Academic Edi o : José An ónio Cou o
Recei ed: 28 No embe 2024
Re ised: 10 Decembe 2024
Accep ed: 20 Decembe 2024
Published: 27 Decembe 2024
Ci a ion: Picon, A.; Campane o, Y.;
Sánchez, C.; Ál a ez, I.; Rod íguez-
Mínguez, E. Valo iza ion o Co ee
Che y By-P oduc s Th ough
Fe men a ion by Human In es inal
Lac obacilli in Func ional Fe men ed
Milk Be e ages. Foods 2025,14, 44.
h ps://doi.o g/10.3390/
oods14010044
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Licensee MDPI, Basel, Swi ze land.
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A icle
Valo iza ion o Co ee Che y By-P oduc s Th ough
Fe men a ion by Human In es inal Lac obacilli in Func ional
Fe men ed Milk Be e ages
An onia Picon 1,* , Yolanda Campane o 1, Ca men Sánchez 1, Inmaculada Ál a ez 2and
E a Rod íguez-Mínguez 1
1
Depa amen o de Tecnología de Alimen os, INIA, CSIC, Ca e e a de La Co uña Km 7, 28040 Mad id, Spain
2Unidad de Se icio de Técnicas Analí icas, ICTAN, CSIC, Calle JoséAn onio No ais 6, 28040 Mad id, Spain
*Co espondence: [email p o ec ed]
Abs ac : Du ing co ee p oduc ion, he emo al and disposal o he co ee bean-
su ounding laye s pose an en i onmen al p oblem. In his wo k, we examined he
e ec s o se e al aqueous co ee che y ex ac s on he g ow h and me abolism, bio ilm
o ma ion, an ioxidan capaci y and an imic obial ac i i y o six lac obacilli om he INIA
collec ion and a comme cial p obio ic Lac obacillus hamnosus GG s ain. G ow h medium
supplemen a ion wi h di e en co ee che y ex ac s (a 40%) s imula ed s ain g ow h
and me abolism. The g ound che y pulp ex ac (CPE) wi h he highes o al polyphenol
con en was selec ed o u he use. This CPE con ained alkaloids, phenolic acids and
la onoids. Upon CPE supplemen a ion, some s ains signi ican ly (p< 0.01) inc eased
bio ilm o ma ion, while all s ains inc eased an ioxidan capaci y and an imic obial ac i i y.
A e p elimina y es s, we de eloped h ee bi unc ional dai y p oduc s, con aining 20%
CPE and e men ed wi h s ains INIA P495, INIA P708 o GG. These s ains main ained
high le els a e manu ac u e, e ige a ed s o age, and h oughou an
in i o
p ocedu e
mimicking gas oin es inal ac condi ions. Compa ed o con ols, CPE-con aining p od-
uc s showed inc eased le els o o al polyphenol compounds, an ioxidan capaci y and
an imic obial ac i i y, oge he wi h posi i e senso y cha ac e is ics. CPE and hese selec ed
s ains could hus be used o elabo a e inno a i e unc ional e men ed milk p oduc s.
Keywo ds: co ee che y pulp; lac obacilli; an ioxidan capaci y; an imic obial ac i i y;
e men ed milk; s a e s; e men ed oods; unc ional oods
1. In oduc ion
Co ee is one o he mos widely consumed be e ages in he wo ld. The es ima ed o al
wo ld co ee p oduc ion in 2022 was 10.8 million ons [
1
]. The co ee sec o is s ill g owing
due o inc easing consump ion in eme ging economies, a s ong in e es in special y co ee
and p oduc inno a ions in de eloped coun ies [
2
]. The wo majo co ee species consumed
wo ldwide a e Co ea a abica (A abica) and Co ea canepho a (Robus a). A abica co ee is
conside ed o be supe io o Robus a due o i s o ganolep ic p ope ies [
3
]. In e es in C.
cane o a has been s eadily g owing, howe e , due o i s conside able esis ance o clima e
change [4].
Rele an ac o s de e mining he co ee quali y include ag icul u al p ocedu es (mos ly
gene ic makeup o he beans and hei ipeness, cul i a ion and ha es ing me hods,
seasonali y, al i ude and clima e), p ocessing me hods, bean oas ing and g inding, as
well as be e age p epa a ion and consump ion [
5
]. Since co ee is p oduced om oas ed
Foods 2025,14, 44 h ps://doi.o g/10.3390/ oods14010044
Foods 2025,14, 44 2 o 18
beans, he su ounding laye s (sil e skin, pa chmen , mucilage, pulp and skin) ha e o be
emo ed and a e disca ded as was e [
6
]. Unsa e disposal o he co esponding was e has
a nega i e impac on he en i onmen in co ee-p oducing coun ies, due o high acidi y
and ca eine, polyphenol and annin concen a ions [
7
]. To imp o e he sus ainabili y o
co ee p oduc ion, bioac i e compounds such as alkaloids, melanoidins and polyphenols
could be ob ained om hese by-p oduc s and used o he de elopmen o nu aceu icals,
unc ional oods o cosme ics [
6
]. Se e al e iews dealing wi h di e en aspec o co ee
by-p oduc alo iza ion ha e been ecen ly published, p o iding composi ional da a, hei
inco po a ion as ing edien s in ood o mula ions and new p oduc de elopmen [
8
],
applica ion o e men a ion in he co ee indus y [
9
], and as p ebio ic ing edien s [
10
].
These e iews highligh he g ea indus ial po en ial o co ee by-p oduc s.
