molecules
A icle
Op imiza ion o Ul asound Assis ed Ex ac ion o Bioac i e
Compounds om Apple Pomace
I zia Egüés1,2, Fabio He nandez-Ramos 2, I án Ri illa 1,3 and Jalel Labidi 1,2,*
Ci a ion: Egüés, I.;
He nandez-Ramos, F.; Ri illa, I.;
Labidi, J. Op imiza ion o Ul asound
Assis ed Ex ac ion o Bioac i e
Compounds om Apple Pomace.
Molecules 2021,26, 3783. h ps://
doi.o g/10.3390/molecules26133783
Academic Edi o s: F ancesco Cacciola
and Pio Paweł Wieczo ek
Recei ed: 2 May 2021
Accep ed: 18 June 2021
Published: 22 June 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 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/).
1
Ma+D-Was e Reco e y Enginee ing, Edi icio Joxe Ma i Ko a—A da Tolosa 72, 20018 Donos ia-San Sebas ián,
Spain; i zia [email p o ec ed] (I.E.); [email p o ec ed] (I.R.)
2
Bio e ine y P ocess Resea ch G oup Chemical and En i onmen al Enginee ing Depa men , Uni e si y o he
Basque Coun y (UPV/EHU), Plaza Eu opa, 1, 20018 Donos ia-San Sebas ian, Spain;
[email p o ec ed]
3IKERBASQUE, Basque Founda ion o Science, 48009 Bilbao, Spain
*Co espondence: [email p o ec ed]
Abs ac :
In he p esen wo k, he op imiza ion o he ex ac ion o an ioxidan compounds om
apple pomace using ul asound echnology as an en i onmen ally iendly and in ensi ica ion p ocess
was de eloped. Di e en sonica ion powe s, ex ac ion empe a u es and ex ac ion imes we e s ud-
ied and hei in luence on ex ac ion yield and cha ac e is ics o he ex ac ed samples ( o al phenolic
compounds, la onoid con en and an ioxidan capaci y) a e p esen ed. The elabo a ed expe imen al
design and he analysis o Pa e o and esponse su ace diag ams allowed us o de e mine he op imal
ex ac ion condi ions. The condi ions ha allow he maximum ex ac ion o phenolic compounds
we e ound a 20 min, 90
◦
C and 50% ul asound ampli ude. Ne e heless, a hese condi ions, he
an ioxidan capaci y measu ed by DPPH dec eased in he ex ac ed samples.
Keywo ds: ul asound; op imiza ion; apple pomace; an ioxidan capaci y
1. In oduc ion
Was e gene a ion and managemen is one o he main conce ns in he indus y. The
was e gene a ed in he ood sec o is gene ally ich in wa e and o ganic compounds and
he e o e hese by-p oduc s can cause en i onmen al p oblems. The manu ac u e s ace
addi ional cos s o he adequa e disposal o ea men o hei esidues. Howe e , ood
was e could be a po en ial aw ma e ial o he ex ac ion o aluable p oduc s. I is cheap
and p oduced in la ge quan i ies as by-p oduc s. In his aspec , mo e comp ehensi e
s udies abou i s alo iza ions could lead o economic and en i onmen al bene i s [1].
Cide is an alcoholic be e age p oduced by he o al o pa ial e men a ion o apple
juice. The manu ac u e o cide is ca ied ou annually and Spain is he second la ges
p oduce in he Eu opean Union a e he Uni ed Kingdom [
2
]. In 2017, he cide indus y
used mo e han 1,000,000 ons o apples du ing i s p oduc ion [
2
]. To p oduce cide , apples
a e ha es ed a e he summe and hen a e p essed o se e al days. A e p essing, apple
pomace was e is gene a ed, which is app oxima ely 25–35% o he ini ial weigh o he
apples [
3
,
4
]. Then, he juice ob ained is ans e ed o a a whe e i unde goes spon aneous
e men a ion. As i can be seen, apple pomace is a was e gene a ed in la ge olume du ing
he cide manu ac u ing p ocess. The e o e, new p ocesses o i s alo iza ion a e cu en ly
being in es iga ed [4].
Apple pomace is composed o a mix u e o pulp, epide mis, seeds and s ems [
5
].
