Rain splash-mediated dispersal of Escherichia coli from fecal deposits to field-grown lettuce in the mid- and south Atlantic U.S. regions is affected by mulch type.

Introduction: Wildlife feces can contaminate vegetables when enteric bacteria are released by rain and splashed onto crops. Regulations require growers to identify and not harvest produce that is likely contaminated, but U.S. federal standards do not define dimensions for no-harvest zones. Moreover, mulching, used to retain soil moisture and maximize crop yield may impact rain-mediated bacterial dispersal from feces. Methods: To assess Escherichia coli dissemination from a fecal point source to lettuce grown on various mulches, lettuce cv. 'Magenta' was transplanted into raised beds with plastic, biodegradable plastic, straw, or left uncovered at field sites in Maryland and Georgia. Eleven days post-transplant, 10 g of rabbit manure spiked with ~8 log CFU g-1 E. coli were deposited in each bed. One day following natural or simulated rain events, lettuce was sampled along 1.5 m transects on either side of fecal deposits. Lettuce-associated E. coli was semi-quantified with an MPN assay and dependence on fecal age (stale or fresh), lettuce age (baby leaf or mature head), distance from point source, mulch and post-rain days were statistically evaluated. Results: Distance (p<0.001), fecal age (p<0.001) and mulch (p<0.01) were factors for E. coli transfer from point source to lettuce. The highest and lowest E. coli estimates were measured from lettuce grown on biodegradable plastic and straw, respectively, with a 2-log MPN difference (p<0.001). Mulch and distance were also significant factors in E. coli recovery 3 days post-rain (both p<0.001), where plastic mulches differed from bare ground and straw (p<0.01). For all treatments, fewer E. coli were retrieved from lettuce at 0.3 m, 3 days post-rain compared to 1 day (p<0.001). Fitting the data to a Weibull Model predicated that a 7-log reduction in E. coli from fecal levels would be achieved at 1.2-1.4 m from the point source on plastic mulches, 0.75 m on bare soil (p<0.05) and 0.43 m on straw (p<0.01). Discussion: Straw and bare ground limited rain-mediated E. coli dispersal from feces to lettuce compared to plastic mulches. Fecal age was negatively associated with E. coli dispersal. These findings can inform harvesting recommendations for measures related to animal intrusion in vegetable production areas.

The effect of emulsifier type and oil inclusion on stress-related gene expression of Salmonella typhimurium in oil-in-water emulsion.

Salmonella has been associated with numerous outbreaks from contaminated food products, including emulsions. Emulsions are influenced by emulsifier type and oil presence, which can have varying degrees of stress or protection on bacteria. Although our previous research has shown that emulsifier solutions, rather than emulsions, provide a protective effect on Salmonella typhimurium after thermal treatment, the underlying mechanism remains unclear. This study selected S. typhimurium as the model microorganism and utilized the same emulsifiers (Tween 20, Tween 80, Triton X-100) to create emulsifier solutions and emulsions with the same oil fraction (60% (v/v)) to examine their effect on the expression of nine selected genes (rpoE, rpoH, otsB, proV, fadA, fabA, dnaK, ibpA, ompC) associated with stress response. Specifically, the study observed variations in gene expression under normal and thermal stress at 55°C. After 20-h incubation, Triton X-100 emulsion caused an upregulation of stress-related genes, rpoE, otsB, and fabA, suggesting stressful environment. After thermal treatment, S. typhimurium in Triton X-100 solution showed a longer 5-log reduction time with increased proV and decreased fabA and ompC expression, suggesting enhanced thermal protection compared to its emulsion. Conversely, Tween 80 solution increased fabA and ompC expression, indicating greater membrane fluidity and passive diffusion, potentially reducing thermal resistance. However, according to the upregulation of ibpA, this effect was likely mitigated by the overproduction of heat shock proteins. Notably, Triton X-100 environments exhibited the most significant gene expression changes after heat treatment, whereas Tween 80 without oil was the most inhospitable for bacterial survival. These findings inform bacterial responses under various conditions, aiding food safety strategies.

The effect of emulsifier type and oil fraction on Salmonella Typhimurium growth and thermal inactivation in oil-in-water emulsion.

