Inactivation of Pathogens by Dense Carbon Dioxide to Enhance Safety of Small Fruits. Name(s), mailing and address(s) of principal investigators

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1 Progress Report Title: Intivtion of Pthogens y Dense Cron Dioxide to Enhne Sfety of Smll Fruits Grnt Code: SRSFC Projet # Proposl Ctegory: _X_ Reserh Outreh Nme(s), miling nd emil ddress(s) of prinipl investigtors Qixin Zhong, Ph.D. (PI) Assistnt Professor in Food Biophysis The University of Tennessee Dept. of Food Siene nd Tehnology 09 River Drive Knoxville, TN 99-9 Phone: () 9-19 Fx: () 9- E-mil: qzhong@utk.edu Dvid A. Golden, Ph.D. (o-pi) Assoite Professor in Food Miroiology The University of Tennessee Dept. of Food Siene nd Tehnology 0 River Drive Knoxville, TN Phone: () 9- Fx: () 9-0 E-mil: dvid.golden@t.edu K.P. Sndeep, Ph.D. (o-pi) Assoite Professor in Food Engineering North Crolin Stte University Deprtment of Food Siene 19 Shu Hll, Box Rleigh, NC 9- Phone: (919) 1-9 Fx: (919) 1-1 E-mil: kp_sndeep@nsu.edu Ojetives: (1). Our primry ojetive is to investigte the effiy of dense phse ron dioxide (DPCD) in intivting pthogens of onern to smll fruits; (). Our seondry ojetive is to evlute the effets of DPCD proessing on the qulity of these fruits. Justifition: Reent outreks of Esherihi oli O1:H in spinh nd lettue hve rekindled the serh for innovtive tehniques to grow, hrvest, hndle, proess, nd store fruits nd vegetles. Some trditionl foods, e.g. strwerry nd lueerry, re onsumed with miniml proessing, nd ny preservtion tehnique, regrdless of its effiy, is inpplile in relity if the resulting produts hve qulity ttriutes tht re uneptle to onsumers. Chlorine wshes re onventionlly used y fruit nd vegetle produers ut this redue miroil ounts y 1 or orders of mgnitude. 1 The potentil internliztion of pthogens in plnt tissue poses n even igger onern in food sfety euse of the inility of wshing nd surfe deontmintion tehniques suh s rdition. Arusvge et l. suggested to reserh snittion prties tht n disinfet proteted sites (suh s wounds where pthogens most likely tth nd proliferte) nd internl plnt tissue more effetive thn wshing. Dense phse ron dioxide (DPCD) is non-therml tehnology tht omines the teriidl effiy of CO nd high pressure t temperture muh lower thn tht during onventionl therml tretments. The teriidl funtion of DPCD ws first notied when the

