Resea ch A icle
Recei ed: 2 Janua y 2025 Re ised: 28 Ma ch 2025 Published online in Wiley Online Lib a y:
(wileyonlinelib a y.com) DOI 10.1002/js a.14316
The mal ex ac ion and cha ac e iza ion o
pec in om semi-solid by-p oduc s o he oli e
oil indus y
A ica Fe nandez-P io ,a,b
*
Luna Ba e a-Chamo o,a,b
El i a Ma quez-Pa adas,a,b Ca ol López-de-Dicas illo,c
Ma ia C. Millan-Lina es,dAl a o Villanue a-Lazodand
Se gio Mon se a -de la Paza,b
Abs ac
BACKGROUND: The alo iza ion o ag o-indus ial by-p oduc s is c ucial o p omo ing sus ainabili y and ci cula economy.
Oli e mill semi-solid by-p oduc s (OMSbP), also known as alpe ujo, con ain aluable bioac i e compounds, including pec in,
which can be ex ac ed and used in ood applica ions. Howe e , he s uc u al p ope ies an an ioxidan po en ial o hese pec-
in equi e u he cha ac e iza ion o assess hei po en ial as unc ional ing edien s.
RESULTS: In he p esen s udy, pec in was ex ac ed om OMSbP using a ci ic acid-based he mal ex ac ion p ocess. The yield
a ied depending on he oli e a ie y and ipeness deg ee, wi h highe ex ac ion e ficiency in mo e ma u e samples
(48.2 g kg
−1
). S uc u al analysis by a enua ed o al eflec ance/Fou ie ans o m-in a ed spec oscopy confi med a high
homogalac u onan con en (∼500 g kg
−1
) and a deg ee o me hyl es e ifica ion o app oxima ely 60%. The monosaccha ide
p ofile indica ed en ichmen in a abinose, hamnose, galac ose and mannose. An ioxidan ac i i y, assessed h ough DPPH
(i.e. 2,2-diphenyl-1-pic ylhyd azyl) adical sca enging, showed a s ong co ela ion wi h mannose con en ( =−0.9967). Addi-
ionally, pec in-based films demons a ed he mal s abili y compa able o comme cial pec in used in ood packaging.
CONCLUSION: This s udy highligh s he po en ial o OMSbP-de i ed pec in as a bio unc ional ing edien wi h an ioxidan ac i -
i y and he mal s abili y. The ex ac ion me hod ensu es a sus ainable app oach o u ilizing oli e indus y by-p oduc s. Fu u e
esea ch should explo e i s bioa ailabili y and applica ion in edible films and coa ings.
© 2025 The Au ho (s). Jou nal o he Science o Food and Ag icul u e published by John Wiley & Sons L d on behal o Socie y o
Chemical Indus y.
Keywo ds: oli e oil semi-solid by-p oduc s; Olea eu opaea; s uc u al cha ac e iza ion; an ioxidan ac i i y; ood packaging
INTRODUCTION
The inc easing en i onmen al challenges humani y aces oday
a e d i ing he de elopmen o mo e sus ainable p oduc ion
and consump ion sys ems. Biomass esou ces, as a esul o hei
abundance, ep esen a aluable oppo uni y o he c ea ion o
high- alue p oduc s.
1
In he oli e oil sec o , he wo-phase ex ac-
ion sys em gene a es a semi-solid by-p oduc known as oli e mill
semi-solid by-p oduc (OMSbP) o alpe ujo.
2-4
This by-p oduc ,
p ima ily composed o oli e peel, pulp, seeds and pi agmen s,
con ains aluable bioac i e compounds such as polyphenols
and ca bohyd a es, including cellulose, hemicellulose and pec-
in.
5
Since he 1990s, OMSbP has played a ole in educing he
en i onmen al impac o oli e oil ex ac ion by minimizing liquid
was e accumula ion. Howe e , i s high mois u e con en p esen s
challenges o e ficien managemen . Recen indus ial he mal
p e- ea men s ha e enhanced he ex ac ion o bioac i e
*Co espondence o: A Fe nandez-P io , Depa men o Medical Biochemis y,
Molecula Biology, and Immunology, School o Medicine, Uni e si y o Se ille,
A . Sanchez Pizjuan s/n, 41009 Se ille, Spain, E-mail: m e [email p o ec ed]
(Fe nandez-P io )
aDepa men o Medical Biochemis y, Molecula Biology and Immunology,
School o Medicine, Uni e si y o Se ille, Se ille, Spain
bIns i u o de Biomedicina de Se illa, IBiS/Hospi al Uni e si a io Vi gen del
Rocio/CSIC/Uni e sidad de Se illa, Se ille, Spain
cPackaging G oup, Ins i u o de Ag oquimica y Tecnologia de los Alimen os,
Spanish Na ional Resea ch Council (IATA-CSIC), Pa e na, Spain
dDepa men o Food & Heal h, Ins i u o de la G asa, Spanish Na ional
Resea ch Council (IG-CSIC), Se ille, Spain
© 2025 The Au ho (s). Jou nal o he Science o Food and Ag icul u e published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and
dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modifica ions o adap a ions a e made.
1
compounds, including hyd oxy y osol and a ious ca bohyd a e
ac ions. This p og ess ma ks a significan s ep owa d es ablish-
ing an OMSbP bio efine y, whe e op imizing ca bohyd a e ex ac-
ion and u iliza ion emains a key objec i e.
Pec in, a complex he e opolysaccha ide, is composed mainly o
homogalac u onan (HG) domains and hamnogalac u onan I
(RG-I) egions wi h a galac u onic acid (GalA) backbone. The GalA
uni s may be subs i u ed wi h me hyl g oups, as some o he C-6
ca boxyl g oups a e me hyl-es e ified. This es e ifica ion influ-
ences he s uc u al cha ac e is ics and physicochemical p ope -
ies o pec in, he eby de e mining i s unc ionali y in ood and
o he indus ial applica ions. Found in he p ima y cell wall
and middle lamella o plan issues, pec in con ibu es signifi-
can ly o plan s uc u e and in eg i y.
