One roadblock to the industrial production of protein hydrolysates from by-products is that most studies with by-products were performed at the laboratory scale, which limits their industrial adaptation [
113]. Some studies attempted pilot trials to confirm the technical feasibility of the laboratory scale at an industrial scale. In this sense, the process of producing peptides from hake by-catches was scaled up from a 0.5 to a 150 L reactor using the optimized hydrolysis conditions (2% enzyme, two h, 50% solids, pH 9, 70 °C) that were identified in the laboratory [
100]. The authors found similar results at the pilot plant scale in terms of protein extraction yield (60.0% pilot and 61.4% lab), antioxidant capacity (172 mg TEAC/g protein in pilot and 224 mg TEAC/g protein in lab), and antioxidant capacity yield (103 mg TEAC/g protein in pilot and 132 mg TEAC/g protein in lab). Furthermore, liquid, solid, and bone yield did not show any significant differences from the results of the laboratory trials. Monkfish by-products (heads and viscera) hydrolysis was scaled up from 100 mL to a 5 L glass reactor at optimized laboratory conditions: 57 °C, pH 8.3, solid to liquid (S/L) ratio of 1:1 (
w/
w), 0.05% Alcalase, and a 200 rpm stirring rate for three hours. Following hydrolysis, the hydrolysates were filtered (100 μm) to remove non-hydrolyzed materials.
3. Antioxidant Activity of Fishery By-Products Protein Hydrolysates and Peptides
The antioxidant peptides had 2–10 amino acids, although some had up to 20 amino acids and had a MW of 0.2 to 2 kDa. The antioxidant activity of peptides is mainly related to the presence and position of specific amino acid residues in the peptide chain. Primary structure, amino acid composition, hydrophobicity, spatial conformation, etc., are characteristics that are affected by enzymatic hydrolysis and determine its antioxidant activity [
114]. Peptides show antioxidant activity due to the presence of one or more hydrophobic (Pro, Ala, Gly, Leu, Ile, Met, Trp, Phe, Val) and aromatic (Tyr, Trp, Phe) amino acids that can quench free radicals via various mechanisms, including hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT), and sequential proton loss by electron transfer (SPLET) mechanisms [
115]. Scavenging free radicals and oxidants using HAT, SET-PT, and SPLET generally leads to the same end results, although the kinetics and potential for side reactions vary. SPLET, SET-PT, and HAT may occur in parallel; however, the dominant mechanism depends on the antioxidant’s conformational and geometrical features, solubility, partition coefficient, and the type of solvents [
116]. The antioxidant activity of hydrophobic amino acids has been attributed to their ability to interact with lipid molecules by increasing the solubility of peptides in lipids and scavenging lipid-derived radicals through electron-donating substituents such as OH and NH
2 on amino acid side chains in peptides. Hydrophobic amino acids can improve the antioxidant activity of peptides by providing a potential pool of free electrons.
Peptides containing proline-rich sequences have been identified to possess antioxidant properties. Proline has an electron-rich nitrogen-containing pyrrolidone ring that stabilizes the radical peptide formed after electron donation [
117,
118]. Peptides with Pro at the C-terminus (e.g., Pox: Tyr-Tyr-His-Pro) were the most potent antioxidant (0.8 TE at 2.5 μM). The modification of the structure by moving Pro into positions X1 (Pro-Tyr-Tyr-His), X2 (Tyr-Pro-Tyr-His), and X3 (Tyr-Tyr-Pro-His), but leaving the other residues in the same order as in Pox, resulted in a significant difference in ORAC, which was 0.2, 0.1, and 0.55 TE at 2.5 μM for X1, X2, and X3, respectively [
119]. Intense antioxidant activity has been reported for small peptides containing amino acid residues such as Tyr, His, and Pro [
120,
121]. The dipeptide Tyr-Tyr at the N-terminal position of Tyr-Tyr-His-Pro and Tyr-Tyr-Pro-His was the portion responsible for stronger antioxidant activity. However, Tyr-Pro-Tyr-His showed the weakest ORAC and inhibited ROS production by 36% at 0.07 μM in human keratinocyte cells (HaCat) after treatment with H
2O
