Factors affecting the efficacy of pressure inactivation of Escherichia coli O157:H7 on alfalfa seeds and seed viability

International Journal of Food Microbiology 131 (2009) 218–223

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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

Factors affecting the efficacy of pressure inactivation of Escherichia coli O157:H7 on alfalfa seeds and seed viability Hudaa Neetoo, Mu Ye, Haiqiang Chen ⁎ Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716-2150, USA

a r t i c l e

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Article history: Received 7 December 2008 Received in revised form 19 February 2009 Accepted 28 February 2009 Keywords: Pressure Alfalfa Seeds Escherichia coli O157:H7 Germination

a b s t r a c t The application of high hydrostatic pressure technology as a seed decontamination technology was evaluated. Alfalfa seeds inoculated with approximately 105 CFU/g of Escherichia coli O157:H7 were subjected to oscillatory pressure treatments at 600 MPa and 20 °C for up to five cycles with a holding time of 2 min/ cycle. However, oscillatory pressurization was not able to eliminate E. coli O157:H7. The application of pressure treatment at 600 MPa for 2 min at 20 °C in the presence of chemicals such as calcium hypochlorite, calcium hydroxide, lactic acid or sodium acid sulfate was subsequently investigated and it was demonstrated that this “multiple hurdle” approach was unable to decontaminate alfalfa seeds. Soaking seeds prior to pressure treatment was found to play a critical role on enhancing the pressure inactivation of E. coli O157:H7; seeds soaked in water for 60 min followed by treatment at 600 MPa for 2 min at 20 °C were decontaminated and had a germination rate of 91% which was 4% lower than that of the untreated seeds (not statistically significant, P > 0.05). It was further demonstrated that a process involving soaking seeds in water for ≥ 10 min followed by treatment at 600 MPa for 15 min at 20 °C was equally effective with respect to E. coli O157:H7 elimination and viability retention of the seeds. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Numerous outbreaks of food-borne diseases linked to consumption of fresh fruits and vegetables clearly point to the fact that these commodities constitute a serious hazard to public health. Seed sprouts are regarded by consumers as a health food because they are low in fat and calories, high in fiber and are rich in anti-cholesterolemic and anti-carcinogenic constituents (Kurtzweil, 1999). However, sprouted seeds continue to be implicated in outbreaks of food-borne illness and hence are considered a significant food safety risk (Anonymous, 1999; Kumar et al., 2006). Since 1995, there have been three outbreaks associated with the consumption of alfalfa and clover sprouts contaminated with E. coli O157 in the United States (Fett, 2002; Ferguson et al., 2005). In the majority of the illness outbreaks, the seeds used for sprout production were proven to be the most likely source of human pathogens (Anonymous, 1999). Alfalfa sprouts are typically eaten raw and therefore intervention treatments must be either applied to the seeds or to the germinated sprouts. Since sprout-directed treatments could be more problematic because of the delicate nature of the sprouts, more research attention has been focused on alfalfa seed decontamination as opposed to treatment on alfalfa sprouts (Lang et al., 2000). In response to the urgent need to curtail sprout-related outbreaks and to protect public safety, the

⁎ 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.2009.02.028

U.S. Food and Drug Administration and the State of California are strongly recommending the treatment of alfalfa seeds with a solution of 20,000 ppm free chlorine from calcium hypochlorite. This treatment is currently considered the “benchmark” and is also recommended by the National Advisory Committee on Microbiological Criteria for Foods (Anonymous, 1999). However, it has been shown that this method may not be robust enough for seed decontamination. It reduces, but does not eliminate pathogens on seeds and consequently outbreaks continue to occur (Brooks et al., 2001; Proctor et al., 2001). As a result, extensive research is currently ongoing to investigate the ability of various physical, chemical and biological preventive strategies to decontaminate seeds from foodborne pathogens. High hydrostatic pressure (HHP) processing is a non-thermal food preservation technology. One of the most important effects of HHP processing is its ability to destroy or reduces the number of foodborne pathogens with minimum impact on food sensory and functional qualities. Previous researchers investigating the application of HHP (Ariefdjohan et al., 2004; Wuytack et al., 2003) to decontaminate alfalfa and mung bean seeds have reported varying degrees of success due to the inability of high pressure to eliminate pathogenic microorganisms from seeds with minimal impact on the seeds germinability. Since these seeds are destined for sprouting, retention of their seed viability is critical to ensure that pressure-treated seeds can consistently meet the yield anticipated by sprouters. A previous study in our laboratory demonstrated that continuous high pressure treatment at 600 MPa for 20 min at 20 °C achieved a ~ 5 log reduction but could not eliminate E. coli O157:H7 (105 CFU/g) on alfalfa seeds

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(Neetoo et al., 2008). Therefore the overall aim of this study was to develop a HHP process to decontaminate alfalfa seeds inoculated with a ~5 log CFU/g of E. coli O157:H7 with minimal adverse impact on the seeds germination potential. The specific objectives were to determine the effect of (1) oscillatory HHP treatments, (2) the combined effect of HHP with antimicrobial agents and (3) the application of HHP on presoaked seeds for varying holding times on seed decontamination and viability. 2. Materials and methods 2.1. Effect of oscillatory pressurization on the inactivation of E. coli O157: H7 on alfalfa seeds 2.1.1. Bacterial strains E. coli O157:H7 strains 1730, 250, 251, Cider and J58, adapted to grow in tryptic soy broth plus 0.6% yeast extract (Difco Laboratories, Sparks, MD, USA) supplemented with 50 µg/ml of nalidixic acid (Fisher Scientific, Hampton, NH, USA) (TSBYE-N) were used. The fivestrain cocktail of E. coli O157:H7 was prepared as described by Neetoo et al. (2008). 2.1.2. Inoculation of seeds The cocktail (10 ml) was mixed with 100 ml of sterile 0.1% peptone water (Fisher). Unscarified and undamaged alfalfa seeds (100 g), obtained from International Specialty Supply (Cookeville, Tenn., USA), were added to the cell suspension (21 ± 1 °C) 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 bio-safety hood at room temperature (21 ± 1 °C) for 24 h to a final aw of 0.622. Dried seeds with an approximate inoculation level of 105 CFU/g of E. coli O157:H7 were placed in sterile pouches and stored at 4 °C until needed. 2.1.3. Pressure treatment Two g of inoculated seeds and 3 ml of sterile DI water were placed in a 3-mil thick pouch (Nylon/Polyethylene, Koch Supplies, Kansas City, MO). To avoid leakage during pressure-treatment, each sample pouch was placed in a larger pouch of an 8-mil thick PVC plastic (McMaster–Carr, Elmhurst, IL) and heat-sealed. HHP treatment of samples was carried out using a high-pressure unit with temperature control (Model Avure PT-1, Avure Technologies, Kent, WA). All the experiments were conducted at 20 °C (initial seed sample temperature prior to pressure treatment) using water as a hydrostatic medium. The temperature of the water-bath surrounding the pressure chamber during pressurization was monitored through K-type thermocouples. The temperature and pressure data were recorded every 2 s (DASYTEC USA, Bedford, NH). The pressure-come-up rate was approximately 22 MPa/s. The pressure-release was < 4 s. Pressurization time reported in this study did not include the pressure come-up or release times. Oscillatory pressurization was performed by alternating atmospheric pressure (0.1 MPa) and high pressure (600 MPa) with 1, 2, 3, 4 or 5 cycles and a holding time of 2 min/cycle. 2.1.4. Microbiological analysis Pouches containing treated seeds were cut open aseptically. The seed mixture consisting of the seeds and immersion water was poured 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, U.K.). The seed slurry was serially diluted in sterile 0.1% peptone and surface-plated in duplicate on tryptic soy agar with 0.6% yeast extract (Difco Laboratories, Sparks, MD, USA) supplemented with nalidixic acid to a final concentration of 50 µg/ml (TSAYE-N). TSAYE-N plates were incubated at 35 °C for 3 days. Presumptive colonies of E. coli O157:H7 formed on the plates were enumerated. One or two colonies were picked at an initial stage

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in the study and confirmed for E. coli O157:H7 identification using either a BAX® system for screening E. coli O157:H7 PCR assay (QualiconDuPont, Wilmington, DE, USA) or Rapid E. coli O157:H7 Test Methods (Strategic Diagnostics Inc., Newark, DE, USA). 2.2. Combined effect of HHP and antimicrobial treatments on inactivation of E. coli O157:H7 on alfalfa seeds The combined application of HHP and antimicrobial treatments on seed decontamination was investigated. Inoculated seeds were either (a) soaked in antimicrobial solutions for varying durations and then subjected to a 2 min-pressure treatment or (b) directly pressuretreated in antimicrobial solutions (without prior soaking) for extended exposure times. In the first approach, 2 g of inoculated seeds were soaked in 20 ml of DI-water (control), calcium hypochlorite (Acros Organics, N.J.) solutions providing approximately 200, 1000, 2000, or 20,000 ppm of free chlorine as determined using chlorine test strips (Fisher), 1% calcium hydroxide (Reheis Inc., Midlothian, TX) with or without 1% Tween 80 (Fisher), lactic acid (1, 2 or 5%) (Fisher), or sodium acid sulfate (0.05, 0.10 or 0.20%) (Jones-Hamilton, Walbridge, OH) for 5, 10 or 15 min at room temperature (21 ± 1 °C). The concentration unit was defined as %w/v. The calcium hypochlorite solutions were prepared in a sterile 0.05 M potassium phosphate buffer solution with pH of 6.8. After soaking, the excess water or chemical solutions was decanted and the seeds were placed into a sterile plastic pouch followed by the addition of 3 ml of the respective treatment solutions and treated at 600 MPa for 2 min at 20 °C. In the second approach, 2 g of the inoculated seeds were placed into sterile pouches and pressure-treated at 600 MPa and 20 °C whilst submerged in 3 ml of various antimicrobial solutions for varying holding times (5, 10 or 15 min). The antimicrobial solutions included 2000 and 20,000 ppm calcium hypochlorite, 1% calcium hydroxide with or without 1% Tween 80 and 1, 2 and 5% lactic acid. Pouches containing treated seeds were cut open aseptically. Seed samples were rinsed with 100 ml of sterile DI-water to remove any residues of the chemical solutions and then poured into a stomacher bag to which 8 ml of sterile 0.1% peptone water was added and subsequently stomached for 2 min. The seed slurry was directly 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 Laboratories, Sparks, MD, U.S.A.) plates supplemented with 50 µg/ml of nalidixic acid. After 24 h incubation, presence of growth exhibiting morphological and biochemical characteristics typical of E. coli O157: H7 were determined by visually inspecting the plates. Colonies were confirmed to be E. coli O157:H7 using Rapid E. coli O157:H7 Test Methods (Strategic Diagnostics Inc., Newark, DE, USA). 2.3. Effect of soaking time in water prior to pressure treatment on inactivation of E. coli O157:H7 on alfalfa seeds and seed viability Two g of inoculated seeds was soaked in sterile DI-water for 0 (without soaking), 30, 60, 90, 120, 180, 240 or 300 min prior to being treated at 600 MPa for 2 min at 20 °C. Seeds were soaked either in a limiting volume (3 ml) or in a large excess (20 ml) of water to determine whether the soaking time as well as the volume of water available were critical factors in the pressure inactivation of E. coli O157: H7 on seeds. For seeds soaked in 3 ml of water, the 2 g inoculated seeds and 3 ml water were directly placed into a pouch and soaked for varying durations before being subjected to the pressure treatment. For seeds soaked in 20 ml of water, the excess water was subsequently decanted and seeds were placed into a pouch in the presence of 3 ml of fresh sterile DI water and pressure-treated. Samples were then microbiologically assayed post-treatment as described previously. Soaked controls were additionally set up by immersing inoculated seeds in 20 ml of sterile DI water for 30, 60, 90, 120, 180, 240 or 300 min before determining the remaining populations of E. coli O157:H7 on the seeds.

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To determine the effect of soaking duration prior to pressure treatment on the seeds germination potential, 2 g of un-inoculated seeds were soaked in 20 ml of water for 0, 30, 60, 90, 120 or 180 min. The water was then decanted and the seeds introduced into a pouch to which 3 ml of DI-water was added. The samples were treated at 600 MPa for 2 min at 20 °C. After HHP, pressure-treated seeds and untreated seeds (control) were soaked in DI-water for a total soaking time of 3 h (including the soaking times before and after the pressure treatment). One hundred seeds were drawn from the soaked seeds and spread evenly on pieces 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 kept at room temperature (21 ±1 °C) for 8 days (as suggested by the seeds provider) and misted daily. 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 seeds were visually evaluated for sprouting 3–8 days after setting up the germination system. To determine the rate of water absorption by seeds, 2 g of uninoculated seeds were immersed in 20 ml of water at room temperature. Soaked seeds were weighed and their weight gain recorded at regular time intervals for up to 19 h. 2.4. Effect of reduced soaking time and extended pressure holding time on the inactivation of E. coli O157:H7 and seed viability Two grams of inoculated seeds were soaked in 20 ml of water for 10 or 15 min. The water was later decanted and seeds mixed with 3 ml of sterile DI-water, packaged and treated at 600 MPa and 20 °C for 5, 10 or 15 min. Samples were then microbiologically assayed as described previously and enriched for the detection of survivors. For the determination of seed germination, 2 g of un-inoculated seeds were soaked for 10 or 15 min and treated at 600 MPa for 15 min at 20 °C. One hundred seeds were drawn from the samples and assayed for germination as described previously.

Fig. 1. Effect of oscillatory pressure treatment on inactivation of E. coli O157:H7 inoculated on alfalfa seeds at a level of 1.4 × 105 CFU/g. Initial sample temperature was 20 °C. Pressure cycled between atmospheric pressure and 600 MPa (2-min hold time/ cycle at 600 MPa). Data are the means of three replicates. Error bars represent ±1 standard deviation.

(1996) also reported greater reductions of Saccharomyces cerevisiae when oscillatory pressure treatments were applied. Enhancement in oscillatory pressure inactivation has also been reported for spores (Hayakawa et al., 1998). It is thought that the effectiveness of oscillatory HHP treatments can be attributed to spore burst which can be promoted by increased spore wall permeability at high pressures (Palou et al., 1998a,b). However, our results are comparable to those reported by Kingsley et al. (2006) for the pressure inactivation of hepatitis A virus (HAV). Oscillatory high pressure processing for 2, 4, 6, and 8 cycles at 400 MPa did not considerably enhance the inactivation of HAV as compared with continuous high pressure application. 3.2. Combined effect of HHP and antimicrobial treatments on inactivation of E. coli O157:H7 on alfalfa seeds

2.5. Statistical analysis All experiments were replicated at least three times. Where appropriate, statistical analyses were conducted using Minitab® Release 15 (Minitab Inc., University Park, PA, USA). One-way analysis of variance (ANOVA) and Tukey's one-way multiple comparisons were used to determine differences in the populations of E. coli O157: H7 recovered on treated alfalfa seeds as well as differences in the germination percentage of seeds. Significant differences were considered at the 95% confidence level (P < 0.05). 3. Results and discussion 3.1. Effect of oscillatory pressurization on the inactivation of E. coli O157: H7 on alfalfa seeds Oscillatory pressure treatment was investigated to determine whether it could be used to enhance pressure inactivation since continuous high pressure treatment at 600 MPa for 20 min at 20 °C could not eliminate E. coli O157:H7 (105 CFU/g) on alfalfa seeds (Neetoo et al., 2008). Results for the oscillatory HHP treatments are summarized in Fig. 1. The degree of pressure inactivation was found to vary as a function of the number of cycles applied, achieving a maximum reduction of 3.7 logs after 5 cycles for a total holding time of 10 min at 600 MPa. Contrary to findings garnered by other researchers, oscillatory pressure treatments did not confer a significant advantage in the inactivation of E. coli O157:H7. Palou et al. (1998a,b) evaluated the inactivation of Zygosaccharomyces bailii by oscillatory pressure and continuous pressure treatments and found that oscillatory pressure treatments increased pressure inactivation. Aleman et al.

Since oscillatory pressurization with as many as 5 cycles could not eliminate 105 CFU/g of E. coli O157:H7, a “multiple hurdle approach” was investigated whereby the simultaneous application of antimicrobials and HHP were evaluated on inoculated alfalfa seeds. Previous research has shown that washing or soaking in antimicrobial agents can reduce the microbial load in seeds (Jaquette et al., 1996; Piernas and Guiraud, 1997). The drawback associated with these chemical treatments however is that high antimicrobial concentrations are usually required to demonstrate any appreciable effect and the treatments cannot eliminate pathogens. In this experiment, the concerted application of HHP and chemical treatments was investigated. Calcium hydroxide was included in this study as an alternative to chlorinated sanitizers while lactic acid and sodium acid sulfate were studied because of their acidulating property, which was thought to enhance pressure inactivation of bacterial pathogens. Since we were interested in determining whether the application of HHP in conjunction with chemical treatments would be able to achieve 100% lethality or not, samples were analyzed qualitatively for presence/absence of survivors after enrichment in non-selective media. Soaking seeds in the different chemical solutions at low, medium or high concentrations prior to pressure treatment did not achieve 100% kill of the pathogen as survivors were consistently detected across all treatments in all three trials (Table 1). Since the 2min pressure holding time did not bring about elimination of E. coli O157:H7, seeds were then pressure-treated with selected antimicrobials consisting of calcium hypochlorite, calcium hydroxide or lactic acid with an extended pressure holding time of up to 15 min. Regardless of the nature or the level of antimicrobials used, survivors were still detected post-enrichment (Table 2).

H. Neetoo et al. / International Journal of Food Microbiology 131 (2009) 218–223 Table 1 Effect of HHP (600 MPa for 2 min at 20 °C) in conjunction with antibacterial agents on inactivation of E. coli O157:H7 on alfalfa seeds inoculated at a level of 2.0 × 105 CFU/g. Fluid Water Ca(OCL)2 Ca(OCL)2 Ca(OCL)2 Ca(OCL)2 Ca (OH)2 Ca (OH)2 Lactic acid Lactic acid Lactic acid Sodium acid sulfate Sodium acid sulfate Sodium acid sulfate

Concentration 0 200 ppm 1000 ppm 2000 ppm 20,000 ppm 1% 1% + 1% Tween 80 1% 2% 5% 0.05% 0.10% 0.20%

Soaking time (min) 0

5

10

15

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

Numbers separated by a “/” represent the number of samples testing positive after enrichment out of a total of three trials.

It is believed that a small population of E. coli O157:H7 was particularly resistant to pressure treatments. Indeed, other authors have shown that vegetative bacteria such as E. coli O157:H7 can be very refractory to high pressure with pronounced “tails” on survival curves (Chen, 2007). These pressure-resistant cells might be sublethally injured during a pressure treatment and with subsequent appropriate conditions conducive to growth, these cells can recover (Earnshaw et al., 1995). Hence sub-lethal damage and subsequent recovery can present a significant problem for alfalfa sprouts that are eaten raw given that these recalcitrant pathogenic cells can multiply during sprouting. Past research by other authors has demonstrated that chemical sanitizers as stand-alone treatments are not effective at eliminating E. coli O157:H7 from alfalfa seeds (Beuchat and Scouten, 2002; Taormina and Beuchat, 1999; Weissinger and Beuchat, 2000). It was thus hypothesized in this study that the simultaneous application of antibacterial agents and HHP could provide a good opportunity for the chemicals to capitalize on the widespread cellular and biochemical damages induced by pressure treatment. The fact that pressurization of the seeds in the presence of very high concentration of these compounds could not eliminate E. coli O157:H7 lends strong credence to the explanation that these pathogens are lodged in deep cracks or crevices on the seed coat or even internalized. As a result, these chemicals cannot permeate the seed coat to target those internalized cells within the time-frame investigated in our study. Microscopy of mung bean seeds has shown that although the seed surface is relatively smooth, the stem scar is porous, allowing bacteria to penetrate into the seed. Microscopic examination of alfalfa seeds has revealed similarities in the seed coat, both being smooth but with areas capable of harboring pathogens, thus protecting them from Table 2 Effect of extended HHP holding times (600 MPa and 20 °C) in conjunction with antibacterial agents on inactivation of E. coli O157:H7 on alfalfa seeds inoculated at a level of 1.2 × 105 CFU/g. Fluid

Concentration

Water Ca(OCL)2 Ca(OCL)2 Ca (OH)2 Ca (OH)2 Lactic acid Lactic acid Lactic acid

0 2000 ppm 20000 ppm 1% 1% + 1% Tween 80 1% 2% 5%

Pressure holding time (min) 5

10

15

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

Numbers separated by a “/” represent the number of samples testing positive after enrichment out of a total of three trials.

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aqueous sanitizers (Delaquis et al., 1999). In addition to its topographic complexity, the surface of alfalfa seeds is known to be covered with a waxy cuticle (cutin) and it is unlikely that aqueous solutions can effectively wet these surfaces. Given the hydrophobic nature of alfalfa seeds, we hypothesized that addition of a surfactant such as Tween 80 as an adjunct to 1% calcium hydroxide could enhance permeation of the surfactant or the sanitizer into the seeds. However, contrary to findings reported by Weissinger and Beuchat (2000), we found that the application of surfactant during pressure treatment did not enhance the antimicrobial activity of calcium hydroxide and was not effective in achieving complete lethality. 3.2.1. Effect of soaking time and immersion volume in water prior to pressure treatment on inactivation of E. coli O157:H7 on alfalfa seeds Since the combined application of antimicrobial treatments with HHP was unsuccessful in achieving elimination of E. coli O157:H7, modification of the HHP strategy to decontaminate seeds was then sought in an attempt to enhance the microbial safety of alfalfa seeds. Seeds soaked in sterile DI water for up to 300 min (soaked controls) did not undergo any significant reduction in the counts of E. coli O157: H7 with final populations ranging from 4.7–5.3 log CFU/g. Table 3 shows that treatment at 600 MPa without prior soaking brought about a 2.7-log reduction. Pre-soaking of the seeds in water was accompanied by a greater extent of inactivation; in fact a direct relationship between the degree of inactivation and soaking time were observed. When seeds were soaked in a limited volume of 3 ml of water, pressure inactivation of the vegetative cells was strongly dependent on the soaking time. This was likely due to the fact that longer soaking times allowed water to permeate deeper into the cracks and crevices of seeds raising their local water activity and hence enhancing pressure inactivation of cells trapped in these spaces. Seeds soaked for 120–180 min had no detectable counts although survivors were still detected post-enrichment. However, soaking for ≥240 min followed by pressure treatment achieved elimination of the 5 log CFU/g initial load. Seeds were also soaked in a large excess of water (20 ml) such that the volume of water for soaking would not be a limiting factor. The same trend was observed i.e., the longer the soaking time, the greater the degree of pressure inactivation. After the seeds were soaked for ≥60 min followed by pressure treatment, elimination of E. coli O157:H7 was consistently achieved. Hence it can be concluded that both the soaking time and water volume are critical factors in ensuring the efficacy of HHP to decontaminate alfalfa seeds. In the light of these results, it can be inferred that in the presence of excess soaking water, there was uninhibited penetration of water into the seeds such that an hour of imbibition was adequate to allow water to access the deepest cracks or crevices of the seed coat. In the presence of limiting volume of water, water uptake was slower; delaying the consequent movement of water into the seed coat spaces. The results from this soaking study reinforce the findings reported in Section 3.2 suggesting that the poor efficacy of HHP with antimicrobial agents might be attributed to the sub-surface location of the pathogen on the seed. It is thought the pre-soaking step is critical in raising the

Table 3 Effect of soaking time prior to treatment at 600 MPa for 2 min at 20 °C on inactivation of E. coli O157:H7 on alfalfa seeds inoculated at a level of 2.5 × 105 CFU/g. Water (ml)

Soaking time prior to HHP (min) 0

30

60

90

120

180

240

300

3 20

2.7 ± 0.8a 2.7 ± 0.8

1.5 ± 0.1b 1/3

1.1 ± 0.4b 0/3

1.4 ± 0.1b 0/3

1/3 0/3

1/3 0/3

0/3 0/3

0/3 0/3

Data are the means of log survivors (CFU/g) ± 1 S.D. Numbers separated by a “/” represent the number of samples testing positive after enrichment out of a total of three trials (The counts were below the detection limit by direct plating (0.8 log CFU/g) in the three trials). Data in the same row followed by the same superscripted letter are not significantly different (P > 0.05).

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water activity of the seeds which is known to have a bearing on the pressure inactivation of vegetative bacterial cells. It is well documented that low water activity levels protect microorganisms against pressure treatment (Oxen and Knorr, 1993; Palou et al., 1997; Kingsley and Chen, 2008). 3.2.2. Effect of soaking time in water prior to pressure treatment on the seed viability To determine the effect of pre-soaking time on seed viability, the germination rate of seeds soaked for varying durations before HHP was determined (Table 4). Soaking for ≤60 min followed by HHP still allowed seeds to retain their viability to a large extent. The process of soaking seeds in 20 ml of water for 60 min followed by pressure treatment for 2 min achieved a final germination yield of 91% which was 4% lower than the control. This difference in seed germination was not statistically significant (P > 0.05). However with prolonged soaking of >60 min, the effect of pressure treatment on the seeds' germinability became more severe. It is not clear what mechanisms are responsible for the progressively lower germination yields but it is possible that denaturation of molecules such as enzymes might have occurred during HHP following their activation during prolonged water imbibition. It is also highly likely that during soaking, seeds imbibe water and as a result of the increased moisture content, the seeds advance into an active physiological state whereby metabolic changes associated with the initial stages of germination start to take place. The effect of high pressure on the delicate early germinative stage of the seeds most probably evoked negative changes in the microstructure of the seeds. Blaszczak et al. (2007) compared the micro-structural and biochemical changes undergone in raw and germinated chickpea seeds after pressure treatment. They demonstrated the occurrence of more pronounced damage to the structural integrity (physical damage) and protoplasmic contents of cells (biochemical damage) after germinated chickpea seeds were pressure-treated compared to raw chickpea seeds. In order to understand the developmental changes undergone in seeds during soaking, the % fresh weight gain profile of alfalfa seeds was determined as shown in Fig. 2. Seeds are known to be quiescent and can be stored for months without harm, but once supplied with water, they become hydrated again and embark on a different stage of activity. This stage results in outgrowth of the root and later the shoot at the expense of the reserve materials. The imbibition profile was thus studied in order to determine the kinetics of this new pattern of development and to understand how HHP possibly interfered with this developmental process. The imbibition curve was found to exhibit a biphasic pattern of water uptake characterized by a rapid increase in fresh weight during the first 3 h of imbibition (Phase I) followed by a much slower rate of water uptake lasting for approximately 15 h (Phase II). Dry seeds by virtue of their low water potentials set up a very high water potential gradient as soon as they become in contact with water resulting in imbibition. This helps to explain the rapid uptake of water shown in Fig. 2 which occurred at a fairly uniform rate

Table 4 Effect of soaking time prior to treatment at 600 MPa for 2 min at 20 °C on the germination rate of alfalfa seeds. Days of control Soaking time prior to HHP (min) germination 0 30 60 90 3 4 5 6 7 8

87 ± 4a 89 ± 3a 91 ± 4a 93 ± 3a 95 ± 3a 95 ± 3a

80 ± 5ac 87 ± 6a 91 ± 7a 93 ± 7a 95 ± 6a 95 ± 6a

75 ± 6ad 79 ± 7a 81 ± 7a 82 ± 7a 84 ± 7a 86 ± 7ac

80 ± 6ae 85 ± 6a 88 ± 6a 89 ± 6a 90 ± 6a 91 ± 5a

71 ± 7a 75 ± 8a 78 ± 8a 80 ± 9a 81 ± 9a 84 ± 9ad

120

180

60 ± 12bcde 67 ± 11ac 73 ± 10ac 76 ± 11ac 79 ± 11ac 82 ± 13ae

40 ± 8b 47 ± 10bc 52 ± 11bc 55 ± 1bc 58 ± 13bc 62 ± 13bcde

Data are the means of % germination ±1 S.D. Data for the same day of germination followed by the same superscripted letter are not significantly different (P > 0.05).

Fig. 2. The % fresh weight gain of imbibing alfalfa seeds. “% fresh weight gain” was calculated as the difference between the final and the initial weight at defined time intervals as a percentage of the initial weight. Data are the means of three replicates. Error bars represent ± 1 standard deviation.

within the first 3 h. Imbibition then slackened off leading to a slower rate of water uptake and plateaued out until no further fresh weight gain could be observed. This point was coincided by the onset of sprouting marked by the protrusion of the radicle from the seed, which occurred 18 h after soaking. As mentioned previously, we observed that an hour of soaking before HHP treatment was optimal and soaking beyond an hour brought about a gradual decrease in the seeds viability after pressure treatment. Reconciling the germination rate data shown in Table 4 with the imbibition profile of Fig. 2, we attribute this critical timedependence to the fact that imbibing seeds may not swell uniformly throughout their tissues. In pea seeds for example, it has been shown that the testa is completely wetted at a relatively early stage of imbibition (Houben, 1966). It is likely that during the first hour of soaking, the testa swells to the extent of leaving a space between the seed coat and the embryo. This space, probably water-filled, acts as a protective cushion for the seed embryo during HHP. More prolonged soaking however may cause water to permeate all the way into the embryo which also swells, eventually occupying the whole volume inside the testa causing the embryo to stretch the slightly elastic testa. This has been demonstrated to happen in pea seeds after 4–5 h of soaking (Houben, 1966). Hence controlling the length of soaking is highly critical in order to ensure complete decontamination of seeds while maintaining a high germination yield. 3.3. Effect of reduced soaking time and extended pressure holding time at 600 MPa on inactivation of E. coli O157: H7 and on the germination rate of seeds Since previous findings showed that the process of soaking seeds for an hour followed by HHP treatment was able to eliminate pathogens at a slight expense on the germination rate, we thus investigated whether seeds could be decontaminated by shortening the soaking time whilst extending the pressure holding time in order to Table 5 Effect of reduced soaking time and extended pressure holding time on inactivation of E. coli O157: H7 on alfalfa seeds inoculated at a level of 2.5 × 105 CFU/g. Holding time at 600 MPa (min)

Soaking time prior to HHP (min) 10

15

5 10 15

3/3 3/3 0/3

2/3 3/3 0/3

Numbers represent the number of samples testing positive after enrichment out of a total of three trials. The counts were below the detection limit by direct plating (0.8 log CFU/g) throughout.

H. Neetoo et al. / International Journal of Food Microbiology 131 (2009) 218–223

Fig. 3. Effect of reduced soaking time followed by treatment at 600 MPa for 15 min at 20 °C on the germination rate of un-inoculated seeds. Data are the means of three replicates. Error bars represent ± 1 standard deviation.

alleviate the structural damage of HHP on pre-soaked seeds. Results for the inactivation and germination experiments with seeds soaked and pressure-treated for various time combinations are shown in Table 5 and Fig. 3, respectively. Elimination was consistently achieved when the soaking time was 10 or 15 min with a pressure exposure time of 15 min. In addition, un-inoculated seeds treated under these conditions still retained their viability to a large extent (Fig. 3). The process of soaking seeds for 15 min followed by pressure treatment of 15 min achieved a final germination yield of 89% which was 4% lower than the control. Differences in seed germination rate between the control and either treatment on the same day of germination were not statistically significant (P > 0.05). 4. Conclusions Results of this study demonstrate that the application of oscillatory pressurization produced little enhancement in pressure inactivation of E. coli O157:H7 on alfalfa seeds. Although the degree of pressure inactivation increased as a function of the number of cycles, oscillatory HHP treatment could not achieve a >5 log reduction even after five 2min cycles at 600 MPa. This work also provides evidence that pressure treatments in conjunction with antimicrobials including sanitizers, acidulants or a combination of sanitizer and surfactant were not sufficient to eliminate E. coli O157:H7 on alfalfa seeds. However, when seeds were soaked in water for 60 min and subsequently treated at 600 MPa for 2 min, elimination of E. coli O157:H7 occurred at a slight expense on the seed germinability. In addition, it was demonstrated that seeds could be alternatively processed by a brief soaking of 10 min in water followed by an extended pressure treatment of 15 min to achieve the same goals with respect to microbial safety and viability of alfalfa seeds. References Anonymous, 1999. Microbiological safety evaluations and recommendations on sprouted seeds. International Journal of Food Microbiology 52, 123–153. Aleman, G.D., Ting, E.Y., Mordre, S.C., Hawes, A.C.O., Walker, M., Farkas, D.F., Torres, J.A., 1996. Pulsed ultra high pressure treatments for pasteurization of pineapple juice. Journal of Food Science 61, 389–390. Ariefdjohan, M.W., Nelson, P.E., Singh, R.K., Bhunia, A.K., Balasubramaniam, V.M., Singh, N., 2004. Efficacy of high hydrostatic pressure treatment in reducing Escherichia coli O157 and Listeria monocytogenes in alfalfa seeds. Journal of Food Science 69, 117–126.

