Dry-inoculation method for thermal inactivation studies in wheat flour using freeze-dried Enterococcus faecium NRRL B-2354

Liquid cultures are commonly used to inoculate low-moisture foods. However, liquid-inoculation may generate varied results due to differences in preparation methods, and the introduction of liquid into a low-moisture matrix may cause stickiness and clumping. The objectives of this study were to develop a dry-inoculation method using freeze-dried Enterococcus faecium NBRL-2354, and to evaluate the stability of freeze-dried inoculum over time (35 days at −20 °C) in terms of survival and heat resistance. Two protectants, freeze-drying buffer and skim milk, mixed separately with liquid E. faecium were freeze-dried and their preservative effects were compared. Heat resistance of freeze-dried inoculum was determined by decimal reduction time at 80 °C (D80) in wheat flour (water activity 0.45 at room temperature). Freeze-drying buffer and skim milk formed slimy layers around bacterial cells and yielded freeze-dried inoculum with high survivability. D80-values of freeze-dried E. faecium protected by freeze-drying buffer or skim milk were 6.67 ± 0.22 min and 5.92 ± 0.39 min, respectively. Freeze-dried inoculum was stable with a high population concentration (>10.0 log CFU/g), while skim milk-protected inoculum showed stable heat resistance throughout storage. In summary, freeze-dried E. faecium protected by skim milk was the optimum dried inoculum and can be used as a substitute for liquid-inoculum in low-moisture foods.