International Journal of Food Microbiology 156 (2012) 197–203

Contents lists available at SciVerse ScienceDirect

International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j foodmicro

High pressure inactivation of Salmonella on Jalapeño and Serrano peppers destinedfor direct consumption or as ingredients in Mexican salsa and guacamole

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

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

0168-1605/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.ijfoodmicro.2012.03.019

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 July 2011Received in revised form 15 March 2012Accepted 18 March 2012Available online 26 March 2012

Keywords:SalmonellaJalapeño peppersSerrano peppersHigh hydrostatic pressureSalsaGuacamole

In summer of 2008, the United States witnessed one of the largest multi-state salmonellosis outbreak linkedto the consumption of Jalapeño and Serrano peppers tainted with Salmonella enterica serovar Saintpaul. Thefirst objective of this study was to assess the application of high hydrostatic pressure (HHP) to decontaminateJalapeño and Serrano peppers from this pathogen. Jalapeño and Serrano peppers were inoculated with a five-strain cocktail of Salmonella to a final level of ca. ~6 log CFU/g and subsequently pressure-treated in the un-wetted, wetted (briefly dipped in water) or soaked (immersed in water for 30 min) state at 300–500 MPa for2 min at 20 °C. The extent of pressure inactivation increased as a function of the pressure level and in theorder of soaked>wetted>un-wetted state achieving population reductions ranging from 1.1 to6.6 log CFU/g. Overall, pressure treatment at 400–450 MPa (soaked) or 450–500 MPa (wetted) for 2 min at20 °C rendered Salmonella undetectable. Since salsa and guacamole are two examples of widely consumedMexican dishes that incorporate raw Jalapeño and Serrano peppers, we subsequently investigated thepressure-inactivation of Salmonella in salsa and guacamole, originating from contaminated peppers used asingredients. The storage time (0, 12 or 24 h) of the condiments prior to HHP as well as the pH (3.8–5.3)and the type of acidulants (vinegar and lemon juice) used all influenced the extent of Salmonella inactivationby HHP. This study demonstrates the dual efficacy of HHP to decontaminate fresh chile peppers destined fordirect consumption and minimally process condiments possibly contaminated with raw peppers to enhancetheir microbiological safety.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Although thought of as a minor crop, peppers form a major worldcommodity. Hot peppers or hot chiles (Capsicum annuum) have beenreported to be an excellent source of vitamins A and C and the com-pound responsible for the irritation (“hotness”) called capsaicin. Theproduction of pepper for spice and vegetables uses has increased bymore than 21% since 1994 (FAO, 2000). North America is one of themajor importing regions of peppers in the world, sourcing 84% of itsimports of fresh peppers from Mexico. The most important chilecultivars in Mexico are “ancho” (wide chile), Jalapeño, Serrano andMirasol, constituting 70 and 80% of chile national production, withJalapeño pepper being the most popular in North America. GreenJalapeños are commonly used fresh in sauces, salads, meats and veg-etable dishes or as spice in condiments (Bosland and Votava, 2000).Serrano is typically the pepper of choice for making relishes such assalsa and Pico de Gallo (Bosland and Votava, 2000).

Unfortunately, peppers (hot and Bell peppers) are increasinglyrecognized vehicles for transmission of foodborne pathogens (Burke,2008). A study conducted on the prevalence of Salmonella on aMexican

+1 302 831 2822.

rights reserved.

farm cultivating chile peppers var. Bell in Culiacán, Sinaloa, showed that10 out of a total of 27 samples from the chile pepper production systemstested positive for Salmonella and were identified as either Salmonellaenterica serovar Typhimurium (91% of cases) or S. enterica serovarEnteritidis (9% of cases) (Gallegos-Robles et al., 2008). In 2008, theUnited States witnessed one of the largest foodborne disease outbreaksattributed to the consumption of fresh Jalapeño and Serrano pepperscontaminated with S. enterica serovar Saintpaul (CDC, 2008a,b,c). It isthought that the contamination of these produce items, grown, har-vested, or packed inMexico, might have occurred on the farm during ir-rigation, processing and/or distribution. Furthermore, Stine et al. (2005)showed that most pathogens including Salmonella have a significantlylower inactivation rate in dry conditions, allowing the bacteria to sur-vive for extended periods of time under conditions of low humidityon the surface of various produce commodities including peppers. Asa result, fresh produce such as peppers can allow foodborne pathogenssuch as Salmonella to persist for long periods by virtue of their dry sur-face making decontamination an imperative (Beuchat, 1995).

Several researchers have investigated the use of chemical andphysical means to improve the microbiological safety and quality ofchile peppers. The use of chlorinated sanitizers (Uyttendaele et al.,2004), trisodium phosphate (Liao and Cooke, 2001), gaseous (Hanet al., 2001) and aqueous (Ketteringham et al., 2006) ozone, calcium

198 H. Neetoo, H. Chen / International Journal of Food Microbiology 156 (2012) 197–203

hydroxide (Alvarado-Casillas et al., 2007), lactic acid (Alvarado-Casillas et al., 2007), hydrogen peroxide (Fallik et al., 1994) and per-oxy acetic acid (Liao et al., 2010) have been investigatedwith reportedvarying degrees of efficacy. In addition, research has also focused onthe use of biological control agents (Liao, 2009) as well as physicalthermal and non-thermal technologies such as the use of hot watertreatments (Fallik et al., 1999), irradiation (Farkas, 1994; Farkas etal., 1997; Farkas, 2001) and UV light (Sommers et al., 2010) to decon-taminate hot or sweet peppers. However to our knowledge, the effec-tiveness of high hydrostatic pressure (HHP) to enhance the safety ofchile peppers (Jalapeño and Serrano peppers) contaminated superfi-cially and internally has not been demonstrated thus far.

Recent statistics have shown that 36% of households in the U.S. fre-quently consume salsa as a condiment. Guacamole is another popularside dish. Unfortunately, both dishes are frequently contaminated bySalmonella (Franco and Simonne, 2009; Franco et al., 2010) and to alesser extent by Campylobacter jejuni (Franco and Simonne, 2009),Shigella (Franco and Simonne, 2009), Staphylococcus aureus (Francoet al., 2010; Simonne et al., 2004), norovirus (Franco and Simonne,2009) or Escherichia coli O157:H7 (Chapman, 2011). Between the pe-riod of 1998 and 2008, therewere 137 salsa and guacamole-associatedoutbreaks reported (CDC, 2010). Mexican foods such as these typicallyfeature multiple ingredients including raw, unprocessed fresh pro-duce (Franco et al., 2010). Therefore, an increase in the frequency offoodborne outbreaks associated with raw produce will also likely bereflected in an increase in outbreaks associated with Mexican dishescomprising these same ingredients (Franco and Simonne, 2009).Similarly, the Salmonella outbreak of 2008 was traced back to contam-inated Jalapeño and Serrano peppers in restaurant-served salsa. Hencethe application of an intervention technology to enhance the safety ofMexican dishes such as salsa and guacamole is warranted. Moreover,the acidity of condiments prepared domestically or commerciallycan vary widely depending on the composition, nature of ingredientsas well as physiological characteristics of fresh vegetables such asthe degree of ripeness of tomatoes (Beuchat andMann, 2008). Variousacidulants can be used in salsa and guacamole to impart flavor andhelp maintain the pH in the desired range. Citric acid which formsthe predominant acid (~5%) of lemon juice (Gulsen and Roose, 2001),and acetic acid present at 5% w/v in vinegar are commonly used. Inthis study, the pH of salsa and guacamole was artificially adjustedusing vinegar or lemon juice to determine the effect of pH on pressureinactivation of Salmonella.

