Metabolic activity of body cells is essential for survival, and by-products from the process, including reactive oxygen species (ROS) and free radicals, are constantly produced (Birben et al., 2012). Moreover, the body is exposed to oxidants such as motor vehicle exhaust, tobacco smoke, oxides of nitrogen, ozone and air pollutants. Continuous interaction of body systems with these oxidants leads to an additional load of free radicals and causes increasing harm to proteins, lipids, DNA, carbohydrates and membranes, resulting in oxidative stress (Chakrabarti et al., 2014; Sah et al., 2015b, 2016c), which may lead to cancer (Sah et al., 2015b) (Figure 2.4).
Free radicals are chemical species that are composed of unpaired electrons in the exterior orbit possessed by molecules (Power et al., 2013). These unpaired reactive electrons allow the capture of electrons from other particles, resulting in free radicals that are able to destroy healthy cells. Free radicals, according to Chakrabarti et al. (2014), have the potential to attack all macromolecules, which in turn, would lead to lipid peroxidation, DNA modification and break down of cellular proteins during an oxidative event (Sah et al., 2016c). Uncontrolled formation of these free radicals has often been linked with the inception of a number of cardiovascular diseases, together with cancer and other disorders such as congestive heart failure, cardiac hypertrophy, cardiomyopathies, hypertension, ischemic heart disease and atherosclerosis (Clarkson & Thompson, 2000; Chakrabarti et al., 2014). The human body contains an endogenous antioxidant structure primarily used as a defence system that comprises a range of enzymes, particularly the superoxide dismutase and catalase, as well as other compounds with antioxidation capabilities. These antioxidants can prevent oxidation by inactivating or preventing generation of ROS (Sah et al., 2016c).
Several naturally occurring antioxidant peptides (e.g. glutathione [GSH: Glu–Cys–Gly], carnosine [β-Ala-L-His], anserine [β-Ala-3 methyl-L-His], homocarnosine [γ aminobutyryl-L-His]) with free radical scavenging activity have been identified in the human body (Marambe & Wanasundara, 2012). Figure 2.4 demonstrates the possibility for food proteins to act as biologically active peptides precursors with diverse physiological characteristics, such as antioxidant activity. Searching for potential antioxidant compounds from natural sources may provide alternative treatments to current synthetic chemicals (Sah et al., 2015a). Peptides derived from proteins originating from milk products are among those that research has linked to natural antioxidants (Sah et al., 2016c) (Table 2.11). They are involved in quenching free radicals by oxidation of AA residues. Casein proteins in bovine milk are thought to possess antioxidant peptides in their primary structure that are released upon hydrolysis. For instance, the peptide Ala– Arg–His–Pro–His–Pro–His–Leu–Ser–Phe–Met released from κ-casein upon milk fermentation with L. delbrueckii ssp. bulgaricus IFO13953 when isolated displays antioxidant activity (Kudoh et al., 2001). Further, the antioxidant activity of whey protein-derived peptides has been reported along with the antioxidant activity of whey itself (Meisel, 2005). For example, the antioxidant peptide Tyr–Tyr–Ser–Leu–Ala–Met– Ala–Ala–Ser–Asp–Ile was released by the action of a commercial protease (corolase® PP) on β-Lg A (Hernández et al., 2005a, b). The bioactivity of antioxidative peptides is associated with AA composition (Suetsuna et al., 2000). Peptides with high amounts of histidine and some hydrophobic AAs have been reported to have more potent antioxidant activity than others (Peña-Ramos et al., 2004). For example, histidine-containing peptide is thought to be connected with hydrogen donation and lipid peroxyl radical trapping (Chan et al., 1994; Sah et al., 2016c). Further, the existence of leucine or proline with histidine–histidine dipeptides at the N-terminus may improve antioxidant activity
(Suarez-Jimenez et al., 2012). The antioxidant activities of 28 peptides were compared to that of the antioxidative peptide (Leu–Leu–Pro–His–His) by Chen et al. (1996), who found that tripeptide (Pro–His–His) exhibited the highest antioxidant activity among all the peptides assessed.
Figure 2.4 Factors that may lead to increased cancer incidence Exogenous sources
Radiation, pollution and inflammation
High level of reactive oxygen species (Oxidative stress)
Gene mutations, altered gene expression, damage to RNA, DNA, Protein and lipids
However, deletion of the histidine C-terminal peptide has been reported to decrease antioxidant activity (Zou et al., 2016), indicating the importance of histidine in antioxidant peptides. Hydrophobicity of a peptide can also be an important determinant of its antioxidant activity and may increase accessibility to hydrophobic targets (e.g., lipophilic fatty acids) (Sah et al., 2016c).
2.10.4.1The Structure–Activity Relationship
The correlation between antioxidant properties of peptides and their structure has not yet been described in full detail. However, some factors, such as AA composition, hydrophobicity and peptide structure, may enhance their bioactivity (Chen et al., 1998; Sah et al., 2016b). For example, antioxidant peptides that contain aromatic ring structure AAs (Trp, Phe and Tyr) can donate protons to electron-deficient radicals (Chen et al., 1998). Further, hydrophobicity has been reported to enhance antioxidant activity (Sah et al., 2016c). Hydrophobic AAs such as valine, leucine and tyrosine contained in antioxidant peptides can boost solubility in phospholipid conditions and facilitate better interaction with hydrophobic radicals (Qian et al., 2008). The hydrophobic properties of AAs at the N- and C-terminus of peptides increase antioxidant activity (Zou et al., 2016). Table 2.9 compares antioxidant peptides released from milk proteins, the majority of which have hydrophobic AAs at their N-terminus (Zou et al., 2016). Also, properties of the second AA adjacent to the C-terminal AA has been reported to enhance antioxidant activity when this AA has low hydrophobicity, strong hydrogen bonding and steric properties. This means that antioxidant activity will be increased, particularly when the second AA is basic, for example, Glu, Asp, Arg, His, Lys or hydrophilic, such as Ser or Thr (Power et al., 2013). Moreover, peptides with Cys at the N-terminal position play a key role in antioxidant activity through the R–SH group, which interacts with radicals by hydrogen donation from the SH group (Elias et al., 2008). Histidine has also been reported
to have antioxidative effects due to the imidazole group in histidine-containing peptides, which has metal chelating, hydrogen donating and lipid peroxyl trapping capabilities (Chan et al., 1994). The presence of hydrophobic residues in His-containing peptides can boost accessibility to hydrophobic radicals (Murase et al., 1993). Chymotrypsin can hydrolyse whey proteins and release antioxidant peptides through its specificity for C- terminal-cleavage bonds containing aromatic or hydrophobic residues (Pihlanto, 2006b), resulting in facilitated electron transfer from these peptides. Suetsuna et al. (2000) showed that a hydrophobic Leu-containing peptide (Tyr–Phe–Tyr–Pro–Glu–Leu) from casein hydrolysed by pepsin exhibited DPPH radical scavenging activity—Glu–Leu residues being the main factor in bioactivity.
2.10.4.2Methods to Evaluate Antioxidant Activity
The importance of natural antioxidants with fewer side effects and lower cost than chemically synthesised drugs cannot be overemphasised. This has led to the investigation of antioxidants in dairy products by food science researchers (Sah et al., 2014; Elfahri et al., 2014, 2015). A number of detection methods has been used and developed to evaluate antioxidant capacity (Sah et al., 2015a). In terms of chemical reactions, the methods for measurement of antioxidant capacity are categorised into two groups: the first is based on hydrogen atom transfer (HAT); and the second, on electron transfer (ET) (Huang et al., 2005; Korhonen, 2009a; Sah et al., 2015a). ET-based assays measure the capacity of an antioxidant in the reduction state, and act as an indicator for monitoring the reaction endpoint (Huang et al., 2005; Dryáková et al., 2010). Ferric ion-reducing antioxidant power and DPPH radical scavenging capacity are the most common methods used. For example, ET-based assays measure the antioxidant activity of peptides during milk fermentation (Sah et al., 2015a, 2016c). However, use of these methods to investigate the
extrapolated to their in vivo antioxidant capacity due to a range of factors such as bioavailability of antioxidants, reactivity, stability, concentration and storage in tissue. Thus, peptides with antioxidant properties may or may not resist proteolytic enzymes in the GIT to reach target sites in the body (Vermeirssen et al., 2002; Mills et al., 2009; Madureira et al., 2010).