Inactivation of Salmonella enterica on tomato stem scars by antimicrobial solutions and vacuum perfusion

International Journal of Food Microbiology 159 (2012) 84–92

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Inactivation of Salmonella enterica on tomato stem scars by antimicrobial solutions and vacuum perfusion Joshua B. Gurtler a,⁎, Amanda M. Smelser b, Brendan A. Niemira a, Tony Z. Jin c, Xianghe Yan d, David J. Geveke a a

Food Safety and Intervention Technologies Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038‐8551, United States Wake Forest University Graduate School of Arts and Sciences, Wake Forest University Baptist Medical Center, Medical Center Boulevard, Winston-Salem, NC 27157, United States c Residue Chemistry and Predictive Microbiology, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, United States d Molecular Characterization of Foodborne Pathogens, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA, United States b

a r t i c l e

i n f o

Article history: Received 24 April 2012 Received in revised form 16 August 2012 Accepted 17 August 2012 Available online 23 August 2012 Keywords: Tomato Sanitizer Stem scar Salmonella Antimicrobial

a b s t r a c t A study was conducted to identify sanitizing solutions effective at inactivating ca. 5 log CFU of Salmonella enterica inoculated onto the stem scar of red round tomatoes during two-minute immersion treatments. Sixty-three antimicrobial combinations were tested. Vacuum perfusion was applied to tomatoes during selected treatments to promote infiltration of sanitizer into porous tomato stem scar tissue. Red round tomatoes were inoculated to ca. 6.9 log CFU/stem scar with a four-serovar composite of Salmonella enterica, air dried, and tomatoes were immersed in circulating sanitizing solutions for 120 s at ca. 22 °C. Stem scars were aseptically excised, macerated in DE neutralizing broth, and the homogenate was spiral plated. Twenty-four washes inactivated ≥3.0 log CFU/stem scar. Seven treatments reduced ≥ 4.8 log (viz., 40% EtOH, sulfuric acid, and organic acid combinations). Log CFU/stem scar reductions for various sanitizers are listed in parenthesis, as follows: 90 ppm peroxyacetic acid (1.31), 200 ppm chlorine (1.53), 190 ppm chlorine + 15″ Hg vacuum perfusion (2.23), 0.2 N sodium hydroxide (NaOH) (3.78), 2% total of lactic + acetic acid (4.35), 3% total of phosphoric + lactic acids (4.51), and 40% ethanol (4.81). Solutions that achieved ≥ 4.95 log reductions were 5.1% total of lactic + acetic + levulinic acids, 49% ethanol, 6% total of lactic + acetic acids, and a 0.2 M H2SO4 (sulfuric acid) solution. The use of vacuum perfusion with 200 ppm chlorine increased inactivation by 0.7 log CFU over chlorine alone, however, P > 0.05. Results from this study provide tomato processers with some sanitization options effective at inactivating Salmonella from the stem scars of tomatoes. These results may also help processors and scientists design future decontamination studies by incorporating combinations of these chemical treatments. Published by Elsevier B.V.

1. Introduction Tomatoes were associated with at least fourteen outbreaks of foodborne illness between 1996 and 2008, accounting for 17% of all produce-related outbreaks in the United States (Gravani, 2009). Between 1990 and 2007, at least 2000 human culture-confirmed cases of salmonellosis were also traced to tomatoes (Bidol et al., 2007). Salmonella serovars implicated in these events included Berta, Baildon, Braenderup, Javiana, Montevideo, Newport, Thompson and Typhimurium (Hanning et al., 2009). It has been reported that Salmonella, inoculated onto tomatoes, is capable of multiplying to populations exceeding 7 log CFU/g under appropriate conditions (Wei et al., 1995; Weissinger et al., 2000; Zhuang et al., 1995). Tomatoes are known to become contaminated with Salmonella by a number of routes, including pathogen-carrying employees, composts and manures, irrigation water, wild and domesticated animal feces, ⁎ Corresponding author. Tel.: +1 215 233 6788; fax: +1 215 233 6406. E-mail address: [email protected] (J.B. Gurtler). 0168-1605/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijfoodmicro.2012.08.014

etc. (Wei et al., 1995). Although a number of Salmonella serotypes have demonstrated the ability to survive on and in tomatoes, the degree of survival and/or persistence may be serotype-dependent. Shi et al. (2007) reported that O antigen Group C serovars (e.g., S. Hadar, Montevideo, Newport) appear to be more adapted for growth on tomatoes than those in Group D, which are more commonly associated with poultry (e.g., S. Enteritidis and Dublin). Several studies have reported the dominance and/or persistence of Salmonella serotype Montevideo in tomatoes, while serovars Poona, Hadar, Michigan, and Newport have also demonstrated the ability to grow within the fruit (Guo et al., 2001, 2002; Shi et al., 2007). Salmonella spp. are also known to grow on the surface of and within tomatoes at temperatures as low as 12 °C, and are able to survive within tomatoes at temperatures as low as 10 °C, seemingly independent of tomato variety or stage of ripeness (Beuchat and Mann, 2008; Ibarra-Sánchez et al., 2004; Iturriaga et al., 2007; Wei, et al. 1995; Zhuang et al., 1995). It has been postulated that citric acid, the primary acid present within tomatoes, may not be capable of inhibiting the survival of Salmonella at pH values as low as 4.0 (Asplund and Nurmi, 1991).

