nu ien s
A icle
E ec s o Pa icle Size and Ex ac ion Me hods on
Cocoa Bean Shell Func ional Be e age
Olga Rojo-Po eda 1,2, Le icia Ba bosa-Pe ei a 1,3,* , Lí ia Ma eus-Reguengo 1,
Ma a Be olino 1, Ca oline S é igny 2and Giuseppe Zeppa 1,*
1Depa men o Ag icul u e, Fo es y and Food Sciences (DISAFA), Uni e si y o Tu in, 10095 G ugliasco,
I aly; [email p o ec ed] (O.R.-P.); [email p o ec ed] (L.M.-R.);
[email p o ec ed] (M.B.)
2RD3 Depa men -Uni o Pha macognosy, Bioanalysis and D ug Disco e y, Facul y o Pha macy,
Uni e si éLib e de B uxelles, 1050 B ussels, Belgium; [email p o ec ed]
3Depa men o Analy ical Chemis y, Nu i ion and Food Science, Facul y o Pha macy, Uni e si y o
San iago de Compos ela, 15782 San iago de Compos ela, Spain
*Co espondence: [email p o ec ed] (L.B.-P.); [email p o ec ed] (G.Z.);
Tel.: +39-011-670-8705 (L.B.-P. & G.Z.)
Recei ed: 15 Ma ch 2019; Accep ed: 15 Ap il 2019; Published: 17 Ap il 2019
Abs ac :
One o he main by-p oduc s in cocoa indus y is he cocoa bean shell (CBS), which ep esen s
app oxima ely 12–20% o he bean. This p oduc has been sugges ed as a ood ing edien because
o i s a oma and high die a y ibe and polyphenol con en s. The pu pose o his wo k was o
e alua e he e ec s o he CBS pa icle size and ex ac ion me hods on he chemical composi ion and
consume accep ance o a unc ional be e age, in o de o ind he bes combina ion o echnological
pa ame e s and heal h bene i s. Fi e pa icle sizes o CBS powde and six home echniques we e
used o be e age p epa a ion. The in luence o hese ac o s on he physico-chemical cha ac e is ics,
me hylxan hine and polyphenolic con en s, an ioxidan and an idiabe ic p ope ies, and consume
accep ance was e alua ed. To al phenolic con en alues up o 1803.83 mg GAE/L we e ob ained
o he be e ages. Phenolic compounds and me hylxan hines we e iden i ied and quan i ied by
HPLC-PDA. These compounds may be ela ed o he high an ioxidan capaci y (up o 7.29 mmol
TE/L) and an idiabe ic p ope ies (up o 52.0% o
α
-glucosidase inhibi ion) obse ed. Fu he mo e,
he consume accep ance esul s indica ed ha CBS may ep esen an in e es ing ing edien o new
unc ional be e ages wi h po en ial heal h bene i s, educing he en i onmen al and economic impac
o by-p oduc disposal.
Keywo ds:
cocoa by-p oduc ; unc ional ood; polyphenols;
α
-glucosidase inhibi ion; an idiabe ic
capaci y; an ioxidan capaci y; me hylxan hines
1. In oduc ion
Acco ding o he In e na ional Cocoa O ganiza ion (ICCO), each yea , mo e han 4000 ons
o cocoa beans a e p ocessed and consumed wo ldwide [
1
]. Conside ing ha he main p oduc s
o cocoa a e ob ained om i s oas ed bean, which ep esen s only 10% o he o al weigh o
he ui , cocoa p ocessing p oduces a la ge amoun o ege al esidue. Besides being expensi e,
he disposal o hese by-p oduc s can be ha m ul o he en i onmen because hey con ain po en ially
phy o oxic polyphenols [
2
] and high concen a ions o heob omine, which may be oxic o non-human
mammals [
3
]. Such unde u iliza ion o esidual biomass can be o e come by he de elopmen
o an added- alue oods u based on cocoa by-p oduc s, pa icula ly he cocoa bean shell (CBS),
which ep esen s 12% o 20% o he cocoa bean [
4
]. CBS has been epo ed o be a conside able sou ce
Nu ien s 2019,11, 867; doi:10.3390/nu11040867 www.mdpi.com/jou nal/nu ien s
Nu ien s 2019,11, 867 2 o 19
o p o eins and die a y ibe , wi h low a con en in compa ison wi h cocoa beans [
5
] bu wi h a simila
p o ile o ola iles [
6
]. Conside ing ha CBS is a inal-s age by-p oduc om cocoa p ocessing, i appea s
o be an economical, o ganolep ic, and nu i ionally ewa ding subs ance o he ans o ma ion o cocoa
bean indus ies. Mos o he esea ch in o CBS u iliza ion is ela ed o animal eeding and, despi e he
p esence o heob omine, CBS has posi i e e ec s on o i ied die s o uminan s, pigs, and poul y [
3
],
among o he animals. Besides, CBS has been applied as an addi i e in o ganic e ilize [
7
], as biomass
o biogas p oduc ion [
8
], o as a pec in sou ce [
5
], among o he applica ions. The applica ion o his
cocoa by-p oduc o ood has a ac ed some a en ion due o i s nu i ional cha ac e is ics and high
concen a ion o phenolic compounds, mainly la onoids [
9
]. In ecen yea s, some s udies in he ood
esea ch ield we e published, sugges ing CBS as a ood ing edien [
4
]. This esea ch in e es can be
linked o he sus ainabili y and bioeconomy amewo k o he mode n ood and ag icul u al indus ies,
leading o he alo iza ion o unc ional oods de eloped om by-p oduc s, as an oppo uni y o make
heal hie oods. Func ional oods a e simila o con en ional ones in ha hey a e pa o a s anda d die
and consumed on a egula basis and in egula amoun s. A unc ional ood is claimed o ha e p o en
bene i s o he main enance o p omo ion o a s a e o well-being o heal h o a educ ion in he isk o
a pa hological p ocess o disease [10]. Al hough his ma ke niche is no well de ined, by in luencing
he da a on global sales, unc ional oods i e u ably ep esen a op end in he ood indus y.
Because o hei an ioxidan capaci y, phenolic compounds may be capable o p o ec ing cell
componen s om oxida i e damage, hus limi ing he isk o se e al diseases associa ed wi h oxida i e
s ess, o ins ance, diabe es [
11
]. The ele ance o polyphenols o he managemen o blood glucose
is mainly due o hei inhibi ion o diges i e enzymes in ol ed in he me abolism o ca bohyd a es
(
α
-glucosidase and
α
-amylase) [
9
]. E en i hese phenolic compounds a e poo ly abso bed, hey can s ill
ac on memb ane-bound enzymes loca ed in he in es inal epi helium. Mo eo e , o he mechanisms
o ac ion o polyphenols on glucose up ake, a e inges ion o ca bohyd a e- ich meals, a e being
s udied [
12
]. The p esence o bioac i e componen s in CBS and i s sus ainable cha ac e may a ouse
in e es in such a p oduc as a po en ial ing edien in he unc ional-be e age indus y. Despi e hei
esou ce- ich ma ix, he e ec i e ex ac ion o plan bioac i e compounds and hei concen a ions in
he inal p oduc can be in luenced by p ocessing o p epa a ion me hods.
