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

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

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Pre-soaking of seeds enhances pressure inactivation of E. coli O157:H7 and Salmonella 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

a r t i c l e

i n f o

Article history: Received 22 August 2009 Received in revised form 17 November 2009 Accepted 25 November 2009 Keywords: High pressure Seeds Salmonella Escherichia coli O157:H7

a b s t r a c t The application of high hydrostatic pressure (HHP) at a level of 600 MPa at 20 °C to decontaminate crimson clover, 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 on broccoli seeds where the trend was reversed. In addition, the application of HHP differentially affected seeds' germinability and the order of pressure tolerance of the seeds was such that red clover N crimson clover ≈ broccoli N radish seeds with final germination percentages ranging from 85–100% while their untreated counterparts had final germination percentages of 99–100%. Pre-soaking the different types of seeds in water for 30, 60 or 90 min at ambient temperature followed by HHP at 600 MPa for 2 or 5 min at 20 °C significantly (P b 0.05) enhanced the pressure inactivation of the inoculated pathogens. Moreover, the ability of HHP-treated seeds to germinate also varied as a function of the pre-soaking duration and the seed type. Pre-soaking radish and broccoli seeds for 30 min prior to HHP (2 or 5 min) resulted in germination percentages of ≤ 1% after 8 days of incubation. On the contrary, red clover seeds displayed higher germination potential when pre-soaked for 60 min at 20 °C prior to HPP (5 min) with final germination percentages of 94%, although their yield was substantially lower than their untreated counterparts. Red clover seeds pre-soaked for 60 min at 4 °C followed by HPP at 600 MPa for 5 min at 20 °C produced germination percentages of 91 and 95% after 3 and 8 days of sprouting compared to 99 and 100% respectively for untreated seeds. In addition, this condition did not significantly (P N 0.05) reduce the sprout yield. The treatment also resulted in a reduction of a 5 log initial load of E. coli O157:H7 and Salmonella to an undetectable level (neither pathogen was detected in 2-g seed samples after enrichment). © 2009 Elsevier B.V. All rights reserved.

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 and naturally occurring sources of vitamins, minerals, enzymes and amino acids known. Sprouts belonging to the Cruciferae family (broccoli and radish sprouts) are particularly renowned for their antioxidant and anticancerous properties (Meyerowitz, 1999a,b). Unfortunately, their rich nutritional content also makes them very supportive of bacterial growth (Thompson and Powell, 2000; Wood, 2000) including the growth of pathogens (Tournas, 2001; Buck et al., 2003). From 1973 to present, more than 40 outbreaks of foodborne illness have been reported worldwide incriminating raw seed sprouts (Waje et al., 2009). Salmonella spp., Escherichia coli O157:H7 and Bacillus cereus were among the pathogens involved (Fett, 2006). The most

⁎ Corresponding author. Tel.: +1 302 831 1045; fax: +1 302 831 2822. E-mail address: [email protected] (H. Chen). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.11.026

prevalent pathogenic microorganisms are enteric pathogens such as Salmonella and E. coli O157:H7 (NACMCF, 1999). Although sprouts can become contaminated during the sprouting, harvesting, packaging and distribution processes, many studies demonstrated outbreaks linked to contaminated seeds (Andrews et al., 1982). Among various seed sprouts implicated in outbreaks were alfalfa (NACMCF, 1999; Taormina et al., 1999), radish (Taormina et al., 1999) and clover sprouts (Taormina et al., 1999; Brooks et al., 2001). The United States Food and Drug Administration thus recommends the treatment of sprouting seeds with 20,000 ppm of free chlorine from calcium hypochlorite or an equivalent antimicrobial (NACMCF, 1999). However, the use of chlorine-based chemicals generates hazardous fumes that have created public health concerns (Beuchat, 1997). As a result, alternative seed decontamination methods have been sought. Researchers studying the efficacy of different chemical, physical and biological methods for reducing or eliminating populations of bacterial pathogens artificially contaminated onto seeds have reported varying degrees of success (Fett, 2006). In our previous studies (Neetoo et al., 2008, 2009a,b), we reported that the application of high pressure either alone or in combination with mild heat was able to successfully inactivate E. coli O157:H7 on

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alfalfa seeds while maintaining seed viability. The objective of this study was to determine the effect of pre-soaking and high-pressure treatment on inactivation of E. coli O157:H7 and Salmonella spp. inoculated on crimson clover, red clover, radish and broccoli seeds and on their germination ability. To simplify sentence construction, the term “elimination” was used in this paper interchangeably with the phrase “reduction of a 5 log initial load of E. coli O157:H7 or Salmonella to an undetectable level (pathogens were not detected in 2-g seed samples after enrichment)”. 2. Materials and methods 2.1. Effect of pressure treatment on the extent of germination of crimson clover, red clover, radish and broccoli seeds Unscarified radish (Raphanus sativus), broccoli (Brassica oleracea var. italica), crimson clover (Trifolium incarnatum) and red clover (Trifolium pratense) seeds were purchased from International Specialty Supply (Cookeville, TN, USA). Two grams of each type of seed were placed in individual 3-mil-thick nylon/polyethylene pouches (Koch Supplies, Kansas City, MO, USA). Deionized (DI) water (3 ml for crimson clover, red clover and broccoli seeds and 4 ml for radish seeds) was added to the pouches and those pouches were heat-sealed. Pressure treatment of samples was carried out using a high-pressure unit with temperature control (Model Avure PT-1, Avure Technologies, Kent, WA, USA). Pressurization was conducted at 600 MPa for 2 min at 20 °C (initial seed sample temperature prior to pressure treatment) using water as a hydrostatic medium. The temperature of the waterbath was monitored with a K-type thermocouple. Temperature and pressure data were recorded every 2 s (DASYTEC USA, Bedford, NH, USA). The pressure-come-up rate was approximately 22 MPa/s. The pressure-release was almost immediate (b 4 s). Pressurization time reported in this study does not include the pressure-come-up or release times. To determine the germination percentage of treated and untreated seeds, seeds were soaked in DI water for 3 h and 100 seeds were spread evenly on layers of wet paper towels on a plastic rack which in turn was placed into a water-filled bucket to provide a moist environment for the seeds. The water level was maintained below the seeds' level. The bucket was covered loosely with a piece of plastic film to allow exchange of air between the inside and outside of the bucket. The bucket was kept at room temperature (∼ 21 °C) for 8 days (suggested by the seeds provider). The seeds were visually evaluated for germination and sprouted seeds were counted after 3, 4, 5, 6, 7 and 8 days and discarded on each day following enumeration. The cumulative germination percentage reached on each day was then determined as the proportion of total number of sprouted seeds to the total number of seeds. 2.2. Determination of the pressure inactivation curves of E. coli O157:H7 and Salmonella spp. inoculated on crimson clover, red clover, radish and broccoli seeds 2.2.1. Test strains and preparation of inoculum A cocktail of five different E. coli O157:H7 strains (250, 251, Cider, 1730 and J58) (Neetoo et al., 2008) and a cocktail of five Salmonella enterica strains (Typhimurium T43, Typhimurium T45, Typhimurium DT 104, Enteritidis E44 and Montevideo Mo57) (Courtesy of Dr. Rolf Joerger, University of Delaware) were used in this study. Cells of E. coli O157:H7 and Salmonella were adapted to grow in tryptic soy broth plus 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). Individual cultures were grown in TSBYE-N for 16–18 h at 35 °C. Cultures were then transferred (one loopful) into 10 ml of fresh TSBYE-N and incubated at 35 °C for 24 h. Equal volumes of individual cultures were

275

mixed to form a five-strain cocktail of Salmonella and a five-strain cocktail of E. coli O157:H7. 2.2.2. Inoculation of seeds A hundred-fold dilution of the five-strain culture cocktails was made in sterile 0.1% peptone water (Fisher). Five hundred grams of each 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 were separated from the cell suspension by pouring the mixture over a double layer of cheesecloth supported by a wire screen and dried inside a biosafety hood at room temperature (21 °C ± 1 °C) for 48 h with intermittent rotation to ensure that seeds were uniformly dry. The mean water activity (aw) values of radish, broccoli, crimson clover and red clover seeds after drying were ca. 0.74, 0.44, 0.52 and 0.67, respectively. Seeds with an approximate inoculation level of 105 CFU/g of E. coli O157:H7 and Salmonella were placed in plastic pouches and stored at 4 °C. 2.2.3. Pressure treatment and microbiological analysis Two grams of inoculated seeds mixed with 3 ml (for crimson clover, red clover and broccoli seeds) or 4 ml (for radish seeds) of sterile DI water were added into a nylon/polyethylene pouch. To avoid leakage during pressure treatment, each sample pouch was placed in a larger pouch of an 8-mil-thick polyvinyl chloride plastic (McMasterCarr, Elmhurst, IL, USA) and heat-sealed. Pressure treatment was carried out at 20 °C at 600 MPa and a treatment time of 2, 4, 6, 8, 10 or 15 min. Pouches containing treated seeds were cut open aseptically. The sample was transferred into a stomacher bag to which 8 ml of sterile 0.1% peptone water was added and subsequently stomached for 2 min at 260 rpm (Seward 400 Stomacher; Seward Medical Co., London, UK). The seed slurry was serially diluted in sterile 0.1% peptone water and surface plated in duplicate on tryptic soy agar with 0.6% yeast extract (Difco) supplemented with nalidixic acid to a final concentration of 50 μg/ml (TSAYE-N). TSAYE-N plates were incubated for 3 days at 35 °C. 2.3. Optimizing the parameters for pre-soaking of seeds required for safety and viability retention after pressure treatment 2.3.1. Effect of the pre-soaking duration on the pressure inactivation of E. coli O157:H7 and Salmonella spp. Two grams of seeds inoculated with E. coli O157:H7 or Salmonella spp. 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 end of the soaking period, the excess water was subsequently decanted and seeds were placed into a pouch in the presence of 3 ml (clover and broccoli seeds) or 4 ml (radish seeds) of fresh sterile DI water and pressure-treated at 600 MPa for 2 min at 20 °C. Samples were then microbiologically assayed on TSAYE-N as described previously. When the bacterial population was below the detection limit for the plating method (b 0.8 log CFU/g), the seed slurry was further enriched in 90 ml of TSBYE-N and incubated for 48 h at 35 °C to allow resuscitation of sub-lethally injured cells. Samples were streaked onto Sorbitol MacConkey agar (Difco) plates supplemented with 50 μg/ml of nalidixic acid for samples inoculated with E. coli O157:H7 or xylose lysine deoxycholate (XLD) agar (Difco) plates supplemented with 50 mg/ml of nalidixic acid for samples inoculated with Salmonella. After 24 h incubation, presence of colorless or faint orange growth typical of E. coli O157:H7 and black-centered or black colonies characteristic 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 red clover seeds were soaked for a maximum of 60 min. Treated seed samples were then microbiologically analyzed as described above.

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Pre-soaked and pressure-treated (2 min) seed samples for which no pathogens were detected in a 2-g sample after enrichment, were not further tested under the extended pressure exposure of 5 min. In addition, treated samples without detectable pathogens after enrichment after a particular soaking period and pressure treatment of 5 min, were not further tested under longer soaking times. 2.3.2. Effect of selected soaking and pressure treatment conditions on the germination percentages of crimson clover, red clover, radish and broccoli seeds To determine the effect of selected soaking and pressure treatment parameters on the seeds' germination potential, two grams of uninoculated seeds were soaked in 20 ml DI water for 30, 60 or 90 min at 4 and/or 20 °C (conditions depending on the seed type). At the end of the soaking period, the excess water was subsequently decanted and seeds were placed into a pouch in the presence of 3 ml of fresh DI water (or 4 ml for radish seeds) and pressure-treated at 600 MPa and 20 °C for 2 or 5 min. Untreated (control) and pressure-treated seeds were then soaked in DI water after pressure treatment for a total soaking time of 3 h. One hundred seeds were drawn from the samples and set to germinate as described previously. The cumulative percentage of germinated seeds was determined after 3, 4, 5, 6, 7 and 8 days from the onset of germination. To determine whether the selected soaking conditions and pressure treatments could eliminate the two pathogens in red clover seeds, 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 °C or (ii) soaked at 4 °C for 90 min and treated at 600 MPa for 2 min at 20 °C. Treated seeds were then assayed post-treatment for the presence of E. coli O157:H7 or Salmonella survivors as previously described. 2.3.3. Effect of selected soaking and pressure treatment conditions on the yield ratio of red clover and crimson clover seeds Red and crimson clover seeds (2 g) were either soaked at 4 °C or 20 °C for 60 min and treated at 600 MPa for 5 min at 20 °C or soaked at 4 °C or 20 °C for 90 min and treated at 600 MPa for 2 min at 20 °C. One hundred seeds from each sample were then randomly picked and allowed to germinate. After 8 days of growth, sprouts were weighed and the yield ratio was calculated by dividing the weight of sprouted seeds by the weight of one hundred dry seeds, a method adapted from Rajkowski and Thayer (2001). 2.4. Statistical analysis All experiments were replicated three times. Where appropriate, statistical analyses were conducted using Minitab release 15 (Minitab Inc., University Park, PA, USA). One-way analysis of variance and Tukey's one-way multiple comparisons were used to determine differences in the populations of E. coli O157:H7 and Salmonella recovered on treated sprouting seeds and differences in the germina-

tion percentages and sprout yield ratio. Differences were considered statistically significant at the 95% confidence level (P b 0.05). 3. Results and discussion 3.1. Effect of high pressure on the extent of germination of crimson clover, red clover, radish, and broccoli seeds The germination percentages for the four different types of seeds after high-pressure treatment are summarized in Table 1. Seed germination response was differentially affected by high pressure and the order of pressure tolerance was such that red clover N crimson clover ≈ broccoli N radish seeds. The germination percentage determined 3–8 days after sprouting was initially lower, but not significantly lower for red clover seeds at all germination times and some treatment and germination times for the other seed types. Red clover seeds exhibited the highest pressure resistance achieving an average seed germination of 97% (after 3 days) compared with 99% for untreated seeds; showing therefore that there was almost no delay in the onset of sprouting. Crimson clover and broccoli seeds were slightly more affected by pressure since they sprouted to ∼85% after 3 days and achieved N90% germination after 4 days while control untreated seeds germinated to 99 % after 3 days. This indicates that the onset of sprouting was slightly delayed. The germination of radish seeds appeared the most desynchronized with an average of 14% after 3 days of germination. By the end of 8 days, the cumulative germination percentages of red and crimson clover and broccoli seeds were 100, 94 and 96% respectively while radish seeds which were the most severely affected, reached a final germination percentage of 85. The pressure resistance of red clover seeds was very similar to those 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 that red clover seeds and alfalfa seeds represent two very popular species of the same large pea family, which may contribute to their similar degree of pressure resistance. The germination results of radish seeds are also in agreement with those of Wuytack et al. (2003). They compared the germination percentages of garden cress, mustard, radish and sesame seeds subjected to levels of high pressure ranging from 250–400 MPa and noted that radish and mustard seeds were highly sensitive to pressure with germination percentages b 10% after 11 days of germination. It is not clear however what mechanism is responsible for the differential pressure sensitivity across the various plant species studied but it is likely due to the intrinsic uniqueness of the embryos of seeds themselves. It could also be due to the specific structural and anatomical characteristics of the seed coat itself. Seed coat acts as an important moisture barrier in certain seeds (Rolston, 1978) such as alfalfa, clover and broccoli seeds. Alfalfa seeds tend to have a relatively impermeable seed coat that effectively slows down water uptake. Lute (1928) showed that the thickened outer wall of palisade cells (and

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

Crimson clover Crimson clover Red clover Red clover Broccoli Broccoli Radish Radish

Treatment

Control HHP Control HHP Control HHP Control HHP

% Germination on different days 3

4

5

6

7

8

99 ± 1a 87 ± 3b 99 ± 1a 97 ± 2a 98 ± 1a 85 ± 3b 98 ± 0a 14 ± 6c

100 ± 0a 91 ± 4b 100 ± 0a 99 ± 1a 99 ± 0a 93 ± 1b 99 ± 0a 31 ± 5c

100 ± 0a 93 ± 3b 100 ± 0a 100 ± 0a 99 ± 0a 94 ± 1b 99 ± 0a 55 ± 5c

100 ± 0a 93 ± 3a 100 ± 0a 100 ± 0a 99 ± 0a 94 ± 1a 99 ± 0a 68 ± 9b

100 ± 0a 93 ± 3a 100 ± 0a 100 ± 0a 99 ± 0a 95 ± 2a 99 ± 0a 71 ± 10b

100 ± 0a 94 ± 2a 100 ± 0a 100 ± 0a 99 ± 0a 96 ± 1a 99 ± 0a 85 ± 5b

Values in the same column followed by the same letter are not significantly different (P N 0.05).

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possibly the cuticle) of alfalfa seeds constitutes a major moisture barrier. Bhalla and Slattery (1984) demonstrated the progressive deposition of callose, a plant polysaccharide at the plasmodesmata of clover 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 on the relative impermeability of their seed coat. McCormac and Keefe (1990) showed that the intact seed coat (testa) of cauliflower seeds were capable of acting as an effective barrier to water influx, thus acting as a protection mechanism for the embryo. Dixon (2007) noted that broccoli morphologically follows similar developmental patterns to cauliflower, hence pointing to the high degree of structural and anatomical similarity between both types of cruciferous seeds. Hence, it is possible that alfalfa, clover and broccoli seeds with relatively impervious coats only imbibe minimal amount of water during the short time frame of pressurization (b 5 min); thus allowing seeds to remain in a physiologically dormant stage. On the contrary, seeds with more 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 to water and hence more susceptible to the effects of high pressure. As Simon (1984) mentioned, water uptake allows seeds to hydrate and advance into a metabolically active phase. As a result, enzymes and other molecules critical for the development of the embryo may be activated and more likely to be denatured under the effects of high pressure. 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 Salmonella for each type of sprouting seed are shown in Fig. 1. For both pathogens, there was a direct relationship between the extent of bacterial inactivation and pressure exposure time; population reductions increased with increasing holding time. After a pressure treatment time of 15 min at 600 MPa at 20 °C, the reductions for Salmonella 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 under the same conditions achieved 2.5, 2.9, 3.0 and 2.5 log CFU/g reduction in the population of E. coli O157:H7 on the four seeds, respectively. E. coli O157:H7 displayed significantly (P b 0.05) higher pressure resistance than Salmonella in the case of broccoli seeds. However overall, Salmonella spp. was more baro-tolerant than E. coli O157:H7 in crimson clover, red clover and radish seeds although the difference was not statistically significant (P N 0.05). In the current study, a composite of five strains of Salmonella including S. Typhimurium DT 104 were used to prepare a cocktail. This strain was included since it has become the object of increasing concern as a result of its rapid unprecedented rapid spread through Britain and the United States over the past 10 to 15 years (Keene, 1999). Humphrey et al. (1997) stated that S. Typhimurium DT 104 exhibited higher resistance to heat compared to other Salmonellae strains. It is possible that the Salmonellae strains used in our study including S. Typhimurium DT 104 were also particularly highly piezotolerant. As far as the substrate is concerned, there was no significant difference (P N 0.05) in the barotolerance of E. coli O157:H7 inoculated on the different sprouting seeds. Unlike E. coli O157:H7, Salmonella was significantly (P b 0.05) more pressure sensitive on broccoli seeds compared to crimson clover and radish seeds after a 15-min treatment. Wuytack et al. (2003) previously pressure-treated garden cress seeds inoculated with seven different bacteria with an inoculum of 107 CFU/ml at 300 MPa for 15 min at 20 °C. The authors observed differential pressure inactivation across bacterial species ranging from 2–6 log CFU/g, with greater population reductions of Gram-negative bacteria than Gram-positive bacteria. The authors noted that garden cress seeds, being members of the Brassicaceae or Cruciferae family,

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 MPa and 20 °C. Error bars represent ± one standard deviation.

are known to produce isothiocyanates that have inherent antimicrobial properties (Isshiki et al., 1992; Delaquis and Massa, 1995; Lin et al., 2000a,b). In fact, both Wuytack et al. (2003) and Ogawa et al. (2000) agreed that Gram-negative bacteria can be sensitized to high pressure in the presence of allyl isothiocyanate (AIT). We thus speculate that broccoli seeds, also members of the Brassicacea (or Cruciferae) family, may also be producing isothiocyanates that may similarly sensitize Gram-negative bacteria such as Salmonella to high pressure. Cruciferous vegetables including broccoli, are rich sources of sulfur-containing compounds called glucosinolates. Isothiocyanates are biologically active breakdown products of glucosinolates produced upon hydrolysis by the endogenous enzyme myrosinase. Several authors had previously identified various isothiocyanate derivatives from broccoli using high performance liquid chromatography 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 high pressure could induce the conversion of glucosinolates to isothiocyanates in broccoli. This might explain the higher reductions in the populations of Salmonella on broccoli seeds treated at 600 MPa for 15 min at 20 °C compared to the other seeds. The fact that E. coli O157:H7 displayed comparatively higher resistance to the application of high pressure in the presence of putative isothiocyanates present in broccoli seeds (compared to Salmonella) is corroborated by findings of Delaquis and Sholberg (1997) who showed that the application of AIT decreased viable S. Typhimurium to a greater extent than E. coli O157:H7. The inactivation curves for Salmonella and E. coli O157:H7 for all tested substrates exhibited a biphasic shape characterized by a rapid

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initial drop followed by tailing caused by a diminishing inactivation rate. Patterson et al. (1995) reported that with the application of hydrostatic pressure, the possibilities of surviving tail populations were more likely. It is not uncommon to observe that a survival curve versus treatment time is concave with a rapid initial decrease in log of survivors followed by a tailing effect, where there is essentially no further inactivation as treatment time increases. Such inactivation curves have also been found with other species such as Vibrio parahaemolyticus, 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 the high pressure elimination of E. coli O157:H7 and Salmonella spp. and seed viability retention Table 2 shows that treatment of 600 MPa for 2 min at 20 °C without prior soaking yielded a reduction of 0.2–2.1 log CFU/g of either pathogen on the various seeds. No significant (P N 0.05) reduction in the counts of E. coli O157:H7 and Salmonella with final populations ranging from 4.9 to 5.3 log CFU/g were observed with crimson clover, red clover, radish and broccoli seeds soaked in sterile DI water for up to 180 min without pressure treatment (soaked controls). However, high pressure preceded by a soaking step delivered significantly (P b 0.05) greater inactivation of E. coli O157:H7. In fact, a direct relationship between the degree of pressure inactivation and soaking time was observed. The same trend was noted with Salmonella; the longer the soaking time, the greater the degree of pressure inactivation (P b 0.05). This phenomenon was possibly because the longer the time the seeds were left in contact with water, the more bacteria that migrated from the inner regions of the seed to the superficial areas of the seed coat (Charkowski et al., 2001), thus making them more vulnerable to pressure inactivation. We speculate that longer soaking times allowed water to permeate deeper into the cracks and crevices of the seeds thereby raising their local water activity and hence enhancing the pressure inactivation of the cells trapped in these spaces. The presence of E. coli O157:H7 on crimson clover seeds soaked for 60 min followed by high-pressure treatment of 2 min was undetectable after enrichment; however, survivors of Salmonella were still detected. When the pre-soaking step was extended to 90 min followed by a 2-min pressure treatment, an initial load of a 5 log CFU/g Salmonella was eliminated in the 2-g seed sample. In the case of radish, broccoli and red clover seeds, soaking for 30, 60 and

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

Crimson clover Crimson clover Red clover Red clover Broccoli Broccoli Radish Radish

Pathogens

E. coli O157:H7 Salmonella E. coli O157:H7 Salmonella E. coli O157:H7 Salmonella E. coli O157:H7 Salmonella

Soaking time prior to pressure treatment (min) 0

30

4.9 ± 0.1 5.1 ± 0.3 5.5 ± 0.2 3.6 ± 0.6 4.1 ± 0.3 3.7 ± 0.2 4.0 ± 0.1 4.1 ± 0.1

1.0 ± 0.3 NDδ 3/3 NDδ 1.1 ± 0.3 NDδ 0/3 0/3

60

90

120

180

0/3 2/3 2/3 NDδ 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3

Data representing mean log survivors (CFU/g) ± standard deviation or number of samples 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 after enrichment under those conditions.

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

Crimson clover Crimson clover Red clover Red clover Broccoli Broccoli Radish Radish

Pathogens

E. coli O157:H7 Salmonella E. coli O157:H7 Salmonella E. coli O157:H7 Salmonella E. coli O157:H7 Salmonella

Soaking time prior to pressure treatment (min) 0

15

3.8 ± 0.1 4.4 ± 0.3 4.5 ± 0.4 3.6 ± 0.6 3.7 ± 0.2 3.8 ± 0.2 3.4 ± 0.1 4.1 ± 0.3

1.7 ± 0.3 NDδ 2/3 NDδ 1.7 ± 0.3 NDδ 3/3 NDδ

30

45

60

2/3 NDδ 2/3 NDδ 0/3 0/3 0/3† 0/3†

0/3 2/3 1/3 NDδ

0/3† 0/3 0/3 0/3

Data representing mean log survivors (CFU/g) ± standard deviation or number of samples 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 after enrichment under those conditions. † : Inferred from data of Table 2 and/or Table 3 rather than experimentally determined.

90 min respectively followed by a high-pressure treatment for 2 min was adequate for elimination of both pathogens. Since the length of pre-soaking negatively impacts on the seeds' germination potential (Neetoo et al., 2009b), we investigated whether shortening the soaking time while extending the pressure holding time could still decontaminate seeds with similar efficacy. Results for this study are presented in Table 3. Overall, an extension in the pressure exposure time at 600 MPa from 2 to 5 min reduced the soaking time requirement to 30, 60 and 60 min for elimination of both pathogens in a 2-g sample of broccoli, crimson and red clover seeds respectively. It is thought that a longer pressure holding time enhanced inactivation of the pathogens as water under high pressure is forced into the deep cracks and crevices of the seed coat allowing water to reach bacteria hidden in sub-surface locations. Table 4 shows the germination percentages achieved with the various seeds soaked and pressure-treated under the different conditions. Seeds soaked followed by pressure treatment germinated to varying extents although their initial (day 3) and final (day 8) germination percentages were lower than the untreated controls across the relevant seed types. Un-soaked pressure-treated seeds retained their viability to a greater extent than their soaked pressuretreated counterparts. Neetoo et al. (2009b) previously demonstrated that the germinability of alfalfa seeds varied with soaking time; the longer the soaking duration, the greater the severity of the treatment on the seeds. We postulate that the longer the imbibition time in water, the greater the extent to which seeds hydrate and become physiologically active and the more delicate and pressure sensitive they become. Simon (1984) mentioned that seeds are usually quiescent and can be stored for months without harm. However, once they are supplied with water, they undergo a gain in fresh weight and embark on a second different phase of activity marked by increased physiological and metabolic reactions as the seeds prepare to germinate. In line with our observation, Blaszczak et al. (2007) compared the structural and physiological changes undergone in raw and sprouted pressure-treated chickpea seeds and observed that pressure treatment was more deleterious to germinated than raw seeds. In addition, we observed that seed species belonging to the Cruciferae family (radish and broccoli) were more severely affected by high pressure than those of the Leguminosae family (red and crimson clover). Overall, the pressure resistance of pre-soaked seeds was in the order of red clover N crimson clover N broccoli ≈ radish seeds. We attribute this difference principally to the characteristics of the seed coat. Barton (1961) stated that the seed coat (testa) plays a

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279

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

Crimson clover Crimson clover Crimson clover Crimson clover Crimson clover Red clover Red clover Red clover Red clover Red clover Broccoli Broccoli Broccoli Radish Radish

Stα (min)

STβ (°C)

tχ (min)

60 60 90 90 Control 60 60 90 90 Control 30 60 Control 30 Control

20 4 20 4

5 5 2 2

20 4 20 4

5 5 2 2

20 20

5 2

20

2

% Germination on different days 3

4

5

6

7

8

22 ± 7d 72 ± 7b 2 ± 1e 52 ± 6c 99 ± 1a 76 ± 4c 91 ± 5ab 8 ± 3d 84 ± 5bc 99 ± 1a 0 ± 0b 0 ± 0b 98 ± 1a 0 ± 0b 98 ± 0a

24 ± 8d 75 ± 6b 4 ± 0e 56 ± 7c 100 ± 0a 79 ± 2c 94 ± 3ab 10 ± 3d 88 ± 2bc 100 ± 0a 0 ± 0b 0 ± 0b 99 ± 0a 0 ± 0b 99 ± 0a

26 ± 10d 75 ± 6b 6 ± 1e 56 ± 7c 100 ± 0a 86 ± 3c 95 ± 3ab 11 ± 2d 88 ± 3bc 100 ± 0a 0 ± 0b 0 ± 0b 99 ± 0a 0 ± 0b 99 ± 0a

27 ± 10d 75 ± 6b 7 ± 2e 56 ± 7c 100 ± 0a 90 ± 3bc 95 ± 2ab 13 ± 3d 88 ± 3c 100 ± 0a 0 ± 0b 0 ± 0b 99 ± 0a 0 ± 0b 99 ± 0a

28 ± 10d 75 ± 6b 7 ± 2e 56 ± 7c 100 ± 0a 92 ± 2bc 95 ± 2ab 14 ± 3d 88 ± 3c 100 ± 0a 0 ± 0b 0 ± 0b 99 ± 0a 0 ± 0b 99 ± 0a

28 ± 11d 75 ± 6b 7 ± 2e 56 ± 7c 100 ± 0a 94 ± 1b 95 ± 2ab 15 ± 3d 88 ± 3c 100 ± 0a 1 ± 1b 0 ± 0b 99 ± 0a 0 ± 0b 99 ± 0a

Yield ratio (w/w) 2.4 ± 0.2c 6.5 ± 0.6b 0.5 ± 0.0d 5.3 ± 0.3b 12.1 ± 0.2a 8.2 ± 0.2b 14.3 ± 0.1a 2.3 ± 0.2c 12.4 ± 0.4a 15.0 ± 0.1a ND ND ND ND 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 (P N 0.05). ND = not done, since % germination of treated seeds was too low.

