The microbiological safety of reconstituted infant formula (RIF) has focused on infectious pathogens, whereas the risk of spore-forming bacteria (SFB) has been limited to spoilage and toxin production. This study suggests an underrecognized niche of SFB as
nitrite producers during the handling of RIF. The production of
nitrite along with the bacterial growth of 133
nitrite-producing SFB isolated from infant formula processing environments and end-products (70 mesophiles and 63 thermophiles) under RIF handling conditions were analysed. Most mesophiles (68 out of 70) and two thermophiles showed
nitrite production during growth at 30 °C or 40 °C. Vigorous producers of
nitrite [Bacillus sp. strains (FHS-PPBM449, 481, 236, 237)] showed a rapid onset of
nitrite production (within 4 h). In particular, FHS-PPBM449 (2-3 log CFU/mL) exhibited the shortest onset time (210 min) and a
nitrite production level up to 521 µM in RIF with 100 ppm
nitrate at 40 °C. Overall, the results of the maximum level of
nitrite produced by vigorous
nitrite producers indicate that infants can consume more than seven times the acceptable daily intake of
nitrite (0.74 mg for 12-month-old infants with an average
body weight), even via a single feeding of RIF. An analysis of the relationship of the onset time of
nitrite production with the bacterial concentration based on predictive models suggests that the growth of SFB up to 5-6 log CFU/mL is regarded as a prerequisite for
nitrite production. This study revealed an underreported source of
nitrite from RIF handling conditions, and the rapid onset of a high level of
nitrite production from SFB should be the major target in the establishment of intervention strategies against
nitrite as a microbial risk.