In Search of Antioxidant Peptides from Porcine Liver Hydrolysates Using Analytical and Peptidomic Approach

  Citation: López-Pedrouso, M.; Lorenzo, J.M.; Borrajo, P.; Franco, D. In Search of Antioxidant Peptides from Porcine Liver Hydrolysates Using Analytical and Peptidomic Approach. Antioxidants 2022,11, 27. https://doi.org/10.3390/ antiox11010027 Academic Editor: David ArráezRomán Received: 14 November 2021 Accepted: 22 December 2021 Published: 24 December 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article In Search of Antioxidant Peptides from Porcine Liver Hydrolysates Using Analytical and Peptidomic Approach María López-Pedrouso 1, JoséM. Lorenzo 2,3 , Paula Borrajo 2and Daniel Franco 2,* 1Departamento de Zooloxía, Xenética e Antropoloxía Física, Universidade de Santiago de Compostela, 15872 Santiago de Compostela, Spain; [email protected] 2Centro Tecnolóxico da Carne de Galicia, Rúa Galicia No. 4, Parque Tecnolóxico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain; jmlor[email protected] (J.M.L.); [email protected] (P.B.) 3Área de Tecnoloxía dos Alimentos, Facultade de Ciencias, Universidade de Vigo, 32004 Ourense, Spain *Correspondence: [email protected] Abstract: The search for antioxidant peptides as health-promoting agents is of great scientific interest for their biotechnological applications. Thus, the main goal of this study was to identify antioxidant peptides from pork liver using alcalase, bromelain, flavourzyme, and papain enzymes. All liver hydrolysates proved to be of adequate quality regarding the ratio EAA/NEAA, particularly flavourzyme hydrolysates. The peptidomic profiles were significantly different for each enzyme and their characterizations were performed, resulting in forty-four differentially abundant peptides among the four treatments. Porcine liver hydrolysates from alcalase and bromelain are demonstrated to have the most antioxidant capacity. On the other hand, hydrophobic amino acid residues (serine, threonine, histidine and aspartic acid) might be reducing the hydrolysates antioxidant capacity. Seventeen peptides from collagen, albumin, globin domain-containing protein, cytochrome β , fructose-bisphosphate aldolase, dihydropyrimidinase, argininosuccinate synthase, and ATP synthase seem to be antioxidant. Further studies are necessary to isolate these peptides and test them in in vivo experiments. Keywords: amino acid profile; alcalase; bromelain; flavourzyme; papain; pork by-products 1. Introduction Novel antioxidant peptides play an increasingly important role in biotechnological applications; consequently, the nutraceutical, functional food, and cosmetic sectors are paying more and more attention to them. In this respect, industrial processing wastes with high content of protein provide an opportunity for preparing hydrolysates with antihypertensive, antioxidant, antimicrobial, antidiabetic, anticancer, and other bio-activities [ 1 ]. Moreover, the field of peptidomics as a part of proteomic is becoming more crucial in search of bioactive peptides. Indeed, from 2015 to 2020, a total of 80 original papers were published according to the Scopus database, using the keywords “peptidomic” and “biopeptide”. More specifically, a systematic search of the literature using the keywords most important of this paper (“bioactive peptides” and “pork liver”) resulted in only five original papers. Therefore, great development of technical capacity is being carried out in recent years, mainly performed by liquid chromatography to separate the peptides followed by mass spectrometry in tandem to identify them. Ultrafiltration, hydrophobic-high-performance liquid chromatography (HPLC), ion-exchange HPLC, and capillary electrophoresis in most cases coupled to electrospray ionization are the main analytical instruments used. Finally, the protein identification is carried out by Q-TOF or MALDI-TOF/TOF instrumentation as a mass spectrometer [2,3]. Protein-rich by-products of animal origin (fish, seafood, milk, and meat products) are valuable