International Journal of Food Microbiology 137 (2010) 274–280

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International Journal of Food Microbiology

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Pre-soaking of seeds enhances pressure inactivation of E. coli O157:H7 andSalmonella spp. on crimson clover, red clover, radish and broccoli seeds

Hudaa Neetoo, Haiqiang Chen ⁎Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716-2150, USA

⁎ Corresponding author. Tel.: +1 302 831 1045; fax:E-mail address: haiqiang@udel.edu (H. Chen).

0168-1605/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2009.11.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 August 2009Received in revised form 17 November 2009Accepted 25 November 2009

Keywords:High pressureSeedsSalmonellaEscherichia coli O157:H7

The application of high hydrostatic pressure (HHP) at a level of 600 MPa at 20 °C to decontaminate crimsonclover, red clover, radish and broccoli seeds inoculated with E. coli O157:H7 and Salmonella were evaluated.Salmonella was generally more pressure-resistant than E. coli O157:H7 on clover and radish seeds except onbroccoli seeds where the trend was reversed. In addition, the application of HHP differentially affectedseeds' germinability and the order of pressure tolerance of the seeds was such that red cloverNcrimsonclover≈broccoliNradish seeds with final germination percentages ranging from 85–100% while theiruntreated counterparts had final germination percentages of 99–100%. Pre-soaking the different types ofseeds in water for 30, 60 or 90 min at ambient temperature followed by HHP at 600 MPa for 2 or 5 min at20 °C significantly (Pb0.05) enhanced the pressure inactivation of the inoculated pathogens. Moreover, theability of HHP-treated seeds to germinate also varied as a function of the pre-soaking duration and the seedtype. Pre-soaking radish and broccoli seeds for 30 min prior to HHP (2 or 5 min) resulted in germinationpercentages of ≤ 1% after 8 days of incubation. On the contrary, red clover seeds displayed highergermination potential when pre-soaked for 60 min at 20 °C prior to HPP (5 min) with final germinationpercentages of 94%, although their yield was substantially lower than their untreated counterparts. Redclover seeds pre-soaked for 60 min at 4 °C followed by HPP at 600 MPa for 5 min at 20 °C producedgermination percentages of 91 and 95% after 3 and 8 days of sprouting compared to 99 and 100% respectivelyfor untreated seeds. In addition, this condition did not significantly (PN0.05) reduce the sprout yield. Thetreatment also resulted in a reduction of a 5 log initial load of E. coli O157:H7 and Salmonella to anundetectable level (neither pathogen was detected in 2-g seed samples after enrichment).

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1. Introduction

Seed sprouts are popular ingredients in many dishes and recently,they have grown in popularity in various cultures from East to West(Wigmore, 1986). Despite being inexpensive and easy to grow,sprouts are known to provide one of the most concentrated andnaturally occurring sources of vitamins, minerals, enzymes and aminoacids known. Sprouts belonging to the Cruciferae family (broccoli andradish sprouts) are particularly renowned for their antioxidant andanticancerous properties (Meyerowitz, 1999a,b). Unfortunately, theirrich nutritional content also makes them very supportive of bacterialgrowth (Thompson and Powell, 2000; Wood, 2000) including thegrowth of pathogens (Tournas, 2001; Buck et al., 2003).

From 1973 to present, more than 40 outbreaks of foodborne illnesshave been reported worldwide incriminating raw seed sprouts (Wajeet al., 2009). Salmonella spp., Escherichia coli O157:H7 and Bacilluscereus were among the pathogens involved (Fett, 2006). The most

prevalent pathogenic microorganisms are enteric pathogens such asSalmonella and E. coli O157:H7 (NACMCF, 1999). Although sproutscan become contaminated during the sprouting, harvesting, packag-ing and distribution processes, many studies demonstrated outbreakslinked to contaminated seeds (Andrews et al., 1982). Among variousseed sprouts implicated in outbreaks were alfalfa (NACMCF, 1999;Taormina et al., 1999), radish (Taormina et al., 1999) and cloversprouts (Taormina et al., 1999; Brooks et al., 2001).

The United States Food and Drug Administration thus recommendsthe treatment of sprouting seeds with 20,000 ppm of free chlorinefrom calcium hypochlorite or an equivalent antimicrobial (NACMCF,1999). However, the use of chlorine-based chemicals generateshazardous fumes that have created public health concerns (Beuchat,1997). As a result, alternative seed decontamination methods havebeen sought. Researchers studying the efficacy of different chemical,physical and biological methods for reducing or eliminating popula-tions of bacterial pathogens artificially contaminated onto seeds havereported varying degrees of success (Fett, 2006).

In our previous studies (Neetoo et al., 2008, 2009a,b), we reportedthat the application of high pressure either alone or in combinationwith mild heat was able to successfully inactivate E. coli O157:H7 on

275H. Neetoo, H. Chen / International Journal of Food Microbiology 137 (2010) 274–280

alfalfa seeds while maintaining seed viability. The objective of thisstudy was to determine the effect of pre-soaking and high-pressuretreatment on inactivation of E. coli O157:H7 and Salmonella spp.inoculated on crimson clover, red clover, radish and broccoli seedsand on their germination ability. To simplify sentence construction,the term “elimination” was used in this paper interchangeably withthe phrase “reduction of a 5 log initial load of E. coli O157:H7 orSalmonella to an undetectable level (pathogens were not detected in2-g seed samples after enrichment)”.

2. Materials and methods

2.1. Effect of pressure treatment on the extent of germination of crimsonclover, red clover, radish and broccoli seeds

Unscarified radish (Raphanus sativus), broccoli (Brassica oleraceavar. italica), crimson clover (Trifolium incarnatum) and red clover(Trifolium pratense) seeds were purchased from International Spe-cialty Supply (Cookeville, TN, USA). Two grams of each type of seedwere placed in individual 3-mil-thick nylon/polyethylene pouches(Koch Supplies, Kansas City, MO, USA). Deionized (DI) water (3 ml forcrimson clover, red clover and broccoli seeds and 4 ml for radishseeds) was added to the pouches and those poucheswere heat-sealed.Pressure treatment of samples was carried out using a high-pressureunit with temperature control (Model Avure PT-1, Avure Technologies,Kent, WA, USA). Pressurization was conducted at 600 MPa for 2 min at20 °C (initial seed sample temperature prior to pressure treatment)using water as a hydrostatic medium. The temperature of the water-bath was monitored with a K-type thermocouple. Temperature andpressure data were recorded every 2 s (DASYTEC USA, Bedford, NH,USA). The pressure-come-up rate was approximately 22 MPa/s. Thepressure-release was almost immediate (b 4 s). Pressurization timereported in this study does not include the pressure-come-up or releasetimes.

