Degree of Hydrolysis
The monitoring of the degree of hydrolysis over time showed an initially high hydrolysis rate, reaching DH values of 5% and 10% within three minutes (0.45 and 2.6 minutes, respectively; Fig. 1).This was followed by a decreased hydrolysis rate, reaching 15% DH at 35.7 minutes and 20% DH at 223 minutes. This decelerated hydrolysis rate over time to reach a stationary phase has been related to the substrate depletion in the enzyme mixture and the potentially adverse effect of reaction products, which might saturate the active site of the enzyme and inhibit its catalytic mechanisms [26-27]. Previous studies have reported the production of protein hydrolysates from fishmeal effluents (mainly wastewater from thawing and cooking processes) using Alcalase as enzyme source, [13, 28-29] attaining 10% to 80% DH with reaction times of 0.5 to 8 hours, with hydrolysis curves and trends similar to those observed in our study.
Real-time monitoring of the degree of hydrolysis allows selecting the most suitable time to stop the enzyme reaction. This is key because the biological activity of hydrolysates depends not only on critical process parameters (CPP) such as protein source, E/S ratio, and the physical and chemical conditions of hydrolysis; also, the bioactive, techno-functional, and organoleptic properties (critical quality attributes)of hydrolysates are influenced by the degree of hydrolysis attained ; however, the effect of DH on the biochemical or bioactive properties of protein hydrolysates from fishmeal effluents has not been examined to date.
Proximate Analyses of Sardine Stickwater and its Protein Hydrolysates
Raw stickwater showed a high moisture content (91.2%). The main components of dissolved matter were a protein fraction, ashes, and lipids (5.9, 1.8, and 1.1% wet weight, respectively) (Table 1). The average crude protein content in stickwater from fishmeal factories is relatively constant, at around 6% of wet matter , similar to the content recorded in our study. Lyophilization of raw stick water attained an almost 15-fold increase in protein content, also in agreement with the results reported by Bechtel . However, centrifugation reduced the lipid content of the effluent used in our study (from 13.9% to 10.8% of total weight), as this process led to the formation of a creamy layer of fat in the uppermost phase, which can be removed easily . Removing this lipid fraction from protein hydrolysates makes the product more stable, as this step eliminates potentially oxidizable biomolecules (e.g., polyunsaturated fatty acids) present in fish used for fishmeal production.
Enzymatic hydrolysis reduced the protein and lipid contents of lyophilized protein hydrolysates (which ranged from 56.6% to 63.3% and from 4.8% to 5.4% of total weight, respectively), with no significant differences (P > 0.05) between hydrolysates showing different DH. Lipid content in the hydrolysates was similar to the levels < 5% reported by several studies . Again, this reduction was due to centrifugating the hydrolysates once the degree of hydrolysis desired was attained. However, the protein content was lower than those reported for other fish protein hydrolysates  and fishmeal effluents , which showed values approximately of about 80% wet weight. The reduced protein content in the hydrolysates is related to a significant (P < 0.001) increase in ash content in parallel with the increase in the degree of hydrolysis. Thus, ash content was 23.55% in the 5% DH hydrolysate and increased to 26.4% in the 20% DH hydrolysate. The high ash content in protein hydrolysates is attributed to the addition of sodium hydroxide to the hydrolysis reaction mixture in order to adjust and keep the medium pH at 9. This has been reported for protein hydrolysates that use the pH-stat technique to determine DH . A high ash content in protein hydrolysates could limit their application. Other authors have reported ash contents above 20% in protein hydrolysates from effluents  and byproducts of fish processing plants [31, 33]. Nevertheless, it is recommended that future studies use methods other than the pH-stat technique to determine the degree of hydrolysis kinetically.
Molecular Weight Distribution
Centrifuged sardine stickwater (CSW) contains a high proportion (94.7% of total peptides) of peptides with molecular weight (MW) lower than 17 kDa, 45.1% of which have MW < 1.35 kDa (Table 2). Fishmeal industry effluents such as stickwater and cooking water from seafood products typically contain peptides with low molecular weight ranging from 15 to 1 kDa [34-35].
The high content of low-MW peptides in these fishmeal industry effluents might result from the physical (particularly thermal) conditions to which the original protein sources are subjected during the production process of fishmeal or precooked foods for human consumption. The high temperatures used to cook fish, crustaceans, or mollusks cause protein denaturation and the ensuing production and release of low-MW peptides and free amino acids. The contents of low‑MW peptides (<1.35 kDa) in stickwater protein hydrolysates of 5, 10, and 15% DH ranged from 47.3% to 48.2% and were not significantly different (P> 0.05) from the content recorded in CSW. On the other hand, when stickwater was hydrolyzed up to 20% DH, the content of low‑MW peptides (<1.35 kDa) increased significantly (P < 0.05) up to 62.4% of total peptides (Table 2). On the contrary, the percentage of peptides with molecular weight between 1.35-17 kDa decreased significantly from 49.6% to 22.4% of total peptides when CSW was hydrolyzed to 20% DH.
The occurrence of low-MW peptides in stickwater protein hydrolysates is related to an increase in their bioactive properties . The degradation of the protein structure by enzymes releases low-MW peptides and oligopeptides; this increases the exposure of reactive amino acid residues compared to their spatial configuration in the native protein, and enhances their potential to interact with other molecules.
Total Amino Acid Content
Table 3 shows the amino acid profiles recorded in stickwater and its protein hydrolysates. It is well-known that the composition and sequence of amino acids in proteins or peptides determine their nutritional value and biological activity [2, 31]. Fishmeal industry effluents typically have a high nutritional value due to their high content of essential amino acids (EAA) ; this is consistent with our results, where EAA accounted for 51.5% of the total AA contained in CSW. The high EAA content of fishmeal effluents has raised interest to use them as substitutes for fishmeal in aquafeed production  and as food supplements for promoting growth  or improving the intestinal health and immune system of aquatic organisms.
The predominant essential amino acid in CSW was His, while Glu and Gly were the predominant non-essential amino acids, with levels of 15, 14.5, and 13.4 g/100 g of protein, respectively. Amino acids such as Glu, Gly, and Pro (the latter was not determined in our study) are commonly predominant in AA profiles of fishmeal effluents such as stickwater [3-4] and cooking water . This is interesting from the nutritional perspective, since AA such as Glu, Gly, His, Ala, and Arg (and other active flavor components) have been related to seafood flavours , with the potential to be used as food attractants.
Protein hydrolysis modified the concentration of some of the amino acids present in CSW. Such changes in amino acid concentration in protein hydrolysates could be due to increased solubility after the enzymatic hydrolysis process. In contrast, in the absence of the hydrolytic process and after a second centrifugation of CSW, some of these AA are removed along with undigested protein remains. For example, Gly concentration increased significantly (P < 0.05) with the degree of hydrolysis until attaining maximum values of 19.6 and 18.6 g/100 g of protein, respectively. On the contrary, His content decreased significantly (P < 0.05) to 3.7 and 2.4 g/100 g of protein, in hydrolysates with 10 and 20% DH, respectively. The reduction in His and the increase in Gly concomitant with the increase in DH led to a significant decrease in the EAA content of hydrolysates (ranging from 34 to 39.4 g/100 g of protein) compared to CSW (51 g/100 g protein).
Contents of other EAA such as Arg, Lys (basic AA), Met (sulfur-containing AA), Phe, Tyr (aromatic AA), Ile, Leu, Val (aliphatic AA), and Thr (hydrophilic AA) did not differ significantly (P > 0.05) between CSW and its protein hydrolysates with 15 and 20% DH (Table 3). This is interesting from a nutraceutical point of view, since ACE-inhibiting peptides are usually rich in hydrophobic amino acids that act on the active site of ACE . In addition, the presence of some basic (His), sulfur-containing (Cys, Met and Tau), and aromatic (Phe, Tyr and Trp) amino acids in peptide structures is closely related to the antioxidant capacity of peptides present in protein hydrolysates . For example, the antioxidant activity of His-containing peptides has been related to hydrogen donation, the chelating capacity of metal ions, the quenching of peroxide radicals, and the scavenging of hydroxyl radicals due to the presence of the imidazole group [43-44]. The phenolic side chains of aromatic AA can act as electron donors contributing to the scavenging of free radicals . The antioxidant effect of Tyr is enhanced by the presence of the hydroxyl group in its structure, which provides a mechanism for transferring hydrogen atoms [42, 45]. In contrast, the -SH group of sulfur-containing amino acids has a crucial antioxidant action due to its direct interaction with radicals .
Therefore, to better understand the action mechanism of protein hydrolysates showing some degree of bioactivity, future studies should make a bio-directed isolation of peptides followed by analysis of their amino acid sequence, since the sequence or location of particular amino acid residues promotes or magnifies their antioxidant activity.
In-vitro Antioxidant Activity of Stickwater Protein Hydrolysates
Fig. 2 shows the effect of varying the degree of hydrolysis of raw sardine stickwater on antioxidant activity as determined through the ability to eliminate DPPH and ABTS radicals measured in terms of EC50. The EC50 of CSW for DPPH could not be determined because the protein fraction of the sample precipitated upon exposure to ethanol.
An effect of degree of hydrolysis on EC50 in the DPPH assay was founded. The hydrolysates with 15 and 20% DH showed the lowest antioxidant activity (DPPH) in terms of EC50 (46.3 and 43.4 mg/mL, respectively). In contrast, a low degree of hydrolysis (5 and 10% DH) had a positive effect on antioxidant activity as measured by DPPH (34.7 and 37 mg/mL, respectively).The analysis of antioxidant activity with the ABTS assay showed that hydrolysis enhanced the antioxidant activity already present in CSW (EC50 = 10.5 mg/mL), reaching the highest activity in the hydrolysate with 15% DH (EC50 = 2.8 mg/mL).
Fig. 3 shows the ferric reducing antioxidant power (FRAP) of stickwater and its protein hydrolysates at different concentrations. Enzymatic hydrolysis of stickwater significantly increased FRAP activity. At the highest concentration evaluated (50 mg/mL), the protein hydrolysate with 15% DH showed the highest activity (TEAC = 1.16 ± 0.03 mM TE/mg) (P < 0.001) compared to centrifuged stickwater (TEAC = 0.23 ± 0.0 mM TE/mg) and the other protein hydrolysates (TEAC = 0.78–0.93 mM TE/mg). Various studies report that the antioxidant activity of peptides obtained by enzymatic hydrolysis of marine proteins is affected by several factors, the most important of which are the hydrolysis conditions (enzyme, protein source, E/S ratio, and DH reached), molecular weight, and amino acid sequence. In this regard, the production of peptides with MW < 3 kDa showing one or more hydrophobic or aromatic amino acids in their sequence, has been described as desirable to achieve an adequate antioxidant cellular activity [46-47]. Therefore, peptides whose amino acid profile meets one or more of these characteristics will exhibit different behaviors in the peptide-radical interaction in the in‑vitro methods used to evaluate the antioxidant properties of protein hydrolysates.
Overall, we observed that the hydrolysis of CSW with Alcalase (Fig. 2) increased its antioxidant activity; the hydrolysate with15% DH stood out in all the in-vitro antioxidant evaluations carried out. Other studies have reported an increased antioxidant activity of fishmeal effluents subjected to enzymatic hydrolysis. For example, the highest antioxidant activity (the lowest EC50 value, 1.31 mg/mL, measured by DPPH) of stick water from kilka (Clupeonella sp.) was attained in the hydrolysate with highest DH (25%) .
Another driver of the antioxidant activity of protein hydrolysates from fishmeal effluents is the selectivity of peptide bond cleavage by the enzyme used. For example, in hydrolysates of tuna cooking water produced using protease XXIII from Aspergillus oryzae, the DPPH scavenging activity of the effluent could be increased from 18% to approximately 85% when DH reached 25.68%. However, hydrolysates with higher DH showed a significant reduction of antioxidant activity . On the other hand, when the subtilisin enzyme from Bacillus subtilis was used to hydrolyze tuna (Thunnus tonggol) cooking water, a 20% DH achieved the highest increase in DPPH radical-scavenging capacity .
