International Journal of Food Microbiology 128 (2008) 348–353

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International Journal of Food Microbiology

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Potential application of high hydrostatic pressure to eliminate Escherichia coli O157:H7 on alfalfa sprouted seeds

Hudaa Neetoo, Mu Ye, 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 © 2008 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2008.09.011

a b s t r a c t

a r t i c l e i n f o

Article history:

Sprouts eaten raw are incr Received 20 June 2008Received in revised form 9 September 2008Accepted 19 September 2008

Keywords:AlfalfaMung beanSeedsSproutsEscherichia coli O157:H7High hydrostatic pressure

easingly being perceived as hazardous foods as they have been implicated inEscherichia coli O157:H7 outbreaks where the seeds were found to be the likely source of contamination.The objective of our study was to evaluate the potential of using high hydrostatic pressure (HHP) technologyfor alfalfa seed decontamination. Alfalfa seeds inoculated with a cocktail of five strains of E. coli O157:H7were subjected to pressures of 500 and 600 MPa for 2 min at 20 °C in a dry or wet (immersed in water) state.Immersing seeds in water during pressurization considerably enhanced inactivation of E. coli O157:H7achieving reductions of 3.5 log and 5.7 log at 500 and 600 MPa, respectively. When dry seeds werepressurized, both pressure levels reduced the counts by b0.7 log. To test the efficacy of HHP to completelydecontaminate seeds whilst meeting the FDA requirement of 5 log reductions, seeds inoculated with a ~5 logCFU/g of E. coli O157:H7 were pressure-treated at 600 and 650 MPa at 20 °C for holding times of 2 to 20 min.A N5 log reduction in the population was achieved when 600 MPa was applied for durations of ≥6 minalthough survivors were still detected by enrichment. When the pressure was stepped up to 650 MPa, thethreshold time required to achieve complete elimination was 15 min. Un-inoculated seeds pressure-treatedat 650 MPa for 15 min at 20 °C successfully sprouted achieving a germination rate identical to untreatedseeds after eight days of sprouting. These results therefore demonstrate the promising application of HHP onalfalfa seeds to eliminate the risk of E. coli O157:H7 infections associated with consumption of raw alfalfasprouts.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

In 1994, the Food and Drug Administration (FDA) and the U.S.Department of Agriculture (USDA) recommended that Americansincrease their consumption of fresh fruits and vegetables (Hu et al.,2004). Fruit and vegetable consumption has since increased over thepast decade as a result of the heightened awareness of their healthbenefits, resulting in a significant growth in the fresh produce industry.However, the incidence of food-borne illness associated with theconsumption of salad vegetables and fruits have also increased. Anincrease in the consumption of raw alfalfa sprouts has been paralleledby an upsurge in the number of food-borne disease outbreaks in recentyears (Sharma et al., 2003). Seeds have been attributed to be the mainsource of contamination of sprouts, although pathogens may beintroduced at various stages of the spout production continuum (Utleeet al., 1998; NACMCF, 1999; Taormina and Beuchat, 1999a,b; Ogawaet al., 2000; Weissinger et al., 2001).

Sanitizing sprouted seeds presents a unique challenge in thearena of produce safety in that even a low residual pathogen po-

1 302 831 2822.

l rights reserved.

pulation remaining post-treatment appears capable of growing tovery high levels (up to 8 log CFU/g) due to favorable conditions ofmoisture, temperature and nutrient availability during seed germina-tion and subsequent sprout growth (Fett, 2006). In addition, after asanitizing procedure seed germination as well as spout yield andquality need to be maintained at commercially acceptable levels. In1999, based on research available at that time, the FDA recommendedthat seeds be disinfested by washing with 20,000 ppm calciumhypochlorite solution prior to sprouting (Rajkowski and Thayer, 2001).This treatment has been shown to only reach pathogenic microorgan-isms on the seed surface and there is still no guarantee that all con-tamination can be removed (Mundt and Hinkle, 1976; Taormina andBeuchat, 1999a,b). Due to the unreliability of the disinfection step, theFDA requires testing of spent irrigation water for the pathogens with-in 24 h after the start of the sprouting procedure (U.S. FDA, 1999;Waddell and Troxell, 2000).

