Fatty Acid Hydroxytyrosyl Esters of Olive Oils Are Bioaccessible According to Simulated In Vitro Gastrointestinal Digestion: Unraveling the Role of Digestive Enzymes on Their Stability

Fa y Acid Hyd oxy y osyl Es e s o Oli e Oils A e Bioaccessible Acco ding o Simula ed In Vi o Gas oin es inal Diges ion: Un a eling he Role o Diges i e Enzymes on Thei S abili y Ca olina Alemán-Jiménez, # Raul Domínguez-Pe les, # Juana I. Gallego-Gómez, Agus ín Simonelli-Munoz, Espé ance Moine, Thie y Du and, Céline C aus e, Fede ico Fe e es, Angel Gil-Izquie do,* and Sonia Medina* Ci e This: J. Ag ic. Food Chem. 2021, 69, 14165−14175 Read Online ACCESS Me ics & Mo e A icle Recommenda ions ABSTRACT: Recen ly, new bioac i e compounds we e iden ified in oli e oil, lipophenols, which a e composed o a a y acid (FA) and a phenolic co e, such as HT (HT-FA). Howe e , hei bioaccessibili y emains unknown. Thus, he p esen s udy unco e s he impac o he sepa a e phases o gas oin es inal diges ion on he elease and s abili y o HT-FAs om oily ma ices unde in i o simula ed condi ions. Acco dingly, i was ound ha he bioaccessibili y o HT de i a i es is la gely dependen on he ype o FA ha es e ifies HT, as well as he ood ma ix. Also, he gene a ion o HT-FAs du ing in es inal diges ion was obse ed, wi h panc ea in being he enzyme esponsible, o a highe ex en , o he de no o o ma ion o lipophenolic de i a i es. These findings p omp us o iden i y new applica ions o oily ma ices and hei byp oduc s as po en ial unc ional ing edien s o he p omo ion o heal h, whe e he possible o ma ion o new lipophenols du ing diges ion should be aken in o conside a ion. KEYWORDS: oli e oil, hyd oxy y osol, lipophenols, in i o gas oin es inal diges ion, bioaccessibili y, lipases ■INTRODUCTION Oli e d upes and de i ed p oduc s a e conside ed pa o he Medi e anean die and co-con ibu e o he heal hy cha ac e - is ics o his die , especially because o hei con en o mono- and polyunsa u a ed a y acids (FAs) and (poly)phenols such as hyd oxy y osol (HT). 1,2 Mo eo e , oleic acid is he mos abundan FA in oli e oil, and HT is i s p incipal phenolic compound. 3 Also, he p esence o lipophenol s uc u es, composed o an FA and a phenolic co e, has been epo ed in edible oils. These molecules esul om he es e ifica ion o HT wi h a y acids (e.g.,α-linolenic, linoleic, and oleic acids (HT-ALA, HT-LA, and HT-OA, co espondingly)). 4,5 To da e, ex a i gin and i gin oli e oils (EVOO and VOO, espec i ely) a e he oily ma ices wi h he highes concen a ion o hese compounds. 5 Lipophenols offe unc ional ad an ages ela i e o hei molecula cons i uen s (HT and FAs). Specifically, hese compounds a e cha ac e ized by a highe lipophilici y, cell memb ane affini y, and imp o ed an ioxidan ac i i ies. 6−8 Howe e , despi e he sugges ion o lipophenol’s biological powe , 9,10 he ac ual unc ions emain almos unexplo ed. Ne e heless, ega ding hei an ioxidan p ope y, i may p og essi ely be augmen ed along wi h he ex ension o he FA chain leng h up o a c i ical poin , when addi ional ex ension could lowe he adical sca enging powe . Because o his phenomenon, he so-called cu offeffec , sho - o medium- chain lipophilic es e s o phenolic compounds (C8−C12) a e mo e effec i e an ioxidan s ela i e o long-chain es e s. 11 In con as , o he s udies ha e associa ed equal o highe ac i i y wi h long-chain es e s (C18−C20). 12,13 Aside om hese conside a ions, he bioaccessibili y o lipophenols emains o be assessed. In his ega d, acco ding o p e ious s udies, in ense hyd olysis o HT ace a e a e panc ea in and bile sal diges ion has been epo ed, 14 while y osol acyl es e s a e hyd olyzed by panc ea ic lipase o p oduce ee y osol, showing a ce ain sus ained- elease beha io . 15,16 These esul s sugges ha he ime ame o e ie ing biological ac i i ies om phenolic compounds could be ex ended due o lipophenol’s bioac i i y, hus allowing us o ob ain addi ional heal h benefi s. 15 These au ho s epo ed ha he s abili y o y osol es e s unde he physicochemical condi ions associa ed wi h he in i o simula ion o gas o- in es inal (GI) diges ion was co ela ed bo h wi h he chain leng h and wi h he numbe o unsa u a ions. 16 None heless, he effec o he diges i e p ocess on he elease o HT-FAs and hei s abili y emains unde explo ed. Also, he expe - imen al app oach implemen ed hus a , based on he analysis o au hen ic s anda ds ins ead o ood ma ices, is negligible because i igno es he effec o he ma ix h oughou he diges ion. 14 To o e come his limi a ion, a simula ed GI Recei ed: Augus 30, 2021 Re ised: No embe 11, 2021 Accep ed: No embe 12, 2021 Published: No embe 19, 2021 A iclepubs.acs.o g/JAFC © 2021 Ame ican Chemical Socie y 14165 h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 Downloaded ia UNIV DE ALMERIA on Decembe 1, 2021 a 19:13:42 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. diges ion by in i o models could be a aluable, ime-sa ing ool, as i equi es ewe esou ces o ob aining p elimina y bu ex emely impo an in o ma ion o he e alua ion o he s uc u al and chemical changes unde GI condi ions. 17 Acco ding o his backg ound in o ma ion and p e ious findings, he p esen wo k aims o gain u he insigh in he esul s o he GI diges ion on HT-ALA, HT-LA, and HT-OA ound in EVOO and VOO o iden i y he lipophenols eleased om he ood ma ix ha emain s able du ing GI diges ion and ha a e a ailable o abso p ion. Also, he e alua ion o he effec o pepsin, panc ea in, and panc ea ic lipase allowed us o disco e he influence o he diges i e enzymes on he s abili y o lipophenols. The majo ou comes e ie ed con ibu ed o se ing up he bioaccessibili y o HT-FAs in he small in es ine compa ed wi h unes e ified HT and o unde s anding he enzyma ic mechanisms esponsible o hem, p o iding he heo e ical basis o he ans o ma ion o lipophenols du ing he diges i e p ocess. ■MATERIALS AND METHODS Chemicals and Reagen s. The au hen ic s anda ds o high-pu i y HT-FAs (HT-ALA, HT-LA, and HT-OA) we e syn hesized and ully cha ac e ized by nuclea magne ic esonance (NMR)-based analysis by he Ins i u des Biomolecules Max Mousse on (IBMM) (Mon pellie , F ance) acco ding o p e iously published p ocedu es. 4 Ace one and bu yla ed hyd oxyanisole (BHA) we e pu chased om Sigma-Ald ich (S . Louis, MO, USA), and all LC−MS-g ade sol en s (deionized wa e , me hanol, and ace oni ile) we e om J.T. Bake (Phillipsbu g, NJ, USA). A ce ified e e ence o FA s anda ds om Sigma-Ald ich was used o hei iden ifica ion and quan ifica ion. Hyd oxy y osol, wi h he ollowing specifica ions: pu i y 99.6%, mois u e 3.7%, and pH 4.26 (1 M aqueous solu ion), was p o ided by Sep ox BIOTECH S.L. (Mu cia, Spain). All lipophenols we e dissol ed in dime hyl sul oxide (DMSO) om Sigma Ald ich (S . Louis, MO, USA) o ob ain a s ock solu ion (a he mM ange o concen a ion), and hen successi e dilu ions we e p epa ed in me hanol/deionized Milli-Q wa e (50:50, / ). All s ock solu ions o lipophenols we e s o ed a −20 °C in he da k. Po cine pepsin (P6887), panc ea in om po cine panc eas (P7545, 8 ×USP), and lipase om po cine panc eas (L3126) we e ob ained om Sigma- Ald ich Co. (S . Louis, MO, USA). All o he eagen s we e o analy ical g ade. Fa y Acid Composi ion Analysis o Edible Oils. Fa y acids o EVOO, VOO, and flaxseed oil (FO) we e ex ac ed acco ding o he me hodology employed p e iously 5 and published by he FAO (FAOLEX No. LEX-FAOC141241, h p://www. ao.o g/ aolex/ esul s/de ails/es/c/%20LEX-FAOC141241/) o he analysis o ege able oils. B iefly, samples (1 g) we e weighed and d ied a 70 ±10 °C o 6 h. The a y acid con en was ob ained by he Soxhle ex ac ion wi h die hyl e he and hep ane and by es e ifica ion wi h me hanolic NaOH (1 M). Fa y acids we e de e mined by gas ch oma og aphy coupled o a flame ioniza ion de ec o (GC-FID) (6890 GC Agilen Technologies, Wald onn, Ge many). The absolu e concen a ion (g/100 g w) o a y acids in he ege able oils (n=3) was calcula ed acco ding o he o mula indi idual a y acid (g/100 g w) = indi idual a y acid (% o a )/100 × o al a (g/100 g w), which was used acco ding o FAO/INFOODS guidelines o con e ing uni s, denomina o s, and exp ession (2012). 18 Vege able Oil Samples and P epa a ion o Analy ical Ex ac s o Hyd oxy y osol Fa y Acid Es e s. Ex a i gin oli e oil (Picual mono a ie al; Olimend os S.L. (Mu cia, Spain)), VOO (Picual mono a ie al; Sal ado Gallego El Jo a S.L. (Albace e, Spain)), and FO (Labo a o ios Almond S.L. (Mu cia, Spain)) we e chosen due o hei dissimila concen a ion o HT es e ified wi h ALA, LA, and OA, wi h FO used as he nega i e con ol sample due o i s lack o HT-FA es e con en . 5 All oil samples we e kep in da k glass bo les, closed wi h sc ew caps, and s o ed a 4 °C o a oid oil oxida ion o he g ea es possible ex en . The HT-FAs we e ex ac ed om ege able oils acco ding o he me hodology desc ibed p e iously. 5 In Vi o Simula ed Gas oin es inal Diges ion. To ep oduce he sepa a e diges ion phases (gas ic, in es inal, and GI), he edible oils (EVOO, VOO, and FO) we e p ocessed based on he ha monized in i o diges ion p o ocol desc ibed in he li e a u e, 19,20 using he simula ed gas ic and in es inal fluids (SGF and SIF, espec i ely) s ock elec oly e solu ion, de eloped acco ding o he in o ma ion p o ided in Table 1. A e diges ion, he samples we e cen i uged a 1600g o 5 min a 4°C o sepa a e he bioaccessible ac ion (BF) and he uppe oily phase o esidual ac ion (RF); he concen a ion o HT and HT-FAs in each ac ion was analyzed. Blanks (nega i e con ols) wi hou enzymes we e p ocessed and analyzed in pa allel unde equal condi ions. All samples we e p o ec ed om ligh o e he en i e p ocess. Bo h BF and RF ac ions we e ozen immedia ely a −80 °C and lyophilized. Fo he HT and HT-FA ex ac ion, he lyophilized samples we e hen dissol ed in 3 mL o an ace one/BHA (99.995:0.005, /w) solu ion, o exed o 1 min, sonica ed o 30 min a 40 kHz, and cen i uged a 8750g o 5 min a 4 °C acco ding o he p ocedu e desc ibed. 5 The samples we e d ied using a SpeedVac concen a o , and he d y ex ac s we e econs i u ed wi h 500 μL o MeOH, sonica ed o 10 min, cen i uged a 8750g o 5 min, and fil e ed h ough a 0.45 μmfil e (Millipo e, Bu ling on, MA, USA). UHPLC-ESI-QqQ-MS/MS Analysis. The sepa a ion and iden i- fica ion o HT and HT-FAs we e pe o med using a UHPLC coupled wi h a iple quad upole MS/MS (Agilen Technologies, Waldb onn, Ge many) acco ding o he me hodology epo ed in he li e a u e. 5 The e en ion imes eco ded o unes e ified HT, HT-ALA, HT-LA, and HT-OA we e 0.6, 3.8, 4.0, and 4.4 min, espec i ely. The analyses we e pe o med by mul iple eac ion moni o ing (MRM) in he nega i e mode, and he quan ifica ion and confi ma ion MRM ansi ions we e as ollows: m/z 153 > 123 a bi a y mass uni s (amu) and m/z 153 > 95 amu, espec i ely, o HT; m/z 413 > 277 amu and m/z 413 > 260 amu, co espondingly, o HT-ALA; m/z 415 > 279 amu and m/z 415 > 262 amu, espec i ely, o HT-LA; and m/z 417 > 281 amu and m/z 417 > 264 amu, co espondingly, o HT- OA. Da a acquisi ion and p ocessing we e done using he MassHun e so wa e e sion B.08.00 (Agilen Technologies, Waldb onn, Ge many). Bo h HT and es e s o HT we e quan ified using e e ence s anda ds, and he concen a ions we e exp essed as nanog ams pe g am o esh weigh (ng/g w). S a is ical Analyses. All he analy ical ex ac ions o he oily ma ices conside ed in he p esen wo k, as well as he p oduc s o he gas ic, in es inal, and GI diges ions, we e analyzed in iplica e (n= 3), and he da a we e exp essed as he mean ±SD. S a is ical analyses we e pe o med a 5% o he significance le el using he SPSS 27.0 Table 1. P epa a ion o Simula ed Gas ic Fluid (SGF) and Simula ed In es inal Fluid (SIF) cons i uen a (mmol/L) simula ed fluids KCl KH2PO4NaHCO3NaCl MgCl2(H2O)6(NH4)2CO3 simula ed gas ic fluid (SGF; pH 3) 6.90 0.90 25.00 47.20 0.10 0.50 simula ed in es inal fluid (SIF; pH 8) 6.80 0.80 85.00 38.40 0.13 a KCl, po assium chlo ide; KH2PO4, po assium phospha e monobasic; NaHCO3, sodium bica bona e; NaCl, sodium chlo ide; MgCl2(H2O)6, magnesium chlo ide hexahyd a e; and (NH4)2CO3, ammonium ca bona e. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14166 Figu e 1. Concen a ion (ng/g w) o HT and i s lipophenolic de i a i es in ex a i gin oli e oil (EVOO) and i s bioaccessible and esidual ac ions (A) and bioaccessibili y esul ing om he indi idual gas ic and in es inal diges ions (B). N.d., no de ec ed. Ba s wi h a diffe en lowe case le e indica e s a is ically significan diffe ences among ma ices a p< 0.05 acco ding o he analysis o a iance (ANOVA) and Tukey’s mul iple ange es . Figu e 2. Concen a ion (ng/g w) o HT and i s lipophenolic de i a i es in i gin oli e oil (VOO) and i s bioaccessible and esidual ac ions (A) and bioaccessibili y esul ing om he indi idual gas ic and in es inal diges ions (B). N.d., no de ec ed. Ba s wi h a diffe en lowe case le e among ma ices indica e s a is ically significan diffe ences among ma ices a p< 0.05 acco ding o he analysis o a iance (ANOVA) and Tukey’s mul iple ange es . Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14167 so wa e package (LEAD Technologies, Inc., Cha lo e, NC, USA). Da a we e subjec ed o a one-way analysis o a iance (ANOVA). The no mal dis ibu ion o he esiduals and he homogenei y o a iance we e es ed wi h he Kolmogo o −Smi no and Le ene es s, espec i ely. When s a is ical diffe ences we e iden ified, he a iables we e compa ed using pos hoc analysis wi h Tukey’s mul iple ange es . ■RESULTS AND DISCUSSION Quan i a i e P ofile o Hyd oxy y osol-Fa y Acids in Ex a Vi gin Oli e Oil and Vi gin Oli e Oil. To unde s and he bioaccessibili y o HT-FAs, fi s , he quan i a i e p ofile o unes e ified and es e ified HT in EVOO and VOO edible oils was se up. As expec ed, he e en ion ime o HT-FAs displayed a e e se co ela ion wi h he deg ee o unsa u a ion o i s lipid pa . Thus, HT-ALA, HT-LA, and HT-OA we e elu ed a 3.8, 4.0, and 4.4 min, espec i ely, which a e consis en wi h p e ious expe imen al esul s 5 and o he lipophenolic de i a i es such as y osol es e s. 16 A e his ask, i was obse ed ha he amoun o unes e ified and es e ified HT o bo h oli e oils was simila (Figu es 1 and 2) bu diffe en om ha co esponding o FO, which exhibi ed an absence o HT FAs (da a no shown). In his way, he unes e ified HT was p esen as 874.74 and 934.09 ng/g w in EVOO and VOO, espec i ely. These concen a ions we e in line wi h hose a ailable in he Phenol- Explo e da abase (h p://phenol-explo e .eu/) on he HT con en o EVOO (0.01−3.47 mg/100 g w) and VOO (<0.01−7.43 mg/100 g w). Rega ding he HT-FAs, alues o 2.80, 1.54, and 20.00 ng/g we e eco ded o HT-ALA, HT- LA, and HT-OA, espec i ely, in EVOO and 4.56, 2.59, and 11.90 ng/g in VOO (Figu es 1 and 2, co espondingly), which a e in ag eemen wi h p e ious epo s on ma ching ood ma ices and analy es, 5 al hough impo an ac o s such as a ie y, geog aphical o igin, ipening s age, and p oduc ion and ex ac ion p ocesses may play an impo an ole in lipophenol con en . 21 Fa y Acid Composi ion o he Edible Oils Assessed. As e e ed o be o e, i is wo h no ing ha he bioaccessibili y o specific bioac i e compounds may be enhanced by he con en o FAs in he ood ma ix h ough dispe sing ood componen s in he diges i e ac o by p omo ing he sec e ion o panc ea ic juice. 22 In his scena io, as one o he objec i es o he cu en s udy was o desc ibe he bioaccessibili y o he o HT-FAs, he quan i a i e p ofile o a y acids o he edible oils unde conside a ion (EVOO, VOO, and FO) was c ea ed, gi en i s cen al impo ance o he o ma ion o he a ge bioac i e compounds, he HT-FAs. As expec ed and acco ding o p e ious desc ip ions in he li e a u e, he FA composi ion o he ege able oils conside ed in he p esen wo k (EVOO, VOO, and FO) in ol ed sa u a ed, monounsa u a ed, and polyunsa u a ed a y acids (SFAs, MUFAs, and PUFAs, co espondingly). 23 Specifically, he oily ma ices s udied con ained a o al o 13 indi idual FAs (Table 2). The SFAs de ec ed we e palmi ic (C16:0) and s ea ic (C18:0) acids, which showed s a is ically significan diffe ences be ween samples (p< 0.05), wi h EVOO being he plan oil wi h he highes con en o palmi ic acid (12.07 g/100 g w) in compa ison wi h VOO and FO (10.97 and 5.77 g/100 g w, espec i ely). On he o he hand, FO p o ided he g ea es concen a ion o s ea ic acid (4.40 g/100 g w) ha su passed by 41.7%, on a e age, he con en s eco ded in EVOO and VOO, which emained in simila bu lowe le els. Mo eo e , significan diffe ences we e eco ded ega ding he con en o palmi oleic and oleic acids (C16:1 n7 and C18:1 n9, espec i ely) be ween he di e se edible oils analyzed (p< 0.05), wi h oleic acid unde lined as ha ing he mos abundan MUFA in all ege able oils conside ed (in he ange o 19.97−80.10 g/100 g w). The highes concen a ion o oleic acid was ound in VOO (80.10 g/100 g w) ollowed by EVOO (3.2% lowe ) and FO (75.1% lowe ) (Table 2). Finally, when analyzing he con en o PUFAs, linoleic (C18:2 n-6) and α-linolenic (C18:3 n-3) acids we e ound a he highes concen a ions in FO (15.27 and 53.53 g/100 g w, espec i ely), whe eas EVOO and VOO displayed alues in a simila lowe le el (4.27 and 0.62 g/100 g w, on a e age, espec i ely) (Table 2). The composi ion esul s ega ding SFAs, MUFAs, and PUFAs a e in acco dance wi h in o ma ion p e iously epo ed in he ame o s udies compa ing he FA composi ion o se e al ypes o edible oils. 5,24 In his ega d, he FA p ofile, oge he join ly wi h he p esence o HT in he ma ices e e ed o in he s udy o HT lipophenolic de i a i es, would p o ide a e y help ul choice o assessing he effec o GI diges ion on he lipophenolic p ofile o ege able oils, which is essen ial o disco e ing he biological in e es o hese compounds. Bioaccessibili y o Fa y Acid Es e s o Hyd oxy y - osol om Ex a Vi gin Oli e Oil and Vi gin Oli e Oil. Fo his fi s desc ip ion o he GI bioaccessibili y o he HT lipophenols, an op imized and s anda dized s a ic me hod was applied o he in i o simula ion o GI diges ion, which allows mimicking he physicochemical condi ions in he GI ac , in i o, as closely as possible. 19,20 The lipophenols o HT, HT- ALA, HT-LA, and HT-OA we e analyzed in he h ee ege able oils u ilized in he p esen s udy. FO was selec ed Table 2. Concen a ion o Fa y Acids (g/100 g w) in he Ex a Vi gin Oli e Oil (EVOO), Vi gin Oli e Oil (VO), and Flaxseed Oil (FO) Analyzed in he P esen Wo k edible oils a y acids a EVOO VOO FO LSD (p< 0.05) C14:0 0.03a b 0.03a 0.03a <0.01 C16:0 12.07a 10.97b 5.77c 0.17 C16:1 n7 0.93a 0.83a 0.10b 0.09 C17:0 0.10a 0.10a 0.10a <0.01 C17:1 n8 0.10a 0.10a 0.10a <0.01 C18:0 2.50c 2.63b 4.40a 0.06 C18:1 n9 77.53b 80.10a 19.97c 0.17 C18:2 n6 4.43b 4.10c 15.27a 0.09 C18:3 n3 0.63b 0.60b 53.53a 0.09 C20:0 0.37a 0.60a 0.33a 0.09 C20:1 0.30a 0.30a 0.10b <0.01 C22:0 0.10a 0.10a 0.10a <0.01 C24:0 0.47a 0.10b 0.10b 0.06 a C14:0: my is ic acid; C16:0: palmi ic acid; C16:1 n7: palmi oleic acid; C17:0: hep adecanoic acid; C17:1 n8: hep adecenoic acid; C18:0: s ea ic acid; C18:1 n9: oleic acid; C18:2 n6: linoleic acid; C18:3 n3: alpha-linolenic acid; C20:0: a achidic acid; C20:1: eicosenoic acid; C22:0: behenic acid; and C24:0: lignoce ic acid. b Means (n= 3) wi hin a ow wi h diffe en lowe case le e s a e significan ly diffe en a p< 0.05 acco ding o he analysis o a iance (ANOVA) and Tukey’s mul iple ange es . Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14168 as a nega i e con ol as i does no con ain ei he HT o i s lipophenolic de i a i es acco ding o ou p e ious wo k. 5 These molecules we e also assessed in he diges a es ob ained a e in i o gas ic, in es inal, and GI diges ion o he ege able oils men ioned abo e. In his ega d, bo h he uppe oily o esidual phase (RF) and he lowe micella o bioaccessible ac ion (BF) esul ing om diges ion we e collec ed and analyzed o e ie e accu a e in o ma ion on he heo e ical and po en ial bioa ailable ac ions o hese new lipophilic phenolic compounds, and also in e con e sions be ween unes e ified HT and i s es e s de i a i es, which would condi ion u u e in i o esea ch aimed a add essing he ac ual bioa ailabili y o HT ( ee and es e ified). The in i o GI diges ion pe o med on EVOO (Figu e 