Polyphenol compounds a e seconda y plan me aboli es wi h se e al p o ec i e oles
o humans. They may ac locally agains oxida i e damage a he gas oin es inal ac
(GIT), and sys emically by educing he isk o se e al ch onic diseases [
11
]. Polyphenols a e
classi ied as p ebio ics, i.e., subs a es ha a e selec i ely u ilized by hos mic oo ganisms
con e ing heal h bene i s by he In e na ional Scien i ic Associa ion o P obio ics and
P ebio ics [
12
]. The e o e, dai y p oduc heal h bene i s could be u he imp o ed by
supplemen a ion wi h polyphenol- ich ex ac s [13].
Co ee mic obio a mos ly comp ise bac e ia (lac obacilli, Bacillus,A h obac e ,Acine-
obac e ,Klebsiella and Weissella) and yeas (Saccha omyces,Pichia,Candida,Rhodo o ula,
Hanseniaspo a and Kluy e omyces) [
14
]. Du ing co ee p ocessing, co ee mic obio a plays a
pi o al ole in e men a ion, signi ican ly impac ing he co ee’s chemical composi ion and
senso y a ibu es [5].
Mo eo e , lac obacilli ha e a p ominen ole in e men ed dai y p oduc s [
15
]. They
a e ecognized as human gu mic obio a commensals and ha e been used as p obio ics o
a long ime [
15
]. Lac obacilli a e able o ans o m complex die a y polyphenol compounds,
inc easing hei bioa ailabili y [
16
]. Depending on indi idual mic obio a composi ion,
howe e , g ea a ia ions in polyphenol ans o ma ion ha e been obse ed [17].
Consume s wi h limi ed endogenous polyphenol- ans o ming abili ies in pa icula
could bene i om he supplemen a ion o dai y p oduc s wi h polyphenol- ich ex ac s.
Fe men a ion wi h ans o ma ion-capable in es inal lac obacilli s ains could hus p o ide
a cos -e ec i e me hod o each a wide public.
The aims o his wo k we e: (1) o examine he e ec s o aqueous co ee che y ex ac s
on se e al lac obacilli s ains (mos o hem om in es inal o igin), and (2) o de elop
bi unc ional dai y p oduc s con aining a co ee che y pulp ex ac (CPE) and one o he
s ains able o inc ease i s polyphenol bioa ailabili y.
2. Ma e ials and Me hods
2.1. Co ee Che y Ex ac P epa a ion and P elimina y Cha ac e iza ion
The co ee (Co ea canepho a) che ies used in his wo k we e collec ed and sun-d ied by
local a me s o Ca éau Ligh G oup S.L. (Mad id, Spain) in Guinea Conak y du ing he
2022 ha es season. A e ai anspo , washing and d ying, hey we e acuum packaged
and s o ed a –30 ◦C un il p ocessing.
A e hawing, pulp and bean we e mechanically sepa a ed. Pa o he pulp was
g ound in a co ee g inde (Ja a, Model ML132, Tudela (Na a a), Spain) o a homogeneous
powde wi h a pa icle size o less han 2 mm. Fou ypes o ex ac ion we e pe o med,
each wi h 10 olumes o s e ile dis illed wa e (in a simila way o p epa ing an he bal ea).
Fo he i s wo, whole pulp o g ound pulp we e mixed wi h boiling wa e and kep a
oom empe a u e o 7 min. Fo he hi d me hod, g ound pulp was mixed wi h s e ile
wa e a 4
◦
C and kep a his empe a u e o 30 min. Fo he las me hod, beans we e mixed
Foods 2025,14, 44 3 o 18
wi h boiling wa e and kep a oom empe a u e o 7 min. Ex ac s we e passed h ough a
s ainless-s eel s aine and il e ed h ough a 0.22
µ
m Cy i a Wha man GD/X il e (Global
Li e Science Solu ions Ope a ions UK L d., Li le Chal on , Buckinghamshi e, UK).
To es ex ac s e ili y, in duplica e, 50
µ
L po ions o each ex ac we e sp ead on
pla e coun aga (PCA, Di co, Beck on, Dickinson & Co, Le Pon de Claix, F ancia) and
yeas glucose chlo amphenicol aga (YGC, Di co, Beck on, Dickinson & Co, Le Pon de
Claix, F ancia) pla es. Incuba ions we e pe o med a 30
◦
C o 48 h o PCA pla es and a
25 ◦C o i e days o YCG pla es.
Ex ac pH and abso bance a 600 nm (A
600
, as an indica ion o colo di e ences) we e
measu ed, in iplica e, wi h a Beckman DU 650 spec opho ome e (Beckman Ins umen s
S.A., Mad id, Spain) and a C ison pH-me e (model Basic 20, C ison Ins umen s, Ba celona,
Spain), espec i ely. Following he me hod o Velioglu e al. [
18
], we used gallic acid and he
Folin-Ciocal eau eagen (bo h om Sigma-Ald ich Co., S . Louis, MI, USA.) o de e mine,
in iplica e, he o al polyphenol con en (TPC). The esul s a e ep esen ed as
µ
g o gallic
acid equi alen s (GAE) pe mL.
2.2. S ains Used and G owing Condi ions
Six s ains, pa o he INIA cul u e collec ion, we e used in his wo k. Fi e s ains,
Lac icaseibacillus (Ls.) pa acasei ssp. pa acasei INIA P495 and INIA P708, Ls. hamnosus INIA
P334, Lac obacillus gasse i INIA P508, and Limosilac obacillus mucosae INIA P459 o igina ed
om b eas - ed in an s [
19
]. These i e s ains su i ed he majo GIT condi ions well and
showed a b oad inhibi o y ac i i y and limi ed coagg ega ion wi h bac e ial pa hogens.
Also, hese s ains we e suscep ible o be a-lac amase inhibi o s and p o ein syn hesis
inhibi o s while no bea ing mucinoly ic ac i i y o p oducing biogenic amines [
19
]. S ain
INIA P459 possessed bile sal hyd olase ac i i y and p oduced hyd ogen pe oxide [
19
].
S ain INIA P708 possessed a opy pheno ype and p oduced exopolysaccha ides (EPS).
The six h s ain, Lac iplan ibacillus (Lp.) plan a um ssp. plan a um INIA TAB84, was a dai y
isola e ha p oduces an uncha ac e ized plan a icin [
20
]. As e e ence, he comme cial
p obio ic Ls. hamnosus GG s ain [21] was included in he s udy.
S ains we e anae obically (Anae ogen, Oxoid, Basings oke, UK) g own a 37
◦
C o
24 h
on pla es o de Man, Rogosa and Sha pe medium (MRS, Di co) wi h 0.05% cys eine-HCl
(Sigma-Ald ich Co.) (MRSC) and 1.5% aga (Di co).
2.3. E ec o Co ee Che y Ex ac s on Lac obacilli Me abolism
To es me abolic ac i i y, we ollowed he p ocedu e de eloped in ou p e ious
s udy [
22
]. B ie ly, b ain hea in usion (BHI, Oxoid) medium wi h 0.05% cys eine-HCl,
0.03% me hyl blue (Sigma-Ald ich Co.) and 1.5% aga (Di co) was dissol ed in 60% o he
o al wa e olume and s e ilized. One o he ex ac s, o wa e (con ol pla es), was added
asep ically o make up he o al olume. Solidi ied pla es we e s o ed a 4 ◦C un il use.
D ops (5
µ
L) o bac e ial cell suspensions (in s e ile 0.2% pep one wa e wi h 0.05%
cys eine-HCl, a app oxima ely 8.5 log CFU/mL) we e placed on pla es con aining indi-
idual co ee che y ex ac s o no ex ac (con ol pla es), in duplica e. A e anae obic
incuba ion a 37
◦
C o 24 h, bac e ial g ow h and dye colo al e a ion we e eco ded,
ollowing he code desc ibed in [
22
]: 0 = no g ow h, 1 = ain g ow h wi hou colo change,
2 = g ow h and dye change o blue, 3 = g ow h and a blue ing o up o 2 mm a ound he
d op and 4 = g ow h and a blue ing o mo e han 2 mm a ound he d op.
2.4. P epa a ion o he Co ee Che y Pulp Ex ac (CPE)
The co ee che y pulp was placed in a ki chen obo (The momix, model TM31,
Vo we k España M.S.L., S.C., Mad id, Spain) and subjec ed o blade speeds o 6 o 60 s and
8 o 30 s. App oxima ely 70 g we e weighed in each o he wo glass beake s, mixed wi h
Foods 2025,14, 44 4 o 18
10 olumes o boiling wa e and incuba ed o 7 min in a s o e a 50
◦
C (Memme GmbH,
Schwabach, Ge many). The mix u e was b ie ly s i ed, and pa icula e ma e ial was
emo ed by il e ing h ough a s e ile gauze. Mic obial s e ili y was achie ed by acuum
il a ion h ough a 0.2
µ
m polye he sul one memb ane (TPP Techno Plas ic P oduc s AG,
T asadingen, Swi ze land). S e ile 50 mL ambe plas ic ubes (Eppendo AG, Hambu g,
Ge many) we e used o s o e ex ac aliquo s a –40 ◦C.
2.5. CPE Cha ac e iza ion
Ex ac s e ili y, pH, TPC and A
600
we e de e mined as desc ibed in Sec ion 2.1. CPE
d y ma e was de e mined as pe cen age o he ini ial weigh . The o al suga con en
was measu ed by he colo ime ic phenol-sul u ic acid me hod [23] using glucose (Sigma-
Ald ich Co.) as a s anda d, and p o ein con en was assayed wi h a Coomasie Plus (B ad-
o d) eagen ki (The mo Scien i ic, Rock o d, IL, USA) and bo ine se um albumin (BSA)
as s anda d [
22
]. The Benzie & S ain me hod [
24
], wi h modi ica ions and T olox (Sigma
Ald ich Co.) as s anda d, was ollowed o de e mine he CPE an ioxidan capaci y [
22
]. All
de e mina ions in his sec ion we e pe o med in iplica e.
In o de o iden i y polyphenol compounds, he CPE was subjec ed o HPLC-PAD/
HPLC-ESI-MS, a he ICTAN (CSIC) Analysis Se ice Uni , ollowing he me hod and
equipmen p e iously desc ibed [22]. Analyses we e ca ied ou in duplica e.
2.6. CPE E ec on Lac obacilli G ow h and Me abolism
G ow h expe imen s we e pe o med in BHI b o h wi h 0.05% cys eine-HCl (BHIC)
o analyze, in a quan i a i e manne , he e ec o he CPE on s ain g ow h and su i al.
BHIC b o h was supplemen ed wi h 40% CPE, while b o h wi hou CPE se ed as con ol.
S ains we e inocula ed in b o h a an app oxima e le el o 7.5 log CFU / mL and incuba ed
unde anae obic condi ions a 37
◦
C o 6 days. Mic obial le els, pH and o al polyphenol
con en we e de e mined a days 1 and 6.
2.7. CPE E ec on Lac obacilli Bio ilm Fo ma ion
To s udy he e ec o CPE on bio ilms, we ollowed he me hod o Lee e al. [
25
],
wi h some modi ica ions [
22
]. B ie ly, 200
µ
L o BHIC b o h con aining 0.02% oxgall
(Sigma-Ald ich Co.) and CPE o wa e (con ol) we e placed in each well o 96-mul iwell
pla es (The mo Fishe Scien i ic, Roskilde, Denma k) and inocula ed wi h 2
µ
L o bac e ial
suspensions (p epa ed as desc ibed in Sec ion 2.3). Fo each s ain, wel e eplica es,
di ided o e wo independen expe imen s, we e p epa ed. Non-inocula ed wells we e
included as nega i e con ols. A e 24 h o anae obic incuba ion a 37
◦
C, he liquid phase
was disca ded, and he wells s ained wi h 0.4% c ys al iole solu ion o 30 minu es. A e
washing h ice wi h dis illed wa e , bio ilms we e esuspended in 30% glacial ace ic acid
and quan i ied by eading A
600
in a mic opla e eade (Mul iskan Spec um, The mo Fishe
Scien i ic Oy, Van aa, Finland).
2.8. CPE E ec on Lac obacilli An ioxidan Capaci ies
The an ioxidan capaci y o he s ains was e alua ed by measu ing he educ ion o
he 2,2-diphenyl-1-pic ylhyd acyl (DPPH, Sigma-Ald ich Co.) adical [
26
]. S ains, g own
on BHIC pla es, supplemen ed o no wi h 40% CPE, we e suspended in 0.85% NaCl and
adjus ed o an OD
600
o 1.2. In iplica e, cell suspensions (400
µ
L) we e mixed wi h DPPH
0.2 M (500
µ
L) and incuba ed p o ec ed om ligh a oom empe a u e o 30 min. A e
cen i uga ion a 6026 g o 3 min, A
517
was measu ed. A 0.85% NaCl solu ion (400
µ
L)
was used as blank. To al an ioxidan capaci y (TAC) o each cul u e was calcula ed as he
pe cen age o DPPH educ ion: TAC = [1 −(A517 (sample)/A517 (blank))] ×100.
Foods 2025,14, 44 5 o 18
2.9. CPE E ec on Lac obacilli An imic obial Ac i i ies
CPE an imic obial ac i i y owa d ou pa hogenic s ains o he INIA collec ion was
s udied, in a simila way as p e iously desc ibed [
22
]. G am-posi i e s ains Clos idium
pe ingens CECT 486 (C) and Lis e ia monocy ogenes Sco A CECT 5672 (L) and G am-
nega i e s ains Klebsiella oxy oca INIA col 108 (K) and Salmonella en e ica se o a En e i idis
CECT 4155 (S) we e es ed. S ains we e g own in BHI a 37
◦
C o 24 h unde anae obic
condi ions (Clos idium pe ingens CECT 486) o as s and cul u es.
The s and-alone inhibi o y ac i i y o he CPE on pa hogenic s ains was e alua ed
ollowing he me hod p e iously desc ibed [
22
]. Fi s , 5 mL o 50
◦
C- empe ed 0.5% BHI
so aga , p e iously inocula ed wi h esh cul u es o each pa hogenic s ain a a 0.1% ( inal
concen a ion, app ox. 5.5–6 log CFU/mL), we e laye ed on op o solidi ied BHI pla es, in
duplica e. Nex , 5 mm-diame e wells we e bo ed wi h a s e ile punch. Finally, each well
was illed wi h he se ially dilu ed CPE ex ac (100%, 50%, 40%, 20%, 10%, 5%, 1%, 0.5%,
0.25%, 0.125% and 0.0625%; 50 µL pe well). Pla es we e incuba ed a 37 ◦C o 24 h.
To s udy he combined an imic obial ac i i y o he selec ed s ains ( ou in es inal
s ains plus he GG e e ence s ain) and CPE agains pa hogenic s ains, suspensions o
cells g own on BHIC (con ol) o BHIC + 40% CPE pla es we e p epa ed as desc ibed in
Sec ion 2.3. D ops (5
µ
L) o each suspension we e deposi ed on a new BHI aga pla e. A e
anae obic incuba ion a 37
◦
C o 24 h, a laye o a 50
◦
C- empe ed inocula ed BHI so aga ,
p e iously inocula ed wi h esh cul u es o each pa hogenic s ain (p epa ed as explained
abo e), was pou ed on op. Pla es we e examined o he p esence o inhibi ion halos a e
24 h incuba ion a 37 ◦C.
2.10. Manu ac u e o CPE Fe men ed Milk P oduc s
Be o e s a ing manu ac u ing, a g oup (n = 8) o ained panelis s pe o med a p e-
limina y es o choose a ex ac concen a ion sui able o senso y e alua ion. UHT milk
(Pascual, Bu gos, Spain) samples supplemen ed wi h 20 o 40% CPE ex ac s we e es ed.
Fo he manu ac u e, wo independen expe imen s we e pe o med. UHT milk
(Pascual, Bu gos, Spain) was supplemen ed wi h 20% CPE o s e ile wa e (con ol) and
dis ibu ed in 15 ml s e ile polyp opylene ubes (VWR, Radno , PA, USA). Con ol and
CPE-supplemen ed milk ubes we e inocula ed a 1% wi h each cell suspension (p epa ed
as in Sec ion 2.3) o he wo selec ed s ains (Ls. pa acasei subsp. pa acasei INIA P495
and INIA P708) and he e e ence s ain, Ls. hamnosus GG, a a inal concen a ion o
app oxima ely 7.5 log CFU/mL. Non-inocula ed con ol ubes we e included. All ubes
we e anae obically incuba ed a 37
◦
C o 24 h. Fe men ed milk p oduc s we e in oduced
in a cold chambe (4 ◦C) and s o ed o up o 20 days.
2.11. CPE Fe men ed Milk P oduc Cha ac e iza ion
Fe men ed milk p oduc s we e analyzed a e 1, 12 and 20 days. Mic obial le els we e
de e mined in duplica e, as desc ibed in Sec ion 2.2. To es s ain su i al
in i o
unde
majo GIT condi ions, he me hod de eloped by Langa e al. [
27
] was applied. B ie ly,
e men ed dai y p oduc samples (1 mL) we e mixed wi h a pH 3.0 bu e ed PBS solu ion
(
9 mL
) a 37
◦
C o 90 s. Mix u es we e anae obically incuba ed a 37
◦
C o 1 h. The ea e ,
mix samples (1 mL) we e added o 9 mL o bile solu ion (0.15%, ox-bile desicca ed; Oxoid)
and kep anae obically a 37
◦
C o 1 h, be o e mic obial enume a ion. P oduc an imic obial
ac i i ies we e es ed as desc ibed in Sec ion 2.9.
Fe men ed dai y p oduc pH alues we e de e mined in iplica e. Following he
me hod o del Olmo e al. [
28
], polyphenol compounds we e ex ac ed. B ie ly, samples
(1 mL) we e subjec ed o a i s ex ac ion wi h an HCl-acidi ied (16 mM) me hanol:wa e
(50:50) solu ion (9 mL), and a second one wi h an ace one:wa e (70:30) solu ion (9 mL).
Foods 2025,14, 44 6 o 18
Bo h ex ac ions we e pe o med unde cons an agi a ion a oom empe a u e o 1 h.
Ex ac s we e mixed o a 1:1 p opo ion and il e ed (0.22
µ
m PVDF memb ane il-
e , Millex-GV, Me ck Millipo e L d., Co k, I eland). Fil a es we e used o de e mine,
in iplica e, e men ed dai y p oduc TPC and an ioxidan capaci y, as desc ibed in
Sec ions 2.1 and 2.5, espec i ely.
A e 24 h o e ige a ed s o age, a g oup o ained panelis s (n = 8) pe o med a
p elimina y senso y es . Panelis s ecei ed in o ma ion abou he samples, p ocedu es and
hei igh s. We collec ed hei signed w i en consen o pa icipa e. Fo he desc ip i e
es , a speci ic senso y lexicon was de eloped [
29
], and he ea e , panelis s we e asked o
desc ibe aspec , odo and la o cha ac e is ics.
2.12. S a is ical Analysis
Two-way analysis o a iance (ANOVA) wi h p esence/absence o CPE and s ain
as main e ec s (SPSS 25.0 s a is ical package, IBM Co po a ion, A monk, NY, USA) was
applied o da a. Tukey’s es by one-way ANOVA, wi h signi icance assigned a p< 0.01,
was hen pe o med o compa e means (p esence/absence o CPE o s ain).
3. Resul s and Discussion
3.1. Co ee Che y Ex ac s: P elimina y Cha ac e iza ion and Selec ion
Fou co ee che y ex ac s, h ee om pulp and one om bean, we e ob ained and
e alua ed (Table 1). Pulp ex ac s displayed signi ican ly (p< 0.01) lowe pH, A
600
and TPC
alues han bean ex ac . Among he ex ac s, he g ound pulp p epa ed wi h boiling wa e
showed he highes TPC.
Table 1. P elimina y cha ac e iza ion o he co ee che y ex ac s 1.
Va iable Ex ac s
Whole Pulp G ound Pulp G ound Pulp 4 ◦C Bean + Sil e skin
pH 3.78 ±0.02 b3.76 ±0.01 b3.74 ±0.01 b5.41 ±0.01 a
A600 0.0268 ±0.0100 bc 0.0460 ±0.0093 b0.0193 ±0.0102 c0.1352 ±0.0098 a
TPC 2136.15 ±1.16 c314.18 ±0.61 a160.48 ±0.45 b70.36 ±0.42 d
1
Co ee che y ex ac s we e p epa ed wi h s e ile dis illed wa e (a a 1:10 a io) a 95
◦
C o 7 min, o a
4◦C
o 30 min.
2
TPC = To al polyphenol con en , in
µ
g GAE/mL. All a iables we e mean
±
SD o iplica e
de e mina ions in wo expe imen s (n = 6). Means wi h lowe -case supe sc ip s di e signi ican ly a p< 0.01.
The esul s on he e ec s o co ee che y ex ac s on s ain me abolism a e shown in
Table 2. Bac e ial g ow h was quali a i ely eco ded in all pla es a e 24 h incuba ion. All
s ains showed limi ed g ow h and dye colo change in con ol pla es and in pla es wi h
he bean ex ac .
Table 2. Beha iou o in es inal s ains g own on BHI aga wi h me hyl blue (con ol, BHIC) and supple-
men ed wi h 40% o he co ee che y ex ac s incuba ed a 37 ◦C o 48 h unde anae obic condi ions.
S ain Con ol Ex ac
Whole Pulp G ound Pulp G ound Pulp 4 ◦C Bean + Sil e Skin
INIA P495 1+ 3 4 4 2
INIA P708 1+ 3 4 4 2
INIA P334 1 2 3 3 1+
INIA TAB84 1 2 3 3 1
INIA P508 1 2 3 3 1
INIA P459 1 3 3 3 1
GG 1+ 3 4 4 1+
Codes: 0 = no g ow h; 1 = ligh g ow h wi hou colo change; 2 = g ow h and change o blue colo ; 3 = blue ing
o up o 2 mm a ound he d op; 4 = blue ing o mo e han 2 mm a ound he d op.
Foods 2025,14, 44 7 o 18
Howe e , he h ee pulp ex ac s s imula ed s ain me abolism, showing blue ings
(due o acid p oduc ion) o bigge diame e s han in he con ol pla es. The bes sco es
we e eco ded o s ains g own wi h g ound pulp ex ac s, i espec i e o ex ac ion
empe a u e. The bigges s imula ions we e eco ded o s ains INIA P495, INIA P708 and
he GG e e ence s ain.
In con as o wha we obse ed in his wo k, wi h 40% concen a ions well ole a ed
by all lac obacilli s ains, ex ac concen a ions o mangos een ui abo e 10% we e
de imen al o he g ow h o hese s ains [22].
Taking in o accoun all hese esul s, he g ound pulp ex ac p epa ed wi h boiling
wa e was selec ed and p epa ed a a la ge scale.
3.2. CPE Cha ac e iza ion
The physico-chemical cha ac e is ics o his co ee che y pulp ex ac a e shown in
Table 3. Bo h pH and A
600
alues we e simila o he ones ob ained o he g ound pulp
ex ac p epa ed wi h boiling wa e . Suc ose is he mos abundan suga p esen in co ee;
i has an impo an con ibu ion o he as e o his be e age [
30
]. A aw il a e o he
we co ee eached a suga con en o 1.67 mg GE/mL [
31
]. Taking in o accoun ha he
a ios used in he we p ocessing we e 1 Kg pulp:80–120 L wa e [
31
], and ha we used a
1:10 a io
, he o al suga con en o ou CPE was lowe han expec ed. These di e ences
could be explained, howe e , by he la ge a ia ions in suc ose ound be ween di e se
species and o igins and e en wi hin beans om he same ba ch [
30
]. In compa ison wi h
p e ious da a on mangos een ex ac s [
22
], CPE had a lowe suga con en han mangos een
pulp ex ac (12.14 mg GE/mL) o e en mangos een ind ex ac (9.91 mg GE/ml).
Table 3. Cha ac e is ics o he co ee che y pulp ex ac .
pH A600 DM 1
(%)
Suga Con en
(mg GE/mL)
P o ein Con en
(µg BSA/mL)
TPC 2
(µg GAE/mL)
An ioxidan Capaci y
(mM TE)
3.81 ±0.01 0.0451 ±0.01 1.98 ±0.03 8.28 ±0.21 47.39 ±4.37 406.43 ±1.50 8.36 ±0.09
1
DM = d y ma e and
2
TPC = To al polyphenol con en . All a iables we e mean
±
SD o iplica e de e mina-
ions in wo expe imen s (n = 6).
The highe TPC o he CPE, in compa ison o he g ound pulp ex ac p e iously p e-
pa ed, migh be due o he highe ex ac ion empe a u e o 50
◦
C s oom empe a u e [
32
].
The TPC o he CPE was lowe han he 1.3 mg GAE / mL ob ained o he aw il a e o
we co ee p ocessing [
31
]. Howe e , TPC on a weigh basis (4.06 mg GAE/g esh pulp)
was in ag eemen wi h he 4.9 mg GAE/g pulp DM [
33
]. Compa ed o he mangos een
ex ac s o ou p e ious wo k [
22
], CPE con ained a much lowe TPC (5.70 and 10.01 mg
GAE / mL o pulp and ind, espec i ely). Acco dingly, he CPE an ioxidan capaci y was
lowe han hose o he mangos een ex ac s (100.95 and 186.60 mM TE o pulp and ind,
espec i ely).
The mos abundan phenolic compounds de ec ed in he CPE by HPLC-PAD/HPLC-
ESI-MS (Figu e 1) we e, in dec easing amoun s (Table 4), he alkaloids igonelline and
ca eine, p o oca echuic and chlo ogenic acids, ca eoylquinic and e uloylquinic acids,
he la an-3-ol ca echin and, in lowe quan i ies, ca eic acid, he la onoid glycoside
u in, se e al dica eoylquinic acid isome s and epica echin. Following he wo k o Cli o d
e al. [
34
], he isome ic o m o each ca eoylquinic, e uloylquinic and dica eoylquinic acids
was assigned. T igonelline and ca eine ha e been p e iously iden i ied in aqueous a abica
and obus a co ee ex ac s [
35
,
36
]. A highe le el o igonelline as compa ed o ca eine
was ound, in ag eemen wi h published esul s [
37
]. T igonelline biosyn hesis occu s in
he pe ica p o co ee be ies, whe eas ca eine is p edominan ly p oduced in he beans [
37
].
In e es ingly, he NAD
+
p ecu so igonelline seems o imp o e muscle unc ion du ing
Foods 2025,14, 44 8 o 18
ageing [
38
]. Compa able o ou indings, p o oca echuic and chlo ogenic acids we e he
dominan phenolic compounds iden i ied in a be e age p epa ed wi h d ied co ee che y
pulp and ho wa e [
33
]. Se e al ca eoylquinic, e uloylquinic, and dica eoylquinic acid
isome s ha e been ound by o he s, oo [
3
,
34
,
36
]. Howe e , ca eoyl e uloylquinic o
p-couma oylquinic acids [3,33] we e no de ec ed in his wo k.
Foods 2025, 14, x FOR PEER REVIEW 8 o 19
[32]. The TPC o he CPE was lowe han he 1.3 mg GAE / mL ob ained o he aw il a e
o we co ee p ocessing [31]. Howe e , TPC on a weigh basis (4.06 mg GAE/g esh pulp)
was in ag eemen wi h he 4.9 mg GAE/g pulp DM [33]. Compa ed o he mangos een
ex ac s o ou p e ious wo k [22], CPE con ained a much lowe TPC (5.70 and 10.01 mg
GAE / mL o pulp and ind, espec i ely). Acco dingly, he CPE an ioxidan capaci y was
lowe han hose o he mangos een ex ac s (100.95 and 186.60 mM TE o pulp and ind,
espec i ely).
The mos abundan phenolic compounds de ec ed in he CPE by HPLC-PAD/HPLC-
ESI-MS (Figu e 1) we e, in dec easing amoun s (Table 4), he alkaloids igonelline and
ca eine, p o oca echuic and chlo ogenic acids, ca eoylquinic and e uloylquinic acids,
he la an-3-ol ca echin and, in lowe quan i ies, ca eic acid, he la onoid glycoside u in,
se e al dica eoylquinic acid isome s and epica echin. Following he wo k o Cli o d e
al. [34], he isome ic o m o each ca eoylquinic, e uloylquinic and dica eoylquinic acids
was assigned. T igonelline and ca eine ha e been p e iously iden i ied in aqueous a a-
bica and obus a co ee ex ac s [35,36]. A highe le el o igonelline as compa ed o ca -
eine was ound, in ag eemen wi h published esul s [37]. T igonelline biosyn hesis oc-
cu s in he pe ica p o co ee be ies, whe eas ca eine is p edominan ly p oduced in he
beans [37]. In e es ingly, he NAD+ p ecu so igonelline seems o imp o e muscle unc-
ion du ing ageing [38]. Compa able o ou indings, p o oca echuic and chlo ogenic acids
we e he dominan phenolic compounds iden i ied in a be e age p epa ed wi h d ied co -
ee che y pulp and ho wa e [33]. Se e al ca eoylquinic, e uloylquinic, and
dica eoylquinic acid isome s ha e been ound by o he s, oo [3,34,36]. Howe e ,
ca eoyl e uloylquinic o p-couma oylquinic acids [3,33] we e no de ec ed in his wo k.
(A)
7
x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
5
5.2
5.4
5.6
5.8
6
6.2
6.4
-ESI EIC(515.1195) Scan F ag=120.0V EC1-4 16-04-24 2ul NEGmsms.d
1
7
Coun s s. Acquisi ion Time (min)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Foods 2025, 14, x FOR PEER REVIEW 9 o 19
(B)
Figu e 1. Ex ac ed ion ch oma og am o majo polyphenol compounds de ec ed in he co ee che y
pulp ex ac . (A): compounds de ec ed a e ope a ion in he posi i e ion mode. (B): compounds
de ec ed a e ope a ion in he nega i e ion mode.
Table 4. LC-MS/MS cha ac e is ics o majo polyphenols de ec ed in he co ee che y pulp ex ac .
Peak
Numbe
Re en ion
Time (min)
Compound
[M-H]-
m/z
MS2 Ions m/z
1
1.9
T igonelline
138.0550 *
138 (100), 94 (63), 53 (27), 78 (18), 65 (13)
2
4.1
3-Ca eoylquinic acid
353.0878
191 (100), 135 (35)
3
4.4
P o oca echuic acid
153.0193
109 (100)
4
6.1
Chlo ogenic acid (5-Ca eoylquinic acid)
353.0878
191 (100)
5
6.3
Ca echin
289.0718
203 (100), 245 (87), 109 (65), 221 (64), 164
(57), 123 (45), 227 (38), 97 (37), 150 (36), 80
(32), 186 (32), 138 (31), 211 (25), 175 (23)
6
6.6
4-Ca eoylquinic acid
353.0878
191 (100), 173 (100), 135 (34)
7
6.7
Ca eine (1,3,7-T ime hylxan hine)
195.0877 *
138 (100) , 195 (41), 110 (19)
8
6.7
3-Fe uloylquinic acid
367.1035
193 (100), 134 (19)
9
8.1
Ca eic acid
179.0350
135 (100), 105 (12), 93 (12)
10
8.1
5-Fe uloylquinic acid
367.1035
367 (100)
11
8.9
Epica echin
289.0718
203 (100), 245 (87), 109 (65), 221 (64), 123
(45), 227 (38), 97 (37), 150 (35), 186 (32), 80
(32), 289 (31), 138 (31), 211 (25)
12
10.5
4-Fe uloylquinic acid
367.1035
191 (100), 173 (24)
13
14.1
Ru in (Que ce in-3-O- u inoside)
609.1461
609 (100), 300 (11)
14
14.5
609.1461
609 (100), 300 (17)
15
17.7
3,4-di-O-ca eoylquinic acid
515.1195
173 (100), 353 (83), 515 (37), 335 (19), 155
(13)
16
18.5
3,5-di-O-ca eoylquinic acid
515.1195
353 (100), 191 (71), 179 (14), 509 (10)
17
21.1
4,5-di-O-ca eoylquinic acid
515.1195
353 (100), 173 (69), 311 (14), 516 (10), 263
(10)
* Majo polyphenol compounds de ec ed in he posi i e ion mode [M+H]+.
Fla onoids such as ca echin, epica echin and u in a e ound in many plan s. These
h ee compounds we e also de ec ed in mangos een ex ac s [22]. In ag eemen wi h ou
6
x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
1.6
1.65
1.7
1.75
-ESI EIC(515.1195) Scan F ag=120.0V EC1-4 16-04-24 2ul NEGmsms.d
2
3
4
5
6
8
9
10 12
11
1314 15 16 17
Coun s s. Acquisi ion Time (min)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Figu e 1. Ex ac ed ion ch oma og am o majo polyphenol compounds de ec ed in he co ee che y
pulp ex ac . (A): compounds de ec ed a e ope a ion in he posi i e ion mode. (B): compounds
de ec ed a e ope a ion in he nega i e ion mode.
Fla onoids such as ca echin, epica echin and u in a e ound in many plan s. These
h ee compounds we e also de ec ed in mangos een ex ac s [
22
]. In ag eemen wi h ou
esul s, all h ee we e epo ed in an 80% me hanol esh co ee pulp ex ac [
39
]. Howe e ,
only u in was de ec ed in co ee che y pulp samples [
30
]. Acco ding o A a ind e al. [
11
],
hese compounds ha e been shown o imp o e he unc ion o endo helial issues, al e
glucose me abolism, educe oxida i e s ess and amelio a e insulin esis ance.
Foods 2025,14, 44 9 o 18
Table 4. LC-MS/MS cha ac e is ics o majo polyphenols de ec ed in he co ee che y pulp ex ac .
Peak
Numbe
Re en ion
Time (min) Compound [M-H]-
m/z MS2 Ions m/z
1 1.9 T igonelline 138.0550 * 138 (100), 94 (63), 53 (27), 78 (18), 65 (13)
2 4.1 3-Ca eoylquinic acid 353.0878 191 (100), 135 (35)
3 4.4 P o oca echuic acid 153.0193 109 (100)
4 6.1 Chlo ogenic acid
(5-Ca eoylquinic acid) 353.0878 191 (100)
5 6.3 Ca echin 289.0718
203 (100), 245 (87), 109 (65), 221 (64), 164
(57), 123 (45), 227 (38), 97 (37), 150 (36), 80
(32), 186 (32), 138 (31), 211 (25), 175 (23)
6 6.6 4-Ca eoylquinic acid 353.0878 191 (100), 173 (100), 135 (34)
7 6.7 Ca eine
(1,3,7-T ime hylxan hine) 195.0877 * 138 (100), 195 (41), 110 (19)
8 6.7 3-Fe uloylquinic acid 367.1035 193 (100), 134 (19)
9 8.1 Ca eic acid 179.0350 135 (100), 105 (12), 93 (12)
10 8.1 5-Fe uloylquinic acid 367.1035 367 (100)
11 8.9 Epica echin 289.0718
203 (100), 245 (87), 109 (65), 221 (64), 123
(45), 227 (38), 97 (37), 150 (35), 186 (32), 80
(32), 289 (31), 138 (31), 211 (25)
12 10.5 4-Fe uloylquinic acid 367.1035 191 (100), 173 (24)
13 14.1
Ru in (Que ce in-3-O- u inoside)
609.1461 609 (100), 300 (11)
14 14.5 609.1461 609 (100), 300 (17)
15 17.7 3,4-di-O-ca eoylquinic acid 515.1195 173 (100), 353 (83), 515 (37), 335 (19), 155
(13)
16 18.5 3,5-di-O-ca eoylquinic acid 515.1195 353 (100), 191 (71), 179 (14), 509 (10)
17 21.1 4,5-di-O-ca eoylquinic acid 515.1195 353 (100), 173 (69), 311 (14), 516 (10), 263
(10)
* Majo polyphenol compounds de ec ed in he posi i e ion mode [M+H]+.
3.3. CPE E ec on Bac e ial G ow h and Me abolism
A e 24 h, all es ed lac obacilli s ains g ew jus as well in con ol as in CPE-
supplemen ed BHIC media (Table S1). S ains INIA P495, P708 and P334 ob ained he
highes alues, wi h le els close o 9 log CFU/mL. Al hough a s imula o y e ec o polyphe-
nols has been desc ibed o lac obacilli [
40
], no signi ican di e ences be ween bo h g ow h
media, ei he a day 1 o 6, we e eco ded. Howe e , pH dec eases we e signi ican ly bigge
(p< 0.01) in CPE-supplemen ed BHIC han in BHIC con ols. The highes dec eases we e
eco ded o s ains INIA P495, INIA P708 and GG, wi h alues a ound 4.3–4.5, in ag ee-
men wi h obse a ions o solid media (Table 2). Al hough pH alues o
4.5–5.0 ypically
esul in a signi ican o comple e inhibi ion o g ow h [
41
], CPE seemed o p o ec cul u es.
We ound no di e ences in g ow h be ween CPE-supplemen ed o con ol media. Simila ly,
Pe ei a e al. [
42
] showed ha inocula ion wi h a Lp. plan a um s ain esul ed in as e and
imp o ed co ee bean e men a ion, con e ing pulp suga s in o o ganic acids and eaching
pH alues o 4.5 in 12 h.
CPE addi ion signi ican ly (p< 0.01) inc eased TPC, by abou 100
µ
g GAE / mL.
Howe e , no inc ease in TPC alues we e eco ded o any o he es ed cul u es. While
lac obacilli a e able o ans o m complex die a y polyphenols, inc easing he bioa ailabili y
o hese compounds [
16
,
43
], he spec oscopic echnique used o de e mine TPC did no
seem o be sensi i e enough o de ec he di e ences obse ed by HPLC-ESI/MS [44].
3.4. Func ional and P obio ic Capaci ies o Selec ed S ains in he P esence o CPE
Fo he nex expe imen s, h ee o he six lac obacilli s ains, each showing di e en
su i al and me abolic beha io , we e selec ed (INIA P495, INIA P708 and INIA P459).
Fo hese isola es, bio ilm o ma ion, an ioxidan capaci y and an imic obial ac i i y we e
compa ed agains he comme cial p obio ic GG s ain.
Foods 2025,14, 44 16 o 18
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