi et l. / Enzymtic Activity nd Metolism of Fresh-Cut s Function of ipening Degree development of off-odour nd off-colours in food nd Lmiknr et l. (2000) suggested tht could sustntilly reduce shelf-life of melons. As it cn e oserved in figure 3, POD ctivity ws doule in unripe melons compred to ripe ones. In oth smples it incresed until the second dy of storge, proly s consequence of the physiologicl response to wounding stress, nd then decresed gin. POD ctivity (/ml) Totl het 24h (J/g) 12 10 5 4 3 2 1 0 8 6 4 2 0 0 1 2 3 4 Time of storge (dys) Fig. 3. Peroxidse (POD) ctivity (/ml) of melon t different ripening degrees during 4 d of storge in controlled conditions. Different letters indicte significnt differences (p-level < 0.05) etween ripe nd unripe fruit for the sme storge time 6 Fig. 4. Totl metolic het production of pple, kiwifruit nd melon t the two ripening stges selected during 24 h t 10 C. Different letters indicte significnt differences (p-level < 0.05) etween ripe nd unripe fruit Metolic het production Metolic profiles otined y isotherml clorimetry hve een integrted in order to clculte the totl metolic het produced y fruit tissues during 24 h. esults re reported in figure 4. Opertions such s peeling nd cutting cuse mechnicl stress to tissues tht rect y strting different metolic processes imed t repiring the dmges. Therefore, s reported y Gomez et l. (2004) nd y Tppi et l. (2013), the het mesured fter wounding is the sum of the norml metolic ctivity nd the one due to wounding. As it cn e oserved, for ll fruits, riper fruits showed lower metolic het production. This my indicte loss of the ility to repir the dmge cused y mechnicl stress. While ll differences were sttisticlly significnt, the highest difference ws oserved for kiwifruit tht showed t the dvnced stge of ripening 50 % decrese in the metolic het production compred to the unripe fruit. CONCLSION The results otined in this reserch showed how the min qulittive nd metolic chrcteristics of the three different fruit were ffected y ripening degree during storge. Degree of ripening influenced significntly flesh colour, hrdness nd enzymtic ctivity in fresh-cut kiwifruit nd melon, ut it showed little effect on fresh-cut pple chrcteristics during the storge time considered. Metolic het decresed upon ripening in ll three fruits, showing strict connection etween the physiologicl stte of the tissue nd the entity of its wounding response. esults highlight how the determintion of the optiml ripening degree for fresh-cut production hs to e crried out specificlly for ech type of fruit. ACKNOWLEDGMENT: The uthors cknowledge the finncil support of the Itlin Ministry for Eduction, niversities nd eserch (FIB, Project BF100CEJ: Innovtive pproch for the study of fresh-cut fruit: qulittive, metolic nd functionl spects). EFEENCES AOAC Interntionl (2000). Officil methods of nlysis (OMA) of AOAC Interntionl, SA. Method numer: 942.15. Aville t http://www.eom.oc.org/ AOAC Interntionl (2002). Officil methods of nlysis (OMA) of AOAC Interntionl, 17th Edition, SA. Method numer: 920.15. Aville t http://www.eom.oc.org/ Britux, O., Amiot, M., ichrd, H., Nicols, J. (1991). Enzymtic rowning of sil (Ocimum silcum L.) studies on phenolic compounds nd polyphenol oxidse. Sciences des Aliments, 11, 49-62. Beulieu, J. C., Ingrm, D. A., Le, J. M., Bett-Grer, K. L. (2004). Effect of Hrvest Mturity on the Sensory Chrcteristics of Fresh-cut Cntloupe. Journl of food science, 69 (7), 250-258. Beirão-d-Cost S., Steiner A., Correi L., Empis J., Moldão-Mrtins M. (2006). Effects of mturity stge nd mild het tretments on qulity of minimlly processed kiwifruit. Journl of Food Engineering, 76, 616 625. Brecht, J.K. (1995). Physiology of lightly processed fruits nd vegetles. Horticulturl Science, 30(1), 18 22. Gil, M.I., Aguyo, E., Kder, A.A. (2006). Qulity chnges nd nutrient retention in fresh cut versus whole fruits during storge. Journl of Agriculturl nd Food Chemistry, 54, 4284-4296. Gómez, F., Toledo,. T., Wdsö, L., Geks, V., Sjöholm, I. (2004). Isotherml clorimetry pproch to evlute tissue dmge in crrot slices upon therml processing. Journl of Food Engineering, 65, 165-173. 222 Journl on Processing nd Energy in Agriculture 19 (2015) 5
Tppi, Silvi et l. / Enzymtic Activity nd Metolism of Fresh-Cut s Function of ipening Degree Hgermn, AE., Austin P. (1986). Continuous spectrophotometric ssy for plnt pectin methyl esterse. Journl of Agriculture nd Food Chemistry, 340, 440 4. Hodges, DM., Toivonen., PMA. (2008). Qulity of fresh-cut fruits nd vegetle s ffected y exposure to iotic stress. Posthrvest Biology nd Technology, 48, 155-162. Lmiknr, O., Wtson, M.A. (2000). Cntloupe melon peroxidse: Chrcteriztion nd effects of dditives on ctivity. Nhrung, 3, 168-172. Lmiknr, O., Bett-Grer, K. L., Ingrm, D. A., Wtson, M. A. (2005). se of Mild Het Pre-tretment for Qulity etention of Fresh-cut Cntloupe. Journl of food science, 70 (1), C53-C57. Lmiknr, O., Chen, J., Bnks, D., nd Hunter, P. (2000). Biochemicl nd microil chnges during the storge of minimlly processed cntloupe. Journl of Agriculturl nd Food Chemistry, 48 (12), 5955-5961. Morles-Blncs E.F., Chindi V.E., Cisneros-Zevllos L. (2002). Therml inctivtion kinetics of peroxidse nd lipoxigense from roccoli, green sprgus nd crrots. Journl of Food Science, 67, 146 54 Silveir, A. C., Aguyo, E., Chisri, M., nd Artés, F. (2011). Clcium slts nd het tretment for qulity retention of freshcut Gli melon. Posthrvest Biology nd Technology, 62 (1), 77-84. Simndjuntk, V., Brrett, D. M., nd Wrolstd,. E. (1996). Cultivr nd mturity effects on muskmelon (Cucumis melo) colour, texture nd cell wll polyscchride composition. Journl of the Science of Food nd Agriculture, 71 (3), 282-290. Tppi, S., Tylewicz,., Cocci, E., omni, S., Dll os M., occuli P. (2013). Influence of ripening stge on qulity prmeters nd metolic ehviour of fresh-cut kiwifruit slices during ccelerted storge. Journl on Processing nd Energy in Agriculture, 17 (4), 149-153. Vroquux, P., Lecendre, I., Vroquux, F., Souty, M. (1990). Chnge in firmness of kiwifruit fter slicing. Science des Aliments, 10 (1), 127 139 Wdsö, L., Gόmez Glindo, F. (2009). Isotherml clorimetry for iologicl ppliction in food science nd technology. Food Control, 20, 956 961. eceived: 27.04.2015. Accepted: 2.10.2015. Journl on Processing nd Energy in Agriculture 19 (2015) 5 223