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.Antioxidants 2020, 9, 117 2 of 24 proteolysis [3]. Many microorganisms are utilized in this process and it is well known that different fermenting strains can generate various patterns of bioactive peptides [4][5][6]. In particular, for dairy products, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Bifidobacterium spp are usually employed [2,7]. Bioactive peptides generated from milk can originate both from whey proteins (β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, lactoferrin and protease-peptone fractions) and from caseins (α-, βand κ-casein) [8][9][10]. Bioactive peptides are studied for their various beneficial activities, for example anti-hypertensive, anti-microbial, opioid and antioxidant [4,[11][12][13][14]. The antioxidant activity of bioactive peptides can depend on their amino acid composition and position in the sequence [15]. Moreover, these compounds can exert their antioxidant activity in cell environment through activation of specific pathways [16,17]. Oxidants and electrophiles are well known molecules recognized to determine the disruption of Keap1/Nrf2 interaction [16,18,19]. However, new series of other compounds are now emerging, such as the bioactive peptides, that with specific protein-protein interactions are able to activate nuclear factor erythroid 2-related factor 2 (Nrf2). The latter, after dissociation from Kelch-like ECH-associated protein 1 (Keap1), migrates to the nucleus where interacts with the antioxidant response element (ARE), activating a large number of genes expressing antioxidant enzymes. Nrf2 translocation is one of the key e...

Advances on Food-Derived Peptidic Antioxidants—A Review

“…Four peptides with high antioxidant activity in both chemistry and cell model evaluations have been purified and identified from the pasteurized milk fermented with Lactobacillus acidophilus NCFM ® , Lactobacillus delbrueckii subs. bulgaricus and Streptococcus thermophilus [129]. Fermented meat products can also be used as a source of PAs [130].…”

Bioactive peptides from food fermentation: A comprehensive review of their sources, bioactivities, applications, and future development

“…The study found that the presence of the hydrophobic amino acids (glycine and proline) and the -SH group of cysteine as an effective hydrogen donor to free radicals in the peptide contributes to its high antioxidative property. In a recent study shown in Table 1, Tonolo et al (2020) identified four novel antioxidative peptides from fermented milk, namely, N-15-M (NTVPAKSCQAQPTTm*), E-11-F (EDELQD-KIHPF), Q-14-R (QGPIVLNPWDQVKR), and A-17-E (APSFSDIPNPIGSENSE), and subjected them to molecular docking analysis to predict the interaction of the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1). Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is known to be the main regulatory system for oxidative stress.…”

“…When an oxidative imbalance takes place, Keap1 and Nrf2 dissociate, followed by the translocation of Nrf2 to the nucleus and bind to antioxidant response element and subsequently activating the overexpression of antioxidant enzymes. Tonolo et al (2020) treated Caco-2 cells with peptides N15-M, Q-14-R, A-17-E, and E-11-F, and found that all cells except the E-11-F-treated cells had increased levels of antioxidant enzymes because the specific amino acid residues in these peptides interact with Keap1 in the binding to the ETGE motif, causing the disruption of Keap1 binding with Nrf2 and subsequent activation of the signaling cascade.…”

Bifunctional peptides with antioxidant and angiotensin‐converting enzyme inhibitory activity in vitro from egg white hydrolysates

“…Antioxidant peptides have been demonstrated to inhibit excessive ROS (Zhang et al., 2019). There are also many antioxidant peptides found in food, such as SSGPPVPGPMGPMGPR from skipjack tuna bone (Ding, Du, Zhang, Zaman, & Huang, 2019), QGPIVLNPWDQVKR from fermented milk (Tonolo et al., 2020), and VYLPR from egg white protein (Zhang et al., 2019). However, most of them were analyzed single biological activity.…”