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

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 effects. 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 effects 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 effect on Keap1/Nrf2 interaction.

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

Figure 1

(A)Purification of the 5–30% ACN fraction with RP-HPLC. Fractions were collected every 2 min. (B) Analysis of antioxidant capacity of the purified fractions in vitro with 2,2′-azinobis(3-ethylbenzo-thiazoline 6-sulfonate) (ABTS) (green) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) (red) scavenging tests. (C) Effects of the purified fractions on cell viability in the presence and absence of TbOOH. Caco-2 cells were treated with the indicated fractions for 24 h and oxidative stress was induced by 200 µM TbOOH. Means of at least three experiments (eight replicates for each experiment) were compared with the treated control. (*** p < 0.001, ** p < 0.01).

Figure 2

Estimation of reactive oxygen species (ROS) production in Caco-2 cells treated with the indicated peptides (0.05 mg/mL) in the absence (red) or presence (green) of 250 µM TbOOH. The values at 5000 s were reported and the means of at least three experiments (four replicates for each experiment) were compared with the treated control. (*** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 3

Nrf2 translocation from cytosol to nucleus in Caco-2 cells in the presence of N-15-M, E-11-F, Q-14-R and A-17-E. (A) Cells were treated with 0.05 mg/mL of each peptide for 24 h. Nuclear fractions were isolated and proteins were subjected to WB detection as indicated in paragraph 2.15. (B) Densitometric analysis of four experiments compared with the control were reported, using PCNA as loading control. (*** p < 0.001, * p < 0.05).

Figure 4

Antioxidant enzymes gene expression analysis. The gene expression of glutathione reductase (GRS, A), thioredoxin reductase (TXNRD1, B), NADPH quinone oxidoreductase (NQO1, C) and superoxide dismutase (SOD1, D) was evaluated in cDNA obtained from Caco-2 cells treated with N-15-M, E-11-F, Q-14-R and A-17-E (0.05 mg/mL) for 24 h. β-actin was used as reference. Means of at least four experiments were compared with the control. (** p < 0.01, * p < 0.05).

Figure 5

Antioxidant enzymes detection by WB analysis. (A) Protein levels of glutathione reductase (GR), thioredoxin reductase (TrxR1), NADPH quinone oxidoreductase (NQO1) and superoxide dismutase (SOD1) in Caco-2 cell lysates treated with the four peptides (0.05 mg/mL) for 24 h. (B–E) Densitometric analysis of four experiments were compared with the control and normalized using GAPDH as loading control. (*** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 6

GR (A) and TrxR1 (B) activities in Caco-2 cells treated with N-15-M, E-11-F, Q-14-R and A-17-E (0.05 mg/mL). The activity of the two antioxidant enzymes was analyzed in Caco-2 cells treated with the four peptides for 24 h. Means of at least four experiments were compared with the control. (* p < 0.05).

Figure 7

Molecular docking analysis of the interaction between peptides and Keap1 Kelch domain. (A–C) Binding geometry of A-17-E, Q-14-R and N-15-M in the pocket of Keap1. (A’–C’) Magnification of the interaction of Keap1 Kelch domain with A-17-E (A’), Q-14-R (B’) and N-15-M (C’). Amino acids involved in the hydrogen bond formation were connected with orange dashed lines and highlighted in Table 4.

Figure 8

Uptake of the peptides by Caco-2 cells monolayer and their detection in AP and BL compartments. Each peptide (75 µg) was administered to the monolayer cells and samples of AP and BL were collected at the indicated time. (A–D) RP-HPLC chromatograms of the four peptides in the apical compartment at 10 and 120 min. (A’–D’) MS analysis of the peptides present in the basolateral side after 120 min.