Alternative methods thus need to be sought to ensure the safety ofsprout consumption without any detriment to their health-relatedqualities. High hydrostatic pressure (HHP) technology could be auseful tool to eliminate pathogenic microorganism like E. coli O157:H7in sprouted seeds. HHP acts instantaneously and uniformly through-out the mass of food independent of the size, shape or geometry(Farkas and Hoover, 2000). Previous research on the application of

349H. Neetoo et al. / International Journal of Food Microbiology 128 (2008) 348–353

HHP to decontaminate alfalfa seeds as carried out by Ariefdjohan et al.(2004) has shown that pressurizing seeds in the dry state greatlyimpaired the seed viability. On the other hand, Penas et al. (2008)soaked alfalfa seeds inwater for 3 h, decanted thewater and pressure-treated the seeds without water immersion and found a subsequentdecrease in the germination yield with respect to untreated seeds. Theoverall objective of our study was to develop a HHP process under-scoring the importance of immersing seeds in water during pres-sure treatment to completely eliminate a population of 105 CFU/g ofE. coli O157:H7 inoculated onto alfalfa seeds while retaining the seedsviability.

2. Materials and methods

2.1. Effect of pressure levels and water-immersion on the germinationrate of alfalfa and mung bean seeds

Alfalfa and mung bean seeds were obtained from InternationalSpecialty Supply (Cookeville, Tenn., USA). One hundred alfalfa ormungbean seeds were placed in individual plastic pouches (Nylon/Polyethylene, Koch Supplies, Kansas City, MO). Water (2 ml for alfalfaseeds and 6ml formung bean seeds)were added to half of the pouchesand those pouches were double heat-sealed. For seeds in the dry state,seeds were vacuum-packaged. To avoid leakage during pressure-treatment, each sample pouch was placed in a larger 8-mil thick PVCplastic pouch (Warp Bros., Chicago, IL, USA). HHP treatment of sam-ples was carried out using a high pressure unit with temperaturecontrol (Model Avure PT-1, Avure Technologies, Kent, WA). The ex-periments were conducted at 20 °C using water as a hydrostaticmedium. Pressurization was conducted at 200–600 MPa for 2 min.The temperature of the water-bath was monitored through a K-typethermocouple. The temperature and pressure data were recordedevery 2 s (DASYTEC USA, Bedford, NH). The pressure-come-up ratewas approximately 22 MPa/s. The pressure-release was almostimmediate (b4 s). Pressurization time reported in this study does notinclude the pressure come-up or release times. Temperature increaseduring pressure treatment due to adiabatic heatingwas 2.5 °C/100MPaat 20 °C (Chen et al., 2005). To determine the germination rate oftreated and un-treated seeds, seeds were soaked in DI-water for 3 hand 100 seeds were spread evenly between two pieces of wet papertowels on a plastic rack which in turn was placed into a water-filledbucket to provide a moist environment for the seeds. The water levelwasmaintained below the seeds' level. The bucketwas covered looselywith a piece of plastic film to allow exchange of air between the insideand outside of the bucket. The bucket was kept at room temperature(~21 °C) for 8 days (suggested by the seeds provider). The seeds werevisually evaluated for germination on days 3–8.

Table 1Effect of pressure treatment and seed wetness on the percentage germination rate of mung

Days ofgermination

Control State Pressure-treatment a

200 MPa

3 94.4±11.8a Dry 5.4±6.2b

Wet 77.7±3.5ac

4 99.2±2.0a Dry 5.4±6.2b

Wet 89.5±5.3ac

5 99.6±1.0a Dry 5.4±6.2b

Wet 91.9±4.0ac

6 99.6±1.0a Dry 5.4±6.2b

Wet 93.8±3.1a

7 99.8±0.5a Dry 5.4±6.2b

Wet 94.5±2.3a

8 100.0±0.0a Dry 5.4±6.2b

Wet 94.5±2.3a

Data are the means of three replicates±one standard deviation.Values for the same day of germination (dry or wet state) followed by the same letter are n