Besides wa e , his mix u e con ains mainly ca bohyd a es, phenolic compounds, p o eins,
a , ash and c ude ibe [
6
]. Howe e , i s composi ion a ies acco ding o he o igin
and he deg ee o ma u a ion [
7
]. In he li e a u e, he composi ion o he apple pomace
co e s a wide ange o alues (% on a d y basis): cellulose 7.2–43.6% [
8
], hemicellulose
4.26–29.90% [
7
,
8
], lignin 15.3–23.5%, pec ins 3.5–14.32% [
8
], p o eins 2.9–5.7% [
8
], lipids
Molecules 2021,26, 3783. h ps://doi.o g/10.3390/molecules26133783 h ps://www.mdpi.com/jou nal/molecules
Molecules 2021,26, 3783 2 o 14
1.20–3.9% [
8
] and ashes 1.47–1.66% [
7
]. The e o e, apple pomace is conside ed as a good
sou ce o ibe , pec in and phenolic compounds [9].
Some s udies ha e used apple pomace as a sou ce o high alue-added p oduc s [
4
,
9
]
o p oduc ion o lac ic acid and ci ic acid [
8
] and he p oduc ion o sus ainable bioma e i-
als [
10
], among o he s. The ex ac ion o bioac i e compounds could be ano he in e es ing
ou e. In ac , some s udies indica ed a possible ad e se e ec o he consump ion o
syn he ic an ioxidan s; he e o e, he ex ac ion o an ioxidan compounds om na u al
esou ces could be a p omising ou e [
11
]. Recen ly, he po en ial applica ion o apple
pomace o an ioxidan ex ac ion was epo ed in se e al wo ks [6,12–15]. Phenolic com-
pounds like que ce in, ca echin, phlo idzin, gallic acid and chlo ogenic acid can educe
ch onic disease isk [
13
]. Ley a-Co al e al. (2016) [
6
] epo ed he p esence o chlo ogenic
acid, epica echin, u in and phlo idzin as phenolic compounds in apple pomace and hei
an ioxidan capaci y was assessed. Ping e e al. (2012) [
12
] also epo ed he chemical
composi ion o polyphenols ex ac ed om apple pomace, whe e ca echin monome s
and phenolic acids wi h an ioxidan capaci y we e ound. Fe en ino e al. (2018) [
14
]
also epo ed he p esence o phlo idzin, epica echin and que ce in and hei an ioxidan
capaci y was e alua ed. In his con ex , he use o apple pomace as a sou ce o phenolic
compounds wi h an ioxidan ac i i y, pa icula ly la onoids, could be a p omising ou e
o i s alo iza ion, as hey emain in he pulp du ing he cide p oduc ion p ocess [
16
,
17
].
In he li e a u e, he ex ac ion o phenols om apple pomace has been pe o med
by di e en echnologies. Among con en ional me hodologies, solid-liquid ex ac ion,
s i ing and shaking [
18
] and he use o di e en o ganic sol en s, namely me hanol,
ace one and e hanol [
19
,
20
] can be ound. The op imiza ion and in ensi ica ion p ocess
a e c ucial s eps o he de elopmen o a easible p ocess a indus ial scale. In addi ion,
he implemen a ion o g een echnologies and he use o mo e en i onmen ally iendly
sol en s a e equi ed. In his con ex , p e ious s udies ha e shown ha wa e could be a
sui able ex ac ion sol en able o ex ac phenolic compounds om apple pomace [17].
As a new en i onmen ally iendly and in ensi ica ion p ocess, sonica ion-assis ed
ex ac ion has gained a en ion. Ul asound-assis ed ex ac ion is an e ec i e and en i on-
men ally iendly me hod o bioac i e compound ex ac ion [
21
,
22
]. I s applica ion could
be an e icien al e na i e o adi ional echniques, since i can imp o e he p oduc i i y,
yield (as i inc eases he con ac a ea be ween he solid and liquid phase) and selec i i y
o he p ocess, educing he cos o ex ac ion ope a ions [
23
,
24
]. Ul asonic p obes can
be used o he applica ion o ul asound, whe e hey can be imme sed in he solu ion
con aining he sample o he ex ac ion o he desi ed componen . Ul asonic p obes
ha e some ad an ages o e an ul asonic ba h, because he ul asound p obe p oduces
mo e ene gy han a con en ional ul asound ba h. This echnology can accele a e chemi-
cal eac ions [
25
]. In ecen s udies, ul asound echnology has also gained a en ion o
an ioxidan ex ac ion om apple pomace [12].
The e o e, he objec i e o his wo k was he op imiza ion o he ul asound-assis ed
ex ac ion o an ioxidan compounds om apple pomace using wa e as a sol en . The aim
was o ind he op imum condi ions ha allow a be e ex ac ion o aluable compounds
such as an ioxidan s using en i onmen ally iendly and in ensi ica ion echnologies. Fo
ha , we show he e ec o ime, empe a u e and sonica ion powe on he ex ac ion yield
and he cha ac e is ics o he ex ac ed samples ( o al phenolic compounds, la onoids
con en and an ioxidan capaci y). Fo a be e co ela ion and in e p e a ion o he ob ained
esul s, di e en analyses and ep esen a ions o Pa e o and su ace esponse diag ams
we e pe o med.