To determine the germination percentage of treated and untreatedseeds, seeds were soaked in DI water for 3 h and 100 seeds werespread evenly on layers of wet paper towels on a plastic rack whichin turn was placed into a water-filled bucket to provide a moistenvironment for the seeds. The water level was maintained below theseeds' level. The bucket was covered loosely with a piece of plasticfilm to allow exchange of air between the inside and outside of thebucket. The bucket was kept at room temperature (∼21 °C) for 8 days(suggested by the seeds provider). The seeds were visually evaluatedfor germination and sprouted seeds were counted after 3, 4, 5, 6, 7and 8 days and discarded on each day following enumeration. Thecumulative germination percentage reached on each day was thendetermined as the proportion of total number of sprouted seeds to thetotal number of seeds.

2.2. Determination of the pressure inactivation curves of E. coli O157:H7and Salmonella spp. inoculated on crimson clover, red clover, radish andbroccoli seeds

2.2.1. Test strains and preparation of inoculumA cocktail of five different E. coli O157:H7 strains (250, 251, Cider,

1730 and J58) (Neetoo et al., 2008) and a cocktail of five Salmonellaenterica strains (Typhimurium T43, Typhimurium T45, TyphimuriumDT 104, Enteritidis E44 and Montevideo Mo57) (Courtesy of Dr. RolfJoerger, University of Delaware) were used in this study. Cells of E. coliO157:H7 and Salmonella were adapted to grow in tryptic soy brothplus 0.6% yeast extract (Difco Laboratories, Sparks, MD, USA)supplemented with nalidixic acid to a final concentration of 50 μg/ml (Fisher Scientific, Hampton, NH, USA) (TSBYE-N). Individualcultures were grown in TSBYE-N for 16–18 h at 35 °C. Cultures werethen transferred (one loopful) into 10 ml of fresh TSBYE-N andincubated at 35 °C for 24 h. Equal volumes of individual cultures were

mixed to form a five-strain cocktail of Salmonella and a five-straincocktail of E. coli O157:H7.

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

made in sterile 0.1% peptone water (Fisher). Five hundred grams ofeach seed type were added to the diluted cell suspension at a seed:inoculum ratio of 5:3 and gently stirred for 5 min. The seeds wereseparated from the cell suspension by pouring the mixture over adouble layer of cheesecloth supported by a wire screen and driedinside a biosafety hood at room temperature (21 °C±1 °C) for 48 hwith intermittent rotation to ensure that seeds were uniformly dry.The mean water activity (aw) values of radish, broccoli, crimsonclover and red clover seeds after drying were ca. 0.74, 0.44, 0.52 and0.67, respectively. Seeds with an approximate inoculation level of105 CFU/g of E. coli O157:H7 and Salmonella were placed in plasticpouches and stored at 4 °C.

2.2.3. Pressure treatment and microbiological analysisTwo grams of inoculated seeds mixed with 3 ml (for crimson

clover, red clover and broccoli seeds) or 4 ml (for radish seeds) ofsterile DI water were added into a nylon/polyethylene pouch. To avoidleakage during pressure treatment, each sample pouch was placed ina larger pouch of an 8-mil-thick polyvinyl chloride plastic (McMaster-Carr, Elmhurst, IL, USA) and heat-sealed. Pressure treatment wascarried out at 20 °C at 600 MPa and a treatment time of 2, 4, 6, 8, 10 or15 min.

Pouches containing treated seeds were cut open aseptically. Thesample was transferred into a stomacher bag to which 8 ml of sterile0.1% peptonewater was added and subsequently stomached for 2 minat 260 rpm (Seward 400 Stomacher; Seward Medical Co., London,UK). The seed slurry was serially diluted in sterile 0.1% peptone waterand surface plated in duplicate on tryptic soy agar with 0.6% yeastextract (Difco) supplemented with nalidixic acid to a final concen-tration of 50 μg/ml (TSAYE-N). TSAYE-N plates were incubated for3 days at 35 °C.

2.3. Optimizing the parameters for pre-soaking of seeds required forsafety and viability retention after pressure treatment

2.3.1. Effect of the pre-soaking duration on the pressure inactivation ofE. coli O157:H7 and Salmonella spp.

Two grams of seeds inoculated with E. coli O157:H7 or Salmonellaspp. were soaked in 20 ml sterile DI water at room temperature (ca.21 °C) for 0 (without soaking), 30, 60, 90, 120 and 180 min. At the endof the soaking period, the excess water was subsequently decantedand seeds were placed into a pouch in the presence of 3 ml (clover andbroccoli seeds) or 4 ml (radish seeds) of fresh sterile DI water andpressure-treated at 600 MPa for 2 min at 20 °C. Samples were thenmicrobiologically assayed on TSAYE-N as described previously. Whenthe bacterial population was below the detection limit for the platingmethod (b 0.8 log CFU/g), the seed slurry was further enriched in90 ml of TSBYE-N and incubated for 48 h at 35 °C to allow resuscitationof sub-lethally injured cells. Samples were streaked onto SorbitolMacConkey agar (Difco) plates supplemented with 50 μg/ml ofnalidixic acid for samples inoculated with E. coli O157:H7 or xyloselysine deoxycholate (XLD) agar (Difco) plates supplemented with50 mg/ml of nalidixic acid for samples inoculated with Salmonella.After 24 h incubation, presence of colorless or faint orange growthtypical of E. coli O157:H7 and black-centered or black coloniescharacteristic of Salmonella were interpreted as a positive result.

In addition, the different types of seeds were also soaked for 0, 15,30, 45 or 60 min and then treated at 600 MPa for 5 min at 20 °C.Broccoli seeds were soaked for up to 30 min while crimson and redclover seeds were soaked for a maximum of 60 min. Treated seedsamples were then microbiologically analyzed as described above.

