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This multi-institutional study assessed the efficacy of Enterococcus faecium NRRL B-2354 as a nonpathogenic Salmonella surrogate for thermal processing of nonfat dry milk powder, peanut butter, almond meal, wheat flour, ground black pepper, and date paste. Each product was analyzed by two laboratories (five independent laboratories total), with the lead laboratory inoculating (E. faecium or a five-strain Salmonella enterica serovar cocktail of Agona, Reading, Tennessee, Mbandaka, and Montevideo) and equilibrating the product to the target water activity before shipping. Both laboratories subjected samples to three isothermal treatments (between 65 and 100°C). A log-linear and Bigelow model was fit to survivor data via one-step regression. On the basis of D80°C values estimated from the combined model, E. faecium was more thermally resistant (P0.05) than Salmonella in nonfat dry milk powder (DEf-80°C, 100.2 ± 5.8 min; DSal-80°C, 28.9 ± 1.0 min), peanut butter (DEf-80°C, 133.5 ± 3.1 min; DSal-80°C, 57.6 ± 1.5 min), almond meal (DEf-80°C, 34.2 ± 0.4 min; DSal-80°C, 26.1 ± 0.2 min), ground black pepper (DEf-80°C, 3.2 ± 0.8 min; DSal-80°C, 1.5 ± 0.1 min), and date paste (DEf-80°C, 1.5 ± 0.0 min; DSal-80°C, 0.5 ± 0.0 min). Although the combined laboratory D80°C for E. faecium was lower (P0.05) than for Salmonella in wheat flour (DEf-80°C, 9.4 ± 0.1 min; DSal-80°C, 10.1 ± 0.2 min), the difference was ∼7%. The zT values for Salmonella in all products and for E. faecium in milk powder, almond meal, and date paste were not different (P0.05) between laboratories. Therefore, this study demonstrated the impact of standardized methodologies on repeatability of microbial inactivation results. Overall, E. faecium NRRL B-2354 was more thermally resistant than Salmonella, which provides support for utilizing E. faecium as a surrogate for validating thermal processing of multiple low-moisture products. However, product composition should always be considered before making that decision.

Recent revisions to U.S. Department of Agriculture, Food Safety and Inspection Service (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, the present study was conducted to evaluate the impact of process humidity on Salmonella lethality at the product core and surface and compliance of the results with 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 eight-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 and 72.8°C, respectively, immediately quenched in liquid nitrogen, and dissected to obtain core and surface samples. Variables included oven temperature (218 and 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, postoven carryover time, and pre- or postoven steam treatments. Salmonella reductions of7 log units were reliably achieved in chicken patties regardless of the processing variables; however, none of the treatments reliably ensured6.5-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) humidity; however, Salmonella inactivation was significantly improved (P0.05) at oven humidities of ≥30% (v/v). Dry oven conditions achieved greater Salmonella lethality at the core than at the surface for multiple products (P0.05). The efficacies of minimal and supplemental critical controls were dependent on product, process, and humidity (P0.05). Overall, process humidity and product variability should be considered in regulatory requirements and process validations.

Isothermal inactivation experiments often are used to investigate the thermal resistance of pathogens, such as Salmonella, in foods; however, little is known about the reproducibility of such experimental methodologies. The objective of this study was to quantify the reproducibility of Salmonella isothermal resistance results via a six-laboratory comparison. Inoculation was performed at a single location and then distributed to each laboratory for isothermal analysis. Salmonella Agona 447967 was inoculated into oat flour, re-equilibrated to a water activity (aw) of 0.45, and then packaged and distributed to each laboratory. Before conducting the inactivation trials, each laboratory was required to verify the inoculated product's aw, enumerate Salmonella population levels, and verify that the isothermal treatment medium was at the target temperature (80°C). All laboratories were required to process at least three replications, collect at least six sample time points with three subsamples at each sampling point, enumerate survivors using an identical plating methodology and media, and verify that the temperature did not substantially change during isothermal treatment. The log-linear model was fit to the Salmonella survivor data, and the resultant D-values were statistically compared via Welch's t test (α = 0.05). Two significant differences in thermal inactivation kinetics were identified as potentially resulting from suspected methodology deviations. Two of the inoculated batches distributed for analysis yielded significantly lower D-values, which likely resulted from a deviation in the inoculation procedures. One laboratory yielded significantly lower D-values, which was likely the result of temperature deviations. Overall, excluding the D-values resulting from deviations, the inactivation results were reproducible, yielding D-values of 30.2 ± 3 min. These results indicate that isothermal inactivation results can be reproducible but that even minor methodology deviations can substantially affect measured Salmonella thermal resistance. HIGHLIGHTS

