Characterization of the nitrite production of mesophilic spore-forming bacteria during the handling of reconstituted infant formula

[Display omitted] •Spore-forming bacteria (SFB) can be an exposure source of nitrite to infants.•SFB produce nitrite as metabolites in reconstituted infant formula (RIF) at 30–40 °C.•SFB showed nitrite production up to 521 µM in RIF containing 100 ppm nitrate at 40 °C.•Nitrite consumption by a single RIF feeding is estimated as exceeding infants’ ADI.•Determinant factors on the rate and the level of nitrite production are identified. 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.