High water activity oil-in-water emulsions can promote survival and growth of Salmonella Typhimurium. Nevertheless, the precise effect of emulsifier type and oil content on bacterial growth and inactivation is not fully understood. Here, emulsions were prepared using different emulsifiers (Tween 20, Tween 80, and Triton X-100) and different oil fractions (20%, 40%, and 60% (v/v)). TSB (control), emulsifier solutions, and emulsions were inoculated with S. Typhimurium. Bacterial growth rate was measured at 7, 22, and 37°C, whereas thermal inactivation was performed at 55°C. Growth and inactivation data was fitted into Logistic and Weibull models, respectively. At an incubation temperature of 37°C, the presence of high amount of oil (60%) in Tween 20 and Triton X stabilized emulsions extended the lag phase (5.83 ± 2.20 and 9.43 ± 1.07 h, respectively, compared to 2.28 ± 1.54 h for TSB, p < 0.05), whereas individual emulsifiers had no effect on growth behavior compared to TSB. This effect was also prevalent but attenuated at 22°C, whereas no growth was observed at 7°C. In thermal inactivation, we observed protective effect in Tween 80 and Triton X-100 solutions, where time required for five-log reduction was 1914.70 ± 706.35 min and 795.34 ± 420.09 min, respectively, compared to 203.89 ± 10.18 min for TSB (p < 0.05). Interestingly, the presence of high amount of oil did not offer protective effect during thermal inactivation. We hypothesize that oleic acid in Tween 80 and lower hydrophobicity value of Triton X-100 help maintain membrane integrity and improve the resistance of bacteria to heat inactivation.

Emulsion matrixes affect Salmonella survival during simulated gastric digestion.

Salmonella enterica subsp. enterica serovar Typhimurium is an emerging prominent foodborne pathogen worldwide. While its acid resistance and pathogenicity have been investigated over the years, there is a necessity to systematically study how food matrices impact Salmonella's resistance to environmental stresses and survivability in the digestive tract. In this study, coarse water-in-oil (W-O) emulsion and oil-in-water (O-W) emulsion matrices were inoculated with Salmonella in the oil and water phase, respectively. The emulsion matrices were then challenged with simulated gastric acid (pH 2 HCl solution with 3 g L-1 pepsin) facilitated with stomacher mixing at 37 °C, and samples were taken at designated time points for bacterial counts. The survival curves suggested that the W-O emulsion offered a significant protective effect against simulated gastric digestion, which achieved 1.55 ± 0.61 log(CFU ml-1) reduction in 60 minutes. However, the same level of protection was not observed in the O-W emulsion, and it showed 4.54 ± 0.69 log(CFU ml-1) reduction in 60 minutes. As for the acid resistance of Salmonella, there was no significant difference between water phase and oil phase inoculation. In addition, the protective effect could be mainly attributed to the W-O emulsion structure but not simply to the high viscosity of the W-O emulsion matrix. Furthermore, the results also indicated that more than 16.3% of bacterial cells were present in the oil phase of the W-O emulsion, which was critical to Salmonella survival. In conclusion, our results revealed an increased health risk of the W-O emulsion against gastric digestion when it is contaminated with foodborne pathogens.

Genetic response of Salmonella Typhimurium to trans-cinnamaldehyde assisted heat treatment and its correlation with bacterial resistance in different low moisture food components.

Our previous study found that water activity (aw)- and matrix-dependent bacterial resistance wasdeveloped in Salmonella Typhimurium during antimicrobial-assisted heat treatment in low moisture foods (LMFs) matrices. To better understand the molecular mechanism behind the observed bacterial resistance, gene expression analysis was conducted on S. Typhimurium adapted to different conditions with or without the trans-cinnamaldehyde (CA)-assisted heat treatment via quantitative polymerase chain reaction (qPCR). Expression profiles of nine stress-related genes were analyzed. The upregulation of rpoH and dnaK and downregulation of ompC were observed during bacterial adaptation in LMF matrices and the combined heat treatment, which likely contributed to the bacterial resistance during the combined treatment. Their expression profiles were partially consistent with the previously-observed effect of aw or matrix on bacterial resistance. The upregulation of rpoE, otsB, proV, and fadA was also observed during adaptation in LMF matrices and might contribute to desiccation resistance, but likely did not contribute to bacterial resistance during the combined heat treatment. The observed upregulation of fabA and downregulation of ibpA could not be directly linked to bacterial resistance to either desiccation or the combined heat treatment. The results may assist the development of more efficient processing methods against S. Typhimurium in LMFs.

Assessment of trans-cinnamaldehyde and eugenol assisted heat treatment against Salmonella Typhimurium in low moisture food components.

Increased thermal resistance of Salmonella at low water activity (aw) is a significant food safety concern in low-moisture foods (LMFs). We evaluated whether trans-cinnamaldehyde (CA, 1000 ppm) and eugenol (EG, 1000 ppm), which can accelerate thermal inactivation of Salmonella Typhimurium in water, can show similar effect in bacteria adapted to low aw in different LMF components. Although CA and EG significantly accelerated thermal inactivation (55 °C) of S. Typhimurium in whey protein (WP), corn starch (CS) and peanut oil (PO) at 0.9 aw, such effect was not observed in bacteria adapted to lower aw (0.4). The matrix effect on bacterial thermal resistance was observed at 0.9 aw, which was ranked as WP > PO > CS. The effect of heat treatment with CA or EG on bacterial metabolic activity was also partially dependent on the food matrix. Bacteria adapted to lower aw had lower membrane fluidity and unsaturated to saturated fatty acids ratio, suggesting that bacteria at low aw can change its membrane composition to increase its rigidity, thus increasing resistance against the combined treatments. This study demonstrates the effect of aw and food components on the antimicrobials-assisted heat treatment in LMF and provides an insight into the resistance mechanism.

Enhancement of Thermal Inactivation of Cronobacter sakazakii in Apple Juice at 58°C by Inclusion of Butyl Para-Hydroxybenzoate and Malic Acid.

ABSTRACT: After studies with powdered infant formula indicated that the enhancement of thermal inactivation of Cronobacter sakazakii by butyl para-hydroxybenzoate (BPB) was blocked by high protein concentrations, we hypothesized that BPB would retain its synergistic activity in foods with limited protein and lipid concentrations. This hypothesis was tested by examining the ability of BPB to enhance the thermal inactivation of C. sakazakii 607 at 58°C in commercial apple juice, including examining the effects of pH and possible synergistic effects with malic acid. Apple juice was adjusted to designated pH values of 3.2 to 9.0, supplemented with selected concentrations of BPB (≤125 ppm), inoculated with early-stationary-phase C. sakazakii 607, and thermally treated (58°C) for 15 min with a submerged coil apparatus. The same methods were used to study the enhancement of thermal inactivation by malic acid. Samples were plated on tryptic soy agar for recovery and enumeration. Survival curves were plotted, and D-values were calculated by linear regression and compared using the Tukey honestly significant difference test. BPB significantly enhanced thermal inactivation in a concentration dependent manner, with D-values of a few seconds at the original pH (3.8). The enhancement of thermal inactivation was pH dependent over the pH range of 3.4 to 9.0. Malic acid enhanced thermal inactivation; the pH was decreased from 3.8 to 3.2. These results support the hypothesis that BPB can enhance the thermal inactivation of C. sakazakii in low-protein and low-lipid foods.

Evaluation of Potential for Butyl and Heptyl Para-Hydroxybenzoate Enhancement of Thermal Inactivation of Cronobacter sakazakii during Rehydration of Powdered Infant Formula and Nonfat Dry Milk.

ABSTRACT: In previous studies, parabens in model systems enhanced the thermal inactivation of foodborne pathogens, including Cronobacter sakazakii, Salmonella enterica serotype Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes. However, few studies have been conducted to evaluate this phenomenon in actual food systems. In the present study, the potential enhancement of thermal inactivation of C. sakazakii by butyl para-hydroxybenzoate (BPB) was evaluated in powdered infant formula (PIF) and nonfat dry milk (NFDM) in dry and rehydrated forms. When PIF was rehydrated with water at designated temperatures (65 to 80°C) in baby bottles, BPB did not enhance thermal inactivation. When rehydrated NFDM and lactose solutions with BPB were inoculated and heated at 58°C, BPB enhancement of thermal inactivation of C. sakazakii was negatively associated with the concentration of NFDM solutions in a dose-dependent manner, whereas thermal inactivation was enhanced in the presence of lactose regardless of its concentration, suggesting an interaction between proteins and BPB. Fluorescence testing further indicated an interaction between BPB and the proteins in PIF and NFDM. In inoculated dry NFDM with and without BPB stored at 24 and 55°C for 14 days, BPB did not substantially enhance bacterial inactivation. This study suggests that BPB is not likely to enhance mild thermal bacterial inactivation treatments in foods that have appreciable amounts of protein.

Synergistic inactivation of bacteria based on a combination of low frequency, low-intensity ultrasound and a food grade antioxidant.

This study evaluated a synergistic antimicrobial treatment using a combination of low frequency and a low-intensity ultrasound (LFU) and a food-grade antioxidant, propyl gallate (PG), against a model gram-positive (Listeria innocua) and the gram-negative bacteria (Escherichia coli O157:H7). Bacterial inactivation kinetic measurements were complemented by characterization of biophysical changes in liposomes, changes in bacterial membrane permeability, morphological changes in bacterial cells, and intracellular oxidative stress upon treatment with PG, LFU, and a combination of PG + LFU. Combination of PG + LFU significantly (>4 log CFU/mL, P < 0.05) enhanced the inactivation of both L. innocua and E. coli O157:H7 compared to PG or LFU treatment. As expected, L. innocua had a significantly higher resistance to inactivation compared to E. coli using a combination of PG + LFU. Biophysical measurements in liposomes, bacterial permeability measurements, and scanning electron microscope (SEM)-based morphological measurements show rapid interactions of PG with membranes. Upon extended treatment of cells with PG + LFU, a significant increase in membrane damage was observed compared to PG or LFU alone. A lack of change in the intracellular thiol content following the combined treatment and limited effectiveness of exogenously added antioxidants in attenuating the synergistic antimicrobial action demonstrated that oxidative stress was not a leading mechanism responsible for the synergistic inactivation by PG + LFU. Overall, the study illustrates synergistic inactivation of bacteria using a combination of PG + LFU based on enhanced membrane damage and its potential for applications in the food and environmental systems.

Assessment of butylparaben (4-hydroxybenzoic acid butyl ester)-assisted heat treatment against Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium in meat and bone meal.

Heat-resistant foodborne pathogens have been a concern in low-moisture foods and ingredients (LMFs). Due to low thermal conductivity of low moisture materials, thermal treatment is not efficient and may cause nutritional loss. This study investigated the enhancement of thermal treatment of meat and bone meal (MBM) at low water activity (aw ) by inclusion of butylparaben (BP) as a model antimicrobial compound. Stationary phase Escherichia coli O157:H7 (Shiga toxin-negative) or Salmonella enterica serotype Typhimurium was inoculated into MBM containing 0-2000 ppm BP and incubated at 55 or 60°C for up to 5 hr. A biphasic inactivation pattern was observed for both pathogens, indicating existence of potentially thermal resistant subpopulations. Addition of 1000 ppm BP to MBM (aw  = 0.4) significantly lowered the D-value at 55°C for E. coli O157:H7 (2.6 ± 0.5 hr) compared to thermal treatment alone (5.1 ± 0.6 h) during the treatment after the first 1 hr (p < 0.05), indicating that addition of BP accelerated the inactivation of thermal-resistant subpopulation of E. coli O157:H7 in MBM. Interestingly, similar enhancement in thermal inactivation upon addition of BP was not observed in either the sensitive or resistant subpopulation of S. Typhimurium at aw of 0.4 or 0.7, which is likely caused by the higher thermal resistance developed by S. Typhimurium within a low aw environment (aw  < 0.85). These results suggest that addition of certain antimicrobial compounds can improve the thermal processing efficiency in LMFs, while their efficiency against different pathogens may vary. PRACTICAL APPLICATION: Addition of appropriate food-grade compounds may help to improve thermal treatment efficiency in low moisture foods with varied efficiency against different pathogens. This approach has the potential to reduce the required heat treatment intensity while minimizing food safety risk.

Synergistic Effects of Butyl Para-Hydroxybenzoate and Mild Heating on Foodborne Pathogenic Bacteria.

ABSTRACT: Although high-temperature heat treatments can efficiently reduce pathogen levels, they also affect the quality and nutritional profile of foods and increase the cost of processing. The food additive butyl para-hydroxybenzoate (BPB) was investigated for its potential to synergistically enhance thermal microbial inactivation at mild heating temperatures (54 to 58°C). Four foodborne pathogenic bacteria, Cronobacter sakazakii, Salmonella enterica Typhimurium, attenuated Escherichia coli O157:H7, and Listeria monocytogenes, were cultured to early stationary phase and then subjected to mild heating at 58, 55, 57, and 54°C, respectively, in a model food matrix (brain heart infusion [BHI]) containing low concentrations of BPB (≤125 ppm). The temperature used with each bacterium was selected based on the temperature that would yield an approximately 1- to 3-log reduction over 15 min of heating in BHI without BPB in a submerged coil system. The inclusion of BPB at ≤125 ppm resulted in significant enhancement of thermal inactivation, achieving 5- to >6-log reductions of the gram-negative strains with D-values of <100 s. A 3- to 4-log reduction of L. monocytogenes was achieved with a similar treatment. No significant microbial inactivation was noted in the absence of mild heating for the same time period. This study provides additional proof of concept that low-temperature inactivation of foodborne pathogens can be realized by synergistic enhancement of thermal inactivation by additives that affect microbial cell membranes.

Inactivation of foodborne pathogens by the synergistic combinations of food processing technologies and food-grade compounds.

There is a need to develop food processing technologies with enhanced antimicrobial capacity against foodborne pathogens. While considering the challenges of adequate inactivation of pathogenic microorganisms in different food matrices, the emerging technologies are also expected to be sustainable and have a minimum impact on food quality and nutrients. Synergistic combinations of food processing technologies and food-grade compounds have a great potential to address these needs. During these combined treatments, food processes directly or indirectly interact with added chemicals, intensifying the overall antimicrobial effect. This review provides an overview of the combinations of different thermal or nonthermal processes with a variety of food-grade compounds that show synergistic antimicrobial effect against pathogenic microorganisms in foods and model systems. Further, we summarize the underlying mechanisms for representative combined treatments that are responsible for the enhanced microbial inactivation. Finally, regulatory issues and challenges for further development and technical transfer of these new approaches at the industrial level are also discussed.

Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing.

Ultrasound has potential to be used for disinfection, and its antimicrobial effectiveness can be enhanced in presence of natural compounds. In this study, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5-2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, as a presentative compound, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within the bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. Findings in this study will be useful to enhance fresh produce safety and shelf-life and design other alternative ultrasound based sanitation processes.

Antimicrobial action of octanoic acid against Escherichia coli O157:H7 during washing of baby spinach and grape tomatoes.

We investigated the antimicrobial efficacy of octanoic acid (OA) against Escherichia coli O157:H7 inoculated on the surface of baby spinach and grape tomatoes during simulated washing processes. 3 mM OA at 45 °C achieved >6 log CFU/g reduction from the surface of tomatoes within 2 min. However, washing baby spinach with 6 mM OA at 5 °C resulted in <1 log CFU/g reduction, highlighting the role of surface properties in inactivation efficacy. OA significantly (p < 0.05) reduced the risk of cross-contamination during washing of spinach as well as tomatoes. Also, total mold and yeast population on surface of spinach was significantly reduced immediately after OA wash and inhibited during following 14 days. Baby spinach and grape tomatoes washed with OA did not cause significant (p > 0.05) difference in color compared to the control and no residual OA was detected in most cases following rinsing of produce in water. OA at the concentrations above 2 mM and temperature higher than 25 °C induced severe membrane damage along with release of ATP and other intracellular constituents resulting in bacterial death. OA can be an attractive natural decontamination agent for washing fresh produce.

UV-C irradiated gallic acid exhibits enhanced antimicrobial activity via generation of reactive oxidative species and quinone.

A novel light-enhanced antimicrobial treatment was developed by exposing gallic acid (GA) to UV-C light. GA (15 mM) solution was exposed to UV-C for 30 min and subsequently incubated with E. coli O157:H7 for 30 min to achieve a 3.2 ±â€¯0.2 log CFU/mL reduction. The antimicrobial activity is affected by the irradiation duration, wavelength, and pH of solution. The addition of benzenesulfinic acid (BSA) to UV-C irradiated GA lowered (P < 0.05) its antimicrobial activity, indicating that quinones contributed to its overall antimicrobial effect. In addition, the attenuated (P < 0.05) antimicrobial activity of UV-C exposed GA in the presence of reactive oxidative species (ROS) quenchers, the generation of hydrogen peroxide, and increased levels of intracellular oxidative stress detected in E. coli O157:H7 illustrated that ROS also played a role in the antimicrobial effect of UV-C irradiated GA. UV-C irradiated GA could be applied as a novel antimicrobial in food systems.

Fog, phenolic acids and UV-A light irradiation: A new antimicrobial treatment for decontamination of fresh produce.

