Academic Edi o : Yonghui Li
Recei ed: 3 Ma ch 2025
Re ised: 28 Ma ch 2025
Accep ed: 1 Ap il 2025
Published: 3 Ap il 2025
Ci a ion: Rod íguez-Muñoz, M.d.R.;
Mo a-Ga ido, A.B.; He edia, F.J.;
Cejudo-Bas an e, M.J.; González-Mi e ,
M.L. Ob en ion and Cha ac e isa ion
o An ioxidan -Rich Pep ides om
De a ed G ape Seed Meal Using
Di e en Enzymes. Foods 2025,14,
1248. h ps://doi.o g/10.3390/
oods14071248
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Ob en ion and Cha ac e isa ion o An ioxidan -Rich Pep ides
om De a ed G ape Seed Meal Using Di e en Enzymes
Ma ía del Rosa io Rod íguez-Muñoz, Ana Belén Mo a-Ga ido , F ancisco J. He edia , Ma ía
Jesús Cejudo-Bas an e * and Ma ía Lou des González-Mi e
Food Colou & Quali y Labo a o y, Facul ad de Fa macia, Uni e sidad de Se illa, 41012 Se illa, Spain;
[email p o ec ed] (M.d.R.R.-M.); [email p o ec ed] (A.B.M.-G.); he [email p o ec ed] (F.J.H.); [email p o ec ed] (M.L.G.-M.)
*Co espondence: [email p o ec ed]
Abs ac : De a ed g ape seed meal (DGSM) is a esidue ob ained om g ape pomace
and is an impo an sou ce o p o ein. The aim o his s udy was o selec pep ides wi h
op imal an ioxidan and colou p ope ies, ob ained using enzymes o di e en o igins
and p o eoly ic cha ac e , o applica ion in winemaking. Fo his pu pose, he assay was
pe o med using no o-P oD (NP), alcalase (AL), no ozym (NZ), pepsin (PE), la ou zyme
(FZ), and papain (PA) enzymes. The pep ide pe cen age, pep ide yield, molecula size o
he pep ide ac ions, o al amino acid, pep ide con en , an ioxidan ac i i y, and CIELAB
colou coo dina es o he hyd olysa es we e de e mined. The pep ide hyd olysa es ob ained
using PE showed he signi ican ly (p< 0.05) highes pe cen ages o pep ides (93%), amino
acid con en (188 mg aa/g hyd olysa e), and ligh ness (L*, 70.3). On he o he hand, NP
pep ide hyd olysa es displayed he signi ican ly (p< 0.05) highes an ioxidan ac i i y
(
154 µmol TE/g
hyd olysa e) and pep ide yield (39%). Rega ding molecula weigh (MW),
PE led o hyd olysa es wi h a lowe p opo ion o low-MW pep ides (MW < 1 kDa). In
conclusion, he pep ide hyd olysa es ob ained by NP and PE exhibi ed he g ea es chemical
cha ac e is ics o u he applica ion, bo h sepa a ely and combined in a ge ed hyd olysis,
as colou s abilise s and an ioxidan capaci y enhance s in wa m clima e winemaking.
Keywo ds: g ape seed pep ide hyd olysa es; enzymes; wa m clima e; molecula weigh
dis ibu ion; an ioxidan pep ides
1. In oduc ion
The ood indus y is g owing as due o globalisa ion and popula ion g ow h. This
gene a es a la ge amoun o was e, 158–298 kg/yea /capi a in he EU alone [
1
], wi h an
impo an en i onmen al impac since he emo al p ocess p oduces emissions and/o
he accumula ion o mic oo ganisms, pa asi es, and pes s [
2
]. Fu he mo e, he e is a
nega i e economic impac because he esidue was ed wo ldwide ep esen s an economic
loss o USD 990 million [
2
]. Fo his eason, he ood indus y is ocusing on ob aining
new alue-added p oduc s om was e, which, in u n, gene a es he de elopmen o a
ci cula economy.
Wi hin he ood indus y, he wine sec o is one o he mos impo an in e ms o
economic, cul u al, and en i onmen al amewo ks in Spain and Eu ope. I is es ima ed
ha 25% o he o al weigh o g apes is disposed o as was e du ing he wine p oduc ion
p ocess [
3
], which is classi ied in o pomace, lees, and was ewa e (a ound 62%, 14% and
12%, espec i ely) [
4
]. Due o i s a ied composi ion o bioac i e compounds and he
amoun gene a ed (a ound 7 /yea wo ldwide), g ape pomace is o g ea in e es . I
Foods 2025,14, 1248 h ps://doi.o g/10.3390/ oods14071248
Foods 2025,14, 1248 2 o 16
cons i u es a ound 10–20% o he weigh o he g ape mass and consis s o seeds, s ems,
and skins [
5
]. In addi ion, g ape pomace can be used in many di e en ields, such as ood,
pha maceu icals, and cosme ics, because i has an ioxidan and an i-in lamma o y e ec s
due o i s high polyphenol con en [
6
]. Mo eo e , a e successi e ex ac ions o ob ain
di e en compounds ha he g ape pomace indus y u he sells, he seeds a e sepa a ed
om he pomace [
7
], d ied, and he oil is ex ac ed, he eby gene a ing de a ed g ape seed
meal (DGSM) as a esidue, which is a ich p o ein sou ce ha can be u he exploi ed [8].
Ha es s o plan s in a wa m clima e, i.e., g ape ine, can be a ec ed by clima ic a i-
ables such as empe a u e and sunligh exposu e [
9
]. Due o global wa ming, empe a u es
a e inc easingly wa me , which may cause he inadequa e phenolic ma u a ion o g apes,
leading o a misma ch be ween he phenolic and echnological (suga s) ma u i y o he
g apes a he ime o ha es ing [
10
]. Among o he e ec s, empe a u es o e 30
◦
C cause a
dec ease in he phenolic con en [
10
], especially an hocyanins and copigmen s ha pa ici-
pa e in colou s abilisa ion phenomena such as copigmen a ion, p oduced by he union o
an hocyanins wi h o he wine componen s such as la onols [
11
]. Due o he imbalance
in phenolic con en be ween pigmen s and copigmen s in wines om wa m clima es, i is
di icul o main ain a s able colou du ing he s o age pe iod [
12
]. Wi h he pu pose o
sol ing his p oblem, di e en s a egies ha e been implemen ed o main ain he phenolic
compound p o ile o hese wines o e ime. One me hod in ol es he addi ion o pep ide
hyd olysa es o wine du ing he s abilisa ion phase, which p oduces a pep ide–phenolic
complex esponsible o colou p ese a ion [13].
The pep ide ac ion mus be soluble in he medium o p oduce he colou -s abilising
complex. Enzyma ic hyd olysis is an op ion o gene a ing pep ides wi h good solubili y;
i also has ad an ages o e chemical hyd olysis, including be e selec i i y, mode a e
eac ion condi ions, educed ood alle genici y, and he capaci y o no deg ade compounds
ha ha e al eady been hyd olysed [
14
,
15
]. Cejudo-Bas an e e al. [
8
] op imised a echnique
o ob aining pep ide hyd olysa es om de a ed g ape seed meal as a sou ce o pep ide
using he enzyme alcalase, wi h posi i e esul s in e ms o wine colou s abilisa ion [
13
].
Se e al ac o s can in luence he p ope ies o pep ide hyd olysa es, such as he subs a e
ype, bu one o he key ac o s is he ype o p o ease used [
14
,
16
]. Thus, he use o ce ain
p o eases, like la ou zyme, can esul in a highe p opo ion o medium- and low-MW
pep ides [
17
], which a e mo e soluble [
14
]. Addi ionally, he amino acid composi ion
o he pep ides may be en iched in glu amine o his idine, po en ially con ibu ing o
imp o ed colou s abilisa ion [
18
] and inc eased an ioxidan ac i i y [
19
]. The e o e, i is
essen ial o conduc s udies using di e en enzymes o p oduce a pep ide hyd olysa e o
use in winemaking ha is no only soluble in wine bu also exhibi s high an ioxidan and
colou -s abilising p ope ies.
This wo k mainly ocuses on de e mining how he p o eoly ic cha ac e and he o igin
o he enzyme used a ec he p ope ies o he pep ide hyd olysa e, wi h he pu pose o
selec ing op imal pep ides o use as colou s abilise s in wa m clima e winemaking. Thus,
an in-dep h chemical cha ac e isa ion based on pep ide con en , pep ide yield, molecula
weigh (MW) dis ibu ion, amino acid con en , pep ide p o ile, an ioxidan ac i i y and he
colou (CIELAB pa ame e s) o he pep ides was conside ed. This is he i s ime such a
s udy has been conduc ed, and i could ep esen an impo an s ep owa ds inno a i e
u u e applica ions in ed wine colou s abilisa ion.
2. Ma e ials and Me hods
2.1. Chemical and Reagen s
The endop o eases alcalase, no ozym, and no o-P oD, and he endo- and exop o ease
la ou zymes we e p o ided by No ozymes (Copenhagen, Denma k). The endop o ease
Foods 2025,14, 1248 3 o 16
pepsin and he endo- and exop o ease papain we e supplied by Sigma-Ald ich (S . Louis,
MO, USA) and Biocon (Ba celona, Spain), espec i ely. T ichlo oace ic acid (TCA) was
supplied by VWR Chemicals (Radno , PA, USA), and azocasein was pu chased om Sigma
Ald ich (S . Louis, MO, USA). The s anda ds we e cy och ome C (Pan eac, Ba celona,
Spain), ap o inin (Sigma-Ald ich, Mad id, Spain), i amin B12 and iglycine (Al a Aesa ,
Ha e hill, MA, USA), and glycine (The mo Fishe Scien i ic, Wal ham, MA, USA).
2.2. Samples
The aw ma e ial, de a ed g ape seed meal (DGSM), a esidue de i ed om he
p ocessing o he g ape pomace indus y, was supplied by Al inesa Na u al Ing edien s
S.A (Daimiel, Ciudad Real, Spain). B ie ly, he g ape pomace was washed in di usion
bands o ex ac componen s o in e es (alcohol, a a ic sal s, phenols, e c.) be o e being
des emmed and d ied. The g ape seeds we e hen sepa a ed om he d ied g ape pomace
by densiome ic ables and we e subsequen ly g ound and g anula ed o p oceed wi h he
seed oil ex ac ion using hexane, gene a ing DGSM as a esidue [
8
]. In he labo a o y, he
DGSM was g ound o inc ease he su ace a ea o achie e be e p o ein ex ac ion and was
inally s o ed in da kness a oom empe a u e.
2.3. Measu emen o P o ease Ac i i y
The p o ease ac i i y was measu ed acco ding o Beynon and Bond [
20
]. B ie ly, a
eac ion mix u e o 0.1 g o azocasein, 0.2 mL o e hanol, and 4.8 mL o phospha e bu e
(0.1 M a pH 7) was p epa ed and hea ed in a wa e ba h (40
◦
C) un il he componen s
we e comple ely dissol ed. To achie e abso bance alues wi hin he ange o 0.3–0.5, he
enzyme solu ions we e p epa ed using dis illed wa e : 1:4 dilu ion o NP, AL, and NZ;
1:8 dilu ion o FZ; 0.5% (w/ ) o PE; and 0.05% (w/ ) o PA. Subsequen ly, 100
µ
L o
each enzyme solu ion was mixed wi h 100
µ
L o he eac ion mix u e and hen incuba ed
o 30 min a 40
◦
C in a wa e ba h. A e s opping he eac ion wi h 5% (w/ ) TCA and
cen i uging he enzyme solu ions (10,000
×
g, 4
◦
C, 4 min), he supe na an s we e collec ed.
The azo dye eleased due o he hyd olysis o azocasein wi h p o eases was measu ed a
440 nm in an Agilen 8453 UV- is spec opho ome e (Agilen Technologies, Palo Al o, CA,
USA) o calcula e he p o ease ac i i y o each enzyme. The samples we e measu ed in
iplica e, and he esul s we e exp essed as U/mL.
2.4. Pep ide Hyd olysa es om DGSM
P o ein ex ac ion was conduc ed acco ding o he me hodology desc ibed by Cejudo-
Bas an e e al. [
8
]. A mix u e o 700 g o DGSM and 3.5 L o dis illed wa e was p epa ed
in a Bio Console ADI 1025 Bio eac o (Applikon Bio echnology, Del , Ne he lands). The
mix u e unde wen cons an agi a ion (180 pm) a pH 10 using ammonia (NH
3
) as a
basic medium o 3 h a 25
◦
C. The esul an p o ein concen a e ob ained was cen i uged
(14,880
×
g, 4
◦
C, 20 min) o sepa a e he p o eins om he non-p o ein p ecipi a e. The
supe na an was hen collec ed and concen a ed app oxima ely o 1.5 L in a o a y e ap-
o a o (1 h, 80
◦
C) o p e en he o ma ion o sal s due o he p esence o NH
3
, and his
was adjus ed o pH 8. Finally, he endogenous enzymes in he p o ein concen a e we e
deac i a ed in a wa e ba h a 80 ◦C o 5 min.
The ea e , he enzyma ic hyd olysis o he p o ein concen a e was ca ied ou in a
bio eac o unde op imised hyd olysis condi ions o each enzyme, as de ailed in Table 1.
Acco ding o Cejudo-Bas an e e al. [
8
], 0.6% ( / ) o each p o ease was added and eac ed
a he op imal hyd olysis ime. The ob ained c ude hyd olysa e was indi idually placed in
a o a y e apo a o (80
◦
C) un il he olume was app oxima ely educed by hal o educe
he pH o he subsequen s ep. Then, he pH o he c ude hyd olysa es was adjus ed o 3.5
( he usual pH o wine) using 32% HCl. In he case o pepsin, he pH was 2.5; hus, i had
Foods 2025,14, 1248 4 o 16
o be inc eased up o 3.5 using NH
3
o p o oke he p ecipi a ion o he non-hyd olysed
pep ides. Finally, he supe na an (soluble pep ides) was sepa a ed om he p ecipi a e
(insoluble pep ides), which was disca ded by cen i uga ion (15,000
×
g, 4
◦
C, 20 min), and
he soluble ac ion (pep ide hyd olysa e) was lyophilised. This p ocedu e was pe o med
in iplica e o each enzyme.
Table 1. The o igin, p o eoly ic cha ac e , and op imal hyd olysis condi ions o he di e en enzymes.
Name
O igin o Enzyme
P o eoly ic Cha ac e pH
T (
◦
C)
Time (h)
Bibliog aphy
NP Mic obial Endop o eoly ic 9 60 2 [21,22]
AL Mic obial Endop o eoly ic 8.5 55 1 [13]
NZ Mic obial Endop o eoly ic 9 50 2 [23,24]
PE Animal Endop o eoly ic 2 33 3.5 [25]
FZ Mic obial Endo-/exop o eoly ic 7 50 2 [21,26]
PA Vege al Endo-/exop o eoly ic 7 50 3.5 [22,25]
NP, no o-P oD; AL, alcalase; NZ, no ozym; PE, pepsin; FZ, la ou zyme; PA, papain.
2.5. Pep ide Con en and Pep ide Yield o Hyd olysa es
The o al ni ogen con en o he pep ide hyd olysa es was de e mined using he
s anda d Kjeldahl me hod [
27
] in an Au oma ic Kjeldahl Dis ille sys em (J.P. Selec a,
Ba celona, Spain). The pe cen age o he pep ide con en in he hyd olysa es was calcula ed
based on he ni ogen concen a ion measu ed using he ollowing o mula:
% N = [0.014 ×(V) ×(N)/(W)] ×100 (1)
whe e % N is he ni ogen pe cen age; V is he olume (mL) o HCl; N is he no mali y o
HCl; and W is he weigh (g) o he sample. Fac o 5.75 was conside ed o he con e sion
o o al ni ogen con en o pep ide con en (%) [8].
The pep ide yield o he hyd olysa es (pep ide con en (%)
×
weigh (g) o he hy-
d olysa e)/(weigh (g) o he s a ing meal) was also de e mined.
2.6. Molecula Weigh by Size-Exclusion Ch oma og aphy (SEC)
The molecula weigh was de e mined acco ding o he me hod desc ibed by Bau is a
e al. [
28
]. In o al, 0.22 g o he pep ide hyd olysa e was dissol ed in 10 mL milli-Q wa e .
A e shaking (2500 pm, 10 min) and cen i uga ion (3220
×
g, 4
◦
C, 5 min), he supe na an
was il e ed h ough a 0.45
µ
m il e . An Agilen 1100 ch oma og aphy sys em equipped
wi h a qua e na y pump, an au oma ic injec o , a UV- is diode a ay de ec o and so wa e
B.04.03 e sion (Agilen Technologies, Palo Al o, CA, USA) was used o he analysis,
using a Supe dexTM 30 Inc ease 10/300 GL column (op imum sepa a ion ange 0.1–7 kDa).
Fo he sepa a ion o pep ide ac ions, a low a e o 0.5 mL/min o 50 mM Na
2
HPO
4
solu ion (pH 7.5) was used in isoc a ic mode a 25
◦
C. Acco ding o he manu ac u e ’s
indica ions, di e en s anda ds we e used o co e he ange 100–7000 Da: cy och ome C,
12,500 Da; ap o inin, 6500 Da; i amin B12, 1355 Da; iglycine, 189 Da; and glycine, 75 Da.
The pep ide ac ions we e moni o ed a 215 and 280 nm. The analysis was pe o med in
iplica e o each pep ide hyd olysa e.
2.7. Amino Acid Analysis
To de e mine he indi idual and o al con en and p o ile o amino acids in he pep-
ide hyd olysa es, ion exchange ch oma og aphy and pos -column de i a isa ion using
ninhyd in we e used [
8
]. A o al o 5 mg o each sample was mixed wi h 1 mL o 6 M
HCl and hyd olysed in a hea ing block a 110
◦
C o 24 h. A e cen i uga ion (
11,357×g
,
4◦C
, 15 min), supe na an s we e collec ed, and pH was adjus ed o 2.0 using 6 M and
1 M
NaOH. In o al, 400
µ
L o each sample was mixed wi h 100
µ
L o he in e nal s anda d
Foods 2025,14, 1248 5 o 16
no leucine a 50
µ
M. Finally, he samples we e analysed using a Bio 30+ Amino Acid
Analyse sys em (Bioch om L d., Camb idge, UK), equipped wi h a high-p essu e PEEK
ca ion exchange column wi h Ul opac 8 ca ion exchange esin and a UV- is de ec o . The
de ec ion wa eleng hs we e 440 nm o p oline and 570 nm o he o he amino acids.
The amino acid con en o each sample was quan i ied in iplica e, and he esul s we e
exp essed in mg/g o pep ide hyd olysa e.
2.8. Pep ide Iden i ica ion
Samples we e desal ed and concen a ed using OMIX Pipe e ips C18 (Agilen Tech-
nologies, Palo Al o, CA, USA). The desal ed pep ide hyd olysa es we e d ied, esuspended
in 0.1% o mic acid (10 mL) and analysed using RP-LC-MS/MS in an EASY-nLC II sys-
em coupled o an ion ap LTQ-O bi ap-Velos-P o hyb id mass spec ome e (The mo
Scien i ic, Wal ham, MA, USA). The pep ides we e concen a ed by e e se-phase ch o-
ma og aphy (0.1 mm
×
20 mm C18 RP P ecolumn, The mo Scien i ic) and sepa a ed using
a 0.075 mm
×
250 mm C18 RP column wi h a low o 0.3
µ
L/min. Then, a 180 min dual
g adien wi h he ollowing p o ile was used o elu e he pep ides: 5–25% sol en B o
135 min, 25–40% sol en B o 45 min, 40–100% sol en B o 2 min, and 100% sol en B o
18 min (sol en A: 0.1% o mic acid in wa e , sol en B: 0.1% o mic acid, 80% ace oni ile
in wa e ). Elec osp ay ionisa ion (ESI) was pe o med using a Nano-bo e emi e made o
s ainless s eel wi h an inne diame e o 30
µ
m (P oxeon, Odense, Denma k) in e aced a a
sp ay ol age o 2.1 kV wi h an S-Lens e iciency o 60%. The O bi ap esolu ion was se o
30,000. Su ey scans we e conduc ed wi hin he ange o 400–1600 a omic mass uni s (amu)
(1
µ
scan), ollowed by 20 sequen ial da a-dependen MS/MS scans (Top 20). The isola ion
wid h o MS/MS was se a 2 u (in mass- o-cha ge a io uni s), wi h a no malised collision
ene gy o 35%, and dynamic exclusion was implemen ed o 60 s in e als. Cha ge-s a e
sc eening was ac i a ed o disca d unassigned and singly cha ged p o ona ed ions. The
esul s we e hen ma ched wi h he UniP o da abase and Peaks X P o so wa e ( e sion
X P o) [8].
2.9. An ioxidan Ac i i y
2.9.1. DPPH Assay
The me hod desc ibed by Sole -Ri as e al. [
29
] was applied o e alua e he an ioxidan
ac i i y o he pep ides. In o al, 0.02 g o each hyd olysa e was dissol ed in 10 mL o
PBS. Then, o o mula e a calib a ion cu e, 300
µ
L o 108
µ
M DPPH me hanolic solu ion
was added o 30
µ
L o he sample, s anda d T olox o 80 % ( / ) blank o me hanol, and
inally, he mix u e was dilu ed wi h 570
µ
L o 80% ( / ) me hanol. A e 30 min in he
da k, abso bance was measu ed a 515 nm in an Agilen 8453 UV- is spec opho ome e
(Agilen Technologies, Palo Al o, CA, USA). The analysis was pe o med in iplica e, and
he esul s we e exp essed as T olox equi alen s (TE)/g pep ide hyd olysa e.
2.9.2. ABTS F ee Radical Sca enging Assay
The an ioxidan ac i i y was de e mined using he ABTS me hod, as desc ibed by Re
e al. [
30
]. Fi s , 7 mM ABTS
•
+ s ock solu ion was p epa ed using pe sul a e 2.45 mM as
he oxidising agen and s o ed in he da k a oom empe a u e o 12–16 h. Subsequen ly,
he wo king eagen o ABTS and PBS (1 mL o ABTS and 79 mL o PBS) was p epa ed,
adjus ing i s abso bance o 0.7
±
0.02 by measu ing he abso bance a 734 nm wi h an
Agilen 8453 spec opho ome e (Agilen Technologies, Palo Al o, CA, USA). Subsequen ly,
a p e-dilu ion es was pe o med on he samples, using dilu ions o 1/10, 1/20 and 1/40
o each sample in iplica e. Finally, 2 mL o he wo king eagen was added o 50
µ
L o
each dilu ion, and he abso bance a 734 nm o each sample was measu ed. The esul s
we e exp essed as T olox equi alen s (TE)/g samples.
Foods 2025,14, 1248 6 o 16
2.10. Colo ime ic Analysis
A CM-5
®
spec opho ome e (Konica Minol a, Tokyo, Japan) was used o measu e he
colou o he pep ide hyd olysa es by di use e lec ance, simila o o he au ho s [
31
]. The
CIELAB colo ime ic pa ame e s (L*, a*, b*, C*
ab
, and h
ab
) we e ob ained ollowing he
ecommenda ions o he Commission In e na ionale de L’Eclai age [
32
] using he CIE 1964
10
◦
S anda d Obse e and he S anda d Illuminan D65 o he calcula ion. Measu emen s
we e pe o med in iplica e o each pep ide hyd olysa e sample (ob ained wi h each
enzyme). F om he colou da a, conside ing he Euclidean dis ance be ween wo poin s
in he h ee-dimensional CIELAB colou space, he colou di e ences (
∆
E*
ab
) be ween
pai s o samples we e calcula ed using he ollowing CIE 1976 colou di e ence o mula:
∆
E*
ab
= [(
∆
L*)
2
+ (
∆
a*)
2
+ (
∆
b*)
2
]
1/2
. Fu he mo e, he ela i e con ibu ions o ligh ness
(%
∆
L), ch oma (%
∆
C), and hue (%
∆
H), which make a gi en colou di e ence (
∆
E*
ab
)
exp essed as pe cen ages, we e calcula ed as ollows:
%∆L= [(∆L*)2/(∆E*ab)2]×100
%∆C= [(∆C*ab)2/(∆E*ab)2]×100
%∆H= [(∆H)2/(∆E*ab)2]×100
∆His deduced ma hema ically om ∆H= [(∆E*ab)2−(∆L)2−(∆C)2]1/2.
2.11. S a is ical Analysis
S a is ica .8.0 so wa e was used o pe o m he s a is ical analysis o he da a [
33
].
A uni a ia e analysis o a iance (ANOVA) using a gene al linea model p og amme
was used o es ablish whe he he means o he sample da a signi ican ly di e ed om
each o he . P incipal componen analysis (PCA) was also conduc ed o iden i y he main
con ibu o s o a iance.
3. Resul s and Discussion
3.1. P o ease Ac i i y
The amoun o enzyme equi ed o he hyd olysis p ocess was calcula ed and ad-
jus ed as a unc ion o i s co esponding p o eoly ic ac i i y o ensu e ha he pa ame e s
measu ed in he hyd olysa es did no depend on his ac o . The p o eoly ic ac i i ies o all
he enzymes we e de e mined (Table 2). The enzyme FZ was ound o p o ide signi ican ly
(p< 0.05) highe p o ease ac i i y, whe eas hose wi h signi ican ly (p< 0.05) lowe ac i i y
we e PE and PA. Finally, conside ing he enzyme AL as a e e ence, enzyme olumes
leading o he same p o eoly ic ac i i y we e adjus ed and added.
Table 2. Mean alues and s anda d de ia ions o p o ease ac i i y (U/mL) o he di e en enzymes.
P o ease Ac i i y (U/mL)
NP 703.84 ±64.81 b
AL 807.53 ±5.03 b
NZ 786.8 ±73.79 b
PE 311.7 ±11.99 c
FZ 1104.19 ±40.5 a
PA 332.56 ±11.04 c
NP, no o-P oD; AL, alcalase; NZ, no ozym; PE, pepsin; FZ, la ou zyme; PA, papain. Di e en le e s in he same
column indica e signi ican (p< 0.05) di e ences acco ding o Tukey’s es .
Foods 2025,14, 1248 7 o 16
3.2. Pep ide Pe cen age and Yield o he Pep ide Hyd olysa es
The pep ide con en o he hyd olysa es anged om 61% o 94% (Table 3). These
esul s a e compa able o hose o o he pep ide hyd olysa es de i ed om di e en oil
seeds [
34
]. Howe e , i was 2.39% when ela ed o DGSMs. As shown in Table 4, he
o igin o he enzymes was an in luen ial ac o in he pep ide con en . In his ega d, he
signi ican ly (p< 0.05) highe % pep ide was a ibu ed o he hyd olysa es ob ained by
animal enzymes (PE). In con as , he hyd olysa es ob ained by he mic obial AL displayed
he signi ican ly (p< 0.05) lowes alues. The speci ici y o alcalase o in e nal bond
clea ages [
35
] could be ela ed o he lowe pep ide pe cen age o he hyd olysa es ob ained
by AL, as he polypep ides ob ained could emain insoluble in he ex ac ion medium due
o hei size.
Table 3. Mean and s anda d de ia ion o pe cen ages o pep ide con en , pep ide yield, molecu-
la weigh (MW) dis ibu ion, and he CIELAB colou pa ame e s (L*, C*
ab
, h
ab
) o he pep ide
hyd olysa es ob ained by he di e en enzymes.
Pep ide
Con en (%)
Pep ide Yield
(%) Molecula Size F ac ions (%) Colou Pa ame e s (CIELAB Uni s)
>5 kDa 5>MW>1
kDa <1 kDa L* C*ab hab
NP
76.61 ±1.62 b 39.18 ±0.83 a 20.07 ±0.11 d 38.20 ±0.71 a
41.73
±
0.67 bc
54.51 ±2.35 b 39.79 ±2.78 b
64.82
±
0.57 cd
AL
65.89 ±4.24 d 30.52 ±1.97 b
22.05
±
0.25 cd
36.00 ±0.50 a
41.95
±
0.27 bc
53.98 ±4.22 b 41.56 ±1.11 b
64.32
±
0.83 de
NZ
77.18 ±0.59 b 31.63 ±0.24 b 20.49 ±1.73 d 38.48 ±2.29 a 41.03 ±0.66 c
52.60
±
4.85 bc
40.23 ±1.44 b 62.26 ±0.18 e
PE
93.17 ±0.34 a 38.29 ±0.14 a 33.84 ±0.57 a 33.23 ±0.66 b 32.92 ±1.23 d 70.29 ±5.42 a
33.42
±
0.27 bc 69.31
±
1.07 ab
FZ 75.66
±
0.76 bc
20.83 ±0.21 c 23.10 ±0.83 c 25.98 ±0.91 c 50.93 ±1.74 a 43.46 ±2.72 c 49.99 ±6.56 a 6.93 ±0.61 bc
PA
71.19 ±0.57 c 17.91 ±0.14 d 27.89 ±0.91 b 28.21 ±0.61 c 43.90 ±0.45 b 80.24 ±1.29 a 31.18 ±0.53 c 70.55 ±1.62 a
NP, no o-P oD; AL, Alcalase; NZ, no ozym; PE, Pepsin; FZ, la ou zyme; PA, papain. Di e en le e s in he
same column indica e signi ican (p< 0.05) di e ences acco ding o Tukey’s es .
Table 4. F- es ANOVA pe o med on all he s udied pa ame e s.
O igin o Enzyme P o eoly ic Cha ac e
FpFp
Pep ide con en 25.11 0.00 * 1.21 0.27
Pep ide Yield 9.33 0.00 * 76.73 0.00 *
MW > 5 kDa 111.58 0.00 * 0.28 0.60
5 > MW > 1 kDa 2.31 0.13 66.71 0.00 *
MW < 1 kDa 10.78 0.00 * 16.08 0.00 *
TAA 28.27 0.00 * 16.19 0.00 *
SAA 4.42 0.03 * 22.82 0.00 *
AAA 73.14 0.00 * 8.07 0.01 *
HAA 57.89 0.00 * 17.34 0.00 *
Asp 46.34 0.00 * 12.13 0.00 *
Th 15.33 0.00 * 15.51 0.00 *
Se 4.24 0.03 * 7.84 0.01 *
Glu 13.47 0.00 * 114.28 0.00 *
Gly 17.53 0.00 * 58.80 0.00 *
Ala 11.69 0.00 * 22.06 0.00 *
Cys 3.13 0.07 13.02 0.00 *
Val 79.62 0.00 * 17.06 0.00 *
Me 5.20 0.02* 21.07 0.00 *
Ile 85.95 0.00 * 13.71 0.00 *
Leu 55.62 0.00 * 19.34 0.00 *
Ty 51.74 0.00 * 9.31 0.00 *
Phe 49.31 0.00 * 2.97 0.10
Foods 2025,14, 1248 8 o 16
Table 4. Con .
O igin o Enzyme P o eoly ic Cha ac e
FpFp
His 26.05 0.00 * 21.93 0.00 *
Lys 46.29 0.00 * 15.30 0.00 *
A g 29.61 0.00 * 18.50 0.00 *
DPPH 1.81 0.20 7.28 0.02 *
ABTS 0.93 0.42 3.72 0.07
L* 45.10 0.00 * 0.36 0.55
C*ab 10.85 0.00 * 0.29 0.60
hab 20.09 0.00 * 7.43 0.01 *
As e isks deno e signi ican di e ences a p< 0.05. MW, molecula weigh ; AAA, a oma ic amino acids; HAA,
hyd ophobic amino acids; SAA, sulphu -con aining amino acids; TAA, o al amino acids.
Conce ning he pep ide yield, bo h he o igin o he enzymes p o eoly ic cha ac e
we e signi ican ly (p< 0.05) in luenced (Table 4). By a , ege al enzymes (PA) and he
endo-exo p o eases (PA and FZ) gene a ed signi ican ly (p< 0.05) lowe amoun s o pep-
ide hyd olysa es (Table 3) and we e he less e icien in e ms o he pep ide ex ac ed.
Acco ding o Ahmahd Nadz i e al. [
36
], papain clea es pep ide bonds p edominan ly
a he C- e minal, causing he o ma ion o a conside able quan i y o amino acids com-
pa ed o ha o pep ides. As a esul , hose o med pep ides could ha e high molecula
weigh s, making hei solubili y diminish and, consequen ly, lead o a low yield o pep ide
hyd olysa e. On he o he hand, he pep ide hyd olysa es by NP and PE showed he
signi ican ly (p< 0.05) highes pep ide yield (Table 3).
Based on he esul s, and bea ing in mind hei subsequen use in winemaking, NP
and PE could be he op imum enzymes o con e ing he highes pep ide con en and
pep ide yield o he de i ed pep ide hyd olysa es.
3.3. Molecula Weigh Dis ibu ion
Table 3shows he pe cen ages o pep ide ac ions o each pep ide hyd olysa e
di ided in o h ee MW anges: la ge han 5 kDa, be ween 5 and 1 kDa, and less han
1 kDa. In gene al, he g ea es p opo ion co esponded o he MW < 1 kDa ac ion o all
samples, showing ha all he hyd olyses we e p ope ly de eloped. Simila esul s ha e
been epo ed, such as by Meinlschmid e al. [
37
], who in es iga ed how o dec ease he
alle genici y o soy p o eins by enzyma ic hyd olysis using di e en enzymes (alcalase,
la ou zyme, papain, colo ase 2TS, and pepsin).
Bo h he o igin o he enzymes and hei p o eoly ic cha ac e in luenced he pe cen -
age o he low-MW ac ion achie ed a e enzyma ic hyd olysis, acco ding o ANOVA
analysis (Table 4). The FZ-de i ed pep ides displayed a signi ican ly (p< 0.05) highe
pe cen age o his ac ion, whe eas he lowes pe cen age was asc ibed o hose ob ained
by PE. This could be suppo ed by he ac ha FZ and PA exhibi ed exopep idase ac i i y,
which a ou s he o ma ion o single amino acids a he han longe polypep ides [
35
].
Rega ding he 1–5 kDa ac ion, he p o eoly ic cha ac e was he mos impo an ac o
ega ding he sample’s di e en ia ion, wi h endop o ease-de i ed pep ides displaying a
highe pe cen age o his ac ion (NP, AL, NZ, and PE). Howe e , he o igin exe ed a
mo e ema kable e ec (p< 0.05) on he MW > 5 kDa pep ide ac ion, wi h he lowes
pe cen ages exp essed in mic obial-de i ed (NP, AL, FZ, and NZ) pep ides.
Based on he esul s, FZ appea ed o be he mos sui able enzyme o po en ially
using pep ide hyd olysa es in winemaking due o i s e ec i eness in p oducing low-MW
pep ide ac ions. Howe e , he o he mic obial enzymes (NP, AL, and NZ) could also be
conside ed, hough hey would yield pep ides wi h medium MW pep ides.
Foods 2025,14, 1248 9 o 16
3.4. Amino Acid Con en
Table 5summa ises he composi ion o soluble amino acid a pH 2 o he pep ide
hyd olysa es ob ained wi h he di e en enzymes, showing he indi idual and o al amino
acids (TAAs) and hei classi ica ion in o a oma ic (AAA: His, Phe, and Ty ), sulphu (SAA:
Me and Cys), and hyd ophobic (HAA: Ala, Val, Leu, Ile, Me , and Phe) amino acids.
Table 5. Mean and s anda d de i a ion o he concen a ion (mg aa/g hyd olysa e) o indi idual and
o al soluble amino acids a pH 2 o he pep ide hyd olysa es o he di e en enzymes.
NP AL NZ PE FZ PA
Asp 8.93 ±2.53 b 11.18 ±0.47 b 11.03 ±0.28 b 16.36 ±1.23 a 8.90 ±0.56 b 4.79 ±0.77 c
Th 3.47 ±0.24 a 3.16 ±0.89 ab 3.52 ±0.14 a 4.10 ±0.80 a 2.89 ±0.33 ab 1.77 ±0.29 b
Se 6.58 ±0.94 a 7.31 ±0.61 a 7.34 ±0.52 a 5.10 ±3.67 a 4.99 ±0.48 a 3.56 ±0.08 a
Glu 52.33 ±5.36 a 53.18 ±4.75 a 54.31 ±3.89 a 54.62 ±1.94 a 34.97 ±2.28 b 24.95 ±0.92 c
Gly 13.06 ±1.16 ab 11.98 ±0.93 ab 11.34 ±0.03 b 13.96 ±1.32 a 8.57 ±0.53 c 6.14 ±0.35 d
Ala 4.79 ±0.99 abc 5.25 ±1.14 ab 6.01 ±1.39 ab 6.97 ±0.84 a 3.66 ±0.49 bc 2.51 ±0.26 c
Cys 0.60 ±0.07 bc 0.88 ±0.07 a 0.69 ±0.04 b 0.61 ±0.10 bc 0.52 ±0.04 bc 0.46 ±0.05 c
Val 6.57 ±0.73 bc 6.77 ±0.57 bc 7.59 ±0.55 b 10.27 ±0.37 a 5.90 ±0.44 c 3.06 ±0.13 d
Me 1.00 ±0.37 ab 1.64 ±0.50 a 1.38 ±0.31 ab 1.69 ±0.25 a 0.65 ±0.17 b 0.54 ±0.14 b
Ile 4.56 ±0.49 b 4.51 ±0.52 b 4.94 ±0.35 b 6.72 ±0.33 a 4.23 ±0.36 b 2.33 ±0.08 c
Leu 7.11 ±0.80 ab 6.66 ±1.17 ab 7.35 ±0.51 b 10.82 ±0.48 a 5.41 ±0.57 c 2.99 ±0.17 d
Ty 4.23 ±0.49 b 4.29 ±0.50 b 4.67 ±0.33 ab 5.55 ±0.22 a 4.39 ±0.33 b 2.63 ±0.15 c
Phe 4.70 ±0.51 b 4.54 ±0.68 b 4.92 ±0.34 b 7.08 ±0.33 a 5.73 ±0.41 b 2.51 ±0.12 c
His 2.52 ±0.19 b 2.22 ±0.23 bc 2.16 ±0.41 bc 3.25 ±0.12 a 1.70 ±0.26 cd 1.27 ±0.05 d
Lys 3.47 ±0.25 b 3.80 ±0.34 b 3.93 ±0.31 b 4.95 ±0.07 a 3.33 ±0.46 b 2.29 ±0.10 c
A g 9.62 ±0.95 b 7.54 ±2.21 bc 9.28 ±0.42 bc 13.27 ±0.62 a 6.52 ±0.35 cd 4.08 ±0.18 d
TAA
157.37
±
16.19 b 155.80
±
11.78 b
163.43 ±10.37 ab 187.97 ±4.10 a 121.14 ±8.39 c 87.75 ±4.61 d
SAA 1.60 ±0.40 abc 2.52 ±0.57 a 2.06 ±0.34 ab 2.31 ±0.35 a 1.17 ±0.14 bc 1.00 ±0.17 c
AAA 11.45 ±1.18 bc 11.06 ±1.40 bc 12.23 ±0.97 b 15.87 ±0.67 a 11.83 ±0.97 c 6.41 ±0.33 d
HAA 28.72 ±3.17 b 29.37 ±4.52 b 33.02 ±2.45 b 43.55 ±1.36 a 25.59 ±2.15 b 13.95 ±0.62 c
NP, no o-P oD; AL, alcalase; NZ, no ozym; PE, pepsin; FZ, la ou zyme; PA, papain. Asp, aspa ic acid; Th ,
h eonine; Se , se ine; Glu, glu amic acid; Gly, glycine; Ala, alanine; Cys, cys eine; Val, aline; Me , me hionine; Ile,
isoleucine; Leu, leucine; Ty , y osine; Phe, phenylalanine; His, his idine; Lys, lysine; A g, a ginine; P o, p oline;
TAA, o al amino acids; SAA, sulphu amino acids; AAA a oma ic amino acids; HAA hyd ophobic amino acids.
Di e en le e s in he same ow indica e signi ican (p< 0.05) di e ences acco ding o Tukey’s es .
In acco dance wi h Cejudo-Bas an e e al. [
8
], i was obse ed ha , ega dless o he
enzyme used, he mos abundan indi idual amino acid in all pep ides was Glu, ollowed by
Gly, which cons i u ed 31% and 7%, espec i ely. On he o he hand, he mino amino acids
p esen in all he pep ides we e Cys (cons i u ed app oxima ely 0.4%) and Me (a ound
0.8%), in ag eemen wi h he esul s achie ed by Mo a-Ga ido e al. [
13
] in g ape seed
pep ide hyd olysa es wi h alcalase.
No ably, he hyd ophobic amino acid amily cons i u ed he la ges p opo ion o
he o al amino acid con en (app oxima ely 22%), ollowed by he a oma ic amino acids,
which accoun ed o app oxima ely 7% (Table 5). The sulphu -con aining amino acids we e
he leas abundan , comp ising only 1%, which is in ag eemen wi h A u ia e al. [
38
],
who also ound ha he SAA concen a ion o o he seeds such as sun lowe , soybean o
apeseed is excep ionally low wi h espec o o he ypes o amino acids. Cejudo-Bas an e
e al. [
8
] ob ained simila pe cen ages o hose g oups o amino acids (HAA, AAA, and
SAA) o g ape seed meal hyd olysa es wi h alcalase.
Acco ding o Table 4, bo h he o igin and p o eoly ic cha ac e o he enzymes we e
ac o s in luencing he indi idual and o al amino acid (TAA) con en , as well as he
dis ibu ion among he di e en amino acid amilies (SAA, HAA, and AAA). Rema kable
a ia ions in he TAA con en we e obse ed among he pep ides, wi h hose ob ained by
PE (animal enzyme) ha ing a signi ican ly (p< 0.05) highe concen a ion, as opposed o
Foods 2025,14, 1248 16 o 16
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au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
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