Deciphe ing he syn hesis in Saccha omyces ce e isiae o he bioac i es
mela onin, se o onin, indoleace ic acid, hyd oxy y osol and y osol om
glucose by using
13
C labelling p ecu so s and UHPLC-MS
Ma ina Gonzalez-Rami ez
a
, Domenico Masue o
b
, Ana B. Ce ezo
a
, Ana M. T oncoso
a
,
U ska V ho sek
b
, M. Ca men Ga cia-Pa illa
a,*
a
Depa amen o de Nu ici´
on y B oma ología, Toxicología y Medicina Legal. Facul ad de Fa macia, Uni e sidad de Se illa, C/P Ga cıa Gonz´
alez No. 2, Se illa 41012,
Spain
b
Me abolomics Uni , Resea ch and Inno a ion Cen e, Fondazione Edmund Mach (FEM), ia E. Mach 1, San Michele all’Adige, I aly
ARTICLE INFO
Keywo ds:
Glucose
Hyd oxy y osol
UHPLC-MS
Ty osol
Yeas
Fe men a ion
Iso opic labelling
ABSTRACT
Saccha omyces ce e isiae p oduces bioac i e compounds such as mela onin (MEL), se o onin (SER), indoleace ic
acid (IAA), hyd oxy y osol (HT), and y osol du ing alcoholic e men a ion. P e ious esea ch shows ha
yp ophan and y osine a e p ecu so s. This s udy aimed o in es iga e whe he S. ce e isiae can syn he ize hese
bioac i es om glucose. Using [U
13
C]-glucose as he e men a ion subs a e and UHPLC-MS o analysis, he
esea ch aced unequi ocally he o ma ion o hese compounds. Resul s con i med ha MEL, SER, IAA, y osol,
and HT a e pa ly de i ed om glucose. Di e en
13
C-labelled o ms o HT, y osol, and y osine we e iden i ied
which allows o p opose a biosyn he ic pa hway leading o he o ma ion o HT h ough eac ions be ween in-
e media es om he pen ose phospha e pa hway, glycolysis, addi ionally o he E lich pa hway. The p oposed
pa hway includes L-DOPA and ca echol which we e de ec ed in hei
13
C-labelled o ms. Unde s anding HT
syn hesis opens oppo uni ies o enhance he bioac i e po en ial o e men ed be e ages.
1. In oduc ion
Du ing alcoholic e men a ion (AF), yeas ans o ms glucose in o
e hanol and simul aneously, i syn he izes many seconda y me aboli es,
such as usel alcohols, aldehydes, o ganic acids, es e s, o ganic sul-
phides, and ca bonyl compounds wi h di e en unc ions. Gene ally,
p ima y me abolism is conside ed essen ial o g ow h, cell di ision, and
su i al whils seconda y me aboli es may se e as de oxi ying agen s o
educe he s ess, o as quo um sensing molecules among o he unc ions
(Mas e al., 2014). In addi ion o he oles ha seconda y me aboli es
may exe o yeas , hey de e mine he senso y cha ac e is ics o he
p oduc ha ing s ong impac on p oduc quali y (Hazelwood e al.,
2008).
Mo e ecen ly, yeas s ha e been s udied o hei abili y o syn hesize
me aboli es wi h biological p ope ies such as mela onin (MEL),
(Fe nandez-C uz e al., 2019) and hyd oxy y osol (HT) (´
Al a ez-
Fe n´
andez e al., 2019). MEL, N-ace yl-5-me hoxy yp amine, is a well-
known neu oho mone ha pa icipa es in he egula ion o human
ci cadian hy hms which is also ound in he plan kingdom whe e i is
widely dis ibu ed and, consequen ly, p esen in se e al oods
(Sza a´
nska e al., 2017). Se e al biological p ope ies ha e been
desc ibed o his compound such as an ioxidan , neu op o ec i e, an i-
in lamma o y and ca dio ascula p o ec ion (Rei e e al., 2000). Due o
i s p esence in wines, Rod iguez-Na anjo e al. (2012) explo ed he
winemaking p ocess and con i med i s p oduc ion du ing he alcoholic
e men a ion by yeas .
Likewise, HT is a bioac i e compound p oduced also du ing he
alcoholic e men a ion p ocess (´
Al a ez-Fe n´
andez e al., 2018).
Al hough i is p esen in wines, i s majo concen a ion is ound in oli es
and oli e oil (Ma eos e al., 2001). As o MEL, HT has shown se e al
bioac i e p ope ies such as an i-in lamma o y, neu op o ec i e, and
an iangiogenic e ec s (Galla do-Fe n´
andez, Ce ezo, e al., 2022; Gal-
la do-Fe n´
andez, Valls-Fonaye , e al., 2022). Addi ionally, HT is a well
-known an ioxidan (Achmon & Fishman, 2015).
I can be conside ed ha , as seconda y me aboli es, hey can be
syn he ized de no o o om p ima y compounds a ailable in he mus
such as amino acids (Baumes, 2009). The knowledge o me abolic
pa hways o syn hesis and he equi ed p ecu so s o hei p oduc ion,
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (M.C. Ga cia-Pa illa).
Con en s lis s a ailable a ScienceDi ec
Food Resea ch In e na ional
jou nal homepage: www.else ie .com/loca e/ ood es
h ps://doi.o g/10.1016/j. ood es.2025.116596
Recei ed 27 Janua y 2025; Recei ed in e ised o m 24 Ap il 2025; Accep ed 4 May 2025
Food Resea ch In e na ional 216 (2025) 116596
A ailable online 21 May 2025
0963-9969/© 2025 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
can help o de elop be e me hods and design s a egies o o ecas ing
he quan i ies o di e en impac me aboli es in inal wines. Indeed,
deciphe ing he mechanism in ol ed in he p oduc ion o MEL and HT
by yeas and i s biological ole, as well as hei p ecu so s such as SER,
and IAA o MEL, o y osol o HT, is o g ea in e es .
