2.1.1. Physical Pre-Treatments

PEF is based on exposing cells to a high-strength electric field, inducing the formation of transient pores in the cell walls in a process known as electroporation [14]. The disruption of the cell walls by PEF can also lead to the formation of permanent or transient pores, allowing the mass transfer of small molecular weight compounds including protein extraction [15] to the outside of the cells and into the solution [16]. Pore sizes may also be further influenced by PEF duration, for instance, the pore radius for short pulses (5 μs) was approximately 0.8–1.6 nm, while when using longer pulses (10 μs) the pore radius increased up to at least approximately 5 nm, which was considered as irreversibly formed [17]. To date, PEF has been primarily used as a method to extract lipids from algae in connection with biofuel manufacture [17]. Vanthoor-Koopmans, Wijffels, Barbosa and Eppink [4] reported that small peptides and free amino acids present in the cell or created during the PEF process may be released from the biomass following PEF, while larger

peptides and proteins were retained within the cell. Thus, according to the authors, adding a protein solubilization step will benefit these extraction processes. This solubilization can be achieved by adding surfactants and/or increasing the pH to untangle proteins, increasing their solubilization and extraction ratios [4,18]. The yield of the extracted protein from *Nannochloropsis* spp. doubled when using alkaline solvents (pH 8) compared to water; however, the recovery of proteins from the biomass was still low compared to other physical extraction procedures, such as UAE [19]. PEF has been described as a promising method for the extraction of proteins from the macroalga *Ulva ohnoi* [20]. The authors achieved a 3-fold increase in the yield of proteins from *U. ohnoi* of with the application of PEF. Similarly, in the case of *Arthrospira platensis*, a microalgae species with high protein content, PEF treatment followed by soaking the biomass in water increased the extraction of the intracellular proteins (c-phycocyanin) 12.7-fold compared to the control [21]. Parniakov, Barba, Grimi, Marchal, Jubeau, Lebovka and Vorobiev [19] emphasized the advantages of PEF in relation to power usage and cost-effectivity of the treatments, although the yields of protein extraction were lower compared to UAE pre-treatments. The power consumption of PEF was approximately 100 kJ/kg at 20 kV/cm for 6 ms, while in the case of UAE it was 250 kJ/kg at 200 W for 10 min [19].

UAE is based on the generation of high-frequency ultrasonic waves in a liquid, which generates a grea<sup>t</sup> number of bubbles that collapse (cavitation) and release a burst of energy that can disrupt the algal cell walls [22]. Advantages of this technology include the possibility to combine its application with various solvents, targeting the extraction of different compounds from the biomass, as well as an increased extraction efficiency in terms of reduced times and energy of extraction compared to conventional solvent extraction processes [23]. For example, the use of UAE with a subsequent alkaline treatment allowed the extraction of 57% of the total proteins from *Ascophyllum nodosum* [24]. Moreover, the use of ultrasounds enabled 2.29-fold higher yields of protein extraction compared to conventional methods from the microalga *A. platensis* [25].

MAE has also been used to extract protein from algae [26]. MAE uses oscillating electric fields which cause vibrational friction of polar molecules in the cells, which allows heating of the sample to occur. The main advantage of MAE is that a small amount of or even no solvents are required, as well as the fast extraction times of this technique compared to conventional methods [27,28]. Juin, Chérouvrier, Thiéry, Gagez, Bérard, Joguet, Kaas, Cadoret and Picot [26] used MAE to extract water-soluble proteins from *Porphyridium purpureum*, focusing mainly on the extraction of phycobilin proteins. The authors reported that MAE improves the extraction yields of compounds and significatively reduced the time of extraction to seconds compared to the hours needed to generate similar extraction yields when using traditional solving extraction methods. Moreover, MAE appeared to be more efficient in protecting thermolabile compounds during the process of extraction, as the application of high temperatures for prolonged periods of time needed during conventional solvent extraction may have a negative impact on these compounds once they are extracted [29].

## 2.1.2. Enzymatic Pre-Treatments

One of the key advantages of enzymatic pre-treatments over other mechanical methods is the relatively low temperatures required, allowing the release of both peptides and proteins from algae with minimal or no damage to their structures [30]. However, the high variability of the composition of the algal cell walls between species requires will require the customization and optimization of these enzymatic treatments [11]. For example, a mixture of trypsin, collagenase, lysozyme, and autolysin was useful as a pre-treatment to disrupt the cell walls of *Chlamydomonas reinhardtii* [31]. Autolysin was the most efficient enzyme as an enzymatic treatment and showed to be preferable to other chemical and mechanical methods such as solvents and sonication [31]. Moreover, the authors also reported that longer pre-treatments resulted in the total lysis of the cells advantageously resulting in increased extraction yields of cellular compounds measured as proteins and

lipids [31]. Other pre-treatments using cellulase followed by protein hydrolysis with bromelain were effective when extracting proteinaceous concentrates from *Fucus spiralis*, increasing its extraction yields by 1.5-fold compared to those achieved by using bromelain alone [32]. The protein content of *Macrocystis pyrifera* and *Chondracanthus chamissoi* was increased by disrupting the carbohydrate matrix of the algae by using cellulase, increasing the yields of protein extraction [33]. As expected, optimum enzymatic conditions also varied between both macroalgae, achieving protein extraction yields of approximately 75% from *M. pyrifera* by an optimized enzymatic treatment with cellulase (1:10, enzyme:seaweed ratio) for 18 h and yields of 36% from *C. chamissoi* using the same enzyme:seaweed ratio for 12 h [33]. Moreover, Fleurence, Massiani, Guyader and Mabeau [13] reported that a mix of carrageenase/cellulase has a 10-fold higher extraction efficiency over the use of just carrageenase alone in *C. crispus*. Similarly, mixtures of agarase/cellulose achieved a 3-fold increase in protein extraction from *G. verrucosa*, while in the case of *P. palmata,* the combined xylanase/cellulase had similar protein extraction yields to those of control [13].

#### *2.2. Generation of Bioactive Peptides*

Once the protein is extracted from the biomass, the classical method for the generation of bioactive peptides uses proteases to break peptide bonds and generate hydrolysates containing a complex mix of peptides [34]. Enzymes exert their action by cleaving sequence motifs within a protein. The preferred cleavage sites of each of these enzymes are summarized in Table 1.


**Table 1.** Characteristics of proteases used for the generation of bioactive peptides.

Amongst all the proteases, trypsin, chymotrypsin and pepsin have been the most widely used enzymes for the generation of bioactive peptides. Trypsin has a specific binding affinity for positively charged side chains of the amino acids lysine and arginine. Trypsin's cleavage site is on the C-terminal side of the amino acid residues. Hydrolysis is decreased with the presence of acidic amino acids on either side of the cleavage site. Cleavage will not occur if a proline residue is present on the carboxyl side of the cleavage site [35]. Chymotrypsin is another serine protease which is itself activated by trypsin cleaving the bond at residues 15 and 16 (arginine and isoleucine). The chymotrypsin enzyme catalyzes the hydrolysis of proteins by cleaving the molecules at hydrophobic amino acid residues, such as the L-isomers of tyrosine, phenylalanine, and tryptophan. It also has the capability of acting on amides and esters of susceptible amino acids [47]. The use of these enzymes can result in the one key advantages of this approach, which is the reproducibility of the process such that similar proteins and hydrolysates products are generated.

Emerging technologies have also been used to hydrolyze protein extracts and generate bioactive peptides from algae and other food products and by-products [48,49]. Amongst them, subcritical water (SCW) processing has been gaining attention as both a green extraction and protein hydrolysis method [50–52]. SCW does not require the use of expensive and lengthy reaction times and it can be a method of extracting compounds from highly insoluble media which are ecologically damaging by-products from industries, such as poultry waste [53], hog fur [54] and fisheries waste [55]. In fact, SCW production of bioactive peptides also significantly reduces the processing time of hydrolysis of collagen by a factor of almost 300 when using enzymatic methods such as collagenase [56] to up 5 min [57]. The use of SCW to produce amino acids and peptides from waste and under-utilized byproducts could give these industries a new revenue stream while mitigating the ecological and economic issues currently associated with the disposal of these by-products [58].

