Two novel strains of Rummeliibacillus sp. and Microbacterium sp. were identified from the intestine of olive flounder (Paralichthys olivaceus) and characterized in vitro as potential probiotics. Feeds without probiotic and with a 50:50 mixture of these two strains (1 × 10⁸ CFU/g feed) were denoted as the control and Pro diets, respectively. Three randomly selected tanks (20 flounders/tank, ~11.4 g each) were used for each diet replication. After 8 weeks of feeding, the growth and feed utilization of the flounder in the Pro group improved (p < 0.05) compared to the control. Among four immune parameters, only myeloperoxidase activity was elevated in the Pro group. Serum biochemistry, intestinal microbial richness (Chao1), and diversity (Shannon index) remained unchanged (p ≥ 0.05), but phylogenetic diversity was enriched in the Pro fish intestine. Significantly lower Firmicutes and higher Proteobacteria were found in the Pro diet; the genus abundance in the control and Pro was as follows: Staphylococcus > Lactobacillus > Corynebacterium and Lactobacillus > Staphylococcus > Corynebacterium, respectively. Microbial linear discriminant scores and a cladogram analysis showed significant modulation. Therefore, the combination of two host-associated probiotics improved the growth and intestinal microbial population of flounder and could be supplemented in the Korean flounder industry.

This study evaluated the effects of dietary host-derived Bacillus strains combined with fructo-oligosaccharide (FOS) on growth performance, basal physiological status, intestinal morphology, and thermal stress responses in juvenile olive flounder (Paralichthys olivaceus). A total of 486 fish with an initial body weight of 7.26 ± 0.04 g were randomly distributed into 27 tanks and fed nine experimental diets for nine weeks. The diets consisted of a basal control, an FOS-only diet, three single-strain synbiotic diets containing FOS and one host-derived Bacillus strain (B. sonorensis, B. subtilis, or B. velezensis), and four multi-strain synbiotic diets containing FOS and combinations of two or three strains. Probiotics were included at 1 × 10⁷ CFU g⁻¹ diet, and FOS was supplemented at 5 g kg⁻¹ diet. After the feeding trial, no significant dietary effects were observed on growth performance, somatic indices, whole-body proximate composition, plasma biochemical parameters, antioxidant enzyme activities, immune-related indicators, stress-related biomarkers, or intestinal morphology. Fish were subsequently subjected to lethal and acute high-temperature challenges to evaluate thermal stress tolerance and associated physiological responses. In the lethal temperature challenge, fish fed the multi-strain diets FOS + B. sonorensis + B. velezensis and FOS + B. sonorensis + B. subtilis + B. velezensis showed numerically higher survival than the other groups; however, these differences were not statistically significant. Following acute heat exposure, dietary treatments did not significantly affect plasma metabolites, and most heat-shock- and energy-metabolism-related genes were not differentially expressed among treatments. Hepatic AMPKβ expression showed a significant dietary treatment effect, with higher expression in the BCF and ACF groups than in the ABF group. Overall, host-derived synbiotic supplementation did not significantly enhance growth performance or basal physiological responses under the present experimental conditions. However, some multi-strain combinations showed a non-significant tendency toward higher survival under lethal thermal stress. Further studies incorporating gut microbiome profiling, metabolomic analysis, and alternative dietary conditions are required to clarify whether host-derived Bacillus–FOS synbiotic formulations can influence thermal stress resilience in olive flounder.