The overall purpose of this study was to investigate the efficacy ofHHP on inactivating Salmonella on raw Jalapeño and Serrano peppersintended for direct consumption or for use as ingredients in salsaand guacamole. Specifically the objectives of this studywere: (i) to de-termine the optimum processing parameters for decontamination ofJalapeño and Serrano peppers directly inoculated with Salmonella,(ii) to assess the efficacy of HHP to inactivate Salmonella originatingfrom artificially contaminated Jalapeño and Serrano peppers in salsaand guacamole, and (iii) to compare the pressure-sensitivity of Salmo-nella in these twoMexican condiments as influenced by the combinedeffects of pH, acidulants and product storage times prior to HHP.

2. Materials and methods

2.1. Jalapeño and Serrano peppers

Jalapeño and Serrano peppers (Capsicum annuum var. annum)were purchased locally and stored at 10±1 °C for no more than aweek (Bosland and Votava, 2000) before use.

2.2. Bacterial strains and inoculum preparation

Five Salmonella strains, three S. typhimurium strains (T43, T45 andTDT 104), S. enterica serovar Worthington W35 and S. enterica serovar

Montevideo Mo57, were used in this study. The cells of Salmonellawere adapted to grow in Tryptic Soy Broth plus 0.6% yeast extract(Difco Laboratories, Sparks, MD, USA) supplemented with 100 μg/mlof nalidixic acid (Fisher Scientific, Hampton, NH, USA) and 100 μg/mlof streptomycin (Sigma) (TSBYE-NS). Briefly, themutantswere selectedby plating 1 ml of the wild-type culture on Tryptic Soy Agar plus 0.6%yeast extract (TSAYE) supplemented with 100 μg/ml of nalidixic acid,picking up of individual colonies and streaking on TSAYE supplementedwith 100 μg/ml of nalidixic acid and 100 μg/ml of streptomycin (TSAYE-NS). Individual cultures were grown in TSBYE-NS overnight at 35 °C.Cultures were then transferred into 10 ml of fresh TSBYE-NS and incu-bated at 35 °C for 24 h. Equal volumes of individual cultures weremixed to form a five-strain cocktail of Salmonella.

2.3. HHP inactivation of Salmonella spp. on un-wetted, wetted or soakedJalapeño and Serrano peppers

2.3.1. InoculationTwo methods of inoculation, spot-inoculation and soak-inoculation,

were compared. Spot-inoculation was carried out to mimic surface-contamination while soak-inoculation was carried out to simulate po-tential infiltration of pathogens during washing or spraying of peppersprior to final processing or packaging. Jalapeño peppers weighing ap-proximately 40 gwere cut into 4 quarters (~10 g/sample)while Serranopeppers weighing approximately 15 g were cut longitudinally into twohalves (~7.5 g/sample). For spot inoculation, 50 μl of a 10-fold dilutionof the cocktail prepared above was inoculated on the external surfaceof Jalapeño and Serrano peppers on the peduncle (stem), calyx and exo-carp of the fleshy pods by depositing the inoculum at equidistant pointsalong the central axis of pepper slices to reach an overall populationdensity of ~6 log CFU/g. The inoculated samples were then air-driedfor 2 h in the biosafety cabinet at ambient temperature (21±1 °C)prior to storage at 10 °C for 1 day. For soak-inoculation, a 5-ml aliquotof the five-strain cocktail wasmixedwith 200 ml of sterile 0.1% peptonewater to reach a level of ~107 CFU/ml. Pepper pieces were then im-mersed into the diluted cell suspension for 10 min after which, the pep-per pieces were separated by pouring over a double layer of cheeseclothsupported by awire screen and dried inside the biosafety cabinet for 2 hprior to storage at 10 °C for 1 day.

2.3.2. Pressure treatmentInoculated samples were (i) directly placed into a sterile stomacher

bag (un-wetted state), or (ii) dipped in sterile De-ionized (DI) water for30 s before being placed into a bag (wetted state) or (iii) immersed insterile DI water for 30 min before being placed into a bag (soakedstate). Thewater-activity of un-wetted,wetted and soaked pepper sam-ples was determined using a water activity meter (Aqualab, DecagonDevices Inc., Pullman, WA, USA). For packaging samples prior to HHP,primary and secondary bags were vacuum-sealed to allow easy inser-tion and withdrawal of samples from the chamber. Double baggingwas done to avoid possible leakage during HHP. The inoculated sampleswere treated at pressure levels ranging from300 to 500 MPa (in 50 MPaincrements) for 2 min at 20 °C (initial sample temperature) using a highpressure unit with temperature control and with water as the trans-mission fluid (Model Avure PT-1, Avure Technologies, Kent, WA). Thepressure-come-up rate was approximately 22 MPa/s. The pressure-release was almost immediate (b4 s). Pressurization time reported inthis study did not include the pressure come-up or release times. Tem-perature increase during pressure treatment of pepper samples was2.8 °C/100 MPa at 20 °C. Untreated inoculated controls were not pack-aged and samples were analyzed directly.

2.3.3. Enumeration of SalmonellaAfter pressure treatments, the treated and un-treated samples

were analyzed by first transferring into a 400 ml stomacher bag (FisherScientific) combined with 30 ml (Serrano pepper) or 40 ml (Jalapeño

199H. Neetoo, H. Chen / International Journal of Food Microbiology 156 (2012) 197–203

pepper) of sterile 0.1% peptone water and then pummeled with aSeward 400 Stomacher (Seward Medical Co., London, England) for2 min. The blended slurry was serially diluted with sterile 0.1% peptonewater. The population of Salmonella on the samples was then deter-mined by spread plating appropriate dilutions of the slurry on TSAYE-NS in duplicate followed by incubation for 72 h at 35 °C prior to colonyenumeration. The pepper samples were also directly enriched in 90 mlof TSBYE-NS and incubated for 48 h at 35 °C to allow resuscitationof sub-lethally injured cells. This enrichment step was conductedwhen the counts were below the detection limit by the platingmethod.Enriched samples were streaked onto Xylose Lysine Desoxycholate(Difco) agar supplemented with 100 μg/ml nalidixic acid (XLD-N). Apreliminary study in our laboratory indicated that XLD-N was an opti-mal selective medium for accurate detection of survivors of Salmonellaantibiotic-resistant strains since it did not yield false positive or falsenegative results. After 24 h incubation, the presence of glossy black orblack-centered pink colonies typical of Salmonellawas recorded as pos-itive (U.S.FDA, 2011).

