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Aims:  To conduct in vitro and in vivo assessments of the safety of two species of Bacillus, one of which, Bacillus subtilis, is in current use as a food supplement. Methods and Results:  Cultured cell lines, Caco‐2, HEp‐2 and the mucus‐producing HT29‐16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc‐diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions. Conclusions:  Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study. Significance and Impact of the Study:  The results support the use of B. subtilis and B. indicus strains as food supplements.

This study demonstrates that pigmented bacilli are ubiquitous and contain new carotenoid biosynthetic pathways that may have industrial importance.

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Spores of Bacillus subtilis including one strain used commercially were evaluated for their potential value as a probiotic and as potential food additives. Two isolates of B. subtilis examined here were HU58, a human isolate and PXN21, a strain used in an existing commercial product. Compared to a domesticated laboratory strain of B. subtilis both isolates carried traits that could prove advantageous in the human gastro-intestinal tract. This included full resistance to gastric fluids, rapid sporulation and the formation of robust biofilms. We also showed that PXN21 spores when administered weekly to mice conferred non-specific cellular immune responses, indicative signs of the stimulation of innate immunity. Spores mixed in wholemeal biscuits were found to survive baking at 235 °C for 8 minutes with only a 1-log reduction in viability. That spores can survive the baking process offers the possibility of using spores as probiotic supplements in a range of novel food products.