In ac , SER and MEL can be syn he ized om yp ophan
(Fe n´
andez-C uz e al., 2017). The e o e, Mu˜
niz-Cal o e al. (2019)
p oposed a pu a i e pa hway o MEL syn hesis ha includes he deca -
boxyla ion o yp ophan in o yp amine, which could be he ea e
hyd oxyla ed o SER.
In o he e ms, o many yea s he Eh lich pa hway has been he mos
s udied and bes -known pa hway o p oduce usel alcohols, which
basically consis s o a h ee-s ep pa hway. Fi s ly, amino acids a e
deamina ed; hen, hey a e deca boxyla ed and inally educed o hei
espec i e alcohol de i a i es, as desc ibed o he syn hesis o y osol
om y osine (Hazelwood e al., 2008; Mas e al., 2014). Addi ionally,
´
Al a ez-Fe n´
andez e al. (2019), highligh ed he p oduc ion o HT by
yeas , possibly h ough he hyd oxyla ion o y osol highligh ing he
in e es in his pa hway and he possibili y o aise he concen a ion o
he bioac i e HT by modula ing he Eh lich pa hway.
In his accoun , Rebollo-Rome o e al. (2020), s udied how he
concen a ion o he co esponding a oma ic amino acid p ecu so in he
ini ial subs a e could in luence he inal HT concen a ion. Fo his
pu pose, di e en ini ial concen a ions o y osine concen a ions we e
added o a syn he ic mus (10 mg L
−1
and 60 mg L
−1
). A e he alcoholic
e men a ion, hey no iced ha a highe y osine concen a ion did no
imply a highe HT con en . In his ega d, Galla do-Fe n´
andez, Ce ezo,
e al. (2022); Galla do-Fe n´
andez, Valls-Fonaye , e al. (2022), s udied
he bioac i e compounds syn he ized by yeas om a oma ic amino
acids by using labelled L-T yp ophan-
15
N
2
and L-Ty osine-(phenyl-
4-
13
C) as he only sou ces o y osine and yp ophan in he mus .
Ne e heless, unlabelled me aboli es we e ound, iden i ying HT, MEL
and SER, labelled and unlabelled, e ealing ha yeas could o m hese
bioac i e compounds ( y osol, HT, MEL o SER) om o he sou ces
di e en om he ini ial amino acids’ p ecu so s p esen in he mus
(Galla do-Fe n´
andez, Ce ezo, e al. (2022); Galla do-Fe n´
andez, Valls-
Fonaye , e al. (2022)). Thei esul s showed ha he e a e o he pa h-
ways in ol ed in he syn hesis o hese bioac i e compounds. One s ep
u he is he s udy o hei syn hesis om glucose ha i is he pu pose
o he p esen wo k.
Nisbe e al. (2014) s udied he p oduc ion o ola ile compounds
om glucose by calcula ing he a io be ween
13
C/
12
C and analysing i
by GC–MS. They e alua ed he signi icance o he anabolic pa hway in
he syn hesis o usel alcohols, hei ace a e es e s and a y acids. They
concluded ha usel alcohols we e 75 % hexoses de i ed. No ably, he
anabolic pa hway could be a s ong con ibu o o usel alcohol p o-
duc ion, h oughou he no o syn hesis om hexoses (Ugliano &
Henschke, 2009).
Fu he mo e, he ole o he ini ial concen a ion o suga (glucose
and uc ose) as a ac o ha in luences HT concen a ion was p e i-
ously explo ed by ou g oup. Alcoholic e men a ion was pe o med
using wo di e en mus glucose concen a ions (100 g L
−1
and 240 g
L
−1
). The s udy concluded ha he highe he glucose concen a ion in
mus , he highe HT p oduc ion by he yeas s (Gonzalez-Rami ez e al.,
2024), e ealing he ele ance o glucose in he media o he inal
concen a ion o he bioac i e HT.
A signi ican numbe o usel alcohols a e belie ed o be o med
du ing he alcoholic e men a ion by one o wo pa hways connec ed
wi h amino acid me abolism: (i) he Eh lich pa hway (Hazelwood e al.,
2008); o (ii) he anabolic pa hway in which he p oduc ion o
α
-ke o
acids du ing amino acid biosyn hesis came om hexoses, which may
hen be deg aded o usel alcohols as desc ibed abo e (Ugliano &
Henschke, 2009; Hazelwood e al., 2008, Ga de-Ce d´
an & Ancín-Azpi-
licue a, 2008).
T ace s’ s udies wi h s able iso opes can be used o analyse he
ela ion and e alua ion o p ecu so -p oduc . Hence, ace s udies ha e
been widely used in ood chemis y (Schiebe le e al., 2005) and ha e
occasionally been ex ended o s udies o he a e and o igin o wine
componen s, such as, o ins ance, deu e a ed isobu y ic acid and e hyl
isobu y a e spikes, used o s udy hei o ma ion in wine (Díaz-Ma o o
e al., 2005). All in all, Su e al. (2020), used
15
N-labelled NH
4
Cl, a gi-
nine, and glu amine, and
13
C-labelled leucine and aline o s udy he
ni ogen me abolism pa e n o wo non-Saccha omyces species. Labelled
glucose has been used o ack he p oduc ion o usel alcohols in g ain
mash e men a ions (Reazin e al., 1973). The e o e, he aim o his
pape is o deciphe whe he se e al bioac i e compounds (MEL, SER,
IAA, HT, y osol and y osine, among o he s) could be syn he ized by
yeas om glucose by using [U
–
13
C]-glucose which is pionee ing o
s udying he syn hesis o hese compounds by S. ce e isiae. Fo his
pu pose, we pe o med alcoholic e men a ion expe imen s wi h syn-
he ic mus ha includes labelled [U-
13
C]-glucose as po en ial p ecu so
o hose bioac i e compounds and analysed hem by UHPLC-MS o an
unequi ocal iden i ica ion.
