Microbial fermentation is an effective strategy to enhance the functional value of plant-derived ingredients. In this study, Stevia rebaudiana leaves were subjected to microbial fermentation to improve their antioxidant potential and functional properties. A composite fermentation system composed of Bacillus subtilis and Candida utilis 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 S. rebaudiana 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 S. rebaudiana 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.

This study evaluated the effects of oligomeric ultrafine-nano hydrogen water (OUHW) on production performance, egg quality, egg nutrient deposition, systemic metabolism, intestinal barrier function, and reproductive status in late-laying hens. Unlike conventional hydrogen-rich water, which primarily delivers molecular hydrogen dissolved in water, OUHW integrates oligomeric water with ultrafine-nano-sized hydrogen bubbles, providing a distinct physicochemical form of hydrogen-water intervention. A total of 288 healthy 67-week-old Jingfen No. 8 laying hens were assigned to 2 treatments with 6 independent replicates per treatment (24 hens per replicate) and provided with either tap water or OUHW for 11 weeks. Compared with the control, OUHW reduced the feed conversion ratio and the rates of manure-spotting while increasing the laying rate, qualified egg rate, and total egg number during weeks 5–8 (p < 0.05). These productive and egg quality improvements were phase-specific effects observed in the late laying stage during the 11-week trial. At week 4, OUHW significantly increased egg weight (p < 0.05). At week 8, OUHW also improved eggshell compressive elastic deformation, eggshell thickness, albumen height, and Haugh unit (p < 0.05). At week 6, eggs from OUHW-treated hens had higher concentrations of glutamate, glycine, alanine, C18:0, C18:1n9c, C20:4n6, and C22:6n3 (p < 0.05). Serum metabolomics showed that 25 differential metabolites from the OUHW treatment group were predominantly enriched in glycerophospholipid, sphingolipid, branched-chain amino acid, histidine, and tryptophan metabolism. In the cecum, OUHW decreased p-cresol and increased isovaleric acid and acetic acid (p < 0.05). Notably, the overall community structures of the cecal and oviductal microbiota exhibited no significant treatment-related alterations, indicating limited structural changes in the microbial communities in response to the OUHW intervention. OUHW also reduced interleukin-2 and tumor necrosis factor-α, increased interleukin-10 in the jejunum and oviductal isthmus (p < 0.05), and upregulated the mRNA expression of ovarian steroidogenic acute regulatory protein and intestinal Occludin, Claudin-1, and Mucin-2 (p < 0.05). These findings indicate that OUHW improved laying performance, egg quality, and egg nutrient deposition, possibly through modulation of the metabolism, intestinal barrier function, ovarian function, and oviductal inflammation.