Extraction, purification, and sequence identification of bioactive peptides from bovine milk with beneficial effects in cellular models

Bioactive peptides are short fragments of proteins (2-20) that exhibit various physiological effects and have gained significant attention in recent years. These compounds are derived from different natural sources such as plants (soy, oat, etc.) and animals (egg, meat, milk, etc.). Bioactive peptides possess the ability to modulate biological processes in the human body, exerting antihypertensive, opioid, antioxidant, and anti-inflammatory effects. One of the most studied sources for bioactive peptides is milk. In this thesis, peptides from bovine milk of both mastitis-affected (M) and healthy (H) cows were extracted, identified, and studied to understand their physiological effects. Firstly, two different peptide extraction techniques were compared aiming to identify a more effective method for obtaining peptides. In particular, this study focuses on using a nanotechnological approach taking advantage of Surface Active Maghemite Nanoparticles (SAMNs) in order to extract peptides from M and H milk. Secondly, solid phase extraction (SPE) was employed in comparison with SAMNs to separate the milk-derived peptides. Moreover, mass spectrometry (MS) analysis was utilized to identify the sequences of the peptides derived from M and H milk in order to understand if the inflammatory process can lead to the release of different peptides in milk. In addition, this analysis aimed to determine the specificity of peptide sequences that were specifically bound to the surface of SAMN nanoparticles and those that were separated using solid surface extraction (SPE). In the end, the pool of peptides isolated with the two different methods was tested in human cells (Caco-2 cells). The peptides showed to exert protective effects against oxidative stress and inflammation induced by TbOOH and TNF-α, respectively. In conclusion, understanding the properties and functions of bioactive peptides opens up new strategies for the development of novel therapeutic interventions and functional products aimed at promoting human health and well-being.