Process control validations require knowledge of the resistance of the pathogen(s) of concern to the target treatment and, in some cases, the relative resistance of surrogate organisms. Selected strains of Escherichia coli O157:H7 (five strains), Listeria monocytogenes (five strains), and Salmonella enterica (five strains) as well as Salmonella Enteritidis phage type (PT) 30 and nonpathogenic Enterococcus faecium NRRL B-2354 were inoculated separately (as individual strains) onto inshell pistachios. The thermal tolerance of each strain was compared via treatment of inoculated pistachios to hot oil (121°C) or hot water (80°C) for 1 min. Survivor curves in hot oil or hot water (0.5 to 6 min, n = 6 to 15) were determined for one or two of the most resistant strains of each pathogen, as well as E. faecium NRRL B-2354 and Salmonella Enteritidis PT 30, and the Weibull model was fit to the data. A pilot-scale air-impingement oven was used to compare the thermal tolerance of E. faecium NRRL B-2354 and Salmonella Enteritidis PT 30 on pistachios with or without a brining pretreatment and at either dry (no steam) or 30% humidity (v/v) oven conditions. No significant difference in the time to a 4-log reduction in hot oil or hot water was predicted for any of the strains evaluated, on the basis of the 95% confidence interval. In the pilot-scale oven, E. faecium NRRL B-2354 was more thermally resistant than Salmonella in a broad set of differing treatments, treatment times, and temperatures. Salmonella is a suitable target pathogen of concern in pistachios for thermal processes because no other pathogen tested was more thermally resistant under the conditions evaluated. E. faecium NRRL B-2354 was at least as thermally resistant as Salmonella under all conditions evaluated, making it a good potential surrogate for Salmonella on pistachios. HIGHLIGHTS

Salmonella continues to be a problem associated with low-moisture foods, particularly given enhanced thermal resistance at lower water activity (aw). However, there is a scarcity of thermal inactivation models accounting for the effect of aw. The objective of this study was to test multiple secondary models for the effect of product (wheat flour) aw on Salmonella enterica Enteritidis phage type 30 thermal resistance. A full-factorial experimental design included three temperatures (75, 80, and 85°C) and four aw values (~0.30, 0.45, 0.60, and 0.70). Prior to isothermal treatment, sample aw was achieved by equilibrating samples in a humidity-controlled conditioning chamber. Two primary models (log linear and Weibull type) and three secondary models (second-order response surface, modified Bigelow type, and combined effects) were evaluated using the corrected Akaike information criterion and root mean squared errors. Statistical analyses of the primary models favored the log-linear model. Incorporat...

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.

Sublethal heating, which can occur during slow cooking of meat products, is known to induce increased thermal resistance in Salmonella. However, very few studies have addressed the kinetics of this response. Although several recent studies have reported improved thermal inactivation models that include the effect of prior sublethal history on subsequent thermal resistance, none of these models were based on cellular-level responses to sublethal thermal stress. The goal of this study was to determine whether a nonlinear model could accurately portray the response of Salmonella to heat stress induced by prolonged exposure to sublethal temperatures. To accomplish this, stationary-phase Salmonella Montevideo cultures were subjected to various heating profiles (held at either 40 or 45°C for 0, 5, 10, 15, 30, 60, 90, 180, or 240 min) using a PCR thermal cycler. Differential plating on selective and nonselective media was used to confirm the presence of cellular injury. Reverse transcription quantitative PCR was used to screen the transcript levels of six heat stress-related genes to find candidate genes for nonlinear modeling. Injury was detected in populations of Salmonella held at 45°C for 30, 60, and 90 min and at 40°C for 0, 5, and 90 min (P0.05), whereas no significant injury was found at 180 and 240 min (P0.05). The transcript levels of ibpA, which codes for a small heat shock protein associated with the ClpB and DnaK-DnaJ-GrpE chaperone systems, showed the greatest increase relative to the transcript levels at 0 min, which was significant at 5, 10, 15, 30, 60, 90, and 180 min at 45°C and at 5, 10, 15, 30, 60, and 90 min at 40°C (P0.05). Using ibpA transcript levels as an indicator of adaptation to thermal stress, a nonlinear model for sublethal injury is proposed. The use of variables indicating the physiological state of the pathogen during stress has the potential to increase the accuracy of thermal inactivation models that must account for prolonged exposure to sublethal temperatures.

