767

Journalof Food Protection, Vol. 66, No. 5, 2003, Pages 767– 774 Copyright q,InternationalAssociation for FoodProtection

Chemical andIrradiation Treatments for Killing Escherichia coli O157:H7 onAlfalfa, Radish, and Mung Bean Seeds

M. L. BARI,1 E. NAZUKA,1 Y. SABINA,1 S.TODORIKI, 1,2 AND K. ISSHIKI1*

1FoodHygiene Teamand 2RadiationInformation Technology Laboratory, National Food Research Institute,Kannondai-2-1-12, Tsukuba305-8642, Japan

MS02-352:Received 26September 2002/ Accepted 3December 2002 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021 ABSTRACT

Inthis study, the effectiveness of dry-heat treatment in combination with chemical treatments (electrolyzed oxidizing [EO] water,califresh-S,200 ppm of active chlorinated water) with and without sonication in eliminating Escherichiacoli O157:H7on laboratory-inoc ulatedalfalfa, radish, and mung bean seeds was compared with that of dry-heat treatment in combinationwith irradiation treatment. The treatment of mung bean seeds with EO waterin combination with sonication followedby a rinsewith sterile distilled water resulted in reductions of approximately 4.0 log 10 CFU of E. coli O157:H7 per g,whereas reductions of ca. 1.52 and 2.64 log 10 CFU/gwereobtained for radish and alfalfa seeds. The maximum reduction (3.70 log10 CFU/g)for mung bean seeds was achieved by treatment with califresh-S and chlorinated water (200 ppm) in combinationwith sonication and a rinse.The combination of dry heat, hot EO watertreatment, and sonication was able to eliminatepathogen populations on mung bean seeds but was unable to eliminate the pathogen on radish and alfalfa seeds. Otherchemical treatments used were effective in greatly reducing pathogen populations on radish and alfalfa seeds without compromisingthe quality of the sprouts, but these treatments did not result in the elimination of pathogens from radish and alfalfaseeds. Moreover ,acombinationof dry-heat and irradiation treatments was effective in eliminating E. coli O157:H7 on laboratory-inoculatedalfalfa, radish, and mung bean seeds. An irradiation dose of 2.0 kGy in combination with dry heat eliminated E. coli O157:H7completely from alfalfa and mung bean seeds, whereas a 2.5-kGydose of irradiationwas required toeliminate the pathogen completely from radish seeds. Dr yheatin combination with irradiation doses of up to2.0 kGy did notunacceptably decrease the germination percentage for alfalfa seeds or the length of alfalfa sprouts but did decrease the lengthsof radish and mung bean sprouts.

Seedsprouts, an elementof traditionaloriental cuisine, hasbeen found to completely eliminate pathogens under havenow gained popularity in many parts of the world, experimentalconditions (18). Hydrogenperoxide, sodium includingEurope and United States, because of theirnutri- hypochlorite,calcium hypochlorite, ethanol, ozonated wa- tivevalue. There has been an increase in consumer demand ter,calcinatedcalcium, and organic acids have been tested for mungbean, alfalfa, radish, and other seed sprouts that for theireffectiveness in killing pathogens on seeds and areprepared either commercially or at home (20). The sprouts (2,5, 16,22). Thesesurface treatments cannot guar- sproutsare grown hydroponically and are usually eaten raw anteethat the interior of the seed is also decontaminated insalads or in sandwiches but can be cooked, as in an (24). Taorminaand Beuchat (23, 24) studiedthe efŽ cacies oriental-stylemeal (15). Increasingnumbers of microbial ofvarious chemical treatments in killing E. coli O157:H7 infectionsassociated with the consumption of sproutshave andreported that none of the chemical treatments evaluated beendocumented in recent years. From 1995and 1998, satisfactorilyreduced populations of E. coli O157:H7 on nineoutbreaks of Escherichiacoli O157:H7 and Salmo- alfalfaseeds and sprouts. These investigators concluded nella infectionsassociated with commercial sprouts were thatprocedures recommended for thesanitization of alfalfa reported (18). Pathogensinvolved in multiple international seedsfail to eliminatethe pathogen. The treatment of mung outbreakslinked to raw sproutsinclude E. coli O157:H7, beanseeds with chlorine was reportedto be ineffective in variousserotypes of Salmonella, and Bacilluscereus (11, eliminating E. coli O157:H7 and Salmonella on mung bean 18, 25). Seedshave been the likely source of contamination sprouts (29). Low-doseirradiation can be usedsuccessfully inmost of theoutbreaks, although contamination may also asan antifungaltreatment for seeds,and there are numerous resultfrom theuse of contaminated water in sprout pro- reportson the use of this treatment for avarietyof seeds, ductionor from mishandlingduring harvesting, packaging, i.e.,soybean, cotton, peanut, maize, onion, barley, chick- ordistribution or in the home. pea,Brazilian bean, and corn (3,4, 7, 8, 10, 11, 19, 27). Researchershave investigated chemical and heat treat- Thislow-dose irradiation treatment was ableto controlfun- mentsfor thereduction or elimination of pathogens inoc- galcontamination without affecting the germination of the ulatedonto seeds and sprouts. However ,nosingletreatment seeds. *Authorfor correspondence. Tel and Fax: 181-298-38-8067;E-mail: Recently,the U.S. Foodand Drug Administration [email protected]. (FDA) gaveits Ž nalapproval for theuse of irradiation at 768 BARI ET AL. J.FoodProt., Vol. 66, No. 5 dosesof up to 8 kGyto control microbial pathogens on cheeseclothand dried in a biosafetycabinet at room temperature seedsto be used for sprouts (26). However,thereare few (21 6 18C)for1 to2 h.Dried seeds with approximately 10 4 to reportson the microbial decontamination of alfalfa, mung 105 CFU of E. coli O157:H7per g weresubjected to dry-heat bean,and radish seeds with low-dose ionizing irradiation treatmentat 50 8Cfor1 hinanincubator,andthese seeds, as well andthe effects of such irradiation on alfalfa, mung bean, asuntreatedseeds, were sealed in sterileplastic bags and used for chemicaland irradiation treatments on the preparation day and/ or andradish seeds. Although good manufacturing practices stored at 48Candused within 2 days.The water content of the andhazard analysis critical control point approaches can seedsfollowing inoculation and before and after the application reducethe risk of contamination, there is still a needto ofdry heat was measured with a wateractivity meter (Novasina develophighly effective methods for theprevention of aW-center,NovasinaAG, Switzerland). sprout-associatedinfections. The objective of thisstudy was todeterminethe effectiveness of dry-heat treatment in com- Preparationof chemical solution. Califresh-S(30% Ca, 45%salt, and 25% dextrin; ABC Techno,T okyo,Japan) was dis- binationwith chemical or irradiationtreatment in the elim- solvedin distilled water (DW) at aconcentrationof 0.5%(wt/ vol) ination of E. coli O157:H7from laboratory-inoculatedal- andŽ lteredthrough double Ž lterpaper (150 mm; Advantec,5C, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021 falfa,radish, and mung bean seeds. ToyoRoshi Ltd., Japan), and the resulting clear solution was used MATERIALS AND METHODS within1 hofpreparation. The chlorine solution was prepared by addingsodium hypochlorite (Wako Chemical Co., T okyo,Japan) Teststrains. Enterohemorrhagic E. coli O157:H7strains solutionto DW (vol/vol). CR-3,MN-28, MY -29,and DT -66,isolated from bovine feces, EO water. Electrolyzedoxidizing (EO) waterwas generated wereused in this study. Dr .NakazawaMuneo (Laboratory of Zoo- witha modelROX-20TA EOwatergenerator (Hoshizaki Electric nosis,National Institute of AnimalHealth, T sukuba,Japan) kindly Co.Ltd., T oyoake,Aichi, Japan). The current passing through the providedthese strains. Nalidixic acid– resistant strains were pre- EOwatergenerator was set to 19.8 A, andthe voltage between paredin our laboratory by chemicalmutagenesis according to the theelectrodes was set to 10 V.A12%solution of sodiumchloride (1). E. coli methodof Adelberg et al. Each strainwas grown in (SigmaChemical Co., St. Louis, Mo.) and deionized water from 5mlof tryptic soy broth (TSB) overnightat 37 8C,transferred thelaboratory supply line were simultaneously pumped into the withthe use of aloopto 5 mlofnewTSB medium,and cultured equipment.The display indicator was activated and observed until at 378Cuntilit reached the mid-log phase. Cells were collected themachine stabilized at 19.8 A. TheEO waterwas collected bycentrifugation (3,000 3 g, 10 min, 208C)andresuspended in fromthe appropriateoutlet in sterilecontainers and wasused with- a 50-mg/ml N-methyl-N9-nitro-N-nitrosoguanidine(NTG) solution in1 hforchemical treatments. Samples to be used for the deter- afterbeing washed twice with phosphate-buffere dsaline(PBS, pH minationof the pH, oxidation-reductio npotential,and free-chlo- 7.2).After 1 hofincubation in the NTG solutionat room tem- rineconcentration were collected at the same time. perature,cells were washed twice with PBS andgrown in 5 ml of TSB at 378Cfor4 h.Theharvested cells were further cultured Irradiationof seeds. Allof the sealed plastic bags contain- inTSB mediumsupplemented with 50 mgofnalidixic acid per ingseeds were irradiated at doses of 1.0, 1.5, 2.0, 2.5, and 3.0 ml at 378Cfor24 h. The nalidixic acid– resistant mutant was iso- kGyat 1.4 kGy/ h(determinedby Fricke dosimetry (12)) with a latedby streakingthe culture on tryptic soy agar (TSA) containing 16,841-Cicobalt-60 gamma source (Gamma Cell-220, Nordion 50 mgofnalidixic acid per ml, and puriŽ ed well-isolatedcolonies InternationalInc., Kanata, Ontario, Canada). The air source and werestored in 20% glycerol at 2808Cpendingfurther use. theroom’ s temperature(21 6 28C)weremaintained during irra- Tominimizethe growth on enumeration media of microor- diation.The absorbed dose was conŽ rmed with an RCF dosimeter ganismsnaturally present on seeds, all test strains of E. coli O157: anda cellulosetriacetate Ž lmdosimeter (FTR-125, Fuji Photo H7were adapted to growin TSB (pH7.3; Nissui Seiyaku, T okyo, Film Co.Ltd., T okyo,Japan) that were attached to the surface of Japan)supplemented with nalidixic acid at 50 mg/ml.Plating on theplastic bag according to McLaughlin et al. (17). mediacontaining nalidixic acid greatly minimized interference Treatmentsand enumerationof pathogens. Ten grams of withcolony development by naturally occurring microorganisms, contaminatedseeds of each type (dry-heat-treat edor untreated) thusfacilitating the detection of the test pathogen on recovery wascombined with 40 ml of sterile deionized water (control) or media. achemicalsolution in a 100-ml askand sonicated (28 kHz) at Preparationof inocula. Eachstrain of E. coli O157:H7 was 21 6 18Cfor20 min in a waterbath. The other set of seedswas culturedin TSB containing50 mgofnalidixicacid per ml at37 8C combinedwith 40 ml of 50 8Cdeionizedwater (control) or chem- withloop transfer at three successive 24-h intervals immediately icalsolution with or without sonication at 50 8Cfor20 min. After beforethe strain was used as an inoculum. Cells of each strain thetreatment solution was decanted, the treated seeds were rinsed werecollected by centrifugation (3,000 3 g, 10 min, 208C) and twicewith 40 ml of sterile DW .Afterthe Ž nalrinse, 40 ml of resuspendedin 5 mlof PBS (pH7.2). Equal volumes of cell 0.1%peptone water was added, and the mixture (radish or alfalfa suspensionsof four strains were combined to produce approxi- seeds)was transferred to a StomacherBag 80 and pummeled in matelyequal populations of eachstrain. The inoculum was main- astomacherfor 1 min.The mixtures of mung bean seeds were tained at 21 6 18Candapplied to seedswithin 1 hofpreparation. transferredto a sterileblender receptacle. The sample was ho- mogenizedfor 20 s witha commercialblender. For seeds treated Seed inoculation. Mungbean and alfalfa seeds were ob- withdry heat and different irradiation doses, 10-g seed samples tainedfrom Salada Cosmo Co. Ltd. (Gifu, Japan), and radish foreach treatment were separately combined with 40 ml of 0.1% seedswere obtained from Murakami Nouen Co. Ltd. (Hiroshima, peptonewater in a StomacherBag 80 and were pummeled in a Japan).Five hundred grams of each type of seed was soaked in a stomacherfor 1 min.As a furthercontrol, unrinsed and untreated four-strainsuspension of E. coli O157:H7(ca. 10 8 CFU/ml)for 1 seedswere also pummeled or homogenized. Serial decimal dilu- minwith gentle agitation. After the inoculum was decanted, seeds tionswere prepared with PBS (pH7.2), and the diluted and un- wereplaced on a sterileperforated tray lined with four layers of dilutedsamples were surface plated in quadruplicate on TSA sup- J.FoodProt., Vol. 66, No. 5 CHEMICALAND IRRADIATION TREATMENTSFOR SEEDS 769

TABLE 1. Wateractivity (a w)valuesfor seeds following inocu- water-saturated90-mm-diameter no. 5 Žlterpaper (Whatman In- lationand before and after dry-heat treatment ternationalLtd. Maidstone, UK) ina 90-mm-diameterpetri dish. Seedswere placed in the dark at room temperature (24 6 18C) a forseeds (mean 6 SD)a w for3 days,and water was applied periodically to maintaina high- After dry-heat moistureenvironment. The number of germinated seeds was de- treatment termined,and the percentage of seeds that germinated was cal- Seed type ControlBefore treatment (508C, 1 h) culated.

Mung bean 0.509 6 0.001 0.551 6 0.002 0.488 6 0.002 Statisticalanalyses. Alltrials were replicated three times. Radish 0.581 6 0.002 0.967 6 0.002 0.882 6 0.001 Reportedplate count data represent the meanvalues obtained from Alfalfa 0.564 6 0.001 0.815 6 0.001 0.641 6 0.002 threeindividual trials, with each of these values being obtained fromduplicated samples. SigniŽ cant differences in plate count a n 5 3. datawere established by the least signiŽ cant difference at the 5%

levelof signiŽ cance. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021 plementedwith 50 mgofnalidixic acid per ml (TSAN), onsor- bitolMacConkey agar (SMAC) (Nissui)supplemented with ceŽ x- RESULTS ime(0.05 mg/ liter)and potassiu mtellurite(2.5mg/ liter) (CT-selectivesupplement, Oxoid) (CT -SMAC, andon CT-SMAC Previousstudies (9, 23, 25) haveshown that traditional containing50 mgofnalidixic acid per ml (CT -SMACN). Inocu- selectivemedia do not support colony development by latedenumeration media were incubated at 37 8Cfor24 to 28 h some of the E. coli O157:H7strains exposed to some of beforepresumptive colonies of each pathogen were counted. E. thechemical and irradiation treatments evaluated in this coli O157:H7was conŽ rmed with the E. coli O157direct im- study.Although nalidixic acid would prevent the growth of munoassaytest kit (Universal Health W atch,Columbia, Md.). mostgram-negative bacteria that might be presenton seeds, Determinationof germination percentage. Approximately thepotential for interferenceby background microbiota of 100control or treated seeds were placed between two pieces of E. coli O157:H7on TSAN exists.For thesereasons, TSAN

TABLE 2. Populationsof Escherichiacoli O157:H7recovered from mung bean seeds following dry-heat treatment and various addi- tionaltreatments

E. coli O157:H7population (logCFU/ g)recoveredon b: Average sprout Germination lengthafter Treatmenta CT-SMACN TSAN Reductionc Ed (%) 4 days (cm)

Control 4.13 4.56 98 9.3 Dry heat 3.46 3.56 1.00 Dry heat 1 hot (508C)chemicalsolution DW 2.47 3.13 1.43 Chlorine 1.28 1.52 3.04 Cal-S 1.04 1.26 3.30 AcEW 0.82 1.14 3.42 Dry heat 1 sonication 1 chemicalsolution DW 2.43 2.69 1.87 Chlorine 0.56 0.84 3.72 Cal-S 0.52 0.82 3.74 AcEW 0.34 0.54 4.02 Dry heat 1 sonication 1 hotchemical solution DW 1.17 1.41 3.15 98 9.2 Chlorine 0.32 0.58 3.98 98 9.1 Cal-S ,1.0 0.31 4.25 97 9.2 AcEW ,1.0 ,1.0 4.56 3 98 9.3 Dry heat 1 irradiationdose 1.0 kGy 1.48 1.97 2.59 98 4.5 1.5 kGy 0.79 1.06 3.50 97 4.4 2.0 kGy ,1.0 ,1.0 4.56 97 4.4 2.5 kGy ,1.0 ,1.0 4.56 98 4.1 3.0 kGy ,1.0 ,1.0 4.56 98 3.9 a DW,distilledwater; AcEW ,acidicelectrolyzed water; Cal-S, califresh-S. b Meanvalue for three replicate experiments ( P , 0.05).CT -SMACN, sorbitolMacConkey agar supplemented with ceŽ xime and potassiumtellurite and nalidixic acid; TSAN, trypticsoy agar supplemented with nalidixic acid; ,1.0,no colonies were recovered. c Log10 reductionrelative to the number recovered from the control on TSAN. d Numberof replicate experiments on which E. coli O157:H7was detected by enrichment. 770 BARI ET AL. J.FoodProt., Vol. 66, No. 5

TABLE 3. Populationsof Escherichiacoli O157:H7recovered from radish seeds following dry-heat treatment and various additional treatments

E. coli O157:H7population (logCFU/ g)recoveredon b: Average sprout Germination lengthafter Treatmenta CT-SMACN TSAN Reductionc % 4 days (cm)

Control 5.43 5.76 98 6.2 Dry heat 4.26 4.86 0.90 Dry heat 1 hot (508C)chemicalsolution DW 4.07 4.53 1.23 Chlorine 3.88 4.12 1.64 Cal-S 3.94 4.26 1.50 AcEW 3.72 4.21 1.55 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021 Dry heat 1 sonication 1 chemicalsolution DW 4.23 4.69 1.07 Chlorine 4.06 4.34 1.42 Cal-S 3.92 4.22 1.54 AcEW 3.84 4.24 1.52 Dry heat 1 sonication 1 hotchemical solution DW 3.87 4.41 1.35 96 6.2 Chlorine 3.62 3.88 1.88 96 6.1 Cal-S 3.82 4.11 1.65 88 6.0 AcEW 3.61 3.82 1.94 96 6.2 Dry heat 1 irradiationdose 1.0 kGy 1.67 1.87 3.84 99 5.7 1.5 kGy 0.67 0.86 4.85 96 5.5 2.0 kGy 0.14 0.22 5.49 95 5.1 2.5 kGy ,1.0 ,1.0 5.71 95 4.6 3.0 kGy ,1.0 ,1.0 5.71 94 4.3 a DW,distilledwater; AcEW ,acidicelectrolyzed water; Cal-S, califresh-S. b Meanvalue for three replicate experiments ( P , 0.05).CT -SMACN, sorbitolMacConkey agar supplemented with ceŽ xime and potassiumtellurite and nalidixic acid; TSAN, trypticsoy agar supplemented with nalidixic acid; ,1.0,no colonies were recovered. c Log10 reductionrelative to the number recovered from the control on TSAN. andCT -SMACN were usedin all experiments for theenu- terheat treatment were 0.551and 0.488, respectively, for meration of E. coli O157:H7on treatedand untreated seeds. mungbean seeds; 0.967 and 0.882, respectively for radish E. coli O157:H7was notrecovered from uninoculated seeds;and 0.815 and 0.640, respectively, for alfalfaseeds. seeds.Regardless of seed conditions or treatments, larger Table2 showsthe sizes of E. coli O157:H7populations populationsof E. coli O157:H7were recoveredon TSAN onmungbean seeds dry heated (13, 21) for 60minat 50 8C thanon CT -SMACN. E. coli O157:H7counts were 0.20to andtreated with various chemical solutions and various 0.60 log10 CFU/ghigherfor samplesfrom seedstreated combinationtreatments. Treatment with hot (50 8C) DW, withwater (control)and plated on TSAN thanfor control DWplussonication, and hot DW plussonication resulted samplesplated on selective media. Larger differences be- inreductions of ca. 1.43, 1.87, and 3.15 log 10 CFU/g, re- tweencounts for samplesplated on TSAN andthose for spectively.Treatment with hot 200-ppm chlorine solution, samplesplated on selective media were observedfor seeds 200-ppmchlorine solution plus sonication, and hot 200- treatedwith different irradiation doses and chemical solu- ppmchlorine solution plus sonication resulted in reductions tions.This Ž ndingis an agreement with other reports de- ofca. 3.04, 3.72, and 3.98 log 10 CFU/g,respectively. The scribingthe poor performance of CT-SMACN inrecovering maximumreduction (4.25 log 10 CFU/g)was achievedwith E. coli O157:H7from chemicallytreated seeds (5, 16). The hotcalifresh-S plus sonication, and the complete elimina- lowercounts obtained for highlyselective media may be tion of E. coli O157:H7was achievedwith hot EO water dueto the inability of cells that have been injured by des- plussonication. The germination percentage and the aver- iccationor by chemical and irradiation treatment to resus- agesprout height were notaffected by thetreatment. How- citate. ever,evenwith these large reductions, the pathogen was Thewater contents of the seeds following inoculation detectedwith the enrichment of treatedseeds (T able2). Dry andbefore and after dry-heat treatment are presented in heatalone was ableto reduce the pathogen population by Table1. The mean water activity (a w)valuesfor control ca. 1.0 log10 CFU/gofseedswithout affecting germination mungbean, radish, and alfalfa seeds were 0.509,0.580, and percentageand average sprout length, whereas dry heat in 0.564,respectively, whereas mean a w valuesbefore and af- combinationwith 2.0-kGy doses of irradiationwere ableto J.FoodProt., Vol. 66, No. 5 CHEMICALAND IRRADIATION TREATMENTSFOR SEEDS 771

TABLE 4. Populationsof Escherichiacoli O157:H7recovered from alfalfa seeds following dry-heat treatment and various additional treatments

E. coli O157:H7population (logCFU/ g)recoveredon b: Average sprout Germination lengthafter Treatmenta CT-SMACN TSAN Reductionc % 4 days (cm)

Control 5.33 5.69 96 4.3 Dry heat 3.57 3.96 1.73 Dry heat 1 hot (508C)chemicalsolution DW 3.27 3.43 2.26 Chlorine 2.38 2.52 3.14 Cal-S 2.64 2.86 2.83 AcEW 1.52 2.14 3.55 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021 Dry heat 1 sonication 1 chemicalsolution DW 3.43 3.79 1.90 Chlorine 3.32 3.54 2.15 Cal-S 3.43 3.82 1.87 AcEW 2.74 3.05 2.64 Dry heat 1 sonication 1 hotchemical solution DW 2.71 3.14 2.55 93 4.3 Chlorine 2.02 2.28 3.41 96 4.2 Cal-S 2.30 2.71 2.98 89 4.2 AcEW 1.00 1.40 4.29 95 4.3 Dry heat 1 irradiationdose 1.0 kGy 2.67 2.87 2.82 93 4.2 1.5 kGy 1.17 1.26 4.43 93 3.7 2.0 kGy ,1.0 ,1.0 5.69 92 3.7 2.5 kGy ,1.0 ,1.0 5.71 93 3.3 3.0 kGy ,1.0 ,1.0 5.71 93 3.3 a DW,distilledwater; AcEW ,acidicelectrolyzed water; Cal-S, califresh-S. b Meanvalue for three replicate experiments ( P , 0.05).CT -SMACN, sorbitolMacConkey agar supplemented with ceŽ xime and potassiumtellurite and nalidixic acid; TSAN, trypticsoy agar supplemented with nalidixic acid; ,1.0,no colonies were recovered. c Log10 reductionrelative to the number recovered from the control on TSAN. eliminatethe pathogen completely in mung bean seeds, and incombination with sonication was mosteffective in elim- thepathogen was notdetected with the enrichment of treat- inating E. coli O157:H7(with a reductionof 4.29log CFU/ edseeds. Although there was noeffecton germinationper- g)without reducing the germination percentage or the av- centage,a substantialdecrease in averagesprout length was eragesprout height (T able4 andFig. 1). For alfalfaseeds, observedthroughout the experiment. 200-ppmchlorine solution was foundto be more effective Theeffects of combinations of dry-heat treatment and thancalifresh-S, with a combinationof hot 200-ppm chlo- chemicalor irradiation treatments on populations of E. coli rinesolution and sonication resulting in a reductionof 3.41 O157:H7in radish seeds are shown in T able3. Sonication logCFU/ gwithoutreducing the quality of thesprouts (Fig. combinedwith hot or unheated DW ,chlorine,or califresh-S 1).Dry heatalone was ableto reduce the E. coli O157:H7 reducedthe population of E. coli O157:H7by 1.0 to 1.8 populationby ca. 1.7 log 10 CFU/gofseeds without affect- log10 CFU/g,but sonication combined with hot water re- ingthe germination percentage and the average sprout sultedin the reduction of populationson radish seeds. The length,whereas dry heat in combination with a 2.0-kGy maximumreduction (1.94 log 10 CFU/g)was achievedwith doseof irradiation eliminated the pathogen completely on EOwater combinedwith sonication and a rinse.Dry heat alfalfaseeds. The average sprout length decreased as the incombination with a 2.5-kGydose of irradiation was re- irradiationdose increased (T able4), but no decrease in the quiredto eliminate the pathogen on radish seeds, and at germinationpercentage was observed.Figure 1 illustrates thisdose level, sprout length was substantiallydecreased. thedecrease in sprout length, which resulted in a lower However,thepercentages of radishseeds germinating were yieldfor higherirradiation doses. Alfalfa sprout length did foundto be more or less similar for alldose levels used. notdecrease unacceptably with a doseof 2.0kGy, whereas E. coli O157:H7populations for dry-heatedalfalfa dosesof 2.0 and 2.5 kGy decreased the lengths of mung seedstreated with irradiation and with different sanitizers beanand radish sprouts, respectively. Mung bean sprouts areshown in T able4. Chemical treatments in combination were foundto be sensitive to irradiation, with the applica- withsonication resulted in signiŽ cant reductions of E. coli tionof even 1.0 kGy of irradiation decreasing their length O157:H7.Overall, treatment with hot or unheatedEO water by50% (4.8 cm) comparedwith radish and alfalfa sprouts 772 BARI ET AL. J.FoodProt., Vol. 66, No. 5 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021

FIGURE 1. Sproutsgrown for 4 daysfrom seeds treated with dry heat followed by different doses of irradiation. (A) Mungbean sprouts.(B) Radish sprouts. (C) Alfalfa sprouts. Each photograph shows (from the left) sprouts treated with dry heat, with dry heat plusa 1.0-kGydose of irradiation, with dry heat plus a 1.5-kGydose of irradiation, with dry heat plus a 2.0-kGydose of irradiation, withdry heat plus a 2.5-kGydose of irradiation, and with dry heat plus a 3.0-kGydose of irradiation.

(Tables2 through4). However ,theapplication of a 3.0- posureto active sanitizer components. Microscopic exam- kGydose of irradiationdecreased the lengths of alfalfaand inationof themung bean seed shows that while the surface radishsprouts by 1.0and 1.9 cm, respectively (T ables3 and ofthe seed is relatively smooth, the stem scar isporous 4).The application of irradiation also shortened the root hairs enoughto allow bacteria to penetrate to deep within the ofthe sprouts compared with those of control sprouts irre- seed (9). Thedrying of seeds after inoculation would in spectiveof thedose applied and the type of sprout (Fig. 1). effectprotect E. coli O157:H7cells that had entered the seedsthrough cracks and crevices against inactivation by DISCUSSION sanitizers.Microscopic examination of the alfalfa seed re- Theinability of sanitizers to completely eliminate the vealsa similarsituation, with the seed coat containing suf- viablecells of pathogenswithout affecting the germination Žcientcracks and crevices to protect the pathogen against percentageis probably the result of cells being trapped in aqueoussanitizers (28). Additionally,seed surfaces are cracksor crevicesand thus being protected from directex- sometimesintentionally roughened by abrasion through a J.FoodProt., Vol. 66, No. 5 CHEMICALAND IRRADIATION TREATMENTSFOR SEEDS 773 processknown as scariŽ cation, which facilitates the inŽ l- 1989.Effect ofgamma irradiationon seed bornefungi and soybean trationof waterthrough the seed coat to thecotyledon, thus seed componentsduring storage. Agric.Res. Rev. 67:619–628. 4.Berezinz, N.M.,A.A.Narimanov,A. F.Revin,and N. Norbeav. enhancinggermination. This process would likely exacer- 1967.Restoration of radiation damages tocotton seeds duringpro- batethe problem of theprotection of bacterialcells against longedstorage. UDC 58:140–145. sanitizercontact. Moreover ,whena sanitizercomes in con- 5.Beuchat, L. R.,T.E.Ward,and C. A.Pettigrew.2001. Comparison tactwith an environment containing a highlevel of organic ofchlorine and a prototypeproduce wash productfor effectiveness matter,thesanitizer’ s potencyis quickly diminished, re- in killing Salmonella and Escherichiacoli O157:H7on alfalfa seeds. sultingin the failure of even high-alkaline or acidic dip J.FoodProt. 64:152–158. 6.Caetano-Anolles, G., G. Favelukes,and W .D.Bauer.1990. Opti- solutionsto inactivate E. coli O157:H7cells in the inŽ l- mizationof surface sterilizationfor legume seed. Crop Sci. 30:708– tratedseeds. Since the 0.1% peptone solution would be ex- 712. pectedto neutralize the activity of any residue remaining 7.Chiou, R. Y.-Y.,C.M.Lin,and S. L.Shyu.1990. Property char- onthe seeds after the decanting of the treatment solution acterization ofpeanutkernels subjected to gamma irradiationand its effect ontheoutgrowth and alfatoxin production by Aspergilluspar- followedby washing,it is presumed that the increase in the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/66/5/767/1674734/0362-028x-66_5_767.pdf by guest on 28 September 2021 asiticus.J. FoodSci. 55:210–217. populationof E. coli O157:H7in thepeptone wash solution 8.Cuero, R. G.,J.E.Smith,and J. Lacey. 1986.The in uence of was dueto the pummeling action, which released E. coli gamma irradiationand sodium hypochlorite sterilization on maize O157:H7cells embedded in cracks in the seed coat or un- seed microora and germination. FoodMicrobiol. 3:107–113. derneathit. There have been reports of decreased germi- 9.Delaquis, P .J.,P .L.Sholberg,and K. Stanich.1999. Disinfection of nationin cases in whichliquid disinfectants come in contact mungbean seeds withgaseous acetic acid. J.FoodProt. 62:953– 957. withthe seeds for anextendedperiod (6). Evenwith liquid 10.Fett, W .F..2002.Reduction of Escherichiacoli O157:H7 and Sal- disinfectionof the seed, there is still no guarantee that the monella spp.on laboratory-inoculated mung bean seed bychlorine resultingsprout is pathogen free (28). TheFDA hasap- treatment. J.FoodProt. 65:848–852. provedthe use of irradiation at doses of up to 8.0 kGy to 11.Harmon, S. M.,D.A.Kautter,andH. M.Solomon.1987. Bacillus controlbacterial pathogens on seeds to be usedfor sprouting. cereus contaminationof seeds andvegetable sprouts grown in a Theresults of this study indicate that treatment of E. homesprouting kit. J.Food.Prot. 50:62–65. 12.International Atomic EnergyAgency. 1982. Training manual on food coli O157:H7–contaminated radish and alfalfa seeds with irradiationtechnology and techniques, 2nd ed. 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