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1 As. J. Foo Ag-In. 21, 3(5), Asin Journl of Foo n Agro-Inustry ISSN Aville online t Reserh Artile Effet of hot wter tretments on survivl of E. oli n Slmonell spp. n physil properties in fresh-ut rooli florets Phni Renumrn, Vrit Srilong, Apiree Uthirtnkij, Sirihi Knlynrt n Pongphen Jitreert* Division of Posthrvest Tehnology n Posthrvest Innovtion Centre, Shool of Bioresoures n Tehnology, King Mongkut s University of Tehnology Thonuri, Bngkok 115, Thiln. Emil: pongphen.jit@kmutt..th This rtile ws originlly presente t the Interntionl Symposium GoOrgni 29, Bngkok, Thiln, August 29. Astrt Hot wter tretments were pplie to fresh-ut rooli (Brssi olere L.) florets to investigte their effets on the survivl of foo-orne pthogens (Esherihi oli n Slmonell spp.) n on the physil properties of the rooli florets uring storge. Brooli florets were trete with hot wter t 5, 55 n 6 C for 3 min n immeitely oole with wter t 4 C. Non-hot wter trete rooli florets serve s ontrols. The trete rooli florets were then ple in PVC oxes for 13 ys t 4 C. Hot wter tretment t 55 n 6 C for 3 min ompletely ontrolle E. oli n Slmonell spp. ut these tretments resulte in tissue softening n sl-like symptoms on the rooli florets. The hot wter tretment t 5 C for 3 min resulte in reution of oth foo-orne pthogens for log 1 CFU.g -1 n mintine freshness, firmness n elye the yellowing of rooli florets for 4 ys. These results suggest tht hot wter tretment t 5 C for 3 min hs the potentil to mintin the physil qulity of rooli florets ut this tretment my not e suffiient to ontrol the growth of foo-orne pthogens. Keywors: post hrvest, vegetles, het tretment, foo-orne pthogens, qulity, Thiln. Introution Brooli hs eome populr vegetle euse of its high nutrition, s it ontins importnt levels of vitmins, ntioxints n nti-rinogeni ompouns [1]. The populrity n sles of fresh-ut rooli hs inrese ue to rising onsumer emn for helthy n onvenient foo. However, one of the most importnt prolems with fresh-ut proue is the potentil miroil ontmintion y foo-orne pthogens whih re use of humn illness. Severl isinfetnt hemils or snitizing tretments hve een use to reue the initil miroil los on fresh proue estine for fresh-ut proessing [2].

2 As. J. Foo Ag-In. 21, 3(5), Hot wter tretment is ommonly use physil tretment to reue foo-orne pthogens n lso to ely senesene [3]. At present there is limite literture eling with miroil ontrol n mintining qulity of fresh-ut rooli florets y hot wter tretment. Thus, the im of the present stuy ws to investigte the optiml hot wter tempertures require to reue foo-orne pthogens (E. oli n Slmonell spp.) n to mintin the physil qulity of freshut rooli florets uring storge t low temperture. Mterils n Methos Plnt mteril n hot wter tretments Fresh rooli (Brssi olere L.) hes were ut into iniviul florets n then iretly trete with hot wter t 5, 55 n 6 C for 3 min. The trete florets were then oole in wter t 4 C for 5 min n ny exess wter ws remove y mnul sl spinner. Trete rooli smples were rnomly tken for nlysis of miroil growth n physil properties to ompre them with the untrete florets s ontrol. A 2 g smple of the rooli florets ws pke in polyvinylhlorie (PVC) ox n kept t 4 C. Eh tretment h 3 replites (oxes). Miroil nlysis A 25 g smple of rooli florets ws homogenize in 225 ml of 1% sterile peptone wter using stomher (IUL Instruments Mstitor, Brelon, Spin) for 2 min. Ten-fol ilution series were me in sterile peptone wter s require for plting. The following ulture mei n onitions were use to enumerte the miroil growth: (1) Plte ount gr (HiMei) inute t 37 C for 24 h for totl teri; (2) Eosin Methylene Blue gr, EMB (HiMei) inute t 37 C for h for E. oli ounts; n (3) Xylose-Lysine Deoxyholte gr, XLD (HiMei) t 37 C for h for Slmonell spp. Miroil ounts were expresse s log 1 CFU g 1 (olony forming units per grm of smple). Colour mesurement Colour of rooli floret smples ws etermine y mesuring L*, *, * vlues n hue ngle with Minolt hrom meter (Moel CR3, Osk, Jpn) whih overe n re of 8 mm 2. Three positions on eh of the florets were mesure for eh tretment n storge time. Internl gs nlysis CO 2 n O 2 onentrtions in PVC oxes of rooli florets were mesure y Gs Anlyzer (OXYBABY 6.). A neele sensor ws penetrte on the silione ruer tthe on the outer surfe (on the top) of the li for 1 se. The CO 2 n O 2 onentrtions were etermine n expresse s perentge. Respirtion rte The respirtion rte of rooli florets ws etermine oring to the metho of Lemoine et l. [4]. Smples of rooli florets (pproximtely 15 g) were ple in 2 L plsti jr, sele n inute t 4 C for 3 h. A 1 ml of hespe gs smple ws withrwn with syringe through septum fitte in the jr li n injete into gs hromtogrph (Shimzu-8A, Tokyo, Jpn) equippe with Porpk Q olumn. The t ws expresse s mg CO 2 g -1 h -1. Sensory evlution The sensory pnel onsiste of 8 1 untrine memers. The sensory ttriutes were evlute y visul hrteristis suh s visul qulity, olour n oour/off-oours. A rting sore inex ws given rnging from 1 eing ompletely eteriorte to 9 representing exellent qulity n freshness of the nlyze smple. The smple ws onsiere s uneptle in terms of sensory hrteristis when the rting sore ws lower thn 5 (limit of slility).