2 when compared to Tyr-Tyr-His-Pro (with the highest ORAC), which showed the similar inhibition of ROS production at 2.5 μM (40%) [
119]. It is believed that besides amino acid composition and sequence, the changes in secondary structure have a significant impact on the capture and dissipation of free radicals. The nanopeptide Val-Leu-Leu-Tyr-Lys-Asp-His-Cys-His (1127 Da) produced using the self-assembly of pine nut Val-Leu-Leu-Tyr (506 Da) and sea cucumber Lys-Asp-His-Cys-His (638 Da) had significantly higher antioxidant activity compared to individual peptides due to changes in the secondary structure as seen in the lower electron paramagnetic resonance (EPR) signal, higher random crimp degree, and increased supply of hydrogen protons (i.e., the higher exposure to active hydrogen) from Raman spectroscopy and
1H NMR spectrum analysis in the nanopeptide than the tetrapeptide and the pentapeptide
The presence of Tyr at the N-terminal position of peptide Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu has been reported as the residue that most contributed to antioxidant activity (3.8 μM TE/mg protein). However, Tyr-Pro-Ile and Tyr-Gln-Ile-Gly-Leu with Tyr at the same position showed lower ORAC (1.6 and 1.7 μM TE/mg protein, respectively), indicating the role of adjacent amino acids and chain length on antioxidant activity [
123]. Tyr-containing peptides from abalone viscera showed strong ABTS radical scavenging activity in the order of Cys-Ile-Gly-Tyr-Asp-Arg (0.144 mg/mL) > Tyr-His-Gly-Phe (0.268 mg/mL) > and Gly-Cys-Tyr-Val-Pro-Lys-Cys (0.389 mg/mL). The first and last peptides, which contained both Tyr and Cys, showed similar trends for scavenging DPPH radicals (IC
50 of 0.207 and 0.405 mg/mL, respectively). Despite the observed high ABTS radical scavenging activity, peptides Met-Glu-Thr-Tyr and Tyr-His-Gly-Phe, which had Tyr at the C- or N-terminal position, respectively, had weak scavenging activity against DPPH radicals (<20%), which was attributed to the lack of Cys in their sequence. Despite having different sizes or amino acids residues, peptides Gln-Cys-Val-Arg and Gln-Ser-Cys-Ala-Arg-Phe showed similar DPPH radical scavenging activity (IC
50 of 0.392 and 0.416 mg/mL, respectively), indicating the complexity of the relationship between the peptides’ structures and function. Regarding the number of amino acid residues within peptide sequence, the peptide Gly-Cys-Tyr-Val-Pro-Lys-Cys, containing two Cys residues, showed lower free radical scavenging activity (IC
50 of 0.389 and 0.405 mg/mL for scavenging ABTS and DHHP radicals, respectively) than Cys-Ile-Gly-Tyr-Asp-Arg, which contained only one Cys (IC
50 of 0.144 and 0.207 mg/mL for scavenging ABTS and DPPH radicals, respectively [
124].
It was shown that the presence of a Tyr, Trp, Cys, or Met residue with electron/hydrogen donating ability was the driving force for dipeptides to scavenge radicals. The presence of Tyr, Trp, and Cys in the sequence was required for dipeptides to scavenge ABTS
•+, while the presence of Tyr, Trp, Cys, and Met was needed for dipeptides to scavenge ROO
• when using the ORAC assay. Structure–activity relationships showed that Tyr- and Trp-containing dipeptides with Tyr/Trp residue at the N-terminus (Tyr/Trp-X; Tyr-Gly, Tyr-Ser, Tyr-Gln, Tyr-Glu) had stronger ORAC and ABTS
•+ scavenging activity than that at the C-terminus (X-Tyr/Trp, Gly-Tyr, and Glu-Tyr) and the steric effects, hydrophobicity, and hydrogen bonding also affected the neighboring AAs. Tyr-containing dipeptides showed higher ABTS
•+ scavenging activity. In contrast, Trp dipeptides (Trp-Gly, Trp-Ser, Trp-Gln, Trp-Glu, Gly-Trp, Glu-Trp) had higher ORAC, and only Cys-containing dipeptides showed moderate reducing power activities [
125]. The calculation of BDE, IP, PA, and ETE of Tyr/Trp-X and X-Tyr/Trp (where X was Gly, Leu, Pro, Phe, Ser, Thr, Asn, Gln, Asp, Glu, Lys, and Arg) showed that there were few differences among dipeptides, indicating that the neighboring AAs did not affect the intrinsic hydrogen or electron-donating ability of the dipeptides studied. Thus, the differences in their radical scavenging activity can be attributed to other factors (such as steric effects and inter/intra-molecular hydrogen bonds).