2. Resul s
2.1. Composi ion o Apple Pomace
The apple pomace we used had he ollowing chemical composi ion (% on a d y basis):
α
-cellulose: 21
±
0.5, hemicelluloses: 23
±
0.5, klason lignin: 19
±
0.4, pec ins: 15.0
±
0.5,
p o eins: 5.00 ±0.3, lipids: 5.55 ±0.3, and ashes con en : 1.7 ±0.1.
Molecules 2021,26, 3783 3 o 14
As desc ibed in he in oduc ion sec ion, he composi ion o he apple pomace is highly
a iable (% on a d y basis): cellulose 7.2–43.6% [
8
], hemicellulose 4.26–29.90% [
7
,
8
], lignin
15.3–23.5%, pec ins 3.5–14.32% [
8
], p o eins 2.9–5.7% [
8
], lipids 1.20–3.9% [
8
] and ashes
1.47–1.66% [
7
]. The cha ac e iza ion echniques used, as well as he o igin o he apple
pomace, may explain he a iabili y o he esul s. In his wo k, he pe cen ages ound
we e in he ange seen in he li e a u e. The e o e, he apple pomace we used demons a es
i s sui abili y o he ex ac ion o biocomponen s.
2.2. E ec o Ex ac ion Time and Ul asound Ampli ude on TPC, TFC and AC
Table 1summa izes he con en o polyphenols (TPC), o al la onoids (TFC) and he
an ioxidan capaci y (AC) pe g am o d ied apple pomace, ex ac ed a 40
◦
C a di e en
imes and a di e en ul asound ampli udes (50% and 70%).
Table 1.
Con en o phenolic compounds (TPC), o al la onoids (TFC) and an ioxidan capaci y
(using DPPH assays) o di e en ex ac ion imes and ul asound powe .
Ampli ude
(%)
Time
(min)
TPC
(mg GAE/g *)
TFC
(mg CE/g *)
AC (DPPH)
(mg TE/g *)
50
5 3.07 ±0.16 2.20 ±1.12 1.29 ±0.32
10 3.51 ±0.22 1.66 ±0.21 1.39 ±0.13
15 3.10 ±0.70 1.92 ±0.12 1.36 ±0.27
20 3.29 ±0.43 1.88 ±0.15 1.36 ±0.13
70
5 2.88 ±0.14 1.60 ±0.30 1.09 ±0.27
10 3.00 ±0.30 1.76 ±0.18 1.29 ±0.20
15 3.54 ±0.33 2.37 ±0.31 1.11 ±0.14
20 3.61 ±0.57 2.34 ±0.17 0.92 ±0.23
* Values p esen ed a e he a e age o iplica e measu emen s.
As i can be seen, he TPC a ies om 2.88 o 3.61 mg GAE/g and TFC anges be ween
1.60–2.37 mg CE/g. The an ioxidan capaci y measu ed in he ex ac ed liquo s anged
om 0.92 o 1.39 mg TE/g.
Çam e al. (2010) [
26
] showed o al phenolic le els be ween 2.23 and 6.18 mg GAE/g
using ul asound ex ac ions and wa e as sol en . Howe e , Cand awina a e al. (2015) [
17
]
showed lowe alues o TPC be ween 0.83 and 1.26 mg GAE/g and an ioxidan ac i i y
be ween 1.00 and 1.65 mg TE/g (by DPPH). In his wo k, he ob ained expe imen al alues
showed accep able le els.
On he o he hand, He nández-Ca anza e al. (2016) [
27
] ob ained a la onoid con en
be ween 4.55 and 6.63 mg o CE/g o apple pomace, using con en ional ex ac ion me hods
(magne ic s i ed ho pla e and cons an agi a ion o 3–12 h). The alues ob ained in his
s udy (1.60 o 2.34 CE/g) we e lowe han hose epo ed in li e a u e. These di e ences
could be a ec ed by he ex ac ion echnique used, he ex ac ion ime, and he o igin o
he aw ma e ial used in he di e en s udies.
The adequacy o he esul s was e i ied by analysis o a iance (ANOVA). The Pa e o
diag ams ob ained in he ANOVA analysis a e shown in Figu e 1. As i can be no ed, he
e ec o he ampli ude a ec s he an ioxidan ac i i y measu ed by he DPPH assay. On
he o he hand, i should be no ed ha he TPC and TFC we e no signi ican ly a ec ed by
any o he a iables s udied.