In our study, CSW exhibited ABTS-radical scavenging activity before the hydrolysis process, thus revealing the presence of peptide fractions (likely generated during thermal processes) with antioxidant capacity. Such activity has been observed in unhydrolyzed shrimp (Penaeus spp.) cooking water, also showing that 1 kDa peptide fractions with high Gly content had the greatest ability to eliminate ABTS radicals . Hydrolysis of CSW with Alcalase further increased this activity (Fig. 2b), which may be related to the release of low-MW peptides (<1.35 kDa) and the likely exposure of the functional amino acid groups such as Gly, Glu, and His, which are associated to in-vitro antioxidant mechanisms of electron or proton transfer .
The higher FRAP activity of all the protein hydrolysates tested (in concentrations ranging from 2.5 to 50 mg/mL), compared to CSW, indicates that the hydrolysis process increased the availability of functional amino-acid groups capable of reducing iron in the TPTZ complex used in this technique. The hydrolysate with 15% DH showed the highest FRAP, which is related to its higher content of Ile (2.2%) and Met (2.9%), compared to the other hydrolysates. According to Nwachukwu and Aluko , sulfur-containing amino acids such as Cys and Met are highly effective in reducing Fe3+ to Fe2+; in addition, hydrophobic amino acids such as Ile, Gly, Pro, and Met contributes to the ferric reducing potential due to the high electron density they generate.
ACE Inhibitory Activity of Stickwater Protein Hydrolysates
Various peptides of protein hydrolysates derived from solid or liquid residues of marine organisms exhibit antihypertensive properties in terms of ACE inhibitory activity . This effect has been mainly attributed to the ACE-inhibitory activity of peptides. Angiotensin converting enzyme plays a key role in regulating blood pressure and hypertension, as it catalyzes the hydrolysis of the decapeptide angiotensin-I into angiotensin-II, a powerful vasoconstrictor that increases blood pressure .
The parameter most commonly used to assess the antihypertensive activity of a compound is IC50, defined as the concentration necessary to inhibit 50% of ACE activity. Table 4 shows the average IC50 of centrifuged sardine stickwater and its hydrolysates. It is evident that enzymatic hydrolysis had a positive effect on antihypertensive activity, as stickwater hydrolysates with 5 and 10% DH showed the lowest IC50 values (0.002 to 0.007 μg/mL) (P < 0.05), corresponding to a higher antihypertensive activity. This effect could be related to the fact that bioactive peptides remain inactive in the amino acid sequence of the intact protein but, upon hydrolysis, these peptides are released and exhibit biological activity [51-52]. Hsu et al.  reported that untreated tuna cooking water was unable to inhibit ACE activity. However, once this effluent was hydrolysed with the endopeptidase subtilisin from Bacillus subtilis, an ACE inhibitory effect was recorded, reaching IC50 values of 46.89 mg/mL.
Stickwater hydrolysates with 15 and 20% DH exhibited significantly higher (P < 0.05) IC50 values than samples with 5 and 10% DH (Table 4). This tendency is opposite to that reported in the literature regarding the effect of hydrolysis on the antihypertensive activity of solid by-products of marine origin. For example, Balti et al.  observed an elevated ACE inhibitory activity in high DH hydrolysates from cuttlefish (Sepia officinalis) muscle, reaching an IC50 of 1120 μg/mL in samples with 18.7% DH, thus indicating that low-MW peptides have a greater capacity to inhibit ACE.
The results of ACE inhibition by sardine stickwater hydrolysates (Table 4) showed that their antihypertensive potential is greater versus other hydrolysates and protein fractions from fishmeal industry effluents. For example, Hsu et al. [8-9] reported that hydrolysates of tuna cooking water produced with the commercial protease subtilisin had an IC50 of 5850 μg/mL. On the other hand, Amado et al.  used ultrafiltration followed by hydrolysis with Alcalase to obtain protein fractions from cuttlefish (Illex argentinus) processing effluents, obtaining IC50 values from 81.6 to 1125 μg/mL. However, as various methods to determine ACE inhibition are available (using different substrates and spectrophotometric or chromatographic detection methods), IC50 values cannot be compared directly. Even for a given analytical test, there are several different methods (linear regression, mathematical modeling, direct interpolation, etc.) to estimate the ACE-inhibition capacity of bioactive compounds .