Purpose: To develop inoculation methods for Enterococcus faecium on whole black peppercorn and cumin seeds that achieve a minimum of 6 log CFU/g on the surface and are stable for a period of 28 d.
Methods: Whole black peppercorns and cumin seeds were inoculated with Enterococcus faecium NRRL B-2354 cells using three inoculation methods: liquid-grown, agar-grown, and biofilm-incorporated cells. Liquid-grown E. faecium were incubated 24 h in tryptic soy broth (TSB) at 37°C, washed and applied to spices. Tryptic soy agar (TSA) grown cells were scraped from TSA plates grown for 24 h at 37°C, and applied directly to spices. E. faecium was incorporated within a native microbiota biofilm on spices over a 24 h period during static incubation in TSB at 37°C. Inoculated spices were dried to aw 0.5, and held for 28 days. Spices (n = 3) were sampled after drying (time 0), and after 1, 7, 14, 21, and 28 days of storage. Cells were enumerated following serial dilution and plating onto bile esculin agar (BEA) and TSA.
Results: Enterococcus faecium recovery was high after 28 d storage, with greater than 6 log CFU/g recovered from peppercorns and cumin seeds. Significant reductions (P < 0.04) in recoverable E. faecium from peppercorns occurred only for the TSB grown cells (0.58-log CFU/g reduction) after 28 d. On cumin seeds, larger reductions after 28 days occurred for TSB grown, TSA grown and biofilm incorporated methods (1.2, 0.5 and 0.6-log CFU/g reductions, respectively).
Significance: The inoculation method influenced the recoverability of Enterococcus faecium from whole peppercorns and cumin seeds after drying. Developing stable inoculation methods is a crucial step toward determining if E. faecium NRRL B-2354 is an appropriate surrogate organism for commercial validation of Salmonella inactivation methods for spices.