3 As. J. Foo Ag-In. 21, 3(5), Sttistil nlysis Anlysis of vrine (ANOVA) n Dunn s new multiple rnge test for omprison of mens n lest signifint ifferenes (P <.5) were performe on the t using the SPSS version 12. softwre. Results n Disussion The effet of hot wter (HW) tretments t ifferent tempertures on E. oli n Slmonell spp. ounts in fresh-ut rooli florets is shown in Figure 1. The initil popultion of E. oli n Slmonell spp. in non-hot wter (non-hw) trete rooli florets verge 3.23 n 3.23 log 1 CFU g -1 respetively. Applition of hot wter on fresh-ut rooli florets reue the numers of E. oli n Slmonell spp. ompre to tht of ontrol. Prtiulrly the HW tretments t 55 n 6 C ompletely inhiite oth foo-orne pthogens on y 1 of storge, while the HW tretment t 5 C for 3 min reue oth foo-orne pthogens y log 1 CFU g -1 (Fig. 1). A similr result ws hieve y Li et l. [5] who reporte tht wrm wter (5 C), with or without 2 mg/l hlorine, signifintly reue the initil popultion of mesophili eroi miroflor in fresh-ut ieerg lettue y log 1 CFU g -1. The popultion ounts of oth pthogens grully inrese throughout the storge time in ll tretments t 4 C. These results suggest tht oth pthogens n survive n multiply t low temperture, with Li et l. [6], Li et l. [7] n Moreir et l. [8] pulishing similr finings. Moreover, the HW tretments were more effiient in ontrolling the growth of E. oli ompre with Slmonell spp. The ifferentil temperture growth response suggests tht Slmonell spp. hs greter tolerne to therml wter tretments thn E. oli (Fig. 1A, 1B). 8 (A) E. oli (log 1 CFU g -1 ) Dys of storge Control 5ºC 55ºC 6ºC

4 As. J. Foo Ag-In. 21, 3(5), (B) Slmonell spp. (log 1 CFU g -1 ) Dys of storge Control 5ºC 55ºC 6ºC Figure 1. Miroil popultion of E. oli (A) n Slmonell spp. (B) in fresh-ut rooli florets trete with hot wter t 5, 55 n 6ºC for 3 min. Fresh-ut rooli florets trete with HW t 55 n 6 C showe lower L* vlue n hue ngle thn florets trete with 5 C n thn non-trete rooli throughout the storge time (Fig. 2A, 2D). The L* vlue n hue ngle of florets trete t 5 C ws lose to tht of nontrete rooli florets. However, HW tretments t 55 n 6 C seeme to erese the florets qulity ue to these tempertures use tissue softening n sl-like symptoms on the rooli florets. The green olour of rooli is inite y * vlue. The fresh-ut rooli trete with HW t 5 C showe lower * vlue thn the other tretments (Fig. 2B). This result inites tht HW t 5 C ws the est tretment to mintin the green olour of rooli. Severl reports hve lso shown tht het tretments suh s HW or hot ir ely the yellowing proess n senesene of rooli florets [3, 9, 1]. A erese in O 2 n n inrese in CO 2 levels in the pkging were oserve in ll tretments (Fig. 3A, 3B). Gses insie the pkge were signifintly ifferent epening on the egree of wter temperture. The lowest levels of O 2 n the highest level of CO 2 were etete in the pkge of fresh-ut rooli trete t 5 C for 3 min. Oserve gs levels were out 13-16% O 2 n % CO 2, wheres the O 2 n CO 2 levels in the pkge of the other tretments rnge out 16-21% n.-4.4%, respetively. Toivonen et l. [11] suggeste tht the gs levels of % O 2 were eptle to keep the rooli hes n when the CO 2 levels rehe out %, this ffete the evelopment of off-oours, whih woul e foun t 4 ys of storge t 1 C. Fonse et l. [12] showe tht 1-2 kp O 2 n 15-2 kp CO 2 in the tmosphere of pkging ws effetive

