Recent revisions to USDA FSIS compliance and safe harbor guidelines for ready-to-eat meat and poultry products addressed process humidity requirements. Given the lack of prior data for impingement-cooked products, this project aimed to evaluate the impact of process humidity on Salmonella lethality at the product core and surface, and compliance of the results with USDA FSIS lethality performance standards. Whole muscle beef strips, ground beef patties, whole muscle chicken breast fillets, and breaded ground chicken patties were inoculated with an 8-serovar cocktail of Salmonella. Beef and chicken samples were cooked in a pilot-scale moist-air impingement oven to a core temperature of 70.0 or 72.8°C, respectively, immediately quenched in liquid nitrogen, and dissected to obtain core and surface samples. Variables included oven temperature (218, 232°C), air velocity (0.7 and 2.8 m/s), and oven humidity (0.7, 15, 30, or 70% moisture by volume (% v/v)). Additional treatments were performed to examine the impact of supplemental critical control processes, such as increased endpoint temperature, post-oven carryover time, and pre- or post-oven steam treatments. Salmonella reductions of >7 log were reliably achieved in chicken patties regardless of the processing variables; however, none of the treatments reliably ensured >6.5 log reductions of Salmonella in ground beef. A majority of whole-muscle samples failed to meet the required performance lethality when processed at 0.7% v/v; however, Salmonella inactivation was significantly improved (P < 0.05) at oven humidities of > 30% v/v. Dry oven conditions achieved greater Salmonella lethality at the core than at the surface for multiple products (P < 0.05). The efficacies of minimal and supplemental critical controls were product-, process-, and humidity-dependent (P < 0.05). Overall, process humidity and product variability should be considered in regulatory requirements and process validations.

ABSTRACT Outbreaks and recalls associated with microbial contamination of powdered foods have raised concern for the safety of the spray-drying process and its products. However, little research on the fate of bacteria during the spray-drying process has been done, leaving much unknown about the risks of contamination in spray dryers. Therefore, quantifying the contamination levels of Salmonella and Enterococcus faecium (as a surrogate) in various locations within a pilot-scale spray dryer can help illustrate the distribution of bacterial contamination, including in the final product. A 10% (w/w) dispersion of water and soy protein isolate was mixed with tryptic soy broth containing yeast extract inoculated with Salmonella enterica serovar Enteritidis phage type 30 (PT30) or E. faecium strain NRRL B-2354. This dispersion was spray dried using a pilot-scale tall-form cocurrent spray dryer at an inlet air temperature of 180, 200, or 220°C. After drying, samples of powder from eight locations within the system were collected or surface swabbed, plated, and enumerated. Spray drying achieved 2.40 to 4.15 and 2.33 to 2.83 log reductions in the concentrations of Salmonella and E. faecium, respectively, in the final powder product accumulated in the dryer's collectors. Salmonella and E. faecium were found in various concentrations in all locations within the spray dryer after a complete drying cycle. Differences in inlet air temperature between 180 and 220°C had no significant effect on the inactivation levels. As a surrogate, E. faecium was more resistant to spray drying than Salmonella . Overall, spray drying is capable of significant bacterial reduction in the final powder product, which can be combined with other hurdle technologies. However, adequate cleaning and sanitization procedures must be taken into consideration to prevent cross-contamination. HIGHLIGHTS Spray drying reduced Salmonella Enteritidis PT30 and E. faecium in soy protein by >99%. Salmonella Enteritidis PT30 and E. faecium were pervasive within the spray dryer. More E. faecium than Salmonella Enteritidis PT30 survived in spray drying of soy protein.