2.4. HHP inactivation of Salmonella spp. in salsa and guacamoleformulated with different acidulants

Jalapeño and Serrano peppers were chopped into dices with ap-proximate dimensions of 1.5 cm×1.5 cm. Diced peppers were soak-inoculated with Salmonella to a level of ~6 log CFU/g and dried as de-scribed in Section 2.3.3. Salsa was made based on a recipe adaptedfrom SimplyRecipes (2011) by mixing chopped tomatoes (200 g),diced inoculated Jalapeño peppers (40 g), diced inoculated Serranopeppers (10 g), Spanish onions (50 g) and fresh garlic (1/2 tsp) andseasoned with salt (1 pinch~¼ tsp). Salsa was then either left un-acidified (pH 4.3) or adjusted to pH 4.0 and 3.8 using lemon juice orvinegar. To make guacamole, a typical recipe comprising the mostcommon ingredients was chosen. Fresh ripe avocadoes were washed,cut in halves and stoned. The pulp was scooped out and puréed usingan electric blender. Pureed avocado pulp (240 g) was thenmixedwithchopped tomatoes (200 g), diced inoculated Jalapeño peppers (40 g),diced inoculated Serrano peppers (10 g) and Spanish onions (75 g).Guacamole was either left un-acidified (pH 5.3) or adjusted to pH4.6 and 4.3 using lemon juice or vinegar. Once prepared, salsa andguacamole were stored at ambient temperature (~22 °C) for 0 (nostorage), 12 or 24 h prior to pressure treatment. Approximately 10 gof salsa and guacamole were filled into individual sterile Stomacherbags, double-sealed and double-bagged and pressure-treated at levelsranging from 300 to 500 MPa for 2 min at 20 °C. After pressure treat-ments, the treated and un-treated samples were mixed with 40 mlof 0.1% peptone water, stomached and microbiologically analyzed onTSAYE-NS as described previously. Enrichment of the samples for de-tection of Salmonellawas also conducted as described above when thecounts were below the detection limit by the plating method.

Table 1Pressure inactivation of Salmonella spot-inoculated at a level of 6.1 and 6.6 log CFU/g onto J

Pressure Population of Salmonella on Jalapeño(MPa)

Un-wetted Wetted Soaked

300 5.0±0.1Aa 4.3±0.3Aa 3.9±0.3350 4.8±0.1Aab 3.4±0.4ABa 3.0±0.9400 3.9±0.3Aab 2.4±0.4ABab 1.8±0.4450 2.8±0.3Ab 0.4±1.0Bbc 1/3Bbc

500 2.6±0.1Ab 1/3Bc 0/3Bc

Data representing mean log survivors (CFU/g)±standard deviation.Numbers in fraction represent the number of samples testing positive after enrichment oubelow the detection limit by the plating method (0.7 log CFU/g).Values in the same row for the same pepper pod type followed by the same upper case letValues in the same column followed by the same lower case letter are not significantly diff

2.5. Statistical analyses

All experiments were replicated at least three times. Where ap-propriate, statistical analyses were performed on data using Minitab®Release 15 (Minitab Inc., University Park, PA, USA). One-way analysisof variance (ANOVA) and Tukey's one-way multiple comparisonswere used to determine differences in the populations of bacterialpopulations. Significant differences were considered at the 95% confi-dence level (Pb0.05). The detection limit for Salmonella by plating is5 CFU/g since the first dilution ratio was 1:4 for all the samples.When the Salmonella counts in those sampleswere below the detectionlimit by plating, but the samples were positive for Salmonella after en-richment, the number of Salmonella might range from 1 CFU in 10 gfor the Jalapeño pepper, salsa and guacamole samples and 1 CFU in7.5 g for the Serrano pepper samples to 5 CFU/g, which means from−1 (Jalapeño pepper, salsa and guacamole samples) or−0.88 (Serranopepper samples) to 0.7 log CFU/g. The upper value of 0.7 log CFU/g wasused in the statistical analysis. When the samples were negative forSalmonella after enrichment, the number of Salmonella was lower than1 CFU in 10 g for the Jalapeño pepper, salsa and guacamole samplesand lower than 1 CFU in 7.5 g for the Serrano pepper samples, whichmeans b−1 log CFU/g for the Jalapeño pepper, salsa and guacamolesamples and b−0.88 log CFU/g for the Serrano pepper samples. Theupper value of −1 or −0.88 was used in the statistical analysis ofthose samples.

3. Results and discussion

3.1. High-pressure inactivation of Salmonella on Jalapeño and Serranopeppers

Results for high-pressure inactivation of Salmonella spot-inoculatedon Jalapeño and Serrano peppers in the un-wetted, wetted and soakedstates are shown in Table 1. The efficacy of pressure inactivation in thedifferent states varied as a function of the pressure magnitude with ca.3.5–6.1 log CFU/g (Jalapeño) and 3.9–6.6 log CFU/g (Serrano) reductionin the population of Salmonella at 500 MPa, compared to 1.1–2.2 log CFU/g (Jalapeño) and 1.0–3.3 log CFU/g (Serrano) inactivationat 300 MPa. For spot inoculation, consistently higher inactivation ratioswere observed in Serrano compared to Jalapeño peppers although thedifferences were not statistically significant (P>0.05).

The inactivation efficacy of HHP also depended on the degree ofwetness of peppers and ranked in the order of soaked>wetted>un-wetted state. This was more noticeable with Jalapeño peppersthan Serrano peppers. When Jalapeño peppers were pressure-treatedin the un-wetted state at levels of up to 500 MPa, a surviving populationof Salmonella (2.6–2.7 log CFU/g) was detected. However, when pep-pers were briefly dipped in water (wetted for 30 s) or subjected to pro-longed soaking (soaked for 30 min) prior to HHP treatment under thesame condition, the population was reduced to below detection limit

alapeño and Serrano peppers respectively in the un-wetted, wetted and soaked states.

Population of Salmonella on Serrano

Un-wetted Wetted Soaked

Aa 5.1±0.5Aa 4.1±0.2Aa 3.3±0.6AaBab 3.8±0.9Aa 3.0±0.6Aab 2.9±0.4AaBb 2.8±0.9Aab 0.8±0.2Abc 0.5±1.1Ab

2.5±0.7Ab 3/3ABbc 1/3Bb

1.5±1.9Ab 1/3Ac 0/3Ab

t of a total of 3 trials. Enrichment was conducted when the counts of Salmonella were

ter are not significantly different (P>0.05).erent (P>0.05).

200 H. Neetoo, H. Chen / International Journal of Food Microbiology 156 (2012) 197–203

(b0.7 log CFU/g by direct plating) for the wetted state and eliminated(negative for Salmonella after enrichment) for the soaked state. Un-wetted Jalapeño and Serrano peppers had a water activity of ca. 0.97just like many fruits and vegetables (Grabowski et al., 2006) while thewater activity of the same peppers in the wetted and soaked stateswere closer to 1 (0.995). This demonstrates that pressure inactivationof Salmonella on Jalapeño peppers was more pronounced at higherwater activity. This is congruent with observations made by Oxen andKnorr (1993) as well as previous findings garnered in our laboratoryon the pressure-inactivation of E. coliO157:H7 and Salmonella on alfalfaseeds and green onions (Neetoo et al., 2008, 2009, 2011). Although asimilar trend was observed for Serrano peppers, the differences in thelog reductions achieved between the different wetness states werenot statistically significant in most cases except for the 450 MPa treat-ment (P>0.05).

