Interlaboratory Evaluation of Enterococcus faecium NRRL B-2354 as a Salmonella Surrogate for Validating Thermal Treatment of Multiple Low-Moisture Foods.

ABSTRACT: 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 (P < 0.05) 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 (P < 0.05) 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 (P > 0.05) 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.

Thermal Inactivation Kinetics of Salmonella and Enterococcus faecium NRRL-B2354 on Whole Chia Seeds (Salvia hispanica L.).

ABSTRACT: Intervention technologies for inactivating Salmonella on whole chia seeds are currently limited. Determination of the thermal inactivation kinetics of Salmonella on chia seeds and selection of an appropriate nonpathogenic surrogate will provide a foundation for selecting and optimizing thermal pasteurization processes for chia seeds. In this study, chia seed samples from three separate production lots were inoculated with a five-strain Salmonella cocktail or Enterococcus faecium NRRL-B2354 and equilibrated to a water activity of 0.53 at room temperature (25°C). After equilibration for at least 3 days, the inoculated seeds were subjected to isothermal treatments at 80, 85, or 90°C. Samples were removed at six time points, and surviving bacteria were enumerated. Whole chia seeds were diluted in a filter bag at 1:30 because bacterial recovery with this method was similar to that obtained from ground seeds. Survivor data were fitted to consolidated models: one primary model (log linear or Weibull) and one secondary model (Bigelow). E. faecium had higher thermal resistance than did Salmonella, suggesting that E. faecium may be a suitable conservative nonpathogenic surrogate for Salmonella. The Weibull model was a better fit for the survivor data than was the log-linear model for both bacteria based on the lower root mean square error and corrected Akaike's information criterion values. Lipid oxidation measurements and fatty acid concentrations were significantly different from those of the control samples, but the overall magnitude of the differences was relatively small. The thermal inactivation kinetics of Salmonella and E. faecium on chia seeds may be used as a basis for developing thermal pasteurization processes for chia seeds.

Integration of ozone with co-immobilized microalgae-activated sludge bacterial symbiosis for efficient on-site treatment of meat processing wastewater.

Direct discharge of high concentration meat processing wastewater (MPW) into municipal sewage system will cause serious shock loading and reduce wastewater treatment efficiency, thus, efficient on-site pretreatment is usually required. Purpose of this study is to integrate ozone with microalgal biotreatment to achieve effective removal of both organic compounds and nutrients with one-step biodegradation and obtain high quality effluent dischargeable to municipal sewage system. Results showed that ozone pretreatment removed 35.0-90.2% color and inactivated 1.8-4.7 log CFU/mL bacteria in MPW. In post biotreatment using microalgae co-immobilized with activated sludge (ACS) bacteria, bacterial growth in ozone pretreated wastewater (7.1-8.1 log CFU/mL) were higher than non-pretreated control (6.0 log CFU/mL) due to enhanced biodegradability of wastewater pollutants. Algal biomass growth in wastewater pretreated with 0.5 (2489.3 mg/L) and 1 (2582.0 mg/L) minute's ozonation were improved and higher than control (2297.1 mg/L). Ozone pretreatment significantly improved nutrients removal. Following ozone pretreatment of 0.5 min, microalgal biotreatment removed 60.1% soluble chemical oxygen demand (sCOD), 79.5% total nitrogen (TN) and 91.9% total phosphate (PO43-) which were higher than control (34.4% sCOD, 63.4% TN, 77.6% total PO43-). Treated effluent contained 342.3 mg/L sCOD, 28.8 mg/L TN, 9.9 mg/L total PO43- and could be discharged into municipal sewage system. However, excessive ozone pretreatment displayed adverse impact on algal growth and sCOD removal. Therefore, integration of 0.5 min's ozone pretreatment with microalgae-based biotreatment is an efficient on-site treatment to simultaneously remove organic compounds and nutrients with one-step biodegradation.

A Comparison of Three Methods for Determining Thermal Inactivation Kinetics: A Case Study on Salmonella enterica in Whole Milk Powder.

