Milk-derived bioactive peptides can improve the treatment of many diseases, behaving as antihypertensive, antimicrobic and antioxidant agents. This study was focused on new peptides evaluated for their antioxidant properties and mechanism of action cell enviroment. Peptide enriched fractions were extracted from fermented milk and purified in order to obtain the sequence of the included bioactive peptides. Twenty-three new peptides were identified and synthesized by solid phase procedure. Their antioxidant properties were analysed in vitro and in a cellular model using Caco-2 cells. Among these peptides, N-15-M, E-11-F, Q-14-R and A-17-E were selected in order to estimate their antioxidant capacity on Caco-2 cells both as protection against oxidative stress and inhibition of ROS production induced by TbOOH. In order to understand the possible action of the peptides on Keap1-Nrf2 pathway, which is involved in the response to oxidative stress, the translocation of Nrf2 from cytosol to the nucleus was taken into account. N-15-M, Q-14-R and A17-E were able to activate the Keap1-Nrf2 system, as shown by the higher amount of Nrf2 found in the nucleus with respect to the control. This modulation led to overexpression and increase of activity of antioxidant enzymes such as TrxR1, GR, NQO1 and SOD1. The interaction of these peptides with Keap1 was also studied through molecular docking analysis. The results confirmed that N-15-M, Q14-R and A-17-E interact with the residues of the Keap1 pocket involved in the binding with Nrf2, acting as disruptor of Keap1-Nrf2 interaction.
Federica Tonolo received her master degree summa cum laude in Sanitary Biology in 2016 at University of Padova, Italy. In 2016, she won a European fellowship as part of an FSE project at University of Padova under the supervision of Prof. Maria Pia Rigobello. Currently, she is a third year PhD student in Biomedical Sciences at University of Padova in the same lab. Her major field of interest is related to the antioxidant bioactive peptides derived from food matrices, in particular milk and soy. Up to now (Jan 08, 2020) the scientific production includes 15 publications divided into 8 full papers and 6 meeting communications