Since wetting and soaking enhanced pressure inactivation ofSalmonella spot-inoculated on peppers, only these two pretreatmentswere considered for the soak-inoculation study. The overall trend forbacterial inactivation on soak-inoculated Jalapeño and Serrano pepperswas fairly similar to their spot-inoculated counterparts (Table 2), withno significant differences observed between the wetting and soakingpretreatments (P>0.05). In addition, no differences were observed be-tween similarly treated soak-inoculated Jalapeño and Serrano peppers(P>0.05).

Comparison of the population reductions achieved for each podtype inoculated under the two different schemes revealed that similarpressure levels were adequate to achieve a satisfactory population re-duction in both cases. This observation thus lends credence to the factthat HHP is a processing technology that relies on the uniform andquasi-instantaneous pressure transmittance allowing both superficialand infiltrated pathogens to be equally and effectively targeted re-gardless of their location or distribution on the plant tissue. Overall,pressure treatment at 500 MPa for 2 min at 20 °C was found to be ad-equate in eradicating a >5 log CFU/g burden of Salmonella on soakedJalapeño and Serrano peppers irrespective of the inoculationmethods.Contamination of fresh produce such as hot peppers with pathogenssuch as Salmonella can occur at multiple steps throughout the farm-to-table food product chain (Lynch et al., 2009). The different stagessuch as cultivation, harvest, processing, and handling can allow path-ogenic cells to contact and possibly attach to inner or outer surfaces.Pre-harvest contamination of peppers in the field due to the use oftainted irrigation water is thought to have been the cause of the2008 Salmonella Saintpaul outbreak associated with Jalapeño andSerrano peppers. Findings from the present study suggest that pep-pers can be completely decontaminated if they are pressure-treatedin themoistened state irrespective of the route or origin of contamina-tion. Pre-packaged pressure-treated sliced green or Jalapeño peppers

Table 2Pressure inactivation of Salmonella soak-inoculated into Jalapeño and Serrano peppersat a level of 5.7 and 6.0 log CFU/g respectively in the wetted and soaked states.

Pressure Population of Salmonellaon Jalapeño

Population of Salmonellaon Serrano(MPa)

Wetted Soaked Wetted Soaked

300 3.9±0.6Aa 3.6±0.5Aa 3.2±0.3Aa 3.6±0.6Aa

350 2.4±0.3Aab 1.2±0.6Ab 1.9±0.5Aa 1.8±0.3Aab

400 3/3Ab 1/3Ab 1.7±0.8Aa 1.0±0.5Ab

450 3/3Ab 1/3Ab 1.0±1.6Aa 3/3Ab

500 0/3Abc 0/3Ab 2/3Ab 0/3Ab

Data representing mean log survivors (CFU/g)±standard deviation.Numbers in fraction represent the number of samples testing positive after enrichmentout of a total of 3 trials. Enrichment was conducted when the counts of Salmonellawerebelow the detection limit by the plating method (0.7 log CFU/g).Values in the same row followed by the same upper case letter are not significantlydifferent (P>0.05).Values in the same column followed by the same lower case letter are not significantlydifferent (P>0.05).

are already being marketed in the United States and they typically aresold along with ready-to-assemble fajita meal kits (Neergaard, 2008).

Various authors have noted that Salmonella has the ability to growin pepper extracts (Nutt et al., 2003), chopped bell pepper (Liao et al.,2010) and chopped Jalapeño peppers (Black et al., 2010; Liao et al.,2010) as well as survive on intact (whole) bell and Jalapeño peppers(Black et al., 2010; Ma et al., 2010). Moreover Liao et al. (2010) andMa et al. (2010) showed that Salmonella inoculated onto Jalapeñoshave the ability to grow at low storage temperatures of 12 °C. Sincea storage temperature of ~10 °C is generally recommended to avoidchilling injury in peppers (Bosland and Votava, 2000), there is a riskof Salmonella survival or growth during storage. As a result, decon-tamination of hot peppers has become an active area of research. Be-sides the use of chemical sanitizers including chlorinated (sodiumhypochlorite, acidified sodium chlorite) or non-chlorinated sanitizers,several authors have investigated the use of physical interventions todecontaminate Jalapeño peppers. Sommers et al. (2010) investigatedthe effectiveness of UV light at doses of 0.5–4.0 J/cm2 to inactivateSalmonella, S. aureus and Listeria monocytogenes surface-inoculatedon peppers and observed a variable reduction of 3.0–3.8 log CFU/gdepending on the UV dose. Unfortunately, the efficacy of UV is oftenlimited by its penetration depth, which in turn is influenced by theproduce morphology and surface topography. On the contrary, pres-sure transmission during high pressure processing is isostatic, makingit better suited to target pathogens located on the surface, subsurfaceor within internal tissues.

3.2. Effect of HHP to inactivate Salmonella in salsa and guacamole

3.2.1. Effect of pH and sampling holding times on the survival ofSalmonella in salsa and guacamole

Table 3 presents the population of Salmonella in unacidified salsaor salsa acidified with vinegar or lemon juice and stored at ambienttemperature for 0, 12 or 24 h prior to pressure-treatment. Salmonellawas unable to proliferate in any of the salsa formulations irrespectiveof pH. Bell and Kyriakides (2002) mentioned that the minimumgrowth pH for Salmonella is 3.8, although most serovars are unableto grow at pH below 4.5. Hence this may partly explain the inabilityof Salmonella strains used in this study to proliferate in salsas withpH of 3.8–4.3. Ma et al. (2010) also compared the growth and survivalpotential of five Salmonella serovars and reported that Salmonellawasunable to grow but could still persist in certain salsa formulations re-gardless of the final pH. Black et al. (2010) on the contrary, showedthat the 2008 Salmonella Saintpaul outbreak isolate was able togrow in Mexican salsa (pH 3.6–3.7) by more than 4–5 log CFU/gduring storage at 23 °C although other outbreak isolates included inthe study such as Salmonella Montevideo, Salmonella Enteritidis,Salmonella Typhimurium and Salmonella Newport increased by only0.5–1 log CFU/g. Campbell et al. (2001) also reported growth of S.enterica serovar Thompson in freshly made salsa samples (pH 3.4)stored at room temperature. Hence, the fate of Salmonella during stor-age depends on the strains used in the inoculation and the nature ofthe ingredients in salsa.