ABSTRACT As demand for fresh-cut produce increases, minimizing the risk of salmonellosis becomes critical for the produce industry. Sanitizers are routinely used during commercial flume washing of fresh-cut produce to minimize cross-contamination from the wash water. This study assessed the efficacy of a novel sanitizer blend consisting of peracetic acid (PAA; OxypHresh 15) with a sulfuric acid–surfactant (SS) antimicrobial (PAA-SS; ProduceShield Plus) against Salmonella during simulated commercial washing of diced tomatoes. Triplicate 9.1-kg batches of Roma tomatoes were dip inoculated in a two-strain avirulent Salmonella cocktail ( Salmonella Typhimurium LT2 and MHM112) to achieve 5 to 6 log CFU per tomato and air dried for 2 h. After mechanical dicing, the tomatoes were washed in a pilot-scale processing line for 60 s with or without an added organic load in 90 ppm of PAA-SS (pH 1.8), SS at pH 1.8, 90 ppm of PAA, 5 or 10 ppm of free chlorine or sanitizer-free water as the control. Overall, PAA-SS (1.75 ± 0.75 log CFU/g) was significantly ( P ≤ 0.05) more effective than water (0.69 ± 0.42 log CFU/g), chlorine (0.35 ± 0.36 log CFU/g), or SS (0.36 ± 0.19 log CFU/g) in reducing Salmonella . After washing for 20 s, PAA-SS was the only sanitizer to show a significant ( P ≤ 0.05) reduction (1.93 ±0.59 log CFU/g) in Salmonella . All wash water samples were negative for Salmonella, except for 5 and 10 ppm of chlorine and the water control. Using PAA-SS with an organic load, yeast and mold populations were below the limit of detection (1.40 log CFU/g) and significantly ( P ≤ 0.05) lower on diced tomatoes after 14 days of refrigerated storage compared with the other treatments (8.37 ± 0.08 log CFU/g), with SS at pH 1.8 (3.91 ± 0.93 log CFU/g) most effective against yeast and mold in the absence of an organic load. On the basis of these findings, the safety and shelf life of commercially washed diced tomatoes can be improved with PAA-SS. HIGHLIGHTS PAA-SS yielded lower Salmonella populations in diced tomatoes than did chlorine ( P ≤ 0.05). Salmonella was not detected in the wash water using PAA-SS. PAA-SS decreased Salmonella 1.93 log after 20 s of washing. PAA-SS yielded lower ( P ≤ 0.05) yeast and mold populations after 14 days of storage.

ABSTRACT Recent outbreaks and recalls of low-moisture foods contaminated with Salmonella have been recognized as a major public health risk that demands the development of new Salmonella mitigation strategies and technologies. This study aimed to assess the efficacy of X-ray irradiation for inactivating Salmonella on or in almonds (kernels, meal, butter), dates (whole fruit, paste), and wheat (kernels, flour) at various water activities (a w ) and storage periods. The raw materials were inoculated with Salmonella Enteritidis PT30, conditioned to 0.25, 0.45, and 0.65 a w in a humidity-controlled chamber, processed to various fabricated products, and reconditioned to the desired a w before treatment. In a storage study, inoculated almond kernels were stored in sealed tin cans for 7, 15, 27, and 103 weeks, irradiated with X ray (0.5 to 11 kGy, targeting up to a ∼2.5-log reduction) at the end of each storage period, and plated for Salmonella survivors to determine the efficacy of irradiation in terms of D 10 -value (dose required to reduce 90% of the population). Salmonella was least resistant ( D 10 -value = 0.378 kGy) on the surface of almond kernels at 0.25 a w and most resistant ( D 10 -value = 2.34 kGy) on the surface of dates at 0.45 a w . The Salmonella D 10 -value was 61% lower in date paste than on whole date fruit. Storage of almonds generally had no effect on the irradiation resistance of Salmonella over 103 weeks. Overall, these results indicate that product structure (whole, meals, powder, or paste), water activity (0.25 to 0.65 a w ), and storage period (0 to 103 weeks) should be considered when determining the efficacy of X-ray irradiation for inactivating Salmonella in various low-water-activity foods. HIGHLIGHTS Salmonella resistance to X ray was significantly different on almonds, wheat, and dates. The structural changes of almonds significantly impacted Salmonella resistance to X ray. Water activity affected the efficacy of X ray for inactivating Salmonella in low-moisture foods. Storing almonds up to 103 weeks had no effect on the X-ray resistance of Salmonella .

