Bioactive peptides in beef: Endogenous generation through postmortem aging

Meat Science 123 (2017) 134–142

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Bioactive peptides in beef: Endogenous generation through postmortem aging Yu Fu, Jette F. Young, Margrethe Therkildsen ⁎ Department of Food Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark

a r t i c l e

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Article history: Received 7 July 2016 Received in revised form 8 September 2016 Accepted 29 September 2016 Available online 30 September 2016 Keywords: Bioactive peptides Postmortem aging Tenderness Beef Collagen

a b s t r a c t The present research was performed to investigate endogenous release of bioactive peptides in beef during postmortem aging times (1, 10 and 20 days). Gradually decreased Warner-Bratzler shear force (WBSF) values of longissimus thoracis (LT) and semitendinosus (ST) muscles were observed and the degradation of structural proteins and collagen led to release of low-molecular weight (b 3 kDa) peptides. These peptides exhibited 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, ACE- and renin-inhibitory activities. The peptide sequences were identified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). In silico analysis (PeptideRanker and BIOPEP) of their bioactivity potentials demonstrated peptides with the predicted bioactivity scores (N0.8) as well as collagen peptides with bioactivity scores (0.6–0.8). The present findings provide insights on development of healthy beef through postmortem aging at 4 °C. © 2016 Published by Elsevier Ltd.

1. Introduction Tenderness, juiciness, color, flavor and aroma are all key attributes of meat palatability with regards to consumers' satisfaction, with tenderness being the most significant factor (Huffman et al., 1996; Miller, Carr, Ramsey, Crockett, & Hoover, 2001; Wu, Fu, Therkildsen, Li, & Dai, 2015). As important as this attribute is, it seems to be the most variable of all meat palatability traits and has been attributed to several factors, including degradation of myofibrillar proteins and to a large extent the connective tissue. Postmortem aging of beef is a very effective method to improve tenderness (Huff-Lonergan, Zhang, & Lonergan, 2010). This process allows the natural enzymatic and biochemical processes to take place resulting in increased tenderness (Nishimura, Hattori, & Takahashi, 1995) due to the weakening of the myofibrils and the intramuscular connective tissue (Dransfield, 1994). During this process, postmortem aging not only contributes to meat tenderness but it may also generate peptide fractions with physiological significance, such as antioxidant and blood pressure lowering effects. Hypertension is a global leading risk factor for cardiovascular diseases (Ahhmed & Muguruma, 2010). The renin-angiotensin system (RAS) is a main pathway responsible for regulating blood pressure and ensuring fluid homeostasis. Moderate hypertension can be controlled through dietary approaches and a number of investigations have reported the antihypertensive and ACE-inhibitory peptides of different food sources (Kim & Wijesekara, 2010). Oxidative stress, an ⁎ Corresponding author at: Department of Food Science, Aarhus University, Blichers Allé 20, Postbox 50, 8830 Tjele, Denmark. E-mail address: [email protected] (M. Therkildsen).

http://dx.doi.org/10.1016/j.meatsci.2016.09.015 0309-1740/© 2016 Published by Elsevier Ltd.

imbalance between oxidants and antioxidants, might further induce and exacerbate hypertension (Kizhakekuttu & Widlansky, 2010). However, administration of food-derived peptidic antioxidants contributes to a lower occurrence of oxidative stress (Samaranayaka & Li-Chan, 2011). In recent years, proteolysis of meat proteins (myofibrillar, sarcoplasmic or collagen) has been documented to release several potential bioactive sequences to exhibit in vitro antihypertensive and antioxidant activities (Ryan, Ross, Bolton, Fitzgerald, & Stanton, 2011; Mora et al., 2014; Escudero et al., 2013; Xing et al., 2016). Although some health-promoting compounds from meat with positive physiological effects have been reported (Decker & Park, 2010; Young et al., 2013), few studies focused on the peptides generated during postmortem aging and their potential as a natural source of antihypertensive and antioxidant peptides to maintain blood pressure and health. Therefore, the aim of this study is to investigate inherent bioactivity developed through aging/tenderization in beef. In this study, beef was aged for 1, 10 or 20 days postmortem and the bioactivity of the released peptides was determined. In addition, their peptide sequences were characterized and bioactivity potentials from the individual peptides were predicted based on in silico analysis in order to estimate their relative contribution to the sample bioactivity. 2. Materials and methods 2.1. Materials and sample preparation 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ACE from rabbit lung were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human recombinant renin inhibitor screening assay kit was purchased from

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Cayman Chemicals (Ann Arbor, MI, USA). Amicon Ultra-15 centrifugal filters (3 kDa molecular weight cut-off) were purchased from Merck Millipore (Cork, Ireland). The Oasis HLB cartridges (1 mL) were obtained from Waters (Dublin, Ireland). Six bulls (3 Danish Holstein +3 Danish Holstein cross breeds; age: 16 ± 2 months; live weight: 245 ± 35 kg) were slaughtered at a Danish Crown slaughter house (Aalborg, Denmark). The animals were stunned by captive bolt pistol, hung and bled. After 15 min postmortem, the carcasses were electrically stimulated with low voltage (78 V, 200 mA) for 35 s. Carcasses were hung in the chiller at 4 °C. After one day postmortem, longissimus thoracis (LT) (from the 1st thoracic vertebrae to the 5th thoracic vertebrae) and semitendinosus (ST) muscles were removed from each carcass and transported for 1 h in insulated shipping boxes filled with ice bags to the lab. The temperatures of the LT and ST muscles after arrival in the lab were determined to be 5.1 ± 0.3 °C and 5.3 ± 0.4 °C with Testo 110 thermometer (Testo GmbH & Co., Lenzkirch, Germany). The pH value was measured in each muscle with a PHM201 pH meter (Radiometer, Denmark) equipped with Metrohm probe type glass electrode (Metrohm, Switzerland). The electrode was calibrated in pH 4.01 and 7.00 IUPAC buffers. Subsequently, each muscle was divided into three (8 cm × 5 cm × 5 cm) blocks, labelled and randomly distributed in three groups of aging (day 1, day 10 and day 20). The samples were packed in the vacuum bags (Lava Vakuumverpackung, Bad Saulgau, Germany), which consists of polyethylene and polyamide (O2 permeability: 50 cm3/m2 ∗ d ∗ bar). The vacuum bags were evacuated and heat-sealed using a vacuum packaging machine (Komet, Stuttgart, Germany) under 1.0 bar of vacuum. Day 1 samples were analyzed for texture at the same day and sub-samples from both raw and cooked samples (described in Section 2.2) at day 1 were stored at −20 °C for further bioactivity analyses, whereas day 10 and 20 samples were stored at 4 °C for additional 9 or 19 days before texture analysis and subsampling for bioactivity measurement as described below.

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2.4. Peptide identification by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) Prior to MS analysis, the extracted peptide samples were subjected to purification using Oasis HLB (C18 solid phase) cartridges in order to remove the salts and impurity. Afterwards, the samples were lyophilized and re-diluted using 0.1% formic acid before injection into LCESI-MS. The LC system (Agilent Technologies, Waldbronn, Germany) was equipped with Jupiter Proteo C18 column of dimensions 150 mm × 0.5 mm (Phenomenex, Denmark). The percentage of solvent B (90% acetonitrile, 0.1% formic acid) in solvent A (0.1% formic acid) was based on a linear gradient. The flow rate was fixed at 100 μL/min for 110 min. Tandem MS spectra were further analyzed by PEAKS Studio 7.5 (Waterloo, ON, Canada) and searched against the customized bovine family (Bos taurus) from UniProt database. The search was implemented using no specific enzyme cleavage sites and an MS/MS mass tolerance of 0.5 Da. The peptides with average local confidence (ALC) over 75% were used for further analysis. The analysis was performed using two individual samples and only peptides positively identified in both samples were acceptable. Peptide sequences, their position in the parent proteins and the observed masses and retention times were collected from the PEAKS. Basic Local Alignment Search Tool (BLAST) was employed to search for regions of local similarities between the identified peptides and the parent protein sequences within the Bos taurus database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All the sequences of the peptides identified in this work were searched and revealed 100% homology with proteins of Bos taurus. 2.5. Peptide concentration The concentration of the extracted peptides was determined by calculating the amount of N-terminal amines using fluorescamine according to Petrat-Melin et al. (2015). The extracted peptide concentration was fixed at 30 mM for further determination of bioactivity.

2.2. Warner-Bratzler shear force (WBSF)

2.6. Bioactivity determination of the extracted peptides

The WBSF of aged beef samples was determined as per the method of Honikel (1998). Briefly, the beef samples were heated in a thermostat bath (GD100, Grant Instruments, Shepreth, UK) after reaching core temperature (63 °C). The heat treatment was terminated by immersing the samples in ice water for 15 min. Thereafter, the samples were stored in a water bath at 4 °C until next day. The next day rectangular blocks (1 ∗ 1 cm thick) were cut parallel to the longitudinal orientation of the muscle fibres and the shear force was measured using a Texture Analyzer TX-T2 (Stable Micro Systems, Godalming, U.K.) with a WarnerBratzler shear blade with a rectangular hole. The blade speed was set to 100 mm/min and the average maximum force (N/cm2) of 6 replicates cut from each sample was used.

2.6.1. DPPH radical scavenging capacity DPPH radical scavenging activity of isolated peptides was determined according to Li, Chen, Wang, Ji, and Wu (2007) with slight modifications. 500 μL test sample (peptide fraction of 30 μM) was mixed with 500 μL of 99.5% ethanol and 125 μL of 99.5% ethanol containing 0.01% DPPH. This mixture was kept in the dark at room temperature for 60 min before determination of absorbance at 517 nm. DPPH radical scavenging activity was calculated as follows:

2.3. Peptide extraction

where Asample is the absorbance of the sample and Acontrol is the absorbance of the control.

The peptides were extracted from beef samples according to Bauchart et al. (2006) with slight modifications. Frozen beef samples of both raw and cooked origin (2.5 g) were homogenized in 12.5 mL of 3% perchloric acid in centrifuge tubes on ice for 2 min using a Polytron PT 2100 homogenizer from Kinematic AG (Luzern, Switzerland). Subsequently, the homogenate was centrifuged at 10,000g for 20 min at 4 °C and the supernatant was collected and filtered using a cellulose acetate filter of 0.2 μm pore size (Frisenette, Denmark). The extracts were neutralized to pH 7 using sodium hydroxide. The salt precipitate was eliminated using the cellulose acetate filter twice. Subsequently, the supernatant was subjected to ultra-filtration using 3 kDa cut-off centrifugal filters at 10,000g for 30 min. The resulting filtrates were lyophilized and stored at −20 °C for further analysis.

DPPH radical scavening capacity ð%Þ ¼

Acontrol −Asample  100 Acontrol

2.6.2. ACE inhibitory-activity ACE-inhibitory activity was determined according to the approach of Petrat-Melin et al. (2015). Briefly, ACE working solution (7.1 U/mL) of 50 μL in borate buffer (50 mmol/L, pH 8.3) was added to wells of a 96well microplate, followed by addition of 50 μL of either sample or control (Milli-Q water). The enzyme reaction was initiated by addition (200 μL) of 0.45 mmol/L Abz-Gly-Phe(NO2)-Pro dissolved in Tris-base buffer (150 mmol/L, pH 8.3). The resultant reagents were immediately mixed and incubated at 37 °C. The generated fluorescence was measured using a microplate reader (BioTek Instruments, Winooski, USA) with excitation and emission wavelengths of 355 and 405 nm, respectively.

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2.6.3. Renin-inhibitory activity Renin-inhibitory activity was determined by fluorescence as per the method of the manufacturers' instructions. Briefly, 20 μL of substrate, 150 μL of assay buffer, and 10 μL of the extracted peptides were added to the wells and the reaction was started by addition of renin (10 μL). Fluorescence intensity was determined at excitation wavelength of 340 nm and emission wavelength of 490 nm. Percentage inhibition was calculated using the following equation: Renin−inhibitory activity ð%Þ ¼

Initial activity−Activitysample  100 Initial activity

2.7. In silico prediction of the identified peptides The identified peptides were assessed for potential of bioactivity by input of the peptide sequences into. PeptideRanker web server using the N-to-1 neural network probability (http://bioware.ucd.ie/~compass/biowareweb/Server_pages/ peptideranker.php) (Mooney, Haslam, Pollastri, & Shields, 2012). The assigned scores ranged from 0 to 1.0 with a threshold of 0.5. The identified peptide sequences were matched up against the published peptide sequences exhibiting ACE-, renin-inhibitory or antioxidant activity in the BIOPEP database (http://www.uwm.edu.pl/biochemia/index.php/ pl/biopep). Data were accessed on May 2016. 2.8. Statistical analysis The shear force and bioactivity data for LT and ST muscles measured after 1, 10 and 20 days of aging were analyzed using SPSS version 20.0 program (Chicago, IL, USA). Differences between groups were analyzed using one-way analysis of variance (ANOVA). Statistical significance among samples was considered at P b 0.05 with post-hoc Duncan test. 3. Results 3.1. pH and Warner-Bratzler shear force (WBSF) values At one day postmortem, the ultimate pH measured of LT muscle was higher than in ST muscle (5.71 ± 0.08 vs. 5.56 ± 0.04, respectively) (P b 0.001). The shear force values of LT and ST muscles are displayed in Fig. 1. In general, high shear force values are observed in both muscles at day 1 postmortem (57.3 and 60.3 N, respectively), with a significant time-dependent decline in shear force values (P b 0.05). After 20 days postmortem, the shear force values of LT and ST muscles were decreased to 37.5 and 41.6 N, respectively. However, there was no

Fig. 1. Effects of postmortem aging on WBSF of LT and ST samples after 1, 10 and 20 days aging postmortem. WBSF values are presented as mean ± SD (n = 6). Different letters indicate significantly different values (P b 0.05) by one-way ANOVA analysis.

significant difference between LT and ST samples at the same aging day (P N 0.05). 3.2. Peptides extracted from aged beef During postmortem aging, the proteolysis of muscle proteins takes place, leading to generation of a considerable amount of peptides. The extracted peptide (b3 kDa) concentrations of raw LT and ST samples at day 1 were 9.0 ± 2.1 and 8.1 ± 2.6 μM, respectively. There was a significant rise (P b 0.05) after 10 and 20 days postmortem, reaching 15.5 ± 4.2 and 32.0 ± 4.8 μM for LT samples and 15.8 ± 1.5 and 30.0 ± 5.4 μM for ST samples. Cooked samples also followed a similar trend. The peptide concentrations of LT and ST samples were remarkably increased (P b 0.05) from the initial 10.0 ± 3.2 μM and 10.8 ± 3.0 μM to 33.7 ± 1.4 μM and 34.3 μM ± 10.8 μM at day 20 postmortem, respectively. However, there was an insignificant rise (P N 0.05) between cooked and raw samples at each aging time. 3.3. Bioactivity of the extracted peptides All the peptides derived from raw and cooked LT and ST samples at different aging days exhibited in vitro DPPH radical scavenging, ACEand renin-inhibitory activities (Figs. 2, 3 & 4). As shown in Fig. 2A, there was a pronounced increase of DPPH radical scavenging activity after 10 days postmortem (P b 0.05) in raw LT samples. The highest DPPH radical scavenging activity in LT (44.6%) was achieved in cooked LT sample at day 20 postmortem (P b 0.05). In ST raw samples, there was no pronounced change (P N 0.05) in DPPH radical scavenging activity from 1 to 10 days of aging, whereas this was not the case for the cooked samples (Fig. 2B). However, from 10 to 20 days of ripening there was a significant increase in DPPH radical scavenging activity, reaching 33.4% and 46.5% in raw and cooked ST samples at 20 days postmortem, respectively (P b 0.05) (Fig. 2B). Overall, the cooked samples possessed higher DPPH radical scavenging activity than raw samples (P b 0.05) and this was increasing with time (except ST samples of day 1, where this was not significant, Fig. 2B). In vitro ACE-inhibitory activities of the extracted peptides from LT and ST muscles are displayed in Fig. 3A & B, respectively. A significant increase in ACE inhibition rate was detected in raw and cooked samples of LT muscle with the progress of aging time (Fig. 3A). The ACE-inhibitory activity was elevated from the initial 14.0% and 17.2% at 1 day postmortem to 34.9% and 40.4% at 20 days postmortem (P b 0.05) in raw and cooked samples, respectively. However, there was no significant

Fig. 2. DPPH radical scavenging activity of the extracted peptides from LT samples (A) and ST samples (B). DPPH radical scavenging activity values are presented as mean ± SD (n = 6).The samples were either raw or cooked (63 °C) and aged for 1, 10 or 20 days postmortem. Different letters indicate significantly different values (P b 0.05) by oneway ANOVA analysis.

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3.4. Peptide identification by LC-ESI-MS

Fig. 3. ACE-inhibitory activity of the extracted peptides from LT samples (A) and ST samples (B). The samples were either raw or cooked (63 °C) and aged for 1, 10 or 20 days postmortem. ACE-inhibitory activity values are presented as mean ± SD (n = 6). Different letters indicate significantly different values (P b 0.05) by one-way ANOVA analysis.

difference (P N 0.05) in ACE-inhibitory activities between raw and cooked LT samples. In case of ST muscle, compared to the ACE-inhibitory activity measured in day 1 samples, the activities of raw samples were significantly increased to 39.8% after 20 days postmortem (P b 0.05) (Fig. 3B). In cooked samples, the highest inhibitory activity (44.9%) was achieved at 10 days postmortem, followed by a remarkable decline in activity of the sample of day 20 (P b 0.05). This decrease was not seen in the raw ST samples. Renin-inhibitory activities of the extracted peptides are shown in Fig. 4A & B. In general, each peptide samples experienced a rise with increase of aging times, except raw LT sample at Day 20. A significant increase in renin activity was detected in raw and cooked samples of LT and ST muscle with the progress of aging time. The cooked samples displayed higher renin-inhibitory activity than raw samples. At 10 days postmortem, a remarkable rise of renin-inhibitory activity was observed in raw LT samples from initial 5.7 to 15.9% (P b 0.05), followed by an insignificant decline (P N 0.05) at day 20. In terms of ST samples, the highest inhibitory rates were achieved at day 20, reaching 24.2% and 39.3% in both raw and cooked samples, respectively.

The identified peptides (b 3 kDa) from LT and ST samples after 1, 10 and 20 days postmortem are presented in Supplementary Excel sheet 1–6 and sheet 7–12, respectively. The identified amino acid sequences, parent proteins, observed m/z, calculated mass, accession No. and post-translational modification (PTM) were listed. In general, the identified peptides from aged beef were dominated by small peptides, ranging from 5 to 26 amino acid residues. Smaller peptides were absent probably due to low concentrations not detectable by LC-ESI-MS. The number of the extracted peptides was substantially increased (N 50) after 20 days postmortem, compared to samples in each muscle after 1 and 10 days postmortem. In raw LT samples, some peptides derived from metabolic enzymes and proteins were found after 1 day postmortem (Supplementary Excel sheet 1), including fructose-1,6bisphosphatase isozyme 2, glutaryl-CoA dehydrogenase, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-4, serine/ threonine-protein phosphatase 2A catalytic subunit alpha isoform and L-2-hydroxyglutarate dehydrogenase. From day 10 onwards, several peptides from proteins closely related to tenderness were detected, such as elastin, actin-related protein 10, putative malate dehydrogenase 1B and collagen alpha-2(I) chain, collagen alpha-2(XI) chain, myosin light chain kinase, DnaJ homolog subfamily B member 6 and heat shock protein 90-alpha (Supplementary Excel sheet 3). As expected more peptides originating from collagen fragments were found in the raw ST richer in collagen (Supplementary Excel sheet 1–12). After 10 and 20 days postmortem, a greater number of peptides were observed, which were derived from several proteins closed related to tenderness, such as calpain small subunit 1, collagen alpha-1(I) and alpha-2(I) chain (Supplementary Excel sheet 9) and tubulin beta-4 A chain, elastin, heat shock 70 kDa protein 1, calpain small subunit 1 and collagen alpha-1(I) chain (Supplementary Excel sheet 11). Cooking can provoke structural changes via proteolysis at specific sites in muscle. The peptides originating from actin filament-associated protein 1-like 2, elastin, collagen alpha-1(I) chain and alpha-1(III) chain could be found in cooked LT sample at day 1 postmortem (Supplementary Excel sheet 2). Several peptides from collagen alpha-1(III) chain, elastin, tubulin-specific chaperone A, myosin light chain kinase 2, tubulin polymerization-promoting protein family member 3 (Supplementary Excel sheet 4) and alpha-actinin-3, myozenin-2, myosin heavy chain 7, myosin light chain kinase (Supplementary Excel sheet 6) were identified in cooked LT samples of day 10 and day 20, respectively. In cooked ST samples, a number of structural proteins were also found, including alpha-actinin-2, collagen alpha-1(II) chain (Supplementary Excel sheet 8), collagen alpha-2(XI), collagen alpha-1(XI), collagen alpha1(III), collagen alpha-1(XVII) chain (Supplementary Excel sheet 10), and myosin regulatory light chain 2, collagen alpha-1(XVII) chain, elastin and tubulin beta-5 chain (Supplementary Excel sheet 12), day 1, 10 and 20 respectively. 3.5. In silico analysis of potential bioactivity of the identified peptides

Fig. 4. Renin-inhibitory activity ability of the extracted peptides from LT samples (A) and ST samples (B). The samples were either raw or cooked (63 °C) and aged for 1, 10 or 20 days postmortem. Renin-inhibitory activity values are presented as mean ± SD (n = 6). Different letters indicate significantly different values (P b 0.05) by one-way ANOVA analysis.

The extracted peptides from LT and ST samples were subjected to in silico analysis for their bioactive potentials. The PeptideRanker scores (N0.5) of bioactive peptides derived from meat peptides are listed in Tables 1 & 2, indicating that these peptides generated from LT and ST samples could be potentially bioactive. A number of promising bioactive peptides (score N 0.8) were observed, including CPSGPGTF from docking protein in raw LD sample of Day 1 (score = 0.91), ARICAF from actin filament-associated protein 1-like 2 in cooked LD sample of Day 1 (score = 0.82), SGAPGPAGSRGPPGP from collagen alpha-1(III) chain in cooked ST sample of Day 10, KQAGFPLGILLL from putative sodium-coupled neutral amino acid transporter 11 in raw LD sample of Day 20 (score = 0.92) (Table 1), RPPKGF from AFG3-like protein 2 in raw ST sample of Day 1 (score = 0.89), WPPLP from secretory carrier-associated membrane protein 3 in raw ST sample of Day 10 (score = 0.98),

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Table 1 The potential of bioactive peptide sequences released from LT muscle predicted by PeptideRanker. No. Raw Peptide sequence

Cooked Parent protein

Score Peptide sequence

Parent protein

Score

Sodium/myo-inositol cotransporter 2 Docking protein 1 Protein CNPPD1 Transcription factor MafF Histone deacetylase 8 Ras-related protein Rab-21 WD repeat-containing protein 91 Reticulon-3 Replicase polyprotein 1ab Immunoglobulin superfamily containing leucine-rich repeat protein Membrane protein FAM174B U1 small nuclear ribonucleoprotein 70 kDa

0.96 0.91 0.89 0.88 0.75 0.65 0.64 0.6 0.59 0.51

ARICAF IPGAPGAIPGIG GPAGDGDAGGR FICPVVGL SPPRPS FGCFQTPE APGTAGLP ACASGP PPPTGK LLLLLLP

Actin filament-associated protein 1-like 2 Elastin Envelope glycoprotein B Protein RTF2 homolog Microtubule-associated protein 10 Serine/threonine-protein kinase N1 Collagen alpha-1(I) chain Semaphorin-4 A Rho-associated protein kinase 2 Protein shisa-5

0.82 0.77 0.7 0.66 0.65 0.62 0.61 0.53 0.59 0.52

Elastin Cadherin-3 Conglutinin Claudin-5 LIM domain-containing protein 1 Palmitoyltransferase ZDHHC5 Actin-related protein 10 N-terminal kinase-like protein Cyclin-C Putative helicase MOV-10 Cadherin-13 Serpin A3–3

0.86 0.84 0.82 0.74 0.71 0.7 0.64 0.63 0.6 0.6 0.59 0.51

CCCCGEGCGEGCGG SGAPGPAGSRGPPGP GAGGPPPLAT GVKPAKPGVGGLVGPG YCAPY GFRVL SEPGCP WSAAGG KGIGKMGLGALVLT QVLACF SQLSLHLPPR LGFSY MADPR

Prosaposin receptor GPR37L1 Collagen alpha-1(III) chain Vacuolar fusion protein MON1 homolog A Elastin Keratin-associated protein 3-1 11-cis retinol dehydrogenase Mitogen-activated protein kinase 7 SCO-spondin Genome polyprotein UPF0686 protein C11orf1 homolog TLD domain-containing protein 2 Serine/threonine-protein kinase Sgk1 Tubulin-specific chaperone A

0.97 0.84 0.78 0.78 0.76 0.76 0.69 0.69 0.67 0.66 0.63 0.63 0.57

Day 20 1 KQAGFPLGILLL

0.92

RACCPGWGG

SCO-spondin

0.95

2

0.87

APGEPLP

DNA (cytosine-5)-methyltransferase 1

0.73

3 4 5 6 7 8 9

Putative sodium-coupled neutral amino acid transporter 11 GGGGGGGGGGGSSLRMSSN Calcium-activated potassium channel subunit alpha-1 GGHMP U1 small nuclear ribonucleoprotein C GAPGGGAAGMAAGFPY CCAAT/enhancer-binding protein beta CCCCGE Prosaposin receptor GPR37L1 LYLPDCC Ficolin-2 GDADSCF Krev interaction trapped protein 1 EACSRPMMN Ubiquitin carboxyl-terminal hydrolase 42 AAPGGKSLALLQCAYP Putative methyltransferase NSUN3

0.83 0.81 0.78 0.76 0.73 0.7 0.69

FSSGTM LLGTF GFVIL FAGGRGG PEGGCCN KPLPQP KLFLA

Replicase polyprotein 1a 78 kDa glucose-regulated protein Microsomal glutathione S-transferase 3 Alpha-actinin-3 ETS translocation variant 1 E3 ubiquitin-protein ligase SH3RF1 GTPase-activating protein and VPS9 domain-containing protein 1

0.69 0.67 0.65 0.63 0.57 0.55 0.53

10 11 12 13 14 15 16 17

FCMSS GPAGPPGVAGEDGDKG FFASV LVDGGGPCGGRV VIGGLLLVVALGPG MAPPAA IIFLLVIGTLL FQKVLM

0.68 0.66 0.63 0.6 0.58 0.56 0.55 0.53

Day 1 1 WPGIL 2 CPSGPGTF 3 ISPCAMMLAL 4 PGPGPAPGPGPAS 5 AGDPMCS 6 AQSVGGGCC 7 KLFAL 9 ACPALGTKSC 10 CACSGDCN 11 LLPLPCSAP 12 13

FRSGK GGGGGGGDM

Day 10 1 RFPGI 2 PSFIP 3 IQGFP 4 MCGGGLVCC 5 LPILPP 6 AGPEPEPPL 7 LPLGG 8 AHKILPVLCGLT 9 SKMPKPKPPP 10 IKPCCN 11 SGKPP 12 RLSPL 13

HMG box-containing protein 1 Collagen alpha-2(XI) chain Nitric oxide synthase, inducible Antigen WC1.1 Surfeit locus protein 4 E3 ubiquitin-protein ligase ICP0 Transmembrane protein 245 Putative uncharacterized protein PXBL-III

0.51 0.5

Note: The scores were obtained based on the prediction by PeptideRanker and peptides (scores N 0.5) were chosen due to the high probability of bioactivity.

PCCAPCPF from phosphatidylserine decarboxylase proenzyme (score = 0.99) in raw ST sample of Day 20 and CSAAGFF from lutropin-choriogonadotropic hormone receptor (score = 0.96) in cooked ST sample of Day 20 (Table 2). In addition, several peptides (LPLGG, FAGGRGG and APPPPAEVP) identified from actin, actinin and troponin may also act as promising bioactive peptides with scores of 0.64, 0.63 and 0.68, respectively. In this work, several peptides identified in the aged beef samples listed in Supplementary Excel sheet 1–12 shared sequences with those reported as antioxidant, ACE- and renin-inhibitory peptides from the BIOPEP database, which further underpinned the experimentally determined ACE-inhibitory activities of aged beef samples. 4. Discussion Postmortem aging is an effective approach to elevate meat tenderness. In this study, beef samples followed the expected pattern that

WBSF values were decreased with postmortem aging (Colle et al., 2015; Lepper-Blilie, Berg, Buchanan, & Berg, 2016; Vitale, Pérez-Juan, Lloret, Arnau, & Realini, 2014). ST muscle is often reported to have higher shear force values compared to LT muscle probably due to higher amount of intramuscular connective tissue (collagen) than ST muscle (Rowe, 1986; Jeremiah & Gibson, 2003). In addition, LT and ST muscles were expected to differ in aging profiles due to different patterns of metabolism in two muscles based on the difference in muscle fiber types (Maltin et al., 2003), also indicated by their differences in ultimate pH in this study, but this did not cause significant variations in WBSF values. Different breakdown patterns of the proteins in the two muscles might also lead to different peptide profiles with different bioactivities. However, no systematic difference between LT and ST was observed in the peptide profiles. Postmortem proteolysis is a dynamic and variable procedure where endopeptidases exert their cleavages randomly to produce a complex

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Table 2 The potential of bioactive peptide sequences released from ST muscle predicted by PeptideRanker and BIOPEP. No. Raw Peptide sequence Day 1 1 RPPKGF 2 FEGMC 3 PPLPAP 4 5 6 7 9 10 11

LLGMP PVPSW FAALR PRLLLLL AGPEPEPPL PPPTGK KAPPGK

12

EPLPPK

Day 10 1 WPPLP 2 SGAGGGGGGGGGGGGGGGG 3 PGPMGPPGLAGP 4 LLGLIILLLW 5 GFRVL 6 DPPFQIT 7 KAPAQLCEGC 8 PGGGGGGAGGRLA 9 PDQDCC 10 APPPPAEVP 11 VFGGCA 12 RALPPAAPL 13 KDTPRLSLLLVIL 14 CCSVCA Day 20 1 PCCAPCPF 2 3 4 5 6 7 8 9 10 11

GRFKRFRKKFKKLFKKLSP QPPLLL GGGALGGGPAL PLPVPPPVG KPLPPSKPRK GGAPSPSSLSLPP ENPFAC LPECALLL VINDCCRGAM EEPSSCSAMAMGR

12 13 14 15

PPAPK KLLSLGKHGRL PLGLTCGMVCPT PPHGEAKAGSSTLPP

Cooked Parent protein

Score Peptide sequence

AFG3-like protein 2 Rhodopsin kinase F-box/WD repeat-containing protein 9

0.89 0.88 0.84

Zinc finger protein 668 Metalloproteinase inhibitor 4 Transmembrane protein 79 (Mattrin) Neuropeptide-like protein C4orf48 homolog Palmitoyltransferase ZDHHC5 Rho-associated protein kinase 2 A disintegrin and metalloproteinase with thrombospondin motifs 4 Putative histone-lysine N-methyltransferase PRDM6

0.79 0.75 0.73 0.7 0.7 0.59 0.53

Secretory carrier-associated membrane protein 3 Calpain small subunit 1 Collagen alpha-1(I) chain Integrin alpha-3 11-cis retinol dehydrogenase Prolyl endopeptidase FAP TBC1 domain family member 1 Neurexin-1-beta Transmembrane protein 183 Troponin T, fast skeletal muscle Kelch domain-containing protein 2 Capsid scaffolding protein Melanoma-associated antigen D4 Insulin-like growth factor-binding protein 2

Phosphatidylserine decarboxylase proenzyme, mitochondrial Cathelicidin-6 Cyclin-dependent kinase 13 Mitotic-spindle organizing protein 2 Retinal guanylyl cyclase 2 AT-rich interactive domain-containing protein 5 A INO80 complex subunit E Zinc finger protein OZF NACHT, LRR and PYD domains-containing protein 5 TRPM8 channel-associated factor 1 DNA-(apurinic or apyrimidinic site) lyase 2 Lymphocyte antigen 6 complex locus protein G6f Telomerase reverse transcriptase Dihydropyrimidine dehydrogenase [NADP(+)] Brefeldin A-inhibited guanine nucleotide-exchange protein 1

16 17 18 19

Parent protein

RACCPGWGG GAQGPMGPAG LSYGPGPL

Score

SCO-spondin Collagen alpha-1(II) chain Alpha-aminoadipic semialdehyde synthase, mitochondrial AGPAALCSPP PWWP domain-containing protein MUM1 GPGSGG Myozenin-1 (Calsarcin-2) SSGPLVP Cathepsin Z SRVAGVLGF N-terminal kinase-like protein LVPPPTLLVP Sine oculis-binding protein homolog CFCQVSGY ERO1-like protein alpha VVGDGAVGKTCLL Cell division control protein 42 homolog

0.95 0.76 0.73

0.98 0.94 0.88 0.81 0.76 0.74 0.73 0.7 0.7 0.68 0.62 0.55 0.55 0.54

PTGAPPGGGAL SPLPPPE EGPQGPPGPVG PGLIGARGPPGP EDPGSML VGAVLPGPLLQ

D site-binding protein Collagen alpha-2(XI) chain Collagen alpha-1(XI) chain Collagen alpha-1(III) chain Limbin Glycerate kinase

0.81 0.77 0.74 0.63 0.52 0.5

0.99

CSAAGFF

Lutropin-choriogonadotropic hormone receptor

0.96

0.85 0.84 0.81 0.77 0.74 0.73 0.72 0.69 0.68 0.66

EPAFM PGAAGGAEDGFF ALAPGHLGGLVL GPGYYNPNGH PADGSMC CFGGAGG LMAPGP MGPCPGE ILLPL LPLPGPTLA

0.81 0.79 0.79 0.74 0.73 0.72 0.68 0.67 0.62 0.6

0.6 0.58 0.56 0.53

HGSGM PEGGCCN ENSGFDGM KIPCIKFSK

Natural killer cells antigen CD94 Coatomer subunit alpha Homeobox protein PKNOX1 O(6)-methylguanine-induced apoptosis 2 Spondin-1 5-oxoprolinase Mitochondrial enolase superfamily member 1 SCO-spondin Collagen alpha-1(XVII) chain Methylmalonic aciduria and homocystinuria type C protein homolog DNA helicase MCM8 ETS translocation variant 1 Ubiquitin carboxyl-terminal hydrolase 37 Mitochondrial inner membrane protein OXA1L

KDTPRLSLLLVIL DLPSPME AICDDGATYC GGGGGGGDM

Melanoma-associated antigen D4 Homeobox protein aristaless-like 4 Complement factor B U1 small nuclear ribonucleoprotein 70 kDa

0.55 0.55 0.54 0.5

0.71 0.63 0.6 0.54 0.54 0.54 0.52

0.53

0.59 0.57 0.57 0.56

Note: The scores were obtained based on the prediction by PeptideRanker and peptides (scores N 0.5) were chosen due to the high probability of bioactivity.

mixture of peptides (Gallego, Mora, Fraser, Aristoy, & Toldrá, 2014). In this work, more peptides were identified after 20 days postmortem mainly due to extensive degradation of meat proteins during extended aging periods (Kemp, Sensky, Bardsley, Buttery, & Parr, 2010). Furthermore, many peptides released during postmortem aging may further be hydrolyzed through the action of endopeptidases to smaller peptides rendering a very dynamic matrix. For example, at any one time peptides are being generated from larger proteins, while other peptides are further degraded to smaller peptides undetectable by the methods applied in the present study. Structural proteins and proteolytic enzymes are two major factors responsible for the meat tenderness during aging (Lana & Zolla, 2016). Tenderization of meat is most likely due to the proteolytic

decomposition of myofibrillar and cytoskeletal proteins or collagen by action of a series of endogenous peptidases (Sentandreu, Coulis, & Ouali, 2002; Christensen & Purslow, 2016; Nishimura et al., 1995; Nishimura, 2015). In the present work, a number of peptide fragments derived from key myofibrillar proteins were identified, such as actin, myosin, actinin, troponin and tubulin, as well as peptide fragments from collagen and elastin including several peptide fragments from proteins identified as markers of tenderness (Weston, Rogers, & Althen, 2002). Heat shock protein 70 kDa, a biomarker of beef tenderness (Jia et al., 2007) was identified in cooked LT and raw ST sample 20 days postmortem. This result was in line with the lowest shear force value (37.5 N and 41.6 N) of LT and ST samples at day 20. Malate dehydrogenase, a biomarker of tenderness for pork (Laville et al., 2007), was found

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in raw LT sample at day 10 postmortem. Furthermore, structural proteins, such as myosin heavy chain 7 (Supplementary Excel sheet 6) and troponin T (Supplementary Excel sheet 9) reported as biomarkers for meat tenderness (Lametsch et al., 2003; Muroya, Ohnishi-Kameyama, Oe, Nakajima, & Chikuni, 2007) were identified in this study together with peptide fragments from proteolytic enzymes. The peptide fragments from calpain small subunit 1 were identified in raw ST muscle at 10 and 20 days postmortem (Supplementary Excel sheet 9 & 11, respectively), suggesting that calpain small subunit autolyzed when exposed to calcium and further exert a regulatory effect on the calpain (Li, Thompson, & Goll, 2004; Pomponio et al., 2008). It is reported that cathepsins capable of catalyzing hydrolysis of collagen (Christensen et al., 2013) are released with increased storage time (Ertbjerg et al., 1999), as reflected in this study by identification of peptide fragments from cathepsin L (Supplementary Excel sheet 10) and cathepsin Z (Supplementary Excel sheet 8) in cooked ST samples. This indicated that cathepsins might exert the weakening effects on muscle proteins and autolyze during cooking (Christensen et al., 2013; Reville, Harrington, & Joseph, 1971). Matrix metalloproteinases (MMPs) participate in degradation of native collagen (Purslow, 2014). In lamb, the active MMP-2 was found at 21 days postmortem (Sylvestre, Balcerzak, Feidt, Baracos, & Bellut, 2002), underpinning the possible autolysis of MMP, as MMP fragments (a disintegrin and metalloproteinase with thrombospondin motifs 2) were identified in raw LT and cooked ST at 10 days postmortem (Supplementary Excel sheet 3 & 10). Molecular weight of peptide is one of the key factors responsible for antioxidant activity (Samaranayaka & Li-Chan, 2011), where the most potent antioxidant peptides typically contain 2–20 amino acid residues per molecule (Elias, Kellerby, & Decker, 2008). In this study, the 3 kDa cut-off centrifugal filter was employed to obtain low-molecular weight peptides, and possibly identify the DPPH radical scavenging activities. Several meat-derived antioxidant peptides have been identified from different proteins through hydrolysis of myofibrillar proteins (Saiga, Tanabe, & Nishimura, 2003), beef brisket sarcoplasmic proteins (Di Bernardini et al., 2011) and duck meat (Wang, Huang, Chen, Huang, & Zhou, 2015) by exogenous enzymes. This work demonstrated that postmortem storage of beef also generated antioxidant peptides most probably due to proteolytic activities by endogenous proteases in skeletal muscles (Sentandreu, Coulis, & Ouali, 2002; Udenigwe & Howard, 2013). It is well documented that the peptides displaying in vitro ACE-inhibitory activities may exert in vivo antihypertensive effects when they reach the blood stream in an active state (Aluko, 2015; Vercruysse, Van Camp, & Smagghe, 2005). Some meat-derived peptides have ACE- and renin-inhibitory inhibitory activities (Udenigwe & Howard, 2013; Vercruysse et al., 2005), and ACE-inhibitory peptides have also been identified in dry-cured ham (Escudero, Aristoy, Nishimura, Arihara, & Toldrá, 2012; Escudero et al., 2013). In the current study, it is clearly evidenced that a number of peptides with ACE-inhibitory activities were present in aged beef. It is worth noting that the highest ACE-inhibitory activity was observed in cooked ST sample of day 10, suggesting that certain potent peptides were generated during this period and made accessible by cooking. The decreased inhibitory activity at day 20 may be due to further degradation of the potent peptides by endopeptidases in meat during aging (Gallego et al., 2014). Limited literature was available regarding the renin-inhibitory peptides from meat sources, but several bioactive hydrolysates from flaxseed protein (Udenigwe, Lin, Hou, & Aluko, 2009), kidney bean protein (Mundi & Aluko, 2014) and hemp seed protein (He et al., 2013) were reported to display renin-inhibitory activity and antihypertensive properties when tested in spontaneously hypertensive rats. Although several antihypertensive and antioxidant peptides from Spanish dry-cured ham and Chinese dry-cured Xuanwei ham were identified (Escudero et al., 2013; Xing et al., 2016), the present study for the first time reports peptides (below 3 kDa) extracted from aged beef with renin-inhibitory activity.

Bioactivities of peptides are related to the amino acid composition, sequence and molecular mass (Matsui & Matsumoto, 2006). The hydrophobic amino acids were reported to be effective amino acids located at the C-terminal of ACE-inhibitory peptides (Aluko, 2015; Wu, Aluko, & Nakai, 2006). In addition, strong hydrophobicity of the N-terminal of peptides can potentiate the antioxidant capacity (Pownall, Udenigwe, & Aluko, 2010; Udenigwe & Howard, 2013). In line with this, the identified peptides extracted from the meat were abundant in Pro, Tyr, Phe, Leu and Ile (Tables 1 & 2). The high amounts of Gly in peptides enhance the antioxidant activity (Di Bernardini et al., 2011; Udenigwe & Howard, 2013; Vercruysse et al., 2005), as also observed by the abundance of Gly in the present study. In silico analysis by PeptideRanker further elucidated the bioactivity of the identified peptides based on the predicted scores. The closer the predicted score is to 1, the higher possibility is that the peptide is bioactive (Mooney et al., 2012). Meat proteins are good precursors for generating bioactive peptides (Udenigwe & Howard, 2013; Vercruysse et al., 2005), as evidenced by the peptides from meat samples in this study with predicted scores over 0.8. Muguruma et al. (2009) identified a pentapeptide (VKAGF) derived from porcine actin as a potent ACE-inhibitor and antihypertensive peptide. Katayama et al. (2008) isolated EKEREQ and KRQKYDI with active ACE-inhibitory activities from pepsin-catalyzed porcine troponin. Minkiewicz, Dziuba, and Michalska (2011) stated that bovine collagen and elastin possess highest frequency of bioactive peptide sequences compared to other meat proteins. Collagen peptides derived from bovine connective tissue have been reported to be potent ACE inhibitors (Fu, Young, Dalsgaard, & Therkildsen, 2015; Fu et al., 2016a; Fu et al., 2016b). Furthermore, collagen-derived peptides containing high amount of Gly and Pro have been suggested to exhibit various healthrelated bioactivities based on in silico analysis (Minkiewicz et al., 2011). Hence, the peptides with moderate predicted scores (0.6–0.8) may still be promising bioactive peptides. For example, in cooked ST sample (day 10), the highest ACE-inhibitory activity was exhibited probably due to the presence of collagen-derived peptides (SPLPPPE, EGPQGPPGPVG and PGLIGARGPPGP). The above mentioned theories underpin the experimentally determined bioactivities of the extracted peptides derived from aged beef. However, the unreported bioactivities and potencies of several promising peptides presented in Tables 1 & 2 remain to be confirmed in vitro using synthetic peptides. Aging time and cooking can influence the generation of peptides and their related bioactivities (Fogle et al., 1982; Christensen et al., 2013). The bioactivity of the extracted peptides derived from the raw and cooked samples were evaluated based on the normalized concentration (30 μM). Therefore, changes in bioactivity with aging time and cooking are ascribed to certain active peptides generated in the different phases of aged or cooked beef. The higher peptide concentration is not taken into account, which would also lead to higher potencies. In addition, 63 °C was selected as the cooking temperature. It is documented that calpains become rapidly inactivated at 55 °C (Christensen et al., 2013), but the remaining endogenous peptidases (e.g. cathepsins, collagenase, and metalloproteinase) in cooked samples may still catalyze the proteolysis in beef during cooking, resulting in higher abundance of small peptides (Escudero et al., 2013). To some extent, this fact explains the increased DPPH radical scavenging and renin-inhibitory activities in some cooked samples, compared to the raw samples. However, it is not guaranteed that higher concentration of the smaller peptides always induces impaired bioactivities as there may be antagonistic effects within a mixture of bioactive peptides (Hartmann & Meisel, 2007; Hernández-Ledesma, Recio, & Amigo, 2008). In the present study, the cooked beef aged for 20 days contained the highest concentration of low-molecular weight peptides (approximately 0.12 μg/g beef) and also exhibited the highest ACE- and renin-inhibitory activities. According to the data from Danish Agriculture & Food Council, the daily intake of red meat in Denmark is around 100–150 g/ day (McAfee et al., 2010). It is reported that a commercialized fermented milk product (Evolus®) containing ACE-inhibitory peptides

Y. Fu et al. / Meat Science 123 (2017) 134–142

(0.14 μg/mL) exerted a mild lowering effect on blood pressure when subjects were given 150 mL milk product per day (Seppo, Jauhiainen, Poussa, & Korpela, 2003; Tuomilehto et al., 2004). Thus, daily consumption of aged beef containing ACE-, renin-inhibitory and antioxidant peptides may likewise play a vital role in maintaining normal level of blood pressure. In addition, we have identified two collagen peptides, VGPV and GPRGF derived from bovine connective tissue, which have been shown to be transported across a monolayer of human intestinal Caco-2 cells (Fu et al., 2016b), contributing to the likelihood that these peptides are bioavailable and could exhibit ACE-inhibitory activity in vivo. However, further in vivo and clinical studies remain to be performed in order to confirm the antihypertensive effects of ingesting aged beef. 5. Conclusions This work presented the endogenous generation of bioactive peptides in beef through postmortem aging. Postmortem aging of LT and ST muscles gave rise to an increased tenderness as well as release of peptides (b3 kDa) in both LT and ST samples which exhibited DPPH radical scavenging, ACE- and renin-inhibitory activities. The differences in bioactivities in the beef samples are the different peptide profiles caused by different muscles or different aging times of beef. The identified peptides were mainly from metabolic enzymes, structural proteins and collagen in muscles. More peptides were found in the samples after 20 days postmortem due to the extensive proteolysis. In silico analysis revealed that peptides with high scores (N0.8) predicted by PeptideRanker as well as collagen peptides with scores (0.6–0.8) may contribute to the measured bioactivities. The present findings provide an insight into the release of bioactive peptides in beef through postmortem aging. Acknowledgements The authors acknowledge the financial support by Future Food Innovation, regional consortium of Central Denmark and the Graduate School of Science and Technology at Aarhus University. The access to meat samples from DC-Ingredients (Flaesketorvet 41, Copenhagen, Denmark) is highly appreciated. The technical assistance from Hanne Søndergaard Møller and Caroline Nebel with LC-ESI-MS at Department of Food Science, Aarhus University are much appreciated. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meatsci.2016.09.015. References Ahhmed, A. M., & Muguruma, M. (2010). A review of meat protein hydrolysates and hypertension. Meat Science, 86, 110–118. Aluko, R. E. (2015). Antihypertensive peptides from food proteins. Annual Review of Food Science and Technology, 6, 235–262. Bauchart, C., Rémond, D., Chambon, C., Mirand, P. P., Savary-Auzeloux, I., Reynes, C., & Morzel, M. (2006). Small peptides (b 5 kDa) found in ready-to-eat beef meat. Meat Science, 74, 658–666. Christensen, S., & Purslow, P. P. (2016). The role of matrix metalloproteinases in muscle and adipose tissue development and meat quality: A review. Meat Science, 119, 138–146. Christensen, L., Ertbjerg, P., Løje, H., Risbo, J., van den Berg, F. W., & Christensen, M. (2013). Relationship between meat toughness and properties of connective tissue from cows and young bulls heat treated at low temperatures for prolonged times. Meat Science, 93, 787–795. Colle, M. J., Richard, R. P., Killinger, K. M., Bohlscheid, J. C., Gray, A. R., Loucks, W. I., ... Doumit, M. E. (2015). Influence of extended aging on beef quality characteristics and sensory perception of steaks from the gluteus medius and longissimus lumborum. Meat Science, 110, 32–39. Decker, E. A., & Park, Y. (2010). Healthier meat products as functional foods. Meat Science, 86, 49–55. Di Bernardini, R., Rai, D. K., Bolton, D., Kerry, J., O'Neill, E., Mullen, A. M., et al. (2011). Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hyrolyzate. Peptides, 32, 388–400.

141

Dransfield, E. (1994). Optimization of tenderization, aging and tenderness. Meat Science, 36, 105–121. Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and peptides. Critical Review Food Science Nutrition, 48, 430–441. Ertbjerg, P., Larsen, L. M., & Møller, A. J. (1999). Effect of prerigor lactic acid treatment on lysosomal enzyme release in bovine muscle. Journal of the Science of Food and Agriculture, 79, 95–100. Escudero, E., Aristoy, M. C., Nishimura, H., Arihara, K., & Toldrá, F. (2012). Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish drycured ham. Meat Science, 91, 306–311. Escudero, E., Mora, L., Fraser, P. D., Aristoy, M. C., Arihara, K., & Toldrá, F. (2013). Purification and identification of antihypertensive peptides in Spanish dry-cured ham. Journal of Proteomics, 78, 499–507. Fogle, D. R., Plimpton, R. F., Ockerman, H. W., Jarenback, L., & Persson, T. (1982). Tenderization of beef: Effect of enzyme, enzyme level, and cooking method. Journal of Food Science, 47, 1113–1118. Fu, Y., Young, J. F., Løkke, M. M., Lametsch, R., Aluko, R. E., & Therkildsen, M. (2016a). Revalorisation of bovine collagen as a potential precursor of angiotensin I-converting enzyme (ACE) inhibitory peptides based on in silico and in vitro protein digestions. Journal of Functional Foods, 24, 196–206. Fu, Y., Young, J. F., Rasmussen, M. K., Dalsgaard, T. K., Lametsch, R., Aluko, R. E., & Therkildsen, M. (2016b). Angiotensin I–Converting enzyme–inhibitory peptides from bovine collagen: Insights into inhibitory mechanism and transepithelial transport. Food Research International. http://dx.doi.org/10.1016/j.foodres.2016. 08.037. Fu, Y., Young, J. F., Dalsgaard, T. K., & Therkildsen, M. (2015). Separation of angiotensin Iconverting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature, pH and digestive enzymes. International Journal of Food Science & Technology, 50, 1234–1243. Gallego, M., Mora, L., Fraser, P. D., Aristoy, M. C., & Toldrá, F. (2014). Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham. Food Chemistry, 149, 121–128. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: From research to food applications. Current Opinion in Biotechnology, 18, 163–169. He, R., Malomo, S. A., Alashi, A., Girgih, A. T., Ju, X., & Aluko, R. E. (2013). Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides. Journal of Functional Foods, 5, 781–789. Hernández-Ledesma, B., Recio, I., & Amigo, L. (2008). β-Lactoglobulin as source of bioactive peptides. Amino Acids, 35, 257–265. Honikel, K. O. (1998). Reference methods for the assessment of physical characteristics of meat. Meat Science, 49, 447–457. Huff-Lonergan, E., Zhang, W. G., & Lonergan, S. M. (2010). Biochemistry of postmortem muscle-Lessons on mechanisms of meat tenderization. Meat Science, 86, 184–195. Huffman, K. L., Miller, M. F., Hoover, L. C., Wu, C. K., Brittin, H. C., & Ramsey, C. B. (1996). Effect of beef tenderness on consumer satisfaction with steaks consumed in the home and restaurant. Journal of Animal Science, 74, 91–97. Jeremiah, L., & Gibson, L. (2003). The effects of postmortem product handling and aging time on beef palatability. Food Research International, 36, 929–941. Jia, X., Ekman, M., Grove, H., Færgestad, E. M., Aass, L., Hildrum, K. I., & Hollung, K. (2007). Proteome changes in bovine longissimus thoracis muscle during the early postmortem storage period. Journal of Proteome Research, 6, 2720–2731. Katayama, K., Anggraeni, H. E., Mori, T., Ahhmed, A. M., Kawahara, S., Sugiyama, M., et al. (2008). Porcine skeletal muscle troponin is a good source of peptides with angiotensin-I converting enzyme inhibitory activity and antihypertensive effects in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 56, 355–360. Kemp, C. M., Sensky, P. L., Bardsley, R. G., Buttery, P. J., & Parr, T. (2010). Tenderness – An enzymatic view. Meat Science, 84(2), 248–256. Kim, S. K., & Wijesekara, I. (2010). Development and biological activities of marine-derived bioactive peptides: A review. Journal of Functional Foods, 2, 1–9. Kizhakekuttu, T. J., & Widlansky, M. E. (2010). Natural antioxidants and hypertension: Promise and challenges. Cardiovascular Therapeutics, 28, e20–e32. Lametsch, R., Karlsson, A., Rosenvold, K., Andersen, H. J., Roepstorff, P., & Bendixen, E. (2003). Postmortem proteome changes of porcine muscle related to tenderness. Journal of Agricultural and Food Chemistry, 51, 6992–6997. Lana, A., & Zolla, L. (2016). Proteolysis in meat tenderization from the point of view of each single protein: A proteomic perspective. Journal of Proteomics, 147, 85–97. Laville, E., Sayd, T., Terlouw, C., Chambon, C., Damon, M., Larzul, C., ... Chérel, P. (2007). Comparison of sarcoplasmic proteomes between two groups of pig muscles selected for shear force of cooked meat. Journal of Agricultural and Food Chemistry, 55, 5834–5841. Lepper-Blilie, A. N., Berg, E. P., Buchanan, D. S., & Berg, P. T. (2016). Effects of post-mortem aging time and type of aging on palatability of low marbled beef loins. Meat Science, 112, 63–68. Li, B., Chen, F., Wang, X., Ji, B., & Wu, Y. (2007). Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization–mass spectrometry. Food Chemistry, 102, 1135–1143. Li, H., Thompson, V. F., & Goll, D. E. (2004). Effects of autolysis on properties of μ- and mcalpain. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1691, 91–103. Maltin, C., Balcerzak, D., Tilley, R., & Delday, M. (2003). Determinants of meat quality: Tenderness. Proceedings of the Nutrition Society, 62, 337–347. Matsui, T., & Matsumoto, K. (2006). Antihypertensive peptides from natural resources. Advances in Phytomedicine, 2, 255–271. McAfee, A. J., McSorley, E. M., Cuskelly, G. J., Moss, B. W., Wallace, J. M., Bonham, M. P., & Fearon, A. M. (2010). Red meat consumption: An overview of the risks and benefits. Meat Science, 84, 1–13.

142

Y. Fu et al. / Meat Science 123 (2017) 134–142

Miller, M. F., Carr, M. A., Ramsey, C. B., Crockett, K. L., & Hoover, L. C. (2001). Consumer thresholds for establishing the value of beef tenderness. Journal of Animal Science, 79, 3062–3068. Minkiewicz, P., Dziuba, J., & Michalska, J. (2011). Bovine meat proteins as potential precursors of biologically active peptides-a computational study based on the BIOPEP database. Food Science and Technology International, 17, 39–45. Mooney, C., Haslam, N. J., Pollastri, G., & Shields, D. C. (2012). Towards the improved discovery and design of functional peptides: Common features of diverse classes permit generalized prediction of bioactivity. PloS One, 7, e45012. Mora, L., Reig, M., & Toldrá, F. (2014). Bioactive peptides generated from meat industry by-products. Food Research International, 65, 344–349. Muguruma, M., Ahhmed, A. M., Katayama, K., Kawahara, S., Maruyama, M., & Nakamura, T. (2009). Identification of pro-drug type ACE inhibitory peptides sourced from porcine myosin B: Evaluation of its antihypertensive effects. Food Chemistry, 114, 516–522. Mundi, S., & Aluko, R. E. (2014). Inhibitory properties of kidney bean protein hydrolysate and its membrane fractions against renin, angiotensin converting enzyme, and free radicals. Austin Journal of Nutrition and Food Sciences, 2, 1–11. Muroya, S., Ohnishi-Kameyama, M., Oe, M., Nakajima, I., & Chikuni, K. (2007). Postmortem changes in bovine troponin T isoforms on two-dimensional electrophoretic gel analyzed using mass spectrometry and western blotting: The limited fragmentation into basic polypeptides. Meat Science, 75, 506–514. Nishimura, T. (2015). Role of extracellular matrix in development of skeletal muscle and postmortem aging of meat. Meat Science, 109, 48–55. Nishimura, T., Hattori, A., & Takahashi, K. (1995). Structural weakening of intramuscular connective tissue during conditioning of beef. Meat Science, 39, 127–133. Petrat-Melin, B., Andersen, P., Rasmussen, J. T., Poulsen, N. A., Larsen, L. B., & Young, J. F. (2015). In vitro digestion of purified β-casein variants A 1, A 2, B, and I: Effects on antioxidant and angiotensin-converting enzyme inhibitory capacity. Journal of Dairy Science, 98, 15–26. Pomponio, L., Lametsch, R., Karlsson, A. H., Costa, L. N., Grossi, A., & Ertbjerg, P. (2008). Evidence for post-mortem m-calpain autolysis in porcine muscle. Meat Science, 80, 761–764. Pownall, T. L., Udenigwe, C. C., & Aluko, R. E. (2010). Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. Journal of Agricultural and Food Chemistry, 58, 4712–4718. Purslow, P. P. (2014). New developments on the role of intramuscular connective tissue in meat toughness. Annual Review of Food Science and Technology, 5, 133–153. Reville, W. J., Harrington, M. G., & Joseph, R. L. (1971). Post-mortem autolysis in bovine muscle. Biochemical Journal, 125, 104. Rowe, R. W. D. (1986). Elastin in bovine Semitendinosus and Longissimus dorsi muscles. Meat Science, 17, 293–312. Ryan, J. T., Ross, R. P., Bolton, D., Fitzgerald, G. F., & Stanton, C. (2011). Bioactive peptides from muscle sources: Meat and fish. Nutrients, 3, 765–791. Saiga, A. I., Tanabe, S., & Nishimura, T. (2003). Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. Journal of Agricultural and Food Chemistry, 51, 3661–3667.

Samaranayaka, A. G. P., & Li-Chan, E. C. Y. (2011). Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications. Journal of Functional Foods, 3, 229–254. Sentandreu, M. A., Coulis, G., & Ouali, A. (2002). Role of muscle endopeptidases and their inhibitors in meat tenderness. Trends in Food Science & Technology, 13, 400–421. Seppo, L., Jauhiainen, T., Poussa, T., & Korpela, R. (2003). A fermented milk high in bioactive peptides has a blood pressure–lowering effect in hypertensive subjects. The American Journal of Clinical Nutrition, 77, 326–330. Sylvestre, M. N., Balcerzak, D., Feidt, C., Baracos, V. E., & Bellut, J. B. (2002). Elevated rate of collagen solubilization and postmortem degradation in muscles of lambs with high growth rates: Possible relationship with activity of matrix metalloproteinases. Journal of Animal Science, 80, 1871–1878. Tuomilehto, J., Lindstrom, J., Hyyrynen, J., Korpela, R., Karhunen, M. L., Mikkola, L., ... Nissinen, A. (2004). Effect of ingesting sour milk fermented using lactobacillus helveticus bacteria producing tripeptides on blood pressure in subjects with mild hypertension. Journal of Human Hypertension, 18, 795–802. Udenigwe, C. C., & Howard, A. (2013). Meat proteome as source of functional biopeptides. Food Research International, 54, 1021–1032. Udenigwe, C. C., Lin, Y. S., Hou, W. C., & Aluko, R. E. (2009). Kinetics of the inhibition of renin and angiotensin I-converting enzyme by flaxseed protein hydrolysate fractions. Journal of Functional Foods, 1, 199–207. Vercruysse, L., Van Camp, J., & Smagghe, G. (2005). ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein: A review. Journal of Agricultural and Food Chemistry, 53, 8106–8115. Vitale, M., Pérez-Juan, M., Lloret, E., Arnau, J., & Realini, C. E. (2014). Effect of aging time in vacuum on tenderness, and color and lipid stability of beef from mature cows during display in high oxygen atmosphere package. Meat Science, 96, 270–277. Wang, L. S., Huang, J. C., Chen, Y. L., Huang, M., & Zhou, G. H. (2015). Identification and characterization of antioxidant peptides from enzymatic hydrolysates of duck meat. Journal of Agricultural and Food Chemistry, 63, 3437–3444. Weston, A. R., Rogers, R. W., & Althen, T. G. (2002). Review: The role of collagen in meat tenderness. The Professional Animal Scientist, 18, 107–111. Wu, J., Aluko, R. E., & Nakai, S. (2006). Structural requirements of angiotensin I-converting enzyme inhibitory peptides: Quantitative structure-activity relationship study of diand tripeptides. Journal of Agricultural and Food Chemistry, 54, 732–738. Wu, W., Fu, Y., Therkildsen, M., Li, X. M., & Dai, R. T. (2015). Molecular understanding of meat quality through application of proteomics. Food Reviews International, 31, 13–28. Xing, L. J., Hu, Y. Y., Hu, H. Y., Ge, Q. F., Zhou, G. H., & Zhang, W. G. (2016). Purification and identification of antioxidative peptides from dry-cured Xuanwei ham. Food Chemistry, 194, 951–958. Young, J. F., Therkildsen, M., Ekstrand, B., Che, B. N., Larsen, M. K., Oksbjerg, N., & Stagsted, J. (2013). Novel aspects of health promoting compounds in meat. Meat Science, 95, 904–911.