As far as guacamole is concerned, Salmonella was also unable togrow in unacidified and acidified guacamole (Table 4). Previous re-search also showed that Salmonella spp. was unable to grow in guaca-mole with a pH of 5.3, and actually decreasing by 1 log CFU/g during a6-day storage (FressureUnderPressure®, 2000). It is possible that cer-tain ingredient(s) present in guacamole could be inimical to Salmonella.This contrasts with findings of Chang et al. (2010) who showed thatSalmonella and E. coliO157:H7were capable of growing in both avocadopulp (pH 6.5) and guacamole (pH 5.3) during a 12-h ambient storageduration. The guacamole formulation used in our study included ingre-dients such as tomatoes and fresh garlic, which were both absent in therecipe used by Chang et al. (2010). We speculate that the addition offresh garlic containing the putative antimicrobial allicin together with

Table 3Population of Salmonella in unacidified salsa or salsa acidified with vinegar or lemon juice and stored at ambient temperature for 0, 12 or 24 h prior to pressure-treatment.

pH Storage time Population of Salmonella treated at pressure levels (MPa) of

0 300 350 400 450 500

No acidulant (pH 4.3) 0 h 6.1±0.4A 4.0±0.1A 2.6±1.1A 1.4±0.3A 0.8±0.1A 3/3A

12 h 5.8 ±0.4A 2.8±0.2A 2.4±0.3A 1.3±0.6A 0.8±0.3A 3/3A

24 h 5.2±0.2A 3.4±0.2A 3/3A 3/3A 1/3A 0/3A

Vinegar (pH 4.0) 0 h 4.8±0.1A 3.0±0.8A 3/3AB 2/3A 1/3A 1/3A

12 h 4.7±0.5A 2.4±0.3A 1.2±0.5A 1/3A 1/3A 1/3A

24 h 4.5±0.1A 1/3B 1/3B 1/3A 1/3A 0/3A

Lemon Juice (pH 4.0) 0 h 5.2±0.1A 3.4±0.3A 1.8±0.2A 1.7±0.8A 1/3A 1/3A

12 h 4.9±0.2A 2.7±0.1A 2.5±0.4A 1/3A 1/3A 1/3A

24 h 4.9±0.1A 2/3B 1/3B 1/3A 1/3A 0/3A

Vinegar (pH 3.8) 0 h 4.1±0.2A 2.1±0.4A 2/3A 0/3A 0/3A 0/3A

12 h 3.9±0.2A 1.8±0.2AB 1/3A 0/3A 0/3A 0/3A

24 h 4.0±0.3A 1/3B 1/3A 0/3A 0/3A 0/3A

Lemon Juice (pH 3.8) 0 h 4.7±0.1A 3.7±0.3A 2.4±0.2A 1/3A 0/3A 0/3A

12 h 4.7±0.1A 1/3B 1/3B 0/3A 0/3A 0/3A

24 h 3.4±0.5A 1/3B 1/3B 0/3A 0/3A 0/3A

Data representing mean log survivors (CFU/g)±standard deviation.Numbers in fraction represent the number of samples testing positive after enrichment out of a total of 3 trials. Enrichment was conducted when the counts of Salmonella werebelow the detection limit by the plating method (0.7 log CFU/g).Values in the same column for the same salsa formulation (i.e. same acidulant and same pH) followed by the same upper case letter are not significantly different (P>0.05).

201H. Neetoo, H. Chen / International Journal of Food Microbiology 156 (2012) 197–203

the acidity conferred by lemon juice and tomatoes could have pre-vented growth of Salmonella during storage at room temperature.Ma et al. (2010) observed an antimicrobial effect exhibited by fresh gar-lic against Salmonellawhen used in combinationwith lime juice in salsapreparation. Hence, the guacamole formulation used in our study likelycomprises ingredients that could have prevented proliferation of thepathogen.

Overall, Salmonella survived well in these condiments. Several au-thors have observed that Salmonella survived better at refrigerated tem-perature than at room temperature in high acid products (Raghubeer etal., 1995). Hence this shows that contaminated salsa or guacamole maypose a food safety risk regardless of the storage temperatures, since theintrinsic characteristics of the products may not present adequate hur-dles to reduce the risk of salmonellosis. As a result, the identification ofappropriate intervention treatments to enhance the safety of salsa andguacamole is warranted.

3.2.2. Survival of Salmonella in pressure-treated salsa and guacamoleduring storage at ambient temperature

Tables 3 and 4 show that the application of HHP at levels of300–500 MPa reduced the population of Salmonella in salsa and

Table 4Population of Salmonella in unacidified guacamole or guacamole acidified with vinegar ortreatment.

pH Storage time Pop

0 300

No acidulant (pH 5.3) 0 h 6.5±0.4A 4.5±0.112 h 5.8±0.4A 3.9±0.424 h 5.5±0.5A 3.0±0.4

Vinegar (pH 4.6) 0 h 4.9±0.2A 3.1±0.112 h 4.7±0.4A 1.6±0.224 h 4.5±0.4A 1/3B

Lemon Juice (pH 4.6) 0 h 5.4±0.3A 3.4±0.312 h 5.0±0.3A 3.7±0.424 h 4.4±0.5A 2/3B

Vinegar (pH 4.3) 0 h 4.7±0.4A 2.3±0.212 h 4.4±0.2A 1.4±0.424 h 4.0±0.4A 0/3B

Lemon Juice (pH 4.3) 0 h 5.2±0.3A 3.8±0.212 h 4.7±0.2A 3.5±0.124 h 3.8±0.5A 2/3B

Data representing mean log survivors (CFU/g)±standard deviation.Numbers in fraction represent the number of samples testing positive after enrichment oubelow the detection limit by the plating method (0.7 log CFU/g).Values in the same column for the same guacamole formulation (i.e. same acidulant and sam

guacamole to varying extents depending on the pressure magnitude,pH, type of acidulants used, and product holding time. Increasing thepressure level resulted in a greater reduction in Salmonella counts. Inaddition, Salmonella became increasingly more pressure-sensitivewith the lowering of pH of the formulations. Reducing the pH ofsalsa from 4.3 to 3.8 or pH of guacamole from 5.3 to 4.3 had a majorinfluence on the effect of pressure inactivation of Salmonella. For ex-ample HHP at 400 MPa reduced the population of Salmonella in gua-camole with pH of 5.3, 4.6 and 4.3 to 2.6, ≤1.2 and ≤1.0 log CFU/g,respectively depending on the acidulants used. The conjunct effectof HHP with acidity to enhance microbial pressure inactivation iswidely acknowledged in the literature. Alpas et al. (1994) demon-strated that when Salmonella cells were suspended in a dilute solu-tion of citric or lactic acid solution, they were highly susceptible topressurization undergoing higher reduction at pH 4.5 compared topH 6.5. Pagan and Mackey (2000) postulated that bacterial cells im-paired by high pressure exhibited increased acid-sensitivity. The au-thors were able to demonstrate that pressure-damaged E. coli O157were impaired in pH homeostasis and consequently more acid sensi-tive than native cells. Overall, this part of the study demonstratedthat the application of HHP in high acid foods (pHb4.6) can be

lemon juice and stored at ambient temperature for 0, 12 or 24 h prior to pressure-

ulation of Salmonella treated at pressure levels (MPa) of

350 400 450 500

A 3.7±0.3A 2.5±0.1A 0.9±0.4A 3/3AA 3.8±0.6A 1.5±0.6B 3/3A 3/3AA 3/3B 3/3C 3/3A 1/3AA 1.3±0.1A 2/3A 2/3A 2/3AAB 3/3A 2/3A 2/3A 1/3A

0/3A 0/3A 0/3A 0/3AA 3.0±0.2A 1.2±0.1A 3/3A 2/3AA 2.7±0.1A 1.2±0.3A 2/3A 2/3A

2/3B 1/3A 1/3A 1/3AA 2/3A 1/3A 1/3A 1/3AA 2/3A 2/3A 1/3A 0/3A

0/3A 0/3A 0/3A 0/3AA 2.0±0.3A 1.1±0.5A 2/3A 2/3AA 2.6±0.2A 2/3A 2/3A 1/3A

1/3B 0/3A 0/3A 0/3A

t of a total of 3 trials. Enrichment was conducted when the counts of Salmonella were

e pH) followed by the same upper case letter are not significantly different (P>0.05).

