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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

Survival during temporary storage and heat inactivation of Shiga toxin-producing Escherichia coli (STEC) were investigated in a selection of four different low moisture foods with water activities between 0.26 and 0.63. A cocktail of nine strains of O157 and non-O157 STEC was selected for this study, giving preference to those strains of public health concern associated with food consumption. The data from previous studies on STEC survival and heat resistance in wheat flour were taken into account for the selection of the relevant target serotypes and strains. No significant difference was observed on STEC viable counts among the four products stored at 16 °C for 7 days. The viable counts decreased by maximum 1 log CFU/g by day 28, as simulation of temporary storage of bulk raw materials before processing. The level of STEC inactivation by dry heat treatments varied with the food formulation. The STEC bacteria showed a lower heat resistance in savory seasoning and pet food formulations compared to confectionery formulation and chicken meat powder. We also identified the minimum heating temperature and time combinations for each food to achieve a 5 log reduction of STEC using linear and Weibull models. Our results show the capability of obtaining a 5 log reduction of STEC in pet food, savory seasoning and confectionery formulations, and chicken meat powder by heating to product temperatures such as 90.9, 92.7, 99.1, and 106.0 °C, respectively. High product temperatures can be achieved by heating food samples in thermal cells hermetically sealed and submerged in a thermo-fluid bath at temperatures of 110 °C or above. The use of thermal cells allowed to simulate in the laboratory the high processing temperatures such as those applied in food extrusion or continuous treatment without moisture loss during heating.

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.