sources of these bioactive compounds. In addition, recycling and reusing these animal wastes lead us to reach a more sustainable industry as a second objective [4]. Antioxidants 2022,11, 27. https://doi.org/10.3390/antiox11010027 https://www.mdpi.com/journal/antioxidants Antioxidants 2022,11, 27 2 of 13 To achieve this purpose, the preferred method to obtain a peptide mixture is enzymatic hydrolysis. Usually, food proteins are hydrolyzed by peptidases and, in most cases, microbial peptidases cause different peptide patterns (Toldrá, Reig, Aristoy, and Mora, 2018), but there are other enzymes from fruit and vegetables. In any case, the enzymatic hydrolysis requires optimization concerning the reaction conditions including the selection of the enzyme, reaction time, and temperature for each enzyme. Regarding pork liver hydrolysates, various enzymes including alcalase, bromelain, flavourzyme, and papain have been employed. In this sense, the optimization of enzymatic reaction was carried out to increase the degree of hydrolysis resulting in 27.5% with alcalase 2.4L ™ and novo Pro-D™ [5]. Within the meat industry, in the last years, there has been a demanding interest in pork liver which is an adequate source of protein (18.54%) with a low-fat percentage (3.38%) and phospholipids, with great benefits for human health [ 6 ]. Furthermore, the amino acid profile is comprised of aromatic (tyrosine and phenylalanine) and hydrophobic (leucine, valine, and isoleucine) amino acids that show high antioxidant capacity [ 7 ]. Other abundant proteins from animal sources as keratin and collagen seem to be inactive molecules because of their high stable protein structures [ 8 ]. In this regard, smaller antioxidant peptides (4–16 amino acids with a molecular weight of 400–2000 Da) have also been found in other animal tissue [9]. Since studies linked to the antioxidant effect of pork liver-derived peptides are still limited, this study aimed to evaluate the effect of several enzymes on pork liver in the generation of antioxidant peptides. To this end, a high number of antioxidant peptides needs to be identified using different techniques and, further, to isolate them for validation of the results. 2. Materials and Methods 2.1. Preparation of Porcine Liver Hydrolysate The fresh porcine livers (n= 6) were provided by a local meat retailer (Cárnicas M. Boo, Ourense, Spain). The connective tissues and external fat were removed from the porcine liver before they were chopped and frozen at − 20 ◦ C till further use. Enzymatic hydrolysis was carried out using bioprotease LA 660 (Alcalase), bromelain 2000 U/g, and Papain 6000 USP provided by Biocon (Barcelona, Spain), meanwhile flavourenzyme ® 1000 L was supplied by Novozymes (Bagsværd, Denmark). The treatments for each enzyme were performed completely separately. Homogenization of samples was performed by mixing with ice (1:1 liver/ice) in a cutter machine (Talsa K3, Valencia, Spain) for 30 min before adding the enzyme. The enzymatic hydrolysis was performed at optimum pH and temperature described for each enzyme: Alcalase (50 ◦ C, pH = 8), bromelain (40 ◦ C, pH = 6 ), flavourenzyme ® (50 ◦ C, pH = 5.5), and papain (37 ◦ C, pH = 6). An enzyme-substrate ratio of 1:100 (w/w) was employed in all incubations for seven hours under agitation in an orbital shaker incubator (125 rpm). During the hydrolysis, periodic adjustments of pH were performed by employing NaOH or HCl 1N. Finally, hydrolysates were heated at 95 ◦ C for 3 min to deactivate the enzymes and, afterwards, liver homogenates were cooled in an ice bath. Hydrolysates were centrifuged (Allegra X-22R Centrifuge, Beckman Coulter, Barcelona, Spain) at 4000 × gfor 10 min. The purified extracts were frozen at − 80 ◦ C until analysis. For each enzymatic treatment, hydrolysis was conducted in triplicate. 