Systems-level characterization of probiotic bifidobacteria - Towards rational optimization of industrial production

Sammanfattning: Probiotic Bifidobacterium strains contribute to a healthy gut microbiota of their hosts. Increasing public awareness of this positive effect has resulted in a growing demand for these microorganisms. During industrial production, probiotic microorganisms encounter environmental stressors, which can negatively impact their viability and health-promoting benefits. In this thesis, the current state of knowledge on robustness, stability, and stress physiology in bifidobacteria is reviewed, and the robust and stable Bifidobacterium animalis subsp. lactis BB-12® and the more sensitive Bifidobacterium longum subsp. longum BB-46 are investigated in detail. The aim of this thesis was to compare the metabolism and physiology of BB-12® and BB-46, and to identify key determinants of growth and viability. The applied approach relied on the integration of constraint-based modeling, classical physiological analyses, and omics analyses. Strain-specific, thoroughly curated, genome-scale metabolic models were built for BB-12® and BB-46, and were applied to identity their nutritional requirements. This allowed for the formulation of a chemically defined medium that supported growth of both strains. The models and medium are valuable tools for optimizing industrial production of these two strains. BB-12® and BB-46 were studied in lab-scale cultivations in the newly formulated medium to identify correlations between cellular characteristics, robustness, and stability of bifidobacteria. Transcriptomic analysis revealed consistently higher expression of several stress-associated genes (e.g., chaperones) in BB-12® as compared to BB-46, which may explain the higher stress tolerance of BB-12®. Upregulation of genes related to DNA repair in BB-46 coincided with increased robustness and stability in stationary compared to exponential phase. The composition of the cultivation medium had a considerable impact on growth and stability of BB-12® and BB-46. The cell membrane fatty acid profile was identified as a key determinant of robustness and stability, by omitting Tween® 80 from the medium. An unsaturated to saturated fatty acid ratio below or around one was found to be beneficial. Moreover, a complex nitrogen source was found to reduce the survival of BB-46, and an increased cell size of BB-12® in complex MRS medium was proposed to contribute to its poor survival under this condition. To assess for possible correlations between gene content and the strain physiology under stress conditions, the genomes of 171 Bifidobacterium strains, including BB-12® and BB-46, were screened for the presence of known stress-associated genes, resulting in the postulation of putative genotype-phenotype correlations. The long-term objective is to use the knowledge gained in this work to guide rational optimization of industrial production processes involving probiotic bifidobacteria.