Application of Pulsed Electric Fields for Obtaining Antioxidant Extracts from Fish Residues

antioxidants Article Application of Pulsed Electric Fields for Obtaining Antioxidant Extracts from Fish Residues Daniel Franco 1, Paulo E. S. Munekata 1, Rubén Agregán1, Roberto Bermúdez 1, María López-Pedrouso 2, Mirian Pateiro 1and JoséM. Lorenzo 1,* 1Centro Tecnolóxico da Carne de Galicia, rúa Galicia n◦4, Parque Tecnolóxico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain; [email protected] (D.F.); [email protected] (P.E.S.M.); rubenagr[email protected] (R.A.); [email protected] (R.B.); mirianpateir[email protected] (M.P.) 2Department of Zoology, Genetics and Physical Anthropology, University of Santiago de Compostela, 15872 Santiago de Compostela, Spain; [email protected] *Correspondence: jmlor[email protected] Received: 31 December 2019; Accepted: 18 January 2020; Published: 21 January 2020   Abstract: Fish processing has serious economic and environmental costs in the food supply chain. It is necessary to find new ways to convert fish residue to added-value products, especially for main aquaculture species. In this study, a pulsed electric field (PEF) process for antioxidant extract production from three residues (gills, bones, and heads) of two commercial species (sea bream and sea bass) was tested. Three methods of extraction using two solvents (water and methanol) and a water extraction assisted by PEF were assessed. Chemical and mineral compositions, as well as amino acid profile of the by-products, were determined. In addition, four in vitro antioxidant methods, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH), 2,2-azinobis-(3-ethyl-benzothiazoline-6-sulphonate radical (ABTS), ferric reducing antioxidant power assay (FRAP), and oxygen radical absorbance capacity assay (ORAC), were used to evaluate the extracts. Antioxidant activity was confirmed by DPPH and ABTS and FRAP tests, obtaining the highest values for residues from the sea bream species. ORAC values were higher in methanol than in water solvent. In general, gills were the residues with the greatest antioxidant activity for the four antioxidant assays employed. For DPPH assay, the extracts of water assisted by PEF from heads, bones, and gills yielded significant increases of 35.8%, 68.6%, and 33.8% for sea bream and 60.7%, 71.8%, and 22.1% for sea bass, respectively, with respect to water extracts. Our results suggest that PEF would be an environmentally friendly and economic choice for antioxidant-extract production from low-value by-products from fish processing. Keywords: sea bream; sea bass; by-products; antioxidant capacity; amino acid 1. Introduction Aquaculture production has been globally increasing and gaining economic importance over the last decade. Particularly for the European market, the main cultured aquaculture species are the Atlantic salmon, rainbow trout, gilthead sea bream, European sea bass, common carp, and turbot [ 1 ]. Due to the large production of ready-to-use products and meals, the fish industry generates a large amount of wastes. These residues in fisheries are mainly composed of heads, skin, and viscera and account for 20–75% of fish weight [ 2 ]. In this sense, great efforts are being made to exploit these fish wastes, ensuring the sustainability of the aquaculture industry. This strategy is supported by the presence of natural antioxidants and other bioactive components in these residues. It has been demonstrated that protein hydrolysates (FPH) obtained from fish by-products have an excellent quality in terms of amino acids composition and antioxidant properties [ 3 , 4 ]. In order Antioxidants 2020,9, 90; doi:10.3390/antiox9020090 www.mdpi.com/journal/antioxidants Antioxidants 2020,9, 90 2 of 14 to extract amino acids and small peptides with more bio-accessibility and bioavailability for health purposes, the use of heating, chemical, and enzymatic treatment have been reported [ 5 ]. However, enzymatic hydrolysis has a great cost in comparison to chemical hydrolysis, which is of very limited use at the industrial level [ 6 ]. Similarly, the other conventional technologies (thermal and chemical) have disadvantages related to extended process time, high energy consumption, and the use of toxic solvents. On the other hand, emerging technologies (ultrasound, high hydrostatic pressure, ohmic heating, and pulsed electric fields, for instance) contribute to the extraction of bioactive compounds from foodstuffs. Particularly for pulsed electric fields (PEF), this technology consists of treating samples with high-voltage electrical pulses for short periods, which disturbs the structure of cell membranes and facilitate the extraction of entrapped bioactive compounds. Moreover, it was demonstrated that PEF treatment can also cause protein hydrolysis [ 5 ]. Extractions using PEF technology have been employed in plant [ 7 ], animal [ 8 ], and fish [ 9 ] products, but the scientific information about their use in the valorization of fish by-products is scarce. Due to characteristics of food, the occurrence and progression of oxidative reactions generates the reactive oxygen species (ROS) by means of enzymatic, chemical, and photochemical reactions. Consequently, the formation of undesirable volatile and carcinogenic compounds is favored, along with changes in the functionalities of proteins, lipids, and carbohydrates, leading to the deterioration of sensory properties and reduced shelf life [ 10 , 11 ]. To tackle this problem, one of the most employed strategies in food systems is the use of antioxidants. Antioxidants are compounds that protect the lipid from oxidation and avoid the formation of rancid flavor and aroma in food products and also extend their shelf life. The synthetic ones are the most common antioxidant additives in food products (butylated hydroxyanisole (BHA), butylated hydroxyltoluene (BHT), propyl gallate (PG), and tert-butyl hydroquinone (TBHQ)). However, some physical properties of BHT and BHA, such as their high volatility and instability at elevated temperature [ 12 ] and their potentially harmful impact on human health, pressure governmental authorities to creation restrict rules in order to limit their use. Therefore, replacing synthetic antioxidants is becoming a main goal in the food industry [13]. There are abundant research studies about the use of natural antioxidants from plants, both terrestrial [ 14 ] and of marine origin [ 15 ], and their active molecules (polyphenols, ascorbic acid, carotenoids, tocopherol, and photosynthetic pigments) in foods. Although fish wastes were also considered to be sources of powerful antioxidants, there is less literature. Taking into account that the PEF approach is an attractive strategy to valorize the residues from fish industry (because of its reduced time process, improved extraction yield, and environmental aspects) and fish by-products can offer interesting bioactive molecules, such as antioxidants (which can be applied in food industry), the aim of the present preliminary study was to evaluate whether sea bream and sea bass by-products (head, bone, and gills) treated with pulsed electric field technology provide antioxidant extracts. 2. Materials and Methods 2.1. Samples The sea bream and sea bass used were purchased from a local supermarket. By-products, heads, bones, and gills were manually obtained from the fishes. Then, these residues were chopped, vacuum-packaged, and stored at −20 ◦C, until further analysis. 2.2. Extraction Procedures 2.2.1. Extraction with Solvents (Water and Methanol) without PEF The heads, bones, and gills from sea bream and sea bass were extracted in a conventional manner, using water and methanol. Briefly, five grams of each one of the residues was mixed with 5 mL of distilled water or methanol. The mixture was intensively crushed and vortexed with an IKA T25 digital ultra-turrax (IKA ® -Werke GmbH & Co. KG, Staufen, Germany), until complete homogenization. Then, Antioxidants 2020,9, 90 3 of 14 ultrasounds were applied for 15 min at room temperature, to increase extraction yield. The obtained extract was centrifuged at 2000 × gfor 10 min, at 4 ◦ C, and the resultant supernatant was passed through 45 µm pore-size filters (Filtros Anoia S. A., Barcelona, Spain). Extracts were stored at −20 ◦C until further analysis. 2.2.2. Extraction with Pulsed Electric Fields (PEF) Fifty milligrams of each one of the residues from sea bream and sea bass, previously defrosted at room temperature, was weighed and mixed with 50 mL of distilled water. The mixture was intensively crushed and vortexed with an IKA T25 digital ultra-turrax (IKA ® -Werke GmbH & Co. KG, Staufen, Germany) until complete homogenization. Then, the homogenates were placed between two electrodes separated by 5 cm, reaching 1.8 cm of height. PEF was generated by using a semiconductor-based positive Marx modulator Epulsus-PM1-10 equipped with a batch treatment chamber (EnergyPulse Systems, Lisbon; Portugal; Figure 1). The PEF working conditions were as follows: 7000 V potential difference, 20 µ s pulse width, 10 Hz frequency, and pulses number of 100. Before starting the PEF treatment, the homogenates’ conductivity was measured in order to know the applicable voltage. The same electrical field was applied for all samples (1.40 kV/cm). The sea bream heads, bones, and gills achieved 26.9, 29.4, and 28.3 kJ/kg, respectively; meanwhile, sea bass head, bone, and gills achieved 26.6, 17.4, and 28.3 kJ/kg, respectively. The entire process was carried out protected from light. Once the PEF treatment was applied, the samples were extracted as described in Section 2.2.1. All treatments were made by triplicate. Figure 1. PEF generator (a) and the batch treatment chamber (b). 2.3. Analytical Determinations 2.3.1. Chemical Composition, Fatty Acid, Amino Acid, and Mineral Profile The International Organization for Standardization (ISO) recommended standards were used to assess moisture [ 16 ], protein [ 17 ], and ash [ 18 ]. Total fat was extracted according to the American Oil Chemists Society (AOCS) Official Procedure Am 5-04 in an extractor Ankom XT10 (ANKOM Technology Corp., Macedon, NY, USA) [ 19 ]. Fatty acid extraction and identification was carried out with gas chromatography (GC-Agilent 7890B, Agilent Technologies, Santa Clara, CA, USA) with a flame ionization detector (FID) and PAL RTC-120 auto sampler, amino acid profile after protein hydrolysis employing high-performance liquid chromatography (Alliance 2695 model, Waters, Milford, MA, USA) with fluorescence detector (model 2475, Waters, Milford, MA, USA), and mineral composition was determined by induced coupling plasma atomic emission spectrometry [20]. 2.3.2. Determination of Antioxidant Capacity DPPH Radical Scavenging Assay The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method was carried out as follows [ 21 ]: the DPPH solution (60 µ M in methanol) was mixed with 100 µ L of sample. The mixture was incubated Antioxidants 2020,9, 90 4 of 14 at 37 ◦ C for 10 min and then the absorbance was measured in a spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at 515 nm. Each extract was analyzed in triplicate, and its antioxidant activity was determined by using Trolox (Acros organics, Morris Plains, NJ, USA) as standard, expressing the results as µg Trolox/g sample. ABTS Radical Cation Decolorization Assay This method was determined according to the procedure previously described by Re et al. [ 22 ], with some modifications. The method is based on the decolorization of blue–green color at 734 nm, since ABTS radical ((2,2-azinobis-(3-ethyl-benzothiazoline-6-sulphonate) is scavenged. This radical was prepared by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate and keeping the mixture in darkness, at room temperature, for 12–16 h before its use. The ABTS solution was added to 20 µ L of sample, and the resultant mixture was mixed and left in darkness for 10 min. Afterward, absorbance was measured in a spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at 734 nm. Each extract was analyzed in triplicate, and its antioxidant activity was determined by using a standard curve of ascorbic acid (AA) in the concentration range 0–150 mg/L AA, expressing the results as mg AA/100 g sample. Ferric-Reducing Antioxidant Power (FRAP) Assay The FRAP assay was based on the Benzie and Strain method [ 23 ], with some modifications. The FRAP reagent was prepared by using 0.3 M acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl 3· 6H 2 O solutions. These three solutions were mixed in a ratio of 10:1:1 (v:v:v). Afterward, 900 µ L of this resultant FRAP solution was added to 30 µ L of properly diluted sample and to 90 µ L of distilled water. The mixture was heated at 37 ◦ C and left to react for 20 min at this temperature. After this time, the absorbance was measured in a spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at 593 nm. Each extract was analyzed in triplicate, and its antioxidant activity was determined by using a standard curve of FeSO 4 in the concentration range 0–400 µM FeSO4, expressing the results as µmol Fe+2/100 g sample. Oxygen Radical Absorbance Capacity (ORAC) Assay The ORAC assay was determined according to the Huang et al. method [ 24 ], with some modifications. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), for a final reaction volume of 200 µ L. Then, 25 µ L of sample was mixed with 150 µ L fluorescein (80 nM of final concentration), and the mixture was pre-incubated at 37 ◦ C for 30 min. Afterward, 25 µ L of AAPH (2,20-azobis (2-methylpropionamidine) dihydrochloride solution (184 mM, final concentration) was added rapidly, using the injectors of a Synergy ™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The plates were immediately placed in the reader and the fluorescence was recorded every minute for 150 min and stirred prior to each reading (excitation wavelength, 485nm, andemissionwavelength, 528nm). Eight calibrationsolutions, intheconcentration range 0–100 µ M, using Trolox as standard, were used in each assay. The phosphate buffer was used as blank. Each extract was analyzed in triplicate, and its antioxidant activity was calculated from the differences in areas under the fluorescein decay curve between the blank and the sample. The results were expressed as mg Trolox/g sample. 2.4. Statistical Analysis Statistical analysis of the obtained data was conducted by using the IBM SPSS Statistics 23.0 program (IBM Corporation, Somers, NY, USA). An analysis of variance (ANOVA), using the General Lineal Model (GLM) procedure, was performed for chemical composition of residues. The following model was used: Yij=µ+Si +Dj +(S ×D)ij +εij (1) where, Yij is the observation of dependent variables, µ is the overall mean, Si is the effect of species, Dj is the effect of residues, (S × D)ij is the interaction term of species and residue, and ε ij is the residual Antioxidants 2020,9, 90 5 of 14 random error associated with the observation. For extract antioxidant capacity, one-way analysis of variance (ANOVA) was applied to all assessed tests (DPPH, ABTS, FRAP, and ORAC), and the results were expressed as mean ( ± ) standard error. The least square means (LSM) were separated by using Duncan’s post hoc test (significance level p<0.05). 3. Results 3.1. Chemical, Mineral, and Amino Acids Composition of Fish Residues In Table 1, the chemical and mineral composition from residues of sea bream and sea bass are depicted. Water content was the main component for the three by-products, with an average value of 55%, reaching a highest percentage above 60% in sea bass gills. However, there was a contradictory trend between species. In sea bass, gills showed the higher content than bones, with values of 62.37% and 51.60%, respectively. Conversely, bones showed higher water content than gills in sea bream (Table 1). In general, there were significant differences (p<0.05) among by-products in both species, but water content in sea bream by-products was narrower (52.52–57.63%) than in sea bass. Fat content showed values significantly higher (p<0.05) in sea bass bone than gills and head; meanwhile, this trend was opposite in the sea bream. This fat composition is expected because there is a strong correlation between water and fat content. Within the fat content, MUFAs were the most abundant fatty acids, with values of approximately 40% of the total FAs, followed by PUFAs, with percentages around 30%. The gills presented the highest percentages of MUFAs, but the lowest of PUFAs, especially in the sea bass, and a significant (p<0.05) increase of SFAs with respect to the other residues. Moreover, the gills showed the highest content in n3 fatty acid, as well as in long chain n3 fatty acids and therefore the most suitable n6/n3 (<1) ratio with respect to the others residues. The protein content presented antagonistic values according to the species, because bone reached the highest protein value (16.39%) in sea bream; meanwhile, bone reached the lowest value (14.24%) in the sea bass. Ash contents were more consistent between species; the ashes were more abundant in the head than in the rest of residues, reaching a value close to 10% in both species. Regarding mineral profile, calcium highlighted over the rest samples with values even higher than 2000 mg/100 g residues in several parts (Table 1). An average value of 2448.2 mg/100 g was the highest content shown in the head from both species. Moreover, phosphorous is presented in substantial quantities, especially in the head, with values higher than 1200 mg/100 g residue in both species. Bones and gills were the most abundant source of potassium and sodium, with mean values of 281.7 mg/100 g and 254.6 mg/100 g residue, respectively. The amino acid profile of the residues from sea bass and sea bream is depicted in Table 2. There were higher significant differences among residues than between species. For all residues, there was a lower content of essential amino acids compared to nonessential ones, showing EAA/NEAA ratios lower than 1 for all cases. In this sense, fish bones were more interesting and became healthier regarding this ratio, with average value of 0.87 (p>0.05) for both species. On the contrary, gills provided the lowest ratio, with an average value of 0.67 (p>0.05) for both species. Among essential amino acids, threonine, valine, lysine, and leucine were highlighted over the rest. The amount of threonine became more important in gills from both species. Specifically, the main essential amino acids were arginine, followed by leucine and lysine for the head and bones in both species. Wide variations were found in arginine, which presented significantly (p<0.05) different values depending on the sample. The head and bone displayed values in the range 700–900 mg/100 g of residue, while the head was around 200 mg/100 g of residue. The sea bream bone showed the highest essential amino acid content. Concerning nonessential amino acids, the more predominant were glutamic acid, glycine, and aspartic acid in both species and residues. Antioxidants 2020,9, 90 6 of 14 Table 1. Chemical composition and mineral profile of residues (gills, heads, and bones) from sea bass and sea bream. Chemical Composition and Mineral Profile Sea Bass Sea Bream SEM Species Residue Species ×Residue Gills Heads Bones Gills Heads Bones Water (%) 62.37a1 58.86b1 51.6c1 55.23B2 52.51C2 57.63A2 0.29 <0.001 <0.001 <0.001 Fat (%) 14.00b1 13.94b1 20.20a1 21.55A2 22.10A2 17.12B2 0.21 <0.001 0.201 <0.001 SFA 26.27a1 21.76b1 21.75b1 20.15B2 20.71A2 20.84A2 0.037 <0.0001 <0.0001 <0.0001 MUFA 46.02a1 43.45c1 44.19b47.61A2 43.45B2 44.19B0.045 <0.0001 <0.0001 <0.0001 PUFA 26.98c1 33.72a32.96b1 31.17B2 33.76A33.75A2 0.067 <0.0001 <0.0001 <0.0001 LCn3 11.79a1 10.22b1 8.87c1 7.43C2 9.77A2 9.34B2 0.049 <0.0001 <0.0001 <0.0001 n3 14.68a1 14.20b1 12.92c1 11.90C2 14.03A2 13.58B2 0.053 <0.0001 <0.0001 <0.0001 n6/n3 0.83c1 1.37b1 1.55a1 1.62A2 1.40C2 1.48B2 0.005 <0.0001 <0.0001 <0.0001 Protein (%) 16.58a1 15.48b1 14.24c1 13.92B2 12.91C2 16.39A2 0.087 <0.001 <0.001 <0.001 Ash (%) 5.57c1 9.96a7.52b6.44B2 9.14A6.23B0.15 0.180 <0.001 0.005 Ca 1382.62c1 2507.15a2093.26b 1873.24 B2 2389.24A1618.82B41.69 0.685 <0.001 <0.001 Fe 1.22a1 0.28c1 0.51b1 2.15A2 0.44C2 0.69B2 0.02 <0.001 <0.001 <0.001 K180.51b1 194.31b262.73a1 134.94C2 184.52B300.67A2 1.96 0.144 <0.001 <0.001 Mg 36.77a1 29.04b24.98c1 47.90A2 28.04B30.70B2 0.44 <0.001 <0.001 <0.001 Mn 500.58a1 266.82b1 270.37b1 585.07A2 211.13B2 206.76B2 11.39 0.612 <0.001 0.006 Na 250.51a162.86b96.03c258.79A159.30B98.00C2.04 0.587 <0.001 0.449 P742.60b1 1277.00a1166.36a955.92B2 1312.27A989.20B20.68 0.567 <0.001 0.01 Zn 1.41b1 2.12a1 1.27c2.12A2 1.71B2 1.39C0.02 <0.001 <0.001 <0.001 Cu 0.09a1 0.03b0.10a0.17A2 0.04B0.14A0.009 0.017 <0.001 0.172 Minerals: Mn is expressed in µ g/100 g, and the others minerals are expressed in mg/100 g. SEM is standard error of mean; mean values followed by a letter (a–c) for sea bass and (A–C) for sea bream display significant differences (p<0.05), within each species; mean values followed by a number (1–2) display significant differences (p<0.05) between residues; LCn3 =long chain n3. Antioxidants 2020,9, 90 7 of 14 Table 2. Amino acid profile of residues (gills, heads, and bones) from sea bass and sea bream. Amino Acid Sea Bass Sea Bream SEM Species Residue Species ×Residue Gills Heads Bones Gills Heads Bones Asp 846.50 891.531875.141790.94B695.72B2 1000.97A2 11.95 0.088 <0.0001 <0.0001 Ser 533.61a1 491.36b1 440.95c1 449.97B2 399.96C2 501.55A2 3.10 <0.0001 <0.0001 <0.0001 Glu 1321.941 1337.3611286.8511270.81B2 1073.06C2 1446.03A2 14.84 0.088 <0.0001 <0.0001 Gli 1447.95a1171.91b956.69c1073.14 1118.67 1110.40 31.73 0.118 0.015 0.007 Ala 896.49a1 657.11b1 618.39b1 702.50A2 586.47 2688.02A2 7.50 <0.0001 <0.0001 <0.0001 