A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria

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 PLOS ONE | DOI:10.1371/jou nal.pone.0166561 No embe 17, 2016 6 / 19 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. 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