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Numerous studies have reported the efficacy of sanitizers in reducing populations of Salmonella on the surface of red or green tomatoes (e.g., Bari et al., 2002, 2003; Beuchat and Brackett, 1991; Beuchat et al., 1998; Bhagat et al., 2010; Chaidez et al., 2007; Chang and Schneider, 2012; Daş et al., 2006; Gündüz et al., 2010; Inatsu et al., 2009, 2010; Kwon et al., 2003; Obaidat and Frank, 2009; Park et al., 2008; Pao et al., 2009; Raiden et al., 2003; Rathinasabapathi, 2004; Sapers and Jones, 2006; Sommers et al., 2010; Valazquez, et al., 2009; Venkitanarayanan et al., 2002; Wei et al., 1995; Yoon, et al., 2004; Zhuang and Beuchat, 1996; Zhuang et al., 1995.) Salmonella inoculated into tomato wounds, growth cracks or stem scars, instead of onto the surface of the fruit, however, appears to have a greater capacity for survival and/or growth, and is also more difficult to inactivate without causing adverse effects on sensory quality (Wei et al., 1995; Yuk et al., 2005). Biofilm formation within the stem scar may also complicate sanitization efforts, as biofilms have been observed on tomato cuticles after 10 days of storage at 22 and 30 °C, respectively (Iturriaga et al., 2007; Iturriaga and Escartín, 2010). The stem-scar region of the tomato has been identified as an important potential source of enteric pathogen contamination due to its highly porous nature, as well as the inability of sanitizers to effectively penetrate these tissues and inactivate pathogens harbored therein (Guo et al., 2002). Nevertheless, few published studies have specifically addressed Salmonella decontamination within the stem scar region of the tomato (Guo et al., 2002). Studies by Wei et al. (1995) and Zhuang et al. (1995), in which tomatoes were immersed for 2 min in 100 ppm and 320 ppm free chlorine, did not completely inactivate Salmonella spp. even on the surfaces of tomatoes, suggesting that more advanced means of decontaminating tomatoes are needed. Yuk et al. (2005) reported that when Salmonella serovars Agona, Gaminara, Michigan, Montevideo and Poona were inoculated onto red round tomato stem scars and immersed for 2 min in sanitizing rinses at 35 °C, Salmonella reductions within the stem scar were 2.5 log (with 200 ppm chlorine from HOCl [hypochlorous acid], pH 6.5), 2.7 log (with 87 ppm peroxyacetic acid), 3.7 log (with 1200 ppm acidified sodium chlorite, pH 2.5), and greater than 5.63 log (with ClO2 gas for 1 h). While the use of chlorine dioxide gas is effective, it may be prohibitive to many producers due to cost, treatment time, and safety implications. Other means of reducing pathogen populations on the fruit are, thus, needed. The goal of the present study, therefore, was to identify economicallyfeasible concentrations of water-soluble chemical compounds effective at inactivating ca. 5 log CFU of a four-serovar composite of Salmonella from the stem scar of red round tomatoes during 2 min, room temperature immersion treatments. 2. Material and methods 2.1. Bacterial strain preparation Four serovars of 100 ppm nalidixic acid-resistant Salmonella enterica were used in this study, including Salmonella Montevideo (Salmonella group C, ATCC # 8387), S. Newport (group C, ERRC culture collection), S. Saintpaul (group B, isolate # 02-517-1 from a cantaloupe outbreak via Bassam Annous, ERRC), and S. Typhimurium (group B, ATCC #14028). Isolates were selected for spontaneous mutants resistant to 100 ppm of nalidixic acid and incubated for 24 h at 37 °C in Tryptic Soy Broth+ 100 ppm nalidixic acid (TSBN), centrifuged for 10 min at 1800 ×g, concentrated four-fold by re-suspending in 25% of the original suspension volume with sterile 0.1% peptone water, and composited in a single test tube. 2.2. Inoculation of tomato stem scars Red round tomatoes were purchased at local supermarkets and stored at 12 °C. One day prior to each experimental repetition, tomatoes

