3.1. Protein Content, Protein Recovery, and DH
As per the results obtained in our previous study that showed the very high solubility of proteins at alkaline pH after enzymatic/alkaline extraction of proteins from P. palmata (> 90 % protein recovery in liquid fractions vs. < 5 % protein recovery in solid fractions) [
11], it was expected that the application of alkaline (or acidic) pH in this study would also contribute to the release and solubility of protein in the liquid fraction. The logic behind this hypothesis was the fact that any deviation from isoelectric point of proteins, whether toward alkaline or acidic conditions, could solubilize proteins in liquid fractions [
24]. At pH values away from isoelectric point, the surface charges of proteins tend to be either negative at alkaline conditions or positive at acidic conditions, which results in the weakened hydrophobic interaction and stronger electrostatic repulsion between proteins contributing to interaction between protein and water and therefore increased protein solubility [
25]. However, the results of this study revealed that neither alkaline nor acidic pH could contribute to the solubilization of proteins in supernatant during aqueous extraction, as evidenced by substantially higher protein concentration and protein recovery in solid fractions after the extraction at varying pH values. One potential reason for this observation might be the presence of polysaccharides in seaweed. These polysaccharides are crucial components of seaweed cell walls and have strong interactions with bioactive compounds such as proteins. [
26]. It seems that although alkaline extraction coupled with enzymatic pretreatment in previous studies yielded liquid fractions with high protein contents compared with solid fractions (xx vs yy %) [
11,
19], alkaline or acidic pH during aqueous extraction cannot contribute to the solubilization of proteins from red seaweed. This could be in part due to the heterogenous nature of seaweed proteins that necessitates the application of more than one extraction method to disintegrate seaweed cell walls and meet the solubility requirements of proteins [
27].
Another explanation for low protein solubility after aqueous extraction at different pH values is the possibility of interactions between proteins and polysaccharides after extraction. Once proteins and peptides are released into the solution, they can reassociate with polysaccharides present in the extract, which can lead to the formation of insoluble complexes. In the solution, hydrogen bonds and electrostatic interactions between proteins and polysaccharides can result in the formation of stable complexes [
28]. When proteins and polysaccharides interact in a solution, they can form complexes through a process known as coacervation. Coacervation involves the associative phase separation between proteins and polysaccharides, resulting in the formation of two distinct phases: a biopolymer-rich phase and a biopolymer-poor phase. The biopolymer-rich phase can exhibit different states, including liquid coacervates and solid precipitates [
29]. Depending on the concentration and types of proteins and polysaccharides, as well as the pH and ionic strength of the solution, these complexes can precipitate out of the solution, contributing to the protein content in the solid fraction [
30]. At certain pH levels, the charges might promote binding and complex formation. For example, at pH 3 or pH 9, depending on their isoelectric point, proteins might have a net positive or negative charge that can interact strongly with charged polysaccharides.
The role of pH in the solubility of electrostatic protein-polysaccharide complexes is defined by considering four critical pH values, i.e. pH
c, pH
φ1, pH
opt, and pH
φ2. To embark on, soluble complexes begin forming, representing the initial interaction. Near or below the protein’s isoelectric point, pH
c initiates soluble complex formation. Above pH
c, proteins and polysaccharides remain co-soluble, whereas below pH
c, charge neutralization causes aggregation, leading to increased turbidity at pH
φ1. Maximum turbidity occurs at pH
opt, the electrically neutral point of proteins and polysaccharides and further pH reduction to pH
φ2 causes complex disassociation into individual biopolymers due to protonation of reactive sites on polysaccharide chains [
31]. However, these effects of pH in the current study were outweighed by other factors since no significant differences were observed in the solubility or precipitation of proteins at varying pH conditions (p > 0.05). It was highlighted that besides pH, other factors such as polysaccharide type, ionic strength, temperature etc. could also determine the solubility of proteins within the complexes [
32].
