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Food Microbiology 28 (2011) 119e127

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Food Microbiology

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Individual and combined application of dry heat with high hydrostatic pressureto inactivate Salmonella and Escherichia coli O157:H7 on alfalfa seeds

Hudaa Neetoo, Haiqiang Chen*

Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716-2150, USA

a r t i c l e i n f o

Article history:Received 2 July 2010Received in revised form1 September 2010Accepted 6 September 2010Available online 16 September 2010

Keywords:High pressureSeedsDry heatSalmonellaEscherichia coli O157:H7

* Corresponding author. Tel.: þ1 302 831 1045; faxE-mail address: haiqiang@udel.edu (H. Chen).

0740-0020/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.fm.2010.09.004

a b s t r a c t

Alfalfa sprouts are recurrently implicated in outbreaks of food-borne illnesses as a result of contami-nation with Salmonella or Escherichia coli O157:H7. In the majority of these outbreaks, the seeds them-selves have been shown to be the most likely source of contamination. The aims of this study were tocomparatively assess the efficacy of dry heat treatments alone or in conjunction with high hydrostaticpressure (HHP) to eliminate a w5 log CFU/g load of Salmonella and E. coli O157:H7 on alfalfa seeds. Dryheat treatments at mild temperatures of 55 and 60 �C achieved �1.6 and 2.2 log CFU/g reduction in thepopulation of Salmonella spp. after a 10-d treatment, respectively. However, subjecting alfalfa seeds tomore aggressive temperatures of 65 �C for 10 days or 70 �C for 24 h eliminated a w5 log population ofSalmonella and E. coli O157:H7. We subsequently showed that the sequential application of dry heatingfollowed by HHP could substantially reduce the dry heating exposure time while achieving equivalentdecontamination results. Dry heating at 55, 60, 65 and 70 �C for 96, 24, 12 and 6 h, respectively followedby a pressure treatment of 600 MPa for 2 min at 35 �C were able to eliminate a w5 log CFU/g initialpopulation of both pathogens. Finally, we evaluated the impact of selected treatments on the seedgermination percentages and yield ratios and showed that dry heating at 65 �C for 10 days did not bringabout any considerable decrease in the germination percentage. However, the sprout yield of treatedalfalfa seeds was reduced by 21%. Dry heating at 60 and 65 �C for 24 and 12 h respectively followed by thepressure treatment of 600 MPa for 2 min at 35 �C did not significantly (P > 0.05) affect the germinationpercentage of alfalfa seeds although a reduction in the sprouting yield was observed.

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1. Introduction

Seed sprouts represent an important component of traditionaloriental cuisine by virtue of their excellent nutritional profiles, andrecently have gained increased popularity in many parts of theworld, including Europe and the United States. Sprouts are believedby many to have anti-cholesterolemic, anti-carcinogenic and anti-oxidative activities (Meyerowitz,1999). Unfortunately, the increasedconsumption of sprouts throughout the world has led to a concom-itant surge in the incidence of food-borne illnesses associated withthis commodity (Fett and Cooke, 2005; Fett et al., 2005).

Salmonella ranks as one of the most frequently reported cause offood-borne outbreaks of gastroenteritis in the United States,making salmonellosis an increasing public health concern (CDC,2008). In the United States alone, there have been at least 29reports of sprout-borne Salmonella outbreaks, involving more than

: þ1 302 831 2822.

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1500 cases since 1995 (Taormina et al., 1999; Chapman, 2009), withmost outbreaks incriminating alfalfa sprouts. However, the safety ofsprouts has also become an international concern with the occur-rence of outbreaks in Canada (Van Beneden et al., 1999), the UnitedKingdom (O’Mahony et al., 1990), Japan (Gutierrez, 1997) andseveral European countries including Finland, Sweden (Ponka et al.,1995; Puohiniemi et al., 1997) and Denmark (Van Beneden et al.,1999). Salmonella has been more frequently identified as the etio-logical agent involved in sprout-related illnesses compared to otherfood-borne pathogens such as Escherichia coli O157:H7 and Bacilluscereus (Proctor et al., 2001; Hora et al., 2007).

Based on the reported isolation of pathogens from implicatedseed lots or on the basis of epidemiological evidence, seedsharboring pathogenic bacteria are the most likely source ofcontamination of sprouts (Fett et al., 2005). Although sproutingseeds represent very low moisture foods with an average wateractivity (aw) of ca. 0.3, contaminated seeds still present a safetyconcern since it is widely known that Salmonella can persist forextended periods of time in low moisture foods (Betts, 2007; Zink,2007). As a result, a few pathogenic cells on seeds can multiply to

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127120

potentially hazardous levels because of the favorable conditions ofmoisture, temperature and nutrient availability during subsequentseed germination and sprout growth.

In response to the urgent need to mitigate the occurrence ofoutbreaks, the U.S. Food and Drug Administration and the CaliforniaDepartment of Health Services enforce that growers treat sproutingseeds with an approved antimicrobial treatment such as20,000 ppm of free chlorine from calcium hypochlorite immedi-ately prior to sprouting (Fett et al., 2005). To date, this treatmentstill remains the yardstick against which the efficacy of otheraqueous sanitizers is compared. However, the limited performanceof calcium hypochlorite along with the fact that organic sproutgrowers are not allowed to treat seeds with such a high level ofchlorinated sanitizer, has spurred a search for alternative seeddecontamination treatments. A variety of physical treatments havebeen tested as stand-alone interventions for decontamination ofsprouting seeds in the form of thermal or non-thermal interven-tions. Thermal treatments investigated have included hot water,dry heat, radio-frequency dielectric heating while non-thermaltechnologies have included gamma irradiation, hydrostatic pres-sure, pulsed UV light and ultrasound.

Thermal treatments used for the decontamination of vegetativematerials, including seeds, have been one of the most heavilystudied intervention methods and has a long history of use sincethe 1920s. Hot water treatments for reducing populations of humanpathogens on sprout seeds were first studied by Jaquette et al.(1996). They found that a 5 min-treatment in hot water at57e60 �C led to a 2.5 log unit decrease in the population ofSalmonella enterica on artificially contaminated seeds with noappreciable reduction in the seed germination rate. However,temperatures that were slightly higher or heating durations thatwere slightly longer brought about significant decrease in seedgerminability. However, the application of dry heat treatment todecontaminate mung bean and alfalfa seeds has been shown toyield more encouraging results (Beuchat and Scouten, 2002; Huet al., 2004; Feng et al., 2007).

