**Identification of New Peptides from Fermented Milk Showing Antioxidant Properties: Mechanism of Action**

**Federica Tonolo 1,**†**, Federico Fiorese 1,**†**, Laura Moretto 1, Alessandra Folda 1, Valeria Scalcon 1, Alessandro Grinzato 1, Stefania Ferro 1, Giorgio Arrigoni 1, Alberto Bindoli 2, Emiliano Feller 3, Marco Bellamio 3, Oriano Marin 1,\* and Maria Pia Rigobello 1,\***


Received: 27 December 2019; Accepted: 27 January 2020; Published: 29 January 2020

**Abstract:** Due to their beneficial properties, fermented foods are considered important constituents of the human diet. They also contain bioactive peptides, health-promoting compounds studied for a wide range of e ffects. In this work, several antioxidant peptides extracted from fermented milk proteins were investigated. First, enriched peptide fractions were purified and analysed for their antioxidant capacity in vitro and in a cellular model. Subsequently, from the most active fractions, 23 peptides were identified by mass spectrometry MS/MS), synthesized and tested. Peptides **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E** were selected for their antioxidant e ffects on Caco-2 cells both in the protection against oxidative stress and inhibition of ROS production. To define their action mechanism, the activation of the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2(Keap1/Nrf2) pathway was studied evaluating the translocation of Nrf2 from cytosol to nucleus. In cells treated with **N-15-M**, **Q-14-R** and **A-17-E,** a higher amount of Nrf2 was found in the nucleus with respect to the control. In addition, the three active peptides, through the activation of Keap1/Nrf2 pathway, led to overexpression and increased activity of antioxidant enzymes. Molecular docking analysis confirmed the potential ability of **N-15-M**, **Q-14-R** and **A-17-E** to bind Keap1, showing their destabilizing e ffect on Keap1/Nrf2 interaction.

**Keywords:** bioactive peptides; Keap1/Nrf2 pathway; natural antioxidants; oxidative stress