2.2. Effect of water-immersion of alfalfa seeds on pressure inactivation ofE. coli O157:H7

2.2.1. Bacterial strainsE. coli O157:H7 strain 1730 and Cider strain (Ibrahim et al., 2004),

strains 250 and 251 (Bhagwat et al., 2005) (Courtesy of Dr. Kniel,University of Delaware) and J58 (Barak et al., 2005) (Courtesy of DrJoerger, University of Delaware) were used. The cells of E. coli O157:H7were adapted to grow in tryptic soy Broth plus 0.6% yeast extract(Difco Laboratories, Sparks, MD, USA) supplemented with 50 µg/mlof nalidixic acid (Fisher Scientific, Hampton, NH, USA) (TSBYE-N). In-dividual cultures were grown in TSBYE-N overnight at 35 °C. Cultureswere then transferred (one loopful) into 10 ml of fresh TSBYE-N andincubated at 35 °C for 24 h. Equal volume of individual cultures wasmixed to form a five-strain cocktail of E. coli O157:H7. Preliminaryexperiments demonstrated that the five-strain wild-type cocktail andthe five strain nalidixic acid-resistant cocktail of E. coli O157:H7 hadcomparable pressure sensitivity (data not shown).

2.2.2. Inoculation of seedsThe cocktail (10 ml) were mixed with 100 ml of sterile 0.1%

peptone water (Fisher). Alfalfa seeds (100 g) was added to the cellsuspension and gently stirred for 5 min. The seeds were separatedfrom the cell suspension by pouring the mixture over a double layer ofcheesecloth supported by a wire screen and dried inside a bio-safetyhood at room temperature (21±1 °C) for 24 h. Dried seeds with anapproximate inoculation level of 109 CFU/g of E. coli O157:H7 wereplaced in sterile pouches and stored at 4 °C.

2.2.3. Pressure treatmentInoculated alfalfa seeds (2 g) were bagged and pressure-treated

either in thewet or dry state. Forwater-immersed seeds, 3ml of sterileDe-ionized (DI) water was added to the seeds. The seeds were treatedat pressures of 500 and 600 MPa for 2 min at 20 °C. The counts ofE. coli O157:H7 in pressure-treated dry and wet seed samples and twountreated controls were determined as described below. The twountreated controls were (i) dry seeds without pressure treatment and(ii) seeds immersed in 3 ml of water and washed under agitation for5 min without pressure treatment.

2.2.4. Microbiological analysisPouches containing treated seeds were cut open aseptically. The 2 g

seeds were poured into a stomacher bag to which 8 ml of sterile 0.1%peptone water was added and subsequently stomached for 2 min at260 rpm (Seward 400 Stomacher, Seward Medical Co., London, U.K.). Theseed slurry was serially diluted in sterile 0.1% peptone and surface-plated(100 μl) in duplicate on tryptic soy agar with 0.6% yeast extract (Difco

bean seeds

t various pressure levels

300 MPa 400 MPa 500 MPa 600 MPa

1.4±0.6b 6.9±9.2b 2.0±1.0b 4.9±1.6b

72.7±7.0c 76.2±4.0c 76.0±4.6c 72.4±2.6c

1.4±0.6b 8.6±12.2b 2.0±1.0b 4.9±1.6b

80.6±3.5c 84.2±3.0c 86.2±0.4ac 85.6±2.7ac

1.7±1.2b 9.7±14.0b 2.7±1.6b 4.9±1.6b

83.9±5.0c 90.0±2.8ac 88.5±1.8ac 86.6±1.7ac

1.7±1.2b 9.7±14.0b 2.7±1.6b 4.9±1.6b

84.9±5.0a 92.7±2.8a 91.1±1.2a 87.2±1.8a

1.7±1.2b 9.7±14.0b 2.7±1.6b 4.9±1.6b

85.2±5.1a 93.3±2.5a 91.8±1.7a 87.2±1.8a

1.7±1.2b 9.7±14.0b 2.7±1.6b 4.9±1.6b

86.2±4.4a 93.3±2.5a 92.4±1.7a 88.1±1.7a

ot significantly different (PN0.05).

Table 2Effect of pressure treatment and seed wetness on the percentage germination rate of alfalfa seeds

Days ofgermination

Control State Pressure-treatment at various pressure levels

200 MPa 300 MPa 400 MPa 500 MPa 600 MPa

3 94.4±11.8a Dry 61.0±17.6a 58.9±18.9a 53.2±15.1a 63.0±23.6a 47.1±12.8a

Wet 71.9±24.9a 71.4±17.9a 68.4±24.7a 71.8±22.7a 92.5±1.2a

4 99.2±2.0a Dry 71.4±13.3a 70.1±13.9a 64.1±13.0a 74.0±21.3a 65.9±14.2a

Wet 84.1±13.0a 80.0±11.6a 78.4±18.5a 83.9±17.7a 94.8±0.6a

5 99.6±1.0a Dry 75.6±12.8a 75.4±12.9a 71.8±13.5a 77.6±19.3a 75.7±13.5a

Wet 87.8±9.3a 85.9±7.0a 84.4±13.9a 90.8±11.6a 97.1±0.9a

6 99.6±1.0a Dry 80.7±13.2a 82.8±7.8a 77.5±11.2a 84.6±14.2a 83.8±11.1a

Wet 93.3±6.0a 89.7±7.0a 88.4±10.7a 94.0±7.1a 98.2±1.3a

7 99.8±0.5a Dry 84.2±15.7a 85.8±7.0a 82.3±10.2a 86.8±13.2a 87.3±10.0a

Wet 96.2±3.4a 93.4±8.0a 92.0±8.7a 95.5±5.3a 98.2±1.3a

8 100.0±0.0a Dry 85.4±15.4a 90.5±7.8a 87.5±6.9a 89.5±11.5a 90.5±7.9a

Wet 97.2±2.5a 94.7±7.4a 94.1±7.5a 96.5±4.7a 98.8±1.2a

Data are the means of three replicates±one standard deviation.Values for the same day of germination (dry or wet state) followed by the same letter are not significantly different (PN0.05).

350 H. Neetoo et al. / International Journal of Food Microbiology 128 (2008) 348–353

Laboratories, Sparks,MD, USA) supplementedwith nalidixic acid to afinalconcentration of 50 μg/ml (TSAYE-N). TSAYE-N plates were incubated at35 °C for 72h. Presumptive colonies ofE. coliO157:H7 formedon theplateswere enumerated. Occasionally, colonies were confirmed to be E. coliO157:H7 using either a BAX® system for screening/E. coli O157:H7 PCRassay (Qualicon-DuPont, Wilmington, DE, USA) or Rapid E. coli O157:H7Test Methods (Strategic Diagnostics Inc., Newark, DE, USA).

2.3. Effect of volume of water added to alfalfa seeds on baro-inactivationof E. coli O157:H7 and viability of pressure-treated seeds

Results from the above experiments demonstrated that addition ofwater to the seeds during pressure-treatment enhanced both bacterialinactivation and seed germination. Therefore, the volume of water requi-red during pressure treatment for optimum bacterial inactivation andsimultaneous seedviability retentionwasdetermined.Variousvolumes (1,2, 3, and 5 ml) of sterile DI water were added to 2 g of inoculated or un-inoculated seeds. The inoculated and un-inoculated seeds immersed inwater were pressure-treated at 600MPa for 2 min at 20 °C and subjectedto microbiological analysis and germination assay as described above.

2.4. Effect of pressure level and treatment time on inactivation of apopulation of ~105 CFU/g of E. coli O157:H7 inoculated on alfalfa seeds

In order to show the decontamination efficacy of HHP on seedsthat are typically subjected to a very low level of field-contaminationwhilst simultaneously demonstrate that the process couldmeet the FDArequirement (N5 log reduction), seeds were inoculated with a lowerinoculation level of ~105 CFU/g. To determine the condition required toachieve complete lethality, 2 g of inoculated seeds immersed in 3 ml ofsterile DI water were subjected to pressure treatment of 600 and650MPa at 20 °C for 2–20min. The 650MPawas theupper limit possiblefor the pressure unit. The counts of E. coli O157: H7 in the treated anduntreated seeds were determined as described above. The remainingseed slurries after plate count determination were enriched in 90 ml of

Table 3Effect of volume of water immersion during pressure treatment at 600MPa for 2min at 20 °C

Water Log Percentage germination rate at different d

Volume (ml) Reductions 3 4

0 −0.4 ± 0.2a 47.1±12.8a 65.9±14.2a

1 −2.3±0.1b 86.4±3.3b 91.8±3.2b

2 −4.3±0.2c 85.0±4.5b 90.7±4.1b

3 −4.4±0.4c 87.0±2.5b 91.6±3.2b

5 −4.4±0.3c 86.3±3.8b 91.6±2.9b

Data are the means of three replicates±one standard deviation.Values in the same column followed by the same letter are not significantly different (PN0.

TSBYE-N and incubated for 48 h at 35 °C to allow resuscitation of sub-lethally injured cells. Samples were streaked onto sorbitol MacConkeyagar (Difco Laboratories, Sparks, MD, U.S.A.) plates supplemented with50 µg/ml of nalidixic acid. After 24 h incubation, sorbitol-negative E. coliO157:H7 typified by pale or colorless colonies were identified on theplates (Hitchins et al., 1995). Colonies were occasionally subjected toconfirmation using the PCR or Rapid Test methods as described above.Since it was found that the treatment of 650MPa for 15min at 20 °Cwasable to completelyeliminate thepathogen, this treatmentwasapplied toun-inoculated seeds and the germination rate of the treated and un-treated seeds were determined.

2.5. Statistical analysis

All experiments were replicated at least three times. Where appro-priate, statistical analyses were conducted using Minitab® Release 15(Minitab Inc., University Park, PA, USA). One-way analysis of variance(ANOVA) and Tukey's one-waymultiple comparisons were used to deter-minedifferences in thepopulations ofE. coliO157:H7 recoveredon treatedalfalfa seeds as well as differences in the germination rate of seeds. Signi-ficant differences were considered at the 95% confidence level (Pb0.05).

3. Results

3.1. Effect of pressure levels and water-immersion on the germinationrate of alfalfa and mung bean seeds

Results are shown inTables1and2.Mungbeanseedshadconsiderablyhigher germination rates when they were immersed in water duringpressurization than when they were pressure-treated in the dry state(Pb0.05). The pressure-treated seeds achievedN85% germination acrossall pressure levels after 8 days of germination. The germination rate ofpressure-treated dry mung-bean seeds was severely impacted at allpressure levels and the best germination yield achieved was 9.7%. Foralfalfa seeds, the difference between untreated and treated wet seeds

on the baro-inactivation of E. coli O157:H7 and the percentage germination rate of seeds

ays

5 6 7 8

75.7±13.5a 83.8±11.1a 87.3±10.0a 90.5±7.9a

95.5±2.6a 97.8±3.0a 98.4±1.9a 99.1±1.6a

94.2±2.3a 97.2±1.7a 98.6±1.3a 99.2±0.8a

95.2±1.6a 96.6±1.4a 98.0±1.8a 98.5±1.4a

94.8±1.1a 96.5±0.4a 96.8±1.0a 96.8±1.0a

05).

Fig. 1. Survival curves of E. coli O157:H7 on alfalfa seeds subjected to pressuretreatments at 20 °C. Data are the means of three replicates. Error bars represent ±1standard deviation.

Fig. 2. Comparison of the germination rates of pressure-treated alfalfa seeds relative tocontrol (untreated) seeds.

351H. Neetoo et al. / International Journal of Food Microbiology 128 (2008) 348–353

across all pressure levels became smaller with increased germinationtime; and eventually disappeared with no significant difference (PN0.05)in thegermination capacity betweenun-treated alfalfa seeds (control) andpressure-treated wet seeds by the eighth day of germination. The ger-mination rates and final germination percentages of treated dry seedswere consistently lower than either the control or their immersed coun-terparts at corresponding pressure levels. It should be noted that treat-ment at pressure as high as 600 MPa did not have any adverse impact onthe germination capability and rate of pressure-treated wet seeds.

3.2. Effect of water-immersion of alfalfa seeds on pressure inactivation ofE. coli O157:H7

Washing the seeds for 5 min in water reduced the counts of E. coliO157:H7 by 0.3 log CFU/gwhilst immersing seeds inwater during pressu-rization considerably enhanced pressure inactivation of E. coli O157:H7.The pressure treatments reduced the counts of E. coli O157:H7 by 3.5 logand 5.7 log at 500 MPa and 600 MPa, respectively when seeds were im-mersed inwater during pressurization.When dry seedswere pressurized,both pressure treatments reduced the counts by ≤0.7 log10 CFU/g.

