30,000 Petri Dishes Later

By Annel K. Greene, PhD, Professor and Center Director
Clemson University Animal Co-Products Research and Education Center


The need for validated thermal processing parameters for rendering continues to grow with implementation of increasing food and feed regulatory actions. In food microbiology terminology, a thermal death rate curve is a graphical depiction showing how a microbial population declines over time when heat is applied. Using a measure of bacterial population on the y-axis and time on the x-axis, bacterial populations are plotted as points through time. As a result, a thermal death rate curve can be established. The “D-value” or decimal reduction time was defined by Banwart (1979) as “the time required to reduce the microbial population by 90 percent at a specified temperature.” In other words, the D-value is the amount of time required to reduce a population of microorganisms by one log colony forming units per gram at a particular time in a particular matrix.

Thermal death time information for various pathogenic microorganisms has been established in thousands of products within the food industry. However, in the hundreds of thermal death time studies conducted over the past 100-plus years, food microbiologists have repeatedly proven that thermal death rate curves of microorganisms are dependent on three factors: (1) the temperature, (2) the organism, and (3) the product (matrix). For instance, in a study conducted by Juneja et al. (2001), the number of minutes required to reduce a Salmonella cocktail was impacted by the percentage of fat. In chicken held at 58 degrees Celsius, it took 7.38 minutes to kill one log of bacteria when the fat content was two percent but it took 7.33 minutes at 6.3 percent fat, 8.54 minutes at nine percent fat, and 9.04 minutes at 12 percent fat to kill the same one log of the bacteria. In turkey held at 58 degrees C, it took 7.50 minutes to kill one log of bacteria when the fat content was two percent but it took 7.71 minutes at 6.3 percent fat, 6.91 minutes at nine percent fat, and 7.41 minutes at 12 percent fat to kill the same one log of the bacteria. At different temperatures, with different microorganisms, and with different products containing different percentages of fat, moisture, and protein, the thermal death time is different and typically unpredictable. Therefore, the food industry has spent the past 100 years testing thermal death times for each combination of conditions in order to validate their thermal processing.

Clemson University Animal Co-Products Research and Education Center (ACREC) researchers have been working on experimentally developing thermal death time validation data for eventual inclusion in a table of values related to product, percentage fat, temperatures, and microorganism. Initial work was centered on developing new laboratory techniques when the researchers discovered that standard methodologies for enumeration of bacteria in rendering materials often did not work due to the high fat content of the products. After learning how to deal with these unique products, the researchers continued their studies on validating thermal death times for pathogens in rendered animal by-products.

Melissa “Lissa” Hayes is conducting thermal death time studies on four of the eight pathogenic strains of Salmonella recognized by the Food and Drug Administration as dangerous for feed. Hayes is a PhD student in microbiology working in Dr. Annel K. Greene’s laboratory at Clemson University on this ACREC-supported project. Using Salmonella choleraesuis, Salmonella Dublin, Salmonella enteriditis, and Salmonella Newport, Hayes is growing each culture individually and concentrating them to produce a final population of at least 10 million bacteria per gram in beef rendering materials adjusted to 50 percent fat content and in poultry rendering materials adjusted to 50 percent fat content. Adding the cultures after the rendering materials have reached a treatment temperature of 240 degrees Fahrenheit (F), Hayes is testing thermal treatment times of zero, 15, 30, 60, 90, 120, 180, 240, and 300 seconds. The chosen treatment temperature of 240 degrees F represents the low end of commercial rendering processing temperatures, and was selected as the starting temperature to begin the thermal death time studies in consultation with the rendering industry members of the ACREC Research Committee.

After thermal treatment of the samples and uninoculated controls, Hayes then conducts a series of tests to determine if each strain of Salmonella survived the heat treatment. Approximately 30,000 Petri dishes will be used to obtain the data for these four Salmonella at this one temperature in 50 percent fat beef and 50 percent fat poultry rendering materials. Initially scheduled for completion at the end of June 2013, the rendering industry asked Hayes to fast-track the project for earlier completion and she is doing so. She has completed the thermal death time analysis at 240 degrees F on Salmonella choleraesuis, Salmonella Dublin, Salmonella enteriditis, and Salmonella Newport in 50 percent fat beef at treatment times of zero, 15, 30, 60, 90, 120, 180, 240, and 300 seconds. Data indicates some variability in Salmonella thermal destruction and there appears to be heat resistant background organisms present. However, at 240 degrees F for Salmonella enteriditis, the last positive sample appeared at 180 seconds and it was destroyed afterwards. For Salmonella Newport and Salmonella Dublin under the same conditions, the last positive sample appeared at 240 seconds and each was destroyed afterwards. For Salmonella choleraesuis, the last positive appeared at 240 seconds on only one day of experimentation; on the other three days of the experiment, the bacteria were destroyed at zero seconds. Beef fat is harder than poultry fat so it will be interesting to see if type of fat has an impact on thermal death of these pathogenic Salmonella. As of early March, Hayes had completed approximately one-third of the poultry project.

Upon completion and preparation and counting of 30,000-plus Petri dishes, Hayes will have eight data points for inclusion in a table of thermal death time values that would show destruction of Salmonella choleraesuis, Salmonella Dublin, Salmonella enteriditis, and Salmonella Newport in 50 percent fat beef and 50 percent fat poultry at 240 degrees F. Further studies will be conducted at the direction of the ACREC Research Committee members whether to move the temperature up and keep the fat percentages the same, or change the fat percentages and continue testing at 240 degrees F. The eventual goal of the researchers is to develop a table where renderers can look up their tissue type (poultry, beef, pork, mixtures), percentage of fat, and processing temperature and find the minimum amount of time required to kill a particular pathogen (each of the pathogenic Salmonella and eventually Clostridium perfringens). This is very time-consuming and expensive work to conduct but the results will be a document of great value for validation of processing conditions within each rendering processing plant.

Hayes, the young woman from Camden, SC, who loves the color pink, is a dedicated and extraordinary student of microbiology. She is rapidly becoming one of the world’s leaders in rendering microbiology. Hayes is scheduled to graduate with her PhD in microbiology later this year and we hope to secure funding to hire her as a post-doctoral candidate to continue her work on the thermal death time studies for the rendering industry.

References:
Banwart, G.J. 1979. Basic Food Microbiology. The AVI Publishing Company, Inc. Westport, CT.
Juneja, V.K., B.S. Eblen, and H.M. Marks. 2001. “Modeling non-linear survival curves to calculate thermal inactivation of Salmonella in poultry of different fat levels.” International Journal of Food Microbiology 70: 37–51.


April 2013 RENDER | back