6
OMSbP-de i ed pec in
has exhibi ed no able biological ac i i ies, including an ioxidan ,
an ip oli e a i e and an i-inflamma o y e ec s, highligh ing i s
po en ial as a unc ional ing edien .
7-12
The inc easing demand o na u al ood addi i es is d i ing
in e es in sus ainable sou ces o pec in.
13
The p esen s udy
in es iga es he ex ac ion and cha ac e iza ion o pec in om
OMSbP using a ci ic acid-based he mal me hod, analyzing i s
s uc u al p ope ies, an ioxidan capaci y and po en ial applica-
ions in ood echnology.
MATERIALS AND METHODS
Raw ma e ial
OMSbP (i.e. alpe ujo), a semi-solid by-p oduc composed o oli e
peel, pulp, seeds, and pi agmen s, was ob ained om Almaza a
de la Subbe ica S.C.A. (Co doba, Spain). OMSbP samples we e
collec ed om wo di e en oli e a ie ies: A bequina and Hoji-
blanca du ing he ea ly ha es season o 2022–2023 (designa ed
as aeOMSbP and heOMSbP, espec i ely). Addi ionally, samples
we e aken om a mix u e o hese a ie ies a wo di e en ip-
ening s ages du ing he same ha es season: an ea ly-s age sam-
ple (meOMSbP) and a ully ma u e sample (mmOMSbP). Samples
we e collec ed di ec ly a he end o he ho izon al cen i uge in
he wo-phase ex ac ion sys em and s o ed a −20 °C un il u -
he p ocessing.
P epa a ion o cell wall ma e ial and pec in ex ac ion
OMSbP samples we e p ocessed ollowing he Rena d me hod
wi h modifica ions o ob ain ex ac ed pec in.
14
B iefly, 1 L o
800 g kg
−1
e hanol was added o 1 kg o esh OMSbP, and he
mix u e was incuba ed o e nigh unde con inuous shaking a
oom empe a u e. A e cen i uga ion a 15000 ×g o 20 min
(RC5C; So all, Mechelen, Belgium), he supe na an was dis-
ca ded, and he emaining alcohol-insoluble esidue (AIR) was
d ied a 40 °C o 24 h. Pec in ex ac ion was pe o med by sus-
pending 30 g o AIR in 300 mL o 30 g kg
−1
ci ic acid solu ion,
adjus ing he pH o 3.0, and s i ing a 95 °C o 95 min.
15
The mix-
u e was hen cen i uged a 3700 ×ga 4 °C o 15 min, and he
supe na an was ea ed wi h e hanol (2:1 / ) and s o ed a 4 °C
o 16 h o p ecipi a e pec in. A e an addi ional cen i uga ion
s ep a 3700 ×g o 30 min a 4 °C, he p ecipi a e was washed
h ee imes wi h anhyd ous e hanol and eeze-d ied.
Chemical cha ac e iza ion o ex ac ed pec in
Pec in yield was calcula ed as he a io o he d y ex ac ed
pec in o he o iginal AIR weigh , exp essed as g kg
−1
d y OMSbP.
The neu al suga con en (NSC) was de e mined using he
An h one me hod, as desc ibed by Dishe.
16
B iefly, 100 μLo
sample ( iplica e) was mixed wi h 200 μL o 0.2% (w/ ) an h one
in concen a ed sul u ic acid, hea ed a 100 °C o 5 min, and mea-
su ed a 630 nm in a mic opla e eade (iMa k; Bio-Rad, He cules,
CA, USA). Glucose was used as he s anda d (0.02–0.2 mg mL
−1
).
U onic acid con en was quan ified using he
m-hyd oxydiphenyl me hod, as desc ibed by Blumenk an z and
Asboe-Hansen.
17
Samples (200 μL, iplica e) was mixed wi h
1.2 mL o 0.0125 Msodium e abo a e in sul u ic acid, hea ed a
100 °C o 5 min, and eac ed wi h 20 μL o 0.15% m-
hyd oxybiphenyl in 0.5% NaOH. Abso bance was measu ed a
520 nm using a galac u onic acid s anda d. Glycosidic composi-
ion was analyzed by gas ch oma og aphy (GC, HP6890 Plus; Hew-
le Packa d, Palo Al o, CA, USA) a e he de i a iza ion in o aldi ol
ace a es, wi h ini ial ifluo oace ic acid hyd olysis using inosi ol
as he in e nal s anda d.
18
Quan ifica ion was pe o med using
s anda d solu ions o L- hamnose (Rha), L-a abinose (A a), D-
galac ose (Gal), D-glucose (Glc), D-mannose (Man), D-Fucose (Fuc)
and D-xylose (Xyl). The ch oma og aphic condi ions we e
desc ibed by Lama-Muñoz e al.
19
The o al phenolic con en
(TPC) was de e mined using he Folin–Ciocal eu spec opho o-
me ic me hod wi h sligh modifica ions.
20
B iefly, 20 μL o each
sample was mixed wi h 100 μL o Folin–Ciocal eu eagen and
80 μL o a 0.70 Msodium ca bona e solu ion in wa e . The mix u e
was s i ed and incuba ed o 10 min o allow colou de elop-
men . Abso bance was measu ed a 655 nm. A calib a ion cu e
was es ablished using gallic acid as he s anda d. The p o ein con-
en o he samples was de e mined using a ni ogen elemen a y
analyze (LECO, S Joseph, MI, USA), based on in e nal combus ion
a 1000 °C ollowed by he mal conduc i i y de ec ion. The p o-
ein con en (g kg
−1
o d y ex ac ) was calcula ed using he con-
e sion ac o 6.25, in acco dance wi h he ISO me hod.