Salmonella can survive in low-moisture, high-protein, and high-fat foods for several years. Despite nationwide outbreaks and recalls due to the presence of Salmonella in low-moisture foods, information on thermal inactivation of Salmonella in these products is limited. This project evaluated the impact of water activity (aw), temperature, and food composition on thermal inactivation of Salmonella enterica serovar Agona in defined high-protein and high-fat model food matrices. Each matrix was inoculated with Salmonella Agona and adjusted to obtain a target aw, ranging from 0.50 to 0.98. Samples were packed into aluminum test cells and heated (52 to 90°C) under isothermal conditions. Survival of Salmonella Agona was detected on tryptic soy agar with 0.6% yeast extract. Complex influences by food composition, aw, and temperature resulted in significantly different ( P < 0.05) thermal resistance of Salmonella for the conditions tested. It was estimated that the same point temperatures at which the D-values of the two matrices at each aw (0.63, 0.73, 0.81, and 0.90) were identical were 79.48, 71.28, 69.62, and 38.42°C, respectively. Above these temperatures, the D-values in high-protein matrices were larger than the D-values in high-fat matrices at each aw. Below these temperatures, the inverse relationship was observed. A correlation between temperature and aw existed on the basis of the level of fat or protein in the food, showing that these compositional factors must be accounted for when predicating thermal inactivation of Salmonella in foods.

Ready-to-eat foods based on dried partially sprouted seeds have been associated with foodborne salmonellosis. Whereas research has focused on the potential for Salmonella initially present in or on seeds to grow and survive during fresh sprout production, little is known about the potential for growth and survival of Salmonella associated with seeds that have been partially sprouted and dried. The goal of this study was to determine the growth of Salmonella during soaking for partial germination of pumpkin, sunflower, and chia seeds and subsequent survival during drying and storage. Pumpkin, sunflower, and chia seeds were inoculated with a four-serotype Salmonella cocktail by the dry transfer method and were soaked in sterile water at 25 or 37°C for 24 h. During the soaking period, Salmonella exhibited growth rates of 0.37 ± 0.26, 0.27 ± 0.12, and 0.45 ± 0.19 log CFU/h at 25°C and 0.94 ± 0.44, 1.04 ± 0.84, and 0.73 ± 0.36 log CFU/h at 37°C for chia, pumpkin, and sunflower seeds, respectively. Soaked seeds were drained and dried at 25, 51, and 60°C. Drying resulted in >5 log CFU/g loss at both 51 and 60°C and ∼3 log CFU/g loss at 25°C on partially sprouted pumpkin and sunflower seeds. There was no decrease in Salmonella during drying of chia seeds at 25°C, and only drying at 60°C provided losses >5 log CFU/g. Dried seeds were stored at 37 and 45°C at 15 and 76% relative humidity (RH) levels. The combination of temperature and RH exerted a stronger effect than either factor alone, such that rates at which Salmonella decreased generally followed this order: 37°C at 15% RH < 45°C at 15% RH < 37°C at 76% RH < 45°C at 76% RH for all seeds tested. Rates differed based on seed type, with chia seeds and chia seed powder having the smallest rate of Salmonella decrease, followed by sunflower and pumpkin seeds. Drying at higher temperatures (50 and 61°C) or storing at elevated temperature and humidity (45°C and 76% RH) resulted in significantly different rates of Salmonella decrease.

Inoculation of a food product for use in subsequent validation studies typically makes use of a high concentration cocktail of microorganisms suspended in aqueous media. However, this inoculation method may prove difficult particularly when the food product is a low-moisture food containing antimicrobial compounds, such as some dried spices. In this study, a dry transfer method for inoculation of clove powder, oregano leaves, ginger powder, and ground black pepper with a five-serovar cocktail of Salmonella was developed and compared with a traditional aqueous inoculation procedure. Spices were inoculated at three levels, 10, 8, and 6 log CFU/g, by using both an aqueous suspension of Salmonella and a dry transfer of Salmonella from previously inoculated silica beads. At the highest inoculation level, the dry transfer method resulted in a significantly higher microbial load (P0.05) for ground cloves and oregano, but not for ginger and ground black pepper. At the intermediate inoculation level, differences were apparent only for ginger and black pepper. Inoculation levels of 6 log CFU/g resulted in recoveries below detection limits for both methods of inoculation. Additional examination on the survival of Salmonella on silica beads after inoculation and in clove powder after dry transfer from silica beads showed linear rates of decline, with a rate of -0.011 log CFU/g/day for beads and -0.015 log CFU/g/day for clove powder. The results suggest that dry transfer of Salmonella via inoculated silica beads is a viable alternative when traditional aqueous inoculation is not feasible.

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 D60°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.