5 As. J. Foo Ag-In. 21, 3(5), L* vlue Dys fter storge -5 * vlue Hue ngle * vlue (B) Dys fter storge Dys fter storge Dys fter storge Figure 2. L*, *, * vlue n hue ngle of fresh-ut rooli florets trete with hot wter t 5, 55 n 6ºC for 3 min.

6 As. J. Foo Ag-In. 21, 3(5), O2 CO2 (%) O2 (%) Respirtion rte rte (mg (mgco2/kg.hr) -1.hr -1 ) Dys fter storge Control 5ºC 55ºC 6ºC Dys fter storge Control 5ºC 55ºC 6ºC Dys fter storge (A) (B) (C) Control 5ºC 55ºC 6ºC Figure 3. Oxygen (A) n ron ioxie (B) onentrtion in pkge n respirtion rte (C) of fresh-ut rooli florets trete with hot wter t 5, 55 n 6ºC for 3 min.

7 As. J. Foo Ag-In. 21, 3(5), to mintin the qulity of shree Gleg kle n exten the storge life for 4-5 ys t 2 C. Serrno et l. [13] reporte tht onentrtion of gs on No-P gs were out 4-12 kp O 2 n kp CO 2, n h extene the storge-life of rooli hes up to 28 ys t 1ºC, with no symptoms of rom n off-flvours oul e etete insie the gs fter opening. Ji et l. [14] reporte tht MAP with no holes pkging h gs levels roun 1-6% O 2 n 8-14% CO 2, whih oul mintin the qulity of rooli florets for 13 ys t 4ºC. High wter tempertures inrese the respirtion rte of fresh-ut rooli florets (Fig. 3C). Florets trete with HW t 6 C for 3 min showe shrply inrese respirtion rtes on the first y of storge ompre to other tretments n followe y rooli florets trete with HW t 55 C. The respirtion rte of the florets trete with HW t 5 C n of non-trete rooli florets grully inrese n peke on y 7 of storge, n florets trete with HW t 5 C h slightly higher respirtion rte thn the ontrol (Fig. 3C). Thus, the inrese of CO 2 n the erese of O 2 gs levels in the pkge were the result of higher respirtion rte of HW trete rooli florets. The evelopment of the gs levels is mostly se on film permeility, surfe re, storge temperture, ultivr, wouning n respirtion tivity of the proue [13, 15]. The visul qulity of HW trete rooli florets ws ompre with tht of non-hw trete rooli. The higher temperture HW tretments showe higher sores of visul qulity n visul olor, n lower sores of oor ttriutes ompre with non-hw tretments (Fig.4A). However, HW tretments t 55-6 C for 3 min re not reommene for fresh-ut rooli floret euse they use tissue softening n sl-like symptoms.

8 As. J. Foo Ag-In. 21, 3(5), (A) Visul Qulity sore (B) Visul Color sore Dys of storge Control 5ºC 55ºC 6ºC 1 (C) Dys of storge Control 5ºC 55ºC 6ºC Oor sore Dys of storge Control 5ºC 55ºC 6ºC Figure 4 Sensory evlution using 9-points heoni sle visul qulity (A), visul olour (B) n oour (C) sore of fresh-ut rooli florets trete with hot wter t 5, 55 n 6ºC for 3 min. Dsh line is representtive of the onsumer eptne, if the sensory sore is lower thn 5 mens the onsumer will not ept the fresh-ut rooli florets.

