This study investigates the postbiotic potential of extracellular products (ECPs) from Bacillus pumilus strains cultivated on microalgae-supplemented media. We assessed enzymatic and antimicrobial activities to select ECPs that enhance the digestive processes in gilthead seabream. Additionally, we explored the in vitro enzymatic capacity of the chosen postbiotics to hydrolyze macromolecules in microalgae. Finally, a feeding trial was conducted to determine the in vivo effects of the ECPs on Sparus aurata. In vitro enzymatic assays demonstrated diverse hydrolytic capacities among ECPs. All conditions exhibited antimicrobial activity against Photobacterium damselae subsp. piscicida, with variation in inhibitory effects against Vibrio harveyi and Tenacibaculum maritimum. Furthermore, in vitro assays revealed differences in protein hydrolysis and soluble protein concentration, influencing amino acid and reducing sugar release from microalgal biomass. These analyses facilitated a selection to test ECPs in vivo. Lastly, the in vivo experiment revealed no differences in the growth performance, nutrient utilization, and general metabolism of S. aurata fed the experimental diets. Dietary inclusion of postbiotics increased the activity of key digestive enzymes in fish compared to the control group, and particularly, values increased significantly when the fish were fed with the ECP-nanoparticulate-supplemented diet. In conclusion, the inclusion of microalgae in the culture media significantly influences the activity of extracellular products from B. pumilus strains, as evidenced in both in vitro and in vivo assays.