In our previous studies, we had demonstrated that the appli-cation of high pressure at 650 MPa for 15 min at 20 �C completelyeliminated a w5 log CFU/g initial burden of E. coli O157:H7 onalfalfa seeds (Neetoo et al., 2008). We subsequently showed thatthe application of high hydrostatic pressure at a reduced level of300e600 MPa for 2 or 5 min in combination with mild heat attemperatures ranging from 40 to 50 �C, was able to successfullyeliminate E. coli O157:H7 (Neetoo et al., 2009) and Salmonella(Neetoo and Chen, 2010) on alfalfa seeds while maintaining seedviability. While the aforementioned treatments did not unaccept-ably decrease the percent germination, a variable reduction in thelengths of the sprouts was observed. The study reported here wasundertaken to compare the influence of dry heat alone and thecombined action of dry heat and HHP on the lethality to Salmonellaand E. coli O157:H7 on alfalfa seeds and seed viability. To simplifysentence construction, the term “elimination” will be used in thispaper with the intended meaning of “reduction of a 5 log initialload of E. coli O157:H7 or Salmonella to an undetectable level in 2-gseed samples after enrichment”.

2. Materials and methods

2.1. Effect of dry heat treatment on the population of Salmonellaand E. coli O157:H7 on alfalfa seeds and the germinationcharacteristics of alfalfa seeds

2.1.1. Bacterial strainsFive Salmonella and five E. coli O157:H7 strains were used in this

study. The Salmonella strains included two S. enterica serovar

Typhimurium strains (T43 and TDT 104), S. enterica serovar Enter-itidis E44, S. enterica serovar Montevideo Mo57 and S. entericaserovar Seftenberg 775W. The E. coli O157:H7 strains includedstrain 1730, Cider strain (Ibrahim et al., 2006), strains 250 and 251(Bhagwat et al., 2005) and J58 (Barak et al., 2005). All the strainswere from the University of Delaware culture collection. The cells ofSalmonella or E. coli O157:H7 were adapted to grow in tryptic soybroth plus 0.6% yeast extract (Difco Laboratories, Sparks, MD)supplemented with nalidixic acid to a final concentration of 50 mg/ml (Fisher Scientific, Hampton, NH) (TSBYE-N). Individual cultureswere grown in TSBYE-N overnight at 35 �C. Cultures were thentransferred (one loopful) into 10ml of fresh TSBYE-N and incubatedat 35 �C for 24 h. Equal volumes of individual cultures of Salmonellaor E. coli O157:H7 were mixed to form a five-strain composite.

2.1.2. Inoculation of seedsA hundred-fold dilution of the five-strain culture cocktails was

made in sterile 0.1% peptone water (Fisher). Five hundred grams ofalfalfa seeds were added to 300 ml of peptone water containing thediluted cell suspension (w7 log CFU/ml) and mixed well ona magnetic stirrer for 5 min. The seeds were separated from the cellsuspension by pouring the mixture over a double layer of cheese-cloth supported by a wire screen and dried inside a biosafety hoodat room temperature (21 � 2 �C) for up to 72 h with intermittentrotation to ensure that seeds were uniformly dry. The inoculatedseeds were placed in plastic pouches and stored at 4�C for at least 3days prior to subjecting them to any treatment. Seeds inoculatedwith Salmonella and E. coli O157:H7 were used within 4 and 2weeks of inoculation respectively, since a preliminary study indi-cated that the populations of Salmonella and E. coli O157:H7 werestable(<0.5 log CFU/g decline)during storage at 4 �C for up to 30and 15 days, respectively. The initial microbial counts of bothSalmonella and E. coli O157:H7 were also determined right beforetreatments and were at an approximate level of 5 log CFU/g ofSalmonella or E. coli O157:H7 as reported in the results.

2.1.3. Dry heat treatmentInoculated and un-inoculated seeds (2g) were placed in capped

glass tubes and subjected to dry heating at temperatures rangingfrom 55 to 70 �C (in 5 �C increments). Samples of seeds wereheated at 55, 60 and 65 �C for up to 10 days and 70 �C for up to24 h. After heat treatment of seeds at 55 and 60 �C, seeds wereallowed to equilibrate to ambient temperature (22e23 �C) for 1 hprior to microbiological analysis (for inoculated seeds) and seedviability test (for un-inoculated seeds). Seeds treated at 65 and70 �C were refrigerated overnight at 4 �C prior to microbiologicalanalysis (for inoculated seeds) and seed viability test (for un-inoculated seeds). These post-treatment storage conditions werechosen in the light of preliminary experiments showing that seedsdry heated at 55 and 60 �C and microbiologically analyzedimmediately after treatment had lower bacterial counts than ifseeds were allowed to “rest” for at least 1 h (data not shown). Noappreciable difference in the microbial counts was observed forplating done 1 h or 24 h post-treatment. In addition, we did notobserve any difference in the counts of treated seeds kept atambient or refrigeration temperature. For seeds treated at 65 and70 �C on the contrary, we observed that a 24-h repair phase at 4 �Cwas optimum for recovery of heat-stressed cells (data not shown)and thus adequate for providing an accurate estimate of numbersof survivors.

2.1.4. Microbiological analysisSeeds were transferred into a stomacher bag to which 8 ml of

sterile 0.1% peptone water was added and subsequently stomachedfor 2 min at 260 rpm (Seward 400 Stomacher; Seward Medical Co.,

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127 121

London, United Kingdom). The seed mixture was serially diluted insterile 0.1% peptone water and surface-plated in duplicate ontryptic soy agar with 0.6% yeast extract (Difco Laboratories, Sparks,MD) supplemented with nalidixic acid to a final concentration of50 mg/ml (TSAYE-N). TSAYE-N plates were incubated for 3 days at35 �C. Presumptive colonies of Salmonella or E. coli O157:H7 formedon the plates were enumerated. In addition, the seed mixture wasalso directly enriched in 90 ml of TSBYE-N and incubated for 48 h at35 �C to allow resuscitation of sub-lethally injured cells whereappropriate. Samples were streaked onto Sorbitol MacConkey agar(Difco) plates supplemented with 50 mg/ml of nalidixic acid forsamples inoculated with E. coli O157:H7 or Xylose Lysine Deoxy-cholate (XLD) agar (Difco) plates supplemented with 50 mg/ml ofnalidixic acid for samples inoculated with Salmonella. After 24 hincubation, presence of colorless or faint orange growth typical ofE. coli O157:H7 and black-centered or black colonies characteristicof Salmonella were interpreted as a positive result.