2 CO pressure ws suddenly relesed from. MP to mient pressure., Most developments of this tehnique however hve ourred in the pst dede. Vrious temperture nd pressure omintions in DPCD hve een reserhed for their effiy in reduing miroorgnisms in different produts., The effiy of DPCD vries signifintly with proessing onditions i.e. temperture, pressure, equipment, tretment time, food mtries, nd teril strins. For fruit juies, uffers, or growth medi, sreening of proessing prmeters my enle the redution of miroorgnisms greter thn orders of mgnitude (regultory stndrds for psteuriztion) t temperture lower thn 0 C. But the fesiility of intivting pthogens in smll fruits hs not een studied. In this projet, we proposed to investigte the effiy of DPCD in intivting Esherihi oli O1:H nd Slmonell spp., ommon foodorne pthogens to fruits nd vegetles, inoulted on strwerries. The effets of DPCD proessing on strwerry firmness will lso e investigted. Methodologies: Strwerry will e used s the model smll fruit in this projet. The tehnique nd protool developed however n e extended to other smll fruits in more extensive future projet. Dr. Zhong will e responsile for proessing inoulted strwerry under different omintions of temperture, pressure, nd tretment time. Dr. Golden will e in hrge of inoultion nd detetion of pthogens. Dr. Sndeep will use his engineering expertise to evlute the effiy of DPCD proessing nd ompre it to other onventionl proesses suh s hlorine wshing so tht the reserhed prmeters n e used in future prodution. Equipment nd proessing protool The superritil CO unit (Thr Tehnologies, Pittsurgh, PA) is housed in the Deprtment of Food Siene nd Tehnology t the University of Tennessee. The system is shemtilly illustrted in Figure 1. Bone-dry grde CO (Airgs, Chigo, IL) from ylinder will e ooled to C nd delivered y CO pump ontinuously t ontrolled mss flow rte. CO will e heted to set temperture y het exhnger efore entering the pressure vessel (00 ml volume). The vessel pressure is regulted y n utomti pressure regultor downstrem nd the vessel temperture is ontrolled y n outer het jket. In typil experiment, three strwerry replites will e ontined in smple holder (see elow), whih will then e pled into the pressure vessel. The pressure vessel p will then e losed nd the vlve to the CO ylinder will e opened. All proessing prmeters re ontrolled y omputer. After tretment, the vessel will e depressurized nd the smple olleted for miroil nd qulity tests. Chiller CO ylinder Exhust P CO pump Smple holder housing strwerry Automti pressure regultor Heter Pressure vessel Figure 1. Shemtis of DPCD proess setup.

3 Sreening of proessing prmeters. Prmeters to e sreened inlude temperture (0-0 C), residene time (10-0 min), nd pressure (1 0 MP). Two prllel tests will e performed. One set of tests will e to evlute totl plte ounts of indigenous teril popultions efore nd fter DPCD proessing. Smples in the other set will e inoulted with Slmonell or E. oli O1:H. Dt will e sttistilly nlyzed using SAS 9.1 (Cry, NC). Miroil inoultion nd detetion tehniques Test orgnisms nd ulture mintenne. Five strins eh of E. oli O1:H nd Slmonell serovrs will e used throughout this study. All test strins re held in ulture olletions t the University of Tennessee. Test ultures will e mintined on tryptone soy gr (TSA) slnts t C nd grown in tryptone soy roth (TSB) t C. A minimum of two suessive h trnsfers will e performed efore experimenttion with h ultures. Prior to experimenttion, the five test strins (for eh orgnism) will e omined to otin mixed popultions of E. oli O1:H or Slmonell onsisting of pproximtely equl proportions of eh test strin. Inoulted, untreted strwerry will serve s the ontrol for eh experimentl proedure. A 100 μl inoulum of E. oli O1:H or Slmonell, t level of log 10 CFU/g of fruit will e deposited on the skin or stem sr tissue of strwerries; the inoulted strwerries will e ir dried for h in iosfety hood nd stored t C for 0 h efore eing proessed y DPCD. Proessed smples will e shken with sonitor nd wshed with 100 ml of phosphte-uffered sline (PBS; ph.) to reover miroorgnisms for exmintion. The protool will lso e used to determine indigenous teril popultions in strwerries. Miroiologil enumertion. E. oli O1:H popultions will e determined y surfe plting (0.1 ml), serilly diluted reovered smples onto modified eosin methylene lue gr (MEMB), whih ontins: 10 g peptone, g potssium phosphte, 0. g eosin Y, 0.0 g methylene lue, 10 g soritol, g sodium hloride, 0.0 g sodium novoioin, nd 1 g gr/l of deionized wter. Roelle et l. 9 demonstrted tht MEMB provides good reovery of sulethlly injured E. oli O1:H. Slmonell popultions will e determined similrly ut y plting onto XLT gr (Difo). Aeroi teril popultions nd yest popultions will e determined using TSA nd YM gr (yest nd mold gr) supplemented with 0.1% hlormpheniol. When E. oli O1:H nd Slmonell popultions re not deteted y diret plting, smples will e enrihed in modified TSB nd tetrthionte roth, respetively, followed y streking onto MEMB nd XLT. Representtive presumptive olonies of E. oli O1:H seleted from MEMB will e onfirmed using the API 0E miniturized dignosti test kit (iomérieux Vitek, In., Hzelwood, MO) nd the E. oli O1 ltex gglutintion test (Oxoid). Representtive presumptive olonies of Slmonell seleted from XLT will e onfirmed using the API 0E miniturized dignosti test kit. Effets of DPCD on strwerry qulity Although the udget is limited in the urrent projet, we pln to use texture nlyzer to ompre the firmness of strwerry efore nd fter DPCD proessing nd Hunter olorimeter to ompre the olor hnges. In future reserh, tste pnels will e onduted to get omplete profile of proessing effets on orgnolepti properties. Kineti nlyses of DPCD proessing Bsed on the deteted miroorgnism popultions fter proessing for different times t onstnt pressure nd temperture, the deiml redution time (D-vlues) will e estimted. The D-vlues t onstnt pressure ut different tempertures will e used to estimte the therml