1A) and VOO (Figu e 2A) e ealed ha ALA-, LA-, and OA-based lipophenols o es e ified HT we e p esen in he BF. The concen a ions o HT-FAs co esponding o EVOO we e 3.68, 4.04, and 239.41 ng/g w, while o he VOO samples, hese we e 8.22, 1.80, and 92.00 ng/g w o HT-ALA, HT-LA, and HT-OA, espec i ely, e idencing ha he es e ified o ms o HT we e bioaccessible. Howe e , he a e o bioaccessibili y depended on bo h he specific FA-based es e ifica ion and he composi ional ea u es o he ood ma ix, as al eady epo ed o ee HT pha macokine ics and bioa ailabili y, in i o, a e inges ion o diffe en oily ma ices. 25 These esul s a e in good ag eemen wi h p e ious epo s on he bioaccessibili y o o he es e s o HT, namely, phospha idyl-hyd oxy y osol (PHT, phospholipid de i a i es o HT wi h phospha idylcholine). 17 In hei wo k, Ma in e al. desc ibed ha he po ion o HT wi hin he BF, ega ding he es e ified o m (PHT), was significan ly highe ela i e o unes e ified HT. 17 In addi ion, al hough a lowe concen- a ions han in BF, bo h unes e ified HT and es e ified HT we e de ec ed in RF (Figu es 1A and 2A). Jus as wi h PHT, he amphiphilic p ope ies o HT es e ified wi h FAs could cause i s dispe sion in he aqueous media, which in u n indi ec ly enabled he dispe sion o he ehicula ed HT. 17 When analyzing he bioaccessibili y a e o HT and i s lipophenol de i a i es, i was ound ha he unes e ified o m p esen ed a bioaccessibili y o 70.9 and 15.4% o EVOO and VOO, espec i ely. These esul s a e in line wi h p e ious desc ip ions conce ning an impo an loss o HT in he duodenal compa men (∼50%, on a e age) e ie ed om alpe ujo (oli e-mill was e) diges ions. 26 These au ho s desc ibed ha when diges ing only he a ge bioac i e compounds in analy ical sol en s (wi hou he p esence o addi ional ood cons i uen s, such as s a ch, casein, and fibe ), he bioaccessibili y o HT was enhanced by ∼20%. This effec could be due o he p esence o he e e ed mac omolecules, which can bind polyphenols and e ain hem wi hin he ood ma ix. Hence, se e al au ho s ha e emphasized he signifi- cance and complexi y o phenolic compound in e ac ions wi hin he ood ma ices associa ed o hei bioaccessibil- i y. 26,27 In ela ion o he concen a ions o lipophenols in EVOO, he concen a ion o HT-FAs in BF inc eased 1.3-, 3.0-, and 11.9- old o HT-ALA, HT-LA, and HT-OA, espec i ely (Figu e 1A). This ac may be due o he in e ac ions be ween ee HT and FAs, whe e HT could ac as a nucleophilic compound able o ap ALA, LA, and OA p esen in he oily ma ix o o m he co esponding es e s ia enzyma ic o chemical means unde GI condi ions, 28 he e o e exe ing ″nega i e″effec s on ee HT bioaccessibili y bu amelio a ing he concen a ion o he es e ified o ms o HT in he diges a e. A simila beha io was obse ed o VOO as a esul o he GI diges ion, which esul ed in a 1.8- and 7.7- old ise in concen a ion o HT-ALA and HT-OA, espec i ely, in he BF when compa ed o hose ound in VOO, wi h he excep ion o HT-LA, whose bioaccessibili y dec eased up o ∼70%, on a e age (Figu e 2A). In addi ion, i should also be s essed ha he mos abundan HT-FA in he RF was HT-OA, which inc eased 2.0- and 5.6- old in compa ison wi h he amoun s ound in EVOO and VOO, espec i ely (Figu es 1A and 2and Table 2). This diffe en ial occu ence o HT-FAs om EVOO and VOO a e GI diges ion could be owed o he in e ac ions o hese compounds wi h he ood ma ix componen s such as amino acids and p o eins, in spi e o hei low con en (0.07− 2.4 mg/kg). The p esence o hese componen s depends on he ipening s age o oli e cul i a , among o he ac o s, 29,30 and may gene a e a y acid-binding p o ein eac ions 31,32 ha can hampe he bioaccessibili y o hese compounds. Con en ionally, he RF has no been conside ed o he e alua ion o he bioaccessibili y o phenolic compounds a e he diges ions. Howe e , his ac ion is o high ele ance because o i s high concen a ion o polyphenols ha a e no abso bed in he small in es ine, which can be me abolized by he gu mic obio a, 33 gi ing ise o addi ional bioac i e de i a i es ha could also con ibu e o he heal hy a ibu es o oods. Thus, bo h ac ions (BF and RF) esul ing om GI diges ion may be esponsible o he posi i e heal h effec s a ibu ed o plan -based oods, including edible oils. 34 In his ma e , he capaci y o he in es inal mic obio a o modi y he quan i a i e phy ochemical p ofile o he diges ion p oduc s and he eby he bioa ailabili y/abso p ion capaci y o bioac i e compounds p esen in oli e and oli e oils ha ha e no been abso bed in he small in es ine is ema kable, and as a esul , he subs a e o hese me abolic eac ions (nonabso bed phenolics) should no be dis ega ded. In ac , he gu mic obio a plays a p ominen ole in he bio ans o ma ion o bo h es e ified and aglycone o ms o phenolic compounds, bu he me abolism o lipophenols by he gu mic obio a is s ill unclea . To da e, he e is only one s udy ha e alua ed he ecal mic obial me abolism o lipophenols u ilizing an in i o e men a ion model as well as he in i o abso p ion and plasma pha macokine ics o y osol es e s in a s, whe e s anda ds o y osol and y osol es e s we e adminis a ed in he d inking wa e . 