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



Ci a ion: López-Ped ouso, M.;
Lo enzo, J.M.; Bo ajo, P.; F anco, D.
In Sea ch o An ioxidan Pep ides
om Po cine Li e Hyd olysa es
Using Analy ical and Pep idomic
App oach. An ioxidan s 2022,11, 27.
h ps://doi.o g/10.3390/
an iox11010027
Academic Edi o : Da id A áez-
Román
Recei ed: 14 No embe 2021
Accep ed: 22 Decembe 2021
Published: 24 Decembe 2021
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A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
an ioxidan s
A icle
In Sea ch o An ioxidan Pep ides om Po cine Li e
Hyd olysa es Using Analy ical and Pep idomic App oach
Ma ía López-Ped ouso 1, JoséM. Lo enzo 2,3 , Paula Bo ajo 2and Daniel F anco 2,*
1Depa amen o de Zooloxía, Xené ica e An opoloxía Física, Uni e sidade de San iago de Compos ela,
15872 San iago de Compos ela, Spain; [email p o ec ed]
2Cen o Tecnolóxico da Ca ne de Galicia, Rúa Galicia No. 4, Pa que Tecnolóxico de Galicia, San Cib ao das
Viñas, 32900 Ou ense, Spain; jmlo [email p o ec ed] (J.M.L.); [email p o ec ed] (P.B.)
3Á ea de Tecnoloxía dos Alimen os, Facul ade de Ciencias, Uni e sidade de Vigo, 32004 Ou ense, Spain
*Co espondence: [email p o ec ed]
Abs ac :
The sea ch o an ioxidan pep ides as heal h-p omo ing agen s is o g ea scien i ic in e es
o hei bio echnological applica ions. Thus, he main goal o his s udy was o iden i y an ioxi-
dan pep ides om po k li e using alcalase, b omelain, la ou zyme, and papain enzymes. All
li e hyd olysa es p o ed o be o adequa e quali y ega ding he a io EAA/NEAA, pa icula ly
la ou zyme hyd olysa es. The pep idomic p o iles we e signi ican ly di e en o each enzyme
and hei cha ac e iza ions we e pe o med, esul ing in o y- ou di e en ially abundan pep ides
among he ou ea men s. Po cine li e hyd olysa es om alcalase and b omelain a e demon-
s a ed o ha e he mos an ioxidan capaci y. On he o he hand, hyd ophobic amino acid esidues
(se ine, h eonine, his idine and aspa ic acid) migh be educing he hyd olysa es an ioxidan capac-
i y. Se en een pep ides om collagen, albumin, globin domain-con aining p o ein, cy och ome
β
,
uc ose-bisphospha e aldolase, dihyd opy imidinase, a gininosuccina e syn hase, and ATP syn hase
seem o be an ioxidan . Fu he s udies a e necessa y o isola e hese pep ides and es hem in
in i o expe imen s.
Keywo ds: amino acid p o ile; alcalase; b omelain; la ou zyme; papain; po k by-p oduc s
1. In oduc ion
No el an ioxidan pep ides play an inc easingly impo an ole in bio echnological
applica ions; consequen ly, he nu aceu ical, unc ional ood, and cosme ic sec o s a e
paying mo e and mo e a en ion o hem. In his espec , indus ial p ocessing was es wi h
high con en o p o ein p o ide an oppo uni y o p epa ing hyd olysa es wi h an ihy-
pe ensi e, an ioxidan , an imic obial, an idiabe ic, an icance , and o he bio-ac i i ies [
1
].
Mo eo e , he ield o pep idomics as a pa o p o eomic is becoming mo e c ucial in sea ch
o bioac i e pep ides. Indeed, om 2015 o 2020, a o al o 80 o iginal pape s we e published
acco ding o he Scopus da abase, using he keywo ds “pep idomic” and “biopep ide”.
Mo e speci ically, a sys ema ic sea ch o he li e a u e using he keywo ds mos impo an
o his pape (“bioac i e pep ides” and “po k li e ”) esul ed in only i e o iginal pape s.
The e o e, g ea de elopmen o echnical capaci y is being ca ied ou in ecen yea s,
mainly pe o med by liquid ch oma og aphy o sepa a e he pep ides ollowed by mass
spec ome y in andem o iden i y hem. Ul a il a ion, hyd ophobic-high-pe o mance
liquid ch oma og aphy (HPLC), ion-exchange HPLC, and capilla y elec opho esis in mos
cases coupled o elec osp ay ioniza ion a e he main analy ical ins umen s used. Finally,
he p o ein iden i ica ion is ca ied ou by Q-TOF o MALDI-TOF/TOF ins umen a ion as
a mass spec ome e [2,3].
P o ein- ich by-p oduc s o animal o igin ( ish, sea ood, milk, and mea p oduc s)
a e aluable sou ces o hese bioac i e compounds. In addi ion, ecycling and eusing
hese animal was es lead us o each a mo e sus ainable indus y as a second objec i e [4].
An ioxidan s 2022,11, 27. h ps://doi.o g/10.3390/an iox11010027 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2022,11, 27 2 o 13
To achie e his pu pose, he p e e ed me hod o ob ain a pep ide mix u e is enzyma ic
hyd olysis. Usually, ood p o eins a e hyd olyzed by pep idases and, in mos cases, mi-
c obial pep idases cause di e en pep ide pa e ns (Told á, Reig, A is oy, and Mo a, 2018),
bu he e a e o he enzymes om ui and ege ables. In any case, he enzyma ic hy-
d olysis equi es op imiza ion conce ning he eac ion condi ions including he selec ion
o he enzyme, eac ion ime, and empe a u e o each enzyme. Rega ding po k li e
hyd olysa es, a ious enzymes including alcalase, b omelain, la ou zyme, and papain
ha e been employed. In his sense, he op imiza ion o enzyma ic eac ion was ca ied
ou o inc ease he deg ee o hyd olysis esul ing in 27.5% wi h alcalase 2.4L
™
and no o
P o-D™ [5].
Wi hin he mea indus y, in he las yea s, he e has been a demanding in e es in po k
li e which is an adequa e sou ce o p o ein (18.54%) wi h a low- a pe cen age (3.38%)
and phospholipids, wi h g ea bene i s o human heal h [
6
]. Fu he mo e, he amino acid
p o ile is comp ised o a oma ic ( y osine and phenylalanine) and hyd ophobic (leucine,
aline, and isoleucine) amino acids ha show high an ioxidan capaci y [
7
]. O he abundan
p o eins om animal sou ces as ke a in and collagen seem o be inac i e molecules because
o hei high s able p o ein s uc u es [
8
]. In his ega d, smalle an ioxidan pep ides
(4–16 amino acids wi h a molecula weigh o 400–2000 Da) ha e also been ound in o he
animal issue [9].
Since s udies linked o he an ioxidan e ec o po k li e -de i ed pep ides a e s ill
limi ed, his s udy aimed o e alua e he e ec o se e al enzymes on po k li e in he
gene a ion o an ioxidan pep ides. To his end, a high numbe o an ioxidan pep ides
needs o be iden i ied using di e en echniques and, u he , o isola e hem o alida ion
o he esul s.
2. Ma e ials and Me hods
2.1. P epa a ion o Po cine Li e Hyd olysa e
The esh po cine li e s (n= 6) we e p o ided by a local mea e aile (Cá nicas M.
Boo, Ou ense, Spain). The connec i e issues and ex e nal a we e emo ed om he
po cine li e be o e hey we e chopped and ozen a
−
20
◦
C ill u he use. Enzyma ic
hyd olysis was ca ied ou using biop o ease LA 660 (Alcalase), b omelain 2000 U/g,
and Papain 6000 USP p o ided by Biocon (Ba celona, Spain), meanwhile la ou enzyme
®
1000 L was supplied by No ozymes (Bags æ d, Denma k). The ea men s o each enzyme
we e pe o med comple ely sepa a ely. Homogeniza ion o samples was pe o med by
mixing wi h ice (1:1 li e /ice) in a cu e machine (Talsa K3, Valencia, Spain) o 30 min
be o e adding he enzyme. The enzyma ic hyd olysis was pe o med a op imum pH and
empe a u e desc ibed o each enzyme: Alcalase (50
◦
C, pH = 8), b omelain (40
◦
C,
pH = 6
),
la ou enzyme
®
(50
◦
C, pH = 5.5), and papain (37
◦
C, pH = 6). An enzyme-subs a e a io
o 1:100 (w/w) was employed in all incuba ions o se en hou s unde agi a ion in an
o bi al shake incuba o (125 pm). Du ing he hyd olysis, pe iodic adjus men s o pH we e
pe o med by employing NaOH o HCl 1N. Finally, hyd olysa es we e hea ed a 95
◦
C
o 3 min o deac i a e he enzymes and, a e wa ds, li e homogena es we e cooled in
an ice ba h. Hyd olysa es we e cen i uged (Alleg a X-22R Cen i uge, Beckman Coul e ,
Ba celona, Spain) a 4000
×
g o 10 min. The pu i ied ex ac s we e ozen a
−
80
◦
C un il
analysis. Fo each enzyma ic ea men , hyd olysis was conduc ed in iplica e.
2.2. F ee Amino Acid P o ile
The ex ac ion o ee amino acids was ca ied ou as ollows: hyd olysa es ex ac
(0.5 g) was homogenized wi h 25 mL o HCl 0.1 M o 8 min employing a dispe se (Ika, T
25 digi al Ul a-Tu ax
®
, S au en, Ge many). A e wa ds, i was cooled and cen i uged a
5000
×
g o 20 min and 200
µ
L o he supe na an was mixed wi h 800
µ
L o ace oni ile o
p ecipi a e possible unhyd olyzed p o eins. Finally, cen i uga ion a 5000
×
g o 5 min was
pe o med and an aliquo was ozen a
−
20
◦
C and s o ed un il analysis. De i a iza ion
and sepa a ion by HPLC we e ca ied ou acco ding o F anco and Lo enzo [
10
]. F ee
An ioxidan s 2022,11, 27 3 o 13
amino acids we e iden i ied by e en ion ime using an amino acid s anda d and exp essed
in mg/100 g o po k li e .
2.3. An ioxidan Ac i i y
2.3.1. DPPH Radical Sca enging Ac i i y
The DPPH assay was pe o med ollowing B and-Williams e al. [
11
] wi h sligh
modi ica ions. A sample o 100
µ
L was mix u e wi h 3900
µ
L o DPPH solu ion (60
µ
M
in me hanol) and incuba ed o 10 min a 37
◦
C. Abso bance was measu ed a 515 nm
(Shimadzu spec opho ome e , Kyo o, Japan). T olox eac i e was he s anda d used and
esul s we e exp essed as µg T olox equi alen s (TE)/g sample.
2.3.2. ABTS Radical Sca enging Ac i i y
ABTS Radical Ca ion Decolo iza ion was pe o med ollowing Re e al. [
12
] wi h sligh
modi ica ions. ABTS was p epa ed by mixing 7 mM ABTS s ock solu ion wi h 2.45 mM
po assium pe sul a e and main aining he mix u e in he da k a oom empe a u e o
12–16 h be o e i s u iliza ion. Be o e use, he ABTS s ock solu ion was dilu ed wi h dis illed
wa e o achie e an abso bance o 0.70 a 734 nm, being equilib a ed a 30
◦
C. The solu ion
(980 mL) was added o an aliquo o 20 mL o each hyd olysa e/s anda d. Abso bance was
measu ed a e 10 min in he da kness. Asco bic acid was he s anda d used and esul s
we e exp essed as mg asco bic acid/100 g sample.
2.3.3. Fe ic Reducing An ioxidan Powe Assay (FRAP)
The FRAP es was ca ied ou ollowing Benzie and S ain [
13
], wi h mino changes.
FRAP eagen was eshly p epa ed om 0.3 M ace a e bu e (pH 3.6), 10 mM 2,4,6-
ipy idyl-s- iazine in 40 mM HCl and 20 mM FeCl
3
:6H
2
O in he a io o 10:1:1 ( : : ). An
aliquo o 900 mL o FRAP solu ion was mixed wi h 30
µ
L o p ope ly dilu ed samples
and 90
µ
L o dis illed wa e . A e incuba ion o 20 min a 37
◦
C in he da kness, he
abso bance was de e mined a 593 nm. The FeSO
4
was he s anda d used and esul s we e
exp essed as µmol Fe+2/100 g sample.
2.3.4. Oxygen Radical Abso bance Capaci y Assay (ORAC)
The ORAC es was pe o med ollowing Huan e al. [
14
] wi h mino modi ica ions.
The eac ion was ca ied ou in 75 mM phospha e bu e (pH 7.4), being 200
µ
L o he inal
olume o he eac ion mix u e. Twen y- i e
µ
L o dilu e sample and 150
µ
L o 0.8
µ
M
luo escein (oxidizable subs a e) we e added in o he in e nal wells o a black 96-well
mic opla e (Bio ek, Syne gy H1, Winooski, VT, USA) and which was immedia ely incuba ed
a 37
◦
C o 30 min in he own luo escence ins umen . A e wa ds, 25
µ
L o 2,2-azobis
(2-me hylp opionamidine) dihyd ochlo ide 184 mM solu ion we e added apidly o each
well o begin he eac ion in he mic opla e. The luo escence was measu ed wi h exci a ion
and emission il e s o 485 nm and 528 nm, espec i ely. The phospha e bu e and T olox
we e used as he blank and s anda d e e ence, espec i ely. The esul s we e es ima ed
based on he di e ences o a eas unde he cu es o luo escence decay o he luo escein
be ween he blank and he sample and exp essed as mg T olox Equi alen (TE)/g sample.
2.4. Pep idomic Analysis
2.4.1. Iden i ica ion o Pep ides by Liquid Ch oma og aphy and Tandem Mass
Spec ome y (LC-MS/MS) Analysis
A pep ide mix u e o 3
µ
L was loaded on o a ap column (3
µ
C18-CL 120
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ORACassays o es ima ingan ioxidan ac i i y omgua a ui ex ac s.J.FoodCompos.Anal.2006,19,669–675.
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112;ISBN9780124158139.

Ᾰ
,
350
µ
m
×
0.5 mm; Eksigen , AB Sciex, Alcobendas, Mad id) and desal ed wi h 0.1%
TFA a 5
µ
L/min o 5 min. A e wa ds, he pep ides we e loaded on o he column (3
µ
C18-CL 120
An ioxidan s2022,11,xFORPEERREVIEW13o 13

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24. Thaipong,K.;Boonp akob,U.;C osby,K.;Cisne os‐Ze allos,L.;HawkinsBy ne,D.Compa isono ABTS,DPPH,FRAP,and
ORACassays o es ima ingan ioxidan ac i i y omgua a ui ex ac s.J.FoodCompos.Anal.2006,19,669–675.
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die .”In .J.FoodSci.Technol.2014,49,2610–2618.
26. Chen,W.;Zeng,Q.F.;Xu,H.P.;Fang,G.F.;Wang,S.D.;Li,C.H.;Wang,Y.D.;Wang,H.;Zeng,Y.Q.Compa isonand ela ionship
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Collagen,Gela in,and heDe i edPep ides omYellow inTuna(Thunnusalbaca es)Skin.Ma .D ugs2020,18,98.
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33. C oom,E.Me abolismo Xenobio icso HumanEn i onmen s,1s ed.;Else ie Inc.:Ams e dam,TheNe he lands,2012;Volume
112;ISBN9780124158139.

Ᾰ
, 0.075
×
150 mm; Eksigen , AB Sciex, Alcobendas, Mad id) equilib a ed in
5% ace oni ile 0.1% FA ( o mic acid). Elu ion was done wi h a linea g adien om 7%
o 45% B in A o 20 min. (A: 0.1% FA; B: ACN, 0.1% FA) a a low a e o 300 nL/min.
Pep ides we e iden i ied in a mass spec ome e nanoESI qQTOF (6600 plus T ipleTOF,
An ioxidan s 2022,11, 27 4 o 13
SCIEX, F amingham, MA, USA) in a da a-dependen mode. Samples we e ionized in a
Sou ce Type: Op i low < 1
µ
L Nano applying 3.0 kV o he sp ay emi e a 200
◦
C. Su ey
MS1 scans we e acqui ed om 350–1400 m/z o 250 ms. The quad upole esolu ion was
se o ‘LOW’ o MS2 expe imen s, which we e acqui ed 100–1500 m/z o 25 ms in ‘high
sensi i i y’ mode using he ollowing swi ch c i e ia: cha ge: +1 o +4; minimum in ensi y;
100 coun s pe second. Up o 50 ions we e selec ed o agmen a ion a e each su ey
scan. Dynamic exclusion was se o 15 s. The sys em sensi i i y was con olled by analyz-
ing 500 ng o K562 ypsin diges ion. In hese condi ions, 2260 p o eins we e iden i ied
(FDR < 1%) in 45 min g adien .
P o einPilo 5.0. (SCIEX) de aul pa ame e s we e employed o gene a e peak lis s
di ec ly om 6600 plus T ipleTOF wi iles. The Pa agon algo i hm [
15
] o P o einPilo
5.0 was used o sea ch he Unip o mammals da abase wi h he ollowing pa ame e s:
none enzyme speci ici y, axonomy es ic ed o pig, and he sea ch e o se o apid.
2.4.2. Label-F ee Rela i e Quan i a i e Analysis by Mass Spec ome y
The quan i ica ion o pep ides was pe o med acco ding o he label- ee me hodology
desc ibed by [
2
]. This app oach is based on he measu emen o ela i e ion in ensi ies
o ex ac ed ion ch oma og ams (XICs) o de e mine he a ios o indi idual pep ides,
employing h ee eplica es pe diges ed hyd olysa e. Pep ides we e quan i ied using
PeakView 1.1 so wa e (AB Sciex, F amingham, MA, USA) and analyzed wi h Ma ke
View 1.3 so wa e (AB Sciex, F amingham, MA, USA). The p o ein g ouping was ca ied
ou by he P o g oup algo i hm. A p o ein g oup in a P o G oup Repo is a se o p o eins
ha sha e some physical e idence. Unlike sequence alignmen analyses whe e ull-leng h
heo e ical sequences a e compa ed, he o ma ion o p o ein g oups in P o G oup is
guided en i ely by obse ed pep ides only. As obse ed pep ides we e iden i ied om
expe imen ally acqui ed spec a, he g ouping can be guided by spec a usage. Then,
unobse ed egions o p o ein sequence play no ole in explaining he da a.
2.5. S a is ical Analysis
S a is ical analysis was pe o med employing he IBM SPSS S a is ics 23.0 p og am
(IBM Co po a ion, Some s, NY, USA). An ANOVA was applied o e alua e he e ec o
each enzyme ea men on he an ioxidan ac i i y o he hyd olysa e. The leas -squa e
means (LSM) o he ou ea men s we e sepa a ed using Duncan’s pos hoc es . All s a is-
ical es s o LSM we e pe o med o a signi icance le el o p< 0.05. Co ela ions among
an ioxidan s es s (p< 0.01) and iden i ied and quan i ied amino acids and pep ides we e
de e mined employing Pea son’s linea co ela ion coe icien . To be e unde s and he
ela ionships among he di e en e ec s o he enzymes o e hyd olysa es an ioxidan ac-
i i y, a clus e analysis based on he unweigh ed pai g oup me hod wi h a i hme ic mean
(UPGMA) dend og am was pe o med using XLSTAT 2021.3.1 (Addinso , Pa is, F ance).
3. Resul s
3.1. Cha ac e iza ion o Po cine Li e Hyd olysa es by Enzyma ic Reac ion
In he p esen s udy, he enzyma ic hyd olysis was ca ied ou using ou enzymes,
alcalase, b omelain, la ou zyme, and papain, o 7 h o each he mos an ioxidan ac i i y.
3.1.1. Amino Acid Composi ion o Po cine Li e Hyd olysa es
The ee amino acids (FAA) p o iles which also p o ide a ough idea o he hyd olysis
p ocess a e shown in Table 1. The highes amoun o FAA was achie ed using la ou zyme
(3065.13 mg/100 g li e ) and he lowes in papain hyd olysa es (1285.73 mg/100 g li e ),
eaching s a is ical di e ences (p< 0.05). On he con a y, be ween hyd olysa es om
alcalase (1572.38 mg/100 g li e ) and b omelain (1904.05 mg/100 g li e ), no signi ican
di e ences (p> 0.05) we e ound on FAA o al con en . All li e hyd olysa es FAA p o iles
om he ou ea men s we e cha ac e ized by a high le el o leucine, lysine, and aline in
he essen ial ac ion. Indeed, leucine anged om 152.95 o 353.05 mg/100 g p o ein a e
An ioxidan s 2022,11, 27 5 o 13
hyd olysis pe o med by papain and la ou zyme, espec i ely. In he case o aline, he
lowe alue (77.80 mg/100 g p o ein) and he highe alue (220.51 mg/100 g p o ein) we e
also p oduced by la ou zyme and papain, espec i ely.
Table 1.
F ee amino acid p o ile o li e (mg/100 g li e ) om hyd olysis ea men wi h di e en
enzymes (alcalase, b omelain, la ou zyme, and papain).
AA Alcalase B omelain Fla ou zyme Papain SEM p-Value
Essen ial
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
Me 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
Th 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
To al EAA 814.65 b935.66 b1647.88 a605.56 c87.86 <0.0001
Non-Essen ial
A g * 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
P o 22.35 c229.35 a53.01 b60.55 b17.51 <0.0001
Se 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
Ty 91.18 a64.36 b86.93 a49.80 c4.07 <0.0001
To al NEAA 757.73 c968.38 b1417.25 a680.16 c66.57 <0.0001
To al FAA 1572.38 bc 1904.05 b3065.13 a1285.73 c153.79 <0.0001
Ra io
EAA/NEAA 1.07 b0.96 c1.16 a0.88 d0.02 <0.0001
* A ginine and au ine a e conside ed as semi-essen ial amino acids; EAA = essen ial amino acids;
NEAA = non-essen ial amino acids; AA = amino acids; SEM = s anda d e o o mean; a o d uppe le e s
indica e signi ican di e ences a p< 0.05 le el using Duncan es .
3.1.2. Pep ide Composi ion o Po cine Li e Hyd olysa es
The pep idomic p o iles in po k li e hyd olysa es we e in e ed using he quan i ica-
ions o LC-MS/MS. A e enzyma ic ea men s, he pep ides om po k li e we e iden i ied
and quan i ied by LC-MS/MS. Fo he clus e analysis, only da a om pep ides iden i ied
wi h di e en ial abundance (44 pep ides) we e conside ed in he UPGMA dend og am
(Figu e 1). As can be obse ed, hyd olysa es o enzyma ic ea men s (alcalase, b omelain,
la ou enzyme, and papain) we e g ouped in o ou g oups. This inding demons a ed ha
he pep ide p o ile is di e en and dis inc i e o each enzyma ic ea men .

An ioxidan s 2022,11, 27 6 o 13
An ioxidan s 2022, 11, x FOR PEER REVIEW 6 o 13
Figu e 1. Clus e analysis using he unweigh ed pai g oup me hod wi h a i hme ic mean (UPGMA)
dend og am based on pep ides quan i ica ions p o ided by he ac ion o alcalase, b omelain, pa-
pain, and la ou yme® on po k li e .
3.2. An ioxidan Capaci y o Po cine Li e Hyd olysa es
The an ioxidan capaci y o po k li e hyd olysa es was measu ed using DPPH,
ABTS, FRAP, and ORAC as shown in Figu e 2. Hyd olysa es om alcalase and b omelain
esul ed in he mos an ioxidan capaci y. Alcalase enzyme gene a ed he highes an ioxi-
dan ac i i y measu ed by ORAC (22.64 mg T olox/g) and ABTS (655.63 mg AA/100 g);
meanwhile, he b omelain enzyme showed he g ea es an ioxidan alues by FRAP (32.55
µmol Fe+2/100 g) and DPPH (283.87 µg T olox/g).
Figu e 2. An ioxidan in i o es s o e alua e an ioxidan capaci y om he ou di e en po k
li e hyd olysa es p oduced by alcalase, b omelain, papain, and la ou yme. a o d uppe le e s
indica e signi ican di e ences a p < 0.05 le el using Duncan es .
3.2.1. An ioxidan E ec o F ee Amino Acids om Po cine Li e Hyd olysa es
The an ioxidan ac i i y o hese hyd olysa es may la gely be due o FAA. As shown
in Table 2 he FAA in ol ed in an ioxidan ac i i y ha e di e en chemical p ope ies in
Figu e 1.
Clus e analysis using he unweigh ed pai g oup me hod wi h a i hme ic mean (UPGMA)
dend og am based on pep ides quan i ica ions p o ided by he ac ion o alcalase, b omelain, papain,
and la ou yme®on po k li e .
3.2. An ioxidan Capaci y o Po cine Li e Hyd olysa es
The an ioxidan capaci y o po k li e hyd olysa es was measu ed using DPPH, ABTS,
FRAP, and ORAC as shown in Figu e 2. Hyd olysa es om alcalase and b omelain esul ed
in he mos an ioxidan capaci y. Alcalase enzyme gene a ed he highes an ioxidan ac i i y
measu ed by ORAC (22.64 mg T olox/g) and ABTS (655.63 mg AA/100 g); meanwhile, he
b omelain enzyme showed he g ea es an ioxidan alues by FRAP (32.55
µ
mol Fe
+2
/100 g)
and DPPH (283.87 µg T olox/g).
An ioxidan s 2022, 11, x FOR PEER REVIEW 6 o 13
Figu e 1. Clus e analysis using he unweigh ed pai g oup me hod wi h a i hme ic mean (UPGMA)
dend og am based on pep ides quan i ica ions p o ided by he ac ion o alcalase, b omelain, pa-
pain, and la ou yme® on po k li e .
3.2. An ioxidan Capaci y o Po cine Li e Hyd olysa es
The an ioxidan capaci y o po k li e hyd olysa es was measu ed using DPPH,
ABTS, FRAP, and ORAC as shown in Figu e 2. Hyd olysa es om alcalase and b omelain
esul ed in he mos an ioxidan capaci y. Alcalase enzyme gene a ed he highes an ioxi-
dan ac i i y measu ed by ORAC (22.64 mg T olox/g) and ABTS (655.63 mg AA/100 g);
meanwhile, he b omelain enzyme showed he g ea es an ioxidan alues by FRAP (32.55
µmol Fe+2/100 g) and DPPH (283.87 µg T olox/g).
Figu e 2. An ioxidan in i o es s o e alua e an ioxidan capaci y om he ou di e en po k
li e hyd olysa es p oduced by alcalase, b omelain, papain, and la ou yme. a o d uppe le e s
indica e signi ican di e ences a p < 0.05 le el using Duncan es .
3.2.1. An ioxidan E ec o F ee Amino Acids om Po cine Li e Hyd olysa es
The an ioxidan ac i i y o hese hyd olysa es may la gely be due o FAA. As shown
in Table 2 he FAA in ol ed in an ioxidan ac i i y ha e di e en chemical p ope ies in
Figu e 2.
An ioxidan
in i o
es s o e alua e an ioxidan capaci y om he ou di e en po k li e
hyd olysa es p oduced by alcalase, b omelain, papain, and la ou yme. a o d uppe le e s indica e
signi ican di e ences a p< 0.05 le el using Duncan es .
3.2.1. An ioxidan E ec o F ee Amino Acids om Po cine Li e Hyd olysa es
The an ioxidan ac i i y o hese hyd olysa es may la gely be due o FAA. As shown in
Table 2 he FAA in ol ed in an ioxidan ac i i y ha e di e en chemical p ope ies in hei
side chains as pola uncha ged (se ine and h eonine), pola cha ged (his idine and aspa ic
An ioxidan s 2022,11, 27 7 o 13
acid), and apola (p oline). Fu he mo e, he pola FAAs dec eases he an ioxidan capaci y
(nega i e co ela ion) in con as o p oline as an apola FAA (posi i e co ela ion). I has
been demons a ed ha FAAs p oduce di e en an ioxidan ac i i ies depending on he
p ope ies o hei side esidues. Speci ically, cys eine, me hionine, yp ophan, y osine,
and his idine ha e p o ed o ha e highe an ioxidan capaci y han o he amino acids
because hey a e ela i ely easily oxidized [
16
]. Howe e , nei he o hese FAA signi ican ly
imp o ed he an ioxidan ac i i y o hyd olysa es in ou s udy.
Table 2. Co ela ions be ween ee amino acids and in i o an ioxidan es .
Aminoacid ABTS DPPH FRAP ORAC
His −0.654 −0.542
Th −0.662 −0.559
Asp −0.687 −0.893
P o 0.704 0.541
Se −0.770 −0.622
Tau −0.727 −0.666 −0.591
Only hose co ela ions signi ican (p< 0.01) in a leas wo es s and wi h a co ela ion coe icien highe han 0.5
a e shown.
3.2.2. Pep ides o Po cine Li e Hyd olysa es wi h An ioxidan Capaci y
F om he pep ides quan i ied wi h signi ican di e ences among he ou enzyma ic
ea men s (Table 3), co ela ion wi h an ioxidan capaci y using ORAC, FRAP, ABTS, and
DPPH es s we e analyzed. F om his ini ial se o pep ides, se en een pep ides showed a
signi ican co ela ion (p≤0.05) wi h a coe icien co ela ion highe han 0.5 (Table 4).
De i ed pep ides o collagen esul ed in a la ge in luence on an ioxidan capaci y
mainly co ela ed wi h DPPH bo h posi i ely and nega i ely. The pep ide SVGPVG-
PAGPI and SP[Oxi]GPDGKTGPP[Oxi]GPAG p oduced by he ac ion o alcalase and b ome-
lain we e posi i ely co ela ed wi h he an ioxidan es o DPPH ( = 0.833 and 0.850,
p< 0.01; espec i ely). On he con a y, GSP[Oxi]GPSGSP[Oxi]GQRGEP[Oxi]GP and
GAP[Oxi]GDKGETGPSGPAGPT by hyd olyza ion wi h papain we e nega i ely co ela ed
( = −0.777 and −0.843, p< 0.05 and 0.01; espec i ely).
I should be no ed ha h ee pep ides om albumin also esul ed in an ioxidan abili y
co ela ed wi h ABTS and ORAC assays. Thus, he pep ides NDNPDIPKLKPDPV and
DNPDIPKLKPDPVAL p oduced by alcalase ac ion was co ela ed wi h ABTS ( = 0.917
and 0.990, p< 0.01; espec i ely) and ORAC ( = 0.729 and 0.788, p< 0.05; espec i ely).
Unlike o he s, he pep ide DFQEDEQKFW hyd olyzed by b omelain was co ela ed wi h
DPPH ( = 0.836, p< 0.05).
Pep ides wi h an ioxidan capaci y, om i on p o eins including cy och ome B and
haemoglobin, we e also de ec ed. As o he pep ides p oduced by alcalase, LVLMILVL
(cy och ome B) was s ongly co ela ed wi h ABTS and ORAC ( = 0.983 and 0.765, p< 0.01
and 0.05; espec i ely). The pep ide o SDGLKHLDNLK (haemoglobin) was ela ed o he
DPPH es ( = 0.753, p< 0.05), as wi h o he pep ides o he b omelain enzyme.
O he pep ides om he p o eins ela ed o me abolic pa hways such as uc ose-
bisphospha e aldolase, dihyd opy imidinase, and a ginosuccina e syn hase we e also
pa icula ly co ela ed wi h ABTS and ORAC. These pep ides o me abolic p o eins we e
pa icula ly gene a ed by alcalase and co ela ed wi h an ioxidan capaci y using ABTS and
ORAC. O e all, he main candida es o an ioxidan pep ides om po cine li e hyd olysa es
esul ing om alcalase ac ion we e pa icula ly sensi i e o ORAC and ABTS. Addi ionally,
b omelain ac ion appea s o inc ease an ioxidan pep ides measu ed by he DPPH es .
An ioxidan s 2022,11, 27 8 o 13
Table 3.
Po k li e pep ides iden i ied and quan i ied by LC-MS/MS om hyd olysis ea men wi h di e en enzymes (alcalase, b omelain, la ou zyme,
and papain).
Pep ide Sequence P o ein o O igin Gen (Unip o
ID) Alcalase B omelain Fla ou zyme Papain SEM p-Value
GVRGPNGDSGRP[Oxi]GEP[Oxi]G Fib illa collagen NC1 domain-con aining p o ein COL1A2 46,984 a237,462 b655,298 c12,167 a97.120 <0.001
GSP[Oxi]GPSGSP[Oxi]GQRGEP[Oxi]GPQ Collagen ype 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 p ep op o ein COL1A1 40,343 a355,355 b45,816 a33,045 a51.950 <0.001
GSP[Oxi]GPSGSP[Oxi]GQRGEP[Oxi]GP Collagen ype 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 p ep op o ein COL1A1 5415 a95,886 b278,639 c107,394 b39.555 0.022
VLSAADKANVK GLOBIN domain-con aining p o ein LOC110259958 7566 a12,638 a10,862 a809,312 b142.092 0.042
AP[Oxi]GDKGETGPSGPAGPTG Collagen alpha-1(I) chain p ep op o ein COL1A1 443 a8189 a151,220 b463,662 c73475 0.009
GKDGEAGAQGPP[Oxi]GPA Collagen alpha-1(I) chain p ep op o ein COL1A1 32,253 a10,023 a833,315 b77,903 a130.966 <0.001
GVQGPP[Oxi]GPAGEEGKRG Collagen alpha-1(I) chain p ep op o ein COL1A1 9582 a19,649 a595,576 b4043 a95.797 <0.001
RKPPTDEESLEK Glu a hione ans e ase GSTO1 372,448 c6332 a29,252 a122,768 b56.928 0.009
M[DTM]GDSRDPASDQMK Ca alase CAT 15,852 203,806 934 428,309 72.553 0.061
GHQGAVGSP[Oxi]GPAGP Collagen ype III alpha 1 chain COL3A1 66,649 199,720 7523 375,109 59.011 0.058
GASGPAGPRGPP[Oxi]GSA Collagen alpha-1(I) chain p ep op o ein COL1A1 9990 a17,658 a308,010 c52,706 b46.659 <0.001
GPVGPSGPP[Oxi]GKDGASG Collagen ype 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 p ep op o ein COL1A1 71,644 a8189 a267,506 b455,180 c69.021 0.011
SGPAGPRGPP[Oxi]GSA Collagen alpha-1(I) chain p ep op o ein COL1A1 429,583 6914 173,859 116,358 64.360 0.051
GLP[Oxi]GTSGPP[Oxi]GENGKP[Oxi]GEP[Oxi]GPK Collagen ype III alpha 1 chain COL3A1 40,928 588,757 285,781 156,852 126.713 0.560
GSP[Oxi]GERGEVGPAGPNG Fib illa collagen NC1 domain-con aining p o ein COL1A2 4779 a11,815 a509,034 b12,027 a81.786 <0.001
DQGPVGRTGETGASGP[Oxi]PG Fib illa collagen NC1 domain-con aining p o ein COL1A2 18,656 b23,656 b309,185 c11,533 a47.713 <0.001
AHHPDDFNPSVH GLOBIN domain-con aining p o ein LOC110259958 88,515 23,406 362,462 889,540 209.499 0.549
GPIGSRGPSGPP[Oxi]GPDGNKGEP[Oxi]G Fib illa collagen NC1 domain-con aining p o ein COL1A2 1213 a3841 a208,112 c17,469 b32.948 <0.001
GPRGPP[Oxi]GAVGAP[Oxi]GPQG Fib illa collagen NC1 domain-con aining p o ein 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-con aining p o ein LOC100515788 1297 a6,029,690 c104,465 ab 831,244 b956.604 0.001
GAGGGAGGGGAGAGAGGGGAGA Glu ama e me abo opic ecep o 5 GRM5 74,644 b300,710 c21,625 a10,893 a44.587 <0.001
GPHEREPTAL AMP-binding domain-con aining p o ein SLC27A5 331,724 7805 22,616 366,344 71.764 0.085
EPDAGDDDSKGSGQ Ras p o ein speci ic guanine nucleo ide eleasing
ac o 2 RASGRF2 951 a164,542 d26,459 b68,937 c23.835 0.001
LSDLHAHKLRVDPVN GLOBIN domain-con aining p o ein LOC110259958 1955 a2686 a1123 a194,505 b32.480 0.006
GPN[Dea]GDSGRP[Oxi]GEP[Oxi]GLM Fib illa collagen NC1 domain-con aining p o ein COL1A2 241,883 23,838 49,764 124,129 38.072 0.145
LANVVALTMEPK 60 kDa chape onin N/A 194,959 43,520 245,393 5966 47.643 0.220
GDAGPP[Oxi]GPAGPTGPP[Oxi]GPIGS Collagen alpha-1(I) chain p ep op o ein 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 p o ein 9 RANBP9 658,925 c84,934 a261,216 b172,460 ab 85.995 0.009
An ioxidan s 2022,11, 27 9 o 13
Table 3. Con .
Pep ide Sequence P o ein o O igin Gen (Unip o
ID) Alcalase B omelain Fla ou zyme Papain SEM p-Value
DPPKTASYPVIIQ Rhodanese domain-con aining p o ein 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-con aining p o ein 5 Nl p5 63,744 285,536 8094 96,696 44.753 0.087
GIIGPLGILGP collagen alpha-1(XXVII) chain iso o m 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 syn hase subuni be a ATPB 439,736 b15,127 a22,057 a66,749 a67.888 0.002
LEGTLLKPNMVT F uc ose-bisphospha e 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-con aining p o ein 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 Ca boxylic es e hyd olase APLE 19,318 b5361 a557,870 c12,771 ab 89.312 <0.001
PGQ.QSFSDGLKHLDNLKGTFAK GLOBIN domain-con aining p o ein LOC100515788 340 530 487 791,589 142.101 0.051
GPPLRPDPSTPDFL Dihyd opy imidinase 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 A gininosuccina e syn hase ASS1 809,850 b12,991 a764 a5895 a131.502 <0.001
DQLHVDPENFRLLG GLOBIN domain-con aining p o ein LOC100515788 33,561 b7863 a603,419 c5475 a96.371 <0.001
AMPDIPVPLTN Aldehyde dehyd ogenase 1 amily membe A1 ALDH1A1 35,049 a188,598 a12,213 a576,409 b92.337 0.037
DQLHVDPENFRLL GLOBIN domain-con aining p o ein LOC100515788 1047 a9104 a1585 a232,710 b40.397 0.035
SVGPVGPAGPI collagen alpha-2(I) chain iso o m X2 LOC101341020 1,210,722 c1,126,713 c476,081 b7974 a187.098 <0.001
PGQ.QLHVDPENFRLLG GLOBIN domain-con aining p o ein LOC100515788 3571 a14,016 a446 a268,209 b45.742 0.025
PGQ.QLHVDPENFRLL GLOBIN domain-con aining p o ein LOC100515788 4500 9124 10,757 702,733 125.698 0.058
LVLMILVL Cy och ome b CYB 427,002 b18,871 a15,799 a582 a68.566 <0.001
Only hose pep ides wi h a concen a ion highe han 150,000 in a leas one ea men g oup a e shown. Abundance mean alues o hose pep ides wi h signi ican di e ences (p< 0.05)
a e no ed by a–d uppe le e s and he highes pep ide alue a e ma ked in bold.