ABSTRACT Dry inoculation (DI) methods using a dry carrier have gained considerable interest for assessing thermal inactivation of Salmonella and other microorganisms in low-moisture foods. However, the effect of carrier residues on microbial resistance to heat remains largely unknown. This study aimed to determine the effect of talc powder on thermal resistance of Enterococcus faecium NRRL-B2354 (a Salmonella surrogate) in almond meal at 0.45 water activity (a w ). Whole almonds were either immersed in an E. faecium suspension for wet inoculation (WI) or mixed with inoculated talc powder for DI. Two additional experimental conditions, inoculation of WI almond meal with added uninoculated talc (WT) and inoculated talc powder alone, were conducted. After WI, DI, and WT, the almonds were equilibrated to 0.45 a w , ground into a meal, and reequilibrated to 0.45 a w . Isothermal treatments were performed by heating almond meal (about 1 g) in aluminum test cells in a water bath at 80°C, with samples collected at more than five sequential time points from triplicate isothermal runs. E. faecium was enumerated by immediately cooling, diluting, and plating the samples on a nonselective or differential medium. E. faecium was more thermally resistant in DI ( D 80°C : 63.5 ± 1.9 min) compared with WI almond meal ( D 80°C : 40.5 ± 1.0 min; P < 0.05), but the resistance in WT almond meal (46.9 ± 0.9 min) was between and different from ( P < 0.05) both DI and WI. E. faecium was less resistant in talc powder alone (20.6 ± 1.1 min) compared with all other almond meal samples. Overall, residual talc affected the thermal resistance of E. faecium . Therefore, when determining thermal resistance or validating commercial processes, carriers such as talc should not be used for inoculation of low-moisture foods without first knowing their impact on the target organism. HIGHLIGHTS E. faecium was more thermally resistant in dry- than in wet-inoculated almond meal. Presence of talc affected thermal resistance of E. faecium in almond meal. Use of dry inoculum carriers for thermal validation studies requires further work.

ABSTRACT The enhanced thermal resistance of Salmonella in low-moisture foods (LMFs) presents a challenge when validating pathogen control processes. Product water is recognized as a controlling factor in thermal inactivation of Salmonella in or on LMFs, such as almonds. Water activity (a w ) describes the state of water in a product; however, a w is temperature dependent and characterized by hysteresis between sorption states. Moisture content (%MC) describes the amount of water in a product; it is not temperature dependent and might be a more convenient metric than a w to account for water in thermal inactivation processes. To test these two metrics independently, Salmonella -inoculated almonds were equilibrated to two %MC levels but the same a w and to two a w levels but the same %MC. Equilibrated products were vacuum packaged and thermally treated in a water bath at 80°C. Survivors were recovered and enumerated. The resulting inactivation curves were used to fit the log-linear inactivation model, and the inactivation kinetics were compared. D -values ranged from 15.7 to 18.0 min, and the root mean square error was 0.25 to 0.69 log CFU/g. No differentiated ( P > 0.05) effect attributable preferentially to a w or %MC was seen in the inactivation kinetics. The separate effects of a w and %MC on the inactivation kinetics of Salmonella in LMFs remain inconclusive, but analyses of data from prior studies strongly suggested an effect of sorption state. Further analysis is needed to identify which metric is best for modeling and validating thermal inactivation processes. HIGHLIGHTS Water affects thermal inactivation kinetics of Salmonella in low-moisture foods. Water activity and moisture content are both feasible predictors of heat resistance. Sorption state of food materials may affect Salmonella heat resistance.

ABSTRACT Temperature is arguably the most important factor affecting microbial proliferation in fresh-cut produce. In this study, growth of Listeria monocytogenes in diced onions and celery and Salmonella Typhimurium in diced tomatoes was determined in modified atmosphere packages and snap-fit containers using three fluctuating temperature scenarios for transport, retail storage, and display. As expected, L. monocytogenes growth in diced onions and celery varied depending on the extent of temperature abuse, with exposure to high and intermediate temperature-abuse scenarios generally being growth supportive. A Baranyi primary model with a square-root secondary model for maximum growth rate, and a linear model for maximum population density, were used to estimate Listeria growth under fluctuating temperature. Accuracy and acceptability of the model prediction were evaluated in terms of root mean square error (RMSE) and acceptable prediction zone (APZ), respectively. Overall, growth predictions for L. monocytogenes were more accurate for celery (RMSE, 0.28 to 0.47) than onions (RMSE, 0.42 to 1.53) under the fluctuating temperature scenarios tested. However, both predictions yielded APZ values that ranged from 82 to 100% for celery and 36 to 78% for onions. In contrast, Salmonella Typhimurium populations increased more than 1 log CFU/g in diced tomatoes under the three fluctuating temperature scenarios studied. Overall, these diced products packaged under a high-oxygen atmosphere showed decreased pathogen growth compared with product stored in a passive modified atmosphere. Findings from this study will be particularly useful in assessing the risk associated with consumption of diced celery, tomatoes, and onions and in designing effective packaging strategies to minimize pathogen growth in fresh-cut produce.