This study evaluates synergistic interactions of food grade phenolic acids (gallic and ferulic acid) and UV-A light to achieve decontamination of fresh produce using a fog to improve dispersion of the phenolic acids on produce surface. Nonvirulent strains of Escherichia coli O157:H7 and Listeria innocua were used as model bacteria and spinach was selected as a model fresh produce. Synergistic combination of a fog deposited phenolic acid and a UV-A light treatment achieved reduction in bacterial plate count up to 2 log CFU/cm2 independently of the initial load of the bacteria (104 or 106 CFU/cm2). Following the treatment, fog deposited gallic and ferulic acid could be easily removed from the surface of produce by immersion in water and the treatment did not significantly alter the total endogenous phenolic content of spinach. The treatment also did not affect the texture, but impacted the color of the spinach leaves on a Hunter's Lab scale although the visual color changes were small. Overall, this technology may aid in developing alternative approaches for decontamination processes using food grade compounds.

Photoirradiated caffeic acid as an antimicrobial treatment for fresh produce.

The antimicrobial efficacy of 400 nm photoirradiated caffeic acid (CA, 5 mM) was evaluated against Escherichia coli O157:H7 and Listeria innocua. A stronger antimicrobial effect was observed on E. coli than on L. innocua where the combined treatment resulted in 4 and 1 log(CFU/mL) reductions, respectively. The treatment's effects on cellular metabolism (resazurin assay), uptake of CA (fluorescence technique) and membrane damage (propidium iodide assay) were studied in both species. CA uptake increased in both species, but membrane damage was only observed in E. coli O157:H7. The treatment had minimal impact on metabolic activity in both species. The treatment applied to the surface of spinach leaves was found to be effective against E. coli O157:H7. The novel treatment proposed in this study has the potential to improve the microbial food safety of fresh produce.

Inactivation of Escherichia Coli O157:H7 and Listeria Innocua by Benzoic Acid, Ethylenediaminetetraacetic Acid and Their Combination in Model Wash Water and Simulated Spinach Washing.

An antimicrobial effect of benzoic acid (BA) and ethylenediaminetetraacetic acid (EDTA) was evaluated as a potential antimicrobial treatment against Escherichia coli O157:H7 and Listeria innocua. A 30 min exposure to the combination of 15 mM BA and 1 mM EDTA at 22 °C resulted in approximately 3 logarithmic reductions in stationary phase E. coli O157:H7. Logarithmic phase E. coli O157:H7 was more sensitive (P < 0.05) to the treatment and 1 mM EDTA alone caused more than 5 logarithmic reductions. L. innocua was also sensitive to a treatment with 15 mM BA alone, which induced 5 logarithmic reductions. By increasing the temperature of the solution containing 15 mM BA and 1 mM EDTA to 40 °C, more than 5 logarithmic reductions in stationary phase E. coli O157:H7 was observed after 5 min of treatment. However, the antimicrobial effect was attenuated (reaching less than 1 logarithmic reductions) at 4 °C. In addition, the combined BA and EDTA treatment retained its antimicrobial effect against E. coli O157:H7 for at least 6 cycles of treatment over 6 days at room temperature (22 °C). In a simulated spinach washing study, 15 mM BA and 1 mM EDTA together were able to prevent cross-contamination of E. coli O157:H7. The results highlight the potential use of combination of BA (15 mM) and EDTA (1 mM) to address microbial risk from E. coli O157:H7 and L. innocua in fresh produce industry. PRACTICAL APPLICATION: This study demonstrates the effectiveness of benzoic acid (BA) and EDTA mixture in inactivating bacteria in the water used for produce washing and reducing the incidence of cross-contamination during washing of fresh produce. Use of BA + EDTA mixture has significant benefits such as: (a) ability to be reused, (b) effectiveness in the presence of organic matter, and (c) reduced need of monitoring wash water conditions such as pH, concentration and organic matter.

Novel sanitization approach based on synergistic action of UV-A light and benzoic acid: Inactivation mechanism and a potential application in washing fresh produce.

Antimicrobial activity of the simultaneous UV-A light and benzoic acid (BA) treatment against stationary phase Escherichia coli O157:H7 was investigated. While 15 mM BA or UV-A light exposure for 30 min alone caused < 1 logarithmic reduction in the bacterial population, > 5 logarithmic reductions were induced by the simultaneous application of UV-A and 15 mM BA in 30 min, demonstrating a synergistic antimicrobial effect. Due to its ability to increase cell membrane permeability, addition of EDTA (1 mM) was able to decrease the required concentration of BA in the simultaneous treatment from 15 to 8 mM. Microbial inactivation was a result of simultaneous membrane damage, intracellular acidification, and intracellular oxidative stress. The simultaneous treatment was effective in the presence of organic load of up to 500 mg/L of chemical oxygen demand (COD) and was able to lower cross-contamination risk during simulated washing of spinach (Spinacia oleracea) without adversely affecting its color.