3.3. Effect of volume of water added to alfalfa seeds on baro-inactivationof E. coli O157:H7 and viability of pressure-treated seeds

Since immersion of seeds inwaterwas critical for baro-inactivationof E. coli O157:H7 and viability of seeds, it was logical to treat seeds at600 MPa immersed in varying volumes of water to determine theoptimumvolume required to immerse seeds. The pressure inactivationof E. coli O157:H7 at different volumes of immersion water are pre-sented in Table 3. When volume of water was increased, there was acommensurate increase in the log reductions of the pathogen with amaximum of 4.4 log at a water volume of 3 ml. Further addition ofwater to the immersed seeds did not enhance pressure inactivation.With respect to seed viability, seeds pressure-treatedwhilst immersedin the various volumes of water did not differ significantly (PN0.05) intheir germination percentages (Table 3).

3.4. Effect of pressure level and treatment time on inactivation of apopulation of ~105 CFU/g of E. coli O157:H7 inoculated on alfalfa seeds

The ability of pressure treatment to completely eliminate a popu-lation of 105 CFU/g of E. coli O157:H7 inoculated on alfalfa seeds wasinvestigated. While population reduction through HHP treatment isdesirable, it is more important to achieve complete elimination of thepathogen since even a few cells can grow explosively to as high as 7–8log CFU/g in the final sprouts during germination. Fig. 1 shows thesurvival curves for E. coli O157:H7 at 600 and 650 MPa for varyingexposure times. There was a close association between bacterialinactivation and treatment duration; population reductions increasedas a function of holding time. Amaximumaverage reduction of ~5.5 log

CFU/g was achieved when the treatment time was ≥6 min for the600 MPa treatments. However, none of the treatments at 600 MPawere adequate in completely eliminating the pathogen as survivorswere detected by enrichment. For pressure treatment at 650 MPa,treatment at ≥4 min was able to achieve a 5 log reduction of the pa-thogen although survivors were still detected by enrichmentwhen theholding time was ≤10 min. Complete elimination was consistentlyobserved for the 15min treatment. Un–inoculated seeds subjected to atreatment of 650 MPa for 15 min at 20 °C retained their germinationpotential with a final germination yield of 93%, which was identical tothe average germination yield of untreated seeds (Fig. 2).

4. Discussion

Increasing consumerdemand forminimally processed, additive-free,shelf-stable products has prompted food scientists to explore otherphysical preservation methods as alternatives to traditional heat treat-ments. The application of HHP as a physical decontamination methodmay bemore promising for use on alfalfa seeds since the use of chemicaldecontamination is difficult to reconcilewith the ‘‘health food’’ image ofseed sprouts (Wuytack et al., 2003). It has been suggested that thebottleneck to most seed decontamination techniques reside in theirinability to reach crevices or cracks where pathogens may be lodged orembryonic and endospermic tissues where they may become inter-nalized (Beuchat, 1996; NACMCF, 1999). However, HHP is thought topresent unique advantages due to the fact that it acts instantaneouslyand uniformly throughout a pressurized sample regardless of size,shape, and geometry (Farkas and Hoover, 2000). Hence, HHP could actuniformly at all sites within the seeds and on the seed surface therebytargeting the superficial as well as the internalized pathogens. Theapplication of HHP to decontaminate seeds from pathogenic micro-organisms such as Salmonella and E. coli O157 has been investigatedpreviously (Wuytack et al., 2003; Ariefdjohan et al., 2004; Penas et al.,2008) with varying degrees of efficacy. Our findings that alfalfa andmung bean seeds subjected to pressure treatment in the dry stategerminated poorly are in agreement with those of Ariefdjohan et al.(2004). They reported that HHP severely impacted the viability of alfalfaseeds pressurized in the dry statewith a significant correlation betweenthe pressure level used and the germination rate. They attributed thelow germination rate to the inability of the structure of dry alfalfa seedsto sustain pressures ranging from 275 MPa to 575 MPa. HHP on drymung bean seeds was more severe than on dry alfalfa seeds withgermination rate varying from1 to10% (Tables1 and2). Pressure-treateddry alfalfa seeds exhibited minimal visible signs of damage whilstdry mung bean seeds appeared to have undergone more extensivedamage. It is thought that the mung bean seed tissues have moreintercellular air spaces which were compressed considerably duringpressure treatment inducing physical damage to the seeds. When seedsare immersed in water, they typically retain their overall characteristicsbetter than when treated in the dry state. Indeed, Michel and Autio(2001) report that pressure acts instantaneously on a plant tissue at the

