Determination and Validation of D-Values for Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli in Cheesemilk

Abstract

Certain cheeses can be legally produced in the U.S. using raw milk, but must be aged for at least 60 days to reduce pathogen risks. However, some varieties, even when aged for 60 days, have been shown to support growth of Listeria monocytogenes or survival of Shiga toxin-producing Escherichia coli (STEC). Thermization, as a sub-pasteurization heat treatment, has been proposed as a control to reduce the risk of pathogens in raw cheesemilk while retaining some quality attributes in the cheese. However, the temperature / time combinations needed to enhance safety have not been well characterized. The objective of this research was to determine and validate D-values for L. monocytogenes and STEC at thermization temperatures 65.6, 62.8, and 60.0°C; a D-value at 57.2°C was also determined for L. monocytogenes only. Non-homogenized whole milk samples (1 ml) were inoculated with 8-log CFU/ml L. monocytogenes or STEC (5- or 7-strain mixtures, respectively), vacuum-sealed in moisture-impermeable pouches and heated via water bath submersion. Duplicate samples were removed at appropriate intervals and immediately cooled in an ice bath. Surviving bacteria were enumerated on Modified Oxford or Sorbitol MacConkey overlaid with Tryptic Soy agar to aid in the recovery of heat-injured cells. Duplicate trials were conducted, and survival data were used to calculate thermal inactivation rates. D65.6°C, D62.8°C, and D60.0°C-values of 17.1 and 7.2, 33.8 and 16.9, and 146.6 and 60.0 s were found for L. monocytogenes and STEC, respectively, and a D57.2°C-value of 909.1 s was determined for L. monocytogenes. Triplicate validation trials were conducted for each test temperature using 100 ml milk inoculated with 3-4 log CFU/ml of each pathogen cocktail. A 3-log reduction of each pathogen was achieved more quickly in larger volumes than what was predicted by D-values (D-values were fail-safe). Data were additionally compared to published results from 21 scientific studies investigating L. monocytogenes and STEC in whole milk heated to thermization temperatures (55.0 to 71.7°C). These data can be used to give producers of artisanal raw milk cheese flexibility in designing thermal processes to reduce L. monocytogenes and STEC populations to levels that are not infectious to consumers.