This project intends to investigate thermal inactivation of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in moist buffer solutions, in simple model low-moisture matrices, and in low-moisture foods such as flour and peanut butter. Using the simple low-moisture simple models as a pre-screening tool, additional thermal resistance testing will be completed of select STEC and Salmonella in low-moisture food matrices in order to increase science-based risk prevention data to estimate lethality. <P>Specific Aim 1. Determine thermal characteristics of Salmonella enterica serovars and Shiga toxin-producing Escherichia coli strains in a moist environment buffer. Salmonella and STEC selection will be finalized 1st quarter FY2013. Preliminary data for Specific Aim 1 have been investigated and will continue 2nd-3rd quarters FY2013. Results from Specific Aim 1 will be used to determine strain selection to progress onto pre-screening using simple low-moisture systems for Specific Aim 2 3rd quarter FY2013. <P>Specific Aim 2. Investigate the thermal resistance of Salmonella enterica serovars and Shiga toxin-producing Escherichia coli in simple matrices that serve as model low-moisture food environments. Inoculation procedures for low-moisture food matrices and evaluation of heating processes occurs 4th quarter FY2013. <P>Specific Aim 3 will be completed by 3rd quarter FY2014. Specific Aim 3. Investigate the thermal resistance of pre-screened Salmonella enterica serovars and Shiga toxin-producing Escherichia coli in multiple low-moisture food matrices. During the 4th quarter FY2014 data analysis will occur resulting in a peer-reviewed publication and presentation at a national scientific conference. <P>Completion of this project will contribute to the specific needs of the regulatory agencies ensuring the safety of food in the United States, especially the FDA in regards to the goals set forth in the Food Safety Modernization Act. Applied research involving thermal resistance of pathogenic microorganisms in low-moisture foods will fill current knowledge gaps needed for science-based preventive controls to assure the safety of foods, indicated through the AFRI Priority Area of "Food Safety, Nutrition, and Health", and the Primary Challenge Area of "Food Safety". As nearly one-third of all reported foodborne diseases in 2008 were attributed to either Salmonella or STEC, it is imperative to eliminate the threat of these pathogens in order to protect public health. This research may result in verifying moist-heat characterization as a rapid, preliminary assessment tool for low-moisture thermal resistance. This research will set the groundwork to validate cleaning and sterilization requirements to eliminate the risk of thermally resistant pathogens in low-moisture foods and processing environments. Knowledge of thermal resistance of Salmonella and STEC in low-moisture foods will provide the food industry with much needed baseline inactivation data needed to validate thermal processes used as "kill steps" in the processing of low-moisture foods.
In the last five years, there have been nine outbreaks associated with low-moisture foods. Some of these foods include: nuts, spices, pet food, cookie dough, and extruded snack foods. Over 1,600 people became sick, and nine people died, after eating these foods contaminated with pathogenic bacteria (Salmonella and Escherichia coli). While pathogenic bacteria cannot grow in these foods, they are able to stay alive over time. Many low-moisture foods, such as flour, are ingredients used in other products. If bacteria get into ready-to-eat food or ingredients people may become sick. Ready-to-eat foods are foods that you eat directly from the store without cooking or heating them at home. This is even more likely to happen if those bacteria do not die very quickly when the foods are cooked or heated. This project will study how quickly these bacteria die at different temperatures. It will also develop simple predictive model systems for the heat resistance of some bacteria known to cause illness. Development of these predictive models will be essential for establishing ways to prevent outbreaks from occurring in the future.
All isolates of Salmonella and STEC used will be obtained from FDA regulated foods or from clinical origins. Screening of moist heat resistance of stationary phase cells of STEC and Salmonella (n > 20) will be completed in a buffered peptone water system via thermal heat survival through residual viable counts for Salmonella and STEC, respectively. Residual viable counts of Salmonella and STEC in buffer at three target temperatures maintained at three holding times will be used to obtain valid inactivation points. Salmonella and STEC with high thermal tolerances will be determined by selecting serovars/strains with D-values = 1.5 x the baseline value, ensuring a minimum of at least a 5-log reduction. Simple model test systems will be identified to mimic thermal resistance behavior of Salmonella serovars in more complex food matrices. Basic studies using simple osmotic solutions will be performed using a minimum of ten test isolates. Solutions consisting of various concentrations of these osmotic agents will be prepared, sterilized and their resulting water activity determined in triplicate. Additionally, simple food systems including evaporated milk, corn syrup, chocolate syrup, and others as needed will also be tested as potential model systems for profiling Salmonella heat resistance. Multiple dry inoculum preparations will be evaluated in terms of reproducibility, ease, and validity. Depending on the composition of the low-moisture food matrix, additional inoculation procedures into the matrix will be evaluated. For food matrices such as peanut butter (aw ~ 0.25), previous research (data not shown) has shown that in cultures grown on sessile environments, harvested, and inoculated into the high lipid emulsion with aid of a surfactant produced the best results by minimizing inherent death of cells through drying effects of the wet inoculum. For low-moisture, low-fat products, such as flour (aw ~ 0.10), best practices for inoculation will be evaluated. Either the inoculum will be dried and applied to the flour or the inoculum will be added to the flour and then dried. Due to inherent properties of low-moisture food matrices, it is imperative to evaluate the heating profiles to ensure rapid and uniform heat transfer throughout the thermal process. Additionally, it is important that the sample be contained for accurate results. Preliminary research will be conducted to ensure uniform heating of the sample for thermal kinetics studies. The STEC and Salmonella test cultures chosen from initial thermal inactivation studies will be propagated and inoculated into low-moisture foods, peanut butter and flour. Thermal inactivation in peanut butter and flour will be evaluated at 70 to 80C at predetermined time intervals for up to one hour. Enumeration of viable cells in the flour before and after the heat treatment will be evaluated in triplicate. Results from low-moisture foods will be correlated with the results from simple systems to determine if low-moisture model systems can be used as a preliminary pre-screening tool to indicate specific thermal resistances in actual food matrices.