ABSTRACT Some thermal processes, such as pistachio roasting, are not yet well characterized with respect to the impact of product and process variables on Salmonella lethality. This study aimed to quantify the effects of process temperature, humidity, and initial product water activity (a w ), on Salmonella lethality for in-shell pistachios. In-shell pistachios were inoculated with Salmonella Enteritidis PT 30 (∼8.5 log CFU/g), equilibrated (0.45 or 0.65 a w ), and heated without soaking (“dry”) or after a pure-water or 27% NaCl brining pretreatment (“presoaked”). Inoculated pistachio samples (15 g) were heated in a laboratory-scale, moist-air convection oven at 104.4 or 118.3°C, humidities of ∼3, 15, or 30%, v/v (∼24.4, 54.4, or 69.4°C dew point), and air speed of 1.3 m/s. Salmonella survivors were quantified at six times during each treatment, targeting total reductions of ∼3 to 5 log. Survivor data were analyzed using analysis of variance to identify main effects (time, temperature, humidity, and initial a w ) and two-term interactions with time. As expected, lethality increased ( P < 0.05) with temperature and humidity. For example, the time to achieve a 4-log reduction decreased 50 to 80% when humidity increased from ∼3 to 30%. When the dry and presoaked treatments were analyzed separately, initial product a w (0.45 versus 0.65 a w or 0.75 versus 0.95 a w ) did not affect lethality ( P > 0.05). However, when comparing dry against presoaked treatments, the time to achieve a 4-log reduction decreased 55 to 85% ( P < 0.05) for presoaked pistachios subjected to the same temperature-humidity treatment. Salt had no effect ( P > 0.05) on lethality outcomes. These results, relative to initial a w , process humidity, brining, and salt effects on process lethality, are critically important and must be considered in the design and validation of thermal processes for Salmonella reduction in pistachio processing.

ABSTRACT Several outbreaks of foodborne illness traced to leafy greens and culinary herbs have been hypothesized to involve cross-contamination during washing and processing. This study aimed to assess the redistribution of Salmonella Typhimurium LT2 during pilot-scale production of baby spinach and cilantro and redistribution of Escherichia coli O157:H7 during pilot-scale production of romaine lettuce. Four inoculated surrogate:uninoculated product weight ratios (10:100, 5:100, 1:100, and 0.5:100) and three inoculation levels (10 3 , 10 1 , and 10 −1 CFU/g) were used for the three commodities. For each of three trials per condition, 5-kg batches containing uninoculated product and spot-inoculated surrogate products at each ratio and inoculation level were washed for 90 s in a 3.6-m-long flume tank through which 890 L of sanitizer-free, filtered tap water was circulated. After washing and removing the inoculated surrogate products, washed product (∼23, 225-g samples per trial) was analyzed for presence or absence of Salmonella Typhimurium or E. coli O157:H7 by using the GeneQuence Assay. For baby spinach, cilantro, and romaine lettuce, no significant differences ( P > 0.05) in the percentage of positive samples were observed at the same inoculation level and inoculated:uninoculated weight ratio. For each pathogen product evaluated (triplicate trials), inoculation level had a significant impact on the percentage of positive samples after processing, with the percentage of positive samples decreasing, as the initial surrogate inoculation level decreased. The weight ratio of contaminated:noncontaminated product plays an important role: positive samples ranged from 0% to 11.6% ± 2.05% and from 68.1% ± 33.6% to 100% among the four ratios at inoculation of 10 −1 and 10 1 CFU/g, respectively. To our knowledge, this study is the first to assess the redistribution of low levels of pathogens from incoming product to leafy greens during processing and should provide important data for microbial risk assessments and other types of food safety analyses related to fresh-cut leafy greens.