ABSTRACT: Different methods for determining the thermal inactivation kinetics of microorganisms can result in discrepancies in thermal resistance values. In this study, thermal resistance of Salmonella in whole milk powder was determined with three methods: thermal death time (TDT) disk in water bath, pouches in water bath, and the TDT Sandwich system. Samples from three production lots of whole milk powder were inoculated with a five-strain Salmonella cocktail and equilibrated to a water activity of 0.20. The samples were then subjected to three isothermal treatments at 75, 80, or 85°C. Samples were removed at six time points and cultures were enumerated for survivors. The inactivation data were fitted to two consolidated models: two primary models (log linear and Weibull) and one secondary model (Bigelow). Normality testing indicated that all the model parameters were normally distributed. None of the model parameters for both consolidated models were significantly different (α = 0.05). The amount of inactivation during the come-up time was also not significantly different among the methods (α = 0.05). However, the TDT Sandwich resulted in less inactivation during the come-up time and overall less variation in model parameters. The survivor data from all three methods were combined and fitted to both consolidated models. The Weibull had a lower root mean square error and a better fit, according to the corrected Akaike's information criterion. The three thermal treatment methods produced results that were not significantly different; thus, the methods are interchangeable, at least for Salmonella in whole milk powder. Comparisons with more methods, other microorganisms, and larger varieties of food products using the same framework presented in this study could provide guidance for standardizing thermal inactivation kinetics studies for microorganisms in foods.

Effect of water activity on the thermal inactivation kinetics of Salmonella in milk powders.

Persistence of Salmonella in milk powders has caused several foodborne outbreaks. The determination of proper pasteurization processing conditions requires an understanding of the thermal inactivation kinetics of Salmonella in milk powders. However, there is a lack of knowledge related to the effects of water activity (aw) and fat content on Salmonella inactivation in milk powder during thermal processing. Two types of milk powders, nonfat dry milk and whole milk powder, with different fat contents (0.62 and 29.46% wt/wt, respectively) were inoculated with a 5-strain cocktail of Salmonella and equilibrated to 3 aw levels (0.10, 0.20, and 0.30) for isothermal treatments at 75, 80, and 85°C to obtain D-values (the time required to achieve a 10-fold reduction of the bacteria at the isothermal treatment temperature) and z-values (the increase in temperature required to achieve a 90% reduction of the decimal reduction time D). Stability tests showed that the inoculation method used in this study provided a high and stable population of Salmonella for thermal inactivation studies. A moisture sorption isotherm was measured to understand the relationship between aw and moisture content of milk powders. The thermal resistance of Salmonella was found to significantly increase as aw decreased, which suggested that a higher temperature or longer processing time would be required at low aw to achieve the desired inactivation of Salmonella. The microbial inactivation kinetics were not significantly different for the 2 milk powders; therefore, data were combined to develop a universal model. A response surface model was compared with a modified Bigelow model. The modified Bigelow model performed well to predict D-values [root mean square error (RMSE) = 1.47 min] and log reductions (RMSE = 0.48 log cfu/g). The modified Bigelow model developed here could be used to estimate D-value as a function of water activity and temperature to design a thermal pasteurization system for milk powders.

TDT Sandwich: An open source dry heat system for characterizing the thermal resistance of microorganisms.

The determination of the thermal death kinetics of microorganisms has traditionally been performed with liquid baths which have some disadvantages such as liquid spillage and liquid infiltration into samples. The TDT Sandwich was developed as a free, open source alternative that utilizes dry heat. The system is capable of heating samples up to 140 °C and maintaining it within 0.2 °C of the target temperature. Other features of the TDT Sandwich include adjustable heating rates up to approximately 100 °C/min, real-time display and recording of temperature readings at a nominal rate of 5 Hz, an optional thermocouple for acquiring temperature of samples, built-in heating timer, and customizable operating parameters. The modular nature of the TDT Sandwich allows multiple units to be connected to a computer or laptop. Operation of the TDT Sandwich is done through a computer program which, along with the build instructions and microcontroller program, are open source and are available for free to the public at https://doi.org/10.17605/OSF.IO/5Q3Y7.

HumidOSH: A self-contained environmental chamber with controls for relative humidity and fan speed.

Relative humidity can affect physical, biological, and chemical changes in biological samples through modification of water activity and is also known to be important in the fabrication of sensitive electronic devices. The HumidOSH is a free, open source, and self-contained system for creating a controlled relative humidity environment within the range of 3 to 97% with a 0.2% tolerance. Each HumidOSH unit also comes with a fan with adjustable fan rotational speed to improve moisture uniformity inside the chamber. The system includes many additional features such as glove ports for manipulating samples, a sample door for transferring objects in and out of the system, ceiling lights for illuminating the work area inside the chamber, and two-point calibration for the relative humidity sensor. While relative humidity and fan rotational speed readings are displayed in real-time on the built-in user-friendly interface, the readings can also be recorded through a USB connection to a laptop or computer and the optional computer program. The design files, source code, and build instructions of the HumidOSH can be accessed at https://dx.doi.org/10.17605/OSF.IO/WCKHM.

A microbial challenge study for validating continuous radio-frequency assisted thermal processing pasteurization of egg white powder.

