Identification of bioaccessible and neuroprotective peptides from fermented casein hydrolysate

Fermented dairy products are beneficial to cognitive health. Fermentation-released bioactive peptides have the potential to contribute to the neuroprotective effects of fermented dairy products. However, known neuroprotective peptides are mostly prepared by enzymatic hydrolysis, and physicochemical screening of food-derived functional peptides typically overlooks the interference of biotransport after ingestion. Thus, we aimed to identify neuroprotective peptides from casein fermented by Lactobacillus delbrueckii ssp. bulgaricus to provide more evidence supporting the contribution of fermentation-released peptides. We first screened bioaccessible peptides from fermented casein hydrolysate by simulating digestion, absorption, and blood-brain barrier penetration using INFOGEST standardized protocols, human colon Caco-2 cells, and human brain microvascular endothelial hCMEC/D3 cells sequentially. Next, we identified peptides of each stage by nano-liquid chromatography tandem MS. The intersections were considered bioaccessible peptides. We performed molecular docking against Kelch-like ECH-associated protein 1 (Keap1) to predict potential bioactive peptides and validated the predicted effects in BV2 microglial cells induced by LPS. As a result, we identified 1,971, 663, 276, and 208 casein peptides from the simulated products at each stage, and 63 bioaccessible peptides were identified during fermentation, underwent simulated digestion, and were transported via the simulated intestinal epithelial barrier and blood-brain barrier. Among these peptides, 7 nontoxic small peptides had relatively high predicted affinities for Keap1 and were verified in LPS-treated BV2 cells. We found that Phe-Val-Ala-Pro-Phe-Pro-Glu (FE7) decreased nitric oxide, IL-1β, reactive oxygen species, and lipid peroxidation levels by 69.6%, 103.6%, 119.3%, and 75.3%, respectively, in LPS-treated BV2 cells. In conclusion, FE7 could be a promising neuroprotective peptide in fermented casein hydrolysate by reducing neuroinflammation and oxidative stress. Our approach provides a feasible paradigm for identifying bioaccessible and neuroprotective peptides from dairy products.