The SCW process maintains water in subcritical conditions inside the reaction chamber by using oven temperatures ranging from >100 ◦C to <374 ◦C and an internal pressure of <22 MPa, stimulating the formation of hydronium (H3O+) and hydroxide ions (HO−) that allow water to interact as a basic or acidic catalyst [52]. The pressure applied during SCW will cause the unfolding and loss of secondary, tertiary, and quaternary structures of the protein, while the ions will interact with the amino acids [49]. The amino acids that are particularly vulnerable to hydrolysis by SCW are aspartic acid and glutamic acid, affected by the weak acidic conditions as their carboxyl group becomes a proton donor for the hydrolysis of the peptide bond next to it [52]. Ahmed, Mulla, Al-Ruwaih and Arfat [57] reported that using sequential pressure pretreatment of 300 MPa for 15 min increased the degree of hydrolysis for proteins when being hydrolyzed with alcalase [57], an enzyme that cleaves the carboxyl side of the amino acids E, L, Y, Q and E [58]. This indicates that the application of high pressure leads to a certain degree of protein unfolding, potentially increasing access of the enzyme to substrate cleavage sites [57]. A similar study using soy protein performed by Meinlschmidt et al. [59] showed similar enhanced digestibility following exposure to a pressure of 100 MPa in the presence of the enzyme flavourzyme for 15 min. Under these conditions, when the pressure exceeds 100 MPa the enzyme itself starts to become denatured by the pressure, and its activity is lost [59].

SCW appears to have some cleavage specificity for bonds adjacent to aspartyl residues, with some 44% of the peptides produced from subcritical water-mediated hydrolysis of BSA containing an N-terminal aspartic [52]. Moreover, peptide production from the microalgae *A. platensis* was optimal at 160 ◦C, while temperatures of over 220 ◦C produced an intense degradation of these proteins and the release of free amino acids rather than peptides, with no distinguishable bands when analyzing the hydrolysates by denaturing protein electrophoresis [49,55]. SCW has also been explored for the production of amino acids at temperatures of 240 ◦C [60,61], while temperatures reaching 260 ◦C will result in the degradation of amino acids to organic acids and ammonia [61]. These data illustrate the need for careful control of temperature during SCW processing to ensure the appropriate release of protein and peptides rather than terminal degradation.

After the proteins have been processed and hydrolyzed to generate bioactive peptides, one or several purification processes are frequently applied to isolate these molecules further. Overall, most authors used one or several steps of molecular weight cut-off filtration (MWCO) to fractionate the compounds of the hydrolysates based on their molecular weight [62]. Thereby, Megías et al. [63] used 5 kDa membranes to remove, concentrate and purify peptides in the hydrolysate by removing larger unhydrolyzed protein fractions and the protease enzymes themselves, as these compounds will be collected and discarded in the retentate. Further purification techniques can also be applied including chromatographic techniques, mainly reversed-phase high-performance liquid chromatography (RP-HPLC) and ultra-performance liquid chromatography (UPLC) depending on the level of purity desired in the final product. Previous studies generating bioactive peptides from the macroalga *Ulva* spp. applied MWCO followed by preparative RP-HPLC at wavelengths of 214 nm, to detect peptide bonds, and 280 nm, indicative of the presence of aromatic amino

acids [64]. These and other purification strategies to isolate bioactive peptides from algae have been recently reviewed in detail by Lafarga et al. [62].

#### **3. Biological Activities and Modes of Action of Algal Peptides**

The generation of bioactive peptides is gaining momentum due to the wide range of biological properties attributed to these compounds that have been extensively reviewed [34,62]. Thus, this section will briefly mention a few examples of the anti-hypertensive, antioxidant, and anti-cancer activities from algae described in the recent scientific literature, also focusing on relating these described activities to their proposed mechanism of action and tools used for these analyses.