critical role in the life and viability maintenance of seeds. We thus surmise that certain seeds (red and crimson clover seeds) respond better to high pressure in the soaked state than others (broccoli and radish seeds) by virtue of the unique characteristics of their seed coat. Rees (1911) provided evidence that waxy (hydrophobic) cuticles common in leguminous seeds such as alfalfa and clover seeds constitute the most impermeable part of the seed coat in many species and the thicker the cuticle is, the longer the imbibition time required to bring about swelling of seeds and the less severe the damage is. Wahlen (1929) showed that in leguminous seeds such as clover seeds, their longevity was critically dependent on the imperviousness of the seed coat. Overall, our study agrees well with our previous findings on alfalfa seeds, alluding to the general conclusion that seeds belonging to the Leguminosae family exhibited greater viability retention after pre-soaking and pressure treatment than seeds belonging to the Brassicacea family. Overall, high-pressure treatment on red clover seeds soaked for 60 min and pressure-treated for 5 min produced higher initial and final germination percentages than the 2-min pressure treatment following soaking for 90 min. It was also the most promising treatment of all, producing a final germination percentage of 94%. Since red and crimson clover seeds produced more satisfactory results after soaking and high-pressure treatment compared to radish and broccoli seeds with respect to their germinability, we subsequently determined whether the extent of germination could be enhanced by lowering the pre-soaking temperature. Results of viability tests for red and crimson clover seeds pre-soaked for 60 or 90 min at refrigeration temperature prior to pressure treatment at 600 MPa for 5 and 2 min, respectively are included in Table 4. Pre-soaking at a lower temperature (4 °C) significantly (P b 0.05) enhanced the germinability of both clover seed types regardless of the pre-soaking duration. As Barton (1961) described, in general, the higher the temperature, the more rapid is the rate of deterioration 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 mentioned that at a low to moderate moisture content, maintaining the temperature in the range of 5–10 °C, will extend the life of seeds beyond that achieved under similar humidity conditions at ordinary room temperature. Overall, the treatments appeared to be more feasible for red clover seeds than crimson clover seeds as the former seed type achieved final germination percentages of 95 and 88% after pre-soaking for 60 and 90 min respectively compared to 75 and 56% for the latter. Moreover, soaking for 60 or 90 min at 4 °C

followed by HHP did not significantly (P N 0.05) affect the yield ratio of red clover seeds (14.3 and 12.1, respectively) compared to control untreated seeds (15.0). However, all other treatment conditions significantly (P b 0.05) reduced the yield ratio of red and crimson clover seeds as shown in Table 4. We then finally evaluated the efficacy of low temperature soaking of red clover seeds for 60 and 90 min followed by HPP at 600 MPa for 5 and 2 min respectively to decontaminate red clover seeds challenged with E. coli O157:H7 and Salmonella. Pre-soaking for 60 min at 4 °C followed by high-pressure treatment for 5 min was able to eliminate both pathogens; while pre-soaking for 90 min followed by a pressure exposure of 2 min resulted in detectable E. coli O157:H7 survivors in one out of three trials. 4. Conclusions The results of our study demonstrated that the application of high hydrostatic pressure inactivated E. coli O157:H7 and Salmonella on various leguminous and cruciferous seeds. We also showed that a pre-soaking step carried out at ambient temperature considerably enhanced pressure inactivation of either enteric pathogen with variable effects on the seed germinability. High-pressure treatment on soaked seeds appeared more promising for leguminous than cruciferous seeds. Finally, we showed that pre-soaking seeds at refrigeration temperature followed by high-pressure treatment decontaminated red clover seeds, achieving a final germination percentage of 95% with minimal impact on the sprout yield. Acknowledgements This study was supported by a start-up fund from the Department of Animal and Food Sciences at the University of Delaware. We wish to thank Dr. Thompson Pizzolato at the University of Delaware for the helpful 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 of Official Analytical Chemists 65, 241–248. Barton, L.V., 1961. Seed preservation and longevity. Interscience Publishers, Inc., New York, U.S.A., pp. 14–28, 30–39. Betz, J.M., Fox, W.D., 1994. High-performance liquid chromatographic determination of glucosinolates in Brassica vegetables. ACS symposium series, vol. 546, pp. 181–196.

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