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were moved from 12 °C storage and equilibrated to room temperature overnight. Stem scar diameters were measured with digital calipers. The four-serovar Salmonella inoculum suspension was deposited in ten-10 μl quantities evenly across the surfaces of each stem scar for a total inoculum volume of 100 μl per stem scar. Tomatoes were placed in a continuously circulating aseptic laminar flow hood to allow inocula to dry for ca. 4 h at 22±2 °C. 2.3. Electron microscopy Salmonella-inoculated and dried stem scars were aseptically excised from each tomato with flame-sterilized knives. The surface of each stem scar was thinly-sliced in a single layer (0.3 mm thick) parallel to the stem scar with a flame-sterilized scalpel to assess the extent of inocula infusion into the porous stem scar tissue. Thin stem scar slices were fixed for scanning electron microscopy (SEM) by immersion in a 2.5% glutaraldehyde-0.1 M imidazole buffer (Electron Microscope Sciences, Hatfield, PA) for 1 h before washing in imidazole buffer and dehydrating in 50%, 80% and absolute ethanol, successively. Samples were critical point dried (Denton Vacuum, Cherry Hill, NJ) with carbon dioxide, mounted with Duco cement (ITW Performance Polymers, Riviera Beach, FL) and colloidal silver adhesive, and sputter-coated with a thin layer of gold using a Scancoat Six Sputter Coater (BOC Edwards, Wilmington, MA). Samples were imaged with a Quanta200 FEG environmental scanning electron microscope (FEI Co., Inc., Hillsboro, OR), with an Everhart Thornley detector, operated in the high vacuum, secondary electron imaging mode at an accelerating voltage of 5 kV. 2.4. Sanitizing immersion treatments Approximately 100 different combinations of sanitizers, as well as sterile deionized water, were tested for inactivating Salmonella on the tomato stem scar, based on compounds reported in Tables 1 and 2. Other compounds that we tested, which are not listed in this report, include those supplied by manufacturers that achieved only minimal levels of inactivation (e.g., b2 log CFU/stem scar), as well as sanitizing combinations that have been withheld from this publication due to patent potential. Sanitizing solutions (700 ml) were prepared in a sterile, 1000 ml beaker containing a magnetic Teflon-coated stir bar and placed on top of a magnetic stir plate. The top half of a transparent, circular polypropylene test tube rack (cut bilaterally) with holes drilled through the side wall (see Fig. 1), was placed in the beaker over a stir bar. Holes in the top of the test tube rack designed to hold 16 mm test tubes, as well as holes drilled through the sidewall permitted circulation of water throughout the beaker during treatments. An inoculated tomato was then immersed in the sanitizing solution on top of the circular rack, while the solution was continuously agitated by the stir bar (Fig. 1). All tomatoes were treated in respective sanitizing solutions for 120 s at 22 ± 2 °C. The pH of the solutions was measured using an Accumet single junction, gelled Ag/AgCl, flat surface electrode (Fisher Scientific, Pittsburgh, PA) connected to a Denver Instrument model UB-5 bench top pH meter (Denver, CO). Duplicate tomatoes were sampled for each respective sanitizing solution in each experimental repetition. 2.5. Vacuum perfusion treatment To determine the efficacy of vacuum perfusion to promote infiltration of sanitizer into the porous tomato stem scar tissue, selected treatments were conducted in a vacuum chamber (Bactron IV Anaerobic Chamber, Sheldon Manufacturing, Cornelius, OR). Tomatoes were immersed in sanitizer solutions, and placed in a vacuum chamber on top of a battery-operated stir plate. A vacuum of 15″ Hg was drawn in the chamber, held for 30 s, and then released to ambient air pressure for a total treatment time of 2 min (Fig. 2). A vacuum of 15″ Hg was chosen

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Table 1 Preliminary sanitizer combinations screened (two replicate tomatoes per antimicrobial treatment) to assess suitability of compounds to decontaminate a four-serovar composite of Salmonella from the stem scars of tomatoes via circulating immersion treatment for 2 min. Sanitizer

Ave. pHa

Supplierb

Abbreviation in this study

1.5% each phosphoric+acetic acid 0.67% each levulinic, lactic, acetic acid 0.5% each levulinic, lactic, acetic acid 0.33% each levulinic, lactic, acetic acid 0.1% levulinic+1% acetic+0.33% lactic acid 0.1% levulinic + 1% acetic + 0.5% lactic acid 0.625% each of phosphoric, levulinic, acetic, lactic acid 0.5% each of levulinic + SDS 90 ppm peracetic + 0.3% lactic acid 3% lactic acid + 3% EDTA 2.7% PABA 2.7% PABA + 3% lactic acid 325 ppm bromine 200 ppm chlorine + 310 bromine 78 ppm peracetic acid + 1% SDS 1% lactic acid + 1% H202 1% acetic acid + 0.3% nisin 1% acetic acid + 1% H202 3% hydrogen peroxide + 1% FeSO4 + 3% SDS 200 ppm Cl + 3% SDS 0.2 N NaOH + 1% SDS 0.4% ammonium hydroxide 0.2% ammonium hydroxide 1% ammonium hydroxide + 1% EDTA 1% sodium metasilicate 0.025 N sodium hydroxide 0.2% calcium oxide 0.4% calcium oxide 1% calcium hydroxide 1% trisodium phosphate 1% SDS + 0.5% calcium oxide 0.2 N NaOH + 1% EDTA

1.20 2.46 2.54 2.41 2.41 2.33 1.59 3.03 2.54 3.43 3.42 2.06 4.84 7.27 3.88 2.45 2.89 2.37 3.50 9.70 13.15 11.02 11.11 10.47 12.58 12.11 12.52 12.55 12.66 12.21 12.76 12.85

MPc and J.T. Baker Acrosd, Acros, J.T. Bakere Acros, Acros, J.T. Baker Acros, Acros, J.T. Baker Acros, J.T. Baker, Acros Acros, J.T. Baker, Acros MP, Acros, J.T. Baker, Acros Acros, MP FMCf, Acros Acros, Sigma Aldrichg Sigmah Sigma, Acros Acros Red Birdi Multiple suppliers, MP Acros, Fisherj J.T. Baker, MP J.T. Baker, Fisher Fisher, Spectrumk, MP Red Bird, MP Mallinckrodtl, MP MP MP MP, Sigma Aldrich MP Mallinckrodt MP MP MP MP MP, MP Mallinckrodt, Sigma Aldrich