2.2. Free Amino Acid Profile The extraction of free amino acids was carried out as follows: hydrolysates extract (0.5 g) was homogenized with 25 mL of HCl 0.1 M for 8 min employing a disperser (Ika, T 25 digital Ultra-Turrax ® , Staufen, Germany). Afterwards, it was cooled and centrifuged at 5000 × gfor 20 min and 200 µ L of the supernatant was mixed with 800 µ L of acetonitrile to precipitate possible unhydrolyzed proteins. Finally, centrifugation at 5000 × gfor 5 min was performed and an aliquot was frozen at − 20 ◦ C and stored until analysis. Derivatization and separation by HPLC were carried out according to Franco and Lorenzo [ 10 ]. Free Antioxidants 2022,11, 27 3 of 13 amino acids were identified by retention time using an amino acid standard and expressed in mg/100 g of pork liver. 2.3. Antioxidant Activity 2.3.1. DPPH Radical Scavenging Activity The DPPH assay was performed following Brand-Williams et al. [ 11 ] with slight modifications. A sample of 100 µ L was mixture with 3900 µ L of DPPH solution (60 µ M in methanol) and incubated for 10 min at 37 ◦ C. Absorbance was measured at 515 nm (Shimadzu spectrophotometer, Kyoto, Japan). Trolox reactive was the standard used and results were expressed as µg Trolox equivalents (TE)/g sample. 2.3.2. ABTS Radical Scavenging Activity ABTS Radical Cation Decolorization was performed following Re et al. [ 12 ] with slight modifications. ABTS was prepared by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate and maintaining the mixture in the dark at room temperature for 12–16 h before its utilization. Before use, the ABTS stock solution was diluted with distilled water to achieve an absorbance of 0.70 at 734 nm, being equilibrated at 30 ◦ C. The solution (980 mL) was added to an aliquot of 20 mL of each hydrolysate/standard. Absorbance was measured after 10 min in the darkness. Ascorbic acid was the standard used and results were expressed as mg ascorbic acid/100 g sample. 2.3.3. Ferric Reducing Antioxidant Power Assay (FRAP) The FRAP test was carried out following Benzie and Strain [ 13 ], with minor changes. FRAP reagent was freshly prepared from 0.3 M acetate buffer (pH 3.6), 10 mM 2,4,6tripyridyl-s-triazine in 40 mM HCl and 20 mM FeCl 3 :6H 2 O in the ratio of 10:1:1 (v:v:v). An aliquot of 900 mL of FRAP solution was mixed with 30 µ L of properly diluted samples and 90 µ L of distilled water. After incubation for 20 min at 37 ◦ C in the darkness, the absorbance was determined at 593 nm. The FeSO 4 was the standard used and results were expressed as µmol Fe+2/100 g sample. 2.3.4. Oxygen Radical Absorbance Capacity Assay (ORAC) The ORAC test was performed following Huan et al. [ 14 ] with minor modifications. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), being 200 µ L of the final volume of the reaction mixture. Twenty-five µ L of dilute sample and 150 µ L of 0.8 µ M fluorescein (oxidizable substrate) were added into the internal wells of a black 96-well microplate (Biotek, Synergy H1, Winooski, VT, USA) and which was immediately incubated at 37 ◦ C for 30 min in the own fluorescence instrument. Afterwards, 25 µ L of 2,2-azobis (2-methylpropionamidine) dihydrochloride 184 mM solution were added rapidly to each well to begin the reaction in the microplate. The fluorescence was measured with excitation and emission filters of 485 nm and 528 nm, respectively. The phosphate buffer and Trolox were used as the blank and standard reference, respectively. The results were estimated based on the differences of areas under the curves of fluorescence decay of the fluorescein between the blank and the sample and expressed as mg Trolox Equivalent (TE)/g sample. 2.4. Peptidomic Analysis 2.4.1. 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Elution was done with a linear gradient from 7% to 45% B in A for 20 min. (A: 0.1% FA; B: ACN, 0.1% FA) at a flow rate of 300 nL/min. Peptides were identified in a mass spectrometer nanoESI qQTOF (6600 plus TripleTOF, Antioxidants 2022,11, 27 4 of 13 SCIEX, Framingham, MA, USA) in a data-dependent mode. Samples were ionized in a Source Type: Optiflow < 1 µ L Nano applying 3.0 kV to the spray emitter at 200 ◦ C. Survey MS1 scans were acquired from 350–1400 m/zfor 250 ms. The quadrupole resolution was set to ‘LOW’ for MS2 experiments, which were acquired 100–1500 m/zfor 25 ms in ‘high sensitivity’ mode using the following switch criteria: charge: +1 to +4; minimum intensity; 100 counts per second. Up to 50 ions were selected for fragmentation after each survey scan. Dynamic exclusion was set to 15 s. The system sensitivity was controlled by analyzing 500 ng of K562 trypsin digestion. In these conditions, 2260 proteins were identified (FDR < 1%) in 45 min gradient. ProteinPilot v 5.0. (SCIEX) default parameters were employed to generate peak lists directly from 6600 plus TripleTOF wiff files. The Paragon algorithm [ 15 ] of ProteinPilot v 5.0 was used to search the Uniprot mammals database with the following parameters: none enzyme specificity, taxonomy restricted to pig, and the search effort set to rapid. 2.4.2. Label-Free Relative Quantitative Analysis by Mass Spectrometry The quantification of peptides was performed according to the label-free methodology described by [ 2 ]. This approach is based on the measurement of relative ion intensities of extracted ion chromatograms (XICs) to determine the ratios for individual peptides, employing three replicates per digested hydrolysate. Peptides were quantified using PeakView v1.1 software (AB Sciex, Framingham, MA, USA) and analyzed with Marker View v1.3 software (AB Sciex, Framingham, MA, USA). The protein grouping was carried out by the Pro group algorithm. A protein group in a Pro Group Report is a set of proteins that share some physical evidence. Unlike sequence alignment analyses where full-length theoretical sequences are compared, the formation of protein groups in Pro Group is guided entirely by observed peptides only. As observed peptides were identified from experimentally acquired spectra, the grouping can be guided by spectra usage. Then, unobserved regions of protein sequence play no role in explaining the data. 2.5. Statistical Analysis Statistical analysis was performed employing the IBM SPSS Statistics 23.0 program (IBM Corporation, Somers, NY, USA). An ANOVA was applied to evaluate the effect of each enzyme treatment on the antioxidant activity of the hydrolysate. The least-square means (LSM) of the four treatments were separated using Duncan’s post hoc test. All statistical tests of LSM were performed for a significance level of p< 0.05. Correlations among antioxidants tests (p< 0.01) and identified and quantified amino acids and peptides were determined employing Pearson’s linear correlation coefficient. To better understand the relationships among the different effects of the enzymes over hydrolysates antioxidant activity, a cluster analysis based on the unweighted pair group method with arithmetic mean (UPGMA) dendrogram was performed using XLSTAT 2021.3.1 (Addinsoft, Paris, France). 3. Results 3.1. Characterization of Porcine Liver Hydrolysates by Enzymatic Reaction In the present study, the enzymatic hydrolysis was carried out using four enzymes, alcalase, bromelain, flavourzyme, and papain, for 7 h to reach the most antioxidant activity. 3.1.1. Amino Acid Composition of Porcine Liver Hydrolysates The free amino acids (FAA) profiles which also provide a rough idea of the hydrolysis process are shown in Table 1. The highest amount of FAA was achieved using flavourzyme (3065.13 mg/100 g liver) and the lowest in papain hydrolysates (1285.73 mg/100 g liver), reaching statistical differences (p< 0.05). On the contrary, between hydrolysates from alcalase (1572.38 mg/100 g liver) and bromelain (1904.05 mg/100 g liver), no significant differences (p> 0.05) were found on FAA total content. All liver hydrolysates FAA profiles from the four treatments were characterized by a high level of leucine, lysine, and valine in the essential fraction. Indeed, leucine ranged from 152.95 to 353.05 mg/100 g protein after Antioxidants 2022,11, 27 5 of 13 hydrolysis performed by papain and flavourzyme, respectively. In the case of valine, the lower value (77.80 mg/100 g protein) and the higher value (220.51 mg/100 g protein) were also produced by flavourzyme and papain, respectively. Table 1. Free amino acid profile of liver (mg/100 g liver) from hydrolysis treatment with different enzymes (alcalase, bromelain, flavourzyme, and papain). AA Alcalase Bromelain Flavourzyme Papain SEM p-Value Essential His 55.28 d137.76 b212.15 a89.31 c12.88 <0.0001 Iso 88.10 b66.38 c149.88 a65.11 c7.70 <0.0001 Leu 192.40 bc 221.65 b353.05 a152.95 c17.40 <0.0001 Lys 189.90 b213.60 ab 239.41 a74.83 c14.94 <0.0001 Met 93.21 b79.38 b142.68 a52.50 c7.33 <0.0001 Phe 83.01 b69.30 b189.71 a63.65 b11.40 <0.0001 Thr 1.08 d68.26 b140.46 a29.40 c11.05 <0.0001 Val 111.65 b79.31 c220.51 a77.80 c12.89 <0.0001 Total EAA 814.65 b935.66 b1647.88 a605.56 c87.86 <0.0001 Non-Essential Arg * 22.36 c81.88 b192.26 a8.71 c15.43 <0.0001 Ala 176.50 a124.43 bc 151.41 ab 98.75 c9.19 0.008 Asp 7.21 c37.96 b98.01 a38.65 b7.22 <0.0001 Cis 153.51 b37.96 c204.36 a25.11 c16.32 <0.0001 Glu 127.08 b59.40 c234.11 a137.01 b13.90 <0.0001 Gli 130.63 b161.50 a131.13 b125.88 b4.04 0.01 Pro 22.35 c229.35 a53.01 b60.55 b17.51 <0.0001 Ser 10.63 d148.00 b234.58 a93.05 c17.58 <0.0001 Tau * 16.25 d23.46 c31.40 b42.63 a2.19 <0.0001 Tyr 91.18 a64.36 b86.93 a49.80 c4.07 <0.0001 Total NEAA 757.73 c968.38 b1417.25 a680.16 c66.57 <0.0001 Total FAA 1572.38 bc 1904.05 b3065.13 a1285.73 c153.79 <0.0001 Ratio EAA/NEAA 1.07 b0.96 c1.16 a0.88 d0.02 <0.0001 * Arginine and taurine are considered as semi-essential amino acids; EAA = essential amino acids; NEAA = non-essential amino acids; AA = amino acids; SEM = standard error of mean; a to d upper letters indicate significant differences at p< 0.05 level using Duncan test. 3.1.2. Peptide Composition of Porcine Liver Hydrolysates The peptidomic profiles in pork liver hydrolysates were inferred using the quantifications of LC-MS/MS. After enzymatic treatments, the peptides from pork liver were identified and quantified by LC-MS/MS. For the cluster analysis, only data from peptides identified with differential abundance (44 peptides) were considered in the UPGMA dendrogram (Figure 1). As can be observed, hydrolysates of enzymatic treatments (alcalase, bromelain, flavourenzyme, and papain) were grouped into four groups. This finding demonstrated that the peptide profile is different and distinctive for each enzymatic treatment. Antioxidants 2022,11, 27 6 of 13 Antioxidants 2022, 11, x FOR PEER REVIEW 6 of 13 Figure 1. Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on peptides quantifications provided by the action of alcalase, bromelain, papain, and flavouryme® on pork liver. 3.2. Antioxidant Capacity of Porcine Liver Hydrolysates The antioxidant capacity of pork liver hydrolysates was measured using DPPH, ABTS, FRAP, and ORAC as shown in Figure 2. Hydrolysates from alcalase and bromelain resulted in the most antioxidant capacity. Alcalase enzyme generated the highest antioxidant activity measured by ORAC (22.64 mg Trolox/g) and ABTS (655.63 mg AA/100 g); meanwhile, the bromelain enzyme showed the greatest antioxidant values by FRAP (32.55 µmol Fe+2/100 g) and DPPH (283.87 µg Trolox/g). Figure 2. Antioxidant in vitro tests to evaluate antioxidant capacity from the four different pork liver hydrolysates produced by alcalase, bromelain, papain, and flavouryme. a to d upper letters indicate significant differences at p < 0.05 level using Duncan test. 3.2.1. Antioxidant Effect of Free Amino Acids from Porcine Liver Hydrolysates The antioxidant activity of these hydrolysates may largely be due to FAA. As shown in Table 2 the FAA involved in antioxidant activity have different chemical properties in Figure 1. Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on peptides quantifications provided by the action of alcalase, bromelain, papain, and flavouryme®on pork liver. 3.2. Antioxidant Capacity of Porcine Liver Hydrolysates The antioxidant capacity of pork liver hydrolysates was measured using DPPH, ABTS, FRAP, and ORAC as shown in Figure 2. Hydrolysates from alcalase and bromelain resulted in the most antioxidant capacity. Alcalase enzyme generated the highest antioxidant activity measured by ORAC (22.64 mg Trolox/g) and ABTS (655.63 mg AA/100 g); meanwhile, the bromelain enzyme showed the greatest antioxidant values by FRAP (32.55 µ mol Fe +2 /100 g) and DPPH (283.87 µg Trolox/g). Antioxidants 2022, 11, x FOR PEER REVIEW 6 of 13 Figure 1. Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on peptides quantifications provided by the action of alcalase, bromelain, papain, and flavouryme® on pork liver. 3.2. Antioxidant Capacity of Porcine Liver Hydrolysates The antioxidant capacity of pork liver hydrolysates was measured using DPPH, ABTS, FRAP, and ORAC as shown in Figure 2. Hydrolysates from alcalase and bromelain resulted in the most antioxidant capacity. Alcalase enzyme generated the highest antioxidant activity measured by ORAC (22.64 mg Trolox/g) and ABTS (655.63 mg AA/100 g); meanwhile, the bromelain enzyme showed the greatest antioxidant values by FRAP (32.55 µmol Fe+2/100 g) and DPPH (283.87 µg Trolox/g). Figure 2. Antioxidant in vitro tests to evaluate antioxidant capacity from the four different pork liver hydrolysates produced by alcalase, bromelain, papain, and flavouryme. a to d upper letters indicate significant differences at p < 0.05 level using Duncan test. 3.2.1. Antioxidant Effect of Free Amino Acids from Porcine Liver Hydrolysates The antioxidant activity of these hydrolysates may largely be due to FAA. As shown in Table 2 the FAA involved in antioxidant activity have different chemical properties in Figure 2. Antioxidant in vitro tests to evaluate antioxidant capacity from the four different pork liver hydrolysates produced by alcalase, bromelain, papain, and flavouryme. a to d upper letters indicate significant differences at p< 0.05 level using Duncan test. 3.2.1. Antioxidant Effect of Free Amino Acids from Porcine Liver Hydrolysates The antioxidant activity of these hydrolysates may largely be due to FAA. As shown in Table 2the FAA involved in antioxidant activity have different chemical properties in their side chains as polar uncharged (serine and threonine), polar charged (histidine and aspartic Antioxidants 2022,11, 27 7 of 13 acid), and apolar (proline). Furthermore, the polar FAAs decreases the antioxidant capacity (negative correlation) in contrast to proline as an apolar FAA (positive correlation). It has been demonstrated that FAAs produce different antioxidant activities depending on the properties of their side residues. Specifically, cysteine, methionine, tryptophan, tyrosine, and histidine have proved to have higher antioxidant capacity than other amino acids because they are relatively easily oxidized [ 16 ]. However, neither of these FAA significantly improved the antioxidant activity of hydrolysates in our study. Table 2. Correlations between free amino acids and in vitro antioxidant test. Aminoacid ABTS DPPH FRAP ORAC His −0.654 −0.542 Thr −0.662 −0.559 Asp −0.687 −0.893 Pro 0.704 0.541 Ser −0.770 −0.622 Tau −0.727 −0.666 −0.591 Only those correlations significant (p< 0.01) in at least two tests and with a correlation coefficient higher than 0.5 are shown. 3.2.2. Peptides of Porcine Liver Hydrolysates with Antioxidant Capacity From the peptides quantified with significant differences among the four enzymatic treatments (Table 3), correlation with antioxidant capacity using ORAC, FRAP, ABTS, and DPPH tests were analyzed. From this initial set of peptides, seventeen peptides showed a significant correlation (p≤0.05) with a coefficient correlation higher than 0.5 (Table 4). Derived peptides of collagen resulted in a large influence on antioxidant capacity mainly correlated with DPPH both positively and negatively. The peptide SVGPVGPAGPI and SP[Oxi]GPDGKTGPP[Oxi]GPAG produced by the action of alcalase and bromelain were positively correlated with the antioxidant test of DPPH (r = 0.833 and 0.850, p< 0.01; respectively). On the contrary, GSP[Oxi]GPSGSP[Oxi]GQRGEP[Oxi]GP and GAP[Oxi]GDKGETGPSGPAGPT by hydrolyzation with papain were negatively correlated (r = −0.777 and −0.843, p< 0.05 and 0.01; respectively). It should be noted that three peptides from albumin also resulted in antioxidant ability correlated with ABTS and ORAC assays. Thus, the peptides NDNPDIPKLKPDPV and DNPDIPKLKPDPVAL produced by alcalase action was correlated with ABTS (r = 0.917 and 0.990, p< 0.01; respectively) and ORAC (r = 0.729 and 0.788, p< 0.05; respectively). Unlike others, the peptide DFQEDEQKFW hydrolyzed by bromelain was correlated with DPPH (r = 0.836, p< 0.05). Peptides with antioxidant capacity, from iron proteins including cytochrome B and haemoglobin, were also detected. As other peptides produced by alcalase, LVLMILVL (cytochrome B) was strongly correlated with ABTS and ORAC (r = 0.983 and 0.765, p< 0.01 and 0.05; respectively). The peptide of SDGLKHLDNLK (haemoglobin) was related to the DPPH test (r = 0.753, p< 0.05), as with other peptides of the bromelain enzyme. Other peptides from the proteins related to metabolic pathways such as fructosebisphosphate aldolase, dihydropyrimidinase, and arginosuccinate synthase were also particularly correlated with ABTS and ORAC. These peptides of metabolic proteins were particularly generated by alcalase and correlated with antioxidant capacity using ABTS and ORAC. Overall, the main candidates of antioxidant peptides from porcine liver hydrolysates resulting from alcalase action were particularly sensitive to ORAC and ABTS. Additionally, bromelain action appears to increase antioxidant peptides measured by the DPPH test. Antioxidants 2022,11, 27 8 of 13 Table 3. Pork liver peptides identified and quantified by LC-MS/MS from hydrolysis treatment with different enzymes (alcalase, bromelain, flavourzyme, and papain). Peptide Sequence Protein of Origin Gen (Uniprot ID) Alcalase Bromelain Flavourzyme Papain SEM p-Value GVRGPNGDSGRP[Oxi]GEP[Oxi]G Fibrillar collagen NC1 domain-containing protein COL1A2 46,984 a237,462 b655,298 c12,167 a97.120 <0.001 GSP[Oxi]GPSGSP[Oxi]GQRGEP[Oxi]GPQ Collagen type III alpha 1 chain COL3A1 11,730 a13,765 a232,011 b22,998 a35.482 <0.001 TDPDAPSRKDPKYR UP PEBP1 266,420 c322,481 d94,672 b10,957 a48.467 0.002 SP[Oxi]GPDGKTGPP[Oxi]GPAG Collagen alpha-1(I) chain preproprotein COL1A1 40,343 a355,355 b45,816 a33,045 a51.950 <0.001 GSP[Oxi]GPSGSP[Oxi]GQRGEP[Oxi]GP Collagen type III alpha 1 chain COL3A1 27,410 a14,226 a198,625 b277,532 c45.123 0.024 GASGPAGPRGPP[Oxi]GSAGAP[Oxi]GKDG Collagen alpha-1(I) chain preproprotein COL1A1 5415 a95,886 b278,639 c107,394 b39.555 0.022 VLSAADKANVK GLOBIN domain-containing protein LOC110259958 7566 a12,638 a10,862 a809,312 b142.092 0.042 AP[Oxi]GDKGETGPSGPAGPTG Collagen alpha-1(I) chain preproprotein COL1A1 443 a8189 a151,220 b463,662 c73475 0.009 GKDGEAGAQGPP[Oxi]GPA Collagen alpha-1(I) chain preproprotein COL1A1 32,253 a10,023 a833,315 b77,903 a130.966 <0.001 GVQGPP[Oxi]GPAGEEGKRG Collagen alpha-1(I) chain preproprotein COL1A1 9582 a19,649 a595,576 b4043 a95.797 <0.001 RKPPTDEESLEK Glutathione transferase GSTO1 372,448 c6332 a29,252 a122,768 b56.928 0.009 M[DTM]GDSRDPASDQMK Catalase CAT 15,852 203,806 934 428,309 72.553 0.061 GHQGAVGSP[Oxi]GPAGP Collagen type III alpha 1 chain COL3A1 66,649 199,720 7523 375,109 59.011 0.058 GASGPAGPRGPP[Oxi]GSA Collagen alpha-1(I) chain preproprotein COL1A1 9990 a17,658 a308,010 c52,706 b46.659 <0.001 GPVGPSGPP[Oxi]GKDGASG Collagen type III alpha 1 chain COL3A1 31,327 a25,684 a14,649 a212,882 b31.988 0.007 GAP[Oxi]GDKGETGPSGPAGPT Collagen alpha-1(I) chain preproprotein COL1A1 71,644 a8189 a267,506 b455,180 c69.021 0.011 SGPAGPRGPP[Oxi]GSA Collagen alpha-1(I) chain preproprotein COL1A1 429,583 6914 173,859 116,358 64.360 0.051 GLP[Oxi]GTSGPP[Oxi]GENGKP[Oxi]GEP[Oxi]GPK Collagen type III alpha 1 chain COL3A1 40,928 588,757 285,781 156,852 126.713 0.560 GSP[Oxi]GERGEVGPAGPNG Fibrillar collagen NC1 domain-containing protein COL1A2 4779 a11,815 a509,034 b12,027 a81.786 <0.001 DQGPVGRTGETGASGP[Oxi]PG Fibrillar collagen NC1 domain-containing protein COL1A2 18,656 b23,656 b309,185 c11,533 a47.713 <0.001 AHHPDDFNPSVH GLOBIN domain-containing protein LOC110259958 88,515 23,406 362,462 889,540 209.499 0.549 GPIGSRGPSGPP[Oxi]GPDGNKGEP[Oxi]G Fibrillar collagen NC1 domain-containing protein COL1A2 1213 a3841 a208,112 c17,469 b32.948 <0.001 GPRGPP[Oxi]GAVGAP[Oxi]GPQG Fibrillar collagen NC1 domain-containing protein COL1A2 970 a248,171 b631,331 c4146 a97.820 <0.001 EQEKQNPDSEFH UP LOC100739741 23,441 124,249 268,476 77,106 40.512 0.131 PGQ.QKNQPMTPEAVK UP N/A 20,703 76,285 153,275 56,800 20.676 0.080 SDGLKHLDNLK GLOBIN domain-containing protein LOC100515788 1297 a6,029,690 c104,465 ab 831,244 b956.604 0.001 GAGGGAGGGGAGAGAGGGGAGA Glutamate metabotropic receptor 5 GRM5 74,644 b300,710 c21,625 a10,893 a44.587 <0.001 GPHEREPTAL AMP-binding domain-containing protein SLC27A5 331,724 7805 22,616 366,344 71.764 0.085 EPDAGDDDSKGSGQ Ras protein specific guanine nucleotide releasing factor 2 RASGRF2 951 a164,542 d26,459 b68,937 c23.835 0.001 LSDLHAHKLRVDPVN GLOBIN domain-containing protein LOC110259958 1955 a2686 a1123 a194,505 b32.480 0.006 GPN[Dea]GDSGRP[Oxi]GEP[Oxi]GLM Fibrillar collagen NC1 domain-containing protein COL1A2 241,883 23,838 49,764 124,129 38.072 0.145 LANVVALTMEPK 60 kDa chaperonin N/A 194,959 43,520 245,393 5966 47.643 0.220 GDAGPP[Oxi]GPAGPTGPP[Oxi]GPIGS Collagen alpha-1(I) chain preproprotein COL1A1 8915 26,500 221,936 111,099 35.314 0.060 IGENIDEKPLPT UP N/A 120,355 142,542 144,051 459,694 61.995 0.119 AGSPGGGAAGPGPAGGGP Ran-binding protein 9 RANBP9 658,925 c84,934 a261,216 b172,460 ab 85.995 0.009 Antioxidants 2022,11, 27 9 of 13 Table 3. Cont. Peptide Sequence Protein of Origin Gen (Uniprot ID) Alcalase Bromelain Flavourzyme Papain SEM p-Value DPPKTASYPVIIQ Rhodanese domain-containing protein TSTD2 237,853 b18,072 a40,463 a29,690 a34.637 0.001 NDNPDIPKLKPDPV Albumin ALB 210,501 c13,125 a49,642 b52,975 b29.353 0.003 ILASCGLTDAACRLL NACHT, LRR and PYD domains-containing protein 5 Nlrp5 63,744 285,536 8094 96,696 44.753 0.087 GIIGPLGILGP collagen alpha-1(XXVII) chain isoform X1 COL27A1 2610 a13,191 a1,506,056 b22,977 a244.419 <0.001 IGAMIGAI ATPase CSA50_09160 124,276 a51,483 a2,046,297 b99,598 a320.834 <0.001 DSGAPIKIPVGPE ATP synthase subunit beta ATPB 439,736 b15,127 a22,057 a66,749 a67.888 0.002 LEGTLLKPNMVT Fructose-bisphosphate aldolase ALDOB 5,032,015 c19,118 a10,580 a214,393 b811.361 <0.001 DNPDIPKLKPDPVAL Albumin ALB 164,367 d55,725 c27,651 b16,727 a22.199 <0.001 PGQ.QSFSDGLKHLDNLK GLOBIN domain-containing protein LOC100515788 9644 a14,854 a2152 a1,049,207 b179.207 0.017 WDGLNPDKLYT UP PEBP1 99,491 a358,524 b32,772 a326,020 b57.198 0.034 SGNPNGEGLPHWP Carboxylic ester hydrolase APLE 19,318 b5361 a557,870 c12,771 ab 89.312 <0.001 PGQ.QSFSDGLKHLDNLKGTFAK GLOBIN domain-containing protein LOC100515788 340 530 487 791,589 142.101 0.051 GPPLRPDPSTPDFL Dihydropyrimidinase DPYS 382,297 c12,485 b1754 a3846 a61.622 <0.001 DFQEDEQKFW Albumin ALB 526,201 b1,619,475 c17,232 a400,233 b226.685 <0.001 GIPIPVTPKNPW Argininosuccinate synthase ASS1 809,850 b12,991 a764 a5895 a131.502 <0.001 DQLHVDPENFRLLG GLOBIN domain-containing protein LOC100515788 33,561 b7863 a603,419 c5475 a96.371 <0.001 AMPDIPVPLTN Aldehyde dehydrogenase 1 family member A1 ALDH1A1 35,049 a188,598 a12,213 a576,409 b92.337 0.037 DQLHVDPENFRLL GLOBIN domain-containing protein LOC100515788 1047 a9104 a1585 a232,710 b40.397 0.035 SVGPVGPAGPI collagen alpha-2(I) chain isoform X2 LOC101341020 1,210,722 c1,126,713 c476,081 b7974 a187.098 <0.001 PGQ.QLHVDPENFRLLG GLOBIN domain-containing protein LOC100515788 3571 a14,016 a446 a268,209 b45.742 0.025 PGQ.QLHVDPENFRLL GLOBIN domain-containing protein LOC100515788 4500 9124 10,757 702,733 125.698 0.058 LVLMILVL Cytochrome b CYB 427,002 b18,871 a15,799 a582 a68.566 <0.001 Only those peptides with a concentration higher than 150,000 in at least one treatment group are shown. Abundance mean values of those peptides with significant differences (p< 0.05) are noted by a–d upper letters and the highest peptide value are marked in bold.