202 H. Neetoo, H. Chen / International Journal of Food Microbiology 156 (2012) 197–203

advantageous since lower pressure can be adequately used to pro-duce injured cells that are unable to repair in the acidic mediumafter HHP.

Vinegar had a greater potentiating effect than lemon juice for bothsalsa (Table 3) and guacamole (Table 4). For example when salsa wasacidified to pH 3.8, and immediately (0 hour holding) subjected topressure levels of 300 MPa, the Salmonella counts in vinegar-acidified salsa (2.1 log CFU/g) was significantly (Pb0.05) lower thansalsa acidified with lemon juice (3.7 log CFU/g). When organic acidsare used as food preservatives, the pH of the medium has a profoundeffect on the proportion of undissociated acids. The undissociatedform of acids represents the uncharged molecular state of the acidswith antimicrobial effect due to its ability to cross the bacterial mem-branes. The proportion of undissociated acids present at any pH variesfrom one organic acid to another depending on its pKa. The active in-gredient of table vinegar is acetic acid at a concentration of 5% whilelemon juice is a mixture of several organic acids with citric acidbeing in highest abundance (6–10%) (Penniston et al., 2008). ThepKa values of acetic and citric acids at 25 °C are 4.76 and 3.14, respec-tively (Sergeant and Dempsey, 1979). As a result, at any pH, acids withthe highest pKa (i.e. weakest acids) will be expected to have the high-est proportion of undissociated acids. For instance, Bell and Kyriakides(2002) showed that at a pH of 4.0, the percentage of acid species inthe undissociated form (i.e. the acid species possessing antibacterialactivity) was 84.5% and 18.9% for acetic and citric acid, respectively.Hence the extent of bacterial inactivation will generally depend onthe type of acid present and other prevailing physico-chemical condi-tions (Bell and Kyriakides, 2002). Jung and Beuchat (2000) reported astudy of survival of 4 strains of Salmonella Typhimurium DT 104against 4 other non-DT 104 Typhimurium strains in media whose pHwas adjusted with various organic acids. They showed that at agiven pH, acetic acid (highest pKa) was the most inhibitory acidulantof those tested. Abdul-Raouf et al. (1993) also compared the antimi-crobial efficacy of three acidulants in inhibiting the growth of E. coliO157:H7 on roasted, ground beef and demonstrated that the orderof effectiveness was in the order of acetic acid>lactic acid≥citric acid.

Holding unacidified and acidified salsa and guacamole samples atroom temperature not only slightly reduced the initial population butalso rendered Salmonella cells more sensitive to HHP. For example, inunacidified salsa (pH 4.3), HHP at 350 MPa reduced the population ofSalmonella to 2.6, 2.4 and b0.7 log CFU/g (i.e. below detectable limitsby plating) after holding the samples for 0, 12 and 24 h, respectively.When the pH of acidified salsa was decreased to 3.8 and held for 24-h,Salmonella was reduced to below detectable limits in salsa followingpressurization at 300 MPa. The same trend was also observed for gua-camole where longer incubation times correlated with greater extentof pressure-inactivation. This shows that holding foods at low pHvalues in combination with HHP enhances microbial inactivation pos-sibly due to prolonged exposure of bacterial cells to high acidity. Pre-viously, Whitney et al. (2008) showed that holding pressure-treatedapple juice and cider (pHb4.1) for 24 h at refrigerated temperatures(4 °C) ensured complete inactivation of E. coli O157:H7 while samplesmicrobiologically analyzed immediately after pressure-treatmentshowed viable cells of the pathogen. Along the same line, Linton etal. (1999) observed that E. coli O157:H7 inoculated in orange juiceunderwent greater inactivation during extended refrigerated storagefollowing HHP due to increased susceptibility of the pathogen to acid-ity. Hence, low pH likely confers bacterial cross-sensitivity to otherhurdles (Smittle, 1977; Zhao et al., 1993) including HHP (Stewart etal., 1997). Cellular injury offers a processing advantage in high acidfoods, as impaired cells are unable to repair and thus die off duringstorage (Patterson et al., 1995).

Generally speaking, commercial processing of salsa and guacamoleare regarded as high cost operations since they are usually carriedout at pressure levels of ~600 MPa (Torres and Velazquez, 2009).Parnell (2003) reported that HHP at 580–600 MPa for ≥2 min could

extend the shelf-life of guacamole while achieving a 5-log reductionof Salmonella and Listeria. Research has also demonstrated the effec-tiveness of generally high-pressure levels of 540 MPa (Farkas, 2007),580 MPa (Raghubeer et al., 2008) and 590–600 MPa (Parnell, 2003)for HHP treatment of salsa for pathogen inactivation depending onthe composition of the product as well as the design and configurationof the pressure systems. Findings of our study underscore the effec-tiveness of low levels of HHP (300–400 MPa) to completely destroySalmonella through careful adjustment of intrinsic (pH, type of acidu-lant) and processing (product holding time, pressure level) parame-ters. Our study highlights the impact of storage of high acid productsas a means to increase the susceptibility of foodborne pathogensto subsequent pressure-inactivation thereby reducing the pressurelevel required for the treatments. Process costs of pressure-treatments are related to the operating pressure, and the capital costof high-pressure equipment generally increases exponentially withincreasing operating pressure. Since process costs are a direct functionof operating costs, lowering the pressure levels required to bring a sat-isfactory inactivation can minimize both operating and process costs.It is hoped that with the rapid advancement of HHP technology andhigh acceptance of and demand for these products by consumers(Bermúdez-Aguirre and Barbosa-Cánovas, 2011), HHP-processedsalsa and guacamole may likely be available in local supermarkets atmore affordable prices.