3%PhosAc 2%LeLaAc 1.5%LeLaAc 1%LeLaAc 1.43%AcLeLac 1.6%AcLeLac 2.5%PhLeAcLa 0.5%Lev&SDS 90paa0.3%Lac 3%LacEDTA 2.7%PABA 2.7%PABA3%Lac 325Bromine 200CL310Br 78paa1%SDS 1%Lac1%H2O2 1%Ac0.3%Nis 1%Ac1%H202 FentonSDS 200Cl3%SDS 0.2NaOH1%SDS 0.4%NH4OH 0.2%NH4OH 1NH4OH1%EDTA 1%NaMet 0.025NaOH 0.2%CaO 0.4%CaO 1%CaOH2 1%TSP 1%SDS0.5%CaO 0.2Na1%EDTA

a b c d e f g h i j k l

The pH values represent averages from experimental repetitions taken prior to immersion treatments of two tomatoes. Chemical suppliers are provided according to the order of compounds listed under the sanitizer column. MP Biomedicals, LLC. Solon, OH. Acros Organics, NJ. J.T. Baker, Phillipsburg, NJ. FMC Corporation, Tonawanda, NY. Sigma Aldrich Inc., St. Louis, MO. Sigma Chemical Co, St. Louis, MO. Red Bird Service, Batesville, IN. Fisher Scientific, Fair Lawn, NJ. Spectrum Chemical MFG. Corp., Gardena, CA. Mallinckrodt Baker, Inc., Paris KY.

based on results from preliminary studies revealing that vacuums higher than 15″ Hg resulted in low pressure-induced cracking and dislocation of stem scar from the tomato (data not shown). 2.6. Stem scar sampling for the presence of Salmonella Tomato stem scars (ca. 5 g each) were aseptically excised from the fruit with kitchen knives by cutting a circular cone-shaped pattern around the stem scar ca. 3 cm deep. Each stem scar was added to 40 ml of Dey Engley Neutralizing Broth (Difco Laboratories, Becton, Dickinson & Company, Sparks, MD), in a Whirl-Pak® filter bag and stem scars were carefully macerated in the bag on the lab bench top with a blunt steel mallet, as well as with a marble rolling pin. Macerated samples in filter bags were then pummeled in a stomacher for 90 s. Sample filtrates were spiral plated (50 μl/plate) with a Wasp II, Don Whitley Spiral Plater (Microbiology International, Fredrick, MD) in duplicate onto Tryptic Soy Agar (Difco) + 100 ppm nalidixic acid+ 0.1% sodium pyruvate (Sigma Aldrich, St. Louis, MO) (TSAPN) to assist in the recovery of injured cells (Gurtler and Beuchat, 2005; Gurtler and Kornacki, 2009; Wesche et al., 2009). Filtrates (undiluted) were also spread-plated (250 μl) in duplicate with sterile plastic plating applicators. All plates were allowed to dry and incubated for 18–24 h at 37 °C before colonies were counted with an automated digital colony

counting system (Synbiosis åCOLyte® Supercount, Microbiology International). Presumptive-positive Salmonella colonies were randomly confirmed by plating on Xylose Lysine Desoxycholate agar (XLD, Difco), as well as by a serological agglutination test (Salmonella O Antiserum Poly A-I & Vi (Difco)). Negative control filtrates from uninoculated stem scars were also plated onto TSAPN to determine the presence of background microflora. 2.7. Statistical analysis Two tomatoes were sampled in each experimental trial that was conducted. For preliminary studies, two replicate tomatoes (duplicate samples) were treated for each single sanitizing solution reported, to assess sanitizer efficacy (Table 1). Chemical compounds deemed antimicrobially suitable, or that otherwise merited further testing underwent, a more extensive evaluation (Table 2). For these studies, numbers of replicate experiments performed for each respective sanitizer are listed in Table 2. Duplicate samples from each experimental trial were transformed to log10 values, and averaged. Data were analyzed by ANOVA, and significant differences (P b 0.05) in the inactivation of Salmonella between treatments were determined by Tukey's Studentized Range HSD test via SAS version 9.1 (SAS Institute, Inc., Cary, NC).