276 H. Neetoo, H. Chen / International Journal of Food Microbiology 137 (2010) 274–280

Pre-soaked and pressure-treated (2 min) seed samples for which nopathogens were detected in a 2-g sample after enrichment, were notfurther tested under the extended pressure exposure of 5 min. Inaddition, treated samples without detectable pathogens after enrich-ment after a particular soaking period and pressure treatment of5 min, were not further tested under longer soaking times.

2.3.2. Effect of selected soaking and pressure treatment conditions onthe germination percentages of crimson clover, red clover, radish andbroccoli seeds

To determine the effect of selected soaking and pressure treatmentparameters on the seeds' germination potential, two grams of un-inoculated seeds were soaked in 20 ml DI water for 30, 60 or 90 min at4 and/or 20 °C (conditions depending on the seed type). At the end ofthe soaking period, the excess water was subsequently decanted andseeds were placed into a pouch in the presence of 3 ml of fresh DIwater (or 4 ml for radish seeds) and pressure-treated at 600 MPa and20 °C for 2 or 5 min. Untreated (control) and pressure-treated seedswere then soaked in DI water after pressure treatment for a totalsoaking time of 3 h. One hundred seeds were drawn from the samplesand set to germinate as described previously. The cumulativepercentage of germinated seeds was determined after 3, 4, 5, 6, 7and 8 days from the onset of germination.

To determine whether the selected soaking conditions andpressure treatments could eliminate the two pathogens in red cloverseeds, seeds inoculated with E. coli O157:H7 and Salmonella were(i) soaked at 4 °C for 60 min and treated at 600 MPa for 5 min at 20 °Cor (ii) soaked at 4 °C for 90 min and treated at 600 MPa for 2 minat 20 °C. Treated seeds were then assayed post-treatment for thepresence of E. coli O157:H7 or Salmonella survivors as previouslydescribed.

2.3.3. Effect of selected soaking and pressure treatment conditions on theyield ratio of red clover and crimson clover seeds

Red and crimson clover seeds (2 g) were either soaked at 4 °C or20 °C for 60 min and treated at 600 MPa for 5 min at 20 °C or soaked at4 °C or 20 °C for 90 min and treated at 600 MPa for 2 min at 20 °C. Onehundred seeds from each sample were then randomly picked andallowed to germinate. After 8 days of growth, sprouts were weighedand the yield ratio was calculated by dividing the weight of sproutedseeds by the weight of one hundred dry seeds, a method adapted fromRajkowski and Thayer (2001).

2.4. Statistical analysis

All experiments were replicated three times. Where appropriate,statistical analyses were conducted using Minitab release 15 (MinitabInc., University Park, PA, USA). One-way analysis of variance andTukey's one-way multiple comparisons were used to determinedifferences in the populations of E. coli O157:H7 and Salmonellarecovered on treated sprouting seeds and differences in the germina-

Table 1Effect of pressure treatment of 600 MPa for 2 min at 20 °C (HHP) on germination of seeds.

Seed type Treatment

3 4

Crimson clover Control 99±1a 100±0a

Crimson clover HHP 87±3b 91±4b

Red clover Control 99±1a 100±0a

Red clover HHP 97±2a 99±1a

Broccoli Control 98±1a 99±0a

Broccoli HHP 85±3b 93±1b

Radish Control 98±0a 99±0a

Radish HHP 14±6c 31±5c

Values in the same column followed by the same letter are not significantly different (PN0

tion percentages and sprout yield ratio. Differences were consideredstatistically significant at the 95% confidence level (Pb0.05).

3. Results and discussion

3.1. Effect of high pressure on the extent of germination of crimsonclover, red clover, radish, and broccoli seeds

The germination percentages for the four different types of seedsafter high-pressure treatment are summarized in Table 1. Seedgermination response was differentially affected by high pressureand the order of pressure tolerance was such that red cloverNcrimsonclover≈broccoliNradish seeds. The germination percentage deter-mined 3–8 days after sprouting was initially lower, but not signifi-cantly lower for red clover seeds at all germination times and sometreatment and germination times for the other seed types. Red cloverseeds exhibited the highest pressure resistance achieving an averageseed germination of 97% (after 3 days) compared with 99% foruntreated seeds; showing therefore that there was almost no delayin the onset of sprouting. Crimson clover and broccoli seeds wereslightly more affected by pressure since they sprouted to ∼85% after3 days and achieved N90% germination after 4 days while controluntreated seeds germinated to 99 % after 3 days. This indicates thatthe onset of sprouting was slightly delayed. The germination of radishseeds appeared the most desynchronized with an average of 14%after 3 days of germination. By the end of 8 days, the cumulativegermination percentages of red and crimson clover and broccoli seedswere 100, 94 and 96% respectively while radish seeds which were themost severely affected, reached a final germination percentage of 85.

The pressure resistance of red clover seeds was very similar tothose of alfalfa seeds as investigated previously by Neetoo et al. (2008,2009a,b). Alfalfa seeds treated at 600 MPa for 2 min at 20 °C had N95%germination after 8 days of incubation. Hayward (1948) noted thatred clover seeds and alfalfa seeds represent two very popular speciesof the same large pea family, which may contribute to their similardegree of pressure resistance. The germination results of radish seedsare also in agreement with those of Wuytack et al. (2003). Theycompared the germination percentages of garden cress, mustard,radish and sesame seeds subjected to levels of high pressure rangingfrom 250–400 MPa and noted that radish and mustard seeds werehighly sensitive to pressure with germination percentages b 10% after11 days of germination.

It is not clear however what mechanism is responsible for thedifferential pressure sensitivity across the various plant speciesstudied but it is likely due to the intrinsic uniqueness of the embryosof seeds themselves. It could also be due to the specific structural andanatomical characteristics of the seed coat itself. Seed coat acts as animportant moisture barrier in certain seeds (Rolston, 1978) such asalfalfa, clover and broccoli seeds. Alfalfa seeds tend to have a relativelyimpermeable seed coat that effectively slows downwater uptake. Lute(1928) showed that the thickened outer wall of palisade cells (and

% Germination on different days

5 6 7 8

100±0a 100±0a 100±0a 100±0a

93±3b 93±3a 93±3a 94±2a

100±0a 100±0a 100±0a 100±0a

100±0a 100±0a 100±0a 100±0a

99±0a 99±0a 99±0a 99±0a

94±1b 94±1a 95±2a 96±1a

99±0a 99±0a 99±0a 99±0a

55±5c 68±9b 71±10b 85±5b

.05).

