Evaluation of the anti-inflammatory activity of milk whey bioactive peptides through in vitro and transcriptomic investigations in Caco-2 intestinal cells.

In the context of growing environmental, economic, and health challenges, the agri-food sector is being asked to adopt circular, sustainable models that reduce waste and add value. In line with the European Green Deal and the Farm to Fork strategy, current research is turning food by-products into high-value bioactive compounds with applications in human health, preventive nutrition, and sustainable food technology. This promotes a circular economy where waste becomes a resource. Among bioactive compounds, bioactive peptides (BAPs) stand out for their ability to modulate physiological pathways involved in oxidative stress and inflammation key mechanisms in noncommunicable diseases (NCDs) such as diabetes, cardiovascular disorders, and cancer. These short amino acid chains (2–20 residues) can be released through fermentation or enzymatic digestion and have been identified in various plant and animal sources and derivatives, including soy, fish, eggs, and milk whey. Whey, a by-product of cheese production, is often discarded despite being rich in nutrients. Even less utilized is whey from mastitis-affected cows, which is excluded from processing due to safety regulations, resulting in economic losses and environmental waste. In this study, two bioactive peptides isolated from mastitic milk, I-14-K and G-14-K, were tested for their biological activity. In detail, this thesis focuses on the in vitro evaluation of the anti-inflammatory potential of the two selected BAPs, using Caco-2 human intestinal epithelial cells, a widely used model for gut barrier studies. The experimental setup simulates a preventive treatment: Caco-2 cells were seeded onto transwell inserts and differentiated for 8 days to form a model of intestinal epithelium; subsequently, cells were exposed to TNF-α for 2.5 hours, with or without prior incubation (21.5 hours) with each peptide. Six experimental conditions were tested: untreated control, TNF-α alone, peptide I-14-K + TNF-α, peptide G-14-K + TNF-α, and each peptide alone. To assess barrier integrity, Transepithelial Electrical Resistance (TEER) was measured. At the end of treatment, total RNA was extracted for transcriptomic analysis (RNA-seq) to identify differentially expressed genes (DEGs). This work shows that food innovation can integrate circular economy principles with scientific evidence for disease prevention, positioning itself at the intersection of sustainability, technological progress, and human health.