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Table 2 Sanitizer combinations used to decontaminate a four-serovar composite of Salmonella from the stem scars of tomatoes. Tomatoes were treated with sanitizers for 2 min under circulating immersion. Sanitizer

Ave. pHa

Supplierb

Number of Repetitionsc

Abbreviation used in this study

6% household bleach solution (3.47 ml diluted into 1000 ml of water) 6% household bleach solution (3.47 ml diluted into 1000 ml of water)+vacuum perfusion 200 ppm free chlorine Vacuum + 190 ppm free chlorine 1% levulinic + 1% SDS 3% each of levulinic + SDS 1% lactic + 1% acetic acid 1.5% lactic + 1.5% acetic 3% lactic + 3% acetic acid 2.5% lactic + 2.5% acetic acid 0.83% each of levulinic, acetic, lactic acids 1% each of levulinic, acetic, lactic acids 1.34% each levulinic, lactic, acetic acids 1.7% each levulinic, lactic, acetic acids 2% each of levulinic, acetic, lactic acids 1.5% each of phosphoric + lactic acid 0.3 M sulfuric acid 0.2 M sulfuric acid 0.15 M sulfuric acid 0.10 M sulfuric acid 3% H2O2 + 1% FeSO4 49% EtOH 40% EtOH 30% EtOH 20% EtOH 0.2 N NaOH 0.1 N NaOH 0.05 N NaOH 90 ppm peroxyacetic acid 2 min in 3% lactic acid followed by a sterile deionized water rinse and then 2 min in 0.2 N NaOH rinse 1% cetylpyridinium chloride

10.22

Cloroxd

3

Bleach

10.22

Clorox

3

Bleach+Vac

a b c d e f g h i j k l

e

10.60 10.37 2.42 2.76 2.11 2.12 1.78 2.00 2.31 2.23 2.10 2.08 1.99 1.31 0.51 0.62 0.66 0.82 2.81 6.38 6.12 5.93 5.86 12.91 12.80 12.48 3.53 1.88/12.94

Red Bird Red Bird Acrosf, and MPg Acros, and MP Acros, and J.T. Bakerh Acros, and J.T. Baker Acros, and J.T. Baker Acros, and J.T. Baker Acros, J.T. Baker, and Acros Acros, J.T. Baker, and Acros Acros, Acros, and J.T. Baker Acros, Acros, and J.T. Baker Acros, J.T. Baker, and Acros MP, and Acros J.T. Baker J.T. Baker J.T. Baker J.T. Baker Fisher Scientifici, Spectrum Chemicalj Warner-Grahamk Warner-Graham Warner-Graham Warner-Graham Mallinckrodtl Mallinckrodt Mallinckrodt Available from many manufacturers Mallinckrodt, Acros

3 3 5 6 5 5 3 2 2 5 2 2 4 4 3 6 4 4 3 3 2 3 3 3 3 2 4 2

200ppmCl 190ppm Cl+Vac 1%Lev1%SDS 3%Lev3%SDS 2%LacAc 3%LacAc 6%LacAc 5%LacAc 2.5%LeAcLac 3%LeLaAc 4%LeLaAc 5%LeLaAc 6%LeLaAc 3%PhosLac 0.3Msulf 0.2Msulf 0.15Msulf 0.10Msulf Fenton 49EtOH 40EtOH 30EtOH 20EtOH 0.2NaOH 0.1NaOH 0.05NaOH 90paa 3%Lac0.2NaOH

5.68

MP

2

1%CPC

The pH values represent averages from experimental repetitions taken prior to immersion treatments of two tomatoes. Chemical suppliers are provided according to the order of compounds listed under the sanitizer column. Each experimental repetition consisted of testing duplicate microbiological samples from each of two tomatoes treated in the same sanitizing solution. Clorox Company, Oakland, CA. Red Bird Service, Batesville, IN. Acros Organics, NJ. MP Biomedicals, LLC. Solon, OH. J.T. Baker, Phillipsburg, NJ. Fisher Scientific, Fair Lawn, NJ. Spectrum Chemical MFG Corp., Gardena, CA. The Warner-Graham Company, Cockeysville, MD. Malinckrodt Baker, Inc., Paris, KY.

3. Results Mean stem scar diameters were 14.22 mm (0.56″), with a standard deviation of ±2.36 mm (0.09″) and an average mass of ca. 5 g/stem scar. Electron micrographs revealed that Salmonella inoculated onto tomato stem scars were capable of infiltrating the porous, cellulosic stem scar tissues, as observed from absorption through the surface to the underside of the thinly-sliced stem scar tissue in Fig. 3. 3.1. Preliminary experiments The goal of the present study was to identify sanitizers capable of inactivating ca. 5 log CFU/stem scar of a four-serovar composite of Salmonella in red round tomatoes. Fig. 4 is a graphical representation of results from preliminary studies, consisting of two replicate tomato treatments, conducted to assess the efficacy of thirty-six sanitizers in inactivating Salmonella from the stem scar of red round tomatoes. Preliminary experiments were conducted to identify compounds suitable for performing further studies. More extensive experiments were conducted on compounds that either achieved high levels of inactivation (ca. 4 log CFU or greater) (see Fig. 5), compounds that are commonly used or of interest to the food industry (e.g., 200 ppm