Fig. 1. Pressure inactivation curves of Escherichia coli O157:H7 and Salmonella spp.inoculated on radish, crimson clover, red clover and broccoli seeds treated at 600 MPaand 20 °C. Error bars represent±one standard deviation.

277H. Neetoo, H. Chen / International Journal of Food Microbiology 137 (2010) 274–280

possibly the cuticle) of alfalfa seeds constitutes a major moisturebarrier. Bhalla and Slattery (1984) demonstrated the progressivedeposition of callose, a plant polysaccharide at the plasmodesmata ofclover seeds particularly in the parenchyma layer of the seed coat,thus increasing the impermeability of seeds to water uptake. Wahlen(1929) also showed that the longevity of clover seeds depended onthe relative impermeability of their seed coat. McCormac and Keefe(1990) showed that the intact seed coat (testa) of cauliflower seedswere capable of acting as an effective barrier to water influx, thusacting as a protection mechanism for the embryo. Dixon (2007) notedthat broccoli morphologically follows similar developmental patternsto cauliflower, hence pointing to the high degree of structural andanatomical similarity between both types of cruciferous seeds. Hence,it is possible that alfalfa, clover and broccoli seeds with relativelyimpervious coats only imbibe minimal amount of water during theshort time frame of pressurization (b 5 min); thus allowing seeds toremain in a physiologically dormant stage. On the contrary, seeds withmore permeable seed coats readily imbibe water. Hayward (1948)described the outer surface of radish seed coats as relatively more“pitted”, possibly rendering the seed coats more permeable towater and hence more susceptible to the effects of high pressure. AsSimon (1984) mentioned, water uptake allows seeds to hydrate andadvance into a metabolically active phase. As a result, enzymes andother molecules critical for the development of the embryo may beactivated and more likely to be denatured under the effects of highpressure.

3.2. Pressure inactivation curve for E. coli O157:H7 and Salmonella spp.on crimson clover, red clover, radish and broccoli seeds

The pressure inactivation curves for E. coli O157:H7 and Salmo-nella for each type of sprouting seed are shown in Fig. 1. For bothpathogens, there was a direct relationship between the extent ofbacterial inactivation and pressure exposure time; population reduc-tions increased with increasing holding time. After a pressuretreatment time of 15 min at 600 MPa at 20 °C, the reductions forSalmonella spp. were 1.9, 2.1, 2.6 and 3.6 log CFU/g for crimson clover,radish, red clover and broccoli seeds, respectively. Processing underthe same conditions achieved 2.5, 2.9, 3.0 and 2.5 log CFU/g reductionin the population of E. coli O157:H7 on the four seeds, respectively. E.coli O157:H7 displayed significantly (Pb0.05) higher pressureresistance than Salmonella in the case of broccoli seeds. Howeveroverall, Salmonella spp. was more baro-tolerant than E. coli O157:H7in crimson clover, red clover and radish seeds although the differencewas not statistically significant (PN0.05). In the current study, acomposite of five strains of Salmonella including S. Typhimurium DT104 were used to prepare a cocktail. This strain was included since ithas become the object of increasing concern as a result of its rapidunprecedented rapid spread through Britain and the United Statesover the past 10 to 15 years (Keene, 1999). Humphrey et al. (1997)stated that S. Typhimurium DT 104 exhibited higher resistance to heatcompared to other Salmonellae strains. It is possible that the Salmo-nellae strains used in our study including S. Typhimurium DT 104were also particularly highly piezotolerant. As far as the substrate isconcerned, there was no significant difference (PN0.05) in the baro-tolerance of E. coli O157:H7 inoculated on the different sproutingseeds. Unlike E. coli O157:H7, Salmonella was significantly (Pb0.05)more pressure sensitive on broccoli seeds compared to crimson cloverand radish seeds after a 15-min treatment.

Wuytack et al. (2003) previously pressure-treated garden cressseeds inoculated with seven different bacteria with an inoculum of107 CFU/ml at 300 MPa for 15 min at 20 °C. The authors observeddifferential pressure inactivation across bacterial species ranging from2–6 log CFU/g, with greater population reductions of Gram-negativebacteria than Gram-positive bacteria. The authors noted that gardencress seeds, being members of the Brassicaceae or Cruciferae family,

are known to produce isothiocyanates that have inherent antimicro-bial properties (Isshiki et al., 1992; Delaquis and Massa, 1995; Linet al., 2000a,b). In fact, both Wuytack et al. (2003) and Ogawa et al.(2000) agreed that Gram-negative bacteria can be sensitized to highpressure in the presence of allyl isothiocyanate (AIT). We thusspeculate that broccoli seeds, also members of the Brassicacea (orCruciferae) family, may also be producing isothiocyanates that maysimilarly sensitize Gram-negative bacteria such as Salmonella to highpressure. Cruciferous vegetables including broccoli, are rich sources ofsulfur-containing compounds called glucosinolates. Isothiocyanatesare biologically active breakdown products of glucosinolates pro-duced upon hydrolysis by the endogenous enzyme myrosinase.Several authors had previously identified various isothiocyanatederivatives from broccoli using high performance liquid chromatog-raphy methods (Betz and Fox, 1994) and the gas chromatography/mass spectrometry methods (Chiang et al., 1998; Jin et al., 1999).Moreover, Van Eylen et al. (2007, 2009) recently showed that highpressure could induce the conversion of glucosinolates to isothiocya-nates in broccoli. This might explain the higher reductions in thepopulations of Salmonella on broccoli seeds treated at 600 MPa for15 min at 20 °C compared to the other seeds. The fact that E. coliO157:H7 displayed comparatively higher resistance to the applicationof high pressure in the presence of putative isothiocyanates present inbroccoli seeds (compared to Salmonella) is corroborated by findingsof Delaquis and Sholberg (1997) who showed that the applicationof AIT decreased viable S. Typhimurium to a greater extent than E. coliO157:H7.