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Beuchat, L.R., Scouten, A.J., 2002. Combined effects of water activity, temperature and chemical treatments on the survival of Salmonella and Escherichia coli O157:H7 on alfalfa seeds. Journal of Applied Microbiology 92, 382–395. Blaszczak, W., Doblado, R., Frias, J., Vidal-Valverde, C., Sadowska, J., Fornal, J., 2007. Microstructural and biochemical changes in raw and germinated cowpea seeds upon high-pressure treatment. Food Research International 40, 415–423. Brooks, J.T., Rowe, S.Y., Shillam, P., Heltzel, D.M., Hunter, S.B., Slutsker, L., Hoekstra, R.M., Luby, S.P., 2001. Salmonella typhimurium infections transmitted by chlorinepretreated clover sprout seeds. American Journal of Epidemiology 154, 1020–1028. Chen, H., 2007. Use of linear, Weibull, and log–logistic functions to model pressure inactivation of seven foodborne pathogens in milk. Food Microbiology 24, 197–204. Delaquis, P.J., Sholberg, P.L., Stanich, K., 1999. Disinfection of mung bean seed with gaseous acetic acid. Journal of Food Protection 62, 953–957. Earnshaw, R.G., Appleyard, J., Hurst, R.M., 1995. Understanding physical inactivation processes: combined preservation opportunities using heat, ultrasound and pressure. International Journal of Food Microbiology 28, 197–219. Ferguson, D.D., Scheftel, J., Cronquist, A., Smith, K., Woo-Ming, A., Anderson, E., Knutsen, J., De, A.K., Gershman, K., 2005. Temporally distinct Escherichia coli O157 outbreaks associated with alfalfa sprouts linked to a common seed source—Colorado and Minnesota, 2003. Epidemiology and Infection 133, 439–447. Fett, W.F., 2002. Reduction of Escherichia coli O157:H7 and Salmonella spp. on laboratoryinoculated mung bean seed by chlorine treatment. Journal of Food Protection 65, 848–852. Hayakawa, I., Furukawa, S., Midzunaga, A., Horiuchi, H., Nakashima, T., Fujio, Y., Yano, Y., Ishikura, T., Sasaki, K., 1998. Mechanism of inactivation of heat-tolerant spores of Bacillus stearothermophilus IFO 12550 by rapid decompression. Journal of Food Science 63, 371–374. Houben, J., 1966. Imbibition des graines de Pisum Sativum 1ere partie Imbibition par Trempage (Imbibition of seeds of Pisum Sativum 1st stage of imbibition by soaking). Planta 71, 87–97. Jaquette, C.B., Beuchat, L.R., Mahon, B.E., 1996. Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage. Applied and Environmental Microbiology 62, 2212–2215. Kingsley, D.H., Chen, H., 2008. Aqueous matrix compositions and pH influence virus inactivation by high pressure processing. Journal of Food Protection 71, 1598–1603. Kingsley, D.H., Guan, D., Hoover, D.G., Chen, H., 2006. Inactivation of hepatitis A virus by high-pressure processing: the role of temperature and pressure oscillation. Journal of Food Protection 69, 2454–2459. Kumar, M., Hora, R., Kostrzynska, M., Waites, W.M., Warriner, K., 2006. Inactivation of Escherichia coli O157:H7 and Salmonella on mung beans, alfalfa, and other seed types destined for sprout production by using an oxychloro-based sanitizer. Journal of Food Protection 69, 1571–1578. Kurtzweil, P., 1999. Questions keep sprouting about sprouts. FDA Consumer 33, 18–22. Lang, M.M., Ingham, B.H., Ingham, S.C., 2000. Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting. International Journal of Food Microbiology 58, 73–82. Neetoo, H., Ye, M., Chen, H., 2008. Potential application of high hydrostatic pressure to eliminate Escherichia coli O157:H7 on alfalfa sprouted seeds. International Journal of Food Microbiology 128, 348–353. Oxen, P., Knorr, D., 1993. Baroprotective effects of high solute concentrations against inactivation of Rhodotorula rubra. LWT Food Science & Technology 26, 220–223. Palou, E., Lopez-Malo, A., Barbosa-Canovas, G.V., Welti-Chanes, J., Swanson, B.G., 1997. Effect of water activity on high hydrostatic pressure inhibition of Zygosaccharomyces bailii. Letters in Applied Microbiology 24, 417–420. Palou, E., Lopez-Malo, A., Barbosa-Canovas, G.V., Welti-Chanes, J., Davidson, P.M., Swanson, B.G., 1998a. Effect of oscillatory high hydrostatic pressure treatments on Byssochlamys nivea ascospores suspended in fruit juice concentrates. Letters in Applied Microbiology 27, 375–378. Palou, E., Lopez-Malo, A., Barbosa-Canovas, G.V., Welti-Chanes, J., Swanson, B.G., 1998b. Oscillatory high hydrostatic pressure inactivation of Zygosaccharomyces bailii. Journal of Food Protection 61, 1213–1215. Piernas, V., Guiraud, J.P., 1997. Disinfection of rice seeds prior to sprouting. Journal of Food Science 62, 611–615. Proctor, M.E., Hamacher, M., Tortorello, M.L., Archer, J.R., Davis, J.P., 2001. Multistate outbreak of Salmonella serovar Muenchen infections associated with alfalfa sprouts grown from seeds pretreated with calcium hypochlorite. Journal Clinical Microbiology 39, 3461–3465. Taormina, P.J., Beuchat, L.R., 1999. Comparison of chemical treatments to eliminate enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds. Journal of Food Protection 62, 318–324. Weissinger, W.R., Beuchat, L.R., 2000. Comparison of aqueous chemical treatments to eliminate Salmonella on alfalfa seeds. Journal of Food Protection 63, 1475–1482. 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 Food Protection 66, 918–923.