chlorine from NaOCl, 90 ppm peroxyacetic acid, etc.) or chemical compounds that were of special interest in replicating for statistical comparative purposes (e.g., ethanol, NaOH, organic acids, etc.). The pH values of respective sanitizer solutions in Fig. 4 are listed in Table 1. All sanitizers in Fig. 4 inactivated ≤ 3.97 log CFU/stem scar of Salmonella, while 29 of 32 (91%) and 16 of 32 (50%) of solutions in Fig. 4 achieved ≤ 3 and ≤2 log CFU/stem scar reductions, respectively. Seven sanitizers (22%) in Fig. 4 effected Salmonella reductions of b1 log per stem scar. Ammonium hydroxide (NH4OH, or ammonia), which has been successfully used as a food decontaminant with other commodities (e.g., Gupta et al., 1988; Stopforth et al., 2004; Jensen et al., 2009), was ineffective for our purposes at levels of up to 1%. The strong aroma produced by this compound would also likely have negative sensory impacts on organoleptic properties of tomatoes. Calcium oxide (CaO, also known as calcinated calcium or quicklime) (Bari et al., 2002) as well as calcium hydroxide [Ca(OH)2, also known as slaked lime or pickling lime] (Neetoo et al., 2009), which has reported antimicrobial action as an alkaline, were ineffective for the purposes of this study at levels of 0.4 and 1%, respectively (Fig. 4). The addition of nisin or EDTA did not appear to increase bacterial inactivation during the two‐minute immersion rinses.

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Fig. 1. Circulating sanitizing immersion treatment of tomato.

3.2. Multiple-replicate experiments The results of multiple replicate experiments with each of thirty-three immersion treatments are displayed in Fig. 5. The pH values of respective sanitizer solutions in Fig. 5 are listed in Table 2. Immersing tomatoes in 200 ppm free chlorine (generated from hypochlorous acid via sodium hypochlorite), induced reductions of only 1.5 log CFU/stem scar. However, when combined with vacuum perfusion, 2.2 log CFU per stem scar of

Salmonella was inactivated by 190 ppm chlorine+vacuum, which was not statistically different (P>0.05) than 200 ppm chlorine without vacuum. Similar results were apparent after rinsing with a diluted household bleach solution. The household bleach dilution used in this study (3.47 ml of 6% bleach into one liter of water) is equivalent to 15 ml (or 1 tablespoon) of 5.25% bleach per gallon of water, which is a common dilution used for kitchen and household surface sanitization. The inactivation of Salmonella on tomatoes when treated with the diluted

Fig. 2. Circulating sanitizing immersion of tomato under vacuum perfusion treatment at 15″ Hg in a vacuum chamber.

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Fig. 3. Scanning electron micrographs of the underside of thinly-sliced sections of tomato stem scars, following outer inoculation with Salmonella and drying. Tissues were visualized at magnifications of (A) 50×, (B) 100×, (C) 250×, (D) 500×, (E) 1000×, (F) 2500×, and (G) 5000×.

household bleach solution increased by 0.9 log CFU per stem scar (from 2.4 up to 3.3 log) when vacuum perfusion was included in the treatment, although the difference was not statistically significant (P>0.05). The use of ethanol as a chemical sanitizer has been reported (Bari et al., 2009). Ethanol treatments in this study resulted in increasing log

reductions of Salmonella as concentrations of the alcohol increased (viz., 1.1 log with 20%, 2.7 log with 30%, 4.8 log with 40% and 5.1 log with 49% ethanol). Relatively high concentrations of five other organic and/or inorganic acids were effective at reducing Salmonella by ca. 5 log CFU/stem scar, including 0.2 and 0.3 M sulfuric acid, 1.7% each,

3.97

Reduction in Population (Log CFU/stem scar)

4.0 3.60

3.5

3.40

3.0

2.91 2.85 2.88 2.88 2.71 2.73 2.75 2.60 2.62 2.45

2.5 2.23

2.29

2.13 1.96

2.0 1.76 1.70 1.74 1.49

1.5

1.55 1.56

1.31 1.20 0.99 0.99

1.0

0.90

0.5

0.42 0.13 0.16 0.00

0. 2%

N

2. H4 7 O 0. %P H 4% A N BA H 0. 4OH 2% 0. Ca 4% O 1% C N H 1% aO 4O N H aM 1% e 32 E t 5B DT r A 1% omi 78 C ne pa aO a1 H2 % 1% SD S 0. DS 1% S 2N 0 T aO .5% SP H Ca 1% O 0. ED 02 T 20 5N A 0 a 1% CL OH A 31 90 c0 0B pa .3% r a0 N .3 is 1% %L 20 Le ac 0C La l3 Ac Fe %S 0. nto DS 5% n L SD 1. ev S 6 & 1% %A SD La cL S c1 eL a 3% %H c La 2O 2 c 1. 5 ED 1. %L TA 43 e 0. % La 2N Ac Ac aO Le H La 1 c 2% %S Le DS La 2. 3 7% % A PA Pho c 1% BA sAc 3 A % 2. c1 La 5% % c Ph H2 Le 02 Ac La

0.0

Fig. 4. Results from preliminary sanitizer screening studies (two replicate tomatoes per antimicrobial treatment), conducted to assess efficacy of sanitizers in inactivating Salmonella from the stem scar of red round tomatoes.

