Microbial Consortium Fermentation Remodels the Metabolite Profile and Enhances the Biological Functionality of Stevia rebaudiana Leaves

Microbial fermentation is an effective strategy to enhance the functional value of plant-derived ingredients. In this study, <i>Stevia rebaudiana</i> leaves were subjected to microbial fermentation to improve their antioxidant potential and functional properties. A composite fermentation system composed of <i>Bacillus subtilis</i> and <i>Candida utilis</i> was established through strain screening, and fermentation conditions were optimized using single-factor and orthogonal experiments, with chlorogenic acid (CA) content and antioxidant activity as evaluation indices. The optimal conditions were determined to be a fermentation temperature of 34 °C, a duration of 36 h, a microbial ratio (Bs:Cu) of 2:1, a moisture content of 55%, and an inoculum level of 3%. Under these optimal conditions, fermentation significantly increased CA content, total phenolic and flavonoid levels, and antioxidant capacity compared with unfermented material. Untargeted metabolomic analysis revealed extensive fermentation-induced remodeling of secondary metabolites, particularly phenolic acids, flavonoids, and terpenoids, including the generation of multiple newly formed bioactive compounds. Functional validation using a laying hen model demonstrated that fermented <i>S. rebaudiana</i> exhibited enhanced antioxidant and anti-inflammatory status and favorable modulation of physiological indicators compared with unfermented samples. Overall, this study demonstrates that microbial consortium fermentation effectively transforms <i>S. rebaudiana</i> from a sweetener-oriented plant into a multifunctional, fermentation-derived functional ingredient. This research is significant as it provides a dual-purpose strategy for developing antioxidant-enriched functional foods for humans and health-promoting natural feed additives for the livestock industry.