Pro 949.47a1 677.66b531.53c1 623.982625.12 626.28216.83 0.008 <0.0001 <0.0001 Tyr 254.75 293.601270.311228.99B204.54B2 306.86A2 6.79 0.062 0.003 <0.0001 NEAA 6280.75a1 5520.56b1 4980.08c1 5140.34B2 4703.57C2 5680.13A2 24.12 <0.0001 <0.0001 <0.0001 His 374.00a323.26b1 300.67b1 311.53B276.50C2 379.58A2 3.82 0.194 <0.0001 <0.0001 Arg 185.28c844.34a1 775.38b1 206.41C701.37B2 870.50A2 6.33 0.486 <0.0001 <0.0001 Thr 694.30a1 453.15b1 445.73b1 567.39A2 386.41C2 516.28B2 5.80 0.001 <0.0001 <0.0001 Val 571.80a471.88b1 454.74b1 494.67A367.85B2 541.70A2 6.46 0.02 <0.0001 <0.0001 Met n.d. 142.20 128.461n.d. 126.45 162.8426.20 0.620 <0.0001 <0.0001 Lys 672.73b747.62ab1 797.34a1 722.25B581.78 C2 903.19A2 15.49 0.911 <0.0001 0.077 Iso 382.16 387.511378.431342.11B277.68B2 450.37A2 7.80 0.105 <0.0001 <0.0001 Leu 663.83 637.021608.121611.99 B469.31C2 742.38A2 11.60 0.228 <0.0001 <0.0001 Phe 474.36a431.84ab1 390.92b1 382.93 B315.45C2 462.85A2 6.38 0.01 <0.0001 <0.0001 EAA 4018.50b4438.86a4279.83ab1 3639.30 B3502.84B2 5029.73A2 48.25 0.058 <0.0001 <0.0001 EAA/NEAA 064b0.80a1 0.86a 0.70B0.74 B2 0.88A0.01 0.647 <0.0001 0.077 Amino acids: mg/100 g of residue. SEM is standard error of mean; mean values followed by a letter (a–c) for sea bass and (A–C) for sea bream display significant differences (p<0.05), within each species; mean values followed by a number (1–2) display significant differences (p<0.05) between residues; NEEA =nonessential amino acids; EEA =essential amino acids; n.d. =not detected. Antioxidants 2020,9, 90 8 of 14 3.2. Antioxidant Activity of EXTRACTs from Residues Fish residues are a source of biological active peptides, with antioxidant-potential activity. To determine the antioxidant capacity of the extracts, the assays often included radical cation scavenging activity; specifically, the DPPH, ABTS, and FRAP assay evaluated the electron transfer capacity, and ORAC assay was employed to evaluate the proton transfer capacity. The use of different treatments affected the extraction of compounds with antioxidant activity in both species (Figure 2). In general, the application of PEF provoked the extraction of compounds with the higher antioxidant activity in all residues. Particularly, the DPPH test displayed significant differences (p<0.001) in all residues analyzed except in sea bass gills. On the contrary, methanolic extraction provided the lowest results in terms of antioxidant activity, except for ORAC values. Regarding the different residues employed, the extracts of gills showed higher antioxidant activity. In the case of water assisted by PEF, values that exceeded 300 µ g Trolox/g gills resulted in the DPPH test. On the contrary, bones extracts showed the lowest results with DPPH values that barely reached 250 µ g Trolox/g bones in water extraction assisted by PEF. Specifically, the antioxidant capacity displayed higher values in gills (196.85–389.62 µ g Trolox/g sample) and heads (102.75–292.47 µ g Trolox/g sample) for sea bream than in gills ( 105.93–313.87 µg Trolox/g sample ) and heads (82.79–238.76 µ g Trolox/g sample) for sea bass. However, the antioxidant capacity of sea bass bones (50.78–256.78 µ g Trolox/g sample) was higher than sea bream bones (41.18–241.43 µ g Trolox/g sample). The differences among heads’, bones’, and gills’ antioxidant activities were more remarkable for the DPPH assay. Figure 2. Cont. Antioxidants 2020,9, 90 9 of 14 Figure 2. Mean values ± SEM. Antioxidant capacities in residues (bones, gills, and heads) of sea bream and sea bass, using methanol (blue), water (red), and water assisted by PEF (green) extractions. In the sea bream species, residue extracts presented higher antioxidant activity than sea bass residues. The solvent and residue variable showed remarkable differences, but the species did not show a clear trend for antioxidant capacity, above all (Figure 3). In this sense, water and methanol do not produce a clear trend in the activity of extracts. Conversely, water assisted by PEF clearly increased the antioxidant activity. Head and gill extracts from sea bream reached values higher than those from sea bass; meanwhile, the sea bass bones’ extract showed antioxidant activity values higher than those of the sea bream bones (Figure 2). Figure 3. Mean values ± SEM. Antioxidant capacities in residues (bones, gills, and heads) of sea bream (blue) and sea bass (gray).