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J.B. Gurtler et al. / International Journal of Food Microbiology 159 (2012) 84–92 6.91 6.91

Reduction in Population (Log CFU/stem scar)

7.0

6.00

6.0 5.53

4.95

5.0

5.05

4.81 4.61 4.35 4.38 4.39

4.0 3.65

3.84 3.73 3.78

3.91 3.95

4.02

4.10

3.34 3.00

3.0 2.65 2.38 2.21 2.23 1.98

2.0 1.47 1.47 1.53 1.31 1.14

1.0 0.61

W 20 ate Et r O 90 H 1% paa 0. C 05 P 3% 20 Na C La 0p OH p 2. c0.2 mC 19 5% Na l 0p Le OH pm Ac C Lac l+ V Bl ac e Fe ach n Bl 30E ton ea tO ch H 3% 0. +Va Le 1N c v3 aO % H 0. SD 4% 2Na S 1% L O Le eL H v1 aA % c 3% S L D 0. ac S 10 Ac 5% Ms L u 2% ac lf L Ac 0. acA 15 c 3% M 3% Le sulf Ph LaA os c 4 La 5% 0E c Le tOH L 49 aAc 6% EtO La H 0 c 6% .2M Ac Le su L lf 0. aA 3M c su lf

0.0

Fig. 5. Inactivation of a four-serovar composite of Salmonella Montevideo, Newport, St. Paul and Typhimurium from the stem scar of red round tomatoes.

or 2% each (5.1% and 6% total, respectively) of acetic, phosphoric and levulinic acids, and 3% each (6% total) of lactic and acetic acids. A 0.15 M H2SO4 (sulfuric acid) solution resulted in reductions of 4.4 log CFU per stem scar of Salmonella, thus 0.2 M H2SO4 was determined to be the minimum concentration of any sanitizing solution capable of achieving a 5 log reduction. 4. Discussion The present study was conducted in order to identify sanitizer treatments sufficient to inactivate ca. 5 log CFU/stem scar of Salmonella on red round tomatoes and, to our knowledge, is only the second study published evaluating the decontamination of Salmonella from tomato stem scars. A previous study with similar objectives (Yuk et al., 2005) reported that treating inoculated tomatoes with sanitizers resulted in Salmonella/stem scar reductions of 2.5 log (with 200 ppm chlorine from HOCl [hypochlorous acid], pH 6.5), 2.7 log (87 ppm peroxyacetic acid), 3.7 log (1200 ppm acidified sodium chlorite, pH 2.5), and greater than 5.63 log CFU/tomato (ClO2 gas for 1 h). While we also tested 200 ppm chlorine generated by HOCl, our results (1.53 log reduction) were lower than those of Yuk et al. (2005) (viz., 2.5 log reduction). Differences between studies might be a function of our higher pH value (10.6). Chlorine is known to be more effective in the neutral pH range versus under alkaline conditions and NaClO (sodium hypochlorite), itself, is innately alkaline. Likewise, we tested 90 ppm peracetic acid as Yuk et al. (2005) did; however, we inactivated only 1.3 log CFU of Salmonella versus their 2.7 log CFU reduction. Differing results between our study and those of Yuk et al (2005) may reflect variations in bacterial strains or inoculum levels. Data from our preliminary experiments (Table 1 and Fig. 4) represent compounds, formulations and treatments tested on two replicate tomatoes, which were not sufficient to achieve suitable reductions of Salmonella on tomato stem scars for the purposes of our study, or otherwise were deemed not to warrant further investigation. As such, these preliminary results do not represent a complete battery of experiments; however, their inclusion in this report may provide useful information for researchers in designing future sanitizer efficacy studies. Further, screening of numerous compounds in this study precluded extensive testing with other variables such as temperature, immersion time and water hardness.

We tested three sanitizer combinations that included both an inorganic and organic acid; namely, (1) 1.5% each (3% total) of phosphoric + acetic acid; (2) 0.625% each (2.5% total) of phosphoric+ levulinic + acetic+ lactic acid; and (3) 1.5% each (3% total) of phosphoric + lactic acid, resulting in Salmonella reductions of 3.97, 2.91, and 4.61 log CFU, respectively. Studies evaluating the antimicrobial efficacy of an organic acid+ inorganic acid combination have been published (Grinstead and Angevaare, 2003; Lemons, 2009; Nou et al., 2011). Combining an organic acid with an organic acid may result in a complementary antimicrobial pair — as a strong inorganic acid reduces the pH of the solution significantly, while the organic acid (with a higher pKa value) is known to be more bacteriocidal at lower concentrations, due to its ability to cross the bacterial membrane, dissociate, and shed a proton within the bacterial cytoplasm (Ahamad and Marth, 1989; Doores, 2005). Ethanol (EtOH) was tested at a maximum concentration of 49% (98 proof) in order to fall below the ignition point (i.e., 50% or 100 proof) for employee safety. Our study has demonstrated that a 49% EtOH solution is capable of inactivating 5 log CFU on the stem scar of tomatoes; however, the practicality of such a treatment might be more suitable for restaurant, kitchen, institutional, and home use, whereas large-scale industrial applications may be cost-prohibitive. Nevertheless, a terminal EtOH treatment in a filtered, recirculating EtOH dump tank might be feasible, considering that EtOH could be re-used, would not have to be changed as often as water in a traditional dump tank, and may serve to extend the shelf life of the fruit by inactivating spoilage bacteria, yeasts and molds. Economic infeasibility, however, would also accompany the use of three other treatments effecting ca. 5 log reductions on stem scars; namely, 1.7% or 2% each (5.1% and 6% total, respectively) of acetic, phosphoric and levulinic acids, and 3% each (6% total) of lactic and acetic acids. The lowest concentration of any compound capable of inactivating ca. 5 log CFU per stem scar was a 0.2 M solution of sulfuric acid. Treatment with 0.2 M H2SO4, while being economically feasible and antimicrobially effective, may present health risks to employees and induce corrosion of stainless steel. Successful adoption of 0.2 M H2SO4 immersion treatments as an intervention for Salmonella on tomatoes would require carefully-designed and continuouslymonitored equipment and processing conditions, as well as organoleptic studies to determine the effects on produce sensory properties.