4. Application of Fish By-Products Protein Hydrolysates to Control Oxidative Deteriorations of Seafood
The oxidation of lipids is often the major cause of the quality loss of foods during storage, as seen in the changes in color, texture, flavor, and aroma, which impairs sensory and nutritional properties and the shelf-life of foods [
28]. The decomposition of the hydroperoxides formed by pro-oxidative metal ions is a driving factor for lipid oxidation, producing highly reactive alkoxyl lipid radicals and hydroxyl ions. Alkoxyl radicals degrade rapidly to form volatile decomposition products, often with off-odors [
29]. Furthermore, protein carbonyls can be introduced into proteins using a covalent linkage of lipid carbonyls (e.g., protein-bound malondialdehyde). Protein oxidation leads to functional property changes such as decreased solubility, digestibility, and water-holding capacity [
30]. The loss of nutrients and myofibrillar water and the changes in texture are inevitable during frozen storage [
133,
134]. The formation of ice crystals, associated with cell membrane rupture and muscle fibers, often leads to protein denaturation and undesirable reactions such as aggregation and decrease in solubility, solute concentration (macromolecular crowding), lipid oxidation, and instability of proteins at the ice–water interphase [
135,
136]. Protein hydrolysates and peptides may be potential antioxidants to reduce oxidation during food storage, thus extending the shelf life [
28]. The antioxidant activity of protein hydrolysates was related to amino acid composition, sequence, size, and the amino acid residues at the C- or N-terminal positions [
114]. Enzymatic hydrolysis disrupts the tertiary structure of food proteins, leading to the increase in the solvent accessibility of peptides to scavenge free radicals and chelate pro-oxidative metal ions. Protein hydrolysates and peptides have been reported to control food oxidation through various mechanisms, including inactivating ROS, scavenging free radicals, chelating pro-oxidative metal ions, reducing lipid hydroperoxides, and changing the physical state of foods.
5. Conclusions
Marine by-products have been studied as a source of antioxidant peptides for food, feed, and nutraceutical applications. Those studies recommended these peptides as potential functional ingredients to enhance health and nutrition. However, the differences in the composition of by-products, the type of proteases used, and the different hydrolysis parameters resulted in various end products from the same protein. To ensure consistency, the process of upgrading at three levels, i.e., by-products, enzymes, and operating parameters must be optimized for each by-product source.
Different peptides were produced by different enzymes. However, it has not been determined which peptide is a more potent antioxidant in controlling oxidation in which food system. Different foods, due to inherent composition differences (i.e., different amounts of pro-oxidants, oxidation-prone substances, and internal antioxidant enzymes), will probably have different reactions to antioxidant peptides and specific structures during storage. Therefore, the effect of peptides with specific structures or protein hydrolysates produced using a specific condition in different food matrices needs to be investigated.
Although the structure of peptides was influenced by the specificity of the proteases used, most studies used fresh by-products with acceptable initial quality. It is less known whether the same peptides structure and function can be obtained using previously stored by-products and how the quality of proteins in refrigerated or frozen by-products, as well as associated chemical reactions during storage, affect hydrolysis and product structure, function, and stability. This area needs to be further investigated, especially in by-products with high lipid and blood contents, such as herring and salmonid by-products, and to understand which fractions are more oxidized and contribute more significantly to undesirable biochemical reactions during hydrolysis. Several researchers have tried to stabilize by-products before upgrading them using antioxidants. There is a price to adding synthetic antioxidants or to maintaining the initial quality and safety of agricultural wastes as sources of antioxidative extracts. The practical ability to undertake this process for large quantities of by-products and the space and energy consumption required to create low temperature storage are among the issues that make this application more complicated.
By-product processing should be carried out near fish production and processing centers so the hydrolysis of by-products can be performed within the shortest possible time. When the quantity of by-products exceeds a center’s capacity, they should be stored frozen. Therefore, the impact of frozen storage at varying lengths of time on the structure of peptides (amino acid composition, sequence, and size) and the occurrence of undesirable oxidative deteriorations and biochemical changes that may affect biological functions needs further study. There is limited information about the effect of initial protein quality due to processing and storage on the functional and biological activities of hydrolysates and peptides for food applications.
Many studies are underway regarding the use of fish protein hydrolysates in food. Nevertheless, the supply of fresh raw materials with acceptable safety, competitive prices with other commercial ingredients from plants and other sources, and the lack of efficient and standardized techniques to transform fish by-products into marketable forms limit their utilization.
Although the role of protein hydrolysates in maintaining the quality of seafood products has been shown, a standard method for its production from a specific source of marine by-products on a pilot or an industrial scale and its industrial application has not yet been undertaken. However, the industry prefers to use synthetic preservatives with lower prices to maintain the quality of seafood products during storage.
Protein hydrolysates may also affect the sensory characteristics of food. So, how to mask or remove the fish smell using encapsulating methods and the cost of such pretreatments on protein hydrolysates and the market must be addressed. Furthermore, hygroscopicity, the development of bitterness during enzymatic hydrolysis, low storage stability, and hydrolysis in the gastrointestinal tract (GIT) may pose several challenges to the application of fish protein hydrolysates in the food industry [
155,
156]. Several encapsulating techniques, including liposomes, nanohydrogels, emulsions, and diphasic gel double emulsions, have been used to improve the storage and gastrointestinal stability of protein hydrolysates [
157,
158].