Bioactive peptides from whey proteins: production, purification, identification, mechanism of action and biofunctionality in foods

Bioactive peptides are specific protein fragments capable of exerting relevant biological activities. In recent years, agro-industrial by-products, such as whey, have been widely explored as sustainable protein sources for the production of these compounds. This narrative review provides an integrated overview of recent advances in the production, extraction, purification, and identification of bioactive peptides derived from whey proteins, with emphasis on their potential applications as antioxidant, antimicrobial, and functional food ingredients. The mechanisms of action underlying the antioxidant and antimicrobial activities of these peptides are also addressed. Enzymatic hydrolysis is the primary strategy for obtaining bioactive peptides due to its efficiency in releasing biologically active sequences; however, exploration of emerging physical technologies remains limited. Chromatographic techniques for the purification, fractionation, and isolation of peptides with defined bioactivities have undergone significant technological advances, ranging from conventional liquid chromatography to ultra-high-performance liquid chromatography (UHPLC). Peptide sequencing and identification are primarily performed using liquid chromatography coupled with mass spectrometry. These processes have also seen significant technological advances with the development of more efficient ionization sources and mass analyzers, such as quadrupole (Q), time-of-flight (TOF), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), often coupled to UHPLC systems. Antioxidant peptides act mainly through free radical scavenging and the chelation of pro-oxidant metals, whereas antimicrobial peptides exert their effects by inhibiting essential extracellular and intracellular structures of microorganisms. In this context, whey-derived bioactive peptides are promising alternatives for food preservation and functional food development.