This beha io was e i ied by s udying he adequacy o he model h ough he s udy o
he F- a io whose signi icance can be e alua ed using he p- alue es ablishing a con idence
le el o 95%. Thus, a p- alue unde 0.05 implies ha he s udied e ec signi ican ly a ec s
he esponse. The alues o he F- a io and p- alues ob ained using he S a g aphics
so wa e a e shown in Table 2. As can be seen, only he independen a iable ampli ude
had a signi ican e ec on he an ioxidan ac i i y (DPPH), con i ming he a o emen ioned.
Molecules 2021,26, 3783 4 o 14
Molecules 2021, 26, x FOR PEER REVIEW 4 o 15
Figu e 1. S anda dized Pa e o diag am o he sc eening o (a) TPC, (b) TFC, and (c) DPPH.
This beha io was e i ied by s udying he adequacy o he model h ough he s udy
o he F- a io whose signi icance can be e alua ed using he p- alue es ablishing a con i-
dence le el o 95%. Thus, a p- alue unde 0.05 implies ha he s udied e ec signi ican ly
a ec s he esponse. The alues o he F- a io and P- alues ob ained using he S a g aphics
so wa e a e shown in Table 2. As can be seen, only he independen a iable ampli ude
had a signi ican e ec on he an ioxidan ac i i y (DPPH), con i ming he a o emen-
ioned.
Table 2. Adequacy o he model h ough he analysis o F- a io and p- alue.
TPC TFC DPPH
F-Ra io p-Value F-Ra io p-Value F-Ra io p-Value
Ampli ude (A) 0.01 0.9303 0.35 0.5951 19.72 0.0212
Time (B) 4.45 0.1255 1.9 0.2622 1.05 0.3816
AB 3.08 0.1775 5.21 0.1067 3.05 0.1793
BB 0.23 0.6674 0.2 0.6844 4.83 0.1154
Fu he mo e, Figu e S1 (supplemen a y da a) shows he impac o a iables on di -
e en esponses. Acco ding o he igu es i can be concluded ha he longe he ex ac-
ion ime, he mo e he esponses (TPC, TFC, DPPH) inc eased, while inc easing he am-
pli ude dec eased he p ope ies o he ex ac s.
Conside ing hese esul s, we es ablished an ampli ude o 50% o ca y ou u he
expe imen al design, as shown in Table 3.
Figu e 1. S anda dized Pa e o diag am o he sc eening o (a) TPC, (b) TFC, and (c) DPPH.
Table 2. Adequacy o he model h ough he analysis o F- a io and p- alue.
TPC TFC DPPH
F-Ra io p-Value F-Ra io p-Value F-Ra io p-Value
Ampli ude (A) 0.01 0.9303 0.35 0.5951 19.72 0.0212
Time (B) 4.45 0.1255 1.9 0.2622 1.05 0.3816
AB 3.08 0.1775 5.21 0.1067 3.05 0.1793
BB 0.23 0.6674 0.2 0.6844 4.83 0.1154
Fu he mo e, Figu e S1 (supplemen a y da a) shows he impac o a iables on di e -
en esponses. Acco ding o he igu es i can be concluded ha he longe he ex ac ion
ime, he mo e he esponses (TPC, TFC, DPPH) inc eased, while inc easing he ampli ude
dec eased he p ope ies o he ex ac s.
Conside ing hese esul s, we es ablished an ampli ude o 50% o ca y ou u he
expe imen al design, as shown in Table 3.
Molecules 2021,26, 3783 5 o 14
Table 3. Expe imen al a iables o he expe imen al design.
Va iable Designa ion Uni s Nomencla u e Value
Fixed
Ampli ude (%) 50
Solid-liquid a io (w/ ) 1:40
Sol en wa e
Pa icle size µm <250
Independen Tempe a u e (◦C) Temp 65–90
Time (min) 10–20
Dependen To al phenolic con en (mg GAE/g) TPC YTPC
To al la onoid con en (mg CE/g) TFC YTFC
An ioxidan capaci y (mg TE/g) DPPH YDPPH
2.3. Op imisa ion o he Ex ac ion Condi ions
Table 4summa izes in de ail he se o expe imen s ob ained using he S a g aphic so -
wa e. In addi ion, i also includes he expe imen al alues co esponding o he dependen
a iables YTPC, YTFC, and YDPPH, and he ex ac ion yield.