4. Conclusions

This study demonstrated that HHP at pressure levels of 500 MPa for2 min at 20 °C was able to effectively decontaminate soaked Jalapeñoand Serrano peppers from Salmonella. Furthermore we demonstratedthat HHP has the ability to target pathogenic cells thatmay contaminatechile peppers either on the surface or internally. We then subsequentlyshowed that peppers contaminated with Salmonella can potentiallyserve as effective vehicles of the pathogen in raw condiments such assalsa and guacamole allowing its persistence in high acid (pHb4.6)and low acid (pH>4.6) formulations. Our study highlights the role ofproduct storage in conjunction with HHP to ensure a >5-log inactiva-tion of Salmonella from salsa and guacamolewith carefullymanipulatedformulations.

Acknowledgment

This project was partially supported by the Agriculture and FoodResearch Initiative Competitive Grants Program of the USDA NationalInstitute of Food and Agriculture, NIFA award no: 2011-68003-30005.

References

Abdul-Raouf, U.M., Beuchat, L.R., Ammar, M.S., 1993. Survival and growth of Escherichiacoli 0157:H7 in ground, roasted beef as affected by pH, acidulants and temperature.Applied and Environmental Microbiology 59, 2364–2368.

Alpas, H., Kalchayandand, N., Sikes, T., Dunne, C.P., Ray, B., 1994. Hydrostatic pressureamong strains of foodborne pathogens. Applied and Environmental Microbiology65, 4248–4251.

Alvarado-Casillas, S., Ibarra-Sanchez, S., Rodriguez-Garcia, O., Martinez-Gonzales, N.,Castillo, A., 2007. Comparison of rinsing and sanitizing procedures for reducingbacterial pathogens on fresh cantaloupes and bell peppers. Journal of Food Protec-tion 70, 655–660.

Bell, C., Kyriakides, A., 2002. Salmonella: A Practical Approach to the Organism And itsControl in Foods. Iowa State University Press, Blackwell Science Company, IA.

Bermúdez-Aguirre, D., Barbosa-Cánovas, G.V., 2011. An update on high hydrostaticpressure, from the laboratory to industrial applications. Food Engineering Reviews3, 44–61.

Beuchat, L., 1995. Pathogenic microorganisms associated with fresh produce. Journal ofFood Protection 59, 204–216.

Beuchat, L.R., Mann, D.A., 2008. Survival and growth of acid-adapted and unadaptedSalmonella in and on raw tomatoes as affected by variety, stage of ripeness, andstorage temperature. Journal of Food Protection 71, 1572–1579.

Black, E., Kirkland, C., Sadowsky, M.J., Diez-Gonzalez, F., 2010. Survival and growth ofSalmonella Saintpaul in fresh mexican salsa stored at room temperature. 2010IAFP Annual Meeting, Anaheim, CA.

203H. Neetoo, H. Chen / International Journal of Food Microbiology 156 (2012) 197–203

Bosland, P.W., Votava, E.J., 2000. Peppers: Vegetable and Spice Capsicums. CABI Pub-lishing, New York, NY, USA.

Burke, G., 2008. Mexican peppers posed problem long before outbreak. AssociatedPress. Yahoo News, 08/18/2008.

Campbell, J.V., Mohle-Boetani, J., Reporter, R., Abbott, S., Farrar, J., Brandl, M., Mandrell,R., Werner, S.B., 2001. An outbreak of Salmonella Serotype Thompson associatedwith fresh cilantro. The Journal of Infectious Diseases 183, 984–987.

CDC, 2008a. Outbreak of Salmonella serotype Saintpaul infections associated with mul-tiple raw produce items — United States, 2008. Morbidity and Mortality WeeklyReport 57, 929–934.

CDC, 2008b. Investigation of outbreak of infections caused by Salmonella Saintpaul.Available at: http://www.cdc.gov/salmonella/saintpaul/.

CDC, 2008c. FDA Salmonella Saintpaul outbreak. Available at: http://www.fda.gov/oc/opacom/hottopics/tomatoes.html.

CDC, 2010. Salsa and guacamole increasingly important causes of foodborne disease.Available at: http://www.cdc.gov/media/pressrel/2010/r100712.html.

Chang, S.-S., Valenza-Martinez, C., Thippareddi, H., 2010. Survival and growth ofEscherichia coli O157:H7 and Salmonella spp. in avocado pulp and guacamole underrefrigeration and abusive temperatures. 2010 IAFP Annual Meeting, Anaheim, CA.

Chapman, B., 2011. Food safety infosheets 07/01/11. Available at:www.foodsafetyinfosheets.com.

Fallik, E., Aharoni, Y., Grinberg, S., Copel, A., Klein, J.D., 1994. Postharvest hydrogen per-oxide treatment inhibits decay in eggplant and sweet red pepper. Crop Protection13, 451–454.

Fallik, E., Grinberg, S., Alkalai, S., Yekutieli, O., Wiseblum, A., Regev, R., Beres, H., Bar-Lev,E., 1999. A unique rapid hot water treatment to improve storage quality of sweetpepper. Postharvest Biology and Technology 15, 25–29.

Farkas, J., 1994. Irradiation as method for decontaminating food. International Journalof Food Microbiology 44, 189–204.

Farkas, J., 2001. Irradiation of minimally processed food. In: Molins, R. (Ed.), Food Irradia-tion: Principles and Applications. John Wiley and Sons, Inc., New York, pp. 273–290.

Farkas, D.F., 2007. Preservation of Food by High Hydrostatic Pressure. Elmhurst Research,Inc. Available at: http://www.elmhurstresearch.com/food_technology1.htm.

Farkas, J., Saray, T., Mohacsi-Farkas, C., Horti, C., Andrassy, E., 1997. Effects of low-dosegamma irradiation on shelf life and microbiological safety of precut/prepared veg-etables. Advances in Food Science 19, 111–119.

Food and Agriculture Organization of the United Nations (FAO-UN), 2000. Chilies andpeppers: green production. Available at: www.fao.org.

Franco, W., Simonne, A.H., 2009. Mexican food safety trends: examining the CDC datain the United States from 1990 to 2006. Food Protection Trends 29, 204–210.

Franco, W., Hsu, W.-Y., Simonne, A.H., 2010. Survival of Salmonella and Staphylococcusaureus in mexican red salsa in a food service setting. Journal of Food Protection 73,1116–1120.

FressureUnderPressure®, 2000. Ultrahigh pressure guacamole. Available at: www.flowcorp.com.

Gallegos-Robles, M.A., Morales-Loredo, A., Alvarez-Ojeda, G., Vega-P, A., Chew-M, Y.,Velarde, S., Fratamico, P., 2008. Identification of Salmonella serotypes isolated fromcantaloupe and chile pepper production systems in Mexico by PCR-restriction frag-ment length polymorphism. Journal of Food Protection 71, 2217–2222.

Grabowski, S., Marcotte, M., Ramaswamy, H., 2006. Dehydrated vegetables: principlesand applications. In: Hui, Y.H. (Ed.), Handbook of food science, technology, and en-gineering., Vol. 3, pp. 103–116.

Gulsen, O.M., Roose, L., 2001. Lemons: diversity and relationships with selected Citrusgenotypes as measured with nuclear genome markers. Journal of the American So-ciety for Horticultural Science 126, 309–317.