9 As. J. Foo Ag-In. 21, 3(5), Conlusions This stuy onfirme tht foo-orne pthogens (E. oli n Slmonell spp.) re foun in rooli florets originting in their generl ultivtion n hnling. HW t 55 n 6 C for 3 min were the most effetive tretments to inhiit E. oli n Slmonell spp. growth in fresh-ut rooli with 4 ys of storge t 4 C, ut the tissue of the rooli florets oul not tolerte these tempertures. A HW tretment t 55 C for 3 min suessfully reue pthogeni teri ontmintion n mintine the physil qulity of fresh-ut rooli florets for 7 ys. Therefore, these results suggest tht HW tretment t 5 C for 3 min hs the potentil to mintin the physil qulity of rooli florets ut this tretment my not e suffiient to ontrol foo-orne pthogen infetions. Aknowlegement The uthors woul like to thnk the Commission on Higher Eution, Thiln for support y grnt fun uner the progrm Strtegi Sholrships for Frontier Reserh Network for the Ph.D. Progrm Thi Dotorl egree for this reserh. Referenes 1. Shpiro, T.A., Fhey, J.W., We, K.L., Stephenson, K.K. n Tlly, P. (21). Chemoprotetive gluosinoltes n isothioyntes of rooli sprouts: metolism n exretion in humns. Cner Epiemiology Biomrkers n Prevention, 1: USFDA, U.S. Foo n Drug Aministrtion. (21). Methos to reue/eliminte pthogens from fresh n fresh-ut proue. Chpter V In: Anlysis n evlution of preventive ontrol mesures for the ontrol n reution of miroil hzrs on fresh n fresh ut proue. U.S. Foo n Drug Aministrtion, Center of Foo Sfety n Applie Nutrition (Online). Aville: ~omm/ift3-5html. 3. Funmoto, Y., Ymuhi, N., Shigeng, T. n Shigyo, M. (22). Effets of het tretment on hlorophyll egring enzymes in store rooli (Brssi olere L.). Posthrvest Biology n Tehnology, 24: Lemoine, M.L., Civello, P., Chves, A. n Mrtínez, G. (29). Hot ir tretment elys senesene n mintins qulity of fresh-ut rooli florets uring refrigerte storge. LWT - Foo Siene n Tehnology, 42(6): Li, Y., Brkett, R.E., Shewfelt, R.L. n Beuht, L.R. (21). Chnges in pperne n nturl miroflor on ieerg lettue trete in wrm, hlorinte wter n then store t refrigertion temperture. Foo Miroiology, 18: Li, Y., Brkett, R.E., Chen, J. n Beuht, L.R. (21). Survivl n growth of Esherihi oli O157:H7 inute onto ut lettue efore or fter heting in hlorinte wter, followe y storge t 5 or 15 egrees C. Journl of Foo Protetion, 64(3): Li, Y., Brkett, R.E., Chen, J. n Beuht, L.R. (22). Mil het tretment of lettue enhnes growth of Listeri monoytogenes uring susequent storge t 5 O C or 15 O C. Journl of Applie Miroiology, 92:

10 As. J. Foo Ag-In. 21, 3(5), Moreir, M.R., Pone, A.G., Vlle, C.E., Pereyr, L. n Rour, S.I. (28). Mil het shoks to exten the shelf life of minimlly proesse lettue. Journl of Applie Hortiulture, 1(2): Tin, M.S., Woolf, A.B., Bowen, J.H. n Ferguson, I.B. (1996). Chnges in olor n hlorophyll fluoresene of rooli florets following hot wter tretment. Journl of the Amerin Soiety for Hortiulturl Siene, 121: Teri, H., Knou, M., Mizuno, M. n Tsuhi, H. (1999). Inhiition of yellowing n ethylene proution in rooli florets following high temperture tretment with hot ir. Foo Preservtion Siene, 25: Toivonen, P.M.A. n DeEll, J.R. (21). Chlorophyll fluoresene, fermenttion prout umultion, n qulity of store rooli in moifie tmosphere pkges n susequent ir storge. Posthrvest Biology n Tehnology, 23: Fonse, S.C., Oliveir, F.A.R., Breht, J.K. n Chu, K.V. (25). Influene of low oxygen n high ron ioxie on shree Gleg kle qulity for evelopment of moifie tmosphere pkges. Posthrvest Biology n Tehnology, 35: Serrno, M., Mrtinez-Romero, D., Guillen, F., Cstillo, S. n Vlero, D. (26). Mintenne of rooli qulity n funtionl properties uring ol storge s ffete y moifie tmosphere pkging. Posthrvest Biology n Tehnology, 39: Ji., C.G., Xu, C.J., Wei, J., Yun, J., Yun, G.F., Wng, B.L. n Wng, Q.M. (29). Effet of moifie tmosphere pkging on visul qulity n gluosinoltes of rooli florets. Foo Chemistry, 114: Bur, S., Klier, R., Hmmes, W.P. n Crle, R. (24). Sensory n miroiologil qulity of shree, pkge ieerg lettue s ffete y pre-wshing proeures with hlorinte n ozonte wter. Innovtive Foo Siene n Emerging Tehnologies, 5: