Antioxidant activity and peptidomic analysis of porcine liver hydrolysates using alcalase, bromelain, flavourzyme and papain enzymes

1 Antioxidant activity and peptidomic analysis of porcine liver 1 hydrolysates using alcalase, bromelain, flavourzyme and 2 papain enzymes 3 4 María López-Pedrouso1, Paula Borrajo2, Mirian Pateiro2, José M. Lorenzo2 and 5 Daniel Franco2* 6 1Department of Zoology, Genetics and Physical Anthropology, University of 7 Santiago de Compostela, Santiago de Compostela -15872, Spain 8 2Centro Tecnológico de la Carne de Galicia, Rúa Galicia Nº 4, Parque 9 Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense 10 11 *corresponding author: [email protected] 12 13 2 Abstract: 14 Porcine liver can be used to prepare hydrolysates with antioxidant activity 15 employing proteolytic enzymes such as alcalase, bromelain, papain and 16 flavourzyme. In this study, the antioxidant activity of these four porcine liver 17 hydrolysates was evaluated by DPPH, ABTS, FRAP and ORAC assays and the 18 identification of bioactive peptides was carried out by SWATH-MS technology. 19 According to the SDS-PAGE pattern, the proteolysis index and the free amino 20 acids amount, the protein degradation was clearly different among the studies 21 enzymes. Indeed, alcalase enzyme produced the release of small peptides, 22 meanwhile flavourzyme produced higher level of free amino acids. The heatmap 23 analysis showed a peptidomic pattern more differentiated for alcalase than for the 24 other enzymes. The peptides most abundant and correlated with antioxidant 25 capacity were APAAIGPYSQAVLVDR from uncharacterized protein, 26 GLNQALVDLHALGSAR, ALFQDVQKPSQDEWGK and LSGPQAGLGEYLFER 27 from ferritin and LGEHNIDVLEGNEQFINAAK from trypsinogen. The production 28 and characterization of biopeptides is a new challenge of meat industry. 29 30 Keywords: proteolytic enzymes, antioxidant peptides, swine industry, peptide 31 mapping, mass spectrometry 32 33 3 1. Introduction 34 Large amount of wastes is generated by meat industry becoming a severe 35 problem for sustainability. Therefore, the improvement and use of these meat by36 products is a major goal for worldwide society. Among animal by-products, the 37 edible fraction (accounting for 55% of the production) can be used and re-process 38 for human consumption, meanwhile the rest of the fraction could be employed for 39 agricultural and industrial applications (Alao, Falowo, Chulayo, & Muchenje, 40 2017). This meat offal are rich sources of proteins resulting a suitable raw 41 material for preparing protein hydrolysates. Animal by-products such as blood, 42 bones, collagen and organs have been employed as source of protein 43 hydrolysates (Borrajo et al., 2019). Food peptides from parent proteins can reach 44 the intestine as peptide or be liberated after digestion. This highly valuable protein 45 hydrolysates are rich in biopeptides, with high digestibility and bio-absorption as 46 well as different biological activities such as antihypertensive, antioxidant or 47 antithrombotic among others (Yu, Hsu, Chang, & Tan, 2017). These bioactive 48 peptides have small molecular weight (400-2000 Da) with sequences of 49 approximately 4-16 amino acids. Accordingly, it has been demonstrated that the 50 bioactivity of the smaller peptides showed the higher activity antioxidant, although 51 this activity is also large extent determined by the amino acid sequence (Liu, Xing, 52 Fu, Zhou, & Zhang, 2016). 53 All these factors result in a very complex issue which can be studied by an Omic 54 strategy. Peptidomic together with proteomic approach can provided a 55 comprehensive vision of proteins/peptides and their bioactivity in a complex food 56 matrix. A main objective of peptidomics is to map of endogenous peptides 57 resulting from food-processing and protein digestion, as well as bioactive 58 4 peptides identification (Dallas et al., 2015). Within animal products, peptidomic 59 studies revealed a great influence of proteolysis on peptide bioactivity from beef, 60 pork, chicken and turkey during digestion (Martini, Conte, & Tagliazucchi, 2019; 61 Zhao et al., 2019). 62 Protein hydrolysates can be obtained from enzymatic, chemical and microbial 63 hydrolysis, but the former one is the most widely selected process for food and 64 pharmaceutical industries to produce bioactive peptides (O’Sullivan, Lafarga, 65 Hayes, & O’Brien, 2017). The most frequently used enzymes come from animal 66 tissues (pepsin and tripsin), plants (papain, ficin, and bromelain), and microbial 67 sources (alcalase®, flavourzyme®, neutrase®, collagenase, or proteinase K) 68 (Marzia, Santillo, Mariangela, Antonella, & Rosaria, 2017). Papain, pepsin or 69 alcalase have been reported as the most successful enzymes, releasing 70 antioxidant and anti-inflammatory biopeptides from animal tissues (O’Sullivan et 71 al., 2017). For instance, myofibrillar proteins of porcine muscle have 72 demonstrated antihypertensive activity through angiotensin-I converting enzyme 73 inhibitory mechanisms (Katayama et al., 2008) as well as antioxidant activity 74 (Saiga, Tanabe, & Nishimura, 2003). Recent investigations have proved that by75 products from porcine organs can be used to prepare appealing hydrolysates. In 76 fact, porcine liver protein hydrolysates from enzymatic hydrolysis with several 77 enzymes such as papain, alcalase, pepsin and trypsin (Verma, Chatli, Kumar, & 78 Mehta, 2019) or microbial suspension of Monascus purpureus (Yu et al., 2017) 79 have showed a significant antioxidant capacity. 80 Therefore, there is a great interest on functional foods field by food and 81 nutraceutical industries. Indeed, these biopeptides could be used both as 82 preservatives in food and beverage providing a functional purpose, due to 83 5 antioxidant peptides might be protect human body from damage of oxidative 84 stress and reduce the risk of degenerative diseases (Liu et al., 2016). 85 The aim of the present study is to assess the antioxidant activity of four porcine 86 liver hydrolysates produced by alcalase, bromelain, papain and flavourzyme, 87 identifying the bioactive peptides sequences by SWATH-MS methodology. 88 89 2. Materials and Methods 90 2.1. Porcine liver samples and chemical composition 91 A total of eight fresh porcine livers were purchased at a local meat market 92 (Cárnicas M. Boo, Ourense). The assessment of moisture, protein, fat and ash 93 was carried out in accordance with Franco & Lorenzo, (2014). The carbohydrate 94 content was calculated based on the difference. The proteolysis index was 95 calculated as the ratio: (non−protein nitrogen/nitrogen total) × 100. Total nitrogen 96 content was determined with Kjeldahl method, meanwhile non-protein nitrogen 97 was determined following protocol described by Lorenzo, García Fontán, Franco, 98 & Carballo, (2008). The liver amino acid profile was performed after protein 99 hydrolysis employing high performance liquid chromatography with fluorescence 100 detector according to Franco & Lorenzo, (2014). Similarly, the same protocol was 101 employed to determinate the total free amino acids without the hydrolysis step. 102 2.2. Enzymatic hydrolysis of porcine liver 103 Livers were cleaned of fat and connective tissues and subsequently were cut into 104 small cubes and frozen at –20 ºC with the aim of reduce liver viscosity. 105 Subsequently, a homogenization mixing with ice (1:1 liver/ice) using a cutter 106 6 machine (Talsa K3, Valencia, Spain) was achieved. Enzymatic hydrolysis of liver 107 was carried out using four different enzymes (Papain 6000 USP, Bromelain 2000 108 U/g and bioprotease LA 660 (alcalase) supplied by Biocon, Spain) and 109 flavourzyme 1000L by (Novozymes, Bagsvaerd Denmark). Liver solutions (1:1, 110 w/w) were preincubated for 30 minutes at the optimal temperature for each 111 protease before addition of the enzyme. The conditions were: (37 ºC and pH=6) 112 for papain, (40 ºC and pH=6) for bromelain, (50 ºC and pH08) for alcalase, and 113 (50 ºC and pH=5.5) for flavourzyme. Enzymes were added in an enzyme114 substrate ratio of 1:100 (w/w). Enzymatic hydrolysis was carried out in an orbital 115 shaker-incubator at 125 rpm for seven hours, adjusting the above pH optimal by 116 addition of NaOH or HCL 1N. After this time, enzymes were heat deactivated at 117 95 ºC for 3 min and the liver hydrolysates were cooled to room temperature using 118 an ice bath. Afterward, they were centrifuged at 10.000g for 10 min using an 119 Allegra X-22R Centrifuge (Beckman Coulter) and supernatants were filtered by 120 0.45 µm and subsequently lyophilized until further analysis. 121 2.3. SDS-PAGE Analysis 122 Peptide extracts from liver hydrolysates were separated under reducing 123 conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS124 PAGE). Fifteen micrograms were loaded on 10% pre-cast gels using a Mini125 Protean Tetra Cell equipment (Biorad Lab., Hercules, CA, USA). The Laemmli 126 buffer (62.5 mM TrisHCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromophenol 127 Blue, 100 mM DTT) was used to dissolve and denature the samples (5 min, 95 128 °C). Staining was carried out using Coomassie Brilliant Blue G-250 solution. The 129 images were acquired using the Gel Doc XR+ system (Bio-Rad Laboratories) and 130 analysed by Image Lab™ software (Biorad Lab., Hercules, CA, USA). 131 7 2.4. Protein extraction and digestion 132 Lyophilized powder (50 mg) of liver hydrolysates was homogenised in RIPA 133 buffer [200 mmol/L Tris/HCl (pH 7.4), 130 mmol/L NaCl, 10% (v/v) glycerol, 0.1% 134 (v/v) SDS, 1% (v/v) Triton X-100, 10 mmol/L MgCl2] and adding anti-proteases 135 and anti-phosphatases (Sigma-Aldrich, St. Louis, MO, USA) using a TissueLyser 136 II (Qiagen, Tokyo, Japan). Afterwards, the hydrolysate was centrifuged at 137 14.000g at 4 ºC for 20 min to obtain peptide solutions. The concentration was 138 quantified by RC-DC kit (Biorad Lab., Hercules, CA, USA). The peptides were 139 concentrated in a gel single in band of 10% SDS-PAGE and excised into pieces. 140 The gel pieces were washed with Milli-Q and 50 mM ammonium bicarbonate in 141 50% methanol followed by dehydration with ACN by a vacuum centrifuge. The 142 resulting peptide extracts were reduced by 10 mM DTT in 50 mM ammonium 143 bicarbonate (60 ºC, 30 min) and then alkylated with 55 mM iodoacetoamide in 50 144 mM ammonium bicarbonate in darkness (room temperature, 30 min). Finally, the 145 peptides were digested with 20 ng/μL trypsin (Promega, Madison, USA) in 20 mM 146 ammonium bicarbonate (37 ºC, 16 h) and dissolved in 0.1% formic acid until 147 analysis. 148 2.5. Generation of the reference spectral library 149 To obtain a pooled sample of each group, 4 μg of peptides from each sample 150 were mixed and combined. The resulting solutions were analysed by shotgun 151 data-dependent acquisition (DDA) approach using micro-LC-MS/MS. To 152 separation of the peptides, the equipment used was a micro-LC system Ekspert 153 nLC425 (Eksigen, Dublin, CA, USA) and an YCM-TriartC18 column (150μm × 0.3 154 mm, 12 nm, s-3 μm) (YMC CO, Japan) at a flow rate of 5 μL/min. Solvent A 155 (water, 0.1% formic acid) and solvent B (ACN, 0.1% formic acid) were used to 156 8 prepare mobile phases in the liquid chromatography. The gradient elution was 157 produced by 5% to 95% B for 30 min, 5 min at 90% B and other 5 min at 5% B 158 for column equilibration, for a total time of 40 min. A quadrupole-TOF mass 159 spectrometer of model Triple TOF 6600 (SCIEX, Framingham, MA, USA) working 160 with data-dependent acquisition system in positive ion mode was used. The 161 selected parameters were: a 250 ms survey scan was performed from 400 to 162 1250 m/z and MS/MS experiments from 100 to 1500 m/z (25 ms of acquisition 163 time) for a total cycle time of 2.8 s. The fragment ion mass spectra of the identified 164 peptides was used to generate the spectral library for SWATH-MS peak 165 extraction using ProteinPilot software v.5.0.1. (SCIEX, Framingham, MA, USA) 166 through Uniprot Swiss-Prot database. 167 2.6. Quantification by SWATH and data analysis 168 Data independent acquisition (DIA) data from SWATH-MS analysis was 169 PeakView v.2.2. (SCIEX, Framingham, MA, USA) matching the reference 170 spectral library (Section 2.5.). Twenty-five samples from 5 groups (control, 171 alcalase, bromelain, papain and flavorzyme) with two technical replicates in each 172 case were analysed. Each peptide sample (4 μg) was analysed by LC as 173 described above with the following settings: acquisition time of 50 ms in a total 174 cycle time of 6.3 s and a cycle consisted of the acquisition of 65 scans per 175 SWATH window of variable width (1 m/z overlap) covering the 400 to 1250 m/z 176 mass range. All DIA files were loaded using the following settings: extraction 177 window (5 min); ion library mass tolerance (30 ppm); ten peptides per peptide; 178 six transitions per peptide; 95% peptide confidence; exclusion of modified and 179 shared peptides. A score and a false discovery rate (FDR) for each assigned 180 peptide were calculated by the software and only peptides with an FDR less than 181 9 1% were used for protein quantification. To compare the groups, a Student’s t182 test was used based on peptides with a p-value scoring above 0.05 and fold 183 change of 1.5 as cut-off. 184 2.7. Determination of antioxidant capacity 185 2.7.1. DPPH Radical Scavenging Activity 186 The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging method was carried out 187 according to Brand-Williams, Cubelier, & Berset, (1995) with slight modifications. 188 Aliquots of 100 μL of samples were added to 3900 μL of DPPH solution (60 μM 189 in methanol) and incubated during 10 min at 37 ºC. Then the absorbance was 190 measured in an UV-180 UV spectrophotometer spectrophotometer (Shimatzu, 191 Kyoto, Japan) at 515 nm. Each liver hydrolysates extract was analysed in 192 triplicate and its antioxidant activity was determined using trolox as standard, 193 expressing the results as μg Trolox equivalents (TE)/g sample. 194 2.7.2. ABTS Radical Scavenging Activity 195 This method was determined according to the procedure previously described by 196 Re et al. (1999) with some modifications. This assay is based on the ability of 197 antioxidants to quench the long-lived ABTS ((2,2-azinobis-(3-ethyl198 benzothiazoline-6-sulphonate) radical cation, a bluish-green chromophore with 199 specific absorption line at 734 nm. This radical was prepared mixing 7 mM ABTS 200 stock solution with 2.45 mM potassium persulfate, keeping the mixture in 201 darkness at room temperature for 12-16 h. Prior to use, the ABTS stock solution 202 was diluted with distilled water to achieve an absorbance of 0.70 at 734 nm, being 203 equilibrated at 30 ºC. A solution of 980 mL was added to an aliquot of 20 mL of 204 each hydrolysate. Afterward, absorbance was measured in an UV-180 UV 205 spectrophotometer at 734 nm. Each hydrolysate was analysed in triplicate and 206 16 hand, SWATH method afforded a label-free quantification of peptide mixtures 356 obtained by alcalase, bromelain, papain and flavourzyme (Table 1). Over 2000 357 peptides were identified and quantified in each sample, but only significantly 358 different peptides among the four enzymatic treatments were considered. Among 359 these 73 peptides, correlations between quantification and antioxidant activity 360 were performed, resulting 35 peptides with significant correlations (P<0.01) and 361 higher correlation coefficient than 0.5 that are depicted in Table 2. 362 The most abundant peptides were APAAIGPYSQAVLVDR from uncharacterized 363 protein (1,694.9; 46,689.5; 57,847.3 and 54,070.6); GLNQALVDLHALGSAR 364 from ferritin (27,606.1; 27,524.9; 10,359.4 and 24,380.3); 365 ALFQDVQKPSQDEWGK from ferritin (18,525.8; 18,502.5; 5,832.1 and 366 18,931.3); LSGPQAGLGEYLFER from ferritin (24,637.8; 22,074.5; 9,058.8 and 367 19,753.6) and LGEHNIDVLEGNEQFINAAK from trypsinogen (92,134.4; 368 51,491.8; 30,478.2 and 36,997.2) for alcalase, bromelain, flavourzyme and 369 papain, respectively. Other peptides were correlated with antioxidant capacity as 370 showed in Table 2, but their quantity was not so high, hence they will not be 371 described in the discussion. Additionally, the gastrointestinal digestion further 372 degrades these peptides in smaller peptides. Three of these peptides were 373 obtained from ferritin which is a main storage protein of iron in vertebrates located 374 in liver and a peptide from trypsinogen which is the proenzyme precursor of 375 trypsin. The iron intake contributes to maintain normal physiological process in 376 human body as well as oxygen transportation, storage and synthesis of 377 cytochromes and metalloenzymes (Heeney & Andrews, 2004). 378 As shown in Table 2, the APAAIGPYSQAVLVDR peptide displayed an important 379 negative correlation for DPPH, ABTS and FRAP (-0.523, -0.724 and –0.562, 380 17 respectively). Thus, the degradation of this peptide may be increased the radical 381 scavenging activity of the pork liver hydrolysate. In this sense, dipeptides which 382 includes amino acid tyrosine (Y) has been proved to be effective radical 383 scavenging because its aromatic amino acid could be contributing to stabilize 384 them (Du et al., 2019). On the contrary, peptides as GLNQALVDLHALGSAR, 385 ALFQDVQKPSQDEWGK and LSGPQAGLGEYLFER from ferritin showed a 386 strong and positive correlation with ORAC assay (0.743, 0.605 and 0.682, 387 respectively). It is possible to hypothesise that the increase in concentration of 388 these peptides could increase an absorption of oxygen capacity and this finding 389 is easily understandable due to ferritin protein can manage iron and oxygen 390 through ferroxidase sites and substrates of iron and oxygen (Liu, Hintze, 391 Lonnerdal, & Theil, 2006). Finally, the peptide LGEHNIDVLEGNEQFINAAK from 392 trypsinogen also demonstrated a high and positive correlation with ABTS, FRAP 393 and ORAC assays (0.789, 0.592 and 0.619, respectively). The ORAC method 394 measures the loss of fluorescence of a protein (β-phycoerythrin) due to a change 395 of its conformation by oxidative damage caused by peroxyl radicals, hence is the 396 most relevant method from a biological perspective integrating both degree and 397 time of antioxidant reaction (Zulueta, Esteve, & Frígola, 2009). This study 398 produced results which corroborate the findings that proved antioxidant peptides 399 from meat products which can protect cells and organisms from oxidative damage 400 as reviewed (Liu et al., 2016). 401 Proteins from animal tissues has also the advantage of a high content 402 methylhistidine and hydroximethyllysine and other essential amino acids in high 403 bioavailability. Moreover, hydrophobic amino acids such as alanine (A), 404 isoleucine (I), leucine (L), proline (P), phenylalanine (F) and tyrosine (Y) which 405 18 are included in these peptides increase the solubility in aqueous solution, 406 enhancing scavenge free radicals (Yu & Tan, 2017). This fact also suggests that 407 these peptides showed antioxidant capacity even higher after digestion. 408 Several of these antioxidant biopeptides contain other dipeptides or tripeptides, 409 which have also been found in the Biopep database (Minkiewicz, Iwaniak, & 410 Darewicz, 2019). Indeed, thirteen peptides contain dipeptides or tripeptides 411 described by another authors (Table 3). Therefore, it can be assumed that these 412 thirteen peptides are contributing to increase the antioxidant capacity of the pork 413 liver hydrolysate. In addition, it can be suggested that the bioactive peptides could 414 be contained in the sequence of parent peptides released by gastric digestion. It 415 is important to highlight that the ferritin peptides GLNQALVDLHALGSAR and 416 ALFQDVQKPSQDEWGK available in large quantities of alcalase, bromelain and 417 papain mixtures included antioxidant peptides as LH, LHA, KP and WG (Chen, 418 Muramoto, Yamauchi, & Nokihara, 1996; Huang et al., 2010; Saito et al., 2003). 419 Another peptides as IYVVDVGTEPR, GGPVQVLEDQELK and 420 AADGTWEPFALGK, which have an intermediate concentration in bromelain, 421 flavorenzyme and papain also included IY, EL, LK, GTW and TW described by 422 other authors as potent antioxidants (Beermann, Euler, Herzberg, & Stahl, 2009; 423 Liu et al., 2015; Suetsuna, Ukeda, & Ochi, 2000). In the remaining cases, the 424 dipeptides or tripeptides were not identified most likely because these databases 425 are not very extensive yet. 426 4. Conclusions 427 This study showed the great value of pork liver for preparing hydrolysates due to 428 its high protein content and adequate amino acid profile. Additionally, the 429 proteolytic enzymes assayed (alcalase, bromelain, papain and flavourzyme) 430 19 differentially affected the hydrolysis, suggesting complex mixtures of biopeptides 431 as final products with antioxidant activity. In this regard, the peptidomic map from 432 alcalase treatment were more differentiated from the other enzymes. The 433 APAAIGPYSQAVLVDR, GLNQALVDLHALGSAR, ALFQDVQKPSQDEWGK, 434 LSGPQAGLGEYLFER and LGEHNIDVLEGNEQFINAAK peptides from an 435 uncharacterized protein, ferritin and trypsinogen are main candidates to explain 436 the antioxidant capacity of the pork liver. 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Asian-Australasian 599 Journal of Animal Sciences, 30(11), 1612–1619. 600 https://doi.org/10.5713/ajas.16.0807 601 Zhao, D., Xu, Y., Gu, T., Wang, H., Yin, Y., Sheng, B., … Zhou, G. (2019). 602 Peptidomic Investigation of the Interplay between Enzymatic Tenderization 603 and the Digestibility of Beef Semimembranosus Proteins. Journal of 604 Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.9b06618 605 Zulueta, A., Esteve, M. J., & Frígola, A. (2009). ORAC and TEAC assays 606 comparison to measure the antioxidant capacity of food products. Food 607 Chemistry, 114(1), 310–316. 608 https://doi.org/10.1016/j.foodchem.2008.09.033 609 610 611 24 CAPTION TO FIGURES 612 Figure 1. SDS-PAGE separation of proteins of pork liver samples obtained from 613 control and enzymatic treatments (alcalase, bromelain, flavourzyme and papain). 614 Figure 2. Free amino acids content (A) and proteolysis index (B) of pork liver 615 samples obtained from control and enzymatic treatments (alcalase, bromelain, 616 flavourzyme and papain). 617 Figure 3. Heat map analysis of 73 differentially abundant peptides (logarithmic 618 scale in base 2) of from control and enzymatic treatments of pork liver samples. 619 Green and red colors represent relatively high and low values of peptide 620 abundance, respectively. 621 622 25 623 Figure 1. 624 625 32 This table only includes the peptides identified by SWATH and correlated with antioxidant capacity (Pearson correlation coefficient higher than 0.5 at P<0.01). 665 Abundance mean values of these peptides with the significant differences noted by a-c upper letter (P<0.05) are showed. 666 667 668 33 Table 3. Correlations of antioxidant peptides identified by SWATH obtained from alcalase, bromelain, flavourzyme and papain 669 treatments determined by DPPH, ABTS, FRAP and ORAC assays. 670 Peptide Protein DPPH ABTS FRAP ORAC AEQDGVPVFK A0A480E686 -0.013 -0.532** -0.304* -0.529** ELGEYGLQAYTEVK Mitochondrial aldehyde dehydrogenase (ALDH2) 0.081 -0.292* -0.215 -0.537** IYVVDVGTEPR Selenium binding protein 1 (SELENBP1) -0.466** -0.671** -0.567** -0.363* GGPVQVLEDQELK Selenium binding protein 1 (SELENBP1) -0.480** -0.667** -0.627** -0.368 FLHNPAASQGFVGC[CAM]ALGSNIQR Selenium binding protein 1 (SELENBP1) -0.324* -0.721** -0.507* -0.436** NTSTEAPDYLATVDVDPK Selenium binding protein 1 (SELENBP1) -0.383** -0.658** -0.527** -0.361* LTGQLFLGGSIVK Selenium binding protein 1 (SELENBP1) -0.481** -0.706** -0.584** -0.374** SPQYC[CAM]QVIHR Selenium binding protein 1 (SELENBP1) -0.405** -0.444** -0.554** -0.285* GAPTTSLISVAVTK Aldehyde dehydrogenase 7 (ALDH7A1) 0.581** 0.549** 0.646** 0.220 VLDASWYSPGTR A0A481AX04 0.209 -0.207 -0.159 -0.526** INEGFELLR PKS_ER domain-containing protein (LOC100512795) 0.506** 0.053 0.198 -0.253 VINSILAFR M20_dimer domain-containing protein 0.708** -0.156 0.647** -0.585** DAVTYTEHAK A0A480KDB1 0.554** 0.682** 0.663** 0.323* DVTLNPDPNEIK Isopentenyl-diphosphate delta isomerase (IDI1) 0.416** 0.749** 0.463** 0.298* APAAIGPYSQAVLVDR Uncharacterized protein (RIDA) -0.523** -0.724** -0.562** -0.433** AAGC[CAM]DFTNVVK Uncharacterized protein (RIDA) -0.585** -0.592** -0.610** -0.344* 34 PASGQLVPGGVVEEAK Uncharacterized protein (RIDA) -0.389** -0.533** -0.504** -0.384** GLNQALVDLHALGSAR Ferritin 0.117** 0.417** 0.377** 0.743** ALFQDVQKPSQDEWGK Ferritin 0.048 0.356* 0.364* 0.605** LSGPQAGLGEYLFER Ferritin 0.147 0.501** 0.438** 0.682** QNYSTEVEAFVNR Ferritin 0.268 0.607** 0.607** 0.768** DDVALEGVSHFFR Ferritin 0.089 0.502** 0.405** 0.718** REATQPEVDTTLGR Carboxylic ester hydrolase (CES3) 0.162 0.774** 0.532** 0.558** MGAPEYGMAEYLFDK Ferritin (FTH1) -0.684** -0.379** -0.514** -0.405** LVNHFVEEFK Heat shock 70kDa protein (HSPA1A) 0.190 0.646** 0.394** 0.393** GNVINISSLVGAIGQSQAVPYVATK Hydroxysteroid 17-beta dehydrogenase 14 0.339* 0.768** 0.590** 0.458** AADGTWEPFALGK A0A481CSP9 -0.501** -0.668** -0.637** -0.317* ALGISPFHEYAEVVFTANDSGR A0A481CSP9 -0.358* -0.566** -0.510** -0.203 TSEFGELHGLTTDEK A0A481CSP9 -0.550** -0.601** -0.555** -0.302* FLEEHPGGEEVLR A0A480SB71 0.038 -0.264 0.238 -0.505** IALTDNALIAR 60S ribosomal protein L7 0.467** 0.749** 0.585** 0.397** LGEHNIDVLEGNEQFINAAK Trypsinogen 0.366* 0.789** 0.592** 0.619** FLEQQNQVLQTK Keratin 1 (KRT1) 0.309* 0.686** 0.628** 0.591** SLNNQFASFIDK Keratin 1 (KRT1) 0.247 0.633** 0.493** 0.483** AGNLGGGVVTIER A0A481CBE4 0.391** 0.629** 0.659** 0.418** 35 This table only includes peptides with at least one significant antioxidant test by Pearson correlation coefficient higher than 0.5 at 671 P<0.01 (bold) 672 673 674 675 36 Table 4. Dipeptides and tripeptides (highlighted in bold) included in pork liver peptides generated from enzymatic treatments with 676 antioxidant capacity according to literature. 677 Peptide References ELGEYGLQAYTEVK (Suetsuna et al., 2000) IYVVDVGTEPR (Beermann et al., 2009) GGPVQVLEDQELK (Suetsuna et al., 2000); (W. Y. Huang et al., 2010) FLHNPAASQGFVGC[CAM] ALGSNIQR (Cheng, Chen, & Xiong, 2010); (Saito et al., 2003); VLDASWYSPGTR (Hernández-Ledesma, Amigo, Recio, & Bartolomé, 2007) INEGFELLR (Suetsuna et al., 2000); DAVTYTEHAK (Cheng et al., 2010) PASGQLVPGGVVEEAK (Suetsuna et al., 2000) GLNQALVDLHALGSAR (Chen et al., 1996); (Saito et al., 2003) ALFQDVQKPSQDEWGK (W. Y. Huang et al., 2010); (Anna et al., 2016) AADGTWEPFALGK (R. Liu et al., 2015) TSEFGELHGLTTDEK (Cheng et al., 2010); (Suetsuna et al., 2000); (Saito et al., 2003) FLEEHPGGEEVLR (Bougatef et al., 2010) 678 679 680 37 681 682