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Possibly due to the porous nature of the stem scar region of the tomato, inter-repetition results fluctuated, apparent in the error bars displayed in Fig. 5. An effective sanitizer must be capable of infiltrating the network of cellulosic tissues that compose the stem scar and, preferably, would be able to penetrate and/or disrupt biofilms and/or other organic matter that may have dried or aggregated, following a liquid contamination event (Iturriaga et al., 2007; Iturriaga and Escartín, 2010). Chang and Schneider (2012) compared flume tank immersion decontamination of tomatoes with an overhead sanitizer spray+ brush roller system. The authors reported that while a 60 s flume tanktreatment achieved only 3.3 and 1.3 log CFU/ml reductions in Salmonella with 100 ppm NaOCl and plain water, respectively, a 60 s spray and brush treatment inactivated 5.5, 5.5, 4.9, and 3.8 log CFU/ml of Salmonella with 100 ppm NaOCl, 80 ppm PAA, 5 ppm ClO2, and plain water, respectively. Differences between our reported results and those of Chang and Schneider (2012) may be accounted for based on the difference in sanitizer application (viz., our immersion treatment versus their spray and brush application) as well as Salmonella inoculation procedures. While we inoculated with nalidixic acid-resistant Salmonella at 6.9 log CFU/tomato for sub-surface absorption into the stem scar tissue, Chang and Schneider (2012) spot-inoculated tomatoes with rifampicinresistant Salmonella in a circle around the blossom scar on the tomato surface with 10 spots of 10 μl inoculum each, for a total of 100 μl inoculum per tomato, or ca. 8.5 log CFU/tomato. A spray and brush system, as reported by Chang and Schneider (2012), in combination with sanitizers that we report in this study may achieve higher reductions than either of our studies report individually. Considerations for future research may include studying the genetic response of Salmonella serovars to various tomato cultivars as well as the influence of extrinsic parameters (Guo et al., 2002) so as to select complimentary inactivation intervention treatments. A number of studies have examined the genetic response of Salmonella and other Gram-negative bacteria when inoculated into tomato tissue (Brown and Allen, 2004; Noel et al., 2010; Tamir-Ariel et al., 2007). Further work in this area could generate useful information related to the resistance or susceptibility of tomatoes to bacterial growth. Growing strategies could be adjusted to take advantage of the genetic propensity of Salmonella to survive and multiply in relation to cultivar, stage of ripeness, rainfall and moisture content, temperature and exposure to sunlight, storage and sanitizing conditions, etc. Future studies may be conducted to discover novel Salmonella regulatory loci controlling oxygen- and acid-response gene expression (Foster and Hall, 1990) on stem scars of various tomato cultivars, as well as in the presence of sanitizing solutions and vacuum perfusion. It is also interesting that, in one regard, tomatoes are more resistant to post-harvest invasion of tissues than other cut produce (e.g., broccoli) due to the natural abscission zone at the stem scar, which precludes the need for cutting the plant during harvesting (Mahovic, 2006). Salmonella may have differential gene expression patterns on the stem scar versus within the tomato, or on other wounding sites. Therefore, a systematic analysis of the tomato and Salmonella transcriptome with a metagenomics approach by deep sequencing is another potential research perspective. All of these approaches may lead to the identification of molecular intervention strategies targeting Salmonella survival and proliferation in and on the fruit, which could be coupled with complimentary terminal sanitization treatments. Further, the depth of potential Salmonella penetration into the tomato tissue during vacuum treatment should also be addressed. Results from this study provide producers with some sanitization options effective at inactivating Salmonella from the stem scar of tomatoes. We have here presented results from sixty-three tomato immersion treatments. This data may also help processors and scientists in designing novel decontamination studies with tomatoes and other fresh produce commodities. Additionally, the use of vacuum

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perfusion, which increased the inactivation of Salmonella by 1 log CFU/stem scar in a diluted household bleach solution (equivalent to 1 tablespoon of 5.25% household bleach into one gallon of water) may be explored for similar synergistic benefits with other sanitizing solutions and/or other fruit and vegetable commodities. Acknowledgments The authors thank Rebecca Bailey, Richard Sims and Anita Paramaswaren for excellent technical laboratory assistance; Mike Haas and Karen Wagner for help with chemical analysis; David Kingsley, Xuetong Fan and Dike Ukuku for critical reviews of this manuscript and providing helpful feedback; John Phillips for assistance with statistical analysis, and Doug Soroka (ERRC Core Technologies Unit) for assistance with electron microscopy imaging. This study was funded by the USDA through ARS National Program 108, ARS CRIS project 1935‐41420‐017. 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