Enhancing digestive and immune functions in Nile tilapia: Effects of Pediococcus acidilactici and Cordyceps militaris spent mushroom substrate extracts and their combination

This study evaluated the effects of Pediococcus acidilactici and Cordyceps militaris spent mushroom substrate (SMS) extract, and their combination, on digestive function, immune responses, and gut microbiota composition in Nile tilapia ( Oreochromis niloticus ). Fish were fed four experimental diets (T0: control, T1: BF-CSE, T2: BF-Pa, and T3: BF-Com) for eight weeks, with three replicates per treatment. After the feeding trial, physiological, immunological, and microbial parameters were evaluated. The P. acidilactici -supplemented diet (BF-Pa: T 2 ) significantly increased the activities of amylase, cellulase, and trypsin, indicating enhanced digestive function, although growth performance remained unchanged. The C. militaris SMS extract diet (BF-CSE: T 1 ) improved innate immune responses, as evidenced by elevated lysozyme activity, respiratory burst, and white blood cell counts. The combined supplementation (BF-Com: T 3 ) produced synergistic effects, significantly upregulating pro-inflammatory cytokines ( IL-1β and IL-8 ) while maintaining stable expression of TGF-β , suggesting a robust but well-regulated immune activation. Gut microbiota analysis revealed an increased abundance of health-associated genera, including Bacillus, Pediococcus , and Cetobacterium , indicating potential prebiotic effects. These findings highlight the potential of combining probiotic and immunomodulatory components in functional feeds to enhance digestive efficiency, immune competence, and microbial stability in Nile tilapia aquaculture. • Pediococcus acidilactici enhanced digestive enzyme activities but did not improve Nile tilapia growth performance. • Cordyceps militaris SMS extract stimulated nonspecific lysozyme and respiratory burst activity immune responses. • Combined supplementation synergistically upregulated IL-1β and IL-8 while maintaining balanced TGF-β expression. • Functional diets enriched Bacillus , Pediococcus , and Cetobacterium beneficial gut microbiota. • The BF-Com (T 3 ) diet showed promise as an integrated functional feed to boost fish health and immunity.