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tissue, cellular and molecular level. A major hurdle for high pressuretreatment of plant tissues is the presence of intercellular air spaces.Because of the high compressibility of air, the tissue is severely com-pressed, resulting in cell wall breakage (if the cell wall is not flexibleenough), membrane disruption, loss of compartmentalization, andliberation of cellular compounds. They recommended circumventingthis problem by filling the intercellular spaces with a liquid of lowcompressibility such as water (which is only compressible by 15% at600 MPa) before HHP treatment (Michel and Autio, 2001). When theseedswere immersed inwater, they probably absorbedwater from theirsurrounding as a result of osmosis and filled up the intercellular airspaces making them better able to withstand the external hydrostaticpressure.

With regard to pressure inactivation of E. coli O157:H7, the po-pulation reductions at 500 and 600 MPa were significantly (Pb0.05)correlated with increasing pressure level when seeds were pressurizedin the immersed state. In addition, the degree of inactivation wassignificantly higher when seeds were immersed in water than whenthey were pressurized in the dry state. Ariefdjohan et al. (2004) alsofound that pressure treatment was not effective against E. coli O157when inoculated alfalfa seeds were pressure treated in the dry state. Apressure treatment of 575 MPa for 2 min at approximately 30 °C onlyreduced the population of E. coliO157 in alfalfa seed by 1.4 log CFU/g. It isthought that water immersion of seeds enhances bacterial inactivationdue to the fact that the pathogens that were previously sequesteredin cracks, crevices or other “microenvironments” of seeds, or trappedbeneath the external coat become exposed at the surface as a result ofimbibition of water during pressure treatment. As a result, these cellswhich previously benefited from some form of physical protection arenow exposed at the surface or released into the surrounding waterrendering them more vulnerable to pressure inactivation. Moreover, alow water activity is known to confer protection to whole organismsagainst heat andpressure (Palou et al.,1999; Smelt et al., 2001). There arenumerous reports showing the protective effects of low water activityagainst pressure (Oxen and Knorr, 1993; Palou et al., 1997; Kingsley andChen, 2008). The lower reductions in the population in dry seeds asopposed to water-immersed seeds at both 500 and 600 MPa may bepartially attributed to the protective effect of the lower water activity inthe dry state of seeds.

Since water was found to be a limiting factor affecting both theextent of population reduction and the viability of seeds, the effect ofvarying the volume of water immersion during pressure treatmentwas investigated. As anticipated, the degree of bacterial inactivationincreased as a function of water availability up to a certain limit. This isprobably because with increased volume of immersion water, morepathogens are released onto the seed surface or into the surround-ing water with maximum release at an immersion volume of 3 ml.Charkowski et al. (2001) reported that bacteria inside seeds may beinternalized and are only released as the seeds imbibe water, swell andexpose previously sequestered regions of the seeds. As this happens, thepathogen might become more prone to inactivation or disruption byhigh pressure. However, unlike pathogen inactivation, the ability ofseeds to germinate after pressure-treatment at 600 MPa was not sig-nificantly different when water was added to varying ratios. Sincepressurization of dry seeds impaired their ability to germinate, we candeduce that for high viability retention of alfalfa seeds, presence ofwater during pressure treatment is much more critical than the actualvolume of water added. This compares well with the study undertakenby Wuytack et al. (2003) who showed that garden cress seeds werefound to achieve 100% germination rate when seeds (1 g) was pressure-treated with 0.25 ml of sterile DI water or a water to seed ratio of 1:4.Moreover, Penas et al. (2008) showed that HHP process lowered thegermination rates of alfalfa and mung-bean seeds when wet/soakedseeds were pressure-treated in the absence of water immersion. Hencein reconciling our findings with those of other authors, we can inferthat presence of water during pressure treatment was critical for seed

viability. Pressurization of soaked seeds without submerging them inwater is likely to cause more structural damage to the integrity of theseeds as a result of softening of their seed coat.