2. Ma e ials and me hod
2.1. Reagen s and ma e ials
HT s anda d was pu chased om Ex asyn hese (Genay, F ance),
y osol, y osine, MEL, SER, IAA om Sigma-Ald ich (S Louis, Uni ed
S a es), and HPLC-MS g ade me hanol and o mic acid we e acqui ed
om Me ck (Da ms ad , Ge many). Uni o mly labelled [U-
13
C]-glucose
was acqui ed om Camb idge Iso ope Labo a o ies (Ando e , MA,
USA).
2.2. Syn he ic mus (SM)
The SM was p epa ed o he expe imen as ins uc ed on Riou e al.
(1997) wi h sligh di e ences. The con en was he same in he
ollowing ing edien s: acids (malic acid 5 g L
−1
, ci ic acid 0.5 g L
−1
and
a a ic acid 3 g L
−1
), mine als (KH
2
PO
4
0.75 g L
−1
, K
2
SO
4
0.5 g L
−1
,
MgSO
4
7H
2
O 0.25 g L
−1
, NaCl 0.2 g L
−1
and CaCl
2
0.155 g L
−1
), 1 mL o
ace elemen s (CoCl
2
⋅ 6H
2
O 0.4 g L
−1
, CuSO
4
⋅ 5H
2
O 1 g L
−1
, H
3
Bo
3
1
g L
-1
, KI 1 g L
−1
, MnSO
4
⋅ H
2
O 4 g L
-1
, (NH
4
)
6
Mo
7
O
24
1 g L
−1
and ZnSO
4
⋅ H
2
O 4 g L
−1
and 10 mL o a i amins solu ion (myoinosi ol 2 g L
−1
,
calcium pan o hena e 0.15 g L
−1
, hiamine hyd ochlo ide 0.025 g L
−1
,
nico inic acid 0.2 g L
−1
, py idoxine 0.025 g L
−1
and bio in 3 mL). The pH
was adjus ed o 3.31 wi h NaOH.
The YAN (Yeas Assimilable Ni ogen) was ob ained by he mix o
ammonium om he amino acids and he ammonium om NH
4
Cl. The
inal YAN was 140 mg L
−1
. F om he o al YAN, 84 mg L
−1
co esponds
o he ni ogen om he amino acids and 56 mg L
−1
o he ammonium
(NH
4
Cl). An amino acids s ock solu ion was p epa ed ollowing he in-
s uc ions p esen ed by Riou e al. (1997): yp ophan 13.4 g L
−1
,
isoleucine 2.5 g L
−1
, aspa ic acid 3.4 g L
−1
, glu amic acid 9.2 g L
−1
,
a ginine 28.3 g L
−1
, leucine 3.7 g L
−1
, h eonine 5.8 g L
−1
, glycine 1.4 g
L
−1
, glu amine 38.4 g L
−1
, alanine 11.2 g L
−1
, aline 3.4 g L
−1
, me hi-
onine 2.4 g L
−1
, phenylalanine 2.9 g L
−1
, se ine 6 g L
−1
, his idine 2.6 g
L
−1
, lysine 1.3 g L
−1
, cys eine 1.6 g L
−1
and p oline 46.1 g L
−1
. The
y osine was added independen ly, 10 mg L
−1
, ollowing Galla do-
Fe n´
andez, Ce ezo, e al. (2022); Galla do-Fe n´
andez, Valls-Fonaye ,
e al. (2022). The suga con en was 200 g L
−1
(glucose- uc ose, 1:1).
Two simul aneous ba ches o e men a ion we e p epa ed, one
ea men se ed as a con ol wi h 100 g L
−1
o glucose (no [U-
13
C]-
glucose added), and a second ba ch in which om he inal concen a-
ion o glucose (100 g L
−1
), he 50 % was non [U-
13
C]-glucose and he
50 % uni o mly labelled [U-
13
C]-glucose (Camb idge Iso ope Labo a-
o ies, Ando e , MA, USA) due o he high cos o labelled compounds.
Each e men a ion condi ion was es ed in lasks o 100 mL wi h 80 mL
o SM. In o de o ensu e he u mos accu acy o ou comes and accoun
o he inhe en biological a iabili y, six biological eplica es we e
aken in o accoun o each e men a ion. The SM was s e ilized wi h
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
2
bo le- op acuum il e s o 0.2
μ
m (Nalgene PES memb ane, The mo
Fishe Scien i ic, Wal ham, Massachuse s, USA).
2.3. Yeas s ains
The alcoholic e men a ion was pe o med wi h S. ce e isiae Red
F ui s ain (Ena is; RF) (Na a e e, La Rioja). They we e selec ed based
on ou p e ious expe imen s in which Red F ui p oduces he highes
quan i y o HT (Rebollo-Rome o e al., 2020).
2.4. Inocula ion o he alcoholic e men a ion
S.ce e isiae Red F ui was p o ided as ac i e d ied yeas s and we e
ehyd a ed o 30 min a 37 ◦C and pla ed on yeas ex ac pep one
dex ose (YPD) aga (2 % pep one, 2 % glucose, 1 % yeas ex ac and 2
% aga ). Then, hey we e incuba ed a 28 ◦C in an o en o 48 h. Sec-
ondly, wo lasks o 250 mL we e used wi h 100 mL o YPD (1 % yeas
ex ac , 2 % glucose and 2 % pep one) and shaken a 150 pm (New
B unswick Inno a® 40/40R, Hambu g, Ge many), 28 ◦C, o e nigh , o
le yeas g ow be o e inocula ion. Once he s ains we e ac i a ed, each
e men a ion lask o 100 mL wi h 80 mL o SM was inocula ed wi h 10
6
cells/mL and capped wi h aps equipped wi h a capilla y o elease
ca bon dioxide. The AF we e elapsed a 25 ◦C and 150 pm (New
B unswick Inno a® 40/40R, Hambu g, Ge many). The wo alcoholic
e men a ions, he one wi h labelled glucose (U
13
C-G- e men a ion) and
he one wi h unlabelled glucose (con ol), we e pe o med in
sex uplica e.
The e men a ion was daily moni o ed; he lasks we e weighed
be o e and a e sampling and he densi y was con olled. The densi y
was measu ed hanks o a densi y me e (An on Paa , G az, Aus ia). The
cou se o he e men a ion, exp essed as a e o CO
2
e sus e men a ion
ime, and he densi y g aphic is gi en in Fig. 1. The cells we e coun ed
wi h a Neubaue Came a and cy ome e (Beckman Coul e s, Pasadena,
Cali o nia, USA). The end o alcoholic e men a ion was eached when
he di e ences be ween daily weigh s we e no signi ican (See Fig. 1).
2.5. Sampling
The alcoholic e men a ion las ed o 10 days, and he samples we e
collec ed acco ding o he exponen ial and s a iona y phase o he
g ow h cu e o he yeas . Samples collec ed co espond o days
1,2,3,4,7 and 10. F om day 1 o 4, 4 mL samples we e collec ed daily,
ma ching wi h he di e en s ages o he exponen ial phase, on day 7
samples we e collec ed as a ep esen a ion o he s a o he s a iona y
phase and a he inal day, day 10. Samples we e cen i uged a 4500
pm (Alleg a X-12-R, Beckman Coul e s, Cali o nia, USA) a a empe -
a u e o 4 ◦C o 3 min. The supe na an s we e p ese ed a −80 ◦C un il
he analysis. A o al o 72 samples we e collec ed. Di e en samples
we e subjec ed o in acellula and ex acellula me hods and a o al o
120 samples we e analysed, 72 ex acellula and 48 in acellula
samples.
2.6. In acellula ex ac ion p ocedu e
The pelle s we e washed wice wi h dis illed wa e and collec ed o
he in acellula me aboli e ex ac ion. The olume collec ed was
calcula ed o ha e 10
9
cells. The cells we e subjec ed o a quenching and
ex ac ion p ocedu e ollowing he p o ocol by ´
Al a ez-Fe n´
andez e al.
(2018). Fi s ly, hey we e esuspended in 1 mL o dis illed wa e and
ans e ed in o a cen i uge ube con aining 4 mL o cold-glyce ol saline
solu ion [3:2 ( ol/ ol) glyce ol: saline solu ion] o s op enzyma ic ac-
i i y. The ea ed samples we e cen i uged a 36,086 g o 20 min a
−20 ◦C (So all LYNK 6000, The mo Fishe Scien i ic, Wal ham, MA
USA). The supe na an was emo ed, and he pelle was washed again
wi h 2.5 mL o cold washing solu ion [1:1 ( ol/ ol) glyce ol/saline so-
lu ion]. A new cen i uge cycle was applied. The supe na an was dis-
ca ded, and he pelle was s o ed a −80 ◦C be o e ex ac ion p ocedu e.
Fo he ex ac ion p ocedu e, 2.5 mL o cold me hanol-wa e solu ion
(50 % / ) was added o he pelle . A e ha , he samples we e sub-
jec ed o wo eeze- haw cycles ( ozen a −80 ◦C o 30 min and hen
hawed in an ice ba h o 5 min). One inished, hey we e subjec ed o a
sonica ion o 1 min in an ice ba h using an ul asonic liquid p ocesso
wi h 20 kHz homogeneous sound (Sonica o Sonoplus HD 2070, Ban-
delin Elec onic GmbH & Co. KG, Be lin, Ge many). A cen i uge cycle
was applied a 36,086 g o 20 min a −20 ◦C. This cycle was epea ed
one mo e ime wi h ano he 2.5 mL o cold me hanol-wa e solu ion,
subsequen ly s o ed a −80 ◦C un il clean up. The aim o his ex ac ion
p ocedu e is o b eak he cell walls in o de o make hem mo e
Fig. 1. Moni o iza ion o alcoholic e men a ion wi h U13C-G labelled and non-labelled glucose. Le : Daily weigh loss; Medium: Daily densi y. Righ : Daily cell
g ow h. Fe men a ions we e ca ied ou in sex uplica e (n =6).
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
3
pe meable o he o ganic sol en and consequen ly, in acellula me-
aboli es can be eleased, while p ese ing hei chemical in eg i y
(Sma e al., 2010).
2.7. Clean-up and concen a ion p ocedu e
All ex acellula ex ac s we e cleaned up as epo ed by ´
Al a ez-
Fe n´
andez e al. (2019) some modi ica ions. C18 SPE ca idges (1 g,
Va ian , Agilen , San a Cla a, Cali o nia, USA) we e condi ioned wi h 2
mL o me hanol and 2 mL o milli Q wa e . Fo cleaning ex acellula
samples, an aliquo o 2 mL o ex acellula sample was loaded, ollowed
by a washing s ep wi h 2 mL o wa e . The analy es we e elu ed wi h 1
mL o 70:30 me hanol: wa e . Sol en s we e e apo a ed un il d yness by
using a acuum concen a o (Hype VAC-LITE, GYOZEN, Ko ea) a
30 ◦C and 2000 pm o 8 h. Each sample was econs i u ed wi h 100
μ
L
o me hanol.
Fo he in acellula samples, hey we e cleaned up using 1 mL zi -
conia coa ed Ph ee™ ca idge (Phenomenex, To ance, Cali o nia,
USA) o a oid he p esence o phospholipids and p o eins due o cell wall
agmen a ion (Ca mical & B own, 2016; an de Res e al., 1995). Fo
he p ocedu e o load he samples, he p o ocol om he manu ac u e
was ollowed. Finally, he samples we e d ied as ex acellula ones and
econs i u ed in 100
μ
L me hanol.
2.8. UHPLC-MSMS ins umen al analysis
The analysis was pe o med in an UHPLC ExionLC sys em coupled
wi h AB6500+Q ap mass spec ome e and an elec osp ay ioniza ion
sys em (ESI) (AB Sciex LLC,F amingham, MA, U.S.A.). Wa e s Acqui y
column (Mil o d, Massachuse s, USA), HSS T3 (2.1 ×150 mm, 1.8
μ
m),
Fig. 2. Labelled Hyd oxy y osol agmen s.
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
4
was used. The column was kep a 40 ◦C. The low a e was se a 0.28
mL/min.
The UHPLC me hod was adap ed om ´
Al a ez-Fe n´
andez e al.
(2019). Mobile phase A was wa e wi h 0.1 % o mic acid; mobile phase
B was me hanol wi h 0.1 % o mic acid. The g adien was p og ammed
as ollows: 5 % B (0 min); 20 % B (2 min); 20 % B (3 min); 28 % B (3.3
min); 42 % B (8 min); 100 % B (8–12 min); 5 % B (14.5 min). The in-
jec ion olume was 2
μ
L.
The MS pa ame e s we e op imized indi idually o each s anda d by
he di ec in usion o hei solu ions (10
μ
g mL
−1
). The mos abundan
agmen s we e iden i ied o each compound o be used as he quan i-
ie , he o he agmen s as quali ie s. F agmen a ions o labelled
compounds we e calcula ed heo e ically based on he monoiso opic
mass o he labelled compound and he possible agmen a ions
achie able based on he posi ion o he labelled elemen .
The cu ain gas was se a 35 psi. The Ion Sp ay Vol age was se a
−4500 V in he nega i e mode and 5000 V in he posi i e mode. The
sou ce empe a u e was se a 500 ◦C, he nebulize gas (Gas1) and
hea e gas (Gas2) a 50 and 60 psi, espec i ely. Declus e ing po en ial
(DP) and en ance po en ial (EP) we e op imized o each p ecu so ion
and collision ene gy (CE) and Collision Cell Exi Po en ial (CXP) o each
p oduc ion.
The e en ion ime o he labelled compounds was he same as he
unlabelled s anda d compound (Wa kins e al., 2019). Fo quan i ica ion
pu poses he e e ence s anda d o he unlabelled compound was used
(Galla do-Fe n´
andez, Ce ezo, e al. (2022); Galla do-Fe n´
andez, Valls-
Fonaye , e al. (2022)). The quan i ica ion o each compound was pe -
o med using ex e nal calib a ion cu es p epa ed in me hanol.
2.9. S a is ical analysis
Da a we e subjec ed o analysis o a iance (ANOVA). Resul s a e
exp essed as mean ±s anda d de ia ion (SD). Di e ences a p <0.05
we e conside ed s a is ically signi ican . S a is ica e sion 14.00 was
used o da a analysis.
3. Resul s and discussion
3.1. Alcoholic e men a ion moni o ing
I was necessa y o check ha yeas could me abolize [U-
13
C]-
Glucose and ha he e men a ions we e simila o hose pe o med wi h
unlabelled glucose. To his end, bo h alcoholic e men a ions (con ol
and [U-
13
C}-Glucose) we e pe o med in sex uplica e, and he moni-
o ing was based on he daily weigh o he lasks, densi y o he mus
and cell g ow h. The lasks we e weigh ed daily be o e and a e sam-
pling, showing a dec ease in weigh in e ms o CO
2
expelled due o he
me abolic eac ion, as displayed in Fig. 1. Du ing he alcoholic
e men a ion, yeas con e s glucose in o e hanol and ca bon dioxide
acco ding o he ollowing chemical equa ion: C₆H₁₂O₆ → 2C₂H₅OH +2
CO₂. This eac ion demons a es ha one molecule o glucose yields wo
molecules o ca bon dioxide. Conside ing he mola masses o glucose
(180 g mol
−1
) and ca bon dioxide (44 g mol
−1
), i can be calcula ed ha
he e men a ion o one g am o glucose esul s in he p oduc ion o
app oxima ely 0.489 g o CO₂. In ou case we ha e ini ially 200 g L
−1
,
and obse ed an a a age o al weigh loss o app oxima ely 42 g L
−1
,
which could co espond o he loss o CO₂. The densi y o he medium
was also measu ed e e y day o sampling as an indica o o he glucose
consumed (Fig. 1). Fu he mo e, cell g ow h was de e mined by low
cy ome y, inc easing a day 1 and emaining cons an du ing alcoholic
e men a ion (Fig. 1). As he igu e shows, bo h e men a ions ook place
in a simila way, las ing 10 days and acco ding o ANOVA no s a is ical
di e ences we e ound (p <0.05) o he same day along he alcoholic
e men a ion be ween he con ol and U-
13
C-G e men a ions o weigh
los , densi y and cell g ow h.
3.2. MRM and MS op imiza ion
Secondly, i was necessa y o op imize he analy ical me hod o he
de e mina ion o he bioac i e compounds. Samples aken a di e en
days o alcoholic e men a ions we e analysed o 6 bioac i e com-
pounds (MEL, SER and 3-IAA, and HT, y osine, and y osol). These
compounds we e de e mined by UHPLC/MS-MS and undoub ably
iden i ied hanks o he compa ison o e en ion imes, exac mass and
he spec a wi h he espec i e comme cial s anda ds. Fo he analysis o
he mass, he MRM me hod was selec ed. The di e en pa ame e s o
MS condi ions we e op imized o each compound in posi i e and
nega i e mode, which a e summa ized in Table S.1 and S.2. Declus e ing
po en ial (DP) and en ance po en ial (EP) we e op imized o each
p ecu so ion and collision ene gy (CE) and Collision Cell Exi Po en ial
(CXP) o each p oduc ion. The op imiza ion was pe o med h ough
he in usion o he s anda ds o each compound. Fo he labelled com-
pounds, a p e ious analysis o he molecule agmen a ion was neces-
sa y. The e en ion ime and he di e en ol ages emained he same.
The MRM me hod o he labelled compounds was s uc u ed based
on he agmen a ion pa e n o he unlabelled compounds. As he me-
dium con ains bo h unlabelled and [U-
13
C]-Glucose, a deepe s udy o
he di e en possible agmen s was equi ed since mul iple numbe and
posi ions o labelled ca bons can be expec ed. All he labelled compound
possibili ies ange om 1 labelled ca bon (
13
C
1
) o he o al numbe o
ca bons o he compound. Fo example,
13
C
1
o
13
C
8
we e explo ed o
HT. A he same ime labelled ca bons may be p esen in di e en po-
si ions in he molecule. In he case o HT, hey can be dis ibu ed be-
ween he ing and he la e al chain (Fig. 2). HT molecula mass is
154.16 Da, he e o e, 153.16 Da co esponds o i s molecula ion in
nega i e mode. Acco ding o he agmen a ion, he mos abundan
ansi ion is m/z a 153 o 123, which supposed a loss o 30 amu, which
co esponds wi h he agmen CH
2
O
−
, pa o he la e al chain.
Table S.1 shows all he ansi ions ound in labelled and unlabelled HT.
Fig. 2 shows he di e en HT ansi ions depending on he numbe and
posi ions o he labelled ca bons. I [
13
C
4
]-HT is conside ed, he ansi-
ions can be as ollows: m/z 157–127, (i he
13
C
4
labelled ca bons a e in
he ing) o m/z 157–126 (i one o he labelled ca bons is in he chain)
(Fig. 2).
Ty osol, he mos abundan agmen co esponded o m/z 137–92,
which explains he loss o he la e al chain (45 amu). The numbe and
posi ions o he possible labelled ca bons a e shown in Fig. S.1. The mos
abundan agmen o y osine was m/z 182–165; he loss occu ing in
he amino g oup (NH
2
) (Table S.1). The ansi ion o he labelled
y osine co esponds o m/z 188–171 since all he ca bons emain in he
agmen s (Table S.1 and Fig. S.2). A simila pa e n is ound o SER
(Table S.2 and Fig. S.3). MEL and IAA mos abundan agmen s we e m/
z 233–174 and m/z 176–130, espec i ely. Fig. S.4 and S.5 show he
di e en labelled agmen s o MEL and IAA (Table S.2).
3.3. Analysis o labelled compounds in e men ed samples and possible
pa hways
As can be seen in Table S.3, he six compounds unde s udy (HT,
y osol, y osine, MEL, SER and 3-IAA) we e de ec ed in hei
13
C-
labelled o ms in U
13
C-G e men ed samples. Indeed, i can be un-
doub edly concluded ha all hese compounds a e in pa glucose-
de i ed, indica ing he impo ance o anabolic pa hways o hei o -
ma ion as he no el con ibu ion o his wo k.
Table S.3 p esen s he di e en concen a ions o each compound
(labelled and unlabelled) a each poin o he e men a ion p ocess.
Resul s a e exp essed as means o he six biological eplica es. The e
we e no signi ican di e ences be ween he concen a ions o HT, MEL,
SER, 3-IAA, y osine and y osol in con ol samples and samples om
13
C-G e men a ions (sum o unlabelled and labelled o ms). Mo eo e ,
he o al amoun o HT p oduced ag ees wi h he esul s p e iously e-
po ed o he same s ain and e men a ion condi ions (Galla do-
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
5
Fe n´
andez, Ce ezo, e al. (2022); Galla do-Fe n´
andez, Valls-Fonaye ,
e al. (2022)). Indeed, i can be concluded ha
13
C labelled glucose
has no a ec ed HT p oduc ion by he yeas ; consequen ly, labelling
does no dis u b yeas me abolism.
Al hough compounds unde s udy anged om
13
C
8
o
13
C
13,
as MEL
has 13 ca bons in o al, only compounds wi h
13
C
1
o
13
C
6
labelled
ca bons we e de ec ed. I is wo h highligh ing ha ce ain labelled
compounds wi h
13
C
1
ca bon we e also de ec ed in he con ol e men-
a ions, since na i e iso opes such as
13
C a e na u ally p esen (Spi zke &
Fauhl-Hassek, 2010). In ac , he e we e no signi ican di e ences in he
concen a ion o
13
C
1
labelled ca bon compounds (HT, y osol, y osine,
MEL, IAA and SER) be ween samples om he
13
C-G and he con ol
e men a ions. The e o e, we disca ded hese da a o u he discussion
and ocused on he di e ences be ween he wo e men a ions.
Unlabelled MEL, SER and IAA we e de ec ed and quan i ied in some
o he samples, i can be concluded ha hese bioac i e compounds can
be syn hesised om glucose in addi ion o o he biosyn hesis pa hways.
Su´
as egui and Shao (2016) concluded ha du ing glycolysis, glucose is
Fig. 3. Di e en pa hway om glucose o bioac i e compounds. PEP, phosphoenolpy u a e; E4P, e y h ose-4-phospha e; DAHP, 3-deoxy-D-a abinohep ulosona e-7-
phospha e; DHQ, dehyd oquina e; DHS, dehyd oshikima e; SA, shikimic acid; S3P, shikimi a e-3-phospha e; EPSP, e-enolpy u yl-3-shikima e phospha e; CA, cho-
ismic acid; AA, an h anilic acid.
Fig. 4. Abundance o he di e en labelled y osine possibili ies excluding 1-
13
C.
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
6
ans o med in o phosphoenolpy u a e (PEP) and also glucose h ough
he pen ose phospha e pa hway can yield e y h ose-4-phospha e (E4P)
(Fig. 3). These wo compounds bond o inally o m an h anilic acid,
which will la e add a phospho yla ed ibose o p oduce yp ophan
(Su´
as egui & Shao, 2016). T yp ophan can yield SER and IAA, SER can
inally p oduce MEL (´
Al a ez-Fe n´
andez e al., 2019). Unlabelled y o-
sine was de ec ed and quan i ied in he in acellula and ex acellula
media. In ac , i is p esen as such in he s a ing syn he ic mus .
Howe e , labelled y osine was de ec ed and quan i ied in he in a-
cellula media bu no in he ex acellula one. Indeed, yeas can syn-
hesize de no o a oma ic amino acids, such as y osine (Su´
as egui &
Shao, 2016). Ou da a show ha his newly syn he ised y osine om
glucose is no exc e ed bu , mos likely, used as p ecu so o o he
compounds. I is well known (Su´
as egui & Shao, 2016) ha du ing
glycolysis, glucose is ans o med in o PEP, and i is bonded o E4P
(Fig. 3). These wo compounds bond o inally o m p ephena e, which
will la e p oduce y osine (Su´
as egui & Shao, 2016). Fu he mo e,
y osine is me abolized o y osol by he Eh lich pa hway which migh
be subsequen ly hyd oxyla ed o HT. Hence, de no o syn he ised y o-
sine was ound wi h di e en labelled ca bons as shown Fig. 4 being
13
C
3
y osine he mos abundan ollowed by
13
C
4
y osine as shown in Fig. 4.
I
13
C E4P akes pa , i will esul in y osine labelled in ou ca bons.
Howe e , i
13
C PEP is in ol ed, hen y osine wi h h ee labelled
ca bons will be o med. In he case y osine is p oduced om wo
13
C
PEP which a e wa ds loses 1
13
C ca bon, i will esul in y osine wi h
i e
13
C (Fig. 5).
Labelled y osol is de ec ed om 13.11 o 221.55 ng ml
−1
as shown
in Table S.3, howe e mos o y osol de e mined ( om 1 o 16.47 mg L
−1
) is unlabelled consis en wi h a majo i y p oduc ion om he ini ial
y osine om he medium. Indeed, Galla do-Fe n´
andez, Ce ezo, e al.
(2022); Galla do-Fe n´
andez, Valls-Fonaye , e al. (2022) in an expe i-
men wi h labelled y osine obse ed ha he majo p opo ion o
y osol (75 %) came om labelled y osine. Ou esul s ein o ce he ac
ha y osol is mos ly o med om he ini ial y osine concen a ion in
he syn he ic mus . Mo eo e , in ag eemen wi h y osine esul s, he
mos abundan y osol o m was he one wi h h ee
13
C ollowed by ou
13
C.
HT was de ec ed and quan i ied in con ol e men a ions in bo h
in acellula and ex acellula media. Labelled HT was de ec ed and
quan i ied in all i s di e en labelled possibili ies bo h in ex acellula
and in acellula media. Galla do-Fe n´
andez, Ce ezo, e al. (2022);
Galla do-Fe n´
andez, Valls-Fonaye , e al. (2022) demons a ed ha HT
can be o med om y osine, bu i was in a low ex ension. Conse-
quen ly, a S. ce e isiae s ain was gene ically modi ied o enhance he
enzyme which hyd oxyla es y osol o HT, ob aining a mode a e in-
c ease o HT concen a ion (Gonzalez-Rami ez e al., 2024). In he
Fig. 5. Di e en possibili ies o he o ma ion o y osine om glucose.
Fig. 6. Abundance o he di e en labelled HT possibili ies excluding 1-
13
C.
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
7
p esen wo k, we compa ed he a io o y osol o HT ob ained in con ol
e men a ion wi h he a io o he labelled compounds in he
13
C-G
e men a ion esul ing ha his a io is 20 imes highe o he labelled
compounds. This ac ein o ces ha an addi ional pa hway o p oduce
HT mus exis , apa om he pu a i e hyd oxyla ion o y osol.
Taking in o accoun ha he mos abundan HT o ms a e he ones
wi h wo and ou
13
C (32 and 30 % espec i ely) (Fig. 6), we
hypo he ised a possible pa hway as shown in Fig. 3 by pu ing oge he
pieces o in o ma ion in li e a u e and he ma ching wi h ou da a. I is
known ha PEP and E4P a e o med h ough glycolysis and he pen ose
phospha e pa hways, espec i ely. Thus, om E4P and PEP, he shikimic
pa hway gi es ise o dehyd oshikima e (DHS), which by he muconic
acid pa hway yields ca echol by losing 1 ca bon a om (Su´
as egui & Shao,
2016). Addi ionally, Min e al., (2014) desc ibed ha ca echol, oge he
wi h ammonium, ha is p esen in he medium, and py u ic acid,
o med by glycolysis, p oduces L-DOPA ((L)-3,4-dihyd ox-
yphenylalanine). Fu he mo e, i was p o en ha S. ce e isiae can
me abolize L-DOPA o inally p oduce HT (Beh inge e al., 2024)
(Fig. 3). These au ho s showed ha nonp o einogenic a oma ic amino
acids such as L-DOPA, can be subs a es o deg ada ion ia he Eh lich
pa hway. 3-(3,4-dihyd oxyphenyl)-py u ic acid is ansamina ed o gi e
L-DOPA and hen con e ed o he inal HT by losing ano he addi ional
ca bon a om. The ca bon a oms los come om PEP and py u ic acid. To
con i m his possibili y, an analysis p o ed ha
13
C ca echol and
13
C L-
DOPA a e p esen . A Pa allel Reac ion Moni o ing (PRM) analysis was
pe o med and
13
C
5
L-DOPA and
13
C
2
ca echol a e de ec ed, suppo ing
he p oposed pa hway which is depic ed in Fig. 3.
I is wo h o emembe ha bo h labelled and no labelled glucose
we e p esen in he medium, he e o e labelled and no labelled p e-
cu so s (E4P, PEP and py u ic acid) a e p oduced, hus, explaining he
di e en possibili ies o combina ion which esul in he di e en
labelled HT o ms ound (Figs. 2 and 7). Fo ins ance, he combina ion o
13
C E4P and
13
C PEP esul s in HT wi h six
13
C in he ing. I
13
C E4P, non
labelled PEP and
13
C py u ic acid a e combined, HT wi h ou
13
C in he
ing and wo
13
C in he la e al chain can be o med. Finally, i jus
13
C
PEP o
13
C py u ic acid a e p esen , i can esul in HT wi h wo
13
C in
he ing o la e al chain espec i ely. This pa hway explains he wo,
ou and six
13
C o ca bons o HT ha a e in he highe p opo ion as
Fig. 6 shows. Fu he mo e, he p esence o HT wi h h ee
13
C can be
explained om he h ee ca bons
13
C y osine syn he ized by yeas om
glucose. Ou da a show ha he sum o HT labelled in wo, ou o six
ca bons explains mos o he HT p oduced e ealing he p oposed
pa hway should be he main a o ed wi h ega d o he pa hway om
y osine. The pen ose phospha e pa hway is up egula ed unde s ess ul
condi ions (Be els e al., 2021), and yeas may p io i ize he p oduc ion
o compounds needed o coun e ac s ess (Pos a u e al., 2023). This
could be he case o HT syn hesis, a well-known an ioxidan , p oduced
om E4P h ough his pa hway.
Fig. 7. Di e en possibili ies o he o ma ion o HT om glucose.
M. Gonzalez-Rami ez e al.
Food Resea ch In e na ional 216 (2025) 116596
8
Rega ding he esul s p esen ed by Galla do-Fe n´
andez, Ce ezo, e al.
(2022); Galla do-Fe n´
andez, Valls-Fonaye , e al. (2022), HT, MEL, and
SER we e ound in highe concen a ions in hei unlabelled o ms;
indeed, i was concluded ha he e was a pa hway ha had mo e impac
han he one om amino acids. Howe e , he labelled y osol om he
labelled y osine was ound in a g ea e concen a ion. These esul s
ag ee wi h ou indings, as we quan i y y osol a low concen a ions and
i is mos ly ound in unlabelled o m coming om ini ial y osine.
4. Conclusions
All in all, his s udy demons a es ha S. ce e isiae can syn hesize
bioac i e compounds such as HT, MEL, SER, IAA and y osol om
glucose du ing alcoholic e men a ion. Al hough yp ophan and y o-
sine a e known p ecu so s, he esul s show ha glucose is a signi ican
sou ce o he syn hesis o hese compounds, especially HT. Using a
UHPLC-MS me hod and [U
13
C]-glucose, he syn hesis pa hways we e
aced and con i med. The s udy p oposes a mechanism whe e HT is
o med om glycolysis (PEP) and pen ose phospha e (E4P) pa hway
in e media es, h ough ca echol and L-DOPA in e media es. Addi ion-
ally, y osol is mainly o med om y osine h ough he shikimic and
Ehli ch pa hways. This inding opens new oppo uni ies o enhance he
bioac i e po en ial o e men ed be e ages by exploi ing hese syn he ic
pa hways.
CRediT au ho ship con ibu ion s a emen
Ma ina Gonzalez-Rami ez: W i ing – o iginal d a , Me hodology,
In es iga ion, Fo mal analysis, Da a cu a ion. Domenico Masue o:
W i ing – e iew & edi ing, W i ing – o iginal d a , Visualiza ion, Su-
pe ision, Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion,
Concep ualiza ion. Ana B. Ce ezo: W i ing – e iew & edi ing, W i ing –
o iginal d a , Visualiza ion, Supe ision, In es iga ion, Fo mal analysis,
Da a cu a ion, Concep ualiza ion. Ana M. T oncoso: W i ing – e iew &
edi ing, W i ing – o iginal d a , Visualiza ion, Supe ision, Resou ces,
P ojec adminis a ion, In es iga ion, Fo mal analysis, Concep ualiza-
ion. U ska V ho sek: W i ing – e iew & edi ing, W i ing – o iginal
d a , Visualiza ion, Supe ision, Me hodology, In es iga ion, Fo mal
analysis, Da a cu a ion, Concep ualiza ion. M. Ca men Ga cia-Pa -
illa: W i ing – e iew & edi ing, W i ing – o iginal d a , Visualiza ion,
Supe ision, Resou ces, P ojec adminis a ion, In es iga ion, Fo mal
analysis, Concep ualiza ion.
Funding
This esea ch was unded by g an PID2022–137807OB-C22 unded
by MICIU/AEI/ 10.13039/501100011033 and by g an PID2019-
108722RB-C32 unded by MICIU/AEI/ 10.13039/501100011033, as
app op ia e, by “ERDF A way o making Eu ope” by he “Eu opean
Union” and Jun a de Andalucía G an P18-RT-3098.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
The au ho hanks he Spanish Go e nmen o i s inancial assis-
ance (PID2022-137807OB-C22) Jun a de Analucía (P18-RT-3098); and
he Uni e si y o Se ille o he M.G.R. p edoc o al con ac PIF and o
he ellowship o s ay ab oad.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. ood es.2025.116596.
Da a a ailabili y
Da a will be made a ailable on eques .
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