**3. Biological activities**

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

**2. Agricultural co-products as a source of bioactive peptides**

ing to allergenicity would degrade within hydrolysis process [8].

production of antioxidant peptides [11].

Agricultural co-products involve a vast variety of materials with great potential as a substrate for bioactive peptide production due to the low value and high protein content. It is estimated that about 9000 tons in dairy, 3 and 9 million tons in seafood and livestock industries, respectively, are annually discarded [4]. Skins, bones and heads are rich in collagen. Collagen is known as a promising source of peptides with several bioactivities which could exert physiological functions. Collagen comprises of hydrophobic amino acids, particularly proline and hydroxyproline, offering a higher chance of being absorbed through epithelial membrane. These amino acids also make them stable against proteases in gastrointestinal digestion tract and brush border, so that the intact peptides could be absorbed and reach the target organ. Blood is also high-protein co-product obtained in a large volume in a slaughterhouse. It has been reported that about 2.5 billion tons of blood are annually generated only in Europe [5]. Hemoglobin in red corpuscles and albumin, globulins, and fibrinogen in plasma are the major proteins [6]. Whey is a predominant co-product of dairy industry with an annual production of approximately 180–190 million tons [7]. The whey-derived peptides have also lower allergenicity beyond their bioactivities, because β-lactoglobulin (β-lg) as a main whey protein contribut-

Production of bioactive peptide from some plan-based materials are restricted due to difficulties in protein recovery from their unique rigid structure of polysaccharides. To meet the challenge, pretreatment of these substrates would improve protein recovery and provide other benefits such as reduction of time and energy consumption, leading to a higher efficacy in bioactive peptide production [9]. Corn gluten meal was immersed in alkali solution, treated with α-amylase and cooked prior to proteolysis [10]. Ultrasound exposure of watermelon seed caused structural changes, leading to production of peptides with higher antioxidant activity than those without any treatment. Ultrasound could degrade interactions in matrix and unfold proteins, so that more hydrophobic residues and reactive sites are exposed, resulting in a higher efficacy in protein hydrolysis [9]. Oat bran polysaccharide was digested by cellulase and viscozyme to ease protein recovery prior to proteolysis and

immunomodulatory activities among others. But these peptides are encrypted in the protein chain and needed to be released to exert bioactivities. Fermentation, enzymatic and chemical (by acid or alkali) hydrolysis could be applied to produce bioactive peptides as well as using solvents which is normally used to extract natural peptides. Among them, enzymatic hydrolysis known to be more effective as it is a mild, ecofriendly, and controllable process. Bioactivities of the released peptides are affected by their size, hydrophobicity, charge, amino acid composition and their sequence which are different based on various enzyme and substrate as well as the hydrolysis conditions. After production of peptides with the certain bioactivity, their structural modification upon gastrointestinal digestion and epithelial transportation and absorption must be taken into consideration to determine their potential bioavailability. Several bioactivities and physiological functions of peptides derived from both animal and plant-based agricultural co-products have been reported, which are mainly focused on their antioxidant, antihypertensive, antidiabetic and antibacterial properties. In this chapter, their main biological activities along with the associated structure will be considered. Besides, the structure-bioavailability relation of the peptides will be demonstrated and procedures to keep them intact upon gastrointestinal digestion and transepithelial transportation will also be proposed.

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Several bioactivities and physiological functions of peptides derived from both animal- and plant- based agricultural co-products have been reported. These activities include antioxidant, antihypertensive, antidiabetic and antibacterial properties.

#### **3.1 Antioxidant activity**

Reactive oxygen species (ROS) are formed during normal cellular metabolism of providing energy, respiration and also when cells are exposed to exogenous oxidative stress [15, 16]. Normally, these products are neutralized by endogenous antioxidant defense systems, such as antioxidant enzymes (superoxide dismutase: SOD, glutathione peroxidase: GPx and catalase), glutathione (GSH) and others. But, the excessive level of ROS could result in many health disorders, such as cancer, cardiovascular, respiratory, neurodegenerative and other diseases [16, 17]. Therefore, antioxidant-containing diet could help to overcome ROS and subsequently their corresponding disorders. Nowadays, attempts have been made to find new antioxidant compounds from natural resources due to their benefits over synthetic compounds. To that direction, antioxidant peptides from agricultural co-products are gaining more attractions, because of their nontoxicity and safety besides their nutritional properties. Antioxidant hydrolysates/peptides production from agricultural coproducts including skins, bones, viscera, whey as animal-based and brans, seeds, leaves, gluten as plant-based co-products have been extensively reviewed [17–21].

Lower molecular weight (MW) peptides with hydrophobic and aromatic amino acids (HAAs and AAA, respectively) have been generally reported to exert good antioxidant activities [22]. The HAAs could improve peptides accessibility toward ROS through binding with lipid and reaching to free radicals, so that the peptides could quench them effectively. The AAAs including Trp, Phe and Tyr are also correlated with the strong antioxidant activity via their high electron transferring capacities of their aromatic rings. Besides HAAs and AAAs, some of hydrophilic amino acids, such as His could improve the activity through its imidazole ring which has been indicated as strong electron donator [23]. Presence of charged amino acids in peptide structure could also improve the activity. A higher negatively charged amino acids (NCAA) have been observed in plasma hydrolysates prepared from

chicken blood that showed higher antioxidant activity than those prepared from blood corpuscles with lower NCAAs [6]. Presence of NCAAs are reported to correlate with the strong antioxidant activity, because they can neutralize free radicals by giving their excess electrons.

Although antioxidant activity of agricultural co-product has been widely evaluated, few studies were conducted to assess the activity *in vivo* and there is still a gap for clinical trial. The chemical *in vitro* studies are not able to reflect the activity in biological systems due to their complicated physiological conditions. In addition, cellular evaluation might be able to provide a comparable environment to biological systems. Antioxidant properties of peptides from fish sauce increased with the increasing of peptides concentration based on chemical assays, while these peptides could act as pro-oxidants in higher concentration (>50 μg L-leucine equivalent/ml) in cellular experiments [24]. Hydrolysates prepared from corn gluten meal and ham seed meal as well as ACFL, a peptide from horse mackerel viscera, increased the level of antioxidant enzymes based on *in vivo* models upon exposure to oxidative stress [10, 25, 26]. In a human trial study, a reduction in plasma malondialdehyde and an increase in SOD level was observed after daily ingestion of 4.5 g black soy derived peptides for 8 weeks [27].
