Enhanced heat tolerance of freeze-dried Enterococcus faecium NRRL B-2354 as valid Salmonella surrogate in low-moisture foods.

In microbial studies of low-moisture foods (LMFs, water activity less than 0.85), freeze-dried bacteria benefit us to inoculate LMFs without introducing extra water or altering food physiochemical properties. However, the freeze-drying process would bring unavoidable damage to bacterial cells and results in less-resistant inoculum that are unlikely to be qualified in microbial studies. Herein, we enhanced bacterial heat tolerance by subjecting the cells to mild heat (42-50 °C) to counteract the reduced heat tolerance and survivability of freeze-dried bacteria. Enterococcus faecium NRRL B-2354 (E. faecium), a Salmonella surrogate in LMFs, was used as the target microorganism because it was widely accepted in microbial validation of thermal pasteurizing LMFs. Three types of LMFs (peanut powder, protein powder, and onion powder) were used as LMFs models to validate the freeze-dried E. faecium in comparison with Salmonella enterica Enteritidis PT 30 (S. Enteritidis) prepared by the traditional aqueous method. The heat tolerance (D value) of E. faecium increased at all treatments and peaked (+31.48 ± 0.13%) at temperature-time combinations of 45 °C-60 min and 50 °C-5 min. Survivability of freeze-dried inoculum and its heat tolerance retained well within 50 d storage. The freeze-dried E. faecium was prepared in this study brought equal or higher heat tolerance (D or D) than S. Enteritidis in tested LMFs models. For instance, the D of freeze-dried E. faecium (heat-treated at 50 °C for 5 min) and S. Enteritidis in whole egg powder are 35.56 ± 1.52 min and 28.41 ± 0.41 min, respectively. The freeze-dried E. faecium with enhanced heat tolerance appears to be a suitable Salmonella surrogate for dry-inoculating LMFs. Our protocol also enables industry-scale production of freeze-dried inoculum by broth-cultivation method combined with mild-heat treatment.