The inactivation curves for Salmonella and E. coli O157:H7 for alltested substrates exhibited a biphasic shape characterized by a rapid

Table 3Effect of soaking time prior to treatment at 600 MPa for 5 min at 20 °C on theinactivation of E. coli O157:H7 and Salmonella spp. on four sprouting seed types. Thepopulation of E. coli O157:H7 on crimson clover, red clover, broccoli and radish seedswere at an initial level of 5.5, 5.2, 5.3 and 5.4 log CFU/g respectively while thepopulation of Salmonella was 5.5, 5.2, 5.3 and 5.7 log CFU/g respectively.

Seed type Pathogens Soaking timeprior topressure treatment(min)

0 15 30 45 60

Crimson clover E. coli O157:H7 3.8±0.1 1.7±0.3 2/3 0/3 0/3†

Crimson clover Salmonella 4.4±0.3 NDδ NDδ 2/3 0/3Red clover E. coli O157:H7 4.5±0.4 2/3 2/3 1/3 0/3Red clover Salmonella 3.6±0.6 NDδ NDδ NDδ 0/3Broccoli E. coli O157:H7 3.7±0.2 1.7±0.3 0/3Broccoli Salmonella 3.8±0.2 NDδ 0/3Radish E. coli O157:H7 3.4±0.1 3/3 0/3†

Radish Salmonella 4.1±0.3 NDδ 0/3†

Data representing mean log survivors (CFU/g)±standard deviation or number ofsamples testing positive after enrichment out of a total of 3 trials.δ: ND = not done, since samples were tested positive for E. coli O157:H7 afterenrichment under those conditions.†: Inferred from data of Table 2 and/or Table 3 rather than experimentally determined.

278 H. Neetoo, H. Chen / International Journal of Food Microbiology 137 (2010) 274–280

initial drop followed by tailing caused by a diminishing inactivationrate. Patterson et al. (1995) reported that with the application ofhydrostatic pressure, the possibilities of surviving tail populationswere more likely. It is not uncommon to observe that a survival curveversus treatment time is concave with a rapid initial decrease in log ofsurvivors followed by a tailing effect, where there is essentially nofurther inactivation as treatment time increases. Such inactivationcurves have also been found with other species such as Vibrioparahaemolyticus, Listeria monocytogenes and Yersinia enterocolitica(Metrick et al., 1989; Earnshaw et al., 1995; Isaacs et al., 1995;Patterson et al., 1995; Chen and Hoover, 2004; Chen, 2007).

3.3. Optimizing the parameters for pre-soaking seeds required for thehigh pressure elimination of E. coli O157:H7 and Salmonella spp. andseed viability retention

Table 2 shows that treatment of 600 MPa for 2 min at 20 °Cwithout prior soaking yielded a reduction of 0.2–2.1 log CFU/g ofeither pathogen on the various seeds. No significant (PN0.05)reduction in the counts of E. coli O157:H7 and Salmonella with finalpopulations ranging from 4.9 to 5.3 log CFU/g were observed withcrimson clover, red clover, radish and broccoli seeds soaked insterile DI water for up to 180 minwithout pressure treatment (soakedcontrols).

However, high pressure preceded by a soaking step deliveredsignificantly (Pb0.05) greater inactivation of E. coli O157:H7. In fact, adirect relationship between the degree of pressure inactivation andsoaking time was observed. The same trend was noted with Salmo-nella; the longer the soaking time, the greater the degree of pressureinactivation (Pb0.05). This phenomenon was possibly because thelonger the time the seeds were left in contact with water, the morebacteria that migrated from the inner regions of the seed to thesuperficial areas of the seed coat (Charkowski et al., 2001), thusmaking them more vulnerable to pressure inactivation. We speculatethat longer soaking times allowed water to permeate deeper into thecracks and crevices of the seeds thereby raising their local wateractivity and hence enhancing the pressure inactivation of the cellstrapped in these spaces.

The presence of E. coli O157:H7 on crimson clover seeds soakedfor 60 min followed by high-pressure treatment of 2 min wasundetectable after enrichment; however, survivors of Salmonellawere still detected. When the pre-soaking step was extended to90 min followed by a 2-min pressure treatment, an initial load of a5 log CFU/g Salmonella was eliminated in the 2-g seed sample. In thecase of radish, broccoli and red clover seeds, soaking for 30, 60 and

Table 2Effect of soaking time prior to treatment at 600 MPa for 2 min at 20 °C on inactivation ofEscherichia coli O157:H7 and Salmonella spp. on four seed types. The population ofE. coliO157:H7 on crimson clover, red clover, broccoli and radish seeds were at an initiallevel of 5.3, 5.7, 5.3 and 5.7 log CFU/g respectively while the population of Salmonellawas 5.5, 5.4, 5.3 and 5.4 log CFU/g respectively.

Seed type Pathogens Soaking time prior to pressure treatment (min)

0 30 60 90 120 180

Crimson clover E. coli O157:H7 4.9±0.1 1.0±0.3 0/3 0/3 0/3 0/3Crimson clover Salmonella 5.1±0.3 NDδ 2/3 0/3 0/3 0/3Red clover E. coli O157:H7 5.5±0.2 3/3 2/3 0/3 0/3 0/3Red clover Salmonella 3.6±0.6 NDδ NDδ 0/3 0/3 0/3Broccoli E. coli O157:H7 4.1±0.3 1.1±0.3 0/3 0/3 0/3 0/3Broccoli Salmonella 3.7±0.2 NDδ 0/3 0/3 0/3 0/3Radish E. coli O157:H7 4.0±0.1 0/3 0/3 0/3 0/3 0/3Radish Salmonella 4.1±0.1 0/3 0/3 0/3 0/3 0/3

Data representing mean log survivors (CFU/g)±standard deviation or number ofsamples testing positive after enrichment out of a total of 3 trials.δ: ND = not done, since samples were tested positive for E. coli O157:H7 afterenrichment under those conditions.

90 min respectively followed by a high-pressure treatment for 2 minwas adequate for elimination of both pathogens.

Since the length of pre-soaking negatively impacts on the seeds'germination potential (Neetoo et al., 2009b), we investigatedwhethershortening the soaking time while extending the pressure holdingtime could still decontaminate seeds with similar efficacy. Results forthis study are presented in Table 3. Overall, an extension in thepressure exposure time at 600 MPa from 2 to 5 min reduced thesoaking time requirement to 30, 60 and 60 min for elimination of bothpathogens in a 2-g sample of broccoli, crimson and red clover seedsrespectively. It is thought that a longer pressure holding timeenhanced inactivation of the pathogens as water under high pressureis forced into the deep cracks and crevices of the seed coat allowingwater to reach bacteria hidden in sub-surface locations.

