Ci a ion: Elejalde, E.; Villa án, M.C.;
Lopez-de-A men ia, I.; Ramón, D.;
Mu illo, R.; Alonso, R.M. S udy o
Unpicked G apes Valo iza ion: A
Na u al Sou ce o Polyphenolic
Compounds and E alua ion o Thei
An ioxidan Capaci y. Resou ces 2022,
11, 33. h ps://doi.o g/10.3390/
esou ces11030033
Academic Edi o s: Kons adinos
Abelio is and Ka ia Lasa idi
Recei ed: 26 Janua y 2022
Accep ed: 16 Ma ch 2022
Published: 21 Ma ch 2022
Publishe ’s No e: MDPI s ays neu al
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Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
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esou ces
A icle
S udy o Unpicked G apes Valo iza ion: A Na u al Sou ce
o Polyphenolic Compounds and E alua ion o Thei
An ioxidan Capaci y
Edu ne Elejalde 1,* , Ma ía Ca men Villa án1, I a xe Lopez-de-A men ia 1, Daniel Ramón1, Raquel Mu illo 1
and Rosa Ma ía Alonso 2
1TECNALIA, Basque Resea ch and Technology Alliance (BRTA), Pa que Tecnológico de Ála a,
c/Leona do Da Vinci 11, 01510 Miñano, Ála a, Spain; [email p o ec ed] (M.C.V.);
[email p o ec ed] (I.L.-d.-A.); [email p o ec ed] (D.R.);
[email p o ec ed] (R.M.)
2Analy ical Chemis y Depa men , Facul y o Science and Technology, Uni e si y o he Basque
Coun y (UPV/EHU), P.O. Box 644, 48080 Bilbao, Vizcaya, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +34-664039345
Abs ac :
E e y yea g ea amoun s o high-quali y wine g apes a e le on he ine unpicked, and
consequen ly los , o con ol he o e p oduc ion in wine a eas wi h limi ed appella ion p oduc ion
yield. In he con ex o ci cula bioeconomy, he alo iza ion o hese g apes as a po en ial sou ce
o na u al an ioxidan s is o g ea in e es . The s udy ca ied ou is ocused on he
polyphenolic
p o ile
cha ac e iza ion
o di e en unpicked g ape a ie ies using he ul asound-assis ed
ex ac ion
echnique
o ex ac he polyphenolic ac ions. Mo eo e , he e alua ion o he an ioxidan
capaci y
by se e al assays was ca ied ou : oxygen adical abso bance capaci y (ORAC), s abili y o 2,2-
diphenyl-1-pic ylhyd azyl adical (DPPH), e ic educing an ioxidan capaci y (FRAP), cup ic
educing
an ioxidan capaci y (CUPRAC) and s abili y o 2,2
0
-azino-bis(3-e hylbenzo hiazoline-6-
sul onic acid) adical (ABTS) assays. The esul s showed a s ong ela ionship be ween o al phenolic
con en and hyd oxycinnamic acids (R
2
= 0.9088) ollowed by la an-3-ols (R
2
= 0.8792) and annins
(
R2= 0.7705
). The an ioxidan capaci y o he g apes was dependen on he o al phenolic con en .
These esul s supply new in o ma ion o a be e unde s anding o he impo ance o gi ing an
added alue o he unpicked g apes due o hei high con en o polyphenols. These indings help he
wine sec o o conside he alo iza ion o he unpicked g apes, classi ied as was es, as an in e es ing
sou ce o na u al an ioxidan s o be used as ood supplemen s and wi h po en ial applica ions in he
pha maceu ical indus y.
Keywo ds:
unpicked g ape; polyphenols; an ioxidan capaci y; ul asound-assis ed ex ac ion;
g ape alo iza ion
1. In oduc ion
Polyphenols a e a g oup o phy ochemicals wi h impo an oles in he p e en ion
o many ch onic noncommunicable diseases [
1
]. They a e widely dis ibu ed in he plan
kingdom, mainly in ui s and ege ables, and mo e han 8000 s uc u es ha e al eady
been iden i ied [
2
]. Polyphenols a e cha ac e ized by he p esence in hei chemical s uc-
u e o a leas one phenolic g oup capable o educing eac i e oxygen species, some
o ganic subs a es and mine als. These edox p ope ies explain he conside able in e es o
polyphenols in he p e en ion o se e al majo ch onic diseases associa ed wi h oxida i e
s ess, such as ca dio ascula diseases, cance , ype II diabe es, neu odegene a i e diseases
o os eopo osis [3,4].
Among oods, g ape is one o he mos popula ui s in he wo ld and one wi h he
highes polyphenol con en [
4
–
8
]. Mo eo e , g ape cul i a ion is one o he mos ex ended
Resou ces 2022,11, 33. h ps://doi.o g/10.3390/ esou ces11030033 h ps://www.mdpi.com/jou nal/ esou ces
Resou ces 2022,11, 33 2 o 14
ag oeconomic ac i i y in he wo ld. In 2020, he o al su ace o 7.3 million hec a es was
unde ineya ds h oughou he wo ld wi h a global g ape p oduc ion o 77.8 million ons:
57% wine g apes, 36% able g apes and 7% d ied g apes [
9
]. Rega ding g apes cul i a ed
o wine making, e e y yea g ea amoun s o high-quali y g apes o di e se a ie ies a e
le on he ine unpicked o con ol he g ape o e p oduc ion in di e en wine a eas wi h
limi ed appella ion p oduc ion yield. Wine sales being down in hese las ew seasons
because o COVID-19 ha e also con ibu ed o his si ua ion [
10
]. In he con ex o ci cula
bioeconomy [
11
–
13
], he alo iza ion o hese g apes gene a ed by he wine sec o as ood
was es is o g ea in e es . I is necessa y o cha ac e ize hese by-p oduc s o e alua e hei
applicabili y as na u al esou ces o bioac i e compounds such as polyphenols o p oduce
added- alue ex ac s o be used as an ioxidan s [14].
G ape phenolic compounds a e di e en ially dis ibu ed in s alk, skin, pulp and
seeds [
7
]. Many s udies a e ocused on he quan i ica ion o polyphenols in di e en pa s
o he g apes, bu in es iga ions analyzing he di e en polyphenolic ac ions in g apes
as a whole a e sca ce [
15
]. Conside ing ha he bene icial heal h e ec s ela ed o g apes
co espond o he whole ui , he analysis o di e en polyphenolic ac ions ha con ibu e
o o al polyphenolic con en and an ioxidan capaci y in he o al g ape is ele an .
The analysis o g ape polyphenols needs an ini ial ex ac ion s ep. Polyphenol ex ac-
ion is complica ed as polyphenols a e con ined o he plan acuoles [
16
]. An ad anced
echnique o polyphenol ex ac ion wi h high eco e y yields is ul asound-assis ed ex-
ac ion (UAE) [
17
]. The b eakdown o cell walls p oduced by ca i a ion in UAE imp o es
di usion a es [
18
]. Fu he mo e, UAE has high ep oducibili y, is simple o be manip-
ula ed and needs low empe a u e, low sol en consump ion and low ene gy inpu [
19
].
The e o e, UAE ep esen s an excellen g een ex ac ion echnique o ex ac unc ional
compounds [20] such as polyphenols.
P ecisely, polyphenols a e included in he g oup o bioac i e compounds due o hei
an ioxidan capaci y. An an ioxidan is a subs ance ha educes he se e i y o oxida i e
s ess. I o ms a less ac i e adical o i quenches he chain eac ion p oduced by ee
adicals on subs a es as p o eins, lipids, ca bohyd a es o DNA [
21
,
22
]. These capaci ies
explain he in e es o an ioxidan s in he p e en ion o majo ch onic diseases associa ed
wi h oxida i e s ess [3,4].
The an ioxidan capaci y o a na u al p oduc has no single “uni e sally accep ed”
assay o quan i a i ely e alua e all ac ions o a pu a i e an ioxidan [
23
]. The e o e, o s udy
he an ioxidan capaci y o a sample, mo e han a unique assay should be conduc ed. Many
in i o
me hods can be ound in he li e a u e o e alua e he e ec i eness o an ioxidan
compounds in di e en ma ices. Two main g oups o me hods a e widely used: (1)
hyd ogen a om ans e eac ions (HAT) and (2) ans e eac ions o a single elec on
(SET) [
24
]. HAT assays include he oxygen adical abso bance capaci y (ORAC), inhibi ion
o lipope oxida ion, c ocin bleaching assay and
β
-ca o ene bleaching assay. Simila ly,
SET me hods a e composed o cup ic-ion educing an ioxidan capaci y (CUPRAC), e ic
educing abili y o plasma (FRAP), Folin–Ciocal eus’ phenol eagen educing abili y,
sca enging e ec s in ela ion o 1,1-diphenyl-2-pic ylhyd azyl (DPPH) and 2,2
0
-azino-
bis (3-e hylbenzo hiazoline-6-sulphonic acid (ABTS) among o he s [
25
,
26
]. Ne e heless,
he mos used me hods o an ioxidan capaci y o polyphenolic ex ac s ob ained om
ege able p oduc s a e DPPH, FRAP and ABTS.
To da e, no esea ch has been ocused on he unpicked wine g apes ha emain unused
and le o o a he ineya d. The e y li le esea ch ela ed o g ape was es has been
p incipally ocused on un ipe g apes de i ed om clus e hinning [
27
–
31
]. The e o e, o
add ess his gap, he cha ac e iza ion o he g apes conside ed as was es as hey a e le
on he ine a e ha es was ca ied ou in his wo k. The aim o his s udy was o gi e
hem an added alue wi h a sus ainable wine y iew and o e alua e hei po en ial use
as a sou ce o bioac i e compounds. Mo eo e , he s udy was ocused on di e en g ape
a ie ies o e alua e he di e ences in he polyphenolic p o ile among a ie ies.
Resou ces 2022,11, 33 3 o 14
This in es iga ion included he p elimina y e alua ion o he polyphenolic p o ile by
simple spec opho ome ic me hods o di e en g ape a ie ies om se e al wine a eas,
he analysis o he an ioxidan capaci y by di e en assays and he s udy o he ela ionship
o he di e en polyphenolic ac ions wi h he ob ained an ioxidan capaci y. Fu he mo e,
his wo k included an easy- o-pe o m ul asound-assis ed ex ac ion me hod capable
o ex ac ing he mos ep esen a i e polyphenolic ac ions om whole ed and whi e
g ape a ie ies.
2. Ma e ials and Me hods
2.1. Chemicals
Folin–Ciocal eu’s phenol eagen , e hanol, sodium ca bona e, hyd ochlo ic acid, am-
monium sulpha e, me hanol, po assium dihyd ogen phospha e, di-po assium hyd ogen
phospha e, sodium ace a e and ammonium ace a e bu e we e analy ical g ade om Scha -
lab (Ba celona, Spain). 2,2
0
-azobis-(2-amidino-p opane) dihyd ochlo ide (AAPH), ace ic
acid, 2,4,6- is (2-py idyl)-s- iazine (TPTZ), e ic chlo ide, cup ic chlo ide, neocup oine
(Nc), luo escein sodium sal , po assium pe sul a e, 4-(dime hylamino) cinnamaldehyde
(DMAC) and me hyl cellulose we e analy ical g ade pu chased om Me ck Li e Science
(Mad id, Spain). 6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid ( olox) wi h 97%
pu i y and 2,2
0
-azino-bis(3-e hylbenzo hiazoline-6-sulphonic acid) adical ca ion (ABTS
•
+)
wi h 98% pu i y we e also pu chased om Me ck Li e Science (Mad id, Spain). The s an-
da ds gallic acid (GA), ca eic acid (CA), que ce in (Q), (+)-ca echin (C) and (
−
)-epica echin
(E) wi h pu i ies
≥
95% we e also pu chased om Me ck Li e Science (Mad id, Spain).
Ca a ic acid wi h >98% pu i y was supplied by Phy oplan (Heidelbe g, Ge many. Finally,
2,2-diphenyl-1-pic ylhyd azyl (DPPH) was p o ided by Fishe Scien i ic (Mad id, Spain).
Aqueous solu ions we e p epa ed using pu i ied wa e .
2.2. Sample P epa a ion
In o al, 13 na i e ed and whi e g ape a ie ies (Vi is ini e a) cul i a ed in he no h
o Spain in 3 di e en wine appella ions we e selec ed o his s udy. The g apes we e
collec ed a season 2020 one week a e he ha es ime s a ing es ablished by he wine y
o each g ape a ie y, conside ing hei op imal g ape ma u i y poin . The samples o his
s udy we e, he e o e, he unha es ed g apes le o o on he ine o obey he limi ed
appella ion p oduc ion yield. All hese g apes we e a hei op imal ma u i y poin and o
high quali y as hey we e ini ially des ined o winemaking. A po ion o each g ape a ie y
was c ushed by hand o ob ain he mus o he physicochemical pa ame e s. The es o
g ape bunches we e immedia ely ozen in liquid ni ogen, he s alks we e emo ed, he
whole g ape be ies we e g ound and lyophilized (Lyobe a 25; Tels a , Te assa, Ba celona,
Spain) un il ul asound-assis ed ex ac ion.
2.3. Ul asound-Assis ed Ex ac ion
The condi ions o he ul asound-assis ed ex ac ion we e es ablished acco ding o
he li e a u e [
32
,
33
] in a Bioblock Scien i ic Vib a Cell VCX 750 sonica o (Sonde s anda d
13 mm, Fishe Scien i ic, Mad id, Spain) a a equency o 20 KHz. In o al, 2 g o each
g ape powde oge he wi h 100 mL o hyd oalcoholic sol en (50% / in e hanol) we e
placed in a beake and sonica ed o 20 min. A e ea men he polyphenolic ex ac was
cen i uged (5810R Eppendo ; Me ck Li e Science, Mad id, Spain) a 4000 g o 5 min,
il e ed h ough 0.45
µ
m il e and s o ed a 4
◦
C be o e analysis. The cha ac e iza ion o
his g ape polyphenolic ex ac included he analysis o he di e en polyphenolic ac ions
and he e alua ion o he an ioxidan capaci y by se e al assays.
2.4. G ape Physicochemical Pa ame e s
pH, o al soluble solids and i a able acidi y we e measu ed as con ol pa ame e s in
he g ape mus s o de e mine he g ape ma u i y. The pH was analyzed wi h a pH-me e
(Basic 20; C ison Ins umen s S.A., Alella, Ba celona, Spain). The o al soluble solids we e
Resou ces 2022,11, 33 4 o 14
analyzed using a e ac ome e (ATAGO N-1E; Tokyo, Japan) and he alues we e exp essed
as B ix deg ee. Ti a able acidi y was es ima ed acco ding o he o icial me hod [
34
]. The
esul s o i a able acidi y we e exp essed as g/L o a a ic acid.
2.5. Polyphenolic F ac ions Analysis
2.5.1. To al Phenolic Con en
The o al phenolic con en (TPC) was es ed using Folin–Ciocal eu assay [
35
]. B ie ly,
20
µ
L o app op ia ely dilu ed sample was mixed ho oughly wi h 100
µ
L o 10% Folin–
Ciocal eu’s phenol eagen in wells o a 96-well mic opla e and 80
µ
L sodium ca bona e
solu ion o 75 g/L. A e 90 min in da kness a oom empe a u e, he abso bance was
measu ed a 750 nm in he mic opla e pho ome e (Mul iskan
™
FC; Fishe Scien i ic,
Mad id, Spain). The concen a ion was exp essed as millig ams o gallic acid equi alen s
(GAE) pe 100 g o d y ma e (dm) g ape sample based on a s anda d cu e o gallic acid.
2.5.2. Hyd oxycinnamic Acid De i a i es and Fla onols
The de e mina ion o his polyphenolic ac ion was ca ied ou acco ding o Mazza e al.,
1999 [
36
] wi h mino modi ica ions. Aliquo s o 0.5 mL o app op ia ely dilu ed samples
we e mixed wi h 0.5 mL o an e hanolic solu ion wi h hyd ochlo ic acid 0.1% and 9.1 mL o
an e hanolic solu ion wi h hyd ochlo ic acid 2%. Abso bance was de e mined a e 15 min a
280 nm and 360 nm o hyd oxycinnamic acid de i a i es and la onols, espec i ely. The
calib a ion cu es we e made o ca eic acid in 10% e hanol solu ion o hyd oxycinnamic
acid de i a i es and o que ce in in 95% e hanol solu ion o la onols. The concen a ion o
hyd oxycinnamic acid de i a i es we e exp essed in mg o ca eic acid equi alen s (CAE) pe
g am o d y ma e g ape sample. The concen a ion o la onols de i a i es we e exp essed
in mg o que ce in equi alen s (QE) pe g am o dm g ape sample.
2.5.3. Fla an-3-ols
The me hod used was based on he eac ion be ween la an-3-ols and 4-(dime hylamino)
cinnamaldehyde (DMAC) desc ibed in bibliog aphy [
37
]. A olume o 1.25 mL o DMAC
solu ion a 5.7 mM was added o 0.25 mL dilu ed sample. Abso bance measu emen s we e
eco ded a 640 nm a e 10 min. The calib a ion cu e was made o (+)-ca echin and esul s
we e exp essed in mg o ca echin equi alen s (CE) pe g am o dm g ape sample.
2.5.4. To al An hocyanins
Fo o al an hocyanin analysis, he g ape ex ac was dilu ed wi h a solu ion o e hanol:
wa e :hyd ochlo ic acid 37% 70:30:1 / / [
38
] and he abso bance was measu ed immedi-
a ely a 540 nm. The inal esul s we e exp essed as mal idin-3-glucoside equi alen s (ME) in mg
pe g am o d y ma e calcula ed om he ollowing equa ion:
C (mg/L) = A540 nm ×26.6 ×d
whe e, A540 nm is he abso bance a 540 nm and d is he dilu ion [38].
2.5.5. Tannins
The me hod was based on he annins p ecipi a ion wi h me hyl cellulose [
39
]. A
olume o 1 mL o app op ia ely dilu ed sample was mixed wi h 6 mL o wa e , 1 mL
o me hyl cellulose 0.04% and 2 mL o ammonium sulpha e. Fo he blank, he sample
aliquo and he me hyl cellulose we e eplaced by wa e . A e 10 min o eac ion a oom
empe a u e, he samples we e cen i uged and he abso bance was de e mined a 280 nm.
The esul s we e exp essed in mg o epica echin equi alen s (EE) pe g am o dm g ape
sample.
2.6. An ioxidan Capaci y De e mina ion
2.6.1. ORAC Assay
The oxygen adical abso bance capaci y alue o he di e en ex ac s was measu ed
acco ding o he li e a u e [
40
]. The assay ex ac s we e dilu ed in ORAC bu e (po assium
phospha e bu e , consis ing o po assium dihyd ogen phospha e and di-po assium hyd o-
Resou ces 2022,11, 33 5 o 14
gen phospha e a pH 7.4) and a olox s anda d cu e (0–100
µ
M) was p epa ed. A he
day o analysis, 175 mM luo escein and 153 mM olox equi alen s (TE) solu ions we e
p epa ed in ORAC bu e . A 96-well black mic opla e was p epa ed con aining 150
µ
L o
luo escein solu ion. Then, 25
µ
L o blank (ORAC bu e ) s anda d o sample was added.
The pla e was incuba ed a 37
◦
C du ing 30 min. A e incuba ion, 25
µ
L o eshly p epa ed
AAPH solu ion was quickly added. Readings o luo escence we e measu ed e e y min
o 1 h using a DTX 880 mul imode de ec o , Beckman Coul e (B ea, CA, USA) (exci a ion
wa eleng h o 485 nm and emission wa eleng h o 530 nm). The esul s we e exp essed as
µmol olox equi alen s (TE)/g dm.
2.6.2. DPPH Radical Sca enging Ac i i y
The sca enging capaci y o he ex ac was e alua ed by DPPH me hod based on he
s abili y o 2,2-diphenyl-1-pic ylhyd azyl adical [
41
] wi h some modi ica ions. An aliquo
o app op ia ely dilu ed 50
µ
L sample was added o 2.950 mL o a 0.1 mM me hanolic (80%)
DPPH adical solu ion, o ex mixed and incuba ed in da k o 30 min a oom empe a u e.
A e incuba ion, he abso bance was measu ed a 515 nm in a spec opho ome e (Lambda
365 UV/VIS; Pe kin Elme , Mad id, Spain). T olox was used as he e e ence compound.
The esul s we e exp essed in µmol olox equi alen s (TE)/g dm.
2.6.3. FRAP Assay
The e ic educing an ioxidan capaci y assay was used acco ding o bibliog aphy [
42
]
wi h mino modi ica ions. FRAP eagen , consis ing o a mix u e o sodium ace a e-ace ic
acid bu e (300 mM, pH 3.6), TPTZ (10 mM in 40 mM hyd ochlo ic acid) and e ic chlo ide
solu ion (20 mM) a a olume a io o 10:1:1, was eshly p epa ed and pu in a wa e ba h
a 37
◦
C be o e use. Aliquo s o 100
µ
L app op ia ely dilu ed sample we e mixed wi h 3 mL
FRAP eagen . A e incuba ion o 4 min, he abso bance o he mix u e was de e mined
a 593 nm. The esul s we e exp essed in µmol olox equi alen s (TE)/g dm.
2.6.4. CUPRAC Assay
The cup ic educing an ioxidan capaci y assay was pe o med using he classical
me hod [
43
] wi h small modi ica ions and conduc ed in a 96-well mic opla e. B ie ly, o
each well was sequen ially added 50
µ
L o cup ic chlo ide (10 mM in wa e ), 50
µ
L o
neocup oine a 7.5 mM in 96% e hanol, 50
µ
L o ammonium ace a e bu e (1 mM in wa e ,
pH 7.0), 25
µ
L o app op ia ely dilu ed sample and 25
µ
L o double dis illed wa e . The
mic opla es we e hen incuba ed in he da k a oom empe a u e o 30 min and a e he
alues o abso bance we e eco ded a 450 nm agains blank (all eagen s excep cup ic
chlo ide) in he mic opla e eade de ailed be o e. A calib a ion cu e was made using he
comme cial compound olox as s anda d and he esul s we e exp essed in
µ
mol olox
equi alen s (TE)/g dm.
2.6.5. ABTS Me hod
Ano he me hod o de e mine he an ioxidan capaci y was he ABTS
•
+ ( adical
ca ion) decolo iza ion assay [
44
] wi h some modi ica ions. The ABTS
•
+ s ock solu ion
was p epa ed by mixing ABTS (7 mM) solu ion and po assium pe sul a e (2.45 mM) in
a olume a io o 1:1. This solu ion was incuba ed in a da k a oom empe a u e o
a leas 16 h. The wo king solu ion was p epa ed dilu ing he ABTS s ock solu ion wi h
e hanol o an abso bance o 0.710
±
0.050 uni s a 734 nm using he same spec opho ome e
as men ioned abo e. Samples we e p e iously dilu ed and 100
µ
L o each sample was
mixed wi h 3.8 mL ABTS wo king solu ion a oom empe a u e and he abso bance o he
mix u e was de e mined a 734 nm a e 6 min. The esul s we e exp essed in µmol olox
equi alen s (TE)/g dm.
Resou ces 2022,11, 33 6 o 14
2.7. S a is ical Analysis
Resul s a e epo ed as mean
±
s anda d de ia ion (SD) alues. Fo each g ape a ie y
h ee independen ex ac s we e pe o med and h ee analyses o each pa ame e we e
made. Da a we e analyzed by one-way ANOVA and he Fishe ‘s leas signi ican di e ence
(LSD) es o es ima e he di e ences be ween alues o he sample es ed, whe e s a is ical
signi icance was decla ed a p alue < 0.05. Co ela ions be ween he polyphenolic ac ions
and he an ioxidan capaci y we e de e mined using linea eg ession analysis. Di e ences
we e conside ed signi ican a p alue < 0.05. Mul i a ia e analysis p incipal componen
analysis (PCA) was also applied o he esul s. S a g aphics Cen u ion XVII so wa e was
used o s a is ical analysis.
3. Resul s and Discussion
3.1. G ape Physicochemical Pa ame e s
In Table 1 he g ape physicochemical con ol pa ame e s a e collec ed.
Table 1. G ape physicochemical pa ame e s in di e en a ie ies.
Va ie y pH B ix Deg ee To al Acidi y 1
Red
Temp anillo 3.18 ±0.02 e24.6 ±0.0 g5.2 ±0.0 g
Ga nacha 3.28 ±0.02 g29.8 ±0.0 l4.4 ±0.0 d
Cabe ne sau ignon 3.47 ±0.01 h25.2 ±0.0 h4.2 ±0.0 c
G aciano 3.21 ±0.02 22.6 ±0.0 d4.9 ±0.0 e
Honda abi bel za 3.17 ±0.02 e17.8 ±0.0 a8.3 ±0.0 j
Ma u ana in a 3.52 ±0.03 i26.4 ±0.0 k4.1 ±0.0 b
Mazuelo 3.11 ±0.02 d21.6 ±0.0 c6.4 ±0.0 h
Whi e
Honda abi zu i 2.80 ±0.01 b20.2 ±0.0 b10.0 ±0.1 k
Pe i cou bu 2.81 ±0.03 b20.2 ±0.0 b10.6 ±0.0 k
Pe i manseng 3.04 ±0.01 c24.4 ±0.0 7.5 ±0.0 i
G oss manseng 2.75 ±0.02 a23.4 ±0.0 d10.9 ±0.0 l
Mal asía3.53 ±0.01 i26.0 ±0.0 i4.0 ±0.0 a
Viu a 3.21 ±0.01 26.2 ±0.0 j5.1 ±0.0
No e: The esul s a e p esen ed as mean
±
SD ( iplica e). Di e en le e s indica e signi ican di e ences (
p< 0.05
)
in each sample ype among he hi een g ape a ie ies. 1To al acidi y is exp essed as g a a ic acid/L.
Al hough g apes we e on he ine unpicked du ing one week a e he ha es ime
s a ing, he physicochemical pa ame e s showed common alues acco ding o heal hy
g apes sui able o winemaking and a hei op imal ma u i y poin . All he g apes we e
bunches ini ially des ined o winemaking, and he e o e o iden ical high quali y. The
esul s show impo an inhe en bu usual di e ences among all he g ape a ie ies which
sugges di e en polyphenolic p o iles and an ioxidan capaci ies. All he samples o his
s udy we e a he op imal ma u i y poin es ablished by he wine ies.
3.2. Polyphenolic F ac ions
The esul s o all he polyphenolic ac ions a e p esen ed in Table 2.
The o al phenolic con en (TPC) anged om 13.87
±
0.43 o 24.95
±
1.07 mg gallic
acid equi alen s (GAE)/g dm o ed g apes and 4.69
±
0.21 o 24.97
±
0.26 mg GAE/g
dm o whi e g apes. Among all he g ape a ie ies, a whi e a ie y had he highes
alue in TPC wi h 24.97
±
0.26 mg GAE/g dm. These esul s ag ee wi h hose p e iously
epo ed [
45
,
46
]. The i s s udy showed TPC alues be ween 92.89 o 100.45 mg GAE/100
g esh g ape o ed a ie ies and a highe o al phenolic con en in whi e g ape a ie ies
wi h alues be ween 50.79 o 141.72 mg GAE/100 g o whi e a ie ies [
45
]. In he o he
s udy he ange o esh g apes wen om 0.294 mg GAE/g esh weigh ( w) o a ed
g ape a ie y o 1.407 mg GAE/g w o a whi e g ape a ie y [
46
]. In any case, i mus be
Resou ces 2022,11, 33 7 o 14
conside ed ha he o al phenolic con en in di e en g apes may a y depending on he
cul i a , he ipening s age o he en i onmen al condi ions [47,48].
Table 2. Polyphenolic ac ions in di e en g ape a ie ies.
Va ie y
To al
Phenolic
Con en
Hyd oxycinnamic
Acids An hocyanins Fla onols Fla an-3-ols Tannins
Red
Temp anillo 20.26 ±0.32 16.56 ±0.11 g5.48 ±0.08 d1.21 ±0.00 g3.05 ±0.05 h23.35 ±0.81 i
Ga nacha 13.87 ±0.43 c11.50 ±0.08 c,d 3.40 ±0.01 c1.14 ±0.01 2.31 ±0.05 d15.88 ±0.38 d,e
Cabe ne sau ignon 21.71 ±0.31 g13.60 ±1.21 e1.35 ±0.06 b1.10 ±0.01 2.92 ±0.02 g16.68 ±0.03
G aciano 22.33 ±0.65 g18.20 ±1.38 h8.12 ±0.12 e1.36 ±0.02 h3.53 ±0.09 i24.68 ±0.46 j
Honda abi bel za 17.49 ±0.30 e11.02 ±0.06 c,d 1.06 ±0.03 a0.61 ±0.01 b2.52 ±0.03 e13.95 ±0.66 c
Ma u ana in a 24.95 ±1.07 h20.40 ±0.16 i11.19 ±0.15 g1.95 ±0.02 j2.72 ±0.05 32.39 ±0.25 k
Mazuelo 14.88 ±0.07 d9.26 ±0.21 b9.27 ±0.22 1.31 ±0.03 h1.47 ±0.01 c20.88 ±0.36 h
Whi e
Honda abi zu i 24.97 ±0.26 h14.71 ±0.50 - 0.85 ±0.01 e4.77 ±0.16 k18.37 ±0.40 g
Pe i cou bu 17.35 ±0.12 e10.49 ±0.44 c-0.61 ±0.02 b
3.00
±
0.02
g,h 9.39 ±0.24 b
Pe i manseng 22.22 ±0.46 g13.06 ±0.22 e-0.73 ±0.00 d4.21 ±0.09 j16.36 ±0.19 e,
G oss manseng 20.88 ±0.63 11.74 ±0.05 d- 0.66 ±0.01 c4.12 ±0.00 j15.57 ±0.14 d
Mal asía 4.69 ±0.21 a2.74 ±0.09 a-0.60 ±0.05 b0.47 ±0.01 a4.81 ±0.06 a
Viu a 7.34 ±0.51 b3.04 ±0.33 a- 0.49 ±0.02 a0.72 ±0.06 b5.03 ±0.19 a
No e: The esul s a e p esen ed as mean
±
SD ( iplica e). The da a a e exp essed as mg/g d ied ma e . Resul s
a e epo ed as mg GAE/g dm o o al phenolic con en , mg CAE/g dm o hyd oxycinnamic acids, mg ME/g
dm o an hocyanins, mg QE/g dm o la onols, mg CE/g dm o la an-3-ols and mg EE/g dm o annins.
Di e en le e s indica e signi ican di e ences (p< 0.05) in each sample ype among he hi een g ape a ie ies.
Among all he polyphenolic ac ions, hyd oxycinnamic acids we e a e he con-
densed annins he mos abundan polyphenols bo h in whi e and ed g ape a ie ies. This
could be due o he UV- isible me hod used ha can o e quan i y he o al hyd oxycin-
namic acids. Ne e heless, hese a e he mos ex ended me hods used o g ape analysis.
Anyway, he da a we e acco ding o he published da a o o he g ape a ie ies a ma u i y
s a e wi h hyd oxycinnamic o al amoun o 221.7–810 mg/g d ied skin [
49
]. Since g ape
skin ep esen s 5–10% o he g ape be y weigh , he esul s a e acco ding o he o al
amoun o hyd oxycinnamic acids. In ou s udy, he con en in ed g apes was be ween
9.26 ±0.21 mg
ca eic acid equi alen s (CAE)/g dm o 20.40
±
0.16 mg CAE/g dm and
2.74 ±0.09 mg CAE/g dm o 14.71 ±0.50 mg CAE/g dm in whi e g apes.
Rega ding an hocyanins, he o al amoun in ed g apes was be ween 1.06
±
0.03 mg
mal idin equi alen s (ME)/g dm o 11.19
±
0.15 mg ME/g dm. As i can be seen, he e
we e quan i a i e di e ences in o al an hocyanins o ed g apes. The concen a ions
a e acco ding o esul s o se e al au ho s wi h a ange o 0.98
±
0.11 o
1.31 ±0.08 mg
an hocyanins/g d y whole g ape [
15
], o 1582.59
±
77.38 o 2271.31
±
50.33 mg o al
an hocyanins/kg [50].
The concen a ion o la onols anged om 0.61
±
0.02 o 1.95
±
0.02 mg que ce in
equi alen s (QE)/g dm o ed g apes and be ween 0.49
±
0.02 o 0.85
±
0.01 mg QE/g dm
o whi e g apes. These concen a ions a e acco ding o esul s ound in li e a u e wi h a
ange o 191.43
±
25.68 o 279.64
±
15.54 mg o al la onols/kg o a ed g ape a ie y [
50
].
Wi h espec o la an-3-ols, he con en in ed g apes anged om 1.47
±
0.01 o
3.53 ±0.09 mg
ca echin equi alen s (CE)/g dm and 0.47
±
0.01 o 4.77
±
0.16 mg CE/g dm
o whi e g apes. The esul s we e acco ding o he li e a u e [
51
] wi h a o al amoun (skin,
lesh and seeds) o la an-3-ols o 3323 mg/Kg esh weigh ( w) o a whi e g ape a ie y
and 3263 mg/Kg w o a ed a ie y.
Conce ning annins, he concen a ion was be ween 13.95
±
0.66 and 32.39
±
0.25 mg
epica echin equi alen s (EE)/g dm o ed g ape a ie ies and be ween 4.81
±
0.06 and
18.37 ±0.40 mg EE/g dm o whi e g ape a ie ies.
Resou ces 2022,11, 33 8 o 14
Rega ding he polyphenolic p o ile, he o al polyphenolic con en and hyd oxycin-
namic acids showed a ela i ely s ong ela ionship (R
2
= 0.9088, wi h a le el o signi icance
o 95%). In ac , he hyd oxycinnamic acids ep esen among he polyphenolic g oups he
one wi h he highes co ela ion wi h TPC. The hyd oxycinnamic acids a e ollowed by
he la an-3-ols (R
2
= 0.8792) and annins (R
2
= 0.7705) wi h a mode a ely s ong ela ion-
ship. Finally, he la onols (R
2
= 0.4968) and an hocyanins (R
2
= 0.3100) ha e a ela i ely
weak ela ionship.
3.3. An ioxidan Capaci ies
The an ioxidan capaci y measu ed by di e en assays is p esen ed in Table 3.
Table 3.
An ioxidan capaci y in di e en g ape a ie ies by ORAC, DPPH, FRAP, CUPRAC and
ABTS assays.
Va ie y ORAC DPPH FRAP CUPRAC ABTS
Red
Temp anillo 335.09 ±25.52 d230.67 ±11.71 j113.14 ±2.86 201.78 ±8.91 i159.79 ±7.10 c,d
Ga nacha 254.94 ±12.06 c134.53 ±3.81 c76.38 ±2.30 d146.85 ±0.60 e206.90 ±0.75 h,i
Cabe ne sau ignon 371.44 ±23.64 e, 164.56 ±14.74 e, 93.27 ±3.36 e154.82 ±4.92 e
169.87
±
10.37
d,e
G aciano 397.90 ±6.23 ,g 193.29 ±14.45 g,h 110.10 ±4.16 174.25 ±4.77 g155.79 ±8.91 b,c
Honda abi bel za 333.26 ±14.62 d144.54 ±18.99 c,d 67.75 ±3.34 c122.72 ±2.67 c196.17 ±2.03 g,h
Ma u ana in a 412.90 ±9.67 g,h 220.78 ±4.23 i,j 124.38 ±2.06 g196.29 ±10.41 h,i 164.35 ±7.68 c,d
Mazuelo 396.31 ±6.73 ,g 72.95 ±2.21 b54.43 ±0.34 b96.24 ±1.23 b220.82 ±4.48 i
Whi e
Honda abi zu i 440.11 ±39.33 h206.80 ±10.45 h,i 114.06 ±10.44 189.49 ±8.21 h129.87 ±13.49 a
Pe i cou bu 383.04 ±7.83 e, 148.47 ±12.85 c,d,e 70.18 ±2.14 c,d 136.07 ±1.12 d191.42 ±4.09 ,g
Pe i manseng 421.47 ±4.24 g,h 175.75 ±15.51 ,g 90.82 ±2.97 e169.62 ±4.70 ,g
142.97
±
13.98
a,b
G oss manseng 365.54 ±10.19 e155.65 ±8.26 d,e 94.20 ±6.80 e163.70 ±4.30 181.34 ±2.27 e,
Mal asía 102.28 ±2.22 a29.50 ±1.57 a14.36 ±0.33 a45.54 ±2.63 a288.75 ±1.26 j
Viu a 146.49 ±6.40 b38.64 ±2.56 a19.38 ±1.43 a50.63 ±2.98 a276.02 ±6.63 j
No e: The esul s a e p esen ed as mean
±
SD ( iplica e), exp essed as
µ
mol olox equi alen s (TE)/g dm.
Di e en le e s indica e signi ican di e ences (p< 0.05) in each sample ype among he hi een g ape a ie ies.
When he ORAC assay was used, he anges we e o 254.94
±
12.06 o
412.90 ±9.67 µmol
oloxequi alen s(TE)/gdm o he edg ape a ie ies and102.28
±
2.22 o
440.11 ±39.33 µmol
TE/g dm o he whi e g ape a ie ies. The highes and he lowes esul we e ound in wo
whi e g ape a ie ies.
The DPPH alues we e in he ange o 72.95
±
2.21 o 230.67
±
11.71
µ
mol olox
equi alen s (TE)/g dm o he ed g ape a ie ies and 29.50
±
1.57 o 206.80
±
10.45
µ
mol
TE/g dm o he whi e g ape a ie ies. The highes o al an ioxidan capaci y by he DPPH
assay was ound in a ed g ape a ie y bu he lowes an ioxidan capaci y was ob ained in
a whi e g ape a ie y. F om his esul can be concluded ha he speci ic composi ion o
he g ape a ie y de e mines he an ioxidan capaci y alue.
Fo he FRAP assay, he anges we e o 54.43
±
0.34 o 124.38
±
2.06
µ
mol olox
equi alen s (TE)/g dm o he ed g ape a ie ies and 14.36
±
0.33 o
114.06 ±10.44 µmol
TE/g dm o he whi e g ape a ie ies. The g ape a ie y wi h he lowes alue o
an ioxidan capaci y by he FRAP assay also ga e he lowes alue when measu ed by
he DPPH assay. Addi ionally, he anking o he an ioxidan capaci y alue by he FRAP
assay o all he g ape a ie ies was e y simila o he anking o he an ioxidan capaci y
measu ed by he DPPH assay.
Rega ding he CUPRAC assay, he esul s we e in he ange o 96.24
±
1.23 and
201.78 ±8.91 µmol
olox equi alen s (TE)/g dm o he ed g ape a ie ies and be ween
45.54
±
2.63 and 189.49
±
8.21
µ
mol TE/g dm o he whi e g ape a ie ies. No esul s by
o he au ho s ha e been ound o he whole g ape. The only esul s ound a e he ones
ob ained in he g ape seeds in he ange o 1357 and 1707 µmol TE/g esh weigh [52].
Resou ces 2022,11, 33 9 o 14
The an ioxidan capaci y by he ABTS assay anged be ween 155.79
±
8.91 o
220.82 ±4.48 µmol
olox equi alen s (TE)/g dm o ed g ape a ie ies and be ween
129.87
±
13.49 o
288.75 ±1.26 µmol
TE/g dm o he whi e g ape a ie ies. In his case,
and when e e ed o he whi e g ape a ie ies, he an ioxidan capaci y ank was he
con a y o he an ioxidan capaci y measu ed by DPPH and FRAP. The highes an ioxidan
capaci y by he DPPH o he FRAP assay ga e he lowes an ioxidan capaci y o he
ABTS assay. Wi h ed g ape a ie ies, he esul s we e simila o he whi e g ape a ie ies.
The lowe an ioxidan capaci y by he DPPH and he FRAP assays, he highe o he
ABTS assay. These esul s a e consis en wi h he li e a u e [
53
], al hough hey a e e e ed
o g ape seeds wi h a ange o 185.2
±
5.9
µ
mol TE/g and 206.3
±
7.7
µ
mol TE/g o
wo Po uguese ed g ape a ie ies. I mus be conside ed ha , as de ailed be o e, each
an ioxidan capaci y assay e alua es a speci ic ac ion.
In any case, bo h whi e and ed g ape a ie ies displayed di e en an ioxidan capac-
i ies ega dless o he assay me hod used o he e alua ion. Thei capaci ies a e ela ed
di ec ly o TPC. These esul s a e acco ding o p e ious li e a u e epo s on o he g ape
a ie ies [
54
]. In ou wo k a s ong co ela ion was obse ed be ween o al polyphenolic
con en and an ioxidan capaci y. Ne e heless, he in e p e a ion o esul s mus be ca -
ied ou wi h ca e, as he da a esul om he combina ion and syne ge ic e ec o all he
cons i uen s o he polyphenolic ex ac s ob ained om g apes. Compounds belonging o
o he chemical g oups may also con ibu e o hese esul s [55].
3.4. Co ela ion o Polyphenolic P o iles and An ioxidan Capaci y
High co ela ion coe icien s we e ound be ween he ORAC s. TPC (R
2
= 0.9119),
DPPH s. TPC (R
2
= 0.9124), as well as be ween FRAP s. TPC (R
2
= 0.9511) wi h a le el
o signi icance o 95% (p< 0.05). High co ela ion coe icien s we e also ound be ween
CUPRAC s. TPC (R
2
= 0.9333) and ABTS s. TPC (R
2
= 0.9576) wi h a le el o signi icance
o 95% (p< 0.05). Da a shown in Figu e 1a–e.
The co ela ion analysis demons a ed ha he an ioxidan capaci y o he g apes, in-
dependen ly he a ie y, was dependen on he o al polyphenolic con en . The co ela ions
we e highe han he esul s epo ed in he li e a u e [
46
]. In all he an ioxidan capaci y
assays highe co ela ions we e ob ained o whi e g ape a ie ies (R
2
= 0.9845 o DPPH s.
TPC, R
2
= 0.9864 o FRAP s. TPC and R
2
= 0.9947 o CUPRAC s. TPC, R
2
= 0.9815 o
ABTS s. TPC) in compa ison o ed g ape a ie ies (R
2
= 0.7836, R
2
= 0.8752, R
2
= 0.7594
and R
2
= 0.8542 o DPPH s. TPC, FRAP s. TPC, CUPRAC s. TPC and ABTS s. TPC,
espec i ely). Ne e heless, hese high co ela ions be ween TPC and an ioxidan capaci y
in g apes as a whole sugges ha i is easible o use TPC o e alua e he an ioxidan
capaci y in whi e and ed g ape a ie ies. The good co ela ion be ween he DPPH, FRAP,
CUPRAC and ABTS and TPC can be a ionalized conside ing ha hese assays ely on
simila eac ion mechanisms in ol ing elec on ans e . These esul s ag ee in bo h ed and
whi e g ape a ie ies. The mechanisms by which phenolic compounds a e able o sca enge
ee adicals a e no exac ly es ablished. Ne e heless, he basic s uc u e o compounds
and o he s uc u al ac o s seem o be essen ial in he sca enging mechanism [56,57].
The di e en g ape a ie ies showed impo an quan i a i e di e ences in he polyphe-
nolic ac ion concen a ion and he an ioxidan capaci y a hei op imal ma u i y poin . To
e alua e hese di e ences, he PCA mul i a ia e analysis was applied (Figu e 2a,b) aking
in o conside a ion hese pa ame e s.
Fo all he ed g ape a ie ies, he cumula i e pe cen age o he o al a iance explained
by he i s and second p incipal componen was 90.089%. The ed g ape a ie ies we e
di e en ly g ouped acco ding o hei phenolic composi ion as shown in he biplo s. Fo ed
g ape a ie ies, he an ioxidan capaci y by CUPRAC, DPPH and FRAP was cha ac e ized
by TPC, hyd oxycinnamic acids and la an-3-ols while he an ioxidan capaci y by ORAC
was cha ac e ized by an hocyanins, annins and la onols. Rega ding whi e g ape a ie ies,
he cumula i e pe cen age o he o al a iance explained by he i s and second p incipal
componen was 99.006%. Excep o wo o he g ape a ie ies wi h he lowes phenolic