Ung ound CBS has al eady been u ilized as one o he ing edien s o comme cialized he bal
in usion bags, such as he “ChocoTea” in usion bags om Valbe be
®
o he “Choco” isane bags om
YogiTea
®
. None heless, o he p epa a ions des ined o he use o his by-p oduc in home-based
be e age-making echniques ha e no been p oposed ye . In his pape , se e al CBS p epa a ions
we e de eloped o be employed in six di used echniques o co ee home-p epa a ion a ailable a a
consume le el, such as he Moka, Neapoli an lip, Ame ican, Esp esso, Capsule, and F ench p ess
co ee make s. Ins ead o co ee powde , CBS a di e en g inding deg ees (GDs) was employed o ind
he op imal GD o each ex ac ion echnique. The aim o his s udy was o ind he bes combina ion
o he CBS GD and he be e age p epa a ion echnique in o de o ob ain a new unc ional be e age
wi h he op imal chemical composi ion, biological e ec s (an ioxidan and an idiabe ic p ope ies),
and senso y cha ac e is ics.
2. Ma e ials and Me hods
2.1. Chemicals
Folin & Ciocal eu’s phenol eagen , sodium ca bona e (
≥
99.5%), 2,2
0
-diphenyl-1-pic ylhyd azyl
(95%) (DPPH), 6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid (97%; olox), anillin (99%),
(+)-ca echin hyd a e (>98%), me hanol (
≥
99.9%), hyd ochlo ic acid ( uming 37%), aluminum
chlo ide (99%), sodium ni i e (
≥
99%),
α
-glucosidase om in es inal ace one powde s om a ,
p-ni ophenyl-
α
-D-glucopy anoside(
≥
99%; p-PNG),aca bose(
≥
95%), po assiumphospha emonobasic
(
≥
99%), o mic acid (
≥
98%), que ce in-3-O-glucoside (
≥
90%; Q-3-G), heob omine (
≥
98.5%), ca eine
(
≥
98.5%), and que ce in (
≥
98.5%) we e p o ided by Sigma-Ald ich (Milan, I aly). Po assium phospha e
Nu ien s 2019,11, 867 3 o 19
dibasic (
≥
98%) was acqui ed om Ca lo E ba (Milan, I aly). Gallic acid, e hanol (
≥
99.9%), sodium
hyd oxide (1 M), (-)-epica echin (>90%), p ocyanidin B1 (
≥
98.5%; PCB1), p ocyanidin B2 (
≥
98.5%;
PCB2), p o oca echuic acid (>97%), ca eic acid (
≥
95%), and anillic acid (
≥
99%) we e supplied by
Fluka (Milan, I aly). Ul apu e wa e was p epa ed in a Milli-Q il e sys em (Millipo e, Milan, I aly).
2.2. Samples
CBS om São Tomécocoa beans (Fo as e o a ie y) was kindly supplied by Pas iglie Leone S. .l.
(Tu in, I aly). The CBS was di ided in o di e en g ain sizes using a BA200N ib a ing sie e (CISA,
Ba celona, Spain). Fi e GDs we e ob ained: abo e 4000
µ
m (GD1), 2000–4000
µ
m (GD2), 1000–2000
µ
m
(GD3), 500–1000 µm (GD4), and 250–500 µm (GD5).
The chemical and nu i ional cha ac e iza ion o CBS was ca ied ou acco ding o
Be olino e al. [13].
2.3. P epa a ion o Be e ages
The be e ages we e p epa ed by six echniques a ailable o consume s, i.e., Moka Exp ess 6 cups
(Biale i, B escia, I aly), Neapoli an lip co ee po (Ilsa, Tu in, I aly), Ame ican co ee make Cucina HD
7502 (Phillips, Milan, I aly), Esp esso Saeco HD 8423/11 (Phillips, Milan, I aly), Capsule LM 3100 (AEG,
Milan, I aly), and F ench p ess Ka e (Ikea, Collegno, I aly). S ill mine al wa e (Valmo a, Luse na San
Gio anni, I aly) was used o he be e age p oduc ion. Each o he GDs was es ed wi h all he co ee
make s, esul ing in 30 be e ages. Th ee p oduc ion ba ches we e gene a ed o each be e age.
The wa e quan i ies employed a ied o each echnique ollowing he es ablished ules o he
use o he di e en machines. Fo Esp esso and Capsule echniques, he ini ial olumes o wa e a e
unknown because bo h machines ope a e wi h a con inuous inle o wa e and he e o e i is possible
o de ine only he inal olume o he ob ained be e age. In addi ion, he quan i ies o CBS powde s
used o he be e age p epa a ions we e adap ed o each echnique and GD o ob ain a echnologically
iable o mula ion.
All he be e ages we e cen i uged on a MPW-260R cen i uge (MPW, Wa saw, Poland) a
3075 ×g
o 10 min and hen passed h ough a 0.45
µ
m cellulose ace a e il e (Ca lo E ba, Milan, I aly)
be o e analyses.
2.4. Analy ical P ocedu es
2.4.1. Physicochemical Analysis
A pH me e MICROpH 2002 (CRISON, Ca pi, I aly) se ed o pH measu emen .
De e mina ion o o al acidi y (exp essed in g ams o ace ic acid pe li e o a be e age) was
pe o med by po en iome ic i a ion o 5 mL o a be e age (dilu ed o 50 mL wi h dis illed wa e ) by
means o 0.01N NaOH up o pH 8.2.
The d y ex ac o each be e age was analyzed g a ime ically o a 5 mL sample o each be e age
d ied in an o en a 110 ◦C un il cons an weigh .
The colo analysis was conduc ed in ansmi ance mode on a CM-5 spec opho ome e (Konica
Minol a, Tokyo, Japan). L*, a*, and b* CIELab pa ame e s we e used o measu e he colo , whe e L* is a
coe icien o ligh ness anging om 0 (black) o 100 (whi e), a* indica es he colo s ed-pu ple (when
posi i e a*) and bluish-g een (when nega i e a*), and b* deno es he colo s yellow (when posi i e b*)
and blue (nega i e b*). The
∆
E pa ame e , which ep esen s he di e ence be ween wo colo s [
14
] and
i s pe cep ibili y by he human eye when ∆E>2.5, was calcula ed acco ding o he equa ion
∆E=q(L∗a−L∗b)2+(a∗a−a∗b)2+(b∗a−b∗b)2
Nu ien s 2019,11, 867 4 o 19
2.4.2. To al Phenolic, Tannin, and Fla onoid Con en s
The o al phenolic (TPC), o al la onoid (TFC), and o al annin (TTC) con en s we e de e mined
acco ding o he me hods desc ibed by Ba bosa-Pe ei a e al. [
15
], in 96-well mic opla es, using a
BioTek Syne gy HT spec opho ome ic mul i-de ec ion mic opla e eade (BioTek Ins umen s, Milan,
I aly). All he measu emen s we e pe o med in iplica e. Fo he TPC analysis, a calib a ion cu e
o gallic acid (20–100 mg/L) was cons uc ed o quan i y he concen a ion, which was exp essed in
millig ams o gallic acid equi alen s pe li e o a be e age (mg GAE/L). The quan i ica ion o bo h TFC
and TTC was pe o med based on a s anda d cu e o ca echin (5–500 mg/L), and he concen a ions
we e exp essed in millig ams o ca echin equi alen s pe li e o a be e age (mg CE/L).
2.4.3. An ioxidan Capaci y
The an ioxidan capaci y o he be e ages was assessed by he 2,2
0
-diphenyl-1-pic ylhyd azyl
(DPPH
•
) adical–sca enging me hod desc ibed by Ba bosa-Pe ei a e al. [
15
]. All he assays we e
conduc ed in iplica e in 96-well mic opla es wi h he BioTek Syne gy HT spec opho ome ic
mul i-de ec ion mic opla e eade (BioTek Ins umen s). An ioxidan capaci y was calcula ed as he
inhibi ion pe cen age (IP) o he DPPH adical as
IP (%)=(A0−A30)
A0
×100
whe e A0is abso bance a he ini ial ime poin , and A30 is he abso bance a e 30 min.
A s anda d cu e o olox was cons uc ed (12.5–300
µ
M) o assessmen o he adical-sca enging
ac i i y alues, which we e exp essed as millimoles o olox equi alen s pe li e o a be e age
(mmol TE/L).
2.4.4. An idiabe ic Capaci y
The an idiabe ic e ec s o he be e ages we e de e mined by he
α
-glucosidase colo ime ic assay
adap ed om he me hod desc ibed by Kwon e al. [16].
An aliquo (50
µ
L) o he sample was mixed wi h 100
µ
L o
α
-glucosidase (10 mg/mL), p epa ed
in 0.1 M phospha e bu e pH 6.9, and incuba ed o 5 min. A e ha , 50
µ
L o subs a e p-PNG
a 4 mM (p epa ed in he phospha e bu e ) was added, and he solu ion was mixed. The solu ion
was incuba ed o 30 min a 37
◦
C, and hen abso bance was measu ed a 405 nm agains a blank
con ol. Aca bose a 0.5 mM (hal -maximal inhibi o y concen a ion, IC
50
) se ed as a posi i e con ol,
and he an idiabe ic capaci y was exp essed as he
α
-glucosidase inhibi ion pe cen age. All o he
measu emen s we e conduc ed in iplica e in 96-well mic opla es, using he BioTek Syne gy HT
spec opho ome ic mul i-de ec ion mic opla e eade (BioTek Ins umen s).
2.4.5. RP-HPLC-PDA Analysis
Cha ac e iza ion o he polyphenols con ained in he be e ages was pe o med by means o
e e sed-phasehigh-p essu eliquidch oma og aphywi hapho odiodea ayde ec o (RP-HPLC-PDA)
The mo-Finnigan Spec a Sys em (The mo-Finnigan, Wal ham, MA, USA). The ins umen was
equipped wi h a P2000 bina y g adien pump, SCM 1000 degasse , AS 3000 au oma ic injec o ,
and Finnigan Su eyo PDA Plus de ec o . Ins umen con ol, da a collec ion, and da a p ocessing
we e pe o med using he Ch omQues so wa e, e sion 5.0 (The mo-Finnigan, Wal ham, MA, USA).
Fo sepa a ion o compounds, a e e se-phase Kine ex Phenyl-Hexyl C18 column (150
×
4.6 mm
in e nal diame e and 5 µm pa icle size; Phenomenex, Cas el Maggio e, I aly) was u ilized a 35 ◦C.
Two sol en s se ed as a mobile phase: wa e con aining o mic acid a 0.1% / (sol en A)
and 100% me hanol (sol en B). The sample injec ion olume was 10
µ
L. To sepa a e he di e en
compounds, g adien elu ion a a low a e o 1 mL/min was conduc ed du ing 45 min as ollows:
minu es 0–2, 90% A and 10% B; minu es 2–18, a linea g adien om 10% o 50% B; minu es 18–40,
Nu ien s 2019,11, 867 5 o 19
a linea g adien om 50% o 80% B; minu es 40–42, a linea g adien om 80% o 90% B; and minu es
42–45, a linea g adien un il 90% A and 10% B we e eached.
De ec ion was ca ied ou ia con inuous scanning o wa eleng hs be ween 200 and 400 nm.
Me hylxan hines ( heob omine and ca eine) we e quan i ied a 272 nm, p o oca echuic acid a
293 nm, ca eic acid a 325 nm, la an-3-ols (ca echin, epica echin, and ca echin-3-O-glucoside),
and p ocyanidins B ( ype B p ocyanidin and p ocyanidin B2) we e quan i ied a 280 nm, and la onols
(que ce in, que ce in-3-O-glucoside, and que ce in-3-O- hamnoside) a 365 nm.
The quan i ica ion was pe o med based on ex e nal linea calib a ion cu es analyzed unde he
same condi ions and he ollowing co ela ion coe icien s we e ob ained: R
2
=0.9995 o heob omine,
R
2
=0.9996 o ca eine, R
2
=0.9999 o ca echin, R
2
=0.9998 o epica echin, R
2
=0.9997 o
p o oca echuic acid, R
2
=0.9999 o ca eic acid, R
2
=0.9998 o p ocyanidin B1 (PB1), R
2
=0.9999 o
p ocyanidin B2 (PB2), R2=0.9996 o que ce in-3-O-glucoside (Q-3-G), and R2=0.9988 o que ce in.
Fo ca echin-3-O-glucoside, ype B p ocyanidin, and que ce in-3-O- hamnoside, concen a ions we e
exp essed as ca echin, p ocyanidin B1, and que ce in-3-O-glucoside equi alen s, espec i ely.
2.5. Consume Accep ance E alua ion
Fo each be e age, a consume es was ca ied ou wi h 20 as e s whe e appea ance, odo , as e,
la o , ex u e, o e all liking, and pu chase p edisposi ion we e e alua ed on a nine-poin hedonic
scale (1 =ex emely dislike, 9 =ex emely like) [
17
]. The es s we e pe o med in an ai -condi ioned
oom wi h whi e ligh a app oxima ely 21◦C.
2.6. S a is ical Analysis
All he ob ained esul s we e subjec ed o analysis o a iance (ANOVA) wi h Duncan’s pos
hoc es a 95% con idence le el and o linea eg ession analysis in he Windows so wa e called
STATISTICA, e sion 13.3 (S a So Inc., Tulsa, OK, USA).
Values ob ained by he consume es we e analyzed by he K uskal–Wallis es ( es H).
3. Resul s and Discussion
3.1. Cocoa Bean Shell—Chemical and Nu i ional Composi ion
Thechemical andnu i ionalcomposi iono hecocoabeanshellemployedinbe e agep epa a ion,
exp essed o 100 g o d ied p oduc , was as ollows: p o ein: 20.9 g, a : 2.3 g, ca bohyd a es: 7.85 g,
die a y ibe : 55.1 g (42.3 g o insoluble ibe and 12.8 g o soluble ibe ), wa e : 5.9 g, and ash: 7.9 g.
3.2. Be e age Yield
Thi y o mula ions we e de eloped, esul ing in 30 be e ages wi h di e en yields, mos ly
depending on he CBS GD (Table 1). Yields anged om 72.0% o 93.3% when GDs abo e 500
µ
m we e
used, and a no able subs an ial dec ease in he eco e y pe cen age was obse ed wi h a educ ion in
he GD, he eby leading o such alues as 29.6% o he be e age p epa ed wi h he Moka and GD5.
This dec ease could be mos ly due o he wa e -holding capaci y o he insoluble ibe p esen in he
CBS; his ibe became mo e a ailable when he su ace- o- olume a io o he CBS powde s inc eased.
In some cases, also he la ge CBS powde quan i ies used in o de o ob ain echnologically ealis ic
p epa a ions in luenced he wa e -holding capaci y. Ne e heless, his in luence may no a ec he
be e ages ob ained wi h o he co ee make s such as he Ame ican po o F ench p ess whe e he CBS
quan i ies emain he same a all he GDs, and he educ ion in he yield is due o he lowe GD only.
Fo he lowes GD (GD5, 250–500
µ
m), he be e age yields dec eased conside ably, and his
pa ame e , in gene al, los he epea abili y obse ed o la ge GDs, mos ly owing o he echnological
p oblems du ing be e age p epa a ion such as machine blockage in cases whe e he CBS abso bed
oo much wa e o wi h ex emely long p epa a ion pe iods. Due o he a o emen ioned p oblems,
he be e ages ob ained by he Moka, Neapoli an, Esp esso, and Capsule echniques wi h GD5 we e
Nu ien s 2019,11, 867 6 o 19
assumed o be no echnologically iable and i was no possible o p oceed wi h u he analyses.
Only he be e ages ob ained wi h he F ench p ess and Ame ican echniques we e conside ed o GD5.
Table 1.
Amoun s o cocoa bean shell (CBS) powde and wa e u ilized o he be e age p epa a ions,
olume o he ob ained be e ages, and p ocess yield egis e ed o each p oduc ion echnique
and GD. Analysis o a iance (ANOVA) was pe o med on he p ocess yields among GDs and
ex ac ion echniques.
Moka Neapoli an Ame ican Esp esso Capsule F ench p ess Sig.
>4000 µm
Wa e (mL) 230.00 400.00 200 n/a n/a 100.00
***
CBS powde (g) 10.00 14.00 8.00 7.00 2.00 6.00
Be e age (mL) 188.00 ±3.46 373.33 ±2.89 172.00 ±1.73 123.67 ±1.53 59.33 ±0.58 82.67 ±0.58
Yield (%) 81.74 ±1.51 bC 93.33 ±0.72 aA 86.00 ±0.87 aB n/a n/a82.67 ±0.58 aC
2000–4000 µm
Wa e (mL) 230.00 400.00 200.00 n/a n/a 100.00
***
CBS powde (g) 18.00 17.00 8.00 13.00 2.80 6.00
Be e age (mL) 190.00 ±2.00 363.33 ±2.89 159.67 ±6.81 125.67 ±2.08 60.33 ±0.58 83.00 ±1.00
Yield (%) 82.61 ±>0.87 abBC 90.83 ±0.72 aA 79.83 ±3.40 bC n/a n/a83.00 ±1.00 aB
1000–2000 µm
Wa e (mL) 230.00 400.00 200.00 n/a n/a 100.00
***
CBS powde (g) 22.00 30.00 8.00 16.00 6.50 6.00
Be e age (mL) 188.33 ±1.53 329.66 ±1.53 152.67 ±1.15 122.67 ±0.29 60.33 ±0.58 78.67 ±1.15
Yield (%) 81.88 ±0.66 bA 82.42 ±0.38 aA 76.33 ±0.58 cC n/a n/a78.67 ±1.15 bB
500–1000 µm
Wa e (mL) 230.00 400.00 200.00 n/a n/a 100.00
***
CBS powde (g) 26.00 30.00 8.00 18.00 8.00 6.00
Be e age (mL) 186.67 ±2.31 316.67 ±5.77 148.67 ±4.16 119.67 ±0.58 60.33 ±0.58 72.00 ±1.73
Yield (%) 81.16 ±1.00 bA 79.17 ±1.44 aA 74.33 ±2.08 cB n/a n/a72.00 ±1.73 cC
250–500 µm
Wa e (mL) 230.00 400.00 200.00 n/a n/a 100.00
***
CBS powde (g) 26.00 30.00 8.00 18.00 8.00 6.00
Be e age (mL) 68.00 ±13.86 200.00 ±81.85 146.67 ±2.31 71.00 ±9.64 36.35 ±0.28 71.33 ±1.53
Yield (%) 29.57 ±6.02 cC 50.00 ±20.46 bB 73.33 ±1.15 cA n/a n/a71.33 ±1.53 cA
Sig. *** ** *** n/a n/a ***
n/a, no applicable. Means ollowed by di e en lowe case supe indexes wi hin he same column (di e en g inding
deg ees) and by uppe case supe indexes wi hin he same ow (di e en echniques) a e signi ican ly di e en a
p<0.05. Signi icance: ** p<0.01; *** p<0.001. Da a a e exp essed as mean alues (n=3) ±s anda d de ia ion.
3.3. Physico-Chemical Cha ac e iza ion
3.3.1. Acidi y, D y Ma e , and Colo
The pH and he i a able acidi y esul s ob ained o he unc ional be e ages a e shown in Table 2.
The be e ages had pH and i a able acidi y anging om 4.84 o 5.19 and om 0.12 o 1.64 g o ace ic
acid equi alen s pe li e o a be e age, espec i ely. In gene al, lowe pH and highe acidi y we e
obse ed in be e ages p oduced wi h a lowe GD, excep o he be e ages p oduced wi h he Moka
and he Neapoli an echniques, which showed nonsigni ican di e ences in pH when he GD was
a ied. Ne e heless, hey s ill showed he same endency o acidi y, which inc eased wi h a dec ease
in pa icle size, p obably owing o a majo acid ex ac ion when he CBS su ace- o- olume a io was
inc eased. This endency was no obse ed o he be e age p oduced wi h he F ench p ess echnique
whe e pH and acidi y we e ound o be independen o he CBS pa icle size.
Fo all he be e ages ob ained by pe cola ion echniques (Moka, Neapoli an, Ame ican, Esp esso,
and Capsule), he quan i y o d y ma e inc eased when he GD was educed (Table 2) because he
ex ac ion seemed o be mo e e ec i e a low GD. Fo F ench p ess, he quan i y o d y ma e did no
co ela e wi h he GD o CBS, sugges ing ha he ex ac ion by his mace a ion echnique was ba ely
a ec ed by he pa icle size o he CBS powde .
Nu ien s 2019,11, 867 7 o 19
Table 2.
pH, i a able acidi y, d y ma e , and CIELab alues o he be e ages ob ained by each ex ac ion echnique and g inding deg ees (GD) o CBS, and ANOVA
among GDs o each echnique.
Technique G inding Deg ee (µm) pH Ti a able acidi y D y weigh L*a*b*
(g ace ic acid eq/L) (%)
Moka
>4000 4.91 ±0.05 a0.40 ±0.05 d0.52 ±0.06 d82.73 ±1.51 a5.69 ±1.36 d50.84 ±3.99 c
2000–4000 4.88 ±0.01 a0.61 ±0.05 c0.84 ±0.11 c75.91 ±0.64 b11.65 ±1.06 c64.77 ±2.83 b
1000–2000 4.89 ±0.03 a0.95 ±0.06 b1.53 ±0.06 b61.20 ±2.83 c25.76 ±1.85 b80.92 ±0.54 a
500–1000 4.93 ±0.01 a1.64 ±0.09 a2.70 ±0.17 a33.21 ±1.96 d37.75 ±1.12 a55.08 ±3.30 c
250–500 n/a n/a n/a n/a n/a n/a
Signi icance ns *** *** *** *** ***
Neapoli an
>4000 4.92 ±0.03 a0.29 ±0.03 c0.41 ±0.01 c82.76 ±0.70 a5.52 ±0.50 c48.74 ±1.33 c
2000–4000 4.91 ±0.04 a0.37 ±0.04 c0.49 ±0.06 c80.56 ±2.80 a7.33 ±2.19 c54.14 ±5.51 c
1000–2000 4.88 ±0.03 a0.95 ±0.08 b1.50 ±0.15 b56.56 ±4.34 b28.45 ±2.89 b79.31 ±0.68 a
500–1000 4.93 ±0.01 a1.23 ±0.04 a2.00 ±0.13 a39.04 ±2.66 c37.24 ±0.70 a63.97 ±4.02 b
250–500 n/a n/a n/a n/a n/a n/a
Signi icance ns *** *** *** *** ***
Ame ican
>4000 4.96 ±0.01 b0.26 ±0.01 d0.36 ±0.01 c87.98 ±0.53 a1.98 ±0.18 e38.34 ±0.68 e
2000–4000 5.07 ±0.04 a0.37 ±0.02 c0.51 ±0.04 bc 85.17 ±0.64 b4.02 ±0.51 d48.39 ±1.86 d
1000–2000 5.07 ±0.01 a0.40 ±0.01 b0.69 ±0.01 b80.12 ±0.34 c8.35 ±0.26 c59.79 ±0.60 c
500–1000 4.99 ±0.02 b0.55 ±0.01 a1.01 ±0.26 a73.99 ±1.33 d14.54 ±1.37 b71.05 ±1.92 b
250–500 4.87 ±0.01 c0.57 ±0.01 a1.00 ±0.04 a64.30 ±0.74 e23.80 ±0.72 a80.12 ±0.31 a
Signi icance *** *** *** *** *** ***
Esp esso
>4000 5.09 ±0.07 a0.20 ±0.02 d0.28 ±0.04 d91.14 ±1.37 a0.58 ±0.69 d29.45 ±3.67 d
2000–4000 4.98 ±0.04 b0.37 ±0.04 c0.51 ±0.05 c86.15 ±1.81 b3.32 ±1.29 c44.39 ±4.08 c
1000–2000 4.98 ±0.01 b0.61 ±0.05 b0.89 ±0.07 b77.63 ±2.16 c10.61 ±2.13 b64.10 ±3.74 b
500–1000 4.87 ±0.02 c1.08 ±0.02 a1.62 ±0.05 a65.87 ±0.52 d22.10 ±0.51 a79.52 ±0.69 a
250–500 n/a n/a n/a n/a n/a n/a
Signi icance ** *** *** *** *** ***
Capsule
>4000 5.19 ±0.04 a0.12 ±0.01 d0.15 ±0.00 d94.73 ±0.12 a−0.47 ±0.05 c18.15 ±0.56 d
2000–4000 5.15 ±0.04 a0.17 ±0.01 c0.24 ±0.00 c92.73 ±0.21 a−0.08 ±0.06 c25.28 ±0.90 c
1000–2000 5.15 ±0.02 a0.43 ±0.00 b0.66 ±0.01 b82.41 ±0.67 b6.22 ±0.58 b53.72 ±1.56 b
500–1000 4.84 ±0.01 b0.93 ±0.03 a1.33 ±0.06 a68.71 ±1.92 c19.06 ±1.73 a75.91 ±1.66 a
250–500 n/a n/a n/a n/a n/a n/a
Signi icance *** *** *** *** *** ***
F ench p ess
>4000 4.91 ±0.05 ab 0.62 ±0.00 a0.91 ±0.06 a77.73 ±1.53 b10.80 ±1.47 a67.09 ±2.30 a
2000–4000 4.94 ±0.02 ab 0.65 ±0.05 a0.91 ±0.05 a76.48 ±2.49 b11.97 ±2.55 a68.31 ±4.37 a
1000–2000 4.94 ±0.01 ab 0.50 ±0.04 b0.86 ±0.10 a77.42 ±2.71 b10.96 ±2.65 a65.83 ±4.68 a
500–1000 4.96 ±0.01 a0.40 ±0.02 c0.66 ±0.05 b82.23 ±1.42 a6.53 ±1.27 b56.03 ±3.50 b
250–500 4.90 ±0.02 b0.49 ±0.03 b0.83 ±0.05 a75.22 ±0.36 b12.75 ±1.40 a67.87 ±3.18 a
Signi icance ns *** ** * * *
n/a, no applicable. Means ollowed by di e en le e s a e signi ican ly di e en a p<0.05. Signi icance: * p<0.05; ** p<0.01; *** p<0.001; ns =no signi ican . Da a a e exp essed as
mean alues (n=3) ±s anda d de ia ion.
Nu ien s 2019,11, 867 8 o 19
Rega ding he ch oma ic pa ame e s (Table 2), gene ally, he b igh ness pa ame e dec eased
wi h he dec easing GD due o he inc ease in he su ace- o- olume a io o he CBS powde , he eby
allowing o be e ex ac ion o colo pigmen s. The be e ages ha ing he lowes alues o L* and
he e o e, da ke be e ages, we e hose ob ained a he GD4 wi h Moka and Neapoli an echniques.
Gene ally, bo h pa ame e s a* and b* ose wi h he dec easing GD, as he be e ages became b owne .
Always ollowing a simila end, a ious be e ages showed signi ican di e ences when he GD
was changed excep o he be e ages p oduced wi h he F ench p ess. This ac can be nume ically
explained by he
∆
E pa ame e (da a no shown), which de e mines whe he wo colo s can be
dis inguished by he human eye (
∆
E>2.5). Conside ing he di e en CBS GDs wi hin each echnique,
he be e ages showed alues o
∆
E highe han 2.5 excep o some be e ages ob ained wi h he F ench
p ess echnique, whe e
∆
E
GD1-GD2
=2.10,
∆
E
GD1-GD3
=1.31, and
∆
E
GD2-GD5
=1.55, which means ha
hese be e ages had colo s indis inguishable o he human eye.
3.3.2. Polyphenolic Con en
TPC, TFC, and TTC da a a e p esen ed in Figu e 1(Figu e 1a, Figu e 1b, and Figu e 1c, espec i ely).
Nu ien s 2019, 11, x FOR PEER REVIEW 8 o 20
Figu e 1. To al phenolic con en (TPC), (a), o al la onoid con en (TFC) (b), and o al annin con en
TTC (c), and an ioxidan capaci y (d) o he be e ages p oduced by he six echniques a di e en
CBS GDs; ANOVA among GDs o each echnique. GAE = gallic acid equi alen , CE = ca echin
equi alen , and TE = olox equi alen . Di e en le e s indica e signi ican di e ences a p < 0.05.
Signi icance: * p < 0.05; ** p < 0.01; *** p < 0.001; ns = no signi ican .
TPC a ied conside ably among he be e ages ob ained by di e en echniques and a a ied
GDs, wi h alues ha anged om 126.86 mg GAE/L o he be e age ob ained by he Capsule
echnique a GD1 o 1803.83 mg GAE/L o he be e age ob ained by he Moka echnique using GD4.
Excep o he be e age ob ained wi h he F ench p ess, la ge signi ican di e ences we e de ec ed
o all he o he be e ages when he GD o he CBS was a ied. Fo he be e ages ob ained by
pe cola ion echniques, TPC inc eased wi h a educ ion in he GD, whe eas o he be e age
p oduced by he mace a ion echnique (F ench p ess), TPC alues we e no in luenced by he GD.
The highes phenolic con en was seen in be e ages p epa ed by he Moka and Neapoli an
echniques, whe e he esul s anged om 276 mg GAE/L a GD1 o 1803.83 mg GAE/L a GD4 and
om 263.67 mg GAE/L a GD1 o 1545.87 mg GAE/L a GD4, espec i ely. The be e age ob ained by
he Capsule echnique mani es ed he lowes alues o his pa ame e , anging om 126.86 mg
GAE/L a GD1 o 671.68 mg GAE/L a GD4. On he o he hand, conside ing he in ake, one cup (200
mL) o he be e age ob ained by he F ench p ess echnique a any GDs o by he Ame ican
echnique using CBS a GDs 3–5, p o ided he same quan i y o polyphenols as one cup (60 mL) o
he be e ages ob ained by he Moka o he Neapoli an echniques wi h CBS a GD4. The di e en
alues o TPC ob ained o he be e ages anged be ween a ious alues p esen ed in p e ious
s udies, whe e be e ages hough o ha e ‘high-polyphenol con en ’ we e e alua ed. The alues o
TPC in he p esen s udy we e in mos cases e en highe han hose ound by Zujko & Wi kowska
[18] o d inking chocola e (600 mg GAE/L) o ho cocoa (300 mg GAE/L), bu also highe in some
cases han hose o di e en ea ypes such as whi e ea (1040 mg GAE/L), g een ea (850 mg GAE/L),
black ea (720 mg GAE/L), and ed ea (380 mg GAE/L). Repo ed TPC alues o ed wine (2410 mg
GAE/L) and whi e wine (260 mg GAE/L) [18] we e also wi hin he ange o he alues ob ained o
dcedc
b
c
cdc
c
b
b
b
cb
b
c
a
a
b
aa
d
a
a
0
500
1000
1500
2000
Moka
***
Neapoli an
***
Ame ican
***
Esp esso
***
Capsule
***
F ench
P ess
**
To al phenolic con en (mg GAE/L)
> 4000μm2000-4000μm1000-2000μm500-1000μm250-500μm
(a)
ccedc
b
c
cdc
c
a
bb
cb
bb
a
a
b
aa
c
a
a
0
100
200
300
400
500
600
Moka
***
Neapoli an
***
Ame ican
***
Esp esso
*
Capsule
***
F ench
P ess
***
To al la onoid con en (mg CE/L)
> 4000μm2000-4000μm1000-2000μm500-1000μm250-500μm
(b)
dc
edc
ab
ccdcc
b
b
b
c
b
bbc
a
a
b
aa
c
a
a
0
100
200
300
400
Moka
***
Neapoli an
***
Ame ican
***
Esp esso
***
Capsule
***
F ench P ess
ns
To al annin con en (mg CE/L)
> 4000μm2000-4000μm1000-2000μm500-1000μm250-500μm
(c)
cc
ddb
b
bbc
cc
b
a
b
b
c
ba
d
a
a
b
aa
c
a
a
0
1
2
3
4
5
6
7
8
Moka
***
Neapoli an
***
Ame ican
***
Esp esso
***
Capsule
***
F ench P ess
**
An ioxidan capaci y (mmol TE/L)
> 4000μm2000-4000μm1000-2000μm500-1000μm250-500μm
(d)
Figu e 1.
To al phenolic con en (TPC), (
a
), o al la onoid con en (TFC) (
b
), and o al annin con en
TTC (
c
), and an ioxidan capaci y (
d
) o he be e ages p oduced by he six echniques a di e en CBS
GDs; ANOVA among GDs o each echnique. GAE =gallic acid equi alen , CE =ca echin equi alen ,
and TE = olox equi alen . Di e en le e s indica e signi ican di e ences a p<0.05. Signi icance:
*p<0.05; ** p<0.01; *** p<0.001; ns =no signi ican .