Table 4 shows the germination percentages achieved with thevarious seeds soaked and pressure-treated under the differentconditions. Seeds soaked followed by pressure treatment germinatedto varying extents although their initial (day 3) and final (day 8)germination percentages were lower than the untreated controlsacross the relevant seed types. Un-soaked pressure-treated seedsretained their viability to a greater extent than their soaked pressure-treated counterparts. Neetoo et al. (2009b) previously demonstratedthat the germinability of alfalfa seeds varied with soaking time; thelonger the soaking duration, the greater the severity of the treatmenton the seeds. We postulate that the longer the imbibition time inwater, the greater the extent to which seeds hydrate and becomephysiologically active and the more delicate and pressure sensitivethey become. Simon (1984) mentioned that seeds are usuallyquiescent and can be stored for months without harm. However,once they are supplied with water, they undergo a gain in freshweight and embark on a second different phase of activity marked byincreased physiological and metabolic reactions as the seeds prepareto germinate. In line with our observation, Blaszczak et al. (2007)compared the structural and physiological changes undergone in rawand sprouted pressure-treated chickpea seeds and observed thatpressure treatment was more deleterious to germinated than rawseeds.

In addition, we observed that seed species belonging to theCruciferae family (radish and broccoli) were more severely affectedby high pressure than those of the Leguminosae family (red andcrimson clover). Overall, the pressure resistance of pre-soaked seedswas in the order of red cloverNcrimson cloverNbroccoli≈radishseeds. We attribute this difference principally to the characteristics ofthe seed coat. Barton (1961) stated that the seed coat (testa) plays a

Table 4Effect of different soaking parameters prior to treatment at 600 MPa and 20 °C on germination of seeds and yield ratio

Seed type Stα STβ tχ % Germination on different days Yield ratio(min) (°C) (min)

3 4 5 6 7 8(w/w)

Crimson clover 60 20 5 22±7d 24±8d 26±10d 27±10d 28±10d 28±11d 2.4±0.2c

Crimson clover 60 4 5 72±7b 75±6b 75±6b 75±6b 75±6b 75±6b 6.5±0.6b

Crimson clover 90 20 2 2±1e 4±0e 6±1e 7±2e 7±2e 7±2e 0.5±0.0d

Crimson clover 90 4 2 52±6c 56±7c 56±7c 56±7c 56±7c 56±7c 5.3±0.3b

Crimson clover Control 99±1a 100±0a 100±0a 100±0a 100±0a 100±0a 12.1±0.2a

Red clover 60 20 5 76±4c 79±2c 86±3c 90±3bc 92±2bc 94±1b 8.2±0.2b

Red clover 60 4 5 91±5ab 94±3ab 95±3ab 95±2ab 95±2ab 95±2ab 14.3±0.1a

Red clover 90 20 2 8±3d 10±3d 11±2d 13±3d 14±3d 15±3d 2.3±0.2c

Red clover 90 4 2 84±5bc 88±2bc 88±3bc 88±3c 88±3c 88±3c 12.4±0.4a

Red clover Control 99±1a 100±0a 100±0a 100±0a 100±0a 100±0a 15.0±0.1a

Broccoli 30 20 5 0±0b 0±0b 0±0b 0±0b 0±0b 1±1b NDBroccoli 60 20 2 0±0b 0±0b 0±0b 0±0b 0±0b 0±0b NDBroccoli Control 98±1a 99±0a 99±0a 99±0a 99±0a 99±0a NDRadish 30 20 2 0±0b 0±0b 0±0b 0±0b 0±0b 0±0b NDRadish Control 98±0a 99±0a 99±0a 99±0a 99±0a 99±0a ND

α: St = soaking time, β: ST = soaking temperature, and χ: t=pressure holding time.Values in the same column within the same seed type followed by the same letter are not significantly different (PN0.05).ND = not done, since % germination of treated seeds was too low.

279H. Neetoo, H. Chen / International Journal of Food Microbiology 137 (2010) 274–280

critical role in the life and viability maintenance of seeds. We thussurmise that certain seeds (red and crimson clover seeds) respondbetter to high pressure in the soaked state than others (broccoli andradish seeds) by virtue of the unique characteristics of their seed coat.Rees (1911) provided evidence that waxy (hydrophobic) cuticlescommon in leguminous seeds such as alfalfa and clover seedsconstitute the most impermeable part of the seed coat in manyspecies and the thicker the cuticle is, the longer the imbibition timerequired to bring about swelling of seeds and the less severe thedamage is. Wahlen (1929) showed that in leguminous seeds suchas clover seeds, their longevity was critically dependent on theimperviousness of the seed coat. Overall, our study agrees well withour previous findings on alfalfa seeds, alluding to the generalconclusion that seeds belonging to the Leguminosae family exhibitedgreater viability retention after pre-soaking and pressure treatmentthan seeds belonging to the Brassicacea family. Overall, high-pressuretreatment on red clover seeds soaked for 60 min and pressure-treatedfor 5 min produced higher initial and final germination percentagesthan the 2-min pressure treatment following soaking for 90 min.It was also the most promising treatment of all, producing a finalgermination percentage of 94%.

Since red and crimson clover seeds produced more satisfactoryresults after soaking and high-pressure treatment compared to radishand broccoli seeds with respect to their germinability, we subse-quently determined whether the extent of germination could beenhanced by lowering the pre-soaking temperature. Results ofviability tests for red and crimson clover seeds pre-soaked for 60or 90 min at refrigeration temperature prior to pressure treatmentat 600 MPa for 5 and 2 min, respectively are included in Table 4.Pre-soaking at a lower temperature (4 °C) significantly (Pb0.05)enhanced the germinability of both clover seed types regardless ofthe pre-soaking duration. As Barton (1961) described, in general,the higher the temperature, the more rapid is the rate of dete-rioration of the seeds' germinability at a given moisture level.Conversely, the lower the temperature, the greater the seeds'tolerance for high moisture content. Barton (1961) also mentionedthat at a low to moderate moisture content, maintaining the tem-perature in the range of 5–10 °C, will extend the life of seedsbeyond that achieved under similar humidity conditions at ordinaryroom temperature. Overall, the treatments appeared to be morefeasible for red clover seeds than crimson clover seeds as theformer seed type achieved final germination percentages of 95 and88% after pre-soaking for 60 and 90 min respectively compared to75 and 56% for the latter. Moreover, soaking for 60 or 90 min at 4 °C

followed by HHP did not significantly (PN0.05) affect the yield ratioof red clover seeds (14.3 and 12.1, respectively) compared tocontrol untreated seeds (15.0). However, all other treatmentconditions significantly (Pb0.05) reduced the yield ratio of redand crimson clover seeds as shown in Table 4.

