Enterococcus faecium NRRL-B2354 as a Surrogate for Salmonella spp. for the Validation of Extrusion

Introduction: Multiple outbreaks of salmonellosis have been associated with the consumption of low-moisture products including extruded products such as breakfast cereals and pet foods.  Although these outbreaks are uncommon, they generally affect large numbers of consumers due to the widespread use of these products.  Therefore, there is a need for a non-pathogenic, surrogate microorganism that can be used to validate extrusion processes for Salmonella spp.

Purpose: The objective of this research was to determine if Enterococcus faecium NRRL B-2354 is an adequate surrogate organism for Salmonella spp during extrusion by comparing the temperature profiles at which each organism is inactivated.

Methods: A balanced carbohydrate-protein mix was formulated to 28% moisture and inoculated with pure cultures of either Enterococcus faecium NRRL-B2354 or a five-strain cocktail of heat resistant Salmonella enterica to a final level of 5 logs per gram or above. In each experiment, the mix was processed in a pilot scale extruder using temperatures from 60 to 110 °C in 5 °C intervals. At each evaluated temperature, the extruder was allowed to equilibrate for 10 minutes before sample collection.  Samples were collected in sterile bags, cooled in dry ice, and stored at 4 °C prior to analysis. These were then enumerated for Salmonella using Tryptic Soy Agar (TSA) with an XLT4 overlay and also in XLT4 alone, followed by incubation at 37 °C for 24 h. E. faecium was enumerated using both TSA and mEnterococcus media followed by incubation at 35 °C for 48 hours. The extrusion process for each organism was replicated twice and from each extrusion, three subsamples were evaluated for microbial counts.  Survival curves were then plotted based on enumeration data (log CFU/g) and temperature to compare the heat resistance of each organism.

Results: According to the data collected, the minimum temperature needed to achieve a 5 log reduction of E. faecium was 73.5 °C in this food matrix. Above 80 °C, E. faecium steadily decreased until undetectable levels (<10 CFU per g) were reached (R² = 0.813).  Salmonella spp were reduced by 5 logs at 61.0 °C, and above 68.0 °C they decreased until undetectable (R² = 0.777).

Significance: The data shows that E. faecium NRRL-B-2354 is inactivated at a higher temperature than Salmonella, indicating that its use as a surrogate would provide an appropriate margin of error in extrusion processes designed to eliminate this pathogen.  With increased regulatory pressure to minimize risk, the industry will be required to validate different formulations in combination with varying thermal treatments, and the use of E. faecium presents itself as a safer alternative for those validation studies.