Furthermore, the presence of salts can shield electrostatic interactions, potentially reducing the formation of complexes. However, at low ionic strengths, these interactions might be stronger, promoting complexation [
33]. Therefore, the presence of naturally occurring salts in P. palmata can influence the electrostatic interactions between proteins and polysaccharides, impacting the formation and stability of insoluble complexes. The seaweed naturally contains a significant amount of minerals, including sodium, potassium, magnesium, and calcium [
34], which can contribute to the overall ionic strength of the seaweed matrix. In this regard, one may consider the protein-polysaccharide complex in the current study as polyelectrolyte multilayers that are formed by the layer-by-layer disposition of oppositely charged polyelectrolytes (e.g. proteins and polysaccharides) and are affected by inherent salt content of the seaweed. However, this proposition should be taken with care because: (i) proteins generally do not possess the flexibility and geometry required to be included in polyelectrolyte-polyelectrolyte systems, (ii) in protein-polyelectrolyte systems, pH modulation primarily impacts protein charge, while ionic strength plays a more complex role, increasing polyelectrolyte’s configurational entropy and affecting the entropy of small ion release, and (iii) when replacing a polyelectrolyte with a protein in the system, the role of configurational entropy is significantly diminished [
30]. Two other factors that could be envisaged as drivers of observed protein dissolubility in the current study regarding the formation of protein-polysaccharide complexes are temperature and polysaccharide type and concentration. Higher temperatures can increase the solubility of proteins but can also increase the kinetic energy, potentially promoting interactions between proteins and polysaccharides and formation of insoluble complexes. Nevertheless, it was reported that temperature rise below the protein's denaturation point decreases the interaction strength between proteins and polysaccharides during complex coacervation [
30]. The temperature applied in the current study for the aqueous extraction was 50 °C, which is below the denaturation threshold of most proteins. Therefore, one ought to think twice before accrediting the role of extraction temperature in the present research on the precipitation of large proportion of proteins in the solid fractions.
A high concentration of polysaccharides can enhance the likelihood of interactions and complex formation with proteins. These polysaccharides can cause structural rearrangements in proteins, leading to the formation of insoluble aggregates [
35]. Carbohydrates make up to 74% of P. palmata’s dry weight, with xylans being the primary component of its cell walls. These xylans consist mainly of β-(1→4)- and β-(1→3)-linked D-xylose units and are largely insoluble. Additionally, minor amounts of cellulose (around 3% dry weight), an insoluble glucan, are present as structural carbohydrates. P. palmata also contains water-soluble, low molecular weight carbohydrates, primarily floridoside (α-D-galactopyranosyl-(1–2)-glycerol), along with smaller amounts of floridean starch. The floridoside content varies seasonally, ranging from less than 5% (dry weight) in winter to up to 25% in summer [
22]. This aligns with the results of the present study, as the biomass used for peptide extraction was harvested in winter when the seaweed contains more insoluble polysaccharides.
We anticipated that the subsequent enzymatic extraction, utilizing the solid fractions obtained from the initial aqueous extraction as substrates, would solubilize proteins. Consequently, we expected the resulting liquid fractions to have significantly higher protein content than LAs. However, our expectations were not met, and once again, most proteins precipitated in the solid fractions. The significant protein precipitation observed in solid fractions after both aqueous and enzymatic extraction stages suggests that the proteins are strongly interacting with other components (possibly polysaccharides) or aggregated in a manner that resists solubilization under the extraction conditions used. One possible explanation for this finding is that the enzymatic hydrolysis with Flavourzyme
® has broken down proteins into peptides of various lengths. These peptides can have exposed amino and carboxyl groups, which can interact with hydroxyl and carbonyl groups present in seaweed polysaccharides [
36]. Protein-polysaccharide conjugates have shown to have varying solubility in terms of the protein biochemistry, type of polysaccharides, and intermolecular disulfide bonds formed upon conjugation and therefore, could be either soluble or insoluble in water [
31]. Future studies are directed toward scrutinizing whether such a conjugation could happen in the presence of seaweed polysaccharides and hydrolyzed proteins. Under certain conditions, especially in complex biological matrices like seaweed extracts, (non-covalent) interactions between hydrolyzed peptides and seaweed carbohydrates can lead to the formation of complexes or conjugates. This presents an important avenue for future research.
The modest DH observed in the solid fractions may reflect that the proteins present have been subject to limited hydrolysis. This could imply either a natural resistance of the proteins in the solid fractions to enzymatic degradation or that the hydrolysis conditions were not conducive to a more complete breakdown of these proteins. It could also imply that these proteins are forming aggregates or complexes that protect them from enzymatic action. Studies have shown that polysaccharides, such as xylan, interact with proteins and proteolytic enzymes, leading to reduced protein hydrolysis in seaweed [
37]. It is noteworthy that the combinational or sequential use of polysaccharidase alongside the protease in our procedure to extract peptides from the seaweed merits further investigation. In addition, the high DH in liquid fractions indicates that the proteins present in these fractions were extensively hydrolyzed. The DH values observed in the present study for the liquid fractions both after the initial aqueous and subsequent enzymatic extractions are higher than those reported in our previously published paper [
11] following enzymatic/alkaline extraction. However, these DH values are comparable to those observed in our ongoing work (currently under preparation for publication) after enzymatic treatment without the subsequent alkaline extraction stage. This discrepancy could be because alkaline conditions can denature proteins, potentially exposing more peptide bonds initially while also causing changes in protein conformation [
38] that render some bonds less accessible to enzymes or the OPA (o-phthaldialdehyde) reagent used for DH measurement. The OPA method is a spectrophotometric assay that relies on the chemical reaction between OPA and primary amines in the presence of a thiol (such as dithiothreitol, DTT) to form a highly fluorescent isoindole derivative [
39]. In details, the OPA molecule reacts with the primary amine group of an amino acid or peptide (from the N-terminal ends of peptides and free amino acids in samples) to form a Schiff base (imine), which involves the nucleophilic attack of the amine nitrogen on one of the aldehyde carbons of OPA. The thiol reacts with the other aldehyde group of OPA, forming a thioacetal intermediate, which facilitates the cyclization process. Finally, the thioacetal intermediate undergoes intramolecular cyclization, resulting in the formation of an isoindole derivative, which is highly fluorescent [
40]. The fluorescence intensity is directly proportional to the concentration of free primary amines in the sample, which corresponds to the extent of protein hydrolysis. Alkaline extraction can cause protein denaturation, which involves the unfolding of protein structures. This can expose hydrophobic regions, leading to the formation of (still soluble) aggregates [
41]. These aggregates might bury free amino groups within their structure, making them less reactive. Alkaline extraction can also induce chemical modifications such as deamidation of aspartic acid and glutamic acid [
42], which can alter the availability and reactivity of amino groups, leading to lower DH values measured by OPA method. Furthermore, enzymes might be susceptible to strongly alkaline conditions [
43] and consequently, any residual enzymatic activity might be lost during the alkaline extraction step, halting further hydrolysis that could have occurred if conditions were maintained for enzyme activity.
3.2. Amino Acid Composition
The general trend observed for the total amino acid composition of liquid and solid fractions corresponded well with the findings of this study regarding the protein content of the samples based on the dry matters. Therefore, readers are referred to the discussion provided in the previous section for clarifications and interpretations on the significant differences between solid fractions from two extraction stages and between solid and liquid fractions in general in terms of amino acid profiles. However, individual differences observed within each group of the samples are interpreted here.
The first notable observation was attributed to the lower content of arginine in SE9 compared to SE3 and SE6. One plausible explanation involves the distinct behavior of arginine counterions under alkaline conditions, leading to the dissociation of arginine from tightly bound micellar aggregates. Consequently, more arginine may diffuse into the soluble fraction, resulting in a lower observed amount in the solid fraction. This trend is reflected by the slightly higher (though not statistically significant) content of arginine in LE9 compared to LE3 and LE6 (
Table 1). At alkaline pH, arginine tends to adopt a zwitterionic state rather than maintaining a net positive charge. This change in charge state can trigger the dissociation of arginine molecules from micellar structures, particularly as the primary amine groups deprotonate. This dissociation process becomes more pronounced at higher pH levels, approaching complete dissociation [
44]. One should also consider the effect of pH on protease activity, which could potentially promote secondary reactions that degrade arginine. However, this hypothesis seems less likely in this case because a similar trend was observed in both liquid and solid fractions obtained after the initial aqueous extraction, where no enzymatic treatment was applied. Moreover, in addition to arginine, our results denoted that histidine, glutamic acid, and aspartic acid were significantly higher in SA3 than in SA9 (
p < 0.05). Since arginine and histidine are both basic amino acids, the above-mentioned explanation regarding the dissociation of the amino acids from micellar aggregates and diffusion into soluble fraction may also be the case for histidine. However, amino acids with acidic side chains like aspartic acid may undergo chemical modifications such as isomerization and racemization under different pH conditions [
45], which can affect the detectable content of these amino acids during analysis.
In addition, histidine was not found in the liquid fractions but was present in the solid samples. This observation may be attributed to the distinctive structural feature of histidine, which includes a basic imidazole group on its side chain [
46]. This characteristic could facilitate robust complexation with macromolecules found in the seaweed extract matrix, such as polyphenols and polysaccharides. Consequently, this interaction may result in histidine predominantly residing in the solid fractions, rendering it undetectable in the liquid fractions. Furthermore, the imidazole group of histidine facilitates molecular interactions (cation-π, π-π stacking, hydrogen-π, coordinate bond, and hydrogen bond interactions) with other amino acids [
47], which might lead to the formation of insoluble complexes that aggregate in the solid fractions. In addition, cystine was detected exclusively in the SE samples, with its concentration being significantly higher in SE3 compared to SE6 and SE9. The emergence of cystine in the solid fractions following the enzymatic process, in contrast to its non-detection in the solids post-aqueous extraction, indicates that the enzymatic hydrolysis was essential in liberating cystine from its formerly attached state. This implies that the pH conditions applied during the aqueous phase were not adequately potent to dissociate cystine from its native structure or the complexes in which it may have been trapped.
3.4. Antioxidant Properties
In both extraction phases, the solid fractions exhibited greater free radical scavenging capabilities than their liquid counterparts, a finding that diverges from our TPC results, where liquid fractions typically had higher levels of phenolic compounds. This observation stands in contrast to earlier studies that emphasized the significant contribution of phenolic compounds to the radical scavenging efficacy of seaweed extracts [
53]. However, the current study suggests that proteins, particularly peptides, free amino acids, and/or their complexes with other macromolecules like polyphenols or carbohydrates, may play a leading role in neutralizing free radicals in seaweed products. The substantial protein concentration in the solid fractions, noted after both the initial aqueous extraction and the subsequent enzymatic treatment, lends support to this hypothesis, marking a stark difference from the protein levels in the liquid fractions. Our results also indicated that the enzymatic hydrolysis of solid residues from the initial aqueous extraction yielded extracts with slightly better radical scavenging properties. This corroborates prior research highlighting the significance of protease treatment in producing peptides that are smaller in size and have improved antioxidant effects [
9]. However, due to the low DH observed in the solid fractions, this conclusion should be approached with prudence. In addition, considering the negligible variance among the solid fractions obtained from various extraction phases at each tested pH level, the practicality of applying additional enzymatic processing should be contemplated, particularly when aiming to obtain fractions with strong free radical neutralizing abilities.
It is important to consider the connection between the DPPH scavenging activity observed in the samples of this study and their amino acid profiles. The lack of histidine in the liquid fractions, contrasted with its presence in the solid fractions, suggests that histidine may play a significant role in neutralizing free radicals. The efficiency of histidine in scavenging DPPH radicals is likely attributed to its imidazole ring structure [
54]. The present study highlights the importance of histidine in the free radical scavenging capabilities of the examined fractions. The notable variance in histidine levels across the samples correlates with the observed differences in DPPH scavenging activity. Specifically, SA9 exhibited a markedly lower DPPH scavenging activity than its counterparts at other two pH values tested, which coincides with its reduced histidine content, especially when compared to SA3 where the disparity was significant (
p < 0.05). A comparable pattern was noted in SE samples, where a decrease in histidine was associated with diminished free radical scavenging activity. This observation extends to glutamic acid and aspartic acid, suggesting their potential involvement in the DPPH radical scavenging process of the solid fractions. Research indicates that sequences of electron-donating units like glutamic acid and aspartic acid within peptide chains enhance the neutralization of free radicals [
55]. Tyrosine may also contribute to the notably greater free radical scavenging capabilities of solid residues relative to liquid fractions, as it is found in higher concentrations within the solids. The presence of a hydroxyl group in tyrosine has been identified as a key factor in its effectiveness as an antioxidant amino acid [
55].
Regarding the metal ion chelating properties of the samples, LA3 was ineffective in chelating Fe²⁺, while SA3 showed moderate activity. This could be due to the protonation of functional groups at low pH, reducing the availability of chelating agents in the liquid fraction. In contrast, in the fraction obtained at alkaline pH, the chelating activity of LA was significantly enhanced, suggesting that higher pH levels favor the solubilization of chelating agents into the liquid fraction. This phenomenon might be partially attributed to the substantial phenolic content in LA9. However, there must be other contributing factors, because if phenolic content were the sole determinant, then LA6, which had a higher phenolic content than LA9, would be expected to exhibit greater chelating activity, yet it did not. This variation may stem from the distinct structural configurations of the phenolic compounds and the differing dynamics of complex formation and stability [
56]. An alternative explanation for LA9’s enhanced Fe²⁺ chelating capability might be that an alkaline environment promotes the release of polysaccharides [
57]. It has been noted that polysaccharides found in red seaweed are effective at chelating metal ions [
58]. Consequently, the increased release of these polysaccharides in an alkaline setting could contribute to the greater metal chelating efficiency of LA9, which could occur either through the direct action of these chelating polysaccharides or by fostering the creation of more effective chelating complexes with proteins and/or polyphenols.
Enzymatic hydrolysis significantly enhanced the metal chelating capacity of the solid fractions across all pH levels, with particularly notable results at pH 3 and 6. This improvement is likely due to the breakdown of proteins by the protease applied, releasing peptides and amino acids with strong metal chelating properties. This is particularly noteworthy considering that total phenolic contents of SE3 and SE6 did not differ significantly from their LE counterparts (p > 0.05), which suggests that the peptides and amino acids play a more dominant role in chelating metals in the solid fraction. The metal chelating capacity has been linked to the size of peptides, indicating that multiple negatively charged groups may improve the binding with metal ions [
55]. Thus, it is reasonable to deduce that the high Fe²⁺ chelating attributes of the SE samples in this study are due to the shorter peptides generated by the protease’s action on the whole or partially broken-down proteins in the solid fractions obtained from the initial aqueous extraction. The elevated chelating activity observed in SE samples may be due, in part, to the notably greater levels of methionine and histidine they contain relative to other samples (
Table 1). Methionine [
59] and Histidine [
60] is recognized for its effective metal ion chelation. As such, its inclusion in peptides can greatly enhance the total metal chelating capacity of the samples. Additionally, the presence of cystine, cysteine’s dimeric variant, exclusively in SE samples, might play a role in their pronounced chelating capacity. Nevertheless, caution is advised when drawing conclusions from this, since cystine’s chelating characteristics may vary from those of cysteine, which is recognized for its strong metal ion chelating properties [
59]. Furthermore, the chelating activity exhibited a steady increase as the pH level was reduced from 9 to 3 in SE samples, although this increase did not present a significant difference (
p > 0.05). Like the outcomes of free radical scavenging, the modest reduction in histidine levels at elevated pH could account for the slightly diminished chelating activity noted in SE samples with a pH of 9.