We then finally evaluated the efficacy of low temperature soakingof red clover seeds for 60 and 90 min followed by HPP at 600 MPa for 5and 2 min respectively to decontaminate red clover seeds challengedwith E. coli O157:H7 and Salmonella. Pre-soaking for 60 min at 4 °Cfollowed by high-pressure treatment for 5 min was able to eliminateboth pathogens; while pre-soaking for 90 min followed by a pressureexposure of 2 min resulted in detectable E. coli O157:H7 survivors inone out of three trials.

4. Conclusions

The results of our study demonstrated that the application of highhydrostatic pressure inactivated E. coli O157:H7 and Salmonellaon various leguminous and cruciferous seeds. We also showed thata pre-soaking step carried out at ambient temperature considerablyenhanced pressure inactivation of either enteric pathogen withvariable effects on the seed germinability. High-pressure treatmenton soaked seeds appeared more promising for leguminous thancruciferous seeds. Finally, we showed that pre-soaking seeds atrefrigeration temperature followed by high-pressure treatmentdecontaminated red clover seeds, achieving a final germinationpercentage of 95% with minimal impact on the sprout yield.

Acknowledgements

This study was supported by a start-up fund from the Departmentof Animal and Food Sciences at the University of Delaware.Wewish tothank Dr. Thompson Pizzolato at the University of Delaware for thehelpful discussion.

References

Andrews, W.H., Mislivec, P.B., Wilson, C.R., Bruce, V.R., Poelma, P.L., Gibson, R., 1982.Microbial hazards associated with bean sprouting. Journal of the Association ofOfficial Analytical Chemists 65, 241–248.

Barton, L.V., 1961. Seed preservation and longevity. Interscience Publishers, Inc., NewYork, U.S.A., pp. 14–28, 30–39.

Betz, J.M., Fox, W.D., 1994. High-performance liquid chromatographic determination ofglucosinolates in Brassica vegetables. ACS symposium series, vol. 546, pp. 181–196.

280 H. Neetoo, H. Chen / International Journal of Food Microbiology 137 (2010) 274–280

Beuchat, L.R., 1997. Comparison of chemical treatments to kill Salmonella on alfalfaseeds destined for sprout production. International Journal of Food Microbiology34, 329–333.

Bhalla, P.L., Slattery, H.D., 1984. Callose deposits make clover seeds impermeable towater. Annals of Botany 53, 125–128.

Blaszczak, W., Doblado, R., Frias, J., Vidal Valverde, C., Sadowska, J., Fornal, J., 2007.Microstructural and biochemical changes in raw and germinated cowpea seedsupon high-pressure treatment. Food Research International 40, 415–423.

Brooks, J.T., Rowe, S.Y., Shillam, P., 2001. Salmonella Typhimurium infections transmittedby chlorine-pretreated clover sprout seeds. American Journal of Epidemiology 154,1020–1028.

Buck, J.W., Walcott, R.R., Beuchat, L.R., 2003. Recent trends in microbiological safety offruits andvegetables. PlantHealth Progress.bhttp://www.plantmanagementnetwork.orgN Accessed August 2009.

Charkowski, A.O., Sarreal, C.Z., Mandrell, R.E., 2001. Wrinkled alfalfa seeds harbor moreaerobic bacteria and are more difficult to sanitize than smooth seeds. Journal ofFood Protection 64, 1292–1298.

Chen, H., 2007. Use of linear, Weibull, and log–logistic functions to model pressureinactivation of seven foodborne pathogens inmilk. FoodMicrobiology 24, 197–204.

Chen, H.Q., Hoover, D.G., 2004. Use of Weibull model to describe and predict pressureinactivation of Listeria monocytogenes Scott A in whole milk. Innovative FoodScience and Emerging Technologies 5, 269–276.

Chiang, W.C.K., Pusateri, D.J., Leitz, R.E.A., 1998. Gas chromatography/mass spectrom-etry method for the determination of sulforaphane and sulforaphane nitrile inbroccoli. Journal of Agricultural and Food Chemistry 46, 1018–1021.

Delaquis, P.J., Massa, G., 1995. Antimicrobial properties of isothiocyanates in foodpreservation. Food Technology 11, 73–84.

Delaquis, P.J., Sholberg, P.L., 1997. Antimicrobial activity of gaseous allyl isothiocyanate.Journal of Food Protection 60, 943–947.

Dixon, G.R., 2007. Vegetable Brassicas and related crucifers. Crop Production Science inHorticulture Series. CABI Publisher, Cambridge, MA, pp. 91–113.

Earnshaw, R.G., Appleyard, J., Hurst, R.M., 1995. Understanding physical inactivationprocesses: combined preservation opportunities using heat, ultrasound andpressure. International Journal of Food Microbiology 28, 197–219.

Fett, W.F., 2006. Interventions to ensure themicrobial safety of sprouts. In: Sapers, G.M.,Gorny, J.R., Yousef, A.E. (Eds.), Microbiology of Fruits and Vegetables. Taylor andFrancis Group, LLC, Boca Raton, FL., pp. 187–210.

Hayward, H.E., 1948. The structure of economic plants. Macmillian Company, Chicago,U.S.A, pp. 309–339.

Humphrey, T.J., Wilde, S.J., Rowbury, R.J., 1997. Heat tolerance of Salmonella TyphimuriumDT104 isolates attached tomuscle tissue. Letters inAppliedMicrobiology25, 265–268.