ABSTRACT Inoculation methods in pathogen inactivation studies ideally represent conditions that might occur in real-world scenarios. Surface contamination in or on low-moisture foods affects Salmonella thermal resistance, which is critically important for process validation applications. The objective of this study was to quantify the effect of inoculation protocol on the thermal resistance of Salmonella Enteritidis PT 30 in fabricated low-moisture foods. Almond meal, almond butter, wheat meal, wheat flour, and date paste were inoculated via prefabrication and postfabrication protocols. In the prefabrication protocol, kernels and fruits were surface inoculated and equilibrated to a target water activity (a w ) (0.40 for almond and wheat products, 0.45 for date products) before fabricating meal, butter, flour, or paste and then reequilibrating the samples to the target a w . In the postfabrication protocol, meal, butter, flour, and paste were fabricated before inoculation and equilibration. All inoculated and equilibrated samples were subjected to isothermal treatment (80°C), pulled sequentially during processing, cooled, serially diluted, and plated to enumerate survivors. Log-linear and Weibull-type models were fit to the Salmonella survivor data and were compared via the corrected Akaike information criterion. Pre- and postfabrication protocols resulted in significant differences ( P < 0.05) in Salmonella thermal resistance in all products. Overall, the thermal resistance of Salmonella Enteritidis PT 30 in almond products was greater ( P < 0.05) than in wheat products, which was also greater ( P < 0.05) than in date paste. Additionally, Salmonella was more thermally resistant in almond products and date paste when inoculated pre- rather than postfabrication; however, the opposite was true for wheat products. These results indicate that the means of inoculation can significantly affect thermal resistance of Salmonella in low-moisture foods.

ABSTRACT Viral foodborne outbreaks are a serious threat to public health, and fresh produce is becoming increasingly recognized as a transmission vehicle. To limit foodborne disease, ready-to-eat leafy greens are typically washed with a chlorine-based sanitizer during commercial production. This study assessed the efficacy of a chlorine-based sanitizer against coliphage MS2, as a potential surrogate for foodborne viruses, on fresh-cut romaine lettuce during simulated commercial production using a small-scale processing line. Before processing, romaine lettuce was inoculated to contain approximately 10 5 and 10 6 PFU/g of MS2 for experiments with and without sanitizer, respectively. Lettuce samples were collected following each stage of processing, which included mechanical shredding, 2 min of flume washing (with or without 25 ppm of free chlorine), shaker table dewatering, and centrifugal drying. In addition, the spent centrifuge water and flume wash water were collected, with the flume water concentrated using hollow-fiber ultrafiltration. MS2 was recovered from lettuce in Tris-glycine buffer and quantified as PFUs in a double-agar overlay assay. The greatest reduction in MS2 occurred between shredding and flume washing, with levels remaining relatively stable following flume washing with or without 25 ppm of free chlorine. Average total reductions of 0.8 and 1.0 log PFU/g were seen after processing with and without the sanitizer, respectively, with no statistical difference observed between the two treatments ( P > 0.05). The average MS2 level in the spent centrifugation water started at 4.0 log PFU/ml for experiments with sanitizer and the average MS2 reduction in the flume wash water was 4 log (PFU) for experiments with sanitizer, demonstrating that removals could be achieved in the water itself. These findings suggest that the currently recommended commercial production practices are unable to effectively decrease viruses once they have attached to leafy greens during commercial processing.

ABSTRACT Limited prior research has shown that inoculation methods affect thermal resistance of Salmonella in low-moisture foods; however, these effects and their repeatability have not been systematically quantified. Consequently, method variability across studies limits utility of individual data sets and cross-study comparisons. Therefore, the objective was to evaluate the effects of inoculation methodologies on stability and thermal resistance of Salmonella in a low-moisture food (wheat flour), and the repeatability of those results, based on data generated by two independent laboratories. The experimental design consisted of a cross-laboratory comparison, both conducting isothermal Salmonella inactivation studies in wheat flour (~0.45 water activity, 80°C), utilizing five different inoculation methods: (i) broth-based liquid inoculum, (ii) lawn-based liquid inoculum, (iii) lawn-based pelletized inoculum, (iv) direct harvest of lawn culture with wheat flour, and (v) fomite transfer of a lawn culture. Inoculated wheat flour was equilibrated ~5 days to ~0.45 water activity and then was subjected to isothermal treatment (80°C) in aluminum test cells. Results indicated that inoculation method impacted repeatability, population stability, and inactivation kinetics (α = 0.05), regardless of laboratory. Salmonella inoculated with the broth-based liquid inoculum method and the fomite transfer of a lawn culture method exhibited instability during equilibration. Lawn-based cultures resulted in stable populations prior to thermal treatment; however, the method using direct harvest of lawn culture with wheat flour yielded different D -values across the laboratories (α = 0.05), which was attributed to larger potential impact of operator variability. The lawn-based liquid inoculum and the lawn-based pelletized inoculum methods yielded stable inoculation levels and repeatable D -values (~250 and ~285 s, respectively). Also, inoculation level (3 to 8 log CFU/g) did not affect D -values (using the lawn-based liquid inoculum method). Overall, the results demonstrate that inoculation methods significantly affect Salmonella population kinetics and subsequent interpretation of thermal inactivation data for low-moisture foods.

ABSTRACT Isothermal inactivation studies are commonly used to quantify thermal inactivation kinetics of bacteria. Meta-analyses and comparisons utilizing results from multiple sources have revealed large variations in reported thermal resistance parameters for Salmonella , even when in similar food materials. Different laboratory or regression methodologies likely are the source of methodology-specific artifacts influencing the estimated parameters; however, such effects have not been quantified. The objective of this study was to evaluate the effects of laboratory and regression methodologies on thermal inactivation data generation, interpretation, modeling, and inherent error, based on data generated in two independent laboratories. The overall experimental design consisted of a cross-laboratory comparison using two independent laboratories (Michigan State University and U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center [ERRC] laboratories), both conducting isothermal Salmonella inactivation studies (55, 60, 62°C) in ground beef, and each using two methodologies reported in prior studies. Two primary models (log-linear and Weibull) with one secondary model (Bigelow) were fitted to the resultant data using three regression methodologies (two two-step regressions and a one-step regression). Results indicated that laboratory methodology impacted the estimated D 60°C - and z -values (α = 0.05), with the ERRC methodology yielding parameter estimates ~25% larger than the Michigan State University methodology, regardless of the laboratory. Regression methodology also impacted the model and parameter error estimates. Two-step regressions yielded root mean square error values on average 40% larger than the one-step regressions. The Akaike Information Criterion indicated the Weibull as the more correct model in most cases; however, caution should be used to confirm model robustness in application to real-world data. Overall, the results suggested that laboratory and regression methodologies have a large influence on resultant data and the subsequent estimation of thermal resistance parameters.

Chemical sanitizers are usually added to dump tank water to minimize cross-contamination during tomato packing. However, the efficacy of sanitizers continues to be questioned. This study assessed the ability of six commonly used sanitizers (40 ppm of peroxyacetic acid, 40 ppm of mixed peracid, 40 ppm of available chlorine alone or acidified to pH 6.0 with citric acid or T-128, and electrolyzed water containing 40 ppm of available chlorine at pH 6.7) to reduce Salmonella on tomatoes, in wash water, and on equipment surfaces using a pilot-scale processing line. Red round tomatoes (11.3 kg) were dip inoculated to contain Salmonella at ~ 6 log CFU/g, air dried for 2 h, treated for 2 min in a 3.3-m-long dump tank and then dried on a roller conveyor, with sanitizer-free water serving as the control. Tomato and water samples were collected at 15-s intervals during washing with additional dump tank, water tank, and roller conveyor surface samples collected after washing. All samples were appropriately neutralized, diluted, and surface plated on Trypticase soy agar containing 0.6% yeast extract, 0.05% ferric ammonium citrate, and 0.03% sodium thiosulfate with or without membrane filtration to enumerate Salmonella . All six sanitizer treatments were more efficacious than the water control ( P ≤ 0.05), with chlorine plus citric acid yielding the greatest Salmonella reduction on tomatoes (3.1 log CFU/g). After processing, all sanitizer wash solutions contained significantly lower ( P ≤ 0.05) levels of Salmonella than the water control (3.0 log CFU/ml). The four chlorine-based sanitizer treatments yielded significantly lower Salmonella populations ( P ≤ 0.05) in the wash solution compared with peroxyacetic acid and mixed peracid. After processing with sanitizers, Salmonella populations decreased to nondetectable levels (< 0.2 log CFU/100 cm 2 ) on the equipment surfaces.