Macauba (Acrocomia aculeata) kernel-derived protein hydrolysates modulate intestinal development, antioxidant responses and hepatic metabolic gene expression in an in ovo (Gallus gallus) model.

Macauba kernel protein isolate (MKPI) is nutrient-rich and ideal for plant-based diets, with potential for generating bioactive peptides upon hydrolysis. This study evaluated the effects of MKPI-derived hydrolysates (HD) on intestinal development, antioxidant defenses, and hepatic glucose metabolism using an in ovo (Gallus gallus) model, combined with peptide characterization and in silico bioactive prediction. Fertile broiler eggs (n = 9/group) were assigned to four groups: (1) non-injected (NI), (2) H₂O, (3) MKPI (IS), and (4) hydrolysates (HD). Intra-amniotic administration was performed on day 17, and tissues were collected at hacth (day 21). HD sequencies were identified by nanoLC-ESI-MS/MS and analyzed using the BIOPEP-UWM database. Hydrolysis reduced protein sizes (<15 kDa) and increased antioxidant capacity for the fractions. Both IS and HD groups induced changes in intestinal morphology, including increased villus height, surface area, and crypt depth compared to NI; however, villus-related improvements were also observed in the H₂O group, indicating a contribution of the injection procedure. In contrast, IS and HD specifically increased crypt depth and goblet cell diameter. Antioxidant-related genes (SOD, GSH-Px, CAT) were upregulated in IS and HD groups. HD further modulated hepatic gene expression (PCK1, G6PD, GAPDH, GLUT2), as well as intestinal SGLT1 expression, while MUC2 and DPP4 were unchanged. A total of 45 peptides were identified in HD, with predicted antioxidant and metabolic regulatory activities. Overall, HD modulated intestinal morphology, enhanced antioxidant responses, and influenced hepatic gene expression in an in ovo model.