Table 4. Se o expe imen s and expe imen al esul s.
Exp.
Independen Va iables No malized Va iables Dependen Va iables Yield
(%)
Tempe a u e(◦C) Time
(min) X1X2YTPC
(mg GAE/g *)
YTFC
(mg CE/g *)
YDPPH
(mg TE/g *)
1 40 10 −1−1 3.51 ±0.22 1.66 ±0.21 1.39 ±0.13 24.8 ±0.07
2 40 15 −1 0 3.10 ±0.70 1.92 ±0.12 1.36 ±0.27 28.6 ±0.10
3 40 20 −1 1 3.29 ±0.44 1.88 ±0.15 1.36 ±0.13 29.5 ±1.10
4 65 10 0 −1 4.68 ±0.36 3.16 ±1.08 0.69 ±0.38 39.5 ±0.05
5 65 15 0 0 4.74 ±1.84 4.30 ±1.05 0.65 ±0.27 55.1 ±0.10
6 65 20 0 1 5.50 ±0.95 4.00 ±1.87 0.71 ±0.16 50.6 ±1.03
7 90 10 1 −1 5.75 ±0.72 4.59 ±0.23 0.61 ±0.11 50.6 ±0.05
8 90 15 1 0 6.02 ±0.28 4.40 ±0.72 0.53 ±0.17 58.4 ±1.15
9 90 20 1 1 6.02 ±0.29 3.53 ±0.82 0.68 ±0.14 38.7 ±1.50
* Values p esen ed a e he a e age o iplica e measu emen s.
Reg ession coe icien s oge he wi h hei co esponding signi icance le els acco ding
o S uden ’s - es , as well as he de e mina ion coe icien (R
2
), i ed R
2
and s a is ical
signi icance (Fishe ’s F es ) a e summa ized in Table 5.
Table 5. Reg ession coe icien s and s a is ical pa ame e s o he expe imen al design.
Reg esion Coe icien s YTPC YTFC YDPPH
b04.86 a3.83 a0.64 a
b11.31 a1.18 a−0.38 a
b20.15 0.00 0.01
b12 0.12 −0.32 0.02
b11 −0.36 −0.67 0.31 a
b22 0.17 −0.25 0.06 b
R20.9731 0.9400 0.9900
R2 i ed 0.9284 0.8600 0.9900
F-exp 21.7720 10.8585 187.0543
F-c i ical 0.0145 0.0387 0.0006
Signi icance le el 98.54 96.12 99.94
a
Signi ican coe icien s a he 99% con idence le el.
b
Signi ican coe icien s om he 90% o 95% con idence le el.
As shown in Table 5, he de e mina ion coe icien s R
2
ob ained o each o he s udied
dependen a iables Y
TPC
, Y
TFC
, and Y
DPPH
we e 0.97, 0.94 and 0.99, espec i ely, indi-
Molecules 2021,26, 3783 6 o 14
ca ing he alidi y o he design. These high de e mina ion coe icien alues indica e
ha a small numbe o he o al a ia ions emain unexplained, conc e ely 0.03%, 0.06%,
and 0.01% o Y
TPC
, Y
TFC
, and Y
DPPH
, espec i ely. Such alues, oge he wi h he high F
alues, indica e ha he selec ed model is adequa e o ep esen he selec ed a iables and
he model esul is s a is ically ep esen a i e.
Acco ding o he eg ession coe icien s epo ed in Table 5, as well as in he Pa e o
diag ams p esen ed in Figu e 2, empe a u e (X
1
) is he mos independen a iable ha
in luences ob aining he phenolics, la onoids and an ioxidan capaci y. On he o he hand,
i should be no ed ha he independen a iable X
2
( ime) did no exhibi a signi ican
in luence o e any o he dependen a iables. Rega ding he quad a ics e ec s (X
12
and
X
22
), i should be no ed ha hey only showed signi ican in luence on he an ioxidan
capaci y o he ob ained ex ac s.
Molecules 2021, 26, x FOR PEER REVIEW 6 o 15
a
Signi ican coe icien s a he 99% con idence le el.
b
Signi ican coe icien s om he 90% o 95%
con idence le el.
As shown in Table 5, he de e mina ion coe icien s R
2
ob ained o each o he s ud-
ied dependen a iables Y
TPC
, Y
TFC
, and Y
DPPH
we e 0.97, 0.94 and 0.99, espec i ely, indi-
ca ing he alidi y o he design. These high de e mina ion coe icien alues indica e ha
a small numbe o he o al a ia ions emain unexplained, conc e ely 0.03%, 0.06%, and
0.01% o Y
TPC
, Y
TFC
, and Y
DPPH
, espec i ely. Such alues, oge he wi h he high F alues,
indica e ha he selec ed model is adequa e o ep esen he selec ed a iables and he
model esul is s a is ically ep esen a i e.