Han, Y., Floros, J.D., Linton, R.H., Nielsen, S.S., Nelson, P.E., 2001. Response surfacemodelingfor the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annuum)by ozone gas treatment. Journal of Food Science 67, 1188–1193.

Jung, Y.S., Beuchat, L.R., 2000. Sensitivity of multi-drug resistant Salmonella TyphimuriumDT104 to organic acids and thermal inactivation in liquid egg products. FoodMicrobi-ology 17, 63–71.

Ketteringham, L., Gausseres, R., James, S.J., James, C., 2006. Application of aqueousozone for treating pre-cut green peppers (Capsicum annuum L.). Journal of FoodEngineering 76, 104–111.

Liao, C.-H., 2009. Control of foodborne pathogens and soft-rot bacteria on Bell pepperby three strains of bacterial antagonists. Journal of Food Protection 72, 85–92.

Liao, C.-H., Cooke, P.H., 2001. Response to trisodium phosphate treatment of SalmonellaChester attached to fresh-cut green pepper slices. Canadian Journal of Microbiolo-gy 47, 25–32.

Liao, C.-H., Cooke, P.H., Niemira, B.A., 2010. Localization, growth and inactivation ofSalmonella Saintpaul on Jalapeño Peppers. Journal of Food Science 75, 377–384.

Linton, M., McClements, J.M., Patterson, M.F., 1999. Survival of Escherichia coli O157:H7during storage in pressure-treated orange juice. Journal of Food Protection 62,1038–1040.

Lynch, M.F., Tauxe, R.V., Hedberg, C.W., 2009. The growing burden of foodborne out-breaks due to contaminated fresh produce: risks and opportunities. Epidemiologyand Infections 137, 307–315.

Ma, L., Zhang, G., Gerner-Smidt, P., Tauxe, R.V., Doyle, M.P., 2010. Survival and growthof Salmonella in salsa and related ingredients. Journal of Food Protection 73,434–444.

Neergaard, L., 2008. Putting the Squeeze on Food. Can It Kill the Pathogens? The AssociatedPress, Washington. Available at: http://findarticles.com/p/news-articles/virginian-pilot-ledger-star-norfolk/mi_8014/is_20080729/putting-squeeze-food-kill-pathogens/ai_n41411269/.

Neetoo, H., Ye, M., Chen, H., 2008. Potential application of high hydrostatic pressure toeliminate Escherichia coli O157:H7 on alfalfa sprouted seeds. International Journalof Food Microbiology 128, 348–353.

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

Neetoo, H., Nekoozadeh, S., Jiang, Z., Chen, H., 2011. Application of high hydrostatic pres-sure to decontaminate green onions from Salmonella and Escherichia coli O157:H7.Food Microbiology 28, 1275–1283.

Nutt, J.D., Li, X., Woodward, C.L., 2003. Growth kinetics response of a Salmonella Typhi-murium poultry marker strain to fresh produce extracts. Bioresources Technology89, 313–316.

Oxen, P., Knorr, D., 1993. Baroprotective effects of high solute concentrations againstinactivation of Rhodotorula rubra. LWT — Food Science and Technology 26,220–223.

Pagan, R., Mackey, B., 2000. Relationship between membrane damage and cell death inpressure-treated Escherichia coli cells: differences between exponential andstationary-phase cells and variation among strains. Applied and EnvironmentalMicrobiology 66, 2829–2834.

Parnell, S., 2003. High pressure processing of fruits and vegetables. 2003 IFT AnnualMeeting. Chicago, IL.

Patterson, M.F., Quinn, M., Simpson, R., Gilmour, A., 1995. Effects of high pressure onvegetative pathogens. In: Ledward, D.A., Johnston, D.E., Earnshaw, R.G., Hasting,A.P.M. (Eds.), High Pressure Processing of Foods. Nottingham University Press,Nottingham, pp. 47–64.

Penniston, K., Nakada, S.Y., Holmes, R.P., Assimos, D.G., 2008. Quantitative assessmentof citric acid in lemon juice, lime juice, and commercially-available fruit juice prod-ucts. Journal of Endourology 22, 567–570.

Raghubeer, E.V., Ke, J.S., Campbell, M.L., Meyer, R.S., 1995. Fate of Escherichia coli O157:H7 and other coliforms in commercial mayonnaise and refrigerated salad dressing.Journal of Food Protection 58, 13–18.

Raghubeer, E., Dunne, P., Farkas, D.F., Ting, E.Y., 2008. Evaluation of batch and semi-continuous application of High Hydrostatic Pressure on foodborne pathogens insalsa. Journal of Food Protection 63, 1713–1718.

Sergeant, E.P., Dempsey, B., 1979. Ionization Constants of Organic Acids in Aqueous So-lution. Pergamon, Oxford.

Simonne, A.H., Nille, A., Evans, K., Marshall Jr., M.R., 2004. Ethnic food safety trends inthe United States based on CDC foodborne illness data. Food Protection Trends24, 590–640.

SimplyRecipes, 2011. Available at: http://simplyrecipes.com/recipes/fresh_tomato_ salsa/.Smittle, R.B., 1977. Microbiology of mayonnaise and salad dressing: a review. Journal of

Food Protection 40, 415–422.Sommers, C.H., Sites, J.E., Musgrove, M., 2010. Ultraviolet light (254 nm) inactivation of

pathogens on foods and stainless steels surfaces. Journal of Food Safety 30,470–479.

Stewart, C.M., Jewett Jr., F.F., Dunne, C.P., Hoover, D.G., 1997. Effect of concurrent highhydrostatic pressure, acidity and heat on the injury and destruction of Listeriamonocytogenes. Journal of Food Safety 17, 23–36.

Stine, S.W., Song, I., Choi, C.Y., Gerba, C., 2005. Effect of relative humidity on pre-harvestsurvival of bacterial and viral pathogens on the surface of cantaloupe, lettuce, andBell peppers. Journal of Food Protection 68, 1352–1358.

Torres, J.A., Velazquez, G., 2009. High hydrostatic pressure processing of foods. In: Jun,S., Irudayaraj, J.M. (Eds.), Food Processing Operations Modeling: Design and Anal-ysis. CRC Press Taylor and Francis Group, pp. 173–212.

U.S.FDA (U.S. Food, Drug Administration), 2011. Bacteriological analytical manual on-line. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070149.htm. Accessed on January 17, 2012.

Uyttendaele, M., Neyts, K., Vanderswalmen, H., Notebaert, E., Debevere, J., 2004. Con-trol of Aeromonas on minimally processed vegetables by decontamination withlactic acid, chlorinated water, or thyme essential oil solution. International Journalof Food Microbiology 90, 263–271.

Whitney, B.M., Williams, R.C., Eifert, J., Marcy, J., 2008. High pressure in combinationwith antimicrobials to reduce Escherichia coli O157:H7 and Salmonella Agona inapple juice and orange juice. Journal of Food Protection 71, 820–824.

Zhao, T.M., Doyle, M.P., Besser, R.E., 1993. Fate of enterohemorrhagic Escherichia coliO157:H7 in apple cider with and without preservatives. Applied and Environmen-tal Microbiology 59, 2526–2530.