Cosmeceutical Potential Of Extracts Derived From Fishery Industry Residues: Sardine Wastes And Codfish Frames Part 1

Abstract: The fishery industry generates large amounts of waste (20–75% (w/w) of the total caught fish weight). The recovery of bioactive compounds from residues and their incorporation in cosmetics represents a promising market opportunity and may contribute to a sustainable valorization of the sector. In this work, protein-rich extracts obtained by high-pressure technologies (supercritical CO2 and subcritical water) from sardine (Sardina pilchardus) waste and codfish (Gadus morhua) frames were characterized regarding their cosmeceutical potential. Antioxidant, anti-inflammatory and antibacterial activities were evaluated through chemical (ORAC assay), enzymatic (inhibition of elastase and tyrosinase), antimicrobial susceptibility (Klebsiella pneumoniae, Staphylococcus aureus, and Cutibacterium acnes)  and cell-based (in keratinocytes-HaCaT) assays. Sardine extracts presented the highest antibacterial activity, and the section obtained using higher extraction temperatures (250 ◦C) and without the defatting step demonstrated the lowest minimum inhibitory concentration (MIC) values (1.17; 4.6; 0.59 mg/mL for K. pneumoniae, S. aureus, and C. acnes, respectively). Codfish samples extracted at lower temperatures (90 ◦C) were the most effective anti-inflammatory agents (a concentration of 0.75 mg/mL reduced IL-8 and IL-6 levels by 58% and 47%, respectively, relative to the positive control). Threonine, valine, leucine,  arginine, and total protein content in the extracts were highlighted to correlate highly with the reported bioactivities (R2 ≥ 0.7). These results support the potential application of extracts from fishery industry wastes in cosmeceutical products with bioactive activities.

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and has the ability to scavenge reactive oxygen species and prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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Keywords: fish waste streams valorisation; antioxidant activity; anti-inflammatory activity; antimicrobial  activity; anti-ageing; anti-hyperpigmentation; cosmeceuticals

1. Introduction

With the constant search for innovation, especially for active ingredients, the cosmetic industry is growing and has demonstrated the intention to replace petroleum-derived components moving forward toward natural compounds [1]. The antioxidant properties of natural active ingredients can help in the prevention of several skin issues caused by oxidative stress and aging [2,3]. Skin aging can be induced by both intrinsic (such as inflammation or telomere shortening) and extrinsic (environmental) factors [4]. Skin aging leads to the loss of mature collagen and alterations at the extracellular matrix (ECM)  which compromises the barrier function, resulting in a dry appearance and susceptibility to external aggressors, increasing the risk for skin disorders [5]. This process can be accelerated by several enzymes, such as elastases, matrix metalloproteinases (MMPs), and hyaluronidases that can induce ECM degradation [6], or even by the accumulation of cessive reactive oxygen species (ROS) that can compromise the normal cell function [7]. Environmental factors, such as exposure to UV radiation, leads to the generation of high quantities of ROS that induces the same molecular and cellular responses as intrinsic aging,  but with amplified effects. Importantly, ROS can intensify the activity of enzymes related to skin aging or skin pigmentation processes [8,9], and thus the presence of antioxidants n plays an important role in the cosmetic field.

In 2018, world fish consumption was estimated by FAO to stand at 20.5 kg per pita [10], which leads to large quantities of by-products, mostly skin, and bones. The generated residues correspond to 20–75% (w/w) of the total caught fish weight, potentially leading to environmental problems [11,12]. However, these residues still contain a  significant amount of lipids, proteins, and minerals and should be adequately valorized. In recent years, extracts from waste generated by the fish industry have shown active properties such as antihypertensive, antioxidative, antimicrobial, neuroprotective,  antihyperglycemic, anti-aging, and anti-inflammatory [13–19]. Atlantic codfish (Gadus rhea) and sardine (Sardina pilchardus) are among the most consumed fish in Portugal and tracts derived from its residues have shown promising nutraceutical potential, such as antioxidant, antiproliferative or anti-inflammatory activities [11,20–22]. However, since the ploitation and valorization of fish industry waste are still in an early stage, there is plenty of room to explore opportunities for the industry to convert this waste into high-value market bioproducts, including cosmetic ingredients.

In previous work, we explored the use of high-pressure technologies (supercritical CO2  and subcritical water), to isolate bioactive fractions from sardine waste and codfish frames with promising health benefits [11,22]. For sardine wastes, we demonstrated that by applying a  first step with supercritical carbon (ScCO2) (to remove lipid fraction) followed by an extraction process with subcritical water (SW) it was possible to obtain protein hydrolysates with high antioxidant potential and antiproliferative effect in colorectal cancer cells [22]. Subcritical water extraction/hydrolysis we also applied to obtain proteins-, peptides- and amino acid enrichedxtracts from codfish frames and we showed that lower processing temperatures (90 ◦C) favor the extraction of compounds with anti-inflammatory potential in a human intestinal epithelial cell model [11]. Most of the proteins present in codfish frame extracts are collagen and collagen fragments. Other compounds include minor quantities of lipids,  ash, and some sugars. Sardine extracts were rich in peptides and amino acids, and lipids,  ash, and sugars were also present. Since fish-derived proteins and peptides may become an important resource for cosmetic industries, the present study aims to evaluate further the active potential of these extracts derived from fish-processing wastes and by-products caused by the assessment of their cosmeceutical potential [23]. For this purpose, a range of chemical, enzymatic, and cell-based assays were applied to explore the antioxidant, antiageing, anti-hyperpigmentation, anti-inflammatory, and antimicrobial effects of the extracted samples. Correlation studies were also performed to identify the main bioactive constituents with cosmeceutical potential.

2. Materials and Methods

2.1. Reagents 

3,4-dihydroxy-l-phenylalanine (L-DOPA), mushroom tyrosinase, porcine pancreatic elastase (PPE) type III, N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN), Tris (2-amino- 2-hydroxymethyl-propane-1,3-diol), 2,20 -azobis (2-methylpropionamidine)dihydrochloride (AAPH), and 20,70 -dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Calcium-adjusted Mueller Hinton broth (CAMHB)  was purchased from BD (Sparks, MD, USA). Brain-heart infusion (BHI) was purchased from Avantor (Radnor, PA, USA). AnaeroGen™ Compact sachets were purchased from Oxoid (Hampshire, UK). PrestoBlue™, Dulbecco’s Modified Eagle Medium (DMEM), heat-inactivated Fetal Bovine Serum (FBS), and Penicillin-Streptomycin were obtained from Invitrogen (San Diego, CA, USA). Human immortalized non-tumorigenic keratinocyte cell line HaCaT was obtained from Cell Line Service (Eppelheim, Germany). Human IL-8 and IL-6 Mini TMB ELISA Development Kits were obtained from Peprotech (London, UK). All other reagents and solvents used in the present study were of analytical grade and purchased from available suppliers.

2.2. Samples

The extracts used in this work were the ones developed in our previous studies focused on process optimization [11,22]. Briefly, codfish frames were supplied by Pascoal and Filhos S.A. (Gafanha da Nazaré, Portugal) and consisted of fish backbone and adhered muscle. Sardine waste, made of heads, spines, and viscera, was supplied by Conservas A Poveira S.A. (Póvoa de Varzim, Portugal). The proximate composition of the raw materials used has been presented in our earlier works [11,22]. Protein (47 wt %) and ash (39 wt %) were the major components of codfish frames, with small quantities of lipids and carbohydrates (2 wt % and 0.3 wt %, respectively). Collagen is found to be the major protein in codfish frames, and in this case, it accounts for ca. 65% of the total protein content of original waste. In contrast, sardine is an oily fish, thus its waste is much richer in lipids than codfish frames. Sardine wastes showed a lipid content of 26 wt % and a protein content of 52 wt %, the rest being ash (17 wt %) and carbohydrates (3 wt %).

The extracts from codfish frames (Cf1, Cf2, Cf3, and Cf4) and sardine wastes (S1, S2, and S3) selected for this work were obtained by high-pressure technology in a lab-scale apparatus as previously described [11,22,24] using the conditions summarized in Table 1. Briefly, 60 g of ground codfish frames or sardine waste (defatted or non-defatted) were loaded into a high-pressure reactor that was put inside an oven. The water pump was switched on at desired flow rate (ca. 10 mL/min) and the pressure was set to 100 bar. As soon as the pressure reached that value, the electrical oven was switched on, and the experiment started. The different extracts were collected for 30 min at different temperatures (90–250 ◦C). S1 and S3 extracts were obtained after a defatting process of the sardine waste by ScCO2  before SW extraction. Subcritical water extraction experiments were duplicated. For each extracted sample, 25 mL were taken in triplicate, lyophilized, and weighed to calculate the corresponding extraction yield. Analytical data—protein content—are expressed as mean ± standard deviation (SD) of triplicates. The information regarding the characterization of these extracts in terms of protein content, amino acid profile, major mineral compounds, or toxic and heavy metals is described in our previous works [11,22].

Stock solutions of Cf1, Cf2, Cf3, Cf4, and S2 were prepared in Milli-Q H2O at a concentration of 100 mg/mL. The other samples, namely S1 and S3, were dissolved in DMSO (300  and 550 mg/mL, respectively) due to their lower solubility in water. Samples were frozen and kept at −20 ◦C until further use. For cellular assays, the samples were previously sterilized by heat (121 ◦C, 15 min) in an autoclave (Tuttnauer 3870 el, Breda, Netherlands).

2.3. Oxygen Radical Absorbance Capacity (ORAC) Assay 

ORAC assay was performed to evaluate the antioxidant capacity of the samples towards peroxyl radicals (ROO• ), following the method developed by Huang et al. [25], with some adjustments as reported previously [26]. Briefly, in a black 96-well microplate, 150 µL disodium fluorescein (0.3 µM) was added to 25 µL of sample dilutions and incubated for 10 min at 37 ◦C. Afterward, the reaction was initiated by the addition of 25 µL of 2,20 - Azobis (2-amidinopropane) dihydrochloride (AAPH, 153 mM), and fluorescence (Ex/Em 485 ± 20/528 ± 20 nm) was measured for 40 min at 37 ◦C in an FLx800 fluorescence microplate reader (FL800 Bio-Tek Instruments, Winooski, VT, USA). A standard curve was prepared using 5, 10, 20, 30, and 40 µM of (6-hydroxy-2,5,7,8-tetramethylchroman-2-  carboxylic acid (Trolox)). All solutions were prepared in phosphate-buffered saline (PBS), 75 mM, pH 7.4. The results are expressed as micromoles of Trolox equivalent antioxidant capacity per gram of extract (µ mol TEAC/g extract).

2.4. Enzymatic Assays

2.4.1. Elastase Inhibition Assay 

This assay was based on the work of Wittenauer et al. [27] with some modifications as described previously [6]. Elastase inhibitory activity is determined by a spectrophotometric method using porcine pancreatic elastase (PPE) and N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN) as the enzyme-substrate, by monitoring the release of p-nitroaniline at 410 nm. PPE was dissolved in 100 mM Tris (2-amino-2-hydroxymethyl-propane-1,3-diol)-HCl buffer (pH = 8.0) to a concentration of 1 mg/mL and stored at 20 ◦C in aliquots. On the day of the assay, an aliquot was taken and diluted in the buffer to a concentration of 0.03 U/mL, 10 µL was loaded in the wells of the microtiter plates together with 100 µL of the Tris-HCl buffer, and 30 µL of each sample. After 20 min of pre-incubation at 25 ◦C, the reaction was initiated by the addition of 40 µL of the substrate AAAPVN (0.55 mM). Absorbance was measured for 20 min after the addition of AAAPVN at a BioTek Instruments EPOCH 2 spectrophotometer microplate reader. The calculations were made as described in Equation (1), where A control and Asample represent the absorbance at 410 nm in the absence or presence of the sample,  respectively. Since DMSO was used to dissolve samples S1 and S3, this solvent was also tested and used as a control for these samples. The potential of the extracts to inhibit elastase was evaluated with increasing concentrations, to determine dose-dependent relations and establish the half-maximal inhibitory concentrations (IC50) values, indicating the capacity of each sample in enzymatic activity inhibition to an extent of 50%.

All results are expressed as IC50 mean values with the lower and upper limits of a 95%  confidence interval, obtained from at least three independent experiments.

2.4.2. Tyrosinase Inhibition Assay

The tyrosinase inhibitory potential of the extracts was evaluated spectrophotometrically using mushroom tyrosinase and L-DOPA as the substrate [28]. Tyrosinase converts L-DOPA to dopaquinone, which will sequentially cyclize to form dopachrome. The dopachrome formation can be observed by measurement of the absorbance at 475 nm. The substrate was added to the enzyme in the presence of the sample dilutions, to a final concentration of 30 U/mL tyrosinase and 2.5 mM L-DOPA. Since DMSO was used to dissolve samples S1 and S3, this solvent was also tested and used as a control for these samples. After incubation at 37 ◦C for 30 min, absorbance was measured at 475 nm on a BioTek Instruments EPOCH 2 microplate spectrophotometer. All the reagents were prepared in sodium phosphate buffer (SPB; 0.1 M, pH 6.8), prepared by mixing sodium phosphate dibasic dihydrate and sodium phosphate monobasic monohydrate, and the calculations were made as described in Equation (1). All results are expressed as IC50 mean values with the lower and upper limits of a 95% confidence interval, obtained from at least three independent experiments.

2.5. Antimicrobial Susceptibility Testing

The target microorganisms selected for the antibacterial activity assays were the gram-negative bacteria Klebsiella pneumoniae CECT 8453 and the gram-positive bacteria Staphylococcus aureus ATCC 6538 and Cutibacterium acnes ATCC 6919T. For K. pneumoniae CECT 8453 and S. aureus ATCC 6538, assays were performed according to the broth microdilution method of CLSI M07-A10 guidelines as previously described by Rodrigues et al. [29]. In short, extract stock solutions were distributed in a round bottom microtiter 96-well plate and 2-fold serially diluted in calcium-adjusted Mueller Hinton broth (CAMHB; BD, Sparks, MD, USA) to obtain a concentration range of solutions. The inoculum was prepared using the growth method to achieve a homogenous suspension in a saline solution. The adjusted inoculum was additionally diluted in CAMHB to guarantee that, following inoculation, each well contained around 5 × 104 CFU. The plates were incubated under aerobic conditions at 37 ◦C for 16 to 20 h. For C. acnes, the assays were performed as previously described with the use of brain-heart infusion (BHI) (Avantor, Radnor, PA, USA)  broth instead of CAMHB and by incubating the microtiter plates for 70–74 h at 37 ◦C in anaerobic jars containing the atmosphere generation system AnaeroGen™ Compact (Oxoid, Hampshire, UK).

For each stock solution analyzed, a positive control (CAMHB or BHI and diluted inoculum), a medium sterility control (uninoculated CAMHB or BHI), and an extract sterility control (uninoculated 2-fold extract stock solution in CAMHB or BHI) were performed accordingly. Minimum inhibitory concentration (MIC) values were the lowest concentration of a sample that visibly inhibited microbial growth after incubation. When needed, MIC  values were confirmed using the cell viability reagent PrestoBlue™ (Invitrogen, San Diego, CA USA) following the manufacturer’s guidelines. An additional MIC value, designated MIC*, was also established and defined as the lowest concentration of a sample at which bacterial growth was visually and differentially affected in comparison to the positive control. Minimum bactericidal concentration (MBC) values were reported as the lowest concentration of a sample leading to at least a 99.9% reduction in viable bacterial counts in comparison to the initial inoculum and for equal incubation time. Results were expressed as a median of the values obtained after three biological replicates. Since DMSO was used to dissolve samples S1 and S3, this solvent was also tested to ensure that the final concentration used did not interfere with the target microorganism, hence the assay.

2.6. Cell-Based Assays

2.6.1. Cell Culture 

Human keratinocyte cell line HaCaT (CLS, Germany) was cultured in a standard Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. The cells were routinely maintained as monolayers in 75 cm2  culture flasks and incubated at 37 ◦C with 5% CO2 in a humidified atmosphere.

2.6.2. In Vitro Cytotoxicity 

Cytotoxicity assays were performed according to previous works [6]. Briefly, HaCaT cells were seeded at a density of 1.4 × 105  cells/cm2  in 96 well plates. After 3 days, cells were incubated with different concentrations of each sample (Cf1; Cf2; Cf3; Cf4; S2—50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39; S1—3, 1.5, 0.75, 0.38, 0.19, 0.09, 0.05, 0.02; S3—5.5, 2.75, 1.38, 0.69, 0.34, 0.17, 0.09, 0.04 mg/mL) diluted in culture medium (DMEM medium containing 0.5% FBS). Wells containing cells incubated only with culture medium supplemented with 0.5% (v/v) of FBS were used as control. Solvent controls with 50% water or 1% DMSO in a culture medium was also performed to exclude solvent toxicity. After 24 h of incubation,  the cell viability was evaluated using PrestoBlue® (5% v/v in culture medium) for 2 h at 37 ◦C, 5% CO2, according to the manufacturer’s instructions. After this, the fluorescence of each well was measured (Ex./Em. 560 ± 20/590 ± 20 nm) in an FLx800 fluorescence microplate reader (BioTek Instruments, Winooski, VT, USA). Cell viability was expressed as the percentage of viable cells relative to the control. Three independent experiments were performed in triplicate.

2.6.3. Cellular Antioxidant Activity

Cellular antioxidant activity was evaluated following previously described methods [6,30],  with some modifications. Briefly, HaCaT cells were seeded at a density of 1.4 × 105  cells/cm2  in 96  well plates and the formation of intracellular ROS was monitored using 20,70 -dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. 72 h after seeding, cells were washed with PBS  and incubated with non-toxic concentrations of the samples (0.1875 mg/mL; 0.375 mg/mL; 0.75 mg/mL) plus 25 µM DCFH-DA in PBS for 1 h. Subsequently, cells were washed again with PBS and incubated with the stress inducer (600 µM AAPH in PBS) for 1 h. After that, fluorescence was measured in an FL800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) (Ex/Em 485 ± 20/528 ± 20 nm). The results are expressed as ROS percentage relative to the untreated control (cells treated with DCFH-DA and AAPH). Three independent experiments were performed in triplicate.

2.6.4. Evaluation of IL-6 and IL-8 Secretion

Experiments were performed as previously described [31], with several modifications. Briefly, HaCaT cells were seeded at a density of 1 × 105  cells/cm2  in 12 well plates. After 3 days, cells were stimulated with 15 µg/mL of lipopolysaccharides (LPS) from Escherichia coli and co-incubated with three different concentrations of each extract (0.1875 mg/mL; 0.375 mg/mL; 0.75 mg/mL) diluted in culture medium (DMEM medium containing 0.5% FBS). Cells incubated only with LPS and cells incubated with only culture media were used as positive and negative controls, respectively. After 24 h, supernatants were collected,  centrifuged for 10 min at 2000 g, and stored at −80 ◦C until further analysis. IL-6 and IL-8  levels were assessed by enzyme-linked immunosorbent assay (ELISA), using commercially available kits (PeproTech; London, UK), according to the manufacturer’s instructions, with absorbance measured at 450 nm with wavelength correction set at 620 nm in a microplate spectrophotometer (EPOCH 2, BioTek Instruments, Winooski, VT, USA). The results are expressed as IL-6 or IL-8 percentage relative to the positive control (cells stimulated with LPS). Three independent experiments were performed in triplicate.

2.7. Statistical Analysis 

ORAC and cell-based assay results are expressed as the mean value ± SD, obtained from at least three independent experiments. For the enzymatic and cytotoxicity assays,  the IC50 values were determined from dose-response curves through log10 plots using GraphPad Prism 8.4.3. software (GraphPad Software, Inc., La Jolla, CA, USA). Results are presented as IC50 with a 95% confidence interval. Statistical analysis of the results was performed using the former software. When homogeneous variance and a normal distribution of the data were verified, the results were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey test for multiple comparisons. In the case of heterogeneous variances or if the data were not normally distributed, an appropriate unpaired Student’s t-test was performed to determine whether the means were significantly different. A p-value ≤ 0.05 was accepted as statistically significant in all cases. Antimicrobial susceptibility testing results are expressed as the median value, obtained from at least three independent experiments.

3. Results and Discussion

This study aims to investigate the cosmeceutical potential of protein-rich extracts that were produced by high-pressure technologies from fishery industry wastes, namely sardine wastes and codfish frames [11,22]. The selection of extracts was based on previous results regarding their characterization and process conditions. For codfish, all extracts were selected aiming at evaluating the impact of the extraction temperature on the recovery of compounds with promising bioactive effects on the skin. For sardine extracts, only three extracts derived from both defatted and non-defatted raw materials, processed at higher temperatures (190  and 250 ◦C) and with the highest extraction yield (>45.7 g/100 g) were chosen. The results regarding the total protein content of each extract are presented in Table 1.

3.1. Antioxidant, Anti-Ageing, and Anti-Hyperpigmentation Activities

The potential cosmeceutical effect of the extracts was initially screened using chemical and enzymatic assays to evaluate their antioxidant, anti-aging, and anti-hyperpigmentation effects. For the antioxidant capacity, the ORAC assay was selected as it measures the ability of samples to scavenge biologically relevant ROS, namely peroxyl radicals, which are considered one of the main inducers of skin aging [2,32]. The anti-aging effect was also evaluated through elastase inhibition since this enzyme is reported to be responsible for the degradation of elastin and other ECM proteins [6]. For the anti-hyperpigmentation effect, the tyrosinase assay was used, to evaluate the capacity of samples to inhibit melanin production. Table 2 summarizes the ORAC and IC50 values of all extracts.