Whilst 600 MPa for 2 min was able to achieve a ~5 log reductionwith seeds inoculated with ~109 CFU/g, seeds inoculated with a lowerE. coli level (~105 CFU/g) underwent only 3.7 log reductions. Indeed,the extent of microbial inactivation achieved at a particular pressuretreatment depends on a number of interacting factors, includingthe initial number of organisms (Palou et al., 1999). It is obvious thatwith a high inoculation level, the pressure inactivation effectwasmoreappreciable, hence studieswith a realistic contamination levelwas alsoneeded. Pressure inactivation of E. coliO157:H7 at 600MPa for 2–6minwas correlated with treatment time with a maximum inactivation at6 min. However, survivors were detected by enrichment at treatmenttimes of N6 min such that even 20 min at 600 MPa could not result incomplete inactivation. Generally, an increase in pressure increasesmicrobial inactivation. However, increasing the process time does notnecessarily increase the lethal effect as noted by Palou et al. (1999).When seedswere treated at 650MPa, the shape of the survival curve ofthe pathogenwas very similar to the one at 600MPawith a rapid initialdrop in bacterial counts. Indeed, there have been many reports show-ing that the inactivation is not always linear (Earnshaw et al., 1995;Palou et al., 1999; Chen, 2007). It is actually not uncommon to find thatthe curve showing log of survivors versus treatment time is concavewith a rapid initial decrease in log of survivors followed by a tailingeffect, where there is essentially no further inactivation as treatmenttime increases. Such inactivation curves have also been found withother species such as Salmonella, Listeria and Yersinia enterocolitica(Metrick et al.,1989; Earnshaw et al.,1995; Isaacs et al.,1995; Pattersonet al., 1995; Chen and Hoover, 2003). These tailing effects have beenreported in thermal resistance studies, but the tails appear to bemore prominent with pressure treatment. It should be noted that thehighest temperatures reached during pressurization were approxi-mately 35 °C and 36 °C for the 600 and 650 MPa treatments, res-pectively. These relatively mild temperatures were not expected tocontribute to the lethality of the process.

E. coli O157:H7 is known to be a highly baro-resistant bacterium(Palou et al., 1999). However, a combination of factors may also com-pound its resistance. It is likely that after the seedswere inoculated andstored, the inoculated pathogen attached itself firmly to the seedsurfaces, underneath it or even permeated into the internal tissues viathe hilum or the micropyle of the seeds. E. coli O157:H7 is known toexpress multiple fimbrial and non-fimbrial adhesions which may beinvolved in adhesion to surfaces (Torres et al., 2005). For example curli-expressing thin aggregative fimbriae have been reported in E. coliO157:H7 to enable them to bind to inert and abiotic surfaces. E. coliO157:H7 may form biofilms on the seed surfaces which makes itmore resistant to processing conditions. Severe pressure treatmentswere therefore needed to eliminate this pathogen from seeds. In thisstudy, a treatment of 650MPa for 15min at 20 °C consistently achievedcomplete inactivation. This therefore demonstrates that excessivelyhigh pressure magnitude in conjunction with long holding time isrequired to achieve 100% lethality. However, this extreme processcondition did not have any deleterious effect on the seed germinationpotential demonstrating therefore that HHP can be a highly feasibletechnology to decontaminate seeds.

5. Conclusion

Decontamination of seeds prior to sprouting is critical in obtaininga safe product. Although several decontamination methods have beenproposed, these methods have proved to be unsatisfactory. Surfaceirregularities, physical damage (e.g. cracks, wrinkles, missing testaparts) to seeds and the presence of pathogen bacterial biofilms on seedsurfaces reduce the effectiveness of chemical sanitizers. The presenceof cracks and openings in the seed coat may also provide a preferential

353H. Neetoo et al. / International Journal of Food Microbiology 128 (2008) 348–353

pathway for entry of pathogens into the seed and may therefore en-hance the survival of pathogens inside the seed. Once pathogensare entrapped inside the seed, targeting them becomes extremelydifficult. In this study, HHP treatment of 650 MPa for 15 min at 20 °Cwas able to completely eliminate a N5 log of E. coli O157:H7 on alfalfaseeds without impairing the germinability of seeds. Since the ini-tial pathogen contamination levels in seeds are typically very low,b1 MPN/g seed (Inami et al., 2001; Stewart et al., 2001), a treatmentgiving a 5-log reduction should be adequate.

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