2.1.5. Determination of seed germination percentageUn-inoculated seeds were dry-heated at temperatures of

55e70 �C as described above. To determine the seed germinationpercentage, heat-treated and control (non heat-treated) un-inoc-ulated seeds were soaked in DI water for 3 h. One hundred seedswere then randomly picked and dispersed evenly on layers of wetpaper towels laid on a plastic rack, which in turn was placed intoa water-filled bucket to provide a moist environment for the seeds.The water level was maintained below the seeds’ level. The bucketwas covered loosely with a piece of plastic film to allow exchange ofair with its surroundings. The bucket was kept at room temperature(21 � 2 �C) for 8 days (suggested by the seeds provider). The seedswere visually evaluated for germination and sprouted seeds werecounted after 3e8 days and discarded. The germination percentagewas determined as the proportion of sprouted seeds to the totalnumber of seeds. The cumulative germination percentage on eachday was then computed.

2.2. Combined effects of dry heat and high-pressure treatment onthe inactivation of Salmonella on alfalfa seeds

The sequential application of dry heat and high hydrostaticpressure (HHP) to inactivate Salmonella was subsequently investi-gated. Inoculated alfalfa seeds were dry-heat treated (2 g) at 55, 60,65 and 70 �C to varying durations depending on the temperature.After the dry heat treatment, seeds dry-heated at 55 and 60 �Cwereallowed to equilibrate to room temperature for 1 h while seedsheated at 65 and 70 �C were refrigerated overnight. Seeds werethen subsequently placed in individual 3-mil thick nylon/poly-ethylene pouches (Koch Supplies, Kansas City, MO, USA). Sterile de-ionized (DI) water (3 ml) was then added to the pouches, whichwere heat-sealed. Pressure treatment of samples was carried outusing a high-pressure unit with temperature control (Model AvurePT-1, Avure Technologies, Kent, WA, USA) with pressurizationconducted at 600 MPa for 2 min at 20 or 35 �C (initial seed sampletemperature prior to pressure treatment) using water as a hydro-static medium. The temperature of the water bath was monitoredwith a K-type thermocouple. Temperature and pressure data wererecorded every 2 s (DASYTEC USA, Bedford, NH, USA). The pressure-come-up rate was approximately 22 MPa/s. The pressure-releasewas almost immediate (<4 s). Pressurization time reported in thisstudy does not include the pressure come-up or release times.

For samples to be pressure-treated at 35 �C, samples were firstsubmerged in the 35 �C water bath surrounding the pressure cellfor 10 min, allowing samples to equilibrate to the water bathtemperature before pressurization. The temperature equilibrationstep and the actual pressure treatment constituted a brief seed-

soaking period lasting ca. 15 min. Immediately after pressuretreatment, samples were cooled in an ice-water mixture. Sampleswere then microbiologically assayed as described above.

2.3. Assessment of the efficacy of selected treatments to achieve w5log reduction of E. coli O157:H7 to below undetectable levels

Two grams of seeds inoculated with an initial burden of w5 logCFU/g of E. coli O157:H7 were dry heated at 55 �C for 96 h, 60 �C for24 h, 65 �C for 12 h and 70 �C for 6 h. After dry heat treatment, seedswere allowed to rest at room or refrigeration temperature asdescribed previously before being mixed with 3 ml of DI water,packaged, and treated at 600 MPa for 2 min at 35 �C. Two grams ofalfalfa seeds inoculated with E. coli O157:H7 were also subjected todry heat at 65 �C for 10 d and 70 �C for 24 h. Samples were thenmicrobiologically assayed on TSAYE-N or enriched as describedpreviously.

2.4. Effect of selected treatments on the germination characteristicsof alfalfa seeds

2.4.1. Determination of the germination percentages and yield ratioof treated seeds

To determine the possible impact of various decontaminationtreatments on the seeds’ germination potential, 2 g of un-inocu-lated alfalfa seeds were subjected to dry heat treatment at 65 �C for10 days or dry heat in combination with high pressure treatment.For the combined treatment, 2 g of seeds was heat treated at 55 �Cfor 96 h, 60 �C for 24 h, 65 �C for 12 h or 70 �C for 6 h. Following dryheating, these seeds were allowed to rest at room or refrigerationtemperature as mentioned before and then mixed with 3 ml of DIwater, packaged, and treated at 600 MPa for 2 min at 35 �C. Afterpressure treatment, seeds were immediately chilled in iced waterfor 10 min and then subsequently soaked in water for 3 h. Onehundred seeds from each treated sample were then randomlypicked and allowed to germinate using the procedure describedabove. Germinated seeds were counted after 3e8 days of germi-nation. In addition, sprouts were weighed after 8 days of growthand the yield ratio was calculated by dividing the weight ofsprouted seeds by the weight of one hundred dry seeds, a methodadapted from Rajkowski and Thayer (2001).

2.4.2. Modification of the germination procedure to improve thesprouting yield of seeds treated by dry heat and HHP

Un-inoculated seeds were dry heated at 60 �C for 24 h and 65 �Cfor 12h as described previously. Following dry heating, seeds werekept at 4 �C for 24 h and then pressure-treated at 600MPa for 2minat 35 �C. Seeds were then chilled, soaked and allowed to germinatefor 3e8 days at 27 �C. We used 27 �C as the incubation temperatureof choice since we observed slightly higher germination rates at27 �C compared to 22 �C and 30 �C (data not shown). Germinatedseeds were enumerated after 3e8 days of germination. In addition,sprouted seeds were weighed after eight days and the sprout yielddetermined as described above.

2.5. Statistical analysis

All experiments were replicated at least three times. Whereappropriate, statistical analyses were conducted using Minitab�

Release 15 (Minitab Inc., University Park, PA, USA). One-wayanalysisof variance (ANOVA) and Tukey’s one-way multiple comparisonswere used to determine differences in the populations of Salmonellaand E. coli O157:H7 recovered on treated alfalfa seeds as well asdifferences in the germination percentage of seeds. Significantdifferences were considered at the 95% confidence level (P < 0.05).

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127122

3. Results and discussion

3.1. Effect of dry heat treatment on the population of Salmonellaand E. coli O157:H7 on inoculated alfalfa seeds

Tables 1e4 indicate that the level of Salmonella decreased tovarying extents and at varying rates during storage at 55, 60, 65 and70 �C respectively. At 55 and 60 �C, the population initially declinedto approximately 3.7 and 3.9 log CFU/g respectively after 2 days andremained fairly stable at a level of ca. 3.0 log CFU/g for theremaining days of the 10-d period. However, when the temperaturewas increased to 65 �C, we observed that there was a steady declinein the population from an initial of w5 log CFU/g to undetectablelevels after 10 days. At a higher temperature of 70 �C, we observeda faster decline in the population of Salmonella to below thedetection limit (<0.7 log CFU/g) after a 24-h exposure. Hence,exposures of seeds to high temperatures of 65 and 70 �C broughtabout a faster decline in the population of Salmonella compared totemperatures of 55 and 60 �Cwhich reduced the populations by< 2log CFU/g after 10 days of exposure. An extended holding period ofup to 30 days at 55 and 60 �C did not further reduce the populationof Salmonella with final population of ca. 3.5 log CFU/g (data notshown). Beuchat and Scouten (2002) also showed that seedsexposed to mild temperatures of 50 and 60 �C underwenta marginal reduction in the population of Salmonella and E. coliO157:H7 of <1.5 log CFU/g after 7 h of exposure while heating athigher temperatures of 70 and 80 �C achieved > 3 log CFU/greduction.

Feng et al. (2007) and Hu et al. (2004), on the other hand, bothreported on the relatively higher efficacy of dry heat treatments at55 �C on mung bean and alfalfa seeds, respectively. Hu et al. (2004)showed that seeds inoculated with cocktails of E. coli O157:H7 (6log CFU/g) or S. enterica (4 log CFU/g) subsequently exposed toa temperature of 55 �C, resulted in undetectable levels of thepathogen after 4 and 5 days respectively. Feng et al. (2007) showedthat an exposure lasting 8 and 6 days at 55 �C were able tocompletely eliminate an initial population of 8 log CFU/g ofSalmonella spp. and E. coli O157:H7, respectively. It is possible thatthe cause of the disparity in our results lies in the variation in thewater activity of inoculated seeds or differences in the heat resis-tance of the strains studied. In our study, a composite of five strainsof Salmonella were used, including S. Typhimurium DT 104 andS. Seftenberg 775W. S. Typhimurium DT 104 is known to exhibithigher tolerance to heat and acid that many other salmonellae andhas also been shown to mount stress responses to conditions of lowstresses (Humphrey, 2001). S. Seftenberg 775W has also beenreported to be fairly resistant to thermal treatments (Ng et al.,1969), with 10e20 times higher thermal resistance than typicalSalmonella strains. Although several authors have commented that

Table 1Effect of dry heat treatment at 55 �C on the populations of Salmonella and E. coli O157:H

Heating time (d) Salmonella E. coli

O157:H7 3

Control 5.1 � 0.2a 5.4 � 0.3a 99 � 1a 101 Not done 2.5 � 0.7b Not done No2 3.7 � 0.3b 0.3 � 0.6c 99 � 1 ab 103 Not done <0.7 (1/3) Not done No4 3.3 � 0.4b <0.7 (0/3) 98 � 1abc 96 3.2 � 0.1b Not done 98 � 1abc 98 3.8 � 0.2b Not done 96 � 1abc 9710 3.5 � 0.2b Not done 94 � 1d 97

Data representing mean log survivors (CFU/g) or mean germination percentage � standThe limit of detection for the platingmethodwas 0.7 log CFU/g. Numbers in parentheses rValues in the same column followed by the same letter are not significantly different (P

this strain exhibits highest resistance at water activity close to 1.00(Goepfert and Biggie, 1968; Riemann, 1968), S. Seftenberg 775W isknown to be generally quite refractory to heat (Baird-Parker et al.,1970). These two thermally resistant strains were not included inthe cocktails used in studies undertaken by Feng et al. (2007) andHu et al. (2004). Doyle and Mazzotta (2000) also acknowledgedthat the heat resistance data acquired by different researchers fordifferent serovars of S. enterica in low moisture food products suchas alfalfa seeds are quite variable, emphasizing that the heatresistance of Salmonella is highly influenced by the strain(s) tested.In recognition of the differential heat tolerance of strains, it is thuspreferable to use strain cocktails in inactivation and validationstudies conducted on food (Balasubramaniam et al., 2004), and toinclude highly heat resistant strains to simulate a worst-casescenario. These would be especially valuable for food processorsandmay help them in designing acceptance limits on critical pointsthat ensure safety against the pathogen on alfalfa seeds.

Moreover, the heat resistance of salmonellae may also beinfluenced by the inoculation and subsequent drying method of theseeds. In our study, alfalfa seeds were inoculated by mixing theminto the inoculum at a ratio of 5:3. The excess liquid was allowed todrain and the seeds were allowed to dry with intermittent turnover for a total period of 72 h. The mean water activity for inocu-lated seeds dried for 24, 48 and 72 h were 0.703, 0.584 and 0.439respectively. Hu et al. (2004) and Feng et al. (2007), on the contrary,only allowed the seeds to dry at room temperature overnight. It ispossible that our inoculation method and subsequent prolongeddrying of seeds for 72 h prior to the actual treatment caused thebacterial cells to become conditioned or “habituated” at a reducedwater activity, resulting in a concomitant increase in their thermalresistance (Mattick et al., 2000). As it is well known, the wateractivity during heat inactivation exerts a profound effect on theheat tolerance of bacteria (Sumner et al., 1991). Kirby and Davies(1990) had previously demonstrated that air-dried Salmonellacells acquired greater heat tolerance in a matrix of reduced aw.Mattick et al. (2000) previously reported that transient condi-tioning of a bacterial cell to a sub-lethal stress (i.e. “habituation”)can confer greater resistance to a more extreme form of the stress.Mattick et al. (2000) further demonstrated that habituation of S.Typhimurium DT 104 to low aw at ambient temperature (w21 �C),can greatly increase its heat tolerance and the impact of habituationon heat tolerance decreases at higher treatment temperaturessuggesting the involvement of additional targets for cell death.Observations made by the aforementioned author(s) may thus helpto explain the relatively higher thermal resistance exhibited bySalmonella at 55 and 60 �C compared to temperatures of 65 and70 �C.

Decline in the population of E. coli O157:H7 on inoculatedseeds was noticeably faster than Salmonella at all temperatures

7 inoculated on alfalfa seeds and seed viability.

Germination (%) at 3e8 days

4 5 6 7 8

0 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a

t done Not done Not done Not done Not done0 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a

t done Not done Not done Not done Not done8 � 1a 99 � 1a 99 � 1a 99 � 1a 99 � 1a

8 � 1a 98 � 1a 99 � 1a 99 � 1a 99 � 1a

� 1 ab 97 � 1 ab 98 � 1a 98 � 1a 98 � 1a

� 2 ab 97 � 2 ab 97 � 2 ab 97 � 2a 97 � 2a

ard deviation.epresent number of samples testing positive after enrichment out of a total of 3 trials.> 0.05).

Table 2Effect of dry heat treatment at 60 �C on the populations of Salmonella and E. coli O157:H7 inoculated on alfalfa seeds and seed viability.

Heating time (d) Salmonella E. coli Germination (%) at 3e8 days

O157:H7 3 4 5 6 7 8

Control 5.2 � 0.2a 5.4 � 0.3a 99 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a

1 4.6 � 0.4ab 1.6 � 0.1b 98 � 1ab 99 � 0ab 99 � 1ab 99 � 0ab 99 � 1ab 99 � 1ab

2 3.9 � 0.2bc 0.2 � 0.4c 95 � 1bc 95 � 0bc 96 � 1bc 96 � 1bc 96 � 0bc 97 � 1ab

3 4.0 � 0.4bc <0.7 (1/3) 95 � 1bc 96 � 1ab 97 � 1abc 97 � 1 ab 97 � 1ab 97 � 1ab

4 4.0 � 0.4bc < 0.7 (0/3) 96 � 1bc 96 � 1ab 96 � 1abc 96 � 1bc 96 � 1bc 96 � 1bc

5 3.5 � 0.4c Not done 96 � 1bc 96 � 1ab 96 � 1bc 96 � 1bc 96 � 1bc 96 � 1bc

6 3.3 � 0.4c Not done 93 � 1cd 95 � 1bc 95 � 2bc 96 � 2bc 96 � 2bc 96 � 2bc

7 3.3 � 0.6c Not done 94 � 2cd 95 � 1bc 95 � 1bc 95 � 1bc 96 � 1bc 96 � 1bc

8 3.0 � 0.2c Not done 95 � 1bc 96 � 1ab 96 � 1bcd 97 � 1ab 97 � 1ab 97 � 1ab

9 3.6 � 0.4c Not done 93 � 1cd 96 � 0ab 97 � 1abc 97 � 1ab 97 � 1ab 97 � 1ab

10 3.1 � 0.3c Not done 94 � 1cd 95 � 1bc 96 � 1bcd 96 � 1bc 96 � 1bc 96 � 1bc

Data representing mean log survivors (CFU/g) or mean germination percentage � standard deviation.The limit of detection for the platingmethodwas 0.7 log CFU/g. Numbers in parentheses represent number of samples testing positive after enrichment out of a total of 3 trials.Values in the same column followed by the same letter are not significantly different (P > 0.05).

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127 123

(Tables 1e4). Feng et al. (2007) and Hu et al. (2004) also observedthat E. coli O157:H7 was much more heat sensitive to dry heat thanSalmonella artificially contaminated onto alfalfa and mung beanseeds, respectively. We speculate that this is probably because ofthe higher heat tolerance exhibited by one or more strains ofSalmonella, such as S. TyphimuriumDT 104 and S. Seftenberg 775W.Moreover, Barak et al. (2002) reported that Salmonella has theability to attach more tightly to seeds and sprout tissue than E. coliO157:H7. Recent findings by Barak et al. (2002) also suggested thatS. enterica may have a survival advantage over E. coli O157:H7 forattachment to sprouting seeds, sprouts and plant tissue in general.The authors explained that the difference could particularly beexplained by the differential ability of S. enterica and E. coli O157:H7to produce aggregative fimbriae (curli). It is thought that S. entericamost readily express curli (Tafi), a structure that plays a funda-mental role in the attachment of S. enterica to seeds and plant tissuein general. Romling et al. (1998) showed that the production of curliwas induced in an environment akin to a plant surface as well aslow temperature and osmolarity. It is possible that the higherthermo-tolerance of Salmonella may be attributed to its strongerattachment to the seed tissue. Cotterill and Glauert (1971) alsoshowed that the heat resistance of salmonellae was much higherthan microorganisms belonging to other genera in low-moisturefoods. The heat resistance of S. Typhimurium, in particular, hasbeen shown to be about 653 times as great in low moisture foodssuch as dried egg white (moisture of about 7%) compared to liquideggs (Liu et al., 1969). Hansen and Riemann (1963) pointed out thatdry heat appears to be less efficient than moist heat in inactivatingmicroorganisms because proteins that are potentially destroyed

Table 3Effect of dry heat treatment at 65 �C on the populations of Salmonella and E. coli O157:H

Heating time (d) Salmonella E. coli

O157:H7 3

Control 5.2 � 0.2a 5.4 � 0.3 99 � 1a 11 4.0 � 0.3ab < 0.7 (1/3) 93 � 2b 92 2.4 � 0.3c < 0.7 (0/3) 92 � 1b 93 2.7 � 0.4bc Not done 93 � 1b 94 2.6 � 0.2c Not done 92 � 1b 95 2.3 � 0.5cd Not done 92 � 1b 96 1.6 � 0.5cde Not done 91 � 1b 97 1.1 � 0.7de Not done 91 � 2b 98 0.7 � 0.7e Not done 92 � 1b 99 0.4 � 0.8e Not done 90 � 1b 910 < 0.7 (0/3) Not done 91 � 2b 9

Data representing mean log survivors (CFU/g) or mean germination percentage � standThe limit of detection for the platingmethodwas 0.7 log CFU/g. Numbers in parentheses reValues in the same column followed by the same letter are not significantly different (P

during (moist) thermal processing remain quite stable in the drystate.

3.2. Effect of dry heat treatment on seed germination percentages

With regard to the viability of seeds, alfalfa seeds exposed to dryheat temperatures of 55e65 �C retained their ability to germinateto a larger extent than when held at 70 �C (Tables 1e4).

Indeed, Farooq et al. (2004) reported that the effect of dry-heattreatment on seed germination depended on both the dry heatintensity and duration of exposure. Martin and Cushwa (1966) alsomade a general observation that under a range of optimal heattreatment temperatures, seeds normally exhibit a consistently highgermination rate but result in decreased germinability at higherlethal temperatures. Farooq et al. (2004) attributed the decreasedgerminability at elevated detrimental temperatures to membranedeterioration. We speculate that the rapid decline in germinationrate for seeds treated at 70 �C to be due to reduced membraneintegrity (Basavarajappa et al., 1991; Farooq et al., 2004) andpossibly to heat-induced damage to plastids, mitochondria or othercellular organelles as proposed by Wang and Berjak (2000).

Extended exposures of alfalfa seeds at 55e65 �C did notsubstantially impact on the seed germinability with a germinationpercentage of >90% after 10 days of holding at 55, 60 and 65 �C.Feng et al. (2007) also showed that when seeds were exposed toa temperature of 55 �C for 4 days or longer, the germinationextent of treated un-inoculated seeds (78%) were not dramaticallylower compared to control untreated seeds (82%). However, thesame authors mentioned that temperatures higher than 55 �C

7 inoculated on alfalfa seeds and seed viability.

Germination (%) at 3e8 days

4 5 6 7 8

00 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a

5 � 2b 95 � 3b 97 � 3ab 97 � 3ab 97 � 3ab

5 � 1b 95 � 2b 95 � 2b 95 � 2b 95 � 2b

5 � 1b 95 � 1b 95 � 1b 95 � 1b 95 � 1b

3 � 1b 93 � 1b 95 � 1b 95 � 1b 95 � 1b

3 � 1b 93 � 1b 93 � 1b 93 � 1b 93 � 1b

2 � 2b 93 � 1b 93 � 1b 93 � 1b 93 � 1b

2 � 2b 93 � 2b 93 � 2b 93 � 2b 93 � 2b

3 � 0b 93 � 1b 94 � 1b 94 � 1b 94 � 1b

3 � 1b 94 � 1b 94 � 1b 94 � 1b 94 � 1b

2 � 2b 94 � 1b 94 � 1b 95 � 1b 95 � 1b

ard deviation.present number of samples testing positive after enrichment out of a total of 3 trials.> 0.05).

Table 4Effect of dry heat treatment at 70 �C on the populations of Salmonella and E. coli O157:H7 inoculated on alfalfa seeds and seed viability.

Heating time (h) Salmonella E. coli Germination (%) at 3e8 days

O157:H7 3 4 5 6 7 8

Control 5.2 � 0.2a 5.4 � 0.3a 99 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a

3 4.7 � 0.0a 1.5 � 0.3b 75 � 3b 80 � 5b 80 � 5b 80 � 5b 80 � 5b 80 � 5b

6 3.3 � 0.4b 0.6 � 0.8b 51 � 9c 62 � 8c 62 � 8c 62 � 8c 62 � 8c 62 � 8c

9 3.3 � 0.6b < 0.7 (0/3) 25 � 2d 31 � 2d 31 � 2d 31 � 2d 31 � 2d 31 � 2d

12 2.7 � 0.2b < 0.7 (0/3) 19 � 3de 21 � 4de 21 � 4de 21 � 4de 21 � 4de 21 � 4de

15 1.8 � 1.0b Not done 11 � 3e 12 � 2ef 12 � 2ef 12 � 2ef 12 � 2ef 12 � 2ef

18 <0.7 (3/3) Not done 5 � 1ef 6 � 1fg 6 � 1fg 6 � 1fg 6 � 1fg 6 � 1fg

21 <0.7 (1/3) Not done 1 � 2ef 1 � 2fg 3 � 2fg 3 � 1fg 4 � 2fg 4 � 2fg

24 <0.7 (0/3) Not done 0 � 1f 0 � 1g 1 � 1g 1 � 1g 1 � 1g 1 � 1g

Data representing mean log survivors (CFU/g) or mean germination percentage � standard deviation.The limit of detection for theplatingmethodwas0.7 logCFU/g.Numbers inparentheses represent thenumber of samples testingpositive after enrichment out of a total of 3 trials.Values in the same column followed by the same letter are not significantly different (P > 0.05).

Table 5Effect of combined application of dry heat treatment with pressure treatment on theinactivation Salmonella spp. with an initial population of 5.8 log CFU/g.

600 MPa for 2 min at

Heat treatment 20 �C 35 �C

1 day at 55 �C 1.5 � 1.2 < 0.8 (3/3)1 day at 60 �C < 0.8 (1/3) < 0.8 (0/3)2 days at 55 �C 1.5 � 1.2 < 0.8 (3/3)2 days at 60 �C < 0.8 (1/3) Not done3 days at 55 �C 1.0 � 0.3 <0.8 (1/3)3 days at 60 �C <0.8 (1/3) Not done4 days at 55 �C <0.8 (1/3) <0.8 (0/3)4 days at 60 �C <0.8 (1/3) Not done3 h at 65 �C 1.6 � 1.1 1.2 � 0.73 h at 70 �C < 0.8 (3/3) < 0.8 (1/3)6 h at 65 �C 0.9 � 0.6 1.1 � 0.66 h at 70 �C <0.8 (3/3) <0.8 (0/3)

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127124

were detrimental to seed germination although they did notspecify the temperatures or temperature ranges studied. Beuchatand Scouten (2002) did not observe any major difference in thegermination percentage between treated (81.6%) and untreatedseeds (86.3%) at 50 �C for 24 h. They also showed that thegerminability of seeds was fairly unaffected even after exposureto a higher temperature of 60 �C for 3 h (87.6%) compared tocontrol untreated seeds (85.0%), although the same authorreported that heating of seeds at 70 �C for � 3 h or at 80 �C for�1 h, resulted in a decrease in germination rate. This observationand the above comment made by Feng et al. (2007) reconcile withour findings, showing that exposures to 55, 60 and 65 �C hadminimal adverse effects on the seed germinability while a highertemperature of 70 �C brought about a noticeable reduction in thegermination percentage to ca. 31% with dry heating times � 9 h.

It is not surprising that alfalfa seeds are quite refractory to dryheat. As a matter of fact, Stewart (1926) previously indicated thatalfalfa seeds heated at temperatures ranging from 60 to 75 �C fora maximum of 1e2 h displayed higher or comparable germinationrates (76e93%) to unheated seeds (79%). They however showedthat seeds underwent a drastic decrease in viability (36%) whenexposed to a brief (10 min) exposure at 80 �C. The author reportedthat alfalfa seeds could be heated for 1 h at 75 �C without injuringthe seed. Staker (1925) found that the killing point of alfalfa seedswas about 90 �C and that temperatures as high as 85 �C resulted inan increase in the percentage germination.

Hence, the above findings garnered by previous researchesshows that there is a certain degree of variability among differentstudies. However, these data are unanimously supportive of the factthat mild temperatures of 55 and 60 �C and slightly highertemperature of 65 �Cdonot result in a dramatic loss in seed viability.

As Beuchat and Scouten (2002) inferred from their study, thereappears to be a correlation between loss of seed viability and loss ofSalmonella viability. This is also evident in our study by comparingthe bacterial population reduction and seed germination percent-ages in Tables 1e4While temperatures of 55 and 60 �C appeared tobe more ‘friendly’ to seeds with minimal adverse impact on seedgermination, the effectiveness of these temperatures at reducingthe population of Salmonella was also comparatively lower. On theother hand, dry heating at 70 �C caused a rapid and steep decline inpathogen inactivation with an accompanying rapid decrease inseed viability.

9 h at 65 �C <0.8 (3/3) <0.8 (3/3)9 h at 70 �C <0.8 (1/3) Not done12 h at 65 �C <0.8 (1/3) <0.8 (0/3)12 h at 70 �C Not done Not done

Data representing mean log survivors (CFU/g) � standard deviation.The limit of detection for the plating method was 0.8 log CFU/g.Numbers in parentheses represent the number of samples testing positive afterenrichment out of a total of 3 trials.

3.3. Combined effect of dry heat and high-pressure treatment onthe inactivation of Salmonella on alfalfa seeds

When seedswith an initial burden ofw5 log CFU/g of Salmonellawere subjected to a dry heat treatment at 55 �C for 2 days followed

by pressure treatment of 600MPa for 2 min at 20 �C, the populationdecreased by ca. 4.3 log CFU/g (Table 5). Extending the dry heatingtime to 4 days followed by the pressure treatment led to a finalpopulation of <0.8 log CFU/g (below the detection limit) althoughsurvivors were detectable after enrichment in one out of threecases. However, when the initial temperature of the pressuretreatment was stepped up to 35 �C following 4 days of dry heatingat 55 �C, the population was consistently undetectable afterenrichment.

When the dry heat temperature was increased to 60 �C, weobserved that a dry heat treatment of 1 day followed by the pres-sure treatment of 600MPa for 2 min at 20 �C was able to reduce thepopulation to below the detection limit although survivors werestill detected in one out of three trials (Table 5). An increase in theinitial temperature of the high pressure processing step from 20 �Cto 35 �C completely eliminated the population with no detectablesurvivors after enrichment. Pressure-treatment of (600 MPa for2 min at 35 �C) seed samples producing no detectable pathogensafter 1 day of dry heating at 60 �C were not further tested underlonger dry heating times.

Similar to the combination treatments of dry heat at 55 and60 �C with HHP, we observed from Table 5 that the sequentialapplication of dry heat at 65 �C for 12 h and 70 �C for 6 h followedby the pressure treatment of 600 MPa for 2 min at 35 �C was alsoable to completely eliminate Salmonella from alfalfa seeds. The

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127 125

results from Tables 5 and 6 show that the pressure sensitivity ofSalmonella had a strong dependence on the HHP treatmenttemperature, with increased sensitivity at a higher temperature of35 �C. Indeed, it is well documented that initial temperatures above30 �C greatly enhance the pressure inactivation of bacteria asreported for Listeria monocytogenes (Chen, 2007), Staphylococcusaureus (Chen et al., 2006), E. coli (Ponce et al., 1998), S. entericaserovar Enteritidis (Ponce et al., 1999), Vibrio vulnificus (Kural andChen, 2008), and Vibrio parahaemolyticus (Kural et al., 2008).Studies conducted by Kingsley et al. (2006) and Chen et al. (2005)demonstrated that temperatures of 30 �C also facilitated thepressure inactivation of hepatitis A virus and feline calicivirus,respectively.

The efficacy of the conjunct application of dry heat with otherhurdles has also been investigated by other authors (Bari et al.,2003, 2009a). Previous research has indicated that dry heat treat-ment (50 �C for 1 h) followed by an exposure to gamma irradiation(2e2.5 kGy) eliminated E. coli O157:H7 from artificially inoculatedalfalfa, radish and mung bean seeds (4e5 log CFU/g reductions)without decreasing seed germination percentages (Bari et al., 2003,2009a). However, one of the problems encountered with gammairradiation is the phenomenon of uneven or patchy absorbance ofirradiation at different locations in the treatment chamber (Fettet al., 2005). As a result, the dose absorbed by seeds located nearthe exterior of the chamber may be different from the doseabsorbed by seeds placed at the center of the chamber (Fett et al.,2005), thus rendering optimization and scale-up of the processdifficult. This problem of heterogeneity could lead to variability inpathogen population reduction within a batch of treated seeds aswell as possible disparate effects on the subsequent seed viability.High pressure processing on the contrary, is governed by the‘Isostatic Principle’, which states that pressure is uniformly trans-mitted throughout a sample under pressure independent of thebatch size (Hogan et al., 2005). Hence, the application of dry heat incombination with high pressure would be expected to yield moreconsistent results with minimal batch-to-batch variation asopposed to other gradient-forming processes such as heat andradiation.

3.4. Assessment of the efficacy of selected treatments to achievecomplete elimination of E. coli O157:H7

Table 6 summarizes the effects of different dry heat time/temperature regimes alone or in conjunction with high pressure atreducing the population of E. coli O157:H7 to undetectable levels.Results presented showed that complete elimination of E. coliO157:H7was possible when alfalfa seeds were heated for 10 days at65 �C and under selected conditions of dry heat exposure time(6 he4 days), dry heat temperature (55e70 �C) and HHP (600 MPafor 2 min at 35 �C). Based on the results presented in Tables 1e4, we

Table 6Effect of single application of dry heat or combined with HHP (600 MPa for 2 min at 35

Dry heat Hurdle E. coli

Td (�C) te (h) type O157:H7 3 4

Control 5.2 � 0.3 99 � 1a 100 � 1a

65 240 Single <0.7 (0/3) 91 � 2b 92 � 2b

60 24 Combined <0.8 (0/3) 89 � 3bc 94 � 2b

65 12 Combined <0.8 (0/3) 90 � 5bc 94 � 3b

55 96 Combined < 0.8 (0/3) 83 � 2c 89 � 2b

70 6 Combined <0.8 (0/3) 62 � 1d 68 � 2c

Values in the same column followed by the same letter are not significantly different (Pd T ¼ Dry heating temperature.e t ¼ Dry heating time.

expected the strains of E. coli O157:H7 to display higher thermalsensitivity than those of Salmonella. In addition, on the basis of ourpreviously developed pressure inactivation curve at 600 MPa forSalmonella (Neetoo and Chen, 2010) and E. coli O157:H7 (Neetooet al., 2008) on alfalfa seeds, we found that Salmonella was morebaro-tolerant than E. coli O157:H7. As anticipated, the pre-deter-mined treatments were able to eliminate E. coli O157:H7 as well.The findings of this study thus indicate that dry heat alone or inconjunction with high pressure can represent effective interven-tions for the decontamination of alfalfa seeds from both entericpathogens.

