IDENTIFICATION OF MICROBIAL FUNCTIONS INTERFERING WITH HOST-CELL PHYSIOLOGY: A QUICK VIEW INTO STREPTOCOCCUS THERMOPHILUS METABOLIC POTENTIAL

Streptococcus thermophilus, long typecast as a dairy workhorse, is ready for a role upgrade. Despite decades of safe use in fermentation in large volume consumer products, this species has been largely sidelined in probiotic research, overshadowed by genera like bifidobacteria and strains belonging to the group formerly classified within lactobacilli, such as Lactiplantibacillus, Lacticaseibacillus, and Limosilactobacillus. This thesis redefines S. thermophilus as a metabolically dynamic, functionally relevant, and therapeutically promising candidate in the probiotic field. In the first part of this work, we present a safe metabolic activation strategy that enhances lactose catabolism in S. thermophilus, starting from the biomass production phase, without compromising cell viability. Within just 30 minutes, activated cells exhibited up to a 30% increase in lactose conversion, compared to not-activated cells. Interestingly, in the presence of both urea and a carbon source, lactic acid release was significantly enhanced, alongside a marked improvement in glycolytic activity, without altering the optimized lactose uptake. This distinctive metabolic behavior of activated cells appears to modulate glycolysis and enhance acidification, positioning S. thermophilus as a promising tool not only for more efficiently supporting lactose digestion but also for additional applications requiring optimized biomass performance, rapid lactose consumption, and controlled glycolytic and lactate output. Importantly, these activated cells are currently being evaluated in a forthcoming in vivo study, where their enhanced metabolic profile is expected to translate into measurable reduced symptoms and improved subject well-being in individuals with lactose intolerance. This therapeutic potential, directed toward specific targets (e.g., lactose maldigestion), reinforces the concept of precision probiotics: microbial products designed not just for general health promotion, but for targeted interventions, tailored to specific conditions and consumer needs. The reproducibility of these effects across different substrates and at production scale underscores both the physiological relevance and industrial feasibility of this strategy, offering a compelling alternative to generalized probiotic strategies in favor of a novel approach in probiotic development. The second study focuses on urease activity, an often overlooked yet highly relevant functional trait in S. thermophilus, closely associated with nitrogen and energy metabolism. Recent findings have linked urease activity in S. thermophilus to a specific beneficial effect on the host: administration of adequate amounts of viable biomass resulted in decreased fecal urease activity, suggesting that this effect may be mediated by the modulation of urea metabolism and a reduction in urease-positive pathogenic bacteria. Building on this and considering that targeting urease in conditions such as Irritable Bowel Syndrome (IBS) could be key to alleviate symptoms, we sought to determine whether this reduction is solely due to S. thermophilus urease contribution or whether additional microbial or host-related factors play a role. To this end, we controlled urease activity levels of S. thermophilus biomass through process-condition control. By fine-tuning fermentation parameters, we successfully modulated urease expression at both laboratory and industrial scales, generating distinct biomasses with tailored metabolic phenotypes. These engineered biomasses are currently being prepared for a forthcoming in vivo study in IBS patients to assess their effects on host symptoms and their influence on gut nitrogen metabolism, based on their urease activity levels. This is particularly relevant given that disruptions in the urea–ammonia balance, arising from the dysbiosis state that characterizes IBS, are known to exacerbate symptoms through altered microbial composition, increased intestinal permeability, and elevated luminal ammonia levels, which can aggravate mucosal inflammation and visceral hypersensitivity, leading to discomfort, abdominal pain, bloating, and other digestive disturbances typical of this syndrome. This work aims to open new avenues for developing targeted probiotic interventions and provides valuable insights into the complex and evolving interplay between urease activity and energy metabolism in S. thermophilus. Most importantly, it highlights a crucial yet often overlooked point: the fermentation process itself can be strategically optimized to predefine specific microbial functionalities. By establishing a direct link between upstream fermentation conditions and downstream functionality, this study reinforces the importance of precision at every stage of probiotic production, from fermentation to functional application, particularly when the effect is driven by the activity level of the administered biomass itself, rather than by downstream growth or adaptation in the host. The final part of the thesis explores an innovative therapeutic application: leveraging S. thermophilus DSM 20617T/ATCC 19258T as a delivery vector of β-galactosidase in situ via prophage-mediated lysis, motivated by the recent evidence that intracellular β-galactosidase from S. thermophilus exerts protective effects against colorectal cancer by inhibiting cancer cell proliferation and tumor development. The strain’s ability to survive gastrointestinal transit, interact with the host microbiota, and potentially release therapeutic molecules in situ represents a novel intersection of microbiology, biotechnology, and therapeutic design. While challenges remain, particularly in controlling prophage induction and ensuring consistent release of bioactive enzymes, such as β-galactosidase, at the site of action in the intestine following administration and cell lysis, this work lays the foundation for using S. thermophilus as a microbial chassis in the design of live biotherapeutics. Together, these studies reposition S. thermophilus as a precision-engineered tool for the future generation of microbiome-based health solutions. In an industry still dominated by one-size-fits-all products and vague probiotic claims, this work challenges the standard narrative. It delivers measurable, mechanism-based evidence that S. thermophilus has been underestimated for too long. As the demand for personalized, effective, and evidence-based microbial therapies continues to rise, this thesis highlights the potential of S. thermophilus and advocates for its greater consideration. Moreover, by expanding the functional framework of S. thermophilus, this research paves the way for formulas designed with precision and purpose, moving probiotic science into a new era.