Isaacs, N.S., Chilton, P., Mackey, B., 1995. Studies on the inactivation by pressure of micro-organisms. In: Ledward, D.A., Johnston, D.E., Earnshaw, R.G., Hasting, A.P.M. (Eds.),High Pressure Processing of Foods. Nottingham University Press, Nottingham,U.K., pp. 65–79.

Isshiki, K., Tokuora, K., Mori, R., Chiba, S., 1992. Preliminary examination of allylisothiocyanate vapor for food preservation. Bioscience, Biotechnology andBiochemistry 56, 1476–1477.

Jin, Y., Wang, M., Rosen, R.T., Ho, C.T., 1999. Thermal degradation of sulforaphane inaqueous solution. Journal of Agricultural and Food Chemistry 47, 3121–3123.

Keene, W.E., 1999. Lessons from investigations of foodborne disease outbreaks. Journalof the American Medical Association 281, 1845–1847.

Lin, C.M., Kim, J., Du, W., Wei, C., 2000a. Bactericidal activity of isothiocyanate againstpathogens on fresh produce. Journal of Food Protection 63, 25–30.

Lin, C.M., Preston, J.F., Wei, C., 2000b. Antibacterial mechanism of ally isothiocyanate.Journal of Food Protection 65, 383–388.

Lute, A.M., 1928. Impermeable seed of alfalfa. Colorado Agricultural Experimental StationBulletin, vol. 326.

McCormac, A.C., Keefe, P.D., 1990. Cauliflower (Brassica oleracea L.) seedvigour: imbibitioneffects. Journal of Experimental Botany 41, 893–899.

Metrick, C., Hoover, D.G., Farkas, D.F., 1989. Effects of high hydrostatic pressure on heat-resistant and heat-sensitive strains of Salmonella. Journal of Food Science 54,1547–1549.

Meyerowitz, S., 1999a. Sprouts: TheMiracle Food. Sproutman Publications,Massachusetts,U.S.A, pp. 57–79.

Meyerowitz, S., 1999b. My favorite greens. Better Nutrition 61, 18–21.NACMCF, 1999. Microbiological safety evaluations and recommendations on sprouted

seeds. International Journal of Food Microbiology 52, 123–153.Neetoo, H., Ye, M., Chen, H., 2008. Potential application of high hydrostatic pressure to

eliminate Escherichia coli O157:H7 on alfalfa sprouted seeds. International Journalof Food Microbiology 128, 348–353.

Neetoo, H., Pizzolato, T., Chen, H., 2009a. Elimination of Escherichia coli O157:H7 fromalfalfa seeds through a combination of high hydrostatic pressure and mild heat.Applied Environmental Microbiology 75, 1901–1907.

Neetoo, H., Ye, M., Chen, H., 2009b. Factors affecting the efficacy of pressure inactivationof Escherichia coli O157:H7 on alfalfa seeds and seed viability. International Journalof Food Microbiology 131, 218–223.

Ogawa, T., Nakatani, A., Matsuzaki, H., Isobe, S., Isshiki, K., 2000. Combined effects ofhydrostatic pressure, temperature and addition of allyl isothiocyanate oninactivation of Escherichia coli. Journal of Food Protection 63, 884–888.

Patterson, M.F., Quinn, M., Simpson, R., Gilmour, A., 1995. Effects of high pressure onvegetative pathogens. In: Ledward, D.A., Johnston, D.E., Earnshaw, R.G., Hasting,A.P.M. (Eds.), High Pressure Processing of Foods. Nottingham University Press,pp. 47–63.

Rajkowski, K.T., Thayer, D.W., 2001. Alfalfa seed germination and yield ratio and alfalfasprout microbial keeping quality following irradiation of seeds and sprouts. Journalof Food Protection 64, 1988–1995.

Rees, B., 1911. Longevity of seeds and structure and nature of seed coats. Proceedings.Royal Society of Victoria 23, 393–414.

Rolston, P., 1978. Water impermeable seed dormancy. Botanical Review 44, 365–389.Simon, E.W., 1984. Early events in germination. In: Murray, D.R. (Ed.), Seed Physiology,

Germination and reservemobilization, vol. 2. Academic Press, Australia, pp. 77–115.Taormina, P.J., Beuchat, L.R., Slutsker, L., 1999. Infections associated with eating seed

sprouts: an international concern. Emerging Infectious Diseases 5, 626–634.Thompson, S., Powell, D.A., 2000. Risks associated with the consumption of fresh

sprouts, Food Safety Network Technical Report # 16, http://www.plant.uoguelph.ca/safe-food/micro-haz/sprouts-risk-sylvanus-juloo.htm.

Tournas, V.H., 2001. Moulds and yeasts in fresh and minimally processed vegetables,and sprouts. International Journal of Food Microbiology 99, 618–622.

Van Eylen, D., Oey, I., Hendrickx, M., Van Loey, A., 2007. Kinetics of the stability ofbroccoli (Brassica oleracea cv. Italica) myrosinase and isothiocyanates in broccolijuice during pressure/temperature treatments. Journal of Agricultural and FoodChemistry 55, 2163–2170.

Van Eylen, D., Bellostas, N., Strobel, B.W., Oey, I., Hendrickx, M., Van Loey, A., Sørensen,H., Sørensen, J.C., 2009. Influence of pressure/temperature treatments onglucosinolate conversion in broccoli (Brassica oleraceae L. cv Italica) heads. FoodChemistry 112, 646–653.

Wahlen, F.T., 1929. Hard seededness and longevity in clover seeds. Proceedings of theInternational Seed Test Association 9–10, 34–39.

Waje, C.K., Han, D.H., Jo, C., Kwon, J.H., Jun, S.Y., Lee, Y.K., Kim, B.N., 2009. Microbialquality assessment and pathogen inactivation by electron beam and gammairradiation of commercial seed sprouts. Food Control 20, 200–204.

Wigmore, A., 1986. The Sprouting Book. Avery, Penguin Group, U.S.A, pp. 5–12.Wood, M., 2000. Safer Sprouts, vol. 48. Agricultural research, Washington, D.C, pp. 16–17.Wuytack, E.Y., Michiels, C.W., Meersseman, K., Diels, A.M., 2003. Decontamination of

seeds for seed sprout production by high hydrostatic pressure. Journal of FoodProtection 66, 918–923.