4 resistne onstnt (z-vlues). These vlues will e used to ompre the results with the use of other proesses suh s therml proessing, hlorine wshing nd will e neessry informtion for prtil prodution. Results: In relevnt projet of PIs Zhong nd Golden, we uilt smple holder (Figure ) to ontin three produe smple replites tht n e pled in the high pressure vessel nd proessed simultneously. Tht projet foused on spinh leves, referred s the spinh projet herefter, nd E. oli strin K-1 inoulted on spinh leves ws intivted to non-detetle level (Figure ). The spinh projet, lthough not diretly relted to this SRSFC projet, provided some preliminry informtion tht n e used in this SRSFC projet. The student ssigned to this projet strted her study in August 00 nd hs lerned fundmentl miroiologil methodologies nd the opertion of the superritil CO system. She will fous on the SRSFC projet in Jn. Mr. 00, sed on the three-month pyments of her slry requested in the proposl. We expet to sumit finl report in April 00. Figure. Custommde smple holder. (A) E. oli K1 Bkground Grm-positive (B) E. oli K1 Bkground Grm-positive. Detetion limit Proessing Time (min) (C). Detetion limit Proessing Time (min) E. oli K1 Bkground Grm-positive. Detetion limit Proessing Time (min) Figure. Redution of E. oli K1 on spinh leves y dense phse ron dioxide t 0 C nd (A),. (B) nd 10 (C) MP. Error rs represent one stndrd devition. Within eh pressure nd miroil ount, dt points leled with different letters re sttistilly different (P < 0.0).

5 Conlusions: Although we do not hve dt to report towrds the speifi ojetives of the SRSFC projet, we hve generted some preliminry informtion from seprte relevnt projet tht provided promising results of developing dense phse ron dioxide into non-therml tehnology to enhne the sfety of produe. We hve uilt smple holder tht n e used for this projet nd trined grdute student redy for this projet. Impt Sttement: Developing novel non-therml psteuriztion tehniques will ensure tht fresh produe produts re sfe nd nutritious. This will redue the outreks of foodorne illness, proteting onsumers nd reduing eonomi losses due to rells, diseses nd lwsuits. Cittion(s) for ny pulitions rising from the projet: No report t this stge. Referenes: 1. Beuht, L.R J. Food Prot. 9, 0.. Arusvge, D.; Lee, K.; Miller, S.; nd LeJeune, J.T. 00. J. Food Si. 1, R9.. Frser, D Nture 1,.. Foster, J.W.; Cown, R.M.; nd Mg, T.A. 19. J. Bteriol. : 0.. Spilimergo, S. nd Bertuo, A. 00. Biotehnol. Bioeng.,.. Dmr, S. nd Bln, M.O. 00. J. Food Si. 1, R1.. Sy, K.V.; MWtters, K.H.; nd Beuht, L.R. J. Food Prot., 11.. Hn, Y.; Linton, R.H.; nd Nelson, P.E. 00. J. Food Prot.,. 9. Roelle, M.; Clvero, S.; nd Beuht, L.R Appl. Environ. Miro. 1,.