Acco ding o he eg ession coe icien s epo ed in Table 5, as well as in he Pa e o
diag ams p esen ed in Figu e 2, empe a u e (X
1
) is he mos independen a iable ha
in luences ob aining he phenolics, la onoids and an ioxidan capaci y. On he o he
hand, i should be no ed ha he independen a iable X
2
( ime) did no exhibi a signi i-
can in luence o e any o he dependen a iables. Rega ding he quad a ics e ec s (X
12
and X
22
), i should be no ed ha hey only showed signi ican in luence on he an ioxidan
capaci y o he ob ained ex ac s.
F-c i ical 0.0145 0.0387 0.0006
Signi icance le el 98.54 96.12 99.94
Figu e 2. S anda dized Pa e o diag am o (a) TPC, (b) TFC, and (c) DPPH.
In addi ion, he ex ac ion yield (Table 4) inc eased as he empe a u e inc eased,
ob aining he maximum yield o 58.44% a a empe a u e o 90
◦
C, while he minimum
yield o 24.8% was ob ained a he lowes empe a u e (40 ◦C).
2.3.1. In luence o he S udied Va iables
The in e ac ion o he independen a iables and hei in luence on he TPC, TFC, and
DPPH a e shown in Figu e 3.
Molecules 2021,26, 3783 7 o 14
Molecules 2021, 26, x FOR PEER REVIEW 7 o 15
Figu e 2. S anda dized Pa e o diag am o (a) TPC, (b) TFC, and (c) DPPH.
In addi ion, he ex ac ion yield (Table 4) inc eased as he empe a u e inc eased, ob-
aining he maximum yield o 58.44% a a empe a u e o 90 °C, while he minimum yield
o 24.8% was ob ained a he lowes empe a u e (40 °C).
2.3.1. In luence o he S udied Va iables
The in e ac ion o he independen a iables and hei in luence on he TPC, TFC,
and DPPH a e shown in Figu e 3.
Figu e 3. Response su ace diag ams showing op imal ex ac ion ime condi ions and empe a u e agains (a) TPC, (b)
TFC, and (c) an ioxidan p ope ies.
To al Phenolic Con en (TPC)
Di e en esea ch indica ed ha he ex ac ion o phenols om ood was e was im-
p o ed wi h he empe a u e and ime [12,27]; howe e , hea deg ada ion may occu a
high empe a u es. In his s udy, he TPC was s ongly a ec ed by he e ec o he em-
pe a u e, while ime exhibi ed a low e ec as indica ed by he eg ession coe icien co -
esponding o he Y
TPC
dependen a iable (Table 5).
Figu e 3a shows he esponse su ace co esponding o he TPC as a unc ion o he
independen a iables X
1
and X
2
. Acco ding o his igu e, i can be app ecia ed ha as he
empe a u e (X
1
) inc eases, he TPC inc eases independen ly o he ex ac ion ime used.
Figu e 3.
Response su ace diag ams showing op imal ex ac ion ime condi ions and empe a u e agains (
a
) TPC, (
b
) TFC,
and (c) an ioxidan p ope ies.
To al Phenolic Con en (TPC)
Di e en esea ch indica ed ha he ex ac ion o phenols om ood was e was im-
p o ed wi h he empe a u e and ime [
12
,
27
]; howe e , hea deg ada ion may occu a high
empe a u es. In his s udy, he TPC was s ongly a ec ed by he e ec o he empe a u e,
while ime exhibi ed a low e ec as indica ed by he eg ession coe icien co esponding
o he YTPC dependen a iable (Table 5).
Figu e 3a shows he esponse su ace co esponding o he TPC as a unc ion o he
independen a iables X
1
and X
2
. Acco ding o his igu e, i can be app ecia ed ha as he
empe a u e (X
1
) inc eases, he TPC inc eases independen ly o he ex ac ion ime used. I
is epo ed in he li e a u e ha he leng h o ime o he ex ac ion was also an impo an
ac o when he ex ac ion is pe o med below he c i ical empe a u e [
17
]. In his wo k,
he ange s udied (10, 15, 20 min) did no ha e s ong in luence.
The TPC con en o he expe imen s we e in he ange o 3.10 o 6.02 (mg GAE/g) as
shown in Table 4. The lowes alue co esponded o he expe imen 2 (40
◦
C and 15 min)
while he highes alue was ob ained o he highes empe a u e and maximum ex ac ion
ime in expe imen 9 (90
◦
C, 20 min). Al hough ime has li le in luence, i can be obse ed
ha a he maximum empe a u e o 90
◦
C and he longe ex ac ion ime, he e was a
sligh imp o emen in he TPC con en . This esul is in ag eemen wi h Cand awina a e al.
(2015) [17], who concluded ha he TPC alues inc eased wi h longe ex ac ion imes.
Molecules 2021,26, 3783 8 o 14
To al Fla onoid Con en (TFC)
Simila ly o TPC, he la onoid con en is mainly a ec ed by he ex ac ion empe a-
u e, while ime has a low in luence due o he low signi icance o i s eg ession coe icien
(Table 5). Howe e , in his case, unlike TPC, he highes TFC alue (4.59 mg CE/g) was
ob ained when he ex ac ion ime was 10 min (expe imen 7). On he o he hand, he
lowes alue was obse ed in expe imen 1, a 40
◦
C and 10 min, as can be shown in
Table 4
and Figu e 3b.
An ioxidan Capaci y (DPPH)
In Figu e 3c, i is shown how empe a u e (X
1
) and ime (X
2
) a iables a ec he an-
ioxidan capaci y o he ex ac s. As in he p e ious cases, ime has no signi ican in luence,
and he an ioxidan capaci y is only a ec ed by he e ec o empe a u e. Howe e , in his
case, empe a u e has a nega i e e ec on he ob ained esponse, i.e., a low empe a u es,
he an ioxidan capaci y was highe . Thus, he highes an ioxidan capaci y is ob ained o
expe imen 1 (1.39 mg TE/g), while a 90
◦
C and 15 min (expe imen 8), he capaci y is
minimal (0.53 mg TE/g).
As i can be seen, he empe a u e signi ican ly imp o ed all esponses excep he
DPPH alues, i.e., a highe ex ac ion empe a u e, highe TPC was ob ained; howe e ,
he an ioxidan capaci y measu ed by DPPH was lowe . These conclusions may be con-
adic o y, since some s udies ha e shown a co ela ion be ween he con en o phenolic
compounds and he an ioxidan capaci y [
17
,
28
]. Ne e heless, i is impo an o highligh
ha he de e mina ion o he o al polyphenol con en by he Folin–Ciocal eu me hod can
gene a e an o e es ima ion o he measu emen s since i can eac wi h o he educing com-
pounds, as suga s [
29
]. On he o he hand, some s udies ha e epo ed ha he an ioxidan
capaci y dec eased wi h empe a u e, because exposu e o phenolic compounds o high
empe a u e can cause hei deg ada ion [30].
2.3.2. Op imiza ion o he Ex ac ion Condi ions and Model Valida ion
The objec i e o he op imiza ion was o de e mine he bes ex ac ion condi ions
o p o ide he highes alues o phenolic and la onoid compounds and he g ea es
an ioxidan capaci y. Fo his pu pose, he desi abili y unc ion was used, employing he
S a g aphics Cen u ion XVI so wa e:
TPC = 4.86117 + 1.31392T+ 0.1457 −0.36155T2+ 0.12155T + 0.1697 2(1)
TFC = 3.82718 + 1.17562T−0.0005333 −0.666217T2−0.32105T −0.245167 2(2)
DPPH = 0.64333 −0.381667T+ 0.01T + 0.305T2+ 0.025T −0.06 2(3)
The op imum condi ions o he s udied independen a iables we e 90
◦
C and he
ex ac ion ime o 18.43 min. Unde he ci ed op imum condi ions, he es ima ed alues
we e: TPC = 6.07 mg GAE/g, TFC = 4.00 mg CE/g and DPPH = 0.62 mg TE/g.
2.4. Rela ion be ween TPC and An ioxidan Capaci y
As commen ed be o e, he e a e s udies ha sugges he exis ence o a ela ionship
be ween he o al con en o polyphenols and he an ioxidan ac i i y o he sample [
17
,
28
].
In Figu e 4, he ela ionship be ween he con en o phenolic compounds (Figu e 4a) and
la onoids (Figu e 4b) agains an ioxidan capaci y de e mined by he DPPH me hod is
shown. I can be obse ed ha a highe concen a ions o polyphenols and la onoids, he
an ioxidan capaci y measu ed by DPPH emained s able o dec eased.
Molecules 2021,26, 3783 9 o 14
Molecules 2021, 26, x FOR PEER REVIEW 9 o 15
The op imum condi ions o he s udied independen a iables we e 90 °C and he
ex ac ion ime o 18.43 min. Unde he ci ed op imum condi ions, he es ima ed alues
we e: TPC = 6.07 mg GAE/g, TFC = 4.00 mg CE/g and DPPH = 0.62 mg TE/g.
2.4. Rela ion be ween TPC and An ioxidan Capaci y
As commen ed be o e, he e a e s udies ha sugges he exis ence o a ela ionship
be ween he o al con en o polyphenols and he an ioxidan ac i i y o he sample [17,28].
In Figu e 4, he ela ionship be ween he con en o phenolic compounds (Figu e 4a) and
la onoids (Figu e 4b) agains an ioxidan capaci y de e mined by he DPPH me hod is
shown. I can be obse ed ha a highe concen a ions o polyphenols and la onoids,
he an ioxidan capaci y measu ed by DPPH emained s able o dec eased.
Figu e 4. Co ela ion be ween (a) TPC and (b) la onoid con en wi h an ioxidan ac i i y es ed by DPPH assay.
These esul s may be due o he ac ha he quali y o he ex ac ed phenolics and
la onoids could be wo se wi h he empe a u e and consequen ly, hey could p o ide
wo se an ioxidan capaci y measu ed by DPPH. In addi ion, o he educing compounds
could in e e e in he Folin–Ciocal eu [29] and la onoid es s. In o de o obse e he
quali y o he ob ained samples, he an ioxidan capaci y o he samples is di ided by he
o al phenols, and he esul s a e shown in Table 6 ( ep esen ed as T olox equi a-
len /GAE). On he o he hand, he adical inhibi ion capaci y o he samples is also demon-
s a ed.
Table 6. The quali y ( ep esen ed as T olox equi alen /GAE) and % o inhibi ion capaci y o apple
pomace ex ac ed samples agains he adical DPPH (a e 15 min) and comme cial an ioxidan
T olox a 0.05 g/L o concen a ion.
Time
(min)
Tempe a u e
(°C)
Quali y
(TE/GAE)
Inhibi ion
(%)
10 0.40 43.84
15 40 0.44 42.94
20
0.41 42.99
10
0.15 22.62
15 65 0.14 21.22
20
0.13 23.35
10
0.11 20.52
15 90 0.09 17.70
20
0.011 22.97
T olox 65.27
Figu e 4. Co ela ion be ween (a) TPC and (b) la onoid con en wi h an ioxidan ac i i y es ed by DPPH assay.
These esul s may be due o he ac ha he quali y o he ex ac ed phenolics and
la onoids could be wo se wi h he empe a u e and consequen ly, hey could p o ide
wo se an ioxidan capaci y measu ed by DPPH. In addi ion, o he educing compounds
could in e e e in he Folin–Ciocal eu [
29
] and la onoid es s. In o de o obse e he
quali y o he ob ained samples, he an ioxidan capaci y o he samples is di ided by he
o al phenols, and he esul s a e shown in Table 6( ep esen ed as T olox equi alen /GAE).
On he o he hand, he adical inhibi ion capaci y o he samples is also demons a ed.
Table 6.
The quali y ( ep esen ed as T olox equi alen /GAE) and % o inhibi ion capaci y o apple
pomace ex ac ed samples agains he adical DPPH (a e 15 min) and comme cial an ioxidan T olox
a 0.05 g/L o concen a ion.
Time
(min)
Tempe a u e
(◦C)
Quali y
(TE/GAE)
Inhibi ion
(%)
10 0.40 43.84
15 40 0.44 42.94
20 0.41 42.99
10 0.15 22.62
15 65 0.14 21.22
20 0.13 23.35
10 0.11 20.52
15 90 0.09 17.70
20 0.011 22.97
T olox 65.27
As i can be seen, he educ ion capaci y shown by he compounds ex ac ed om
apple pomace a 40
◦
C and 50% ampli ude was a ound 40%, whe e his alue dec eased
un il 23% a 65
◦
C and 90
◦
C ex ac ion empe a u e. These alues indica e ha he highes
pe cen age o educ ion was egis e ed in he samples ex ac ed a lowe empe a u e,
which is in ag eemen wi h he p e ious ob ained esul s. On he o he hand, he an-
ioxidan capaci y o he comme cial an ioxidan compound T olox was sligh ly highe ,
abou 65%.
2.5. Suga s Cha ac e iza ion o he Ex ac ed Samples
In Table 7, he suga analysis and he ob ained by-p oduc s in he ex ac ed samples
a di e en empe a u es and ex ac ion imes is shown.