Spray dried egg white powder (EWP) is traditionally processed by hot room treatment for a prolonged period of time (67 °C for 15 days) to enhance its functionality (foaming and gelling) and to improve microbial safety of EWP. Our prior research demonstrated that radio-frequency (RF) assisted thermal processing can considerably reduce the processing time, without compromising the functional properties of EWP. In this study, continuous RF processing was evaluated for pasteurization of EWP. EWP samples were inoculated with a 5-strain Salmonella cocktail or Enterococcus faecium NRRL B-2354 for the microbial challenge studies. To evaluate the inoculation method, stability and homogeneity tests were conducted for both Salmonella and E. faecium in EWP. Continuous RF heating of EWP was conducted in a 6-kW, 27.12 MHz pilot-scale parallel-plate RF heating system. RF-assisted thermal processing of EWP at 80 °C for 2 h provided >6.69 log reduction for Salmonella. E. faecium was found to be a suitable surrogate for Salmonella due to its higher resistance and similar inactivation kinetics during RF heating of EWP. The validated RF-assisted thermal process can be scaled up for use in the egg industry.

Radiofrequency pasteurization process for inactivation of Salmonella spp. and Enterococcus faecium NRRL B-2354 on ground black pepper.

Salmonella persistence in ground black pepper has caused several foodborne outbreaks and created public concern about the safety of low water activity (aw) foods. In this study, radiofrequency (RF) processing was evaluated for pasteurization of ground black pepper. Stability and homogeneity tests were done for both Salmonella spp. and E. faecium during moisture equilibration before RF heating to evaluate the inoculation method. Moisture content of samples were conditioned such that the final moisture content after RF heating reached the optimal storage moisture. RF heating was shown to provide more than 5.98 log CFU/g reduction for Salmonella spp. and the reduction of 3.89 log CFU/g for E. faecium with a 130 s of treatment time. The higher thermal resistance of E. faecium indicated its suitability as surrogate for Salmonella spp. during RF heating of ground black pepper. Piperine, total phenolics, volatile compounds, and antioxidant activity were assessed as quality parameters for ground black pepper. The results demonstrated that the RF processing provided effective inactivation of Salmonella spp. with insignificant (p > 0.05) quality deterioration.

Radio-Frequency Processing for Inactivation of Salmonella enterica and Enterococcus faecium NRRL B-2354 in Black Peppercorn.

Several Salmonella outbreaks linked to black pepper call for effective inactivation processes, because current decontamination methods result in quality deterioration. Radio-frequency (RF) heating provides a rapid heating rate and volumetric heating, resulting in a shorter come-up time. This allows for choosing a high-temperature and short-time combination to achieve the desired inactivation with minimal quality deterioration. The objectives of this study were to evaluate RF heating for inactivation of Salmonella enterica and Enterococcus faecium in black peppercorn and evaluate quality changes of RF-treated black peppercorn. Black peppercorns were inoculated with a five-strain cocktail of Salmonella or E. faecium to attain initial population levels of 6.8 and 7.3 log CFU/g, respectively, and were then adjusted to a moisture content of 12.7% (wet basis) and a water activity of 0.60 at room temperature. A stability test was performed to quantify the microbial reduction during inoculation and equilibration before RF heating inactivation. During RF heating, the cold spot was determined to be at the center on the top surface of the treated sample. In addition to inoculating the entire sample, an inoculated packed sample was placed at the cold spot of the tray. An RF heating time of 2.5 min provided a 5.31- and 5.26-log CFU/g reduction in the entire sample contained in the tray for Salmonella and E. faecium, respectively. Color parameters (L*, a*, b*), piperine content, total phenolics, scavenging activity, and most of the volatile compounds of 2.5-min RF-treated samples were not significantly different from those of the control samples. These data suggest that RF heating is a promising thermal inactivation treatment for Salmonella without significant quality deterioration, and E. faecium seems to be a suitable surrogate for Salmonella to validate the efficacy of RF heating of black peppercorn.

Response Surface Methodology for Salmonella Inactivation during Extrusion Processing of Oat Flour.

An increase in the number of foodborne outbreaks and recalls due to Salmonella in low-moisture foods has resulted in the need for the development and validation of process controls to ensure their microbiological safety. Furthermore, the Food Safety Modernization Act Preventive Controls for Human Food final rule requires food processors to validate their process controls to ensure food safety. The objective of this study was to develop a response surface model to predict Salmonella inactivation in oat flour, as affected by moisture, fat content, screw speed, and temperature. Oat flour was adjusted to different moisture (14 to 26% wet basis) and fat (5 to 15% [w/w]) contents and was then inoculated with a five-strain cocktail of Salmonella. Inoculated material was extruded through a single-screw extruder running at different screw speeds (75 to 225 rpm) and temperatures (65 to 85°C), without a die. Once steady-state conditions were attained, extruded samples were collected, cooled, and stored under refrigeration, and Salmonella survivors were enumerated. A split-plot central composite second-order response surface design was used, with the central point replicated six times. Temperature showed a significant ( P < 0.0005) positive effect on microbial reduction. Moisture content showed significant linear ( P = 0.0014) and quadratic ( P = 0.0005) effects, whereas higher fat content showed a significant ( P < 0.0001) protective effect on Salmonella destruction. The screw speed did not play a major role in inactivating Salmonella, but it had a significant ( P = 0.0004) interactive effect with temperature. Results indicated that a >5.5-log reduction was achieved in oat flour extruded at a temperature above 85°C at all moisture and fat contents evaluated at a screw speed of 150 rpm. The developed response surface model can be used to identify the extrusion process conditions to achieve a desired reduction of Salmonella based on the moisture and fat contents of the product.

Evaluation of Enterococcus faecium NRRL B-2354 as a Surrogate for Salmonella During Extrusion of Low-Moisture Food.

Salmonella in low-moisture foods is an emerging challenge due to numerous food product recalls and foodborne illness outbreaks. Identification of suitable surrogate is critical for process validation at industry level due to implementation of new Food Safety Modernization Act of 2011. The objective of this study was to evaluate Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during the extrusion of low-moisture food. Oat flour, a low-moisture food, was adjusted to different moisture (14% to 26% wet basis) and fat (5% to 15% w/w) contents and was inoculated with E. faecium NRRL B-2354. Inoculated material was then extruded in a lab-scale single-screw extruder running at different screw speeds (75 to 225 rpm) and different temperatures (75, 85, and 95 °C). A split-plot central composite 2nd order response surface design was used, with the central point replicated six times. The data from the selective media (m-Enterococcus agar) was used to build the response surface model for inactivation of E. faecium NRRL B-2354. Results indicated that E. faecium NRRL B-2354 always had higher heat resistance compared to Salmonella at all conditions evaluated in this study. However, the patterns of contour plots showing the effect of various product and process parameters on inactivation of E. faecium NRRL B-2354 was different from that of Salmonella. Although E. faecium NRRL B-2354 may be an acceptable surrogate for extrusion of low-moisture products due to higher resistance than Salmonella, another surrogate with similar inactivation behavior may be preferred and needs to be identified. PRACTICAL APPLICATION: Food Safety Modernization Act requires the food industry to validate processing interventions. This study validated extrusion processing and demonstrated that E. faecium NRRL B-2354 is an acceptable surrogate for extrusion of low-moisture products. The developed response surface model allows the industry to identify process conditions to achieve a desired lethality for their products based on composition.

Radiofrequency Heating for Enhancing Microbial Safety of Shell Eggs Immersed in Deionized Water.

Radiofrequency (RF) heating was simulated for improving the microbial safety of a shell egg immersed in deionized water using a finite element model. A regression equation that relates the top electrode voltage to the gap between the electrodes and vertical position of the egg was also developed. The root mean squared errors between the simulation and validation results ranged from 0.76 to 2.86 °C. Concentrated heating occurred in the yolk in all the investigated configurations, with some configurations reaching close to 60 °C in the yolk after 20 min of RF heating. The cooling effect of the water along with lower electric field intensity in the egg caused the focused heating in the yolk. Extrapolation of the model revealed that a scaled-up RF heating process (10.5 kV at top electrode) followed by a hot water immersion process can achieve a minimum of 3 log reductions of Salmonella in the yolk within 37 min. PRACTICAL APPLICATION: Radiofrequency (RF) heating with the assistance of deionized water was shown to have the potential of improving the microbial safety of shell eggs. The validated model results revealed that this effect was due to a combination of focused heating and surface cooling.

Challenges in Radiofrequency Pasteurization of Shell Eggs: Coagulation Rings.

A total of 50 different configurations of simple radiofrequency (RF) heating at 27.12 MHz of a shell egg were simulated using a finite element model with the purpose of pasteurizing the egg. Temperature-dependent thermal and dielectric properties of the yolk, albumen, and shell were measured, fitted, and introduced into the model. A regression equation that relates the top electrode voltage to the gap between the electrodes and vertical position of the egg was developed. Simulation and experimental results had good agreement in terms of temperature deviation (root mean squared error ranged from 0.35 °C to 0.48 °C) and both results demonstrated the development of a "coagulation ring" around the air cell. The focused heating near the air cell of the egg prevented pasteurization of the egg due to its impact on quality (coagulation). Analysis of the electric field patterns offered a perspective on how nonuniform RF heating could occur in heterogeneous food products. The results can be used to guide development of RF heating for heterogeneous food products and further development of RF pasteurization of eggs.