35 The au ho s concluded ha y osol es e de i a i es ac ed longe in i o han na i e y osol by obse ing a second abso p ion peak in pha macokine ic p ofiles, possibly due o mic obio a deg ada ion, wi h he es e ified molecules exhibi ing an imp o ed bioa ailabili y as compa ed o ha o ee y osol. The incuba ion o y osol es e s in he e men a ion solu ion libe a ed ee y osol in a ime- dependen manne , indica ing he occu ence o hyd olysis. 35 Howe e , his finding should be es ed wi h a s ed wi h na u al sou ces o hese lipophenols, no wi h s anda ds, and in he na u al concen a ions in which hey a e ound. Con ibu ion o he Gas ic and In es inal Diges ion Phases o he Bioaccessibili y o HT-FAs. To unde s and he impac o he diges ion p ocess on he bioaccessibili y o HT-FAs, he gas ic diges a es om EVOO and VOO we e also analyzed (Figu es 1B and 2), which p o ided aluable in o ma ion on he elease o he a ge lipophenols on each diges ion s age. Upon his analysis, i was obse ed ha unes e ified HT and i s lipophenol de i a i es we e p esen a Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14169 high concen a ions in he RF ob ained om he gas ic diges ion o bo h EVOO and VOO. When e alua ing he impac o gas ic diges ion on he EVOO lipophenols, low concen a ions o HT and HT-FAs we e obse ed in he BF o all indi idual analy es (no de ec ed, no de ec ed, and 1.77 and 0.33 ng/g o HT-ALA, HT-LA, HT-OA, and HT) (Figu e 1B). Simila esul s we e ob ained ega ding VOO (no de ec ed and 0.13, 0.82, and 0.52 ng/g o HT-ALA, HT-LA, HT-OA, and HT) (Figu e 2B). The low concen a ions eco ded could be due o he labili y o lipophenols agains he physicochemical condi ions and he enzyma ic ac i i y p ope o he gas ic diges ion, which would be associa ed wi h a low elease o s abili y o he a ge analy es. This would be consis en wi h p e ious epo s on he bioaccessibili y o HT in oli e lea es, 36 u he suppo ing he influence o he ood ma ix, he gas ic enzymes, o acidic condi ions p e iously desc ibed. 37 None heless, he de elop- men o in es inal diges ion on he chyme p o ided a high concen a ion o ee HT in he BF. This could be due o he hyd olysis o oleu opein and i s aglycone du ing in es inal diges ion ha depends on he lipase ac i i y, hus leading o he appea ance o HT, as p e iously s a ed by Rocche i e al. 38 In e es ingly, as a esul o in es inal diges ion, he concen- a ion o HT-FAs significan ly inc eased in he BF o e he en i e GI p ocess, en a i ely due o he pu a i e enzyma ic ac i i y o panc ea ic lipase ha may ca alyze bo h he hyd olysis o syn hesis o es e s. 39 As a as we know, o da e, mos esea ch on he bioaccessibili y o lipophenols has been pe o med on solu ions o au hen ic s anda ds exposed o he GI physicochemical condi ions. The e o e, despi e he e e ed a ibu es o he syn hesis o lipophenols as a esul o he diges i e enzyma ic ac i i y, addi ional wo ks ha e e idenced ha HT de i a i es, such as hyd oxy y osol ace a e, unde go in ense hyd olysis du ing in es inal diges ion mainly due o he enzyma ic ac i i y o panc ea in. 14 Simila ly, i has been epo ed ha y osol acyl es e s a e hyd olyzed by panc ea ic lipase o p oduce ee y osol. 15,16 Mo eo e , he s abili y o es e a ol es e s wi h cap ylic acid du ing diges ion is nega i ely co ela ed wi h he deg ee o subs i u ion o he FA moie y, gi en ha a e 120 min o incuba ion a 37 °C, ∼54 and ∼11% o monoes e s and dies e s a e hyd olyzed, espec i ely, while no hyd olysis o he ies e s has been no iced. 40 Howe e , hese esul s ha e been associa ed wi h an ″a ificially″ob ained (only wi h chemically o enzyma ically syn hesized s anda ds) highe bioaccessibili y ela i e o he effec s ha occu du ing he GI diges ion o oods because hese models do no allow moni o ing he ex ac i e capaci y o he p ocess, he in e con e sion be ween molecules, o he de no o syn hesis o he a ge analy es ( om he complex pool o molecules p esen in he ood ma ix). This gap in knowledge has been o e whelmed by a s udy on he bioaccessibili y o HT wi h alpe ujo (solid byp oduc o oli e oil ex ac ion). This s udy desc ibed a lowe bioacces- sibili y o HT when applying GI diges ion on a ege able ma ix due o he p esence o fibe s and suga s ha could in e ac wi h HT and he diges i e enzymes, modula ing he bioaccessibili y a e. 26 Acco dingly, he assessmen o he bioaccessibili y o HT o HT-FAs needs o include a ood ma ix o ob ain obus and nonspecula i e conclusions. The ele ance o he in es inal diges ion phase o he p esence o HT lipophenols in he p oduc o he GI diges ion p omp ed us o explo e he effec o he in es inal diges ion conside ed indi idually on he in ac ood ma ix, a oiding he unce ainly de imen al effec ha could be occu ing du ing he gas ic s age (Table 3). Thus, when he in es inal diges ion was pe o med on bo h EVOO and VOO (wi hou he p e ious gas ic phase), he concen a ion o HT lipophenols obse ed in he BF was highe ela i e o ha ob ained ollowing he physiological wo kflow (Table 