Complementary and Synergistic Effects of Dietary Bacillus subtilis natto and Lactobacillus plantarum on Growth, Immunity and Intestinal Microflora Restructuring in Juvenile Japanese Flounder (Paralichthys olivaceus)

Prophylactic antibiotic use in intensive aquaculture promotes antimicrobial resistance, necessitating the development of microbial-based interventions. This study evaluated the individual, complementary, and synergistic effects of Bacillus subtilis natto (BSN) and Lactobacillus plantarum (LP) on the physiological performance and intestinal microecology of juvenile Japanese flounder (Paralichthys olivaceus). Over a 60-day trial, juveniles (initial weight: 5.81 ± 0.03 g) received a basal diet (CON) or a diet supplemented with 107 CFU/g of BSN, LP, or both (BSN+LP). The BSN+LP consortium elicited complementary improvements in final body weight (21.39 ± 0.75 g vs. 18.66 ± 0.44 g in CON) and feed conversion efficiency (p < 0.05). Transcriptomic analysis revealed synergistic upregulation of digestive proteases (trypsin, chymotrypsin). Notably, an in pro-inflammatory markers (IL-1β, TNF-α) was counterbalanced by substantial upregulation of anti-inflammatory cytokines (IL-10, 5.65-fold; TGF-β1, 4.48-fold), suggesting the induction of mucosal tolerance rather than pathological enteritis. High-throughput 16S rRNA sequencing showed that the control cohort had a potential baseline microbial, characterized by a high relative abundance of Proteobacteria. BSN+LP administration significantly altered this microbial community into a fermentative eubiosis enriched in Firmicutes and Bacteroidota. Correlation network analyses confirmed negative interaction dynamics: increased abundance of Lactobacillus, Bacteroides, and Muribaculaceae was negatively correlated with baseline pathobiont abundance. These findings indicate that co-administration of BSN and LP hypothetically enhances metabolic energy harvest via short-chain fatty acid-producing taxa, strengthens the gut–immune axis, and competitively mitigates opportunistic pathogens.