Novel bioavailable hypocholesterolemic peptides derived from milk fermented with Lactococcus lactis NRRL B-50572

The aim of this study was to identify bioavailable peptides with 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitory activity derived from milk fermented with Lactococcus lactis NRRL B-50572 (FM-572) by integrating in vitro, ex vivo, and in silico studies. In this sense, FM-572 was subjected to simulated gastrointestinal digestion (SGD) and an ex vivo intestinal absorption process, and the HMGCR inhibitory activity was evaluated in peptide fractions. The results showed that the abundance of absorbed peptides from FM-572 was not different (p > 0.05) compared to unfermented milk. However, the HMGCR inhibitory activity (IC₅₀) of FM-572 peptide fractions was significantly higher after SGD and the ex vivo absorption process (p < 0.05) compared to unfermented milk. Afterwards, absorbed peptides (bioavailable) were isolated by RP-HPLC, and HMGCR was evaluated in the chromatographic fractions (CF). After this analysis, 106 peptides were identified by LC-MS/MS in CF from FM-572. However, only 76 peptides that presented IC₅₀ values ≤41.6 μg/mL were selected from CF. Comprehensive bioinformatic analysis was carried out on these selected peaks to identify HMGCR inhibitory peptides combining 3D-QSAR modeling, molecular docking, and molecular dynamic simulations. These analyses revealed ten potent peptides with HMGCR inhibitory activity. Moreover, four peptides, VAFVK (Lactotransferrin, 225-229), FPGPI (β-CN, 77-81), PVEPF (β-CN, 130-34), and APFPE (αS1-CN, 41-45), showed the best affinity interaction and stability in the catalytic pocket of the enzyme, resulting from the formation of intermolecular hydrogen bonds. Henceforth, these findings suggest that novel peptides released from FM-572 are bioavailable and capable of inhibiting the HMGCR enzyme, suggesting a cholesterol-lowering effect by the reduction of cholesterol synthesis.