3). The con en o HT-ALA, HT-LA, and HT-OA in BF o he in es inally diges ed EVOO was ∼64-, ∼8-, and ∼15- old highe , espec i ely, in compa ison wi h he BF ob ained a e a comple e GI diges ion (gas ic plus in es inal diges ions). This end was simila o VOO, whe e he con en o lipophenols in he BF inc eased ∼4-, ∼3-, and ∼13- old o HT-ALA, HT-LA, and HT-OA, espec i ely, ela i e o BF ob ained om he sequen ial gas ic and in es inal diges ions (Table 3). On he con a y, he highes concen a ion o unes e ified HT was ound in he BF om in es inal diges ions de eloped on gas ic diges a es o EVOO (619.88 ng/g w) in compa ison wi h he di ec in es inal diges ion o he in ac ood ma ix (55.0% lowe , p< 0.001) (Table 3), while ega ding VOO, no s a is ically significan diffe ences we e ound, ein o cing he Table 3. Concen a ion (ng/g w) o he Bioaccessible and Residual F ac ions (BF and RF, Respec i ely) o HT and I s Lipophenolic De i a i es as a Resul o he Only In es inal Diges ion on he Diffe en Subs a es (Ex a Vi gin Oli e Oil, Vi gin Oli e Oil, and Thei Gas ic Diges a es) concen a ion (ng/g w) subs a e o he in es inal diges ion gas ic diges a e oli e oil oil ma ix analy e a oli e oil BF RF BF RF EVOO a HT 874.47 ±41.88a b 619.88 ±144.42b 64.46 ±12.70d 279.05 ±63.95c 542.00 ±34.15b HT-ALA 2.80 ±0.95b 3.68 ±2.30b 0.61 ±0.06b 235.96 ±23.77a 0.64 ±0.02b HT-LA 1.54 ±0.49bc 4.04 ±1.97b N.d. 34.19 ±1.61a N.d. HT-OA 20.00 ±8.11c 237.64 ±76.94b 12.68 ±2.72c 3599.94 ±107.20a 102.35 ±0.41bc VOO HT 934.09 ±29.53a 143.76 ±25.08bc 39.58 ±1.27c 207.75 ±131.67b 251.66 ±3.38b HT-ALA 4.56 ±1.52b 8.22 ±1.79b 5.12 ±1.25b 32.44 ±5.17a 4.59 ±0.54b HT-LA 2.59 ±0.46b 1.98 ±0.44b N.d. 5.87 ±1.51a N.d. HT-OA 11.90 ±4.90b 91.18 ±2.97b 63.90 ±4.21b 1205.44 ±160.57a 85.81 ±7.79b a HT, hyd oxy y osol; HT-ALA, hyd oxy y osol es e ified wi h α-linolenic acid; HT-LA, hyd oxy y osol es e ified wi h linoleic acid; and HT-OA, hyd oxy y osol es e ified wi h oleic acid. b Da a a e shown as means ±SD (n= 3). Da a wi hin a single ow ollowed by he same lowe case le e a e no significan ly diffe en a p< 0.001 acco ding o he analysis o a iance (ANOVA) and Tukey’s mul iple ange es . N.d., no de ec ed. Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14170 ele ance o he ood ma ix composi ion o he final bioaccessibili y esul s. The dis inc beha io in he diges i e hallma k o HT and i s lipophenol de i a i es could be due o he spa ing solubili y o HT in oli e oil and i s consequen loca ion in he aqueous phase (>99%), while, boos ed by he amphiphilic na u e, HT-FAs a e dis ibu ed be ween bo h he oily and aqueous phases. 41 The eby, as expec ed, HT-OA was he mos abundan lipophenol in he ood ma ix and he GI diges a es (Table 3). This lipophenol should be o med h ough he enzyma ic es e ifica ion o HT wi h OA, which is he mos abundan FA in he oily ma ices conside ed in he p esen wo k (EVOO and VOO) (Table 2). Based on he findings om he o ma ion o HT-FAs unde in es inal diges i e condi ions, he use o p o ec o s o HT agains he gas ic condi ions migh be o in e es o ake ad an age o he bioac i i ies o HT-FAs om biological o echnological poin s o iew. Rela i e Influence o he In es inal Enzymes on he Gene a ion o Hyd oxy y osol Lipophenols. Gi en he ele ance o he in es inal s age o he bioaccessibili y o HT and i s lipophenolic de i a i es, he sepa a e and join effec s o in es inal enzymes (panc ea in and panc ea ic lipase) on hese compounds we e u he in es iga ed o gain u he unde - s anding o hei ole on he elease o HT-FAs in o he in es inal lumen and hei s abili y. Wi h his objec i e, EVOO was diges ed using SIF con aining indi idual and combined in es inal enzymes a 37 °C o 120 min. The incuba ion o EVOOin heSIFwi hou anydiges i eenzymewas conside ed as a nega i e con ol. This app oach allowed e ie ing c i ical in o ma ion on hei ela i e influence on he bioaccessibili y o HT-FAs. Lipases ( iacylglyce ol acyl hyd olases, EC 3.1.1.3.) a e bioca alys s wi h a high selec i i y and s abili y ha ca alyze hyd olysis, es e ifica ion, anses e ifica ion, and alcoholysis eac ions. 42 To da e, lipases ha e been applied o he p oduc ion o FA de i a i es by he ag o- ood and nu aceu ical indus ies because o hei po en ial uses as fla o ing es e s, a y acid es e s o an ioxidan s, and s uc u ed lipids wi h high egio- and s e eo-selec i i ies. 43 Up o now, he biosyn hesis o HT-FAs has been done using comme cial enzymes on pu e s anda d compounds. 