TPC a ied conside ably among he be e ages ob ained by di e en echniques and a a ied GDs,
wi h alues ha anged om 126.86 mg GAE/L o he be e age ob ained by he Capsule echnique a
GD1 o 1803.83 mg GAE/L o he be e age ob ained by he Moka echnique using GD4. Excep o he
be e age ob ained wi h he F ench p ess, la ge signi ican di e ences we e de ec ed o all he o he
be e ages when he GD o he CBS was a ied. Fo he be e ages ob ained by pe cola ion echniques,
Nu ien s 2019,11, 867 9 o 19
TPC inc eased wi h a educ ion in he GD, whe eas o he be e age p oduced by he mace a ion
echnique (F ench p ess), TPC alues we e no in luenced by he GD. The highes phenolic con en was
seen in be e ages p epa ed by he Moka and Neapoli an echniques, whe e he esul s anged om
276 mg GAE/L a GD1 o 1803.83 mg GAE/L a GD4 and om 263.67 mg GAE/L a GD1 o 1545.87 mg
GAE/L a GD4, espec i ely. The be e age ob ained by he Capsule echnique mani es ed he lowes
alues o his pa ame e , anging om 126.86 mg GAE/L a GD1 o 671.68 mg GAE/L a GD4. On he
o he hand, conside ing he in ake, one cup (200 mL) o he be e age ob ained by he F ench p ess
echnique a any GDs o by he Ame ican echnique using CBS a GDs 3–5, p o ided he same quan i y
o polyphenols as one cup (60 mL) o he be e ages ob ained by he Moka o he Neapoli an echniques
wi h CBS a GD4. The di e en alues o TPC ob ained o he be e ages anged be ween a ious
alues p esen ed in p e ious s udies, whe e be e ages hough o ha e ‘high-polyphenol con en ’ we e
e alua ed. The alues o TPC in he p esen s udy we e in mos cases e en highe han hose ound by
Zujko & Wi kowska [
18
] o d inking chocola e (600 mg GAE/L) o ho cocoa (300 mg GAE/L), bu also
highe in some cases han hose o di e en ea ypes such as whi e ea (1040 mg GAE/L), g een ea
(850 mg GAE/L), black ea (720 mg GAE/L), and ed ea (380 mg GAE/L). Repo ed TPC alues o ed
wine (2410 mg GAE/L) and whi e wine (260 mg GAE/L) [18] we e also wi hin he ange o he alues
ob ained o he be e ages s udied in he p esen wo k. Rega ding he alues epo ed o some ui
juices by Ga dne e al. [
19
] such as o ange (755 mg GAE/L), apple (339 mg GAE/L), o pineapple juice
(358 mg GAE/L), he le els o o al phenols in CBS be e ages we e always be ween hese alues o
e en highe .
Fla onoids we e he main compounds ha con ibu ed o TPC, cons i u ing om 20.8% o 34.7%
o his alue, depending on he p epa a ion echnique bu hese con ibu ions emained cons an a
di e en GDs wi hin each echnique. TFC and TPC we e highly co ela ed ( =0.9965), and hus he
o me ollowed he same endencies o abundance depending on he echnique and GD. In his way,
he highes alue o TFC was seen in he be e ages ob ained by he Moka echnique a GD4 (566.42 mg
CE/L), ollowed by he be e age ob ained by he Neapoli an echnique wi h GD4 (489.43 mg CE/L).
Be e ages ob ained by he wo echniques had highe alues a each GD compa ed wi h he o he
echniques excep o he F ench p ess, o which he be e ages showed sligh di e ences wi h he
a ia ion o he GD. The TFC alues o he be e ages p oduced by he F ench p ess echnique anged
be ween 97.98 and 155.63 mg CE/L and he e o e had he highes alues when la ge CBS pa icle
sizes we e chosen. Again, he lowes alues we e obse ed o he Capsule be e age, which had TFC
be ween 8.78 and 198.47 mg CE/L, which inc eased wi h a educ ion in he CBS pa icle size.
Values o TTC accoun ed o 3.4% o 19.2% o TPC wi h a signi ican ly high co ela ion ( =0.9968).
Con a y o wha was obse ed be ween TPC and TFC, he pe cen age o TTC’s con ibu ion o TPC
inc eased wi h a educ ion in he GD wi hin he alues ob ained o each echnique, excep o he
be e ages p oduced by he mace a ion echnique, which main ained he con ibu ion o he annin
con en (12.0% o 13.9%) o TPC alues independen ly o he GD. This ac can be no iced as he
di e ences be ween big and small CBS pa icle sizes in TTC alues become highe han hose o TPC
and TFC. CBS pa icle size could ha e an in luence on selec i e ex ac ion o some polyphenol g oups
o he pe cola ion echniques, as could be he case o annins. Tannins a e no mally la ge molecules
han la onoids and he e o e, a dec ease in he pa icle size could acili a e hei ex ac ion compa ed o
ha o la onoids, which we e al eady ex ac ed using he CBS powde wi h high pa icle sizes. In his
way, g ea e inc eases we e obse ed o TTC wi hin he same echnique when he pa icle size was
educed in compa ison wi h hose obse ed o TPC and TFC. Ne e heless, he highes concen a ions
o annins we e again de ec ed in he Moka and Neapoli an be e ages a GD4 (334.64 and 296.06 mg
CE/L, espec i ely) and he lowes alues o TTC we e obse ed in he Capsule be e age, which anged
om 5.18 o 99.86 mg CE/L.
Mo e han 30 polyphenolic compounds ha may con ibu e o he abo e alues we e de ec ed
and quan i ied by HPLC analysis. Only he concen a ions de e mined o he main cocoa ma ke
phenolic compounds and hose showing he highes concen a ions a e gi en in Table 3. Belonging
Nu ien s 2019,11, 867 16 o 19
3.6. Technological E iciency o Polyphenol Ex ac ion
To iden i y he echnique allowing highe ex ac ion yield o polyphenols, no maliza ion o he
TPC alues was pe o med acco ding o he di e en CBS powde amoun s and wa e quan i ies
employed. The new TPC alues, exp essed in millig ams o gallic acid equi alen s pe g am o CBS
anged be ween 2.72 and 16.32 mg GAE/g (Table 5). A signi ican inc ease in TPC was obse ed o he
be e ages ob ained by he o ced pe cola ion echniques (Moka, Esp esso, and Capsule) a GDs below
1000
µ
m and o he be e ages p oduced by he Neapoli an me hod (na u al pe cola ion) a GDs below
2000
µ
m. Fo he be e age p oduced by he Ame ican echnique (na u al pe cola ion), TPC inc eased
p og essi ely wi h he dec easing GDs. On he con a y, o he be e age p oduced wi h he F ench
p ess, no in luence o he GDs was obse ed, he eby leading o he conclusion ha he di e ence in GDs
had only a mino impac on he polyphenol ex ac ion by his echnique based on a mace a ion p ocess.
Neapoli an was always he mos e ec i e echnique when o al phenol ex ac ion was compa ed
among he be e ages ob ained by all he echniques a he same GD, ollowed by hose p oduced by
he Ame ican, F ench p ess, o Moka echniques. The lowes TPC alues belong o he be e ages
p oduced by he Capsule and Esp esso. On he o he hand, hese echniques usually a o ded highe
ex ac ion o essen ial oils because hey p oduce mo e a oma ic co ee. The highes TPC obse ed
(16.32 mg GAE/g o he be e age ob ained wi h Neapoli an using GD4) was signi ican ly highe han
hose epo ed o o he ypes o CBS ex ac ion in p e ious wo ks. He n
á
ndez-He n
á
ndez e al. [
23
]
ob ained a TPC alue o 3 mg GAE/g when ex ac ing 1 g o CBS a 500 µm in 6 mL o wa e a 70 ◦C.
Manzano e al. [
33
] obse ed a TPC alue o 6.04 mg GAE/g using 2 g o CBS sc eened a 75
µ
m,
which was p ocessed in 50 mL o wa e wi h 5-min e lux ex ac ion. Ne e heless, in o he s udies in
which assis ed ex ac ion was ca ied ou , highe alues o TPC we e ob ained as compa ed o hose
ob ained o he be e ages in he p esen wo k, as expec ed. Nso -A indana e al. [
32
] epo ed a alue
o 17.21 mg GAE/g a e he ex ac ion o 2 g o CBS g ound up a 250
µ
m in 50 mL o wa e using
mic owa es. In any case, i is impo an o no e ha he solid/liquid a io, which was di e en in all
s udies, could also ha e some in luence on hese esul s.
Table 5.
To al phenolic con en o he be e ages a e no maliza ion conside ing CBS and wa e
quan i ies. ANOVA among GDs and ex ac ion echniques. Values a e exp essed in millig ams o gallic
acid equi alen s o each g am o CBS powde employed o he be e age p epa a ion (mg GAE/g CBS).
Moka Neapoli an Ame ican Esp esso Capsule F ench P ess Sig.
>4000 µm5.21 ±0.54 cB 7.03 ±0.47 cA 4.84 ±0.28 dB 3.12 ±0.30 bC 3.76 ±0.25 bC 6.68 ±0.64 aA ***
2000–4000 µm6.37 ±2.25 bcAB 7.14 ±1.06 cA 5.26 ±0.09 dB 2.72 ±0.39 bC 3.25 ±0.30 cC 6.74 ±0.72 aA ***
1000–2000 µm6.92 ±0.72 bB
10.00
±
1.33
bA 7.32 ±0.34 cB 3.41 ±0.33 bD 3.03 ±0.11 cD 5.78 ±0.30 bC ***
500–1000 µm12.94 ±0.96 aB
16.32
±
1.04
aA 9.39 ±0.92 bC 5.10 ±0.15 aD 5.06 ±0.25 aD 4.10 ±0.31 cE ***
250–500 µm n/a n/a
13.45
±
0.86
aA n/a n/a6.29 ±0.26 aB ***
Sig. *** *** *** *** *** *** ***
n/a, no applicable. Means ollowed by di e en lowe case supe indexes wi hin he same column (di e en g inding
deg ees) and by uppe case supe indexes wi hin he same ow (di e en echniques) a e signi ican ly di e en a
p<0.05. Signi icance: *** p<0.001. Da a a e exp essed as mean alues (n=3) ±s anda d de ia ion.
4. Conclusions
The a ious ex ac ion echniques used o CBS g ound o di e en deg ees allowed us o
ob ain be e ages wi h di e en chemical cha ac e is ics and consume - ela ed pa ame e s. Se e al
compounds we e iden i ied and quan i ied by HPLC (phenolic acids, la an-3-ols, que ce in-glycosides,
ca echin-glycosides, and p ocyanidins), which may unde lie he high adical sca enging capaci y
and signi ican
α
-glucosidase inhibi ion esul s shown by he be e ages. The GD was op imized
o each ex ac ion echnique; he smalles GDs allowed us o ob ain he mos unc ional be e ages
when using pe cola ion echniques, whe eas he mace a ion echnique (F ench p ess) in gene al,
showed no dependence on he CBS pa icle size. This inding may be o g ea in e es as wi h he
F ench p ess echnique, no u he CBS g inding ea men s will be needed o ob ain a be e age
ha ing high-po en ial biological ac i i ies. In e ms o consume accep ance, i was ound ha , in
Nu ien s 2019,11, 867 17 o 19
gene al, he mos ac i e be e ages we e he leas app ecia ed as a as as e and la o a e conce ned,
p obably because o he bigge p esence o polyphenols and me hylxanhines. This ac could open
a possibili y o u he esea ch in o de o op imize hese be e ages, aiming a a highe consume
accep ance. Such op imiza ion could be achie ed by bioac i e compounds encapsula ion o by adding
new pleasan ing edien s, among o he op ions.
Fo he i s ime, i was demons a ed ha he Moka and Neapoli an echniques may be he mos
e ec i e me hods o polyphenol ex ac ion, a o ding he highes adical sca enging ac i i y and
α
-glucosidase inhibi ion capaci y, whe eas he be e age p oduced by he Capsule echnique showed
he poo es ex ac ion. The e o e, his wo k indica es ha CBS may be an op imal ing edien o
home-made unc ional be e ages wi h po en ial heal h bene i s o consume s, he eby educing he
en i onmen al and economic impac o by-p oduc disposal.
Au ho Con ibu ions:
W i ing—o iginal D a P epa a ion, O.R.-P.; In es iga ion, O.R.-P. and L.M.-R.; Re iew
and Edi ing, L.B.-P., G.Z., C.S., and M.B.; Concep ualiza ion, L.B.-P., G.Z., and M.B.; Supe ision, L.B.-P., G.Z.,
and C.S.; P ojec Adminis a ion, L.B.-P. and G.Z.; Funding acquisi ion, L.B.-P. and G.Z.
Funding:
The p esen wo k has been suppo ed by COVALFOOD “Valo isa ion o high added- alue compounds
om cocoa indus y by-p oduc s as ood ing edien s and addi i es”. This p ojec has ecei ed unding om he
Eu opean Union’s Se en h F amewo k p og am o esea ch and inno a ion unde he Ma ie Skłodowska-Cu ie
g an ag eemen No. 609402-2020 esea che s: T ain o Mo e (T2M).
Acknowledgmen s:
O. Rojo-Po eda g a e ully acknowledges he Uni e si y o Tu in o he awa d o a PhD
s uden ellowship. L. Ba bosa-Pe ei a is g a e ul o he Eu opean Union’s Se en h F amewo k p og am o he
Ma ie Skłodowska-Cu ie g an .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Abb e ia ions
CBS, cocoa bean shell; GD, g inding deg ee; TPC, o al phenolic con en ; TFC, o al la onoid con en ; TTC,
o al annin con en , RSA, adical sca enging ac i i y; GAE, gallic acid equi alen s; CE, ca echin equi alen s;
TE, olox equi alen s; RP-HLPC-PDA, e e sed-phase high-p essu e liquid ch oma og aphy equipped wi h
pho odiode a ay de ec o ; PB1, p ocyanidin B1; PB2, p ocyanidin B2; Q-3-G, que ce in-3-O-glucoside; ANOVA,
analysis o a iance.
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