RESEARCH ARTICLE
A S aigh o wa d Access o New Families o
Lipophilic Polyphenols by Using Lipoly ic
Bac e ia
Ley e Sa
´nchez-Ba ionue o
1,2☯
, Alejand o Gonza
´lez-Benjumea
3☯
, Almudena Escoba -
Niño
1,2
, Ma ı
´a Te esa Ga cı
´a
2
, O
´sca Lo
´pez
3
, Ine
´s Maya
3
, Jose
´G. Fe na
´ndez-Bolaños
3
,
Da id Ca
´no as
1
*, Enca nacio
´n Mellado
2
*
1Depa men o Gene ics, Facul y o Biology, Uni e si y o Se ille, Se ille, Spain, 2Depa men o
Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o Se ille, Se ille, Spain, 3Depa men o
O ganic Chemis y, Facul y o Chemis y, Uni e si y o Se ille, Se ille, Spain
☯These au ho s con ibu ed equally o his wo k.
*[email p o ec ed] (EM); da [email protected] (DC)
Abs ac
The chemical syn hesis o new lipophilic polyphenols wi h imp o ed p ope ies p esen s
echnical di icul ies. He e we desc ibe he selec ion, isola ion and iden i ica ion o lipoly ic
bac e ia om ood-p ocessing indus ial was es, and hei use o ailo ing a new se o com-
pounds wi h g ea in e es in he ood indus y. These bac e ia we e employed o p oduce
lipoly ic supe na an s, which we e applied wi hou u he pu i ica ion as bioca alys s in he
chemoselec i e and egioselec i e syn hesis o lipophilic pa ially ace yla ed phenolic com-
pounds de i ed om oli e polyphenols. The chemoselec i i y o polyphenols acyla ion/dea-
cyla ion was analyzed, e ealing he p e e ence o he lipases o phenolic hyd oxyl g oups
and phenolic es e s. In addi ion, he alcoholysis o pe ace yla ed 3,4-dihyd oxyphenylglycol
esul ed in a se ies o lipophilic 2-alkoxy-2-(3,4-dihyd oxyphenyl)e hyl ace a e h ough an
unexpec ed lipase-media ed e he i ica ion a he benzylic posi ion. These new compounds
a e mo e lipophilic and e ained hei an ioxidan p ope ies. This app oach can p o ide
access o unp eceden ed de i a i es o 3,4-dihyd oxyphenylglycol wi h imp o ed
p ope ies.
In oduc ion
The bene icial e ec s o he Medi e anean die a e pa ly due o i s high con en in an ioxidan
compounds [1]. In pa icula , he polyphenols p esen in he i gin oli e oil display a s ong
an ioxidan ac i i y in i o [2] and in i o [3,4], which has impelled a g owing in e es in hese
compounds, especially hose ha can be ob ained om by-p oduc s o he ood indus y [5].
Se e al epidemiological s udies ha e shown he bene icial heal h e ec s a ising om consump-
ion o oods ich in an ioxidan s, p e en ing he damage caused by p olonged oxida i e s ess
in ce ain biomolecules (nucleic acids, lipids, p o eins), which is associa ed wi h an inc eased
isk o ch onic diseases [6]. The acyla ed polyphenols display imp o ed p ope ies as
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 1 / 19
a11111
OPEN ACCESS
Ci a ion: Sa
´nchez-Ba ionue o L, Gonza
´lez-
Benjumea A, Escoba -Niño A, Ga cı
´a MT, Lo
´pez O
´,
Maya I, e al. (2016) A S aigh o wa d Access o
New Families o Lipophilic Polyphenols by Using
Lipoly ic Bac e ia. PLoS ONE 11(11): e0166561.
doi:10.1371/jou nal.pone.0166561
Edi o : Willem J.H. an Be kel, Wageningen
Uni e si ei , NETHERLANDS
Recei ed: June 15, 2016
Accep ed: Oc obe 30, 2016
Published: No embe 17, 2016
Copy igh : ©2016 Sa
´nchez-Ba ionue o e al. This
is an open access a icle dis ibu ed unde he
e ms o he C ea i e Commons A ibu ion
License, which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium,
p o ided he o iginal au ho and sou ce a e
c edi ed.
Da a A ailabili y S a emen : The nucleo ide
sequences epo ed in his wo k ha e been
deposi ed unde accession numbe s KP212109 o
KP212128 in he GenBank da abase.
Funding: We hank he Jun a de Andalucı
´a (P08-
NMR-3515, P11-CVI-7427 MO, FQM134 and BIO-
213) and he Eu opean Regional De elopmen
Fund (FEDER) o inancial suppo . AGB hanks
he Spanish Minis e io de Economı
´a y
Compe i i idad o he awa d o a g an .
unc ional ing edien s compa ed o he na u al polyphenols [7] as hey a e lipophilic an ioxi-
dan s wi h imp o ed esis ance agains me abolic deg ada ion, and hey can also be inco po-
a ed in o lipid ood ma ices such as a s and oils, p ocessed oods, and ma ga ines [8,9].
P o ec ion and dep o ec ion o unc ional g oups a e commonly employed in o ganic
chemis y o ca y ou he syn hesis o pa ially acyla ed de i a i es [10]. Howe e , despi e o
he many ad ances in o ganic syn hesis du ing he las decades, he con en ional chemical
syn hesis o mono acyla ed de i a i es o polyphenols p esen s se ious di icul ies due o he
high densi y o e y simila unc ional g oups, which equi es ex ensi e p o ec ion and dep o-
ec ion sequences [10]. Thus, al hough some chemical app oaches ha e been de eloped o
accessing pa ially acyla ed polyphenols in a chemoselec i e way [11–13], mos o such p oce-
du es a e pu e chemical syn hesis, and in ol e he use o haza dous eagen s o non-g een
condi ions. As an al e na i e, he bio ech indus ies ha e adi ionally p oduced compounds
o comme cial in e es by mic obial e men a ion, o by e men a ion ollowed by subsequen
chemical modi ica ion o imp o e speci ic p ope ies, such as ac i i y, solubili y, abso p ion,
pha macokine ics o s abili y. In his con ex , enzymes and mic oo ganisms ha e been e i-
cien ly used as bioca alys s in chemo-, egio- and s e eoselec i e syn hesis o bioac i e
compounds.
We eason ha an enzyme-ca alyzed app oach could o e a iable al e na i e o adi ional
e men a ion p ocedu es and con en ional chemical me hods o he chemo- and egioselec-
i e syn hesis o hese new lipophilic compounds [14]. In his wo k, special a en ion has been
ocused on hyd oxy y osol (HT) and 3,4-dihyd oxyphenylglycol (DHPG), which p edomina e
in lea es and ui s o oli e ees (Olea eu opea), ei he ee o as acyl de i a i es, and display
an ioxidan ac i i y [15]. We ha e de eloped a no el enzyma ic me hod o ob ain a ge ed
mono- o di-acyla ed de i a i es o he polyphenols.
Ma e ials and Me hods
Si e desc ip ion and sample collec ion
The bac e ial s ains used in his s udy we e isola ed om loca ions in he p o inces o Badajoz
and Huel a (Spain) in 2010. Samples HR11 and HR12 con ained semisolid a s om a mea
cu ing ac o y (38.151216˚N, -6.684258˚E). Sample HR11 was ob ained by collec ing he d ip-
ping a om he loo o he ac o y p emises. Sample HR12 was ob ained om a ank con-
aining a le o e s. Sample HR21 consis ed o he ish dus ha esul s a e cu ing ish in o
pieces be o e canning in a ish canning ac o y (37.20994˚N, -7.26167˚E). All samples we e col-
lec ed in 50 ml s e ile plas ic ubes and s o ed a 4˚C un il use.
Sc eening o de ec lipoly ic mic oo ganisms (hyd olysis)
Fish sawdus samples (7.28 g) we e suspended in 25 ml o s e ile saline solu ion (NaCl 0.85%
w/ ). In he case o he sample om cu ed mea oil, 5 ml o each sample o a we e suspended
in 20 ml o s e ile saline solu ion (NaCl 0.85% w/ ). Sc eening o lipoly ic mic oo ganisms
was pe o med as p e iously desc ibed [16].
T anses e i ica ion assay o lipoly ic ac i i y
T anses e i ica ion ac i i y o lipase was es ed by a colo ime ic me hod wi h mino modi ica-
ions [16]. This me hod is based on he elease o he yellow-colo ed compound p-ni ophenol
(p-NP) a e he anses e i ica ion o p-ni ophenyl palmi a e (p-NPP; Sigma-Ald ich) wi h
e hanol, and he subsequen de ec ion by using UV-Vis spec opho ome y. S ains p oducing
he maximum lipase ac i i y we e selec ed o u he s udies.
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 2 / 19
Compe ing In e es s: AEN, LSB, DC, EM, AGB, OL,
IM and JGFB ha e iled a PCT pa en applica ion
i led “Bac e ial s ains and he uses he eo in
acyla ion and/o deacyla ion eac ions” (PCT/
ES2015/000062; WO/2015/169980) ha desc ibe
pa s o he esea ch in his manusc ip . This does
no al e he au ho s’ adhe ence o all o he PLOS
ONE policies on sha ing da a and ma e ials
p esen ed in his manusc ip .
Op imiza ion o bac e ial g ow h condi ions and lipase p oduc ion
In o de o op imize he p oduc ion o he bac e ial lipases, he s ains we e g own in wo di -
e en media (PYB o LB) in he p esence o in he absence o 2% ibu y in (PYBT o LBT
media). PYB medium con ains 1% (w/ ) pep one, 0.5% (w/ ) yeas ex ac , 0.1% (w/ )
K2HPO4, 0.02% (w/ ) MgSO47H2O. pH was adjus ed o 7.5. LB medium (1% (w/ ) yp one,
0.5% (w/ ) yeas ex ac , 0.5% (w/ ) NaCl) was supplemen ed wi h 2% (w/ ) glucose. Bac e ia
we e g own in 500 ml E lenmeye lasks con aining 100 ml o medium. G ow h was moni-
o ed by measu ing he abso bance a 600 nm (OD
600
) in a Beckman DU640 spec opho ome-
e . The lipase ac i i y was assayed employing he p-NPP me hod desc ibed a 37˚C.
Pu i ica ion o he supe na an s
The cell- ee supe na an was ob ained by cen i uga ion o bac e ial cul u es a 4,500 pm o
5 min a 4˚C. These supe na an s we e concen a ed in dialysis bags (12 kDa, Sigma) agains
polye hylene glycol (8 kDa) a 4˚C o e nigh , and hen, hey we e dialyzed in 0.05 M po as-
sium phospha e bu e (pH 7.6). Finally, hey we e eeze-d ied. The d y supe na an s we e
employed as enzyma ic cock ails o he lipase assays.
Isola ion o DNA and 16S RNA gene sequence analysis
Bac e ial DNAs we e isola ed and used o he ampli ica ion o he 16S RNA by PCR using
he uni e sal p ime s 16F27 (50-AGAGTTTGATCMTGGCTCAG-30) and 16R1488 (50-
CGGTTACCTTGTTAGGACTTCACC-30) as p e iously epo ed [16]. 16S RNA sequences co -
esponding o posi ions 53 o 667 o he 16S RNA gene om Esche ichia coli we e ob ained
and analyzed as p e iously desc ibed [16].
Nucleo ide sequence accession numbe s
The nucleo ide sequences we e deposi ed unde accession numbe s KP212109 o KP212128 in
he GenBank da abase.
Gene al me hods o he chemoenzyma ic syn heses o acyla ed
polyphenols
NMR spec a we e eco ded a 25˚C on a B uke A ance 300 spec ome e , on a B uke
A ance III 500 MHz, and on a B uke A ance III 700 MHz ins umen s equipped wi h a c yo-
genically cooled 5 mm TCI g adien p obe. Chemical shi s a e epo ed in ppm (δ) and spec-
a we e e e enced o he esidual p o ona ed sol en (3.31 and 49.0 ppm o CD
3
OD, 7.26
and 77.2 ppm o CDCl
3
, 2.05 and 29.8 o (CD
3
)
2
CO, o
1
H and
13
C NMR, espec i ely).
Coupling cons an s (J) a e exp essed in He z (Hz). The assignmen s o
1
H and
13
C signals
we e con i med by 1D and 2D NMR expe imen s (COSY, HSQC, HMBC). High esolu ion
mass spec a we e ob ained by LSIM using a Hewle Packa d 5989 A spec ome e coupled o
a Hewle Packa d 5990 II gas ch oma og aphe and a Mic omass Au oSpec-Q spec ome e
wi h a esolu ion o 1,000 o 10,000 (10% alley de ini ion); a cesium gun, 1- hioglice ol as
ma ix and NaI as addi i e we e used. Column ch oma og aphy was pe o med using Me ck
silica gel 60 (230–400 mesh). TLCs we e pe o med on silica-coa ed aluminum shee s om
Me ck (silica gel 60 F254) using mix u es o CH
2
Cl
2
−MeOH and E OAc−hexane as eluan s;
spo s we e isualized by UV ligh and by s aining wi h anillin/H
2
SO
4
in E OH (1.5 g o anil-
lin in 100 mL o 95% E OH/conc H
2
SO
4
100:1).
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 3 / 19
Lipase-media ed ace yla ions o polyphenols 1, 4 and 8
The O-ace yla ions o he phenolic compounds we e pe o med using he ou lipoly ic bac e-
ial ex ac s, in a subs a e-lipase ex ac 1:1 a io in weigh (40 mg). Isop openyl ace a e was
used as sol en and acyla ing agen , 40 equi (0.52–0.63 mL). The mix u e was s i ed a 40˚C
o 24 h in da kness. In he case o glycol de i a i e 4, DMF was used as co-sol en because o
solubili y p oblems, isop openyl ace a e-DMF 1:1 in olume (1.0 mL).
Lipase-media ed deacyla ion o compounds 2, 5 and 9
Fo he deacyla ion eac ions o pe ace yla ed polyphenols, o a solu ion o 2,5and 9(50 mg)
in a p ima y linea alipha ic alcohol (MeOH, E OH, p opan-1-ol o bu an-1-ol) was added sil-
ica gel and he lipase ex ac ( om Bacillus sp. HR21-6 o Te ibacillus sp. 2B122), in a sub-
s a e-alcohol-lipase ex ac -silica gel 1:50:2:2 a io in weigh . The mix u e was hea ed a 60˚C
o 24–168 h un il o al con e sion (in he case o Te ibacillus sp. 2B122 lipase a second addi-
ion o lipase ex ac was needed when he eac ion a e is ma kedly educed). Finally, he sol-
en was e apo a ed and he esidue was pu i ied by column ch oma og aphy o gi e 3,6a-d
and 10, espec i ely, using E OAc-hexane o CH
2
Cl
2
-MeOH g adien s as eluan s (see S1
Supplemen a y Expe imen al P ocedu es).
Hyd oxy y osyl ace a e (3) om compound 2
Elu ed wi h E OAc-hexane 1:1 ga e 3(90% wi h Bacillus sp. HR21-6 o Te ibacillus
sp. 2B122) as a colo less sy up. Spec oscopic da a o 3a e in ag eemen wi h hose epo ed
in li e a u e [17].
Deace yla ion eac ions o compounds 6a-d
To a solu ion o 6(40 mg) in d y MeOH (1 mL) we e added Cs
2
CO
3
(2 equi .) and sodium
asco ba e (1 equi .). The mix u e was s i ed in he da kness and in he p esence o A a oom
empe a u e o 2–3 h. Then, he p oduc was pu i ied wi hou e apo a ion o he sol en by
column ch oma og aphy o gi e 7a-d, using CH
2
Cl
2
−MeOH, CH
2
Cl
2
−E OH, and E OAc
−hexane g adien s as eluan s (see S1 Supplemen a y Expe imen al P ocedu es).
4-(Me hoxyme hyl)benzene-1,2-diol (10) om compound 9
Elu ed wi h E OAc-hexane 1:1 ga e 10 (quan . wi h Bacillus sp. HR21-6) as a colo less sy up.
Spec oscopic da a o 10 a e in ag eemen wi h hose epo ed in li e a u e [18]. Supplemen-
a y expe imen al da a p ocedu es include addi ional in o ma ion o he compounds 6a-d
and 7a-d.
DPPH adical sca enging ac i i y
The an i adical ac i i y o 6a-d and 7a-d has been e alua ed by he DPPH me hod [19,20].
Resul s
Lipase-media ed O-deacyla ion o pe ace yla ed HT
In o de o ob ain lipophilic phenolic es e s i is essen ial o achie e he es e i ica ion o p i-
ma y alcoholic g oups wi hou a ec ing he ca echol moie y, which is known o be essen ial
o he an ioxidan e ec s. This p ocess equi es a chemoselec i e p ocedu e.
In a p e ious wo k we isola ed bac e ia capable o pe o ming anses e i ica ion eac ions in
a egioselec i e ashion [16]. We easoned ha hose bac e ial isola es could also be used o he
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 4 / 19
egioselec i e acyla ion/deacyla ion o polyphenols. Fo an ini ial analysis, HT (1) was i s ly
pe ace yla ed wi h ace ic anhyd ide in py idine o ob ain 2, as p e iously desc ibed [21]. The
deace yla ion o he pe ace yla ed HT was e icien ly achie ed using he supe na an s o a bac e-
ial cul u e ob ained om ou bes pe o ming s ain (Te ibacillus sp. 2B122) and an alipha ic
alcohol as sol en (MeOH). The anses e i ica ion eac ion was comple ely chemoselec i e in
he phenolic posi ions, and ga e a monoace yla ed de i a i e in he alipha ic posi ion (com-
pound 3, [22] as depic ed in Fig 1, a na u al compound p esen in ex a i gin oli e oil [23]).
Sc eening o addi ional mic oo ganisms showing lipoly ic ac i i y
In he p e ious eac ion, he deace yla ion was comple ely selec i e owa ds he phenolic
es e s, bu we did no obse e any selec i i y owa ds he alipha ic es e moie ies wi h his o
o he s ains p e iously isola ed. The e o e, we designed a new sc eening aiming a inding
mic oo ganisms capable o anses e i ica ion he alipha ic es e selec i ely. We easoned ha
ood indus ies in ol ed in he p ocessing o oods wi h high a con en would p o ide he
bes en i onmen . Since a ege able oil- ich loca ion was sc eened p e iously, a ish and a
mea indus ies loca ed nea Isla C is ina (Huel a, Spain) and Higue a la Real (Badajoz,
Spain), espec i ely, we e selec ed o sampling his ime. A e a i s ound o selec ion o e
4,000 colonies ( ungi and bac e ia) we e isola ed om ish sawdus and om cu ed mea a .
Ou o hose, 459 we e conside ed as posi i e (S1 Table). In e es ingly, no lipoly ic bac e ial
isola es we e selec ed om he sample HR12 ob ained om a mea cu ing ac o y, whe e he
numbe o mic oo ganisms ob ained was 378. The lipoly ic bac e ia we e g own in a non-
selec i e medium (i.e. in he absence o ibu y in), and he lipoly ic ac i i y was hen con-
i med on LB supplemen ed wi h 0.5% ibu y in. These samples we e ans e ed o he sec-
onda y sc eening.
Sc eening o mic oo ganisms capable o pe o ming anses e i ica ion
eac ions among he selec ed lipoly ic s ains
The second s ep o he sc eening was pe o med using eeze-d ied supe na an s o he 66 bac-
e ial isola es selec ed a e he i s s ep o he sc eening. The supe na an s we e assayed o
anses e i ica ion ac i i y by quan i ica ion o he yellow-colo ed p-ni ophenol (p-NP) ha is
eleased a e he anses e i ica ion o p-ni ophenyl palmi a e (p-NPP) wi h e hanol o gi e
ace yl-palmi a e es e and p-NP. We pe o med con ol assays as p e iously desc ibed [16].
The maximum alue ob ained in hese con ol eac ions was 0.119 and i was se as he h esh-
old. Ou o he 66 lipoly ic bac e ial s ains in his second sc eening, 20 supe na an s p oduced
abso bance alues highe han he cu -o o 0.119 (S2 Table). Mos o he supe na an s om
he bac e ial isola es displayed alues be ween he cu -o and 0.300 and only e y ew we e
capable o displaying highe ac i i ies (4 isola es).
Phylogene ic classi ica ion o he selec ed lipoly ic bac e ia
Phylogene ic s udies we e conduc ed using pa ial 16S RNA sequences wi h he aim o iden i-
ying he bac e ial isola es. Signi ican ly, all s ains isola ed belong o he G am-posi i e bac e-
ia and we e closely ela ed o membe s o he genus Bacillus exhibi ing simila i y alues
anging om 98% o 100%. The phylogene ic econs uc ion ca ied ou wi h di e en me h-
ods was consis en and sugges ed ha all he s ains (HR21-1, HR21-3, HR21-6, HR21-12,
HR21-13, HR21-17, HR21-18, HR21-20, HR21-23, HR21-26, HR21-28, HR21-29, HR21-30,
HR21-52, HR21-59, HR21-60, HR21-62, HR21-63, HR11-64 and HR11-65) clus e ed oge he ,
exhibi ing a high simila i y (98% o 100%) o he 16S RNA sequences o Bacillus ae ophilus,
Bacillus s a osphe icus and Bacillus al i udinis (Fig 2).
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 5 / 19

O-Deacyla ion o pe ace yla ed HT by Bacillus lipases
Since mos o he isola es belonged o he Bacillus genus and we e closely ela ed, we only
es ed wo andom isola es o deacyla ion o HT. Howe e , he esul s we e simila o hose
ob ained p e iously in Fig 1. Since he phylogene ic econs uc ion using pa ial 16S RNA
sequences did no clea ly di e en ia e be ween se e al species belonging o he genus Bacillus,
we selec ed one o hose wo isola es o u he s udies and sequenced he comple e 16S RNA.
A e u he analysis, HR21-6 appea ed o clus e oge he wi h B.pumilus and no wi h B.
s a osphe icus,B.ae ophilus o B.al i udinis (S1 Fig).
Op imiza ion o he bac e ial g ow h condi ions o he p oduc ion o
lipases
The s udy was con inued wi h ou isola es belonging o di e en gene a o es s in acyla ion
and deacyla ion eac ions o na u al polyphenols. Th ee o he s ains we e p e iously isola ed
by ou esea ch g oup ( he G am nega i e Pseudomonas sp. 2B120 and En e obac e sp. 1B89,
and he G am posi i e Te ibacillus sp. 2B122) [16] and he ou h s ain, Bacillus HR21-6, was
isola ed du ing his wo k (as desc ibed abo e).
An impo an ac o in his s udy was o de ine he op imal condi ions o he p oduc ion
o he lipase- ich supe na an s. Two pa ame e s we e selec ed o designing he op imiza ion
a iables: medium and incuba ion ime. A his s age i was no known whe he he lipases
esponsible o he anses e i ica ion ac i i y obse ed we e induced by lipidic subs ances o
cons i u i ely exp essed. The e o e, we also added he lipidic subs a e ibu y in (p e iously
used o he sc eening) o he media. The e ec o he di e en cul u e media in lipase ac i i y
a a ious ime in e als is shown in Fig 3. In gene al, he ac i i y o he supe na an s o Bacil-
lus sp. HR21-6 was highe when he mic oo ganism was g own in PYB han in LB medium.
The maximum ac i i y was ob ained a 48 h in PYB medium, which co esponded wi h he
la e s a iona y phase. In LB medium, he maximum ac i i y was eached a e 24 h o g ow h
(Fig 3A). In bo h cases, addi ion o ibu y in p oduced a dec ease in he lipase ac i i y.
Fo Pseudomonas sp. 2B120 he lipase p oduc ion s a ed a la e s a iona y phase o he bac-
e ial g own. The maximum lipase ac i i y was ob ained in PYB medium a e 72 h o g ow h
(Fig 3B). The addi ion o ibu y in did no inc ease he lipase ac i i y o he supe na an s. Fo
Te ibacillus sp. 2B122 he maximum lipase ac i i y was also de ec ed in PYB medium a 72 h
(Fig 3C). Su p isingly he esul s o he Te ibacillus sp. 2B122 lipase ac i i y in LB medium
om 48 h o 72 h showed a signi ican ly high e o , which may be due o he cell lysis expe i-
enced by his s ain in LB and LBT a a ound 24 h o cul i a ion. Op imal condi ions o he
lipase ac i i y o En e obac e sp. 1B89 we e de e mined o be maximal in LBT medium a 48 h
cul i a ion. In his case, addi ion o ibu y in o he cul u e medium allowed an inc ease in
he lipase ac i i y (Fig 3D).
Fig 1. Regioselec i e deace yla ion o pe ace yla ed HT (2) ca alyzed by he supe na an s om bac e ial cul u es.
doi:10.1371/jou nal.pone.0166561.g001
New Families o Lipophilic Polyphenols
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Lipase-media ed O-deacyla ion o polyphenols
Du ing his wo k we also aimed a s udying ano he in e es ing polyphenol, 3,4-dihyd oxy-
phenylglycol (DHPG), which is also p esen in oli es and shows excellen an ioxidan cha ac-
e is ics. In a i s s ep, he pe ace yla ed DHPG (compound 5) was p epa ed om DHPG 4
[24]. The phenolic hyd oxyl g oups we e egioselec i ely deace yla ed by a ansace yla ion
p ocess wi h a p ima y linea alipha ic alcohol (me hanol, e hanol, p opan-1-ol o bu an-1-ol)
in a eac ion mix u e con aining he bac e ial supe na an s and silica gel, using a 1:50:2:2 sub-
s a e−alcohol−lipase ex ac −silica gel a io. The ace oxy g oup a he p ima y posi ion
emained unchanged, whe eas he ace oxy g oup a benzylic posi ion was, unexpec edly,
subs i u ed by he co esponding alkoxy g oup (Figs 4and 5A). The p og ess o he eac ions
was moni o ed by TLC, and i esul ed in he syn hesis o monoace yla ed e he i ied de i a-
i es 6a-d (see S1 Supplemen a y Expe imen al P ocedu es) wi h a yield anging om 40 o
90% depending on he bac e ial isola e (Fig 5B). This p ocedu e cons i u es he i s syn hesis
o 3,4-dihyd oxyphenylglycol e he s 6. The enzymes did no only ca alyze he alcoholysis o
he ace oxy g oups on he a oma ic ing, bu also he subs i u ion o he ace oxy g oup a ben-
zylic posi ion by an alkoxy g oup (–OR). The co esponding deace yla ed de i a i es 7a-d
Fig 2. E olu iona y ela ionships o he selec ed s ains. The phylogene ic ee shows he posi ion o he
isola es displaying lipoly ic ac i i y wi h espec o o he ype s ains o genus Bacillus and an ex e nal bac e ial
g oup. The dis ances we e calcula ed using Maximum Composi e Likelihood. 16S RNA gene sequences
om he isola es co espond o 614 bps.
doi:10.1371/jou nal.pone.0166561.g002
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 7 / 19
Fig 3. Op imiza ion o g ow h condi ions o he selec ed s ains o he de ec ion o lipase ac i i y. To
de e mine de lipase ac i i y he bac e ial s ains, Bacillus sp. HR21-6 (A), Pseudomonas sp. 2B120 (B),
Te ibacillus sp. 2B122 (C) and En e obac e sp. 1B89 (D) we e g own in PYB o LB media wi h (PYBT o
LBT) o wi hou (PYB o LB) ibu y in o he indica ed imes. Lipase ac i i y in he supe na an s was
quan i ied by using he p-NPP me hod. Da a shown a e he a e age o a leas 3 independen expe imen s
and he s anda d de ia ion o he mean.
doi:10.1371/jou nal.pone.0166561.g003
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 8 / 19
we e p epa ed by a basic me hanolysis in he p esence o Cs
2
CO
3
as a weak base (wi h a yield
o 31–65%, see S1 Supplemen a y Expe imen al P ocedu es). Due o he easy deg ada ion o
he ca echol g oup a he high pH equi ed o he deace yla ion eac ion, sodium asco ba e (1
equi ) was added o he eac ion mix u e o p e en ex ensi e deg ada ion.
The p e ious eac ion on DHPG cons i u ed an ou s anding case o an enzyma ic con e -
sion o an es e in o an e he . In o de o u he check his eac ion, we assayed pe ace yla ed
p o oca echuic alcohol 9, which is a compound e y simila o pe ace yla ed DHPG 5, ha -
bou ing a benzylic alcohol bu lacking he ace oxyme hyl g oup a he end o he alipha ic side
chain. P o oca echuic alcohol 8is a e y po en an ioxidan molecule, also ound in i gin
oli e oil [25]. As desc ibed abo e, we i s ob ained he pe ace yla ed de i a i e o p o oca e-
chuic alcohol (compound 9[26]), and hen he deace yla ion eac ion p oceeded in he p es-
ence o he bac e ial supe na an s and MeOH as sol en (Fig 6). Simila ly o he eac ion
desc ibed o pe ace yla ed DHPG 5in Fig 4, he ace oxy g oup a he benzylic posi ion was
subs i u ed by a me hoxy g oup, gi ing e he 10.
Analysis o lipase-media ed O-acyla ion o polyphenols
All he p e ious eac ions we e based on he deace yla ion o a pe ace yla ed de i a i e o he
co esponding polyphenol. In o de o check he selec i i y o he enzymes in he bac e ial
supe na an s du ing es e i ica ion eac ions, we also es ed he di ec O-acyla ion o polyphe-
nols 1,4and 8. In his case, a comme cial lipase ob ained om Candida an a c ica (No ozyme
435) used p e iously in his ype o eac ions [27] was also s udied in pa allel wi h ou sub-
s a es o compa a i e pu poses. The O-acyla ions o he phenolic compounds 1,4and 8we e
ca ied ou wi h he ou lipoly ic bac e ial ex ac s and isop openyl ace a e, as sol en and
Fig 4. Regioselec i e deace yla ion o pe ace yla ed DHPG (5) wi h bac e ial supe na an s.
doi:10.1371/jou nal.pone.0166561.g004
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 9 / 19
In conclusion, his s udy opens new a enues o he ood indus ies o ob ain unp ece-
den ed de i a i es o an ioxidan s wi h expanded physico-chemical p ope ies and u ili ies.
Families o DHPG de i a i es could be mo e sui ed o hese pu poses han he ex ensi ely
s udied HT. Consequen ly, biological and pha macological s udies o bo h se ies o DHPG
de i a i es will be unde aken, in o de o elucida e he mo e e ec i e compounds in cance
and ch onic degene a i e disease p e en ion, and as an i-in lamma o y compounds.
Suppo ing In o ma ion
S1 Fig. Phylogene ic maximum-likelihood ee showing he posi ion o Bacillus sp HR21-6
wi h espec o o he ype s ains o he genus Bacillus.The 16S RNA sequence o S ep o-
coccus pneumoniae was used as an ex e nal g oup. 16S RNA gene sequences om he isola es
co espond o 1380 bps. The phylogene ic econs uc ion ca ied ou wi h di e en me hods
was consis en , and consequen ly only he ee ob ained wi h Neighbo -Joining, o he e olu-
iona y his o y, and Maximum Composi e Likelihood, o e olu iona y dis ances is shown.
(TIF)
S2 Fig. Lipase ca alized ace yla ion o polyphenols HT (1), DHPG (4) and p o oca echuic
alcohol (8). Pa ial
1
H-NMR spec a o he lipase-media ed ace yla ion o HT (A) o gi e
mono- and di-ace yla ed de i a i es o HT (C), compa ed o he pe ace yla ed HT (B). Pa ial
1
H-NMR spec a o he lipase-media ed ace yla ion o DHPG (D) o gi e mono- and di-ace y-
la ed de i a i es o DHPG (E). Pa ial
1
H-NMR spec a o he lipase-media ed ace yla ion o
p o oca echuic alcohol (F) o gi e mono- and di-ace yla ed de i a i es o p o oca echuic alco-
hol (H), compa ed o he pe ace yla ed p o oca echuic alcohol (G).
(TIF)
S1 Supplemen a y Expe imen al P ocedu es. 2-(3,4-Dihyd oxyphenyl)-2-me oxye hyl ace-
a e (6a) om compound 5; 2-(3,4-Dihyd oxyphenyl)-2-e hoxye hyl ace a e (6b); 2-(3,4-Dihy-
d oxyphenyl)-2-p opoxye hyl ace a e (6c); 2-Bu oxy-2-(3,4-dihyd oxyphenyl)e hyl ace a e
(6d); 2-(3,4-Dihyd oxyphenyl)-2-me hoxye hanol (7a); 2-(3,4-Dihyd oxyphenyl)-2-e hox-
ye hanol (7b); 2-(3,4-Dihyd oxyphenyl)-2-p opoxye hanol (7c); 2-Bu oxy-2-(3,4-dihyd oxy-
phenyl)e hanol (7d); Spec oscopic da a o c ude eac ion depic ed in Fig 7; Spec oscopic
da a o c ude eac ion depic ed in Fig 8; Spec oscopic da a o c ude eac ion depic ed in Fig
9.
(PDF)
S1 Table. Selec ion o lipoly ic mic oo ganisms in he p ima y sc eening (hyd oly ic).
(PDF)
S2 Table. Abso bance alues ob ained in he anses e i ica ion assay. In ed, he isola es
selec ed wi h abso bance alues highe han he highes hyd olysis con ol.
(PDF)
Acknowledgmen s
We would like o hank he Se icio de Resonancia Magne
´ ica Nuclea , CITIUS (Uni e si y o
Se ille) o he pe o mance o NMR expe imen s.
Au ho Con ibu ions
Concep ualiza ion: MTG OL IM JGFB DC EM.
Fo mal analysis: MTG OL IM JGFB DC EM.
New Families o Lipophilic Polyphenols
PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 16 / 19

Funding acquisi ion: JGFB EM.
In es iga ion: LSB AGB AEN.
Me hodology: MTG OL IM JGFB DC EM.
P ojec adminis a ion: JGFB EM.
Supe ision: OL JGFB DC EM.
Valida ion: OL JGFB DC EM.
W i ing – o iginal d a : MTG OL IM JGFB DC EM LSB.
W i ing – e iew & edi ing: MTG OL IM JGFB DC EM.
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