21
DPPH adical sca enging capaci y
Lyophilized pec in samples we e dissol ed in wa e a
10 mg mL
−1
and fil e ed h ough a 0.45-μm nylon fil e . The an i-
oxidan ac i i y was assessed using he 2,2-diphenyl-
1-pic ylhyd azyl (DPPH) adical sca enging me hod,
22
whe e
5μL o each ex ac was added o 195 μL o 3.8 mg pe 50 mL
DPPH solu ion in me hanol, incuba ed o 30 min a oom empe -
a u e, and measu ed a 490 nm. The esul s we e exp essed as
EC
50
alues (mg mL
−1
), ep esen ing he concen a ion equi ed
o educe adical ac i i y by 50%.
Deg ee o me hyl es e ifica ion by ATR/FT-IR and
1
H NMR
spec oscopy
ATR/FT-IR spec oscopy was conduc ed o de e mine he deg ee
o me hyl es e ifica ion o he ex ac ed pec in. Th ee eplica es
o each sample we e analyzed, and peak in ensi y was used o
measu emen . This me hod p o ides a eliable quali a i e and
semi-quan i a i e assessmen o pec in es e ifica ion, as epo ed
by Muñoz-Almag o e al.
23
The samples we e g ound in an aga e
mo a o p ope homogeniza ion be o e analysis. Spec a we e
eco ded using a FT/IR-4700 spec opho ome e (Jasco, Tokyo,
Japan) ac oss he ange 400–4000 cm
−1
. Deg ee o me hyl es e -
ifica ion (DM) was calcula ed using he a io o he abso p ion
bands a 1730 and 1620 cm
−1
:DM=A
1730
/(A
1730
+A
1620
).
1D
1
H NMR spec oscopy o meOMSbP and mmOMSbP sam-
ples was also ca ied ou o de e mine hei s uc u al cha ac e is-
ics. Each pec in samples was dissol ed in D
2
O, and he
1
H NMR
spec a we e eco ded a 25 °C using an A ance NEO 500 MHz
spec ome e (B uke , Bille ica, MA, USA) equipped wi h a Onebay
www.soci.o g A Fe nandez-P io e al.
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Jou nal o he Science o Food and Ag icul u e published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y.
J Sci Food Ag ic 2025
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console ea u ing h ee adio equency channels and a BBFO
5 mm dual esonance Z-g adien p obe.
Su ace mo phology analysis by scanning elec on
mic oscopy (SEM)
Lyophilized pec in samples we e analyzed by SEM o examine su -
ace mo phology. The samples we e spu e -coa ed wi h a hin
laye o gold–palladium unde acuum using an EM ACE600
ins umen (Leica Mic osys ems, We zla , Ge many) o imp o e
conduc i i y. Imaging was pe o med wi h a Zeiss C ossbeam
550 scanning elec on mic oscope (Zeiss, Mad id, Spain) a an
accele a ing ol age o 2.00 kV and di e en magnifica ions.
24
The mog a ime ic analysis o pec in films
Pec in films om meOMSbP and mmOMSbP samples we e
ob ained ia he cas ing echnique, whe e 20 g kg
−1
pec in dis-
pe sions in dis illed wa e we e pou ed in o Pe i dishes and d ied
a 37 °C o e nigh . The esul ing films ( hickness: 45–55 μm) we e
analyzed o he mal s abili y using he mog a ime ic analysis
(TGA) Q-500 ins umen (TA Ins umen s, New Cas le, DE, USA).
App oxima ely 5–8 mg o each sample was placed in alumina
capsules and subjec ed o a empe a u e ange o 30–800 °Ca
a hea ing a e o 10 °C min
−1
unde ni ogen a mosphe e
(50 mL min
−1
). The maximum deg ada ion empe a u es (T
d
)
was de e mined.
S a is ical analysis
Pea son's co ela ion coe ficien s we e calcula ed o den i y sig-
nifican ela ionships be ween pec in yield, chemical compo-
nen s, molecula ea u es and an ioxidan ac i i y. Co ela ion
hea maps we e gene a ed using he ggplo 2 package
(Wickham H, ggplo 2: Elegan G aphics o Da a Analysis.
Sp inge -Ve -lag, New Yo k (2016)). ( 3.4.2). h ps://link.sp inge .
com/book/10.1007/978-3-319-24277-4. Linea eg ession models
we e used o e alua e he p edic i e powe o specific a iables
o an ioxidan ac i i y, wi h he coe ficien o de e mina ion (R
2
)
used o assess model fi . All alues a e exp essed as he mean
±SD. The numbe o eplica es used o each measu emen is
explici ly s a ed o ensu e anspa ency and ep oducibili y. S a is-
ical analyses we e pe o med using P ism, e sion 8.01
(G aphPad So wa e Inc., San Diego, CA, USA). Significance
be ween sample g oups was de e mined using a pai ed - es
o equal a iances. P<0.05 was conside ed s a is ically
significan .
RESULTS AND DISCUSSION
Chemical cha ac e iza ion o ex ac ed pec in om OMSbP
Ex ac ed pec in om OMSbP using ci ic acid a 95 °C om di e -
en oli e a ie ies (i.e., aeOMSbP e sus heOMSbP) and ipeness
s ages (i.e., meOMSbP e sus mmOMSbP) we e ully cha ac e ized
o i s chemical composi ion, as shown in Table 1. F eezing he
Table 1. Yield, chemical composi ion and molecula ea u es o OMSbP pec in om wo di e en oli e a ie ies (aeOMSbP and heOMSbP) and wo
di e en ipeness s ages (meOMSbP and mmOMSbP)
Ex ac ed pec in om oli e mill semi-solid by-p oduc (OMSbP)
aeOMSbP heOMSbP P- alue meOMSbP mmOMSbP P- alue
Yield (g kg
−1
)
a,b
24.80 ±1.90 34.90 ±2.90 0.007 40.00 ±3.10 48.20 ±3.90 0.046
P o eins
b
27.40 ±1.90 33.10 ±0.40 0.007 18.50 ±1.50 12.20 ±0.10 0.002
Phenols
b
7.80 ±0.10 6.40 ±0.60 0.016 07.20 ±0.20 5.30 ±0.10 <0.001
To al ca bohyd a es
b
715.40 ±9.70 570.10 ±7.10 <0.001 570.70 ±20.60 504.90 ±0.90 0.005
Monosaccha ide composi ion
c
Galac u onic acid (GalA) 62.48 ±0.17 52.20 ±0.07 <0.001 51.63 ±0.29 50.03 ±0.16 0.001
Rhamnose (Rha) 1.59 ±0.10 1.71 ±0.04 0.126 1.95 ±0.24 0.69 ±0.03 0.001
Fucose (Fuc) 0.34 ±0.02 0.65 ±0.07 0.002 0.58 ±0.06 0.54 ±0.18 0.734
A abinose (A a) 7.55 ±0.48 9.05 ±1.41 0.156 10.30 ±0.28 13.58 ±3.04 0.136
Xylose (Xyl) 4.70 ±0.37 4.37 ±1.03 0.629 4.75 ±0.83 3.68 ±0.58 0.141
Mannose (Man) 2.81 ±0.14 3.89 ±0.34 0.007 4.24 ±0.24 4.79 ±1.49 0.562
Galac ose (Gal) 10.71 ±0.96 9.81 ±1.62 0.454 12.97 ±2.10 13.04 ±1.25 0.963
Glucose (Glc) 9.82 ±0.67 10.62 ±1.67 0.484 13.58 ±1.07 13.65 ±1.20 0.943
Mola a io
d
Homogalac u onan (HG) 60.89 ±0.10 50.49 ±0.04 <0.001 49.68 ±0.24 49.34 ±0.03 0.072
Rhamnogalac u onan I (RGI) 21.45 ±1.39 22.27 ±3.15 0.701 27.18 ±2.84 27.99 ±4.31 0.799
R1, linea i y 1.80 ±0.11 1.47 ±0.24 0.096 1.18 ±0.12 1.11 ±0.11 0.498
R2, RG con ibu ion 0.03 ±0.00 0.03 ±0.00 >0.999 0.04 ±0.00 0.01 ±0.00 <0.001
R3, leng h o side chains 11.48 ±0.17 11.01 ±1.57 0.633 11.93 ±0.40 38.59 ±5.97 0.002
DM (%)
e
58.67 ±2.08 54.67 ±1.53 0.055 61.33 ±1.53 59.00 ±0.33 0.061
Da a a e exp essed as he mean ±SD (n=3).
a
Based on AIR.
b
Exp essed as g kg
−1
o d y ex ac .
c
Rela i e %.
d
Mola a ios: exhibi ing he p ima y s uc u al p ope ies o pec in molecules; HG =GalA –Rha, homogalac u onan; RGI =2(Rha) + A a + Gal,
R1 =GalA/(Fuc + Rha + Glu + A a + Gal+Xyl), he linea i y o pec in; R2 =Rha/GalA, he con ibu ion o RG o pec in popula ion; R3 =(Gal+A a)/
Rha, he leng h o side chains a ached o RGI.
e
Deg ee o me hyl es e ifica ion (DM) de e mined by ATR/FT- IR.
Cha ac e iza ion o pec in om by-p oduc s o he oli e oil indus y www.soci.o g
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aw ma e ial be o e ci ic acid ex ac ion was c ucial o p e en
pec inoly ic enzyme ac i i y. The pec in ex ac ion yield anged
om 24.8 g kg
−1
(aeOMSbP) o 48.2 g kg
−1
(mmOMSbP). A signi -
ican ly highe yield was ob ained om mmOMSbP (48.2 g kg
−1
)
compa ed o meOMSbP (40.0 g kg
−1
), ep esen ing a 20%
dec ease in ex ac ion e ficiency a an ea lie ipeness s age. How-
e e , hese yield we e lowe han hose epo ed o pec in om
o ange peel (app oxima ely 200 g kg
−1
), which is he main
ag oindus ial was e used o he comme cial p oduc ion o
pec in,
25
pomeg ana e peel (110 g kg
−1
),
26
young apples
(80 g kg
−1
)
27
and suga bee (160 g kg
−1
).
28
The p o ein con en
o he ex ac ed pec in was wi hin he ange epo ed o com-
me cial apple pec in (app oxima ely 30 g kg
−1
)
29
and highe han
ha ound in co ee pulp (app oxima ely 10 g kg
−1
).
30
Addi ion-
ally, he phenolic con en o OMSbP pec in was highe han ha
o ci us pec in (1.5–1.8 g kg
−1
), bu he simila o alues epo ed
o apple pec in (up o 6 g kg
−1
).
31
To assess he ca bohyd a e composi ion and deg ee o b anch-
ing, he monosaccha ide con en was de e mined using gas ch o-
ma og aphy. Significan di e ences we e obse ed be ween
di e en ipeness s ages, wi h ea ly-ha es ed eOMSbP samples
showing a highe con en o bo h u onic acids and neu al suga s.
This esul ed in a p e e en ial loss o o al ca bohyd a es om he
polyme ic cell wall in la e-ha es ed samples, pa icula ly Rha and
Xyl, which con ibu e o a abinoxylan s uc u es in he cell wall. As
shown in Table 1, OMSbP samples con ained se en p ima y
monosaccha ides: Rha, A a, Gal, Glu, Xyl, Fuc and Man, in a ying
p opo ions. GalA was he dominan monosaccha ide, wi h le els
anging om 500.3 g kg
−1
(mmOMSbP) o 624.8 g kg
−1
(aeOMSbP), confi ming i s high HG con en .
The GalA a io exceeded 50% in all analyzed pec in samples,
which is highe han he 35% epo ed by p e ious s udies.
32
Rha, Gal and A a oge he accoun ed o o e hal o he o al neu-
al suga s, sugges ing a high con ibu ion o RG-I o he pec in
s uc u e. The HG con en (49.34–60.43%) was g ea e han ha
o RG-I (21.45–27.99%), indica ing ha he linea backbone s uc-
u e p edomina ed o e side chains. Pec in con aining mo e han
74% GalA has been epo ed o influence gu mic obio a compo-
si ion and i s mic obial me aboli es,
33
pa icula ly h ough he
ac i i y o RG-I, which plays a ole in modula ing immune
esponse.
34
In he p esen s udy, he R1 alues o OMSbP pec in
samples (1.11–1.80) we e ound lowe han hose epo ed o
g ape ui peel pec in (2.52)
35
and pis achio g een hull pec in
(1.97),
36
indica ing ha OMSbP pec in has a highe deg ee o
b anching. Howe e , he R1 alue o aeOMSbP pec in (1.80) was
highe han o swee lemon peel (1.53),
37
highligh ing a ia ions
in s uc u al complexi y among di e en sou ces. The R2 and R3
alues u he suppo ed ha he majo con ibu ions o pec in
s uc u e we e om RG-I domains, wi h mmOMSbP exhibi ing
he highes deg ee o b aching (38.59) compa ed o o he sam-
ples (app oxima ely 11).
To be e unde s and he ela ionship be ween he s uc u al
componen s and pec in p ope ies, Pea son co ela ion coe fi-
cien s we e calcula ed (Fig. 1). S ong co ela ions (>0.95) we e
obse ed be ween mul iple a iables, including yield and o al
ca bohyd a es (−0.96), yield and a abinose (0.97), yield and man-
nose (0.99), yield and R1 (−0.97), and yield and DPPH (−0.98).
Addi ionally, significan co ela ions we e ound be ween p o ein
con en and galac ose (−0.96), phenols and xylose (0.95), o al
ca bohyd a es and galac u onic acid (0.98), o al ca bohyd a e
and mannose (−0.98), o al ca bohyd a e and glucose (0.96), o al
ca bohyd a e and DPPH (0.99), hamnose and R2 (0.99), hamnose
and R3 (−0.96), xylose and R2 (0.95), mannose and R1 (−0.97), glu-
cose and hamnogalac u onan I (0.99), glucose and R1 (−0.96),
homogalac u onan and DPPH (0.96), and R1 and DPPH (0.97).
The s uc u al complexi y and unc ionali y o pec in a e closely
ela ed.
38
Recen s udies ha e modeled he co ela ion be ween
pec in s uc u e and i s echno- unc ional p ope ies. Xie e al.
39
es ablished ela ionships be ween pec in cha ac e is ics and
unc ional a ibu es using Pea son co ela ion coe ficien s,
highligh ing p ope ies such as heology, chewiness, adhesion,
eeze– haw s abili y and ha dness. These esul s ein o ced he
no ion ha pec in con o ma ion, monosaccha ide composi ion,
and molecula weigh a e he p ima y ac o s influencing i s phys-
icochemical beha io . Fu he s udies ha e demons a ed he
impac o pec in s uc u e and i s DM on gela iniza ion p ope -
ies.
40
A s udy by Luo e al.
41
alida ed a mul iple eg ession
model demons a ing how a ia ions in GalA con en , molecula
weigh , DM and amida ion deg ee influence gela iniza ion cha -
ac e is ics such as peak and alley iscosi y, as well as se back
and b eakdown alues. These findings unde sco e he signifi-
cance o s uc u al pa ame e s in de e mining pec in unc ional-
i y, u he suppo ing he complex in e play be ween chemical
composi ion and echno- unc ional a ibu es.
An ioxidan ac i i y and co ela ion wi h s uc u al
componen s
Figu e 2A p esen s he an ioxidan ac i i y o he ex ac ed pec in,
measu ed h ough DPPH adical sca enging. The EC
50
alues indi-
ca e ha meOMSbP exhibi ed a 6% highe ee adical sca enging
abili y compa ed o mmOMSbP. This inc ease is di ec ly p opo -
ional o he 7% highe phenolic con en ound in meOMSbP, con-
fi ming ha he an ioxidan ac i i y o hese ex ac s is p ima ily
a ibu ed o hei phenolic compounds a he han he pec in
s uc u e i sel . Phenols eadily bind o suga s, ans e ing hei
an ioxidan p ope ies o hese molecules.
7
The EC
50
alues
ob ained o he ex ac ed pec in de i ed om oli e pomace
by-p oduc s we e lowe han hose epo ed o Spi ulina sp., a
mic oalga known o i s no able an ioxidan capaci y. The mos
e ec i e concen a ion was 2.5 mg mL
−1
, and his ac i i y has
been mainly a ibu ed o he p esence o bioac i e compounds
such as phenolic acids, phycocyanins and ocophe ols.
42
Suga s can also enhance he bioa ailabili y o phenols, c ea ing
a syne gis ic unc ional ela ionship be ween he wo compound
g oups.
43
This in e ac ion is o high nu i ional in e es because i
makes his ex ac ed pec in om OMSbP mo e aluable o he
o mula ion o heal h-o ien ed ood p oduc s compa ed o com-
me cial pec in ha lack phenolic associa ions.
To u he in es iga e whe he specific cha ac e is ics o he
OMSbP-de i ed pec in, such as yield, chemical composi ion and
molecula s uc u e, influence i s an ioxidan capaci y, Pea son
co ela ion coe ficien s we e calcula ed. Figu e 2B demons a es
ha DPPH exhibi ed s ong co ela ions (>±0.5) wi h mul iple
a iables, bo h posi i e and nega i e. The s onges co ela ion
was ound be ween DPPH and mannose con en ( =−0.9967).
To assess he significance o his ela ionship, a simple linea
eg ession model was applied, yielding he equa ion
y=2.3805–0.1991x, whe e ‘y’ ep esen s an ioxidan ac i i y and
‘x’ ep esen s mannose concen a ion. The model confi med ha
mannose had a significan impac (P<0.05) on DPPH, explaining
99% o he a iance in he da a (R
2
=0.9901). This sugges ha
inc easing mannose con en in ex ac ed pec in could enhance
hei an ioxidan p ope ies. P e ious s udies ha e shown ha
he an ioxidan ac i i y o oligogalac u onides is closely ela ed
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o hei s uc u al a ibu es, including monosaccha ide composi-
ions and molecula weigh .
44-47
The an ioxidan mechanisms o
pec in a e in ica e, and u he esea ch is needed o explo e
he ela ionship be ween polysaccha ide s uc u e and an ioxi-
dan unc ion in mo e dep h.
48
Deg ee o me hyl es e ifica ion o ex ac ed pec in om
OMSbP
The ATR/FT-IR spec a we e analyzed o de e mine whe he he
lyophilized OMSbP pec in ex ac ed om di e en oli e a ie ies
and ipeness s ages a ec ed unc ional g oups and bonding con-
figu a ions (Fig. 3).
O e all, sligh a ia ions we e obse ed in he spec al p ofiles
o ex ac ed pec in om he A bequina a ie y (aeOMSbP)
(Fig. 3A, g een line) and he Hojiblanca a ie y (heOMSbP)
(Fig. 3A, o ange line). Addi ionally, di e ences we e no ed
be ween ea ly-season (meOMSbP) (Fig. 3B, pink line) and la e-
season (mmOMSbP) (Fig. 3B, blue line) pec in samples. The b oad
abso p ion band a 3400 cm
−1
, co esponding o O H s e ching
ib a ion o hyd oxyl g oups, appea ed sha pe in mmOMSbP
compa ed o meOMSbP, likely because o a lowe phenolic con-
en (Table 1).
49
No significan di e ences we e de ec ed be ween
he wo oli e a ie ies, aligning wi h p ecious findings, and po en-
ially a ibu ed o he high dissocia ion cons an o he ci ic acid
used du ing ex ac ion.
50
The abso p ion band a 2925 cm
−1
a ibu ed o C H(—CH,
—CH
2
,—CH
3
), emained consis en ac oss all pec in samples,
sugges ing ha ipeness did no influence hese unc ional
g oups. Howe e , aeOMSbP exhibi ed a highe in ensi y in his
egion compa ed o heOMSbP. No ably, ex ac ed pec in om
la e-season (mmOMSbP) and Hojiblanca oli es (heOMSbP) dis-
played a dec ease in he band nea 1730 cm
−1
, co esponding
o C O s e ching o he me hyles e ified ca boxyl g oups
(COO-R), whe eas he e was an inc ease a 1620 cm
−1
, associa ed
wi h ca boxyla e ion (COO
−
) s e ching, indica ing sligh a ia-
ions in he deg ee o DM, as eflec ed in Table 1. The abso p ion
bands a 1146 cm
−1
and 1405 cm
−1
, associa ed wi h C—OH
s e ching in side g oups and C—O—C glycosidic bond ib a ion,
espec i ely, emained unchanged ac oss all samples, indica ing
consis ency in glycosidic linkages.
51,52
Simila ly, cha ac e is ic
bands a 1250 cm
−1
,(—CH
3
CO s e ching), 957 cm
−1
(C O bend-
ing), 912 cm
−1
( ocking mode o CH
3
) and 820 cm
−1
(—CCH and
—COH bending a he C-6 posi ion) we e main ained, ega dless
o ipeness s age o oli e a ie y, ein o cing he s uc u al s abil-
i y o ex ac ed pec in.
1
H NMR analysis
The
1
H NMR spec a (Fig. 4) o he ex ac ed pec in om
meOMSbP and mmOMSbP we e acqui ed a 500 MHz o u he
Figu e 1. Pea son co ela ion coe ficien hea map showing ela ionships among s uc u al, con o ma ional cha ac e is ics and an ioxidan capaci y o
pec in. The colo g adien ep esen s he Pea son co ela ion coe ficien , wi h alues displayed wi hin each squa e.
Cha ac e iza ion o pec in om by-p oduc s o he oli e oil indus y www.soci.o g
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co obo a e and cla i y hei s uc u es ea u es. Spec a we e
eco ded a 60 °C using samples dissol ed in D
2
O, aiming o
educe sample iscosi y. Measu ing a his empe a u e also had
he added benefi o shi ing he esidual wa e signal o app ox-
ima ely 4.4 ppm, a oiding o e lap wi h ele an pec in signals.
The mos ele an signals appea in he 3.0–5.5 ppm egion, co -
esponding o he skele al and anome ic p o ons o he
monosaccha ides. Pec in samples wi h a high deg ee o me hyla-
ion, such as hose analyzed, ypically display anome ic p o on
peaks a ound 5 ppm. In pa icula , anome ic H-1 and H-5
(COO
−
) p o ons a e obse ed a ⊐5.3 ppm and ⊐4.8 ppm, espec-
i ely, cha ac e is ics o GalA esidues linked ia ⊍-1,4 glycosidic
bonds. Addi ionally, a peak a 4.6 ppm co esponds o H-4 p o-
ons, whe eas signals a app oxima ely 4.2 and 4.1 ppm a e
Figu e 2. (A) DPPH adical sca enging ac i i y (EC
50
) o pec in samples. Values (mg mL
−1
) a e exp essed as he mean ±SD (n=3). S a is ical signi -
icance be ween a ie ies and ipeness s ages is indica ed by P- alue. (B) Co ela ion analysis be ween chemical composi ion and an ioxidan capaci y
(EC
50
,mgmL
−1
). The ba leng h ep esen s he Pea son co ela ion coe ficien , wi h alues displayed wi h he ba s.
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Figu e 3. Fou ie ans o m-in a ed spec oscopy (ATR/FT-IR) spec a o lyophilized OMSbP pec in ex ac ed om (A) di e en oli e a ie ies and
(B) di e en ipeness s ages. A bequina a ie y (aeOMSbP, g een line), hojiblanca a ie y (heOMSbP, o ange line), ea ly-s age ipeness (meOMSbP, pink
line) and ma u e-s age ipeness (mmOMSbP, blue line).
Figu e 4. 1D
1
HNMR spec a o ex ac ed pec in om meOMSbP and mmOMSbP eco ded a 500 MHz in D₂Oa 60°C.
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a ibu ed o H-3 and H-2 p o ons, espec i ely, in ag eemen wi h
a p e ious s udy.
53
The egion be ween 3.0 o 4 ppm shows o e -
lapping signals assigned o galac ose, a abinose and hamnose
ing p o ons, sugges ing he p esence o a abinogalac an s uc-
u es o pec ic side chains. Mo eo e , signals a ound 1.0–1.5 ppm
a e likely associa ed wi h he me hyl g oups o hamnose, indica-
i e o b anched egions wi hin he pec in s uc u e. No ably, a
se ies o double o double s o double s (ddd) signals obse ed
be ween ⊐2.7 and ⊐2.9 ppm may be ela ed o he binding o
pec in o he ci ic acid used du ing ex ac ion. These signals a e
a ibu ed o me hylene p o ons o ci ic acid, as p e iously sug-
ges ed by Ke mani e al.
54
This spec al p ofile confi ms he com-
plex composi ion o he ex ac ed pec in, which comp ise a GalA
backbone wi h po en ial subs i u ions by neu al suga .
Su ace mo phology analysis by SEM
The su ace mo phology o lyophilized OMSbP pec in ex ac ed
om di e en oli e a ie ies (Fig. 5A–D) and a di e en ipeness
s ages (Fig. 5E–H) was analyzed using SEM (Fig. 5). The SEM
images e ealed s uc u al di e ences be ween pec in samples.
In he eOMSbP samples, deg ada ion in o smalle agmen s was
obse ed a e he ha es season, leading o a educ ion in he
pa icle size unde iden ical imaging condi ions (Mag =50×and
AV =2.0 kV/Mag =250×and AV =2.0 kV). Ex ac ed pec in om
aeOMSbP exhibi ed a smoo h su ace, likely because o i s highe
HG con en and ewe s uc u al dis up ions. By con as ,
mOMSbP pec in displayed a dis inc s uc u e wi h isible long
c acks on he su ace. This he e ogeneous a chi ec u e, cha ac e -
ized by ough, compac and flaky s uc u es, may be a ibu ed o
a lowe con en o acidic suga side chains, which influences he
o ganiza ion and mechanical p ope ies o he ex ac ed pec in.
23
The mal s abili y o OMSbP pec in-based films
TGA and i s de i a i e (DTGA) cu es (Fig. 6A,B) p o ide c i ical
insigh s in o he he mal s abili y and decomposi ion beha io
o pec in-based films de i ed om OMSbP, specifically om di -
e en ipeness s ages. Unde s anding hese he mal ansi ions
is essen ial no only o e alua ing p ocessabili y unde hea , bu
also o p edic ing hei beha io in end-use applica ions such as
ood packaging, edible films o con olled- elease sys ems.
As shown in Fig. 6A, bo h pec in samples exhibi ed a simila he -
mal deg ada ion p ofile, wi h mino di e ences mo e e iden in
he DTGA cu es (Fig. 6B). The deg ada ion ollowed a ypical
h ee-s age pa e n, cha ac e is ic o polysaccha ide-based
ma e ials.
55,56
The ini ial mass loss, obse ed a ound 93 °C, co -
esponded o he e apo a ion o ee and bound mois u e,
eflec ing he hyg oscopic na u e o pec in and he p esence o
hyd oxyl g oups capable o o ming hyd ogen bonds wi h
wa e .
35
This s ep is pa icula ly impo an in edible films
because mois u e e en ion can a ec mechanical in eg i y and
shel -li e pe o mance. The second deg ada ion s age, occu ing
a app oxima ely 233 °C, was associa ed wi h he depolyme iza-
ion and b eakdown o he HG backbone, which is mainly com-
posed o GalA uni s. Addi ional decomposi ion p ocessed we e
ela ed o he clea age o glycosidic linkages and he deca box-
yla ion o GalA. The mal esis ance in his phase may be influ-
enced by he deg ee o es e ifica ion, molecula weigh and
p esence o impu i ies o esidual phenolic compounds co-
ex ac ed om he oli e by-p oduc ma ix.
57,58
A hi d majo
decomposi ion peak, obse ed a ound 443 °C, likely co e-
sponds o he b eakdown o mo e he mally s able ac ions
and he oxida i e deg ada ion o ca bonaceous esidues. The
p esence o a mino shoulde in he DTGA cu es sugges s pa -
ial ca boniza ion and esidual cha o ma ion, po en ially as a
esul o lignocellulosic agmen s o phenolic esidues com-
monly e ained in ex ac ed pec in om ag o-indus ial esi-
dues.
58
These findings indica e ha OMSbP pec in exhibi
he mal s abili y compa able o ha o comme cial pec in, sup-
po ing i s po en ial use in he mally p ocessed applica ions
such as low- empe a u e d ying, ex usion o lamina ion.
59
CONCLUSIONS
The p esen s udy highligh s he po en ial o ex ac ed pec in
om OMSbP as a unc ional and sus ainable ing edien o ood
applica ions. Pec in ob ained om ea ly-s age ipeness OMSbP
samples (eOMSbP) exhibi ed a highe GalA con en and deg ee
o me hyla ion, along wi h dis inc monosaccha ide composi ion
di e ences. Despi e chemical a ia ions among ex ac ed pec in
om di e en oli e a ie ies and ipeness s ages, hey displayed
significan an ioxidan p ope ies, likely a ibu ed o hei pheno-
lic con en and a s ong co ela ion wi h mannose concen a ion.
The he mal s abili y o OMSbP pec in-based films was compa a-
ble o ha o comme cial pec in, mee ing key heological
Figu e 5. Su ace mo phology o lyophilized OMSbP pec in ex ac ed om di e en oli e a ie ies and di e en ipeness s ages. (A, B) A bequina a ie y
(aeOMSbP), (C, D) Hojiblanca a ie y (heOMSbP), (E, F) ea ly-s age ipeness (meOMSbP) and (G, H) ma u e-s age ipeness (mmOMSbP), analyzed by SEM a
wo magnifica ions (Mag.). Uppe SEM images we e aken a Mag =50x and AV =2.0 kV (scale ba =100 μm), while lowe SEM images we e aken a
Mag =250x and AV =2.0 kV (scale ba =30 μm).
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p ope ies equi ed in he ood indus y. The applica ion o he -
mal ea men , simila o hose being inc easingly adop ed by
oli e oil pomace p ocesso s, combined wi h ci ic acid ex ac ion,
enables he p oduc ion o bioac i e pec in wi h film- o ming and
hea - esis an p ope ies. These cha ac e is ics suppo hei
po en ial use in ood p ese a ion and o mula ion. The findings
o he p esen s udy confi m ha he mal p e- ea men s a e an
e ec i e s a egy o enhancing he ex ac ion o bioac i e com-
pounds om oli e pomace, con ibu ing o he ull alo iza ion
o his by-p oduc . The pec in ob ained demons a ed s uc u al
in eg i y, unc ional s abili y and an ioxidan ac i i y, ein o cing
i s sui abili y o inco po a ion in o ood p oduc s a he same
ime as p omo ing sus ainabili y in he oli e oil indus y. Fu u e
esea ch should ocus on op imizing ex ac ion condi ions o u -
he enhance pec in yield and bioac i i y. Addi ionally, in es iga -
ing he bioa ailabili y and heal h e ec s o OMSbP-de i ed pec in
in unc ional ood applica ions could expand i s po en ial in nu a-
ceu icals and clean-label o mula ions.
AUTHOR CONTRIBUTIONS
SM-dlP was esponsible o s udy concep ualiza ion. AF-P, LB-C,
CL-dD and AV-L we e esponsible o he me hodology. AF-P,
EM-P and SM-dlP we e esponsible o o mal analysis.
AF-P, CMM-L and SM-dlP we e esponsible o in es iga ions.
AF-P and SM-dlP we e esponsible o esou ces. AF-P and
SM-dlP we e esponsible o w i ing he o iginal d a . AF-P
and SM-dlP we e esponsible o e iewing and edi ing. AF-P
and SM-dlP we e esponsible o supe ision. SM-dlP was
esponsible o unding acquisi ion. All au ho s ha e ead and
app o ed he final e sion o he manusc ip submi ed o
publica ion.
FUNDING
This s udy was suppo ed by he TED2021-130521A-I00 esea ch
p ojec (Minis y o Science and Inno a ion, Go e nmen o
Spain) in o he Reco e y, T ans o ma ion, and Resilience Plan
unding by Nex Gene a ionEU.
ACKNOWLEDGEMENTS
A ica Fe nandez-P io has he benefi o a Juan de la Cie a pos -
doc o al ellowship FJC2021-047485-I om he Spanish Minis y
o Science and Inno a ion. Luna Ba e a-Chamo o has he ben-
efi o a PIF doc o al ellowship suppo ed by he VII P og am o
Inne Ini ia i e o Resea ch and T ans e o Uni e si y o Se ille
(VII-PPIT-US). El i a Ma quez-Pa adas has he benefi o a FPU
doc o al ellowship (FPU22/01097) om he Spanish Minis y o
Science and Inno a ion. C. López de Dicas illo acknowledges
he awa d o he Spanish go e nmen MCIU/AEI o he IATA-CSIC
as Cen e o Excellence Acc edi a ion Se e o Ochoa (CEX2021-
001189- S/MCIU/AEI/10.13039/501100011033).
CONFLICTS OF INTEREST
The au ho s decla e ha hey ha e no conflic s o in e es .
DATA AVAILABILITY
Da a a e a ailable om he co esponding au ho upon eason-
able eques .
REFERENCES
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Figu e 6. (A) The mog a ime ic analysis (TGA) cu es and (B) hei co esponding de i a i e TGA (DTGA) o OMSbP pec in-based films ex ac ed om
ea ly-s age ipeness (meOMSbP, pink line) and ma u e-s age ipeness (mmOMSbP, blue line).
Cha ac e iza ion o pec in om by-p oduc s o he oli e oil indus y www.soci.o g
J Sci Food Ag ic 2025 © 2025 The Au ho (s).
Jou nal o he Science o Food and Ag icul u e published by John Wiley & Sons L d on behal o Socie y o Chemical Indus y.
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