39 These eac ions ha e been epo ed as being closely dependen on he acyl chain leng h and he deg ee o unsa u a ion o FAs ha affec he con e sion yield o he es e ifica ion p ocess due o diffe ences in hei spa ial configu a ion. 44,45 In he cu en wo k, he gene a ion o HT es e s wi h an equi alen ca bon alkyl chain (C18) bu wi h diffe en deg ees o unsa u a ion, C18:1, C18:2, and C18:3 co esponding o HT es e ified wi h OA, LA, and ALA, espec i ely, was moni o ed, o he fi s ime, a e he in es inal diges ion o EVOO wi h indi idual and combined in es inal enzymes (panc ea in and panc ea ic lipase). The con e sion yield o HT-FAs was mo e p onounced when he eac ion was ca alyzed only by panc ea in han when panc ea ic lipase was Figu e 3. Rep esen a i e UHPLC-ESI-QqQ-MS/MS-ex ac ed ion ch oma og ams co esponding o he bioaccessible lipophenols o hyd oxy y osol (HT-ALA, hyd oxy y osol-α-linolenic acid; HT-LA, hyd oxy y osol-linoleic acid; and HT-OA, hyd oxy y osol-oleic acid) in diges a es ob ained a e applying diffe en enzyme combina ions on ex a i gin oli e oil du ing simula ed in i o in es inal diges ion using he quan ifica ion MRM ansi ions. CPS, cha ges pe second. Table 4. Concen a ion (ng/g w) o Hyd oxy y osol Es e ified wi h Fa y Acids a e In es inal Diges ion o Ex a Vi gin Oli e Oil wi h Diffe en Combina ions o Diges i e Enzymes analy e a con ol (no enzyme) panc ea in panc ea ic lipase panc ea in + panc ea ic lipase HT-ALA 4.58 ±1.44b b 228.85 ±14.87a 193.94 ±27.88a 173.40 ±4.55 a HT-LA 0.68 ±0.17c 25.77 ±2.25a 21.33 ±7.50ab 14.54 ±0.42b HT-OA 31.83 ±2.02c 4853.34 ±69.57a 2235.37 ±244.55b 2876.17 ±315.30b a HT-ALA: hyd oxy y osol es e ified wi h α-linolenic acid; HT-LA: hyd oxy y osol es e ified wi h linoleic acid; and HT-OA: hyd oxy y osol es e ified wi h oleic acid. b Mean ±s anda d de ia ion (n= 3) ollowed by he same lowe case le e a e no s a is ically significan a p< 0.05 acco ding o he analysis o a iance (ANOVA) and Tukey’s mul iple ange es . Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14171 p esen (alone o in combina ion wi h panc ea in) (Figu e 3). HT-OA was he lipophenol wi h he highes concen a ion in he in es inal diges a e, independen ly o he enzymes o hei combina ion applied (4853.34, 2235.37, and 2876.17 ng/g wi h panc ea in, panc ea ic lipase, and combina ions o he wo enzymes, espec i ely), and showed no s a is ical diffe ences be ween panc ea ic lipase and he combina ion o bo h enzymes (p> 0.05) (Table 4). Rega ding HT-LA and HT- ALA, he final concen a ions achie ed in he in es inal diges a e, as well as he diffe ences be ween enzyma ic condi ions, a ied o a lesse ex en ela i e o HT-OA (Figu e 3and Table 4). This may be due o he deg ee o unsa u a ion o he FAs in ol ed in he es e ifica ion o HT, which may affec hei con o ma ion and he eby he eac ion ime, as s a ed by p e ious esea ch. 44 The gene a ion o HT-FAs has al eady been desc ibed in oily ood ma ices a e o i ying ood ma ices wi h HT, 5 as well as in oli e oil byp oduc s du ing p ocessing and s o age, while his was no de ec able in in ac oli es. 46 A hypo hesis suppo ing hese findings sugges s ha HT es e s could be o med independen ly as a esul o he es e ase ac i i y om lipase-posi i e yeas s, which a e p esen in plan -based oods. Howe e , he molecula mechanisms esponsible o he de no o o ma iono lipophenols,aswellas heop imal condi ions needed o he success ul de elopmen o such eac ions, emain unclea . Likewise, ecen ly, he o ma ion o y osol es e s, pa icula ly wi h OA, has been desc ibed, iden i ying c ushing and kneading p ocesses when enzymes a e ac i a ed and he eac ions igge ed. 47 Wi hin his ame, he p esen s udy con ibu es o he unde s anding o he gene a ion o lipophilic hyd oxy y osyl es e s du ing GI diges ion in oily ma ices, wi h a discussion o some possible scena ios being necessa y. The diffe en efficiency ega ding he o ma ion o he sepa a e lipophenols (HT-ALA, HT-LA, and HT-OA) may be associa ed wi h he deg ee o unsa u a ion, which in u n modifies he FA spa ial configu a ion and he lipase selec i i y. Mo eo e , he concen a ion o he p ecu so s in he ood ma ix (bo h unes e ified HT and FAs) seems o be c i ical o he syn hesis o HT-FAs du ing diges ion as well since he con en o ee OA in EVOO and VOO was, on a e age, ∼99 and ∼95% in compa ison wi h he ALA and LA concen a ion, espec i ely (Table 2). This is in ag eemen wi h ecen esea ch, which u ilized nanopa icles wi h immobilized lipase om The momyces lanuginosus and employed oli e lea aqueous ex ac o he syn hesis o bioac i e hyd oxy y osyl FA es e s. The s udy epo ed ha he con e sion yields (%) o OA (C18:1), LA (C18:2), and my is ic acid (C14:0) we e 55.0, 50.4, and 64.3%, espec i ely, es ablishing ha he numbe o double bonds had a di ec co ela ion wi h he molecula s uc u e o he FA, which, as a esul , would affec he ad ance o he acyla ion eac ions. 45 On he con a y, addi ional s udies wo king wi h he pu e s anda d o long-chain unsa u a ed FAs indica ed ha he e was no ela ionship be ween chain leng h and con e sion, as he highes con e sion was ob ained wi h eicosapen aenoic acid (EPA) (68.0%) ela i e o OA (46.0%). The a ia ion in hese findings may be caused by he selec i i y o lipase o he FAs, wi h he deg ee o unsa u a ion being a mo e ele an ac o a he han FA chain leng h. 39 In addi ion, lipase pe o ms he e ogeneous eac ions, and i s ca aly ic ac i i y is maximum when he enzyme is adso bed a an oil− wa e in e ace. Gene ally, long-chain es e s a e mainly dispe sed in he oil d ople , while sho -chain es e s a e ound mos ly in he wa e d ople . 48 Fo lipophