Oxygen exposure and diffusion are key formulation barriers in the microencapsulation of extremely oxygen-sensitive...

Extremely oxygen-sensitive (EOS) next-generation probiotics are increasingly recognised as promising microbial biotherapeutics; however, their strict intolerance to oxygen poses major challenges for formulation and long-term stability as oral therapies. Many established bioencapsulation approaches, including prilling-based microencapsulation, were developed for the controlled delivery of aerotolerant probiotic strains, and such methods may be unsuitable for these next-generation EOS organisms. This study systematically evaluated whether prilling-based hydrogel bioencapsulation, a platform conventionally developed and optimised for aerotolerant probiotic strains, is suitable for the formulation of EOS probiotics. Using Anaerobutyricum hallii as an EOS organism and Lactobacillus johnsonii as an aerotolerant comparator, we show that oxygen exposure during all stages of microencapsulation, in addition to post-encapsulation storage, compromises EOS survival, while aerotolerant strains remained unaffected. When this conventional prilling workflow was modified to maintain oxygen-free conditions, EOS viability was preserved through the early processing stages; however, recovery remained low following complete MC formation, even under oxygen-free conditions (2.8-11.6%). Post-encapsulation storage revealed rapid EOS viability loss under oxygen-replete conditions and progressive decline even under anaerobic storage. Direct physical measurements of oxygen transport within MCs demonstrated that calcium cross-linking slowed, but did not prevent, oxygen diffusion, with oxygen ingress occurring in a particle-size-dependent manner. These findings establish oxygen exposure as a critical design constraint in the formulation of EOS probiotics and demonstrate that prilling-based bioencapsulation workflows developed for conventional probiotic delivery are not directly transferable to these organisms. Future EOS microbiome therapeutics will require oxygen-controlled manufacturing workflows and biomaterial systems specifically engineered to preserve viability from processing through gastrointestinal delivery.