Ontogenetic shifts in digestive enzyme kinetics drive age-dependent in vitro digestibility outcomes for fungal biomass and conventional ingredients in Oncorhynchus mykiss

Abstract Aquaculture needs alternative protein sources to meet the growing fish feed demands. Fungal protein is a promising alternative ingredient to common sources like fishmeal and soybean meal; however, the evaluation of this replacement via lengthy in vivo trials is resource-intensive. Therefore, this study employed an in vitro approach to compare the degree of hydrolysis of Neurospora intermedia, Aspergillus oryzae, and Rhizopus oligosporus against conventional ingredients (fishmeal, wheat gluten and soybean meal) using enzyme models from three distinct rainbow trout ( Oncorhynchus mykiss ) size classes (0 + , 1 + , and 2 + ). Gastric and intestinal extracts were prepared for each class, and their proteolytic activities were kinetically modeled against varying substrate concentrations to standardize the assay parameters. Tested ingredients underwent a two-phase in vitro digestion simulating gastric (2-hour) and intestinal (4-hour) digestion phases at physiological pH and temperatures. The kinetic parameters of digestive enzymes fluctuated non-linearly across life stages. Gastric enzymes followed Michaelis-Menten patterns, yielding V max values of 11.02, 7.85, and 15.71 µmol mg -1 min -1 for 0 + , 1 + , and 2 + size classes, respectively. Conversely, intestinal enzymes exhibited substrate inhibition, with K i values of 0.55%, 3.99%, and 2.02%, respectively. In 0 + and 1 + fish, fishmeal achieved the highest hydrolysis (49–55%), followed by N. intermedia (39–43%) and A. oryzae (~ 40%), whereas soybean meal and wheat gluten showed the lowest hydrolyzed (21-29%). Notably, in 2 + fish, N. intermedia and A. oryzae became the most extensively hydrolyzed ingredients (27% and 28%), significantly outperforming fishmeal (14%). These findings revealed that protein digestibility is a dynamic, age-dependent variable. Clinical Trial Registration Clinical trial number: not applicable.