Academic Edi o : I ana
Gene ali´c Mekini´c
Recei ed: 31 Oc obe 2024
Re ised: 19 Decembe 2024
Accep ed: 22 Decembe 2024
Published: 25 Decembe 2024
Ci a ion: Fe nández-Pé ez, R.;
Ayuso, S.; Mo e a, C.; Saiz-Abajo, M.-J.;
Gas ón-Lo en e, M.; Ruiz-La ea, F.;
Teno io, C. Chemical P o ile and
An ibac e ial Ac i i y o Vi is ini e a L.
c G aciano Pomace Ex ac s Ob ained
by G een Supe c i ical CO2Ex ac ion
Me hod Agains Mul id ug-Resis an
Esche ichia coli S ains. Foods 2025,14,
17. h ps://doi.o g/10.3390/
oods14010017
Copy igh : © 2024 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
Chemical P o ile and An ibac e ial Ac i i y o Vi is ini e a L. c
G aciano Pomace Ex ac s Ob ained by G een Supe c i ical CO2
Ex ac ion Me hod Agains Mul id ug-Resis an
Esche ichia coli S ains
Rocío Fe nández-Pé ez 1, Sil ia Ayuso 1, C is ina Mo e a 1, Ma ía-JoséSaiz-Abajo 2, Miguel Gas ón-Lo en e 2,
Fe nanda Ruiz-La ea 1,* and Ca men Teno io 1
1Ins i u o de Ciencias de la Vid y del Vino (ICVV) (Uni e sidad de La Rioja,
Consejo Supe io de In es igaciones Cien í icas (CSIC), Gobie no de La Rioja), 26007 Log oño, Spain
2Na ional Cen e o Food Technology and Sa e y (CNTA), 31570 San Ad ian, Spain
*Co espondence: e nanda. [email p o ec ed]
Abs ac : The objec i es o his s udy we e o ob ain and cha ac e ise polyphenolic ex ac s
om ed g ape pomace o Vi is ini e a L. c G aciano ia con en ional sol en ex ac ion
(SE) and g een supe c i ical luid ex ac ion (SFE) and o e alua e hei
in i o
an ibac e-
ial ac i i y agains suscep ible and mul id ug- esis an Esche ichia coli s ains o in es inal
o igin. The SE and SFE me hods we e op imised, and ul a-pe o mance liquid ch oma og-
aphy/mass spec ome y (UPLC/QqQ-MS/MS) analysis e ealed 38 phenolic compounds
in he SE sample, wi h an hocyanins being he p edominan polyphenols, and 21 phenolic
compounds in he SFE samples, among which hyd oxybenzoic acids and la onols we e he
p edominan compounds. The SE and SFE samples showed an ibac e ial ac i i y agains
bo h an ibio ic-suscep ible and - esis an E. coli s ains, and minimal inhibi o y concen a ion
alues we e in he ange o 1–4 mg/mL. The ac i i y was bac e ios a ic in all cases, and i
was shown ha a highe con en o o al polyphenols co ela ed wi h a highe an ibac e-
ial ac i i y o he ex ac s. This s udy shows ha ed g ape pomace o Vi is ini e a L. c .
G aciano is a ich sou ce o bioac i e phenolic compounds ha can become an impo an
eeds ock o addi i es and o he upg aded p oduc s o bio echnological in e es , which can
help o modula e in es inal mic obio a and comba bac e ial an ibio ic esis ance.
Keywo ds: g ape pomace; Vi is ini e a; polyphenols; supe c i ical luid ex ac ion;
Esche ichia coli; an ibio ic esis ance; an ibac e ial ac i i y
1. In oduc ion
The winemaking p ocess gene a es g ea amoun s o g ape pomace, whose e icien
exploi a ion is cu en ly a challenge in he con ex o he ci cula economy, and which may
become a sui able candida e o de eloping iable, sus ainable, eco- iendly and bio-based
p oduc s. G ape pomace, which is made up mainly o skins and seeds, is es ima ed o
ep esen a ound 20–30% o he o iginal g ape weigh used o he winemaking [
1
], and
app oxima ely 1 kg o g apes is equi ed o p oduce 0.75 L o wine [
2
]. Acco ding o he
In e na ional O ganisa ion o Vine and Wine [
3
], 237 million hec oli es o wine was he
global wine p oduc ion in 2023. Taking in o accoun hese igu es and 25% as he midway
poin in he es ima ed ange ( he a o emen ioned 20–30%) o he p opo ion o g ape weigh
composed o pomace, 7.9 million ons was he wo ld p oduc ion o g ape pomace las yea .
Foods 2025,14, 17 h ps://doi.o g/10.3390/ oods14010017
Foods 2025,14, 17 2 o 21
G ape pomace can be used o ob ain e hanol o g ape seed oil; ne e heless, subs an ial
amoun s o phenolic compounds emain in he was e p oduc .
Phenolic compounds a e seconda y me aboli es syn he ised by plan s o igh agains
en i onmen al s ess ac o s [
4
] such as UV adia ion, empe a u e shi s, wa e de ici s
o in ec i e and pa hogenic agen s. This cell p o ec ion unc ion agains a ious abio ic
and bio ic s esso s has been mainly associa ed wi h he an ioxidan ac i i y o phenolic
compounds and hei in e ac ion wi h eac i e oxygen species [
5
]. This ac i i y has been
associa ed wi h a numbe o bene i s o human heal h, and cu en ly, phenolic compounds
a e used as he apeu ic agen s and nu i ional supplemen s (see e e ences [
5
,
6
] o ecen
e iews o plan polyphenols). The di e si y o plan phenolic compounds, also e e ed
o as polyphenols, is g ea , and hey can be b oadly di ided in o ou classes: phenolic
acids, la onoids, s ilbenes and lignans [
7
]. Phenolic acids include hyd oxybenzoic and
hyd oxycinnamic acids. Fla onoids can be subdi ided acco ding o hei deg ee o unsa -
u a ion and oxida ion in o subclasses, among which la anols, la ones (which include
la onols and o he la ones) and an hocyanins a e included. S ilbenes con ain wo phenyl
ings connec ed by a wo-ca bon e hylene b idge, and lignans con ain wo phenyl ings in
a 2,3-diphenylbu ane s uc u e [7].
No el ex ac ion echniques o polyphenols om g ape pomace ha e been he subjec
o conside able in e es among esea che s, and ecen e iews can be ound on eme ging
echnologies o he ex ac ion o polyphenols om g ape pomace [
8
,
9
]. Con en ional
ex ac ion om solid ma e ial is based on he use o sol en s o mace a e he ma e ial and
achie e he mass ans e and solubilisa ion o he compounds o in e es , whe eas g een
ex ac ion echniques need less sol en and a oid he use o o ganic sol en s. Among g een
ex ac ion echniques, he ex ac ion based on supe c i ical ca bon dioxide (CO
2
) is ega ded
as an en i onmen ally iendly ex ac ion me hod as i does no u ilise o ganic sol en s
and no sol en esidues a e gene a ed. CO
2
is subjec ed o high p essu es and pumped
in o he ex ac ion chambe illed wi h he plan ma e ial. The p essu ised CO
2
possesses
cha ac e is ic luid p ope ies, such as he absence o su ace ension and e y low iscosi y,
which enhance i s pene abili y in o he small po es o he solid ma e ial, a ou ing he
ex ac ion o compounds, especially hose wi h some hyd ophobic cha ac e [
10
]. This
supe c i ical luid ex ac ion (SFE) has been applied o ed g ape pomace, mainly o he Vi is
ini e a Me lo a ie y ( e iewed in [
8
,
9
,
11
–
13
]), and also o he pomace o he ollowing
a ie ies: Ga nacha [
14
], Pe i Ve do [
15
], Cabe ne Sau ignon [
16
] and Vi is lab usca [
17
],
which do no include G aciano. These p e ious s udies ocused mainly on he phenolic
con en , composi ion and an ioxidan ac i i y o he SFE ex ac s.
The G aciano g ape is a Vi is ini e a ed a ie y au och honous o he Spanish No h-
e n egion o La Rioja [
18
]. I is o icially named by a numbe o synonyms, and hus, i is
known as Mo as el in F ance, Cagnula i in I aly, Tin a Miúda in Po ugal and Xe es in
Cali o nia [
19
]. Acco ding o he In e na ional O ganisa ion o Vine and Wine, he G aciano
a ie y is no included among he en mos widely cul i a ed g ape a ie ies. Cu en ly, i
is mainly g own in Spain (2080 ha, in La Rioja and Na a a), ollowed by I aly (437 ha),
Po ugal (326 ha) and a ew o he coun ies ha dedica e less han 50 ha o his cul i a [
20
].
