The use of bacterial spores in probiotics over viable loads of bacteria has many advantages, including the durability of spores, which allows spore-based probiotics to effectively traverse the various biochemical barriers present in the gastrointestinal tract. However, the majority of spore-based probiotics developed currently aim to treat adults, and there is a litany of differences between the adult and infant intestinal systems, including the immaturity and low microbial species diversity observed within the intestines of infants. These differences are only further exacerbated in premature infants with necrotizing enterocolitis (NEC) and indicates that what may be appropriate for an adult or even a healthy full-term infant may not be suited for an unhealthy premature infant. Complications from using spore-based probiotics for premature infants with NEC may involve the spores remaining dormant and adhering to the intestinal epithelia, the out-competing of commensal bacteria by spores, and most importantly the innate antibiotic resistance of spores. Also, the ability ofBacillus subtilisto produce spores under duress may result in lessB. subtilisperishing within the intestines and releasing membrane branched-chain fatty acids. The isolateB. subtilisBG01-4TMis a proprietary strain developed by Vernx Biotechnology through accumulating mutations within the BG01-4TMgenome in a serial batch culture. Strain BG01-4TMwas provided as a non-spore-formingB. subtilis, but a positive sporulation status for BG01-4TMwas confirmed throughin vitrotesting and suggested that selection for the sporulation defective genes could occur within an environment that would select against sporulation. The durability of key sporulation genes was ratified in this study, as the ability of BG01-4TMto produce spores was not eliminated by the attempts to select against sporulation genes in BG01-4TMby the epigenetic factors of high glucose and low pH. However, a variation in the genes in isolate BG01-4-8 involved in the regulation of sporulation is believed to have occurred during the mutation selection from the parent strain BG01-4TM. An alteration in selected sporulation regulation genes is expected to have occurred from BG01-4TMto BG01-4-8, with BG01-4-8 producing spores within 24 h, ~48 h quicker than BG01-4TM.