Our results show that sardine and codfish extracts presented antioxidant activity and inhibition effects on elastase and tyrosinase enzymes’ activity. Among sardine samples, S1  showed the highest ORAC value (1.94 ± 0.08 µmol TEAC/mg extract) followed by S3 and S2. These results are by a previous antioxidant evaluation through an alternative method (2,2-diphenyl-1-picrylhydrazyl-DPPH assay) where the extract S1 presented the lowest IC50 values [22]. The higher scavenging capacity towards peroxyl radicals of the S1 sample could be derived from peptides with different amino acid sequences present in the extracts since interactions among them can influence the radical scavenging ability [33]. Additionally, compounds generated in Maillard or other thermo-oxidation reactions might influence the antioxidant activity of the samples [34]. Despite the lowest ORAC value,  samples S2 and S3 were the ones with the highest capacity in inhibiting tyrosinase and elastase activities, respectively.

Among codfish extracts, Cf4 was shown to have the highest antioxidant and antihyperpigmentation activities. SEC-GPC analysis of the extracts has shown that peptides of decreasing molecular weight were obtained when increasing the extraction temperature up to 250 ◦C [11]. The elastase inhibition capacity of this sample is within the range of values found for other Cf extracts, namely Cf1 and Cf3. However, for the concentrations tested,  these two extracts showed no inhibition activity towards tyrosinase.

In the literature, some reports are showing that extracts from marine by-products present relevant antioxidant activity and inhibition of tyrosinase. For instance, extracts derived from marine (Scophthalmus maximus) by-products by alkaline hydrolysis, have demonstrated antioxidant activity accessed by different chemical assays: 1,1-diphenyl-2-  picrylhydrazyl (DPPH) radical-scavenging ability (36.12% about control), ABTS (2,20 -azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid) method (12.81 µg BHT/mL) and crocin bleaching assay (8.03 µg Trolox/mL) [35]. In another study, enzymatic extraction of alum-salted jellyfish (Lobonema smithii) showed to produce hydrolysates with high antioxidant activity (IC50 = 0.9 mg/mL for ABTS e DPPH assays) and tyrosinase inhibitory potential, with IC50 values ranging between 14.1 and 24.5 mg/mL [36], which are in the same order of magnitude as the ones obtained in this work. Additionally, extracts with similar antioxidant values (ORAC values – 0.4 to 3.5 µmol TEAC/mg extract) were obtained from another type of food industry residues, namely winemaking waste streams [6]. However, these winery residues extracts presented higher tyrosinase (IC50 from 4.0 to 0.14 mg extract/mL) and elastase (IC50 from 3.4 to 0.1 mg extract/mL) inhibitory capacities than those obtained in this work, probably due to the presence of phenolic compounds that are recognized to have several bioactivities. It is important to mention that in our study we used mushroom tyrosinase to screen the anti-hyperpigmentation effect of extracts, as this enzyme has been widely used in high throughput assays [37]. Nevertheless, since there are some controversies regarding the similarity and homology of this enzyme with mammalian/human tyrosinase [38–40], future studies involving mammalian cell lines should be considered to evaluate the potential anti-hyperpigmentation effect of sardine and codfish extracts.

To identify which compounds could be responsible for the bioactive response of extracts, correlation studies between bioactivity data and the extracts’ amino acid composition reported previously [11,22] were performed (Table S1). For antioxidant activity, the highest correlations (R2 ≥ 0.7) were obtained between ORAC value and total threonine, free valine,  as well as free and total leucine for all extracts. In the case of sardine samples, a high correlation (R2 ≥ 0.94) was also obtained for free and total tryptophan content. Accordingly,  all these amino acids have been previously reported to have antioxidant properties in several model systems [41–43]. For elastase and tyrosinase inhibition activities, the highest correlation coefficients (R2 ≥ 0.8) were obtained for total protein content and free arginine,  respectively, suggesting that these compounds could have an important role in the potential anti-aging and anti-hyperpigmentation effect of fish industry waste streams extracts.

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