I is wo h no ing ha i s cul i a ion is cu en ly ex ending [
21
] due o i s d ough esis ance,
which is an essen ial ai o wi hs and he cu en global wa ming.
Esche ichia coli is a ele an G am-nega i e species o sap ophy es o he gu mic obio a
o humans and animals; ne e heless, i may become an oppo unis ic pa hogen, mainly
when he hos ’s immune de ence is impai ed, and i may de elop p oblema ic an ibio ic
esis ances ha hinde ea men s agains bac e ial in ec ions. E. coli is also a common
indica o o aecal con amina ion, an ibio ic esis ance and dissemina ion h ough he
ood chain [
22
]. Global dissemina ion o bac e ial esis ance o an ibio ics is cu en ly an
Foods 2025,14, 17 3 o 21
issue o majo conce n. This p oblem is due o bac e ia’s na u al abili y o mu a e apidly
and acqui e new an ibio ic esis ance genes, adding hem o hei own genome, and hus
e ading he e ec s o an ibio ics. The sp ead o bac e ial esis ance was d i en by he
misuse o an ibio ics [
23
]. Some o he majo cu en measu es o ackling his p oblem a e
he p uden use o an ibio ics and he de elopmen o al e na i e an imic obials ha do no
elici an ibio ic esis ance. Aqueous soluble polyphenols pu i ied om a a ie y o plan s
ha e been shown o possess an imic obial ac i i ies, among which es e a ol and ca eic
acid we e ound [
6
], and ed wine was he epo ed na u al sou ce o hese wo bioac i es.
As men ioned abo e, ed g ape pomaces cons i u e a ich sou ce o polyphenolic bioac i e
compounds, and hey a e cu en a ge s o alo isa ion as aw ma e ials o alue-added
p oduc s [
24
]. Ne e heless, o ou knowledge, o da e, no s udy has been epo ed on he
ac i i y o SFE samples om g ape pomace, esh skins o seeds on he g ow h o an ibio ic-
esis an E. coli s ains. A p e ious s udy o ou esea ch g oup epo ed an imic obial
ac i i y o polyphenolic ex ac s om esh ed g ape skins and seeds o Vi is ini e a o he
G aciano a ie y agains mul id ug- esis an E. coli s ains [
25
]. The objec i es o his wo k
we e o ob ain and cha ac e ise polyphenolic ex ac s om G aciano ed g ape pomace
ia con en ional sol en ex ac ion and g een supe c i ical CO
2
ex ac ion using e hanol
as co-sol en and o e alua e hei
in i o
an ibac e ial ac i i y agains suscep ible and
mul id ug- esis an E. coli s ains o animal in es inal o igin.
2. Ma e ials and Me hods
2.1. Chemicals and Reagen s
S anda ds o phenolic compounds we e as ollows. Ca a ic acid (99.5%), ans-piceid
(98%), cyanidin-3-O-glucoside chlo ide (99%), p ocyanidin B1 (99.5%), p ocyanidin B2 (98%),
kaemp e ol-3-O-glucoside (99%), iso hamne in (99.1%), my ice in (99.7%), axi olin 3-O-
hamnoside (99.8%), p ocyanidin ime C1 (98.4%) and que ce in-3-O-glucu onide (98%)
we e om Biopu i y Phy ochemicals (Chengdu, China). Mal idin-3-O-glucoside chlo ide,
g ade o high-pe o mance liquid ch oma og aphy (
HPLC ≥95%
), peonidin-3-O-glucoside
chlo ide (HPLC
≥
95%), pe unidin-3-O-glucoside chlo ide (
HPLC ≥95%
), delphinidin-3-O-
glucoside chlo ide (HPLC
≥
95%), iso hamne in-3-O-glucoside (
HPLC ≥98%
), my ice in-3-O-
galac oside (97%), lu eolin-7-O-glucoside (97%), p ocyanidin dime A2 (92%) and sy inge in-
3-O-glucoside (HPLC
≥
99%) we e om Ex asyn hese (Genay, F ance). Ma ai esinol
(
HPLC ≥85%
), secoisola ici esinol (HPLC
≥
95%), ca eic acid (98%), ans-couma ic
acid (98%), ans- e ulic acid (99%), gallic acid (97.5%), 4-hyd oxybenzoic acid (99%),
4-hyd oxyphenylace ic
acid (98%), p o oca echuic acid (97%), sy ingic acid (95%), anillic acid
(97%), ans- es e a ol (99.5%), ca echin (98%), epica echin (95%) and que ce in (97%) we e
om Sigma-Ald ich (S . Louis, MO, USA). Na ingenin (95%), couma ic acid e hyl es e (97%),
e ulic acid e hyl es e (99.87%) and phlo izin (98%) we e om Fluo ochem (Had ield, UK).
Kaemp e ol (98.3%) and apigenin (98.3%) we e om Glen ham Li e Science (Co sham, UK).
Ca eic acid e hyl es e (92.9%), apigenin-7-O-glucoside (99.79%) and galloca echin (99.09%)
we e om Ta ge mol Chemicals (Bos on, MA, USA). Ty osol (98%), p o oca echuic acid e hyl
es e (98.8%) and 3-O-ca eoylquinic acid (99.58%) we e om Apollo Scien i ic (B edbu y, UK).
T ans-cou a ic acid (97%) and ans- e a ic acid (90%) we e om Phy oLab (Ves enbe gsg eu h,
Ge many). Lu eolin (98%) was om biosyn h-ca bosyn h (B a isla a, Slo akia), phlo e in
(99.8%) was om TCI (Tokyo Chemical Indus y) (Tokyo, Japan) and hyd oxy y osol (99.5%)
was om Sep ox Bio ech (Mu cia, Spain). Mos o he compounds we e acqui ed h ough he
in e media y Cymi Química (Ba celona, Spain).
Rega ding chemical eagen s needed o he ele an analy ical analyses (ex ac ion
and chemical analysis), ace oni ile high-pe o mance liquid ch oma og aphy/mass spec-
ome y (HPLC/MS) g ade, me hanol HPLC/MS g ade and o mic acid HPLC/MS g ade
Foods 2025,14, 17 4 o 21
(99%) we e pu chased om Ca lo E ba Reagen s S.A.S. (Val-de-Reuil, F ance). DPPH
(Sigma-Ald ich) (2,2-diphenyl-1-pic ylhyd azyl) and T olox (Sigma-Ald ich) (6-hyd oxy-
2,5,7,8- e ame hylch oman-2-ca boxylic acid; 97%) we e used o an ioxidan ac i i y
de e mina ion. E hanol 96% (VWR In ., Ba celona, Spain) and CO
2
(Ai Liquide Co.,
Mad id, Spain) we e used o SFE. A Milli-Q
®
In eg al de ice equipped wi h an LC-Pak
Ca idge a ached o Millipak®Exp ess 40 inal il e o 0.22 µm (Me ck Millipo e, Me ck
KGaA, Da ms ad , Ge many) was employed o ob ain ul apu e wa e o UPLC-MS.
2.2. G ape Pomace
The aw ma e ial was ed g ape pomace ha was kindly p o ided by one wine y loca ed
in he No he n Spanish egion o Rioja. I was collec ed a e adi ional ini ica ion o ed
Vi is ini e a L. c . G aciano g apes du ing in age 2021 and comp ised mainly g ape skins
and seeds. A educed numbe o sho s ems always emain a ached o some o he g apes
a e he ini ial p ocess o c ushing and des emming he g apes om he s alks. Red g ape
pomace was ozen and s o ed a −20 ◦C un il i was used o ex ac ion p ocedu es.
2.3. To al Phenolic Con en (TPC)
The Folin–Ciocal eu assay [
26
] was pe o med o calcula e he TPC o ex ac ion
samples. B ie ly, a 100
µ
L p ope ly dilu ed ex ac o a s anda d sample o a ying concen-
a ion was dissol ed in o 1.4 mL o deionised wa e , and 2.5 mL Folin–Ciocal eu solu ion
(1:10 / dilu ion in me hanol) and 1 mL o ca bona e solu ion (20% w/ ) was added. The
mix u e was ho oughly mixed and kep in he da k o 90 min. The abso bance was mea-
su ed a 725 nm in a spec opho ome e (730 UV–Visible spec opho ome e Jasco, Mad id,
Spain) and compa ed wi h a calib a ion cu e p epa ed wi h known concen a ions o
gallic acid, which was used as he s anda d o quan i y he TPC o samples. The calib a ion
cu e was p epa ed wi h a s ock solu ion o gallic acid in me hanol, and s anda d samples
we e p epa ed in he ange 0–750
µ
g/mL. Resul s we e exp essed as
µ
g o equi alen gallic
acid (GAE) pe g o d ied sample (µgGAE/gDW).
2.4. An ioxidan Ac i i y (AA)
The DPPH assay [
27
] was used o de e mine AA. The s ock solu ion o 2,2-diphenyl-
2-pic ylhyd azyl (DPPH) was 0.05 g/L o me hanol. This s ock solu ion was u he
dilu ed 1:5 in me hanol o each an abso bance o 0.75
±
0.05 a 515 nm (Jasco 730 UV-Vis
spec opho ome e ) on he day o he analysis. B ie ly, iplica es we e p epa ed combin-
ing a 40
µ
L p ope ly dilu ed ex ac o s anda d sample o a ying concen a ions wi h
1960
µ
L o he DPPH wo king solu ion, hey we e incuba ed o 1 h a oom empe a u e
in da kness, and abso bance was measu ed a 515 nm. A 100
µ
M s ock solu ion o T olox
(6-hyd oxy-2,5,7,8- e ame hylch omane-2-ca boxylic acid) was used as s anda d o p epa e
he calib a ion cu e in he ange 5–60
µ
M and quan i y AA. Resul s we e exp essed as mg
o equi alen T olox pe g o d ied sample (mgT olox/gDW).
2.5. Con en ional SE
The i s p ocedu e be o e ex ac ion was g inding he ozen pomace in a G indomix
GM 200 kni e mill (Re sch GmbH, Düsseldo , Ge many) un il a homogenous mix u e was
ob ained. The ini ial mois u e con en o he sample was 57.97 ±0.93%.
Condi ions o con en ional SE wi h an acid hyd oalcoholic solu ion o e hanol and
0.01% HCl we e op imised ollowing he Box–Behnken design me hod using he Nem od-
W
®
so wa e ( e sion 2007, LPRAI, Ma seille, F ance). This design leads o he op imisa ion
o and he educ ion in he numbe o expe imen s, and i also conside s he possible
in e ac ions be ween he s udied ac o s and hei e ec s on he esul s. Following p e ious
obse a ions and epo ed SE me hods in he bibliog aphy, h ee independen a iables
Foods 2025,14, 17 5 o 21
and hei co esponding alue anges we e chosen o s udy he e ec s on TPC and AA o
ex ac s: e hanol concen a ion, empe a u e and ex ac ion ime. The so wa e gene a ed
an expe imen al design o 27 assays wi hin he ollowing design space: om 30% o
70% e hanol; om 30
◦
C o 50
◦
C; and om 1 h o 5 h ex ac ion ime (Table 1). B ie ly, 3 g o
he g ound and de os ed pomace was weighed and mixed wi h 30 mL o he app op ia e
acid hyd oalcoholic solu ion in a glass lask. The mix u e was o exed un il homogenei y
and incuba ed in a wa e ba h unde he es ablished condi ions o empe a u e and ime
o incuba ion. A e wa ds, incuba ion samples we e spun a 2425
×
g o 10 min. Finally,
supe na an s we e submi ed o TPC and AA analyses.
Table 1. Design a angemen o op imising con en ional SE.
Assay N . E hanol (%) Tempe a u e (◦C) Time (h)
1 30 30 3
2 30 30 3
3 70 30 3
4 70 30 3
5 30 50 3
6 30 50 3
7 70 50 3
8 70 50 3
9 30 40 1
10 30 40 1
11 70 40 1
12 70 40 1
13 30 40 5
14 30 40 5
15 70 40 5
16 70 40 5
17 50 30 1
18 50 30 1
19 50 50 1
20 50 50 1
21 50 30 5
22 50 30 5
23 50 50 5
24 50 50 5
25 50 40 3
26 50 40 3
27 50 40 3
The esul ing op imised condi ions o SE (55% e hanol, 45
◦
C, 5 h) we e used o ob ain
250 mL o polyphenolic ex ac , which was eeze-d ied (Tels a LyoAl a 6-80 eeze-d ye ,
Ba celona, Spain) o ob ain he SE sample named Es, which was weighed, assayed o TPC
and AA and chemically analysed, as indica ed in Sec ions 2.3,2.4 and 2.7.
2.6. SFE
A SFE pilo plan (SFF model, Ibe Fluid/PID Eng and Tech. Mad id, Spain) equipped
wi h an ex ac ion essel, wo sepa a o s in se ies and a s o age ank o CO
2
(
≥
99.9% pu e
CO
2
) was used o supe c i ical CO
2
ex ac ion using e hanol as he co-sol en . E hanol
was chosen as co-sol en o inc ease he supe c i ical CO
2
abili y o dissol e polyphenols.
E hanol pola i y is highe han ha o CO
2
, and i inc eases he luid sol a ing powe in
SFE [
9
]. Wa e was no included as co-sol en , as p e ious epo s indica ed ha pec ic
subs ances accompanied phenolic compounds in he ex ac when wa e was included
Foods 2025,14, 17 6 o 21
as co-sol en ; mo eo e , sligh ly highe empe a u es we e equi ed o he eco e y o
polyphenols when compa ed wi h e hanol as co-sol en [28].
P io o SFE, he ozen g ape pomace was eeze-d ied and hen g ound in he
G indomix kni e mill. Pa icles we e classi ied acco ding o pa icle size using a digi al
sie e shake (RP200N model, CISA, Ba celona, Spain) wi h he ollowing mesh sizes:
1250
µ
m, 1000
µ
m, 500
µ
m and 200
µ
m o pa icle diame e . Sepa a ions we e pe o med
o 3 min a 3000 ib a ions pe minu e, 2.3 mm o ib a ion ampli ude, in a discon inuous
mode (in e als: 7.5 s on/2.5 s o ).
Two a iables we e chosen o SFE: ex ac ion o al p essu e and pa icle size. An
expe imen al design, shown in Table 2, was gene a ed wi hin he ollowing design space:
om 150 o 600 ba and wi h wo pa icle sizes.
Table 2. Design a angemen o SFE.
Assay N . Pa icle Size
(µm o Diame e )
P essu e in he Fi s
Ex ac ion Vessel (ba )
P essu e in
Sepa a o A (ba )
P essu e in
Sepa a o B (ba )
To al P essu e in he
Assay (ba )
SF1 200–500 150 110 80 340
SF2 200–500 300 200 100 600
SF3 1000–1250 150 110 80 340
The ex ac ion essel was illed wi h 50 g o he g ound and si ed sample, and glass
beads (mean diame e o 5 mm) we e added. Supe c i ical CO
2
ex ac ions we e ca ied
ou a he ollowing ixed pa ame e s: CO
2
low a e (2000 mL/h), e hanol concen a ion
(10%), ex ac ion ime (3 h) and ex ac ion empe a u e (40
◦
C). Samples we e eco e ed in
he h ee ypes o assays (SF1, SF2, SF3) om each o he h ee essels: ex ac o (samples
SF1.1, SF2.1, SF3.1), sepa a o A (samples SF1.2, SF2.2, SF3.2) and sepa a o B (samples
SF1.3, SF2.3, SF3.3). A e emo al o he co-sol en in a o a y e apo a o , he samples
we e weighed, analysed o TPC and AA and selec ed o subsequen an imic obial and
chemical analyses.
2.7. Chemical Analysis o he Ex ac s by UPLC/QqQ-MS/MS
Chemical analysis o he ob ained polyphenolic ex ac s was pe o med by he Se ice
o Ins umen al Analysis o he Ins i u e o Science o Vine and Wine (ICVV, Log oño,
Spain). Phenolic p o iles o he ob ained ex ac s we e iden i ied by UPLC/QqQ-MS/MS
using a liquid ch oma og aph (Shimadzu Nexe a, Shimadzu Co po a ion, Kyo o, Japan)
coupled wi h a QTRAP mass spec ome e (AB Sciex 3200QTRAP
®
, Sciex, F amingham,
MA, USA) equipped wi h an ESI elec osp ay ionisa ion sou ce (ESI Tu bo V™ Sou ce). The
ch oma og aphic sepa a ion was pe o med on a Wa e s Acqui y UPLC BEH C18 column
(100 mm
×
2.1 mm, 1.7
µ
m pa icle size; Wa e s, Mil o d, MA, USA) equipped wi h a
VanGua dTM AcQui y BEH C18 P e-Column (5 mm
×
2.1 mm, 1.7
µ
m pa icle size; Wa e s,
Mil o d, MA, USA).
Two ch oma og aphic me hods we e employed, one o he analysis o an hocyanins
and ano he o he analysis o he es o he phenolic compounds. In bo h me hods,
he low a e was 0.45 mL/min, he injec ion olume was 2.5
µ
L and he au osample
and o en empe a u es we e 8 and 40
◦
C, espec i ely. The mobile phases we e wa e
(sol en A) and ace oni ile (sol en B) acidi ied wi h a 2% ( / ) o o mic acid o sepa a e
an hocyanins and acidi ied wi h 0.1% ( / ) o mic acid o sepa a e he es o he analy es.
In bo h me hods, he ch oma og aphic sepa a ion ook place in less han 15 min. The
g adien elu ion p og am was as ollows: 0–0.5 min, 1% B; 0.5–1.5 min, 1–8% B; 1.5–4.0 min,
8% B; 4.0–5.0 min, 8–12% B; 5.0–5.5 min, 12% B; 5.5–6.0 min, 12–14% B; 6.0–7.0 min, 14% B;
7.0–9.0 min, 14–22% B; 9.0–12.0 min, 22–30% B; 12.0–13.7 min, 30–50% B; 13.7–14.0 min,
Foods 2025,14, 17 7 o 21
50–90% B; 14.0–15.0 min, 90% B; and inally, e u ning o he ini ial condi ions in 0.5 min
and main ained 3 min be o e he nex injec ion.
The elu ed compounds we e analysed by using a iple-quad upole mass spec ome e .
The elec osp ay in e ace was se in posi i e ion mode (ESI+) wi h an ion sp ay ol age o
+4.5 kV o he analysis o he an hocyanins and in nega i e ion mode (ESI
−
) wi h an ion sp ay
ol age o
−
3.5 kV o he analysis o he es o he analy es. Ni ogen (degasi ied liquid
ni ogen > 99.99% pu i y, Ai Liquide, Mad id, Spain) was used as he sou ce and collision
gas. The sou ce empe a u e was 700
◦
C, and he gas p essu es we e cu ain gas 50 psi, GS1
50 psi and GS2 60 psi. The da a we e acqui ed by mul iple eac ion moni o ing (MRM). The
dwell ime es ablished o each ansi ion (and numbe o da a poin s ac oss he UPLC peak)
was op imised h ough he ch oma og am employing he Scheduled MRM™ Algo i hm by
means o he e en ion ime wi h an MRM de ec ion window o 60 s and a a ge scan ime
o 1.2 s. The e en ion ime and MRM ansi ions o quan i ica ion and iden i ica ion, in
addi ion o he indi idual declus e ing po en ial (DP), en ance po en ial (EP), collision cell
en ance po en ial (CEP), collision ene gy (CE) and collision cell exi po en ial (CXP) o each
phenolic compound a e shown in Supplemen a y Table S1. Some o he phenolic compounds
we e iden i ied and quan i ied using pu e comme cial s anda ds. Analy es wi h no a ailable
s anda ds we e iden i ied by s udying and compa ing MS agmen a ion pa e ns and ela i e
e en ion imes desc ibed in he li e a u e. Those analy es we e en a i ely quan i ied using
he calib a ion cu es o s anda ds wi h simila chemical s uc u es. The s anda d selec ed
o each analy e is also shown in Supplemen a y Table S1.
Samples we e p epa ed by dilu ing app oxima ely 10 mg o each ex ac in 800 µL o
MeOH/wa e / o mic acid (60:39:1 / / ). When u bidi y was obse ed, he sample was
sonica ed o 5–10 min in an ice-cold ul asonic ba h ( empe a u e less han 20
◦
C). Samples
we e cen i uged a 4500
×
g(10 min, 4
◦
C) and il e ed h ough a 0.22
µ
m PTFE il e (Scha -
lab, Ca alonia, Spain) be o e he ch oma og aphic analysis. Due o he wide ange o con-
cen a ions o phenolic compounds in samples, a 1:10 dilu ion wi h MeOH/wa e / o mic
acid (20:79:1 / / ) was also injec ed. The inal concen a ions o phenolic compounds in
each sample we e de e mined by a e aging he con en a e h ee consecu i e injec ions,
and he esul s a e exp essed as ng/mg o d y ex ac .
Da a p ocessing was pe o med wi h he so wa e package Analys 1.6.2 and
Mul iQuan TM 3.0.2 (AB Sciex, F amingham, MA, USA).
2.8. Mic obial Cul u e Condi ions and An imic obial Assays
Fou E. coli s ains (Table 3) o animal in es inal o igin we e ob ained om p e ious
s udies pe o med by he OneHeal h-UR esea ch g oup. Two s ains we e suscep ible o
an ibio ics, and he o he wo we e ex ended-spec um be a-lac amase (ESBL)-p oducing
s ains and showed esis ance o a leas wo amilies o an ibio ics.
Table 3. An ibio ic-suscep ible and mul id ug- esis an E. coli s ains included in his s udy.
S ain Resis ance Pheno ype Resis ance Geno ype O igin
C7023 An ibio ic suscep ible Cow
C7067 An ibio ic suscep ible Rabbi
C7577 Resis an o: AMP, CAZ, CTX, NAX, TET, SXT SHV-12 Dee
C6840 Resis an o: AMP, CTX, NAX, TET, SXT CTX-M-14a Coa í
AMP (AMP ampicillin), CAZ (ce azidime), CTX (ce o axime), NAX (nalidixic acid), TET ( e acycline), SXT
( ime hop im-sul ame hoxazole). SHV-12: geno ype o ex ended-spec um
β
-lac amases; CTX-M-14a: geno ype
o ex ended-spec um β-lac amases.
S ains we e es ed by he mic o i e dilu ion me hod [
29
] o e alua e he an imic obial
ac i i y o he polyphenolic ex ac s o ou s udy: Es, SF1.3, SF2.3 and SF3.3. Cul u es
we e pe o med in b ain hea in usion (BHI) b o h (Condalab, Mad id, Spain) in 96-well
Foods 2025,14, 17 8 o 21
mic odilu ion pla es, and he mul imode mic opla e eade Tecan Spa k 10M (Tecan T ading
AG, Männedo , Swi ze land) was used o measu e bac e ial g ow h by op ical densi y a
600 nm. All assays we e pe o med in iplica e, and nega i e and posi i e con ol samples
we e included in all he assays. Nega i e con ols wi hou bac e ial inoculum we e used
o e i y ha he e was no c oss-con amina ion. Posi i e con ols o bac e ial g ow h in
he absence o polyphenolic ex ac we e used o e i y he bac e ial inoculum iabili y
and i s adequa e cell g ow h in each assay. The op ical densi y alues o posi i e con ols
we e he e e ence o de e mine bac e ial g ow h inhibi ion in each assay. Consecu i e
2- old dilu ions o each ex ac we e p epa ed ac oss each ow o he mic odilu ion pla e. A
o al olume o 100
µ
L in each well con ained 25
µ
L inoculum o he bac e ial suspension
(10
5
cells/mL), 25
µ
L o ex ac sample wi h he app op ia e se ial dilu ion and BHI b o h.
Incuba ions we e pe o med a 30
◦
C o 10–24 h. Con ols we e included in all he assays.
MIC was de ined as he lowes concen a ion o he ex ac ha inhibi ed bac e ial g ow h
a e 24 h incuba ion. Bac e icide ac i i y was de e mined by subcul u ing on o BHI
aga pla es wi hou he es ed ex ac o 36 h, and MBC was de ined as he minimal
concen a ion o he an imic obial agen ha had killed mo e han 99.9% o he ini ial
inoculum a e 48 h incuba ion on o BHI aga pla es. The es ed concen a ion ange o
he ex ac s was 8.0–0.13 mg/mL (double dilu ions), and all he bac e ial assays we e
pe o med in iplica e.
2.9. S a is ical Analysis
Mean alues and s anda d de ia ions o h ee epea s we e calcula ed. Analysis o
a iance (ANOVA) was applied as he da a showed no mal dis ibu ion and homogeneous
a iances. Tukey’s ange es was used as he mean compa ison me hod o da a ob ained in
all he pe o med physicochemical analyses a a signi icance le el o p= 0.05. The IBM-SPSS
S a is ics 22.0 o Windows (IBM-SPSS Inc., Chicago, IL, USA) s a is ical package was used
o da a p ocessing.
3. Resul s and Discussion
Pomace o Vi is ini e a ed g apes o he G aciano a ie y was chosen o his s udy
because his a ie y s ands ou o i s high con en o phenolic compounds [
30
], and
hese molecules a e well known o playing a ele an ole in plan s ess esponse o a
a ie y o ex e nal ha assmen s such as d ough , as men ioned abo e. This high con en
o phenolic compounds explains why he G aciano a ie y is ega ded as a d ough -
ole an ed g ape ine a ie y, as men ioned, and a candida e o a enua e he dele e ious
e ec s o clima e change on g ape ines. Rega ding G aciano a ie al ed wines, hei
con en o la onoid compounds has been epo ed o be among he highes [
31
]. The
s a ing ma e ial o G aciano ed g ape pomace in ou s udy showed indeed a high TPC
(
68,220 ±3190 mgGAE/gDW
). This pomace was submi ed o bo h he SE and SFE ex ac ion
me hods desc ibed abo e.
3.1. Con en ional SE
The esul s o he Box–Behnken design expe imen s o con en ional solid–liquid
ex ac ion wi h measu ed esponses o TPC a e shown in Figu e 1.
Figu e 1A,B show ha he maximal ex ac ion o polyphenols was achie ed in he
in e als wi h 45–60% e hanol and 40–50
◦
C. Figu e 1C,D show ha he maximal al-
ues o ex ac ion we e achie ed wi h 45–55% e hanol and 5 h ex ac ion ime. Finally,
Figu e 1E,F
show he maximal alues a 40–50
◦
C and ha ime did no signi ican ly a ec
he ex ac ion yield.
Foods 2025,14, 17 9 o 21
Foods 2025, 14, x FOR PEER REVIEW 9 o 22
ma e ial o G aciano ed g ape pomace in ou s udy showed indeed a high TPC (68,220 ±
3190 mgGAE/gDW). This pomace was submi ed o bo h he SE and SFE ex ac ion me hods
desc ibed abo e.
3.1. Con en ional SE
The esul s o he Box–Behnken design expe imen s o con en ional solid–liquid ex-
ac ion wi h measu ed esponses o TPC a e shown in Figu e 1.
Figu e 1. Th ee-dimensional con ou and esponse su ace plo s o he a ia ions in he o al phenolic
con en (
µ
g
GAE
/g
DW
) o SE samples. (A,B) Resul s a ixed ex ac ion ime o 3 h. (C,D) Resul s a
40 ◦C ixed empe a u e. (E,F) Resul s a ixed e hanol concen a ion o 50%.
Simila ly, he esponse o AA unde he designed assays was also s udied. The esul s
o hese Box–Behnken design expe imen s a e shown in Figu e 2.
Foods 2025,14, 17 16 o 21
E. coli s ains [
51
,
52
]. Sanhueza e al. [
51
] ob ained one ex ac om ed g ape pomace by
con en ional SE and a second liquid–liquid ex ac ion. The majo componen s o he ex ac
we e phenolic acids and la onols (78.8%) and, unlike ou SE ex ac , no an hocyanin was
de ec ed in i s composi ion. They epo ed MIC alues o 1.5–3.0 mg/mL agains hei
an ibio ic- esis an E. coli s ains, alues in he same ange as he MIC alues o ou ex ac s.
Peixo o e al. [
52
] epo ed wo hyd ome hanolic ex ac s om ed g ape pomace and om
seeds whose MIC alues we e 20 mg/mL agains one an ibio ic- esis an E. coli s ain wi h
a geno ype o ex ended-spec um
β
-lac amases. The composi ion o hei con en ional
SE samples included la an 3-ols, p ocyanidins, la onols, phenolic acids and negligible
concen a ions o an hocyanins. One SE sample ob ained om whi e g ape pomace was
also epo ed o possess an imic obial ac i i y agains ano he an ibio ic- esis an E. coli
s ain wi h a geno ype o ex ended-spec um
β
-lac amases [
53
]. The majo componen s
o hei ex ac we e la an 3-ols, la onols and p ocyanidins, and he epo ed MBC and
IC50 (inhibi o y concen a ion ha inhibi s 50% o mic obial g ow h) alues o he ex ac s
agains he an ibio ic- esis an E. coli s ain we e 15% and 4.65% ( / ), espec i ely. In hese
las wo s udies [
52
,
53
], he epo ed an imic obial ac i i ies o he SE ex ac s we e a leas
10- old lowe han he ones shown in ou s udy.
Rega ding he en e ohaemo hagic E. coli O157:H7 s ain esponsible o oodbo ne
in ec ions, some s udies o con en ional SE samples om ed g ape pomace [
54
], whi e
and ed g ape skins [
55
] and g ape seeds [
56
] epo ed an imic obial ac i i y agains his
pa icula E. coli s ain, and he epo ed IC50 alues and MBC we e on he o de o 25 mg
o solid ex ac pe mL o cul u e b o h [
54
,
56
]. Some o he epo s o an imic obial ac i i y
o con en ional SE samples agains an ibio ic-sensi i e E. coli s ains we e epo ed om
g ape pomace [
57
–
59
], g ape seeds [
60
,
61
] and ex ac s o g ape juice and wines [
62
,
63
].
Ne e heless, he epo ed an ibac e ial ac i i ies a y ex ensi ely due o he di e si y
o he s a ing aw ma e ial, he Vi is ini e a a ie y, he di e en me hods and cul u e
condi ions u ilised o ac i i y de ec ion and quan i ica ion o he phenolic con en o
he ex ac s.
The MIC alues o he g ape pomace ex ac s o ou s udy, shown in Table 6, a e in
he ange o p e iously epo ed alues (6.25 mg/mL) o G aciano skin ex ac s om esh
g apes [
25
] and a e also in acco dance wi h s udies on he ac i i y o polyphenols agains
E. coli s ains ha epo ed MIC alues in he ange o 6–18 mg/mL o la an-3-ols and
sligh ly highe alues, in he ange o 25–100 mg/mL, o an hocyanins om a a ie y o
ege able sou ces [64].
Ou esul s wi h he ex ac s o he G aciano ed g ape pomace indica e ha his
by-p oduc o he wine y indus y is s ill a ich sou ce o bioac i e compounds and ha he
mos po en inhibi o o bac e ial g ow h, Es, was also he ex ac wi h he highes con en
o all he phenolic amilies. Es was ollowed in an ibac e ial ac i i y and phenolic con en by
he SF2.3 ex ac . A di ec co ela ion be ween bo h pa ame e s, an ibac e ial ac i i y and
phenolic con en , is e idenced by hese esul s. Figu e 4shows some ep esen a i e g ow h
cu es o he an imic obial ac i i y assays o he ou SE and SFE polyphenolic samples
agains he collec ion o E. coli s ains. Addi ional esul s o he an ibac e ial ac i i y o
he ou SE and SFE ex ac s agains he whole collec ion o E. coli s ains a e shown in
Supplemen a y Table S2.
In summa y, he o e all esul s o his s udy show ha he s udied polyphenolic
ex ac s exe an inhibi o y e ec on he bac e ial g ow h o in es inal E. coli s ains, bo h
hose suscep ible o and esis an o an ibio ics, and ha i is no a bac e icidal bu a
bac e ios a ic e ec . A highe con en o o al polyphenols is shown o co ela e wi h highe
an ibac e ial ac i i y o he ex ac s, whe eas he ela i e abundance o phenolic amilies
shows no co ela ion wi h he an ibac e ial ac i i y, sugges ing ha he mechanisms by
Foods 2025,14, 17 17 o 21
which he inhibi ion o bac e ial g ow h is exe ed by he polyphenolic ex ac s a e mul i-
a ge ed. Fu he esea ch will be equi ed o iden i y he molecula mechanisms and he
speci ic e ec o s ha igge bac e ial g ow h inhibi ion. In addi ion, his s udy shows ha
ed g ape pomace o Vi is ini e a L. c . G aciano is a ich sou ce o bioac i e polyphenolic
ex ac s, and i can become an impo an eeds ock o addi i es and o he upg aded
p oduc s o bio echnological in e es ha can help o modula e in es inal mic obio a and
comba an ibio ic- esis an bac e ia.
Foods 2025, 14, x FOR PEER REVIEW 18 o 22
Figu e 4. G ow h cu es o he mul id ug- esis an E. coli C6840 s ain in he p esence o inc easing
concen a ions (mg/mL) o each o he polyphenolic ex ac s: (A) SF1.3, (B) SF2.3, (C) SF3.3 and (D)
Es. Each do ep esen s he mean alue o iplica es, and ba s indica e s anda d de ia ions; 0
mg/mL: op ical densi y o con ol samples o bac e ial g ow h wi hou inhibi o s.
In summa y, he o e all esul s o his s udy show ha he s udied polyphenolic ex-
ac s exe an inhibi o y effec on he bac e ial g ow h o in es inal E. coli s ains, bo h
hose suscep ible o and esis an o an ibio ics, and ha i is no a bac e icidal bu a bac-
e ios a ic effec . A highe con en o o al polyphenols is shown o co ela e wi h highe
an ibac e ial ac i i y o he ex ac s, whe eas he ela i e abundance o phenolic amilies
shows no co ela ion wi h he an ibac e ial ac i i y, sugges ing ha he mechanisms by
which he inhibi ion o bac e ial g ow h is exe ed by he polyphenolic ex ac s a e mul i-
a ge ed. Fu he esea ch will be equi ed o iden i y he molecula mechanisms and he
speci ic effec o s ha igge bac e ial g ow h inhibi ion. In addi ion, his s udy shows ha
ed g ape pomace o Vi is ini e a L. c . G aciano is a ich sou ce o bioac i e polyphenolic
ex ac s, and i can become an impo an eeds ock o addi i es and o he upg aded p od-
uc s o bio echnological in e es ha can help o modula e in es inal mic obio a and com-
ba an ibio ic- esis an bac e ia.
Supplemen a y Ma e ials: The ollowing supplemen a y in o ma ion can be downloaded a :
www.mdpi.com/xxx/s1, Table S1: (MRM condi ions employed o he analysis and quan i ica ion o
he phenolic compounds de e mined in he ex ac s); Table S2: (G ow h moni o ing o E. coli s ains
Figu e 4. G ow h cu es o he mul id ug- esis an E. coli C6840 s ain in he p esence o inc easing
concen a ions (mg/mL) o each o he polyphenolic ex ac s: (A) SF1.3, (B) SF2.3, (C) SF3.3 and
(D) Es. Each do ep esen s he mean alue o iplica es, and ba s indica e s anda d de ia ions;
0 mg/mL: op ical densi y o con ol samples o bac e ial g ow h wi hou inhibi o s.
Supplemen a y Ma e ials: The ollowing supplemen a y in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/ oods14010017/s1, Table S1: (MRM condi ions employed o he
analysis and quan i ica ion o he phenolic compounds de e mined in he ex ac s); Table S2: (G ow h
moni o ing o E. coli s ains in absence (mg/mL = 0) o polyphenolic ex ac s, and in p esence o
inc easing concen a ions o each polyphenolic ex ac ).
Au ho Con ibu ions: Concep ualisa ion, F.R.-L., R.F.-P. and C.T.; me hodology, C.M., M.-J.S.-A.
and M.G.-L.; so wa e, C.M., M.-J.S.-A. and M.G.-L.; alida ion, S.A., C.M. and M.-J.S.-A.; o mal
analysis, F.R.-L., R.F.-P., S.A. and C.T.; esou ces, F.R.-L. and C.T.; in es iga ion, S.A.; da a cu a ion,
R.F.-P. and S.A.; w i ing—o iginal d a p epa a ion, F.R.-L., R.F.-P. and C.M.; w i ing— e iew and
edi ing, F.R.-L., R.F.-P., C.M., M.-J.S.-A., M.G.-L. and C.T.; supe ision, F.R.-L., R.F.-P. and M.-J.S.-A.;
Foods 2025,14, 17 18 o 21
p ojec adminis a ion, F.R.-L.; unding acquisi ion, C.T. and F.R.-L. All au ho s ha e ead and ag eed
o he published e sion o he manusc ip .
Funding: This esea ch was unded: by he Agency o Economic De elopmen o he Au onomous
Communi y o La Rioja (ADER) o Spain and he Eu opean Regional De elopmen Fund (ERDF) o
he E.U., g an numbe 2019-I-IDD-00048; by he Regional Go e nmen o La Rioja (Spain), g an
numbe A ianza2023/04, A ianza2021/07, PR-10-20; and by he Uni e si y o La Rioja, g an numbe
REGI2022/26, REGI2020/35.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : The o iginal con ibu ions p esen ed in he s udy a e included in he
a icle/Supplemen a y Ma e ial, u he inqui ies can be di ec ed o he co esponding au ho .
Con lic s o In e es : The au ho s decla e no con lic s o in e es . The unde s had no ole in he design
o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip ; o
in he decision o publish he esul s.
Re e ences
1. Dwye , K.; Hosseinian, F.; Rod, M. The Ma ke Po en ial o G ape Was e Al e na i es. J. Food Res. 2014,3, 91. [C ossRe ]
2.
Be es, C.; Cos a, G.N.S.; Cabezudo, I.; da Sil a-James, N.K.; Teles, A.S.C.; C uz, A.P.G.; Mellinge -Sil a, C.; Tonon, R.V.; Cab al,
L.M.C.; F ei as, S.P. Towa ds In eg al U iliza ion o G ape Pomace om Winemaking P ocess: A Re iew. Was e Manag. 2017,68,
581–594. [C ossRe ]
3.
OIV 2023. A ailable online: h ps://www.oi .in /si es/de aul / iles/2024-04/OIV_STATE_OF_THE_WORLD_VINE_AND_
WINE_SECTOR_IN_2023.pd (accessed on 4 Oc obe 2024).
4.
Ray, A.; Kundu, S.; Mohapa a, S.S.; Sinha, S.; Khosh u, B.; Keswani, C.; Mi a, D. An Insigh in o he Role o Phenolics in Abio ic
S ess Tole ance in Plan s: Cu en Pe spec i e o Sus ainable En i onmen . J. Pu e Appl. Mic obiol. 2024,18, 64–79. [C ossRe ]
5.
Zagoskina, N.V.; Zubo a, M.Y.; Nechae a, T.L.; Kazan se a, V.V.; Goncha uk, E.A.; Ka anskaya, V.M.; Ba ano a, E.N.; Akseno a,
M.A. Polyphenols in Plan s: S uc u e, Biosyn hesis, Abio ic S ess Regula ion, and P ac ical Applica ions (Re iew). In . J. Mol.
Sci. 2023,24, 13874. [C ossRe ]
6.
Mandal, M.K.; Domb, A.J. An imic obial Ac i i ies o Na u al Bioac i e Polyphenols. Pha maceu ics 2024,16, 718. [C ossRe ]
[PubMed]
7.
Pandey, K.B.; Riz i, S.I. Plan Polyphenols as Die a y An ioxidan s in Human Heal h and Disease. Oxid. Med. Cell Longe . 2009,2,
270–278. [C ossRe ] [PubMed]
8.
Cas o, L.E.N.; Sganze la, W.G.; Sil a, A.P.G.; John, O.D.; Ba oso, T.L.C.T.; Ros agno, M.A.; Fo s e -Ca nei o, T. Sus ainable Ex ac-
ion Me hods o he Reco e y o Polyphenolic Compounds om G ape Pomace and I s Biological P ope ies: A Comp ehensi e
Re iew. Phy ochem. Re . 2024, 1–28. [C ossRe ]
9.
Wani, T.A.; Majid, D.; Da , B.N.; Mak oo, H.A.; Allai, F.M. U iliza ion o No el Techniques in Ex ac ion o Polyphenols om
G ape Pomace and Thei The apeu ic Po en ial: A Re iew. J. Food Meas. Cha ac . 2023,17, 5412–5425. [C ossRe ]
10.
Yang, Y.; Hu, B. Bio-Based Chemicals om Bio e ining: Lipid and Wax Con e sion and U iliza ion. In Ad ances in Bio e ine ies;
Else ie : Ams e dam, The Ne he lands, 2014; pp. 693–720. ISBN 978-0-85709-521-3.
11.
Ab eu, T.; Sousa, P.; Gonçal es, J.; Hon man, N.; Teixei a, J.; Câma a, J.S.; Pe es elo, R. G ape Pomace as a Renewable Na u al
Biosou ce o Value-Added Compounds wi h Po en ial Food Indus ial Applica ions. Be e ages 2024,10, 45. [C ossRe ]
12.
Ka as e giou, A.; Gancel, A.L.; Jou des, M.; Teissed e, P.L. Valo iza ion o G ape Pomace: A Re iew o Phenolic Composi ion,
Bioac i i y, and The apeu ic Po en ial. An ioxidan s 2024,13, 1131. [C ossRe ] [PubMed]
13.
Peña-Po illo, G.C.; Acuña-Nelson, S.M.; Bas ías-Mon es, J.M. F om Was e o Weal h: Explo ing he Bioac i e Po en ial o Wine
By-P oduc s—A Re iew. An ioxidan s 2024,13, 992. [C ossRe ]
14.
Pinelo, M.; Ruiz-Rod íguez, A.; Sinei o, J.; Seño áns, F.J.; Regle o, G.; Núñez, M.J. Supe c i ical Fluid and Solid–Liquid Ex ac ion
o Phenolic An ioxidan s om G ape Pomace: A Compa a i e S udy. Eu . Food Res. Technol. 2007,226, 199–205. [C ossRe ]
15.
Cejudo-Bas an e, C.; A jona-Muda a, P.; Fe nández-Ponce, M.T.; Casas, L.; Man ell, C.; Ma ínez de la Ossa, E.J.; Pe ey a, C.
Applica ion o a Na u al An ioxidan om G ape Pomace Ex ac in he De elopmen o Bioac i e Ju e Fibe s o Food Packaging.
An ioxidan s 2021,10, 216. [C ossRe ]
16.
Zhabaye a, A.N.; Velyamo , M.T.; Nakypbeko a, N.E.; Dolgikh, S.G.; Adekeno , S.M. Supe c i ical Fluid Ex ac ion in Res e a ol
Isola ion Technology. Eu asian Chem.-Technol. J. 2021,23, 119–124. [C ossRe ]
Foods 2025,14, 17 19 o 21
17.
Gha oo , K.; Pa k, J.; Choi, Y.-H. Op imiza ion o Supe c i ical Fluid Ex ac ion o Bioac i e Compounds om G ape (Vi is Lab usca
B.) Peel by Using Response Su ace Me hodology. Inno . Food Sci. Eme g. Technol. 2010,11, 485–490. [C ossRe ]
18.
Núñez, V.; Monagas, M.; Gomez-Co do és, M.C.; Ba olomé, B. Vi is ini e a L. C . G aciano G apes Cha ac e ized by I s
An hocyanin P o ile. Pos ha es Biol. Technol. 2004,31, 69–79. [C ossRe ]
19. A ailable online: h ps://www.oi .in /wha -we-do/ i icul u e-da abase- epo ?oi = (accessed on 4 Oc obe 2024).
20.
Ande son, K.; Nelgen, S. Which Wineg ape Va ie ies A e G own Whe e? A Global Empi ical Pic u e (Re ised Edi ion); Uni e si y o
Adelaide P ess: Adelaide, Aus alia, 2020; ISBN 978-1-925261-86-8.
21.
Mezei, L.V.; Johnson, T.E.; Goodman, S.; Collins, C.; Bas ian, S.E.P. Mee ing he Demands o Clima e Change: Aus alian
Consume Accep ance and Senso y P o iling o Red Wines P oduced om Non-T adi ional Red G ape Va ie ies. OENO One 2021,
55, 29–46. [C ossRe ]
22.
Sanz, S.; Ola e, C.; Hidalgo-Sanz, R.; Ruiz-Ripa, L.; Fe nández-Fe nández, R.; Ga cía-Vela, S.; Ma ínez-Ál a ez, S.; To es,
C. Ai bo ne Dissemina ion o Bac e ia (En e ococci, S aphylococci and En e obac e iaceae) in a Mode n B oile Fa m and I s
En i onmen . Animals 2021,11, 1783. [C ossRe ] [PubMed]
23.
Rol , J.; Bonhoe e , S.; Klo , C.; Leis ne , R.; Regoes, R.; Hochbe g, M.E. Fo ecas ing An imic obial Resis ance E olu ion. T ends
Mic obiol. 2024,32, 736–745. [C ossRe ] [PubMed]
24.
Niculescu, V.-C.; Ione e, R.-E. An O e iew on Managemen and Valo isa ion o Wine y Was es. Appl. Sci. 2023,13, 5063.
[C ossRe ]
25.
Fe nández-Pé ez, R.; Teno io, C.; Ruiz-La ea, F. E ec o Wine Polyphenol Ex ac s on he G ow h o Esche ichia coli. In Explo ing
Mic oo ganisms: Recen Ad ances in Applied Mic obiology; B own Walke P ess: I ine, CA, USA; Uni e sal Publishe s, Inc.: Boca
Ra ón, FL, USA, 2018; pp. 226–230. ISBN 978-1-62734-623-8.
26.
Single on, V.L.; O ho e , R.; Lamuela-Ra en ós, R.M. Analysis o To al Phenols and O he Oxida ion Subs a es and An ioxidan s
by Means o Folin-Ciocal eu Reagen . In Me hods Enzymology; Academic P ess: Camb idge, MA, USA, 1999; Volume 299, pp.
152–178.
27.
Molyneux, P. The Use o he S able F ee Radical Diphenylpic yl-Hyd azyl (DPPH) o Es ima ing An ioxidan Ac i i y. J. Sci.
Technol. 2004,26, 212–219.
28.
Hay ape yan, G.; T chounian, K.; Buon, L.; No e , L.; Pinel, B.; Lag ue, J.; Assi aoui, A. Sequen ial Ex ac ion o High-Value
Added Molecules om G ape Pomaces Using Supe c i ical Fluids wi h Wa e as a Co-Sol en . RSC Sus ain. 2023,1, 2014–2023.
[C ossRe ]
29.
Rojo-Beza es, B.; Sáenz, Y.; Za azaga, M.; To es, C.; Ruiz-La ea, F. An imic obial Ac i i y o Nisin agains Oenococcus oeni and
O he Wine Bac e ia. In . J. Food Mic obiol. 2007,116, 32–36. [C ossRe ]
30.
Po u, J.; Rosa Gu ié ez-Vigue a, A.; González-A enzana, L.; San ama ía, P. Cha ac e iza ion o he Colo Pa ame e s and
Monome ic Phenolic Composi ion o ‘Temp anillo’ and ‘G aciano’ Wines Made by Ca bonic Mace a ion. Food Chem. 2023,406,
134327. [C ossRe ] [PubMed]
31.
Monagas, M.; Suá ez, R.; Gómez-Co do és, C.; Ba olomé, B. Simul aneous De e mina ion o Nonan hocyanin Phenolic Com-
pounds in Red Wines by HPLC-DAD/ESI-MS. Am. J. Enol. Vi ic. 2005,56, 139–147. [C ossRe ]
32.
Bon igli, M.; Godoy, E.; Reinheime , M.A.; Scenna, N.J. Compa ison be ween Con en ional and Ul asound-Assis ed Techniques
o Ex ac ion o An hocyanins om G ape Pomace. Expe imen al Resul s and Ma hema ical Modeling. J. Food Eng. 2017,207,
56–72. [C ossRe ]
33.
Casque e, R.; Beni o, M.J.; Ma ín, A.; Ma ínez, A.; de los Angeles Ri as, M.; de Guía Có doba, M. In luence o Di e en
Ex ac ion Me hods on he Compound P o iles and Func ional P ope ies o Ex ac s om Solid By-P oduc s o he Wine Indus y.
LWT 2022,170, 114097. [C ossRe ]
34. Fon ana, A.; An oniolli, A.; D’Ama io Fe nández, M.A.; Bo ini, R. Phenolics P o iling o Pomace Ex ac s om Di e en G ape
Va ie ies Cul i a ed in A gen ina. RSC Ad . 2017,7, 29446–29457. [C ossRe ]
35.
Spigno, G.; De Fa e i, D.M. An ioxidan s om G ape S alks and Ma c: In luence o Ex ac ion P ocedu e on Yield, Pu i y and
An ioxidan Powe o he Ex ac s. J. Food Eng. 2007,78, 793–801. [C ossRe ]
36. FAO S.L.231.41. A ailable online: h ps:// aolex. ao.o g/docs/pd /ml 41702.pd (accessed on 4 Oc obe 2024).
37.
EC Di ec i e 2009/32. A ailable online: h ps://eu -lex.eu opa.eu/legal-con en /EN/TXT/?u i=CELEX:02009L0032-20100916
(accessed on 4 Oc obe 2024).
38.
Pin a´c, D.; Majki´c, T.; To o i´c, L.; O ˇci´c, D.; Bea a, I.; Simin, N.; Mimica–Duki´c, N.; Lesjak, M. Sol en Selec ion o E icien
Ex ac ion o Bioac i e Compounds om G ape Pomace. Ind. C ops P od. 2018,111, 379–390. [C ossRe ]
39.
Da Po o, C.; Na olino, A. Policosanols om G ape Ma c: A New S ep owa ds a Sus ainable Bio e ine y o he Wine Indus y by
SC-CO2Ex ac ion. J. CO2 U il. 2024,82, 102762. [C ossRe ]
40.
Oli ei a, D.A.; Sal ado , A.A.; Smânia, A.; Smânia, E.F.A.; Ma aschin, M.; Fe ei a, S.R.S. An imic obial Ac i i y and Composi ion
P o ile o G ape (Vi is ini e a) Pomace Ex ac s Ob ained by Supe c i ical Fluids. J. Bio echnol. 2013,164, 423–432. [C ossRe ]
Foods 2025,14, 17 20 o 21
41.
O e o-Pa eja, M.; Casas, L.; Fe nández-Ponce, M.; Man ell, C.; Ossa, E. G een Ex ac ion o An ioxidan s om Di e en Va ie ies
o Red G ape Pomace. Molecules 2015,20, 9686–9702. [C ossRe ]
42.
Pazi , F.; Koçak, E.; Tu an, F.; O a, G. Ex ac ion o An hocyanins om G ape Pomace by Using Supe c i ical Ca bon Dioxide. J.
Food P ocess. P ese . 2021,45, e14950. [C ossRe ]
43.
Da Po o, C.; Na olino, A.; Deco i, D. Ex ac ion o P oan hocyanidins om G ape Ma c by Supe c i ical Fluid Ex ac ion Using
CO2as Sol en and E hanol–Wa e Mix u e as Co-Sol en . J. Supe c i . Fluids 2014,87, 59–64. [C ossRe ]
44.
Rue, E.A.; Rush, M.D.; an B eemen, R.B. P ocyanidins: A Comp ehensi e Re iew Encompassing S uc u e Elucida ion ia Mass
Spec ome y. Phy ochem. Re . 2018,17, 1–16. [C ossRe ]
45.
Mosele, J.; da Cos a, B.S.; Bobadilla, S.; Mo il a, M.-J. Phenolic Composi ion o Red and Whi e Wine Byp oduc s om Di e en
G ape ine Cul i a s om La Rioja (Spain) and How This Is A ec ed by he Winemaking P ocess. J. Ag ic. Food Chem. 2023,71,
18746–18757. [C ossRe ] [PubMed]
46.
Po u, J.; López, R.; Ewald, P.; San ama ía, P.; Win e hal e , P.; Ga de-Ce dán, T. E alua ion o G enache, G aciano and Temp anillo
G ape S ilbene Con en a e Field Applica ions o Elici o s and Ni ogen Compounds. J. Sci. Food Ag ic. 2018,98, 1856–1862.
[C ossRe ]
47.
Monagas, M.; Gómez-Co do és, C.; Ba olomé, B.; Lau eano, O.; Rica do da Sil a, J.M. Monome ic, Oligome ic, and Polyme ic
Fla an-3-Ol Composi ion o Wines and G apes om Vi is ini e a L. C . G aciano, Temp anillo, and Cabe ne Sau ignon. J. Ag ic.
Food Chem. 2003,51, 6475–6481. [C ossRe ] [PubMed]
48.
Ribei o, L.F.; Ribani, R.H.; S a ussa, A.P.; Maka a, C.N.; B anco, I.G.; Maciel, G.M.; Haminiuk, C.W.I. Explo a o y Analysis o
Bioac i e Compounds and An ioxidan Po en ial o G ape (Vi is ini e a) Pomace. Ac a Sci. Technol. 2022,44, e56934. [C ossRe ]
49.
Sil a, V.; Ig ejas, G.; Falco, V.; San os, T.P.; To es, C.; Oli ei a, A.M.P.; Pe ei a, J.E.; Ama al, J.S.; Poe a, P. Chemical Composi ion,
An ioxidan and An imic obial Ac i i y o Phenolic Compounds Ex ac ed om Wine Indus y by-P oduc s. Food Con ol 2018,
92, 516–522. [C ossRe ]
50.
Sil a, V.; Ribei o, J.; Singh, R.K.; Ai es, A.; Ca alho, R.; Falco, V.; Pe ei a, J.E.; Ig ejas, G.; Poe a, P. Explo ing Winemaking
By-P oduc s o Tin o Cão G apes: An ioxidan and An imic obial Ac i i y agains Mul i esis an Bac e ia. Med. Sci. Fo um 2024,
24, 7. [C ossRe ]
51.
Sanhueza, L.; Melo, R.; Mon e o, R.; Maisey, K.; Mendoza, L.; Wilkens, M. Syne gis ic In e ac ions be ween Phenolic Compounds
Iden i ied in G ape Pomace Ex ac wi h An ibio ics o Di e en Classes agains S aphylococcus au eus and Esche ichia coli.PLoS
ONE 2017,12, e0172273. [C ossRe ] [PubMed]
52.
Peixo o, C.M.; Dias, M.I.; Al es, M.J.; Calhelha, R.C.; Ba os, L.; Pinho, S.P.; Fe ei a, I.C.F.R. G ape Pomace as a Sou ce o Phenolic
Compounds and Di e se Bioac i e P ope ies. Food Chem. 2018,253, 132–138. [C ossRe ]
53.
Manso, T.; Lo es, M.; Rama, J.L.R.; Villa ino, R.-A.; Cal o, L.G.; Cas illo, A.; Celei o, M.; de Miguel, T. An ibac e ial Ac i i y
agains Clinical S ains o a Na u al Polyphenolic Ex ac om Alba iño Whi e G ape Ma c. Pha maceu icals 2023,16, 950.
[C ossRe ] [PubMed]
54.
Cheng, V.J.; Bekhi , A.E.-D.A.; McConnell, M.; M os, S.; Zhao, J. E ec o Ex ac ion Sol en , Was e F ac ion and G ape Va ie y on
he An imic obial and An ioxidan Ac i i ies o Ex ac s om Wine Residue om Cool Clima e. Food Chem. 2012,134, 474–482.
[C ossRe ]
55.
Ka alini´c, V.; Možina, S.S.; Sk oza, D.; Gene ali´c, I.; Ab amo iˇc, H.; Miloš, M.; Ljubenko , I.; Piske nik, S.; Pezo, I.; Te pinc, P.; e al.
Polyphenolic P o ile, An ioxidan P ope ies and An imic obial Ac i i y o G ape Skin Ex ac s o 14 Vi is ini e a Va ie ies G own
in Dalma ia (C oa ia). Food Chem. 2010,119, 715–723. [C ossRe ]
56.
Bayda , N.G.; Sagdic, O.; Ozkan, G.; Ce in, S. De e mina ion o An ibac e ial E ec s and To al Phenolic Con en s o G ape (Vi is
ini e a L.) Seed Ex ac s. In . J. Food Sci. Technol. 2006,41, 799–804. [C ossRe ]
57.
Se a, A.T.; Ma ias, A.A.; Nunes, A.V.M.; Lei ão, M.C.; B i o, D.; B onze, R.; Sil a, S.; Pi es, A.; C espo, M.T.; San Romão, M.V.;
e al. In Vi o E alua ion o Oli e- and G ape-Based Na u al Ex ac s as Po en ial P ese a i es o Food. Inno . Food Sci. Eme g.
Technol. 2008,9, 311–319. [C ossRe ]
58.
Sagdic, O.; Oz u k, I.; Kisi, O. Modeling An imic obial E ec o Di e en G ape Pomace and Ex ac s on S. au eus and E. coli in
Vege able Soup Using A i icial Neu al Ne wo k and Fuzzy Logic Sys em. Expe Sys . Appl. 2012,39, 6792–6798. [C ossRe ]
59.
Faisal, K.; Mosammad, S.S.; He i, K. An imic obial Ac i i ies o G ape (Vi is ini e a L.) Pomace Polyphenols as a Sou ce o
Na u ally Occu ing Bioac i e Componen s. A . J. Bio echnol. 2015,14, 2157–2161. [C ossRe ]
60.
Jayap akasha, G.K.; Sel i, T.; Saka iah, K.K. An ibac e ial and An ioxidan Ac i i ies o G ape (Vi is ini e a) Seed Ex ac s. Food
Res. In . 2003,36, 117–122. [C ossRe ]
61.
Pozzo, L.; G ande, T.; Ra aelli, A.; Longo, V.; Weidne , S.; Ama owicz, R.; Ka ama´c, M. Cha ac e iza ion o An ioxidan and
An imic obial Ac i i y and Phenolic Compound P o ile o Ex ac s om Seeds o Di e en Vi is Species. Molecules 2023,28, 4924.
[C ossRe ] [PubMed]
62.
Filocamo, A.; Bisignano, C.; Mandala i, G.; Na a a, M. In Vi o An imic obial Ac i i y and E ec on Bio ilm P oduc ion o a
Whi e G ape Juice (Vi is ini e a) Ex ac . E id. Based Complemen . Al e na . Med. 2015,2015, 1–5. [C ossRe ] [PubMed]
Foods 2025,14, 17 21 o 21
63.
Rado ano i´c, A.; A si´c, B.; Rado ano i´c, V.; Jo anˇciˇce i´c, B.; Nikoli´c, V. B oad-Spec um o An imic obial P ope ies o
Comme cial Wines om Di e en Vi is ini e a L. Va ie ies. Wo ld J. Mic obiol. Bio echnol. 2017,33, 18. [C ossRe ] [PubMed]
64.
Ma, Y.; Ding, S.; Fei, Y.; Liu, G.; Jang, H.; Fang, J. An imic obial Ac i i y o An hocyanins and Ca echins agains Foodbo ne
Pa hogens Esche ichia coli and Salmonella. Food Con ol 2019,106, 106712. [C ossRe ]
Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
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
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