3.5. Effect of selected treatments on the germination characteristicsof alfalfa seeds

3.5.1. Determination of the germination percentages and yieldratios of seeds treated under selected conditions

The germination percentages as determined 3e8 days from theonset of sprouting showed that seeds pressure-treated under thedifferent conditions were affected to variable extents depending onthe conditions of the treatment (Table 7). Application of dry heat for10 days at 65 �C appeared as the most promising treatment withrespect to seed germination percentage (95%) and yield (12.2 w/w)compared to untreated seeds, with a germination percentage of100% and yield of 15.5 w/w (Table 7). This represents an approxi-mate 21% reduction in yield ratio, which may still be consideredacceptable by the commercial sprout grower (Rajkowski andThayer, 2001).

The sequential application of dry heat at 70 �C and HHP wasdeleterious to seed viability with a significantly (P < 0.05) reducedgermination percentage and yield. Although seeds dry heated at 60and 65 �C and treated by HHP did not undergo an appreciabledecrease in their germination rates, we observed a significantdecrease (P < 0.05) in the yield with final sprouting yield ratios ofw9 w/w. Other authors have also observed that while certain seedtreatment regimes may have little to no effect on seed germination,the impact on the sprouting yield could be considerable. Rajkowskiand Thayer (2001) previously showed that the application ofgamma irradiation at doses ranging from 1 to 5 kGy had littleapparent effect on seed germination although the yield ratio ofalfalfa sprouts decreased with increasing dose. Bari et al. (2003)previously showed that the sequential application of dry heat at50 �C for 1 h followed by irradiation at doses of up to 2.0 kGy hadlittle to no effect on the germination rate of radish and mung beanseeds although a significant decrease in sprout lengths wereobserved. Bari et al. (2009b) also recently showed that irradiation ofmung bean seeds at 1.0 kGy subsequent to dry heating at 50 �C for17 h did not affect the percentage germination although the authorreported a significant decrease in the average sprout length.We can

�C) on E. coli O157:H7 population and viability indices of alfalfa seeds.

Germination (%) at 3e8 days Yield ratio (w/w)

5 6 7 8

100 � 1a 100 � 1a 100 � 1a 100 � 1a 15.5 � 0.5a

94 � 1b 94 � 1b 95 � 1b 95 � 1b 12.2 � 0.3b

96 � 2ab 98 � 1ab 99 � 1ab 99 � 1ab 9.2 � 1.9c

95 � 4ab 96 � 3ab 96 � 3ab 96 � 3ab 9.3 � 1.1c

91 � 1b 93 � 1b 94 � 3b 94 � 3b 7.9 � 0.6c

75 � 2c 76 � 2c 78 � 3c 80 � 1c 7.0 � 0.7d

> 0.05).

Table 7Effect of modifications to the germination procedure on the viability indices of alfalfa seeds subjected to dry heating and HHP (600 MPa for 2 min at 35 �C).

Dry heat Germination (%) at 3e8 days Yield ratio (w/w)

Tc (�C) td (h) 3 4 5 6 7 8

Control 99 � 2a 100 � 1a 100 � 1a 100 � 1a 100 � 1a 100 � 1a 15.7 � 0.3a

60 24 h 96 � 2a 97 � 2a 97 � 2a 97 � 2a 97 � 2a 97 � 2a 11.4 � 0.3b

65 12 h 96 � 2a 97 � 1a 97 � 1a 97 � 1a 97 � 1a 97 � 1a 10.9 � 0.6b

Values in the same column followed by the same letter are not significantly different (P > 0.05).c T ¼ Dry heating temperature.d t ¼ Dry heating time.

H. Neetoo, H. Chen / Food Microbiology 28 (2011) 119e127126

thus ascertain that sprout yield ratio is an important index of seedviability in addition to the percentage germination.

3.5.2. Modification of the germination procedure to improve thesprouting yield of pressure-treated seeds

Previously, we showed that the viability of seeds that were dryheated at 60 and 65 �C prior to HHP was adversely affected,resulting in an appreciable decrease in their yield. In this experi-ment (Table 7), we showed that when seeds were dry heated at60 �C for 24 h, chilled at 4 �C for 24 h prior to HHP, and subsequentlyallowed to germinate at an incubation temperature of 27 �C (ratherthan ambient temperature), a sprouting yield of 11.4 w/w wasrecorded, compared to the yield of 9.2 w/w determined previously.When seeds were dry heated at 65 �C for 12 h, chilled at 4 �C for24 h prior to HHP and then allowed to germinate under the sameconditions, only a slight improvement in the yield was observedwith a final sprouting yield ratio of 10.9 w/w compared to 9.2 w/wrecorded previously. Hence, modification to the germinationconditions only brought about amarginal improvement in the yieldratio. It is likely that although certain seed decontaminationregimes may have minimal adverse effect on the processes leadingto radicle emergence (as measured by percentage germination),seedling growth (as measured by sprout yield) appears to be morevariably impaired. Future studies are thus warranted to determinewhether seeds subjected to dry heating and high pressure couldundergo a restoration of viability upon storage.

4. Conclusions

The results of this study indicate that dry heating alone atmoderate temperatures (55 and 60 �C) represented an ineffectiveintervention to decontaminate alfalfa seeds from Salmonella.However, exposure to a relatively more aggressive temperature of65 �C with a holding time of 10 days, was able to simultaneouslyeliminate Salmonella as well as E. coli O157:H7 at little expense ofthe seed germinability. The study also stressed on various intrinsicand extrinsic factors impacting on the dry heat inactivationincluding the choice and type of strains used, the aw of thematrix ofinterest and the treatment temperatures. This should be of partic-ular interest to seed companies or sprout growers wishing toimplement a dry heat treatment step. In addition to a stand-aloneintervention, we also demonstrated that a ‘multiple-hurdle’approach consisting of a dry heating step (60�C/24h) in combina-tion with high pressure (600 MPa/2 min/35 �C) fully decontami-nated alfalfa seeds but also led to a >20% reduction in the sproutyield.

Acknowledgements

This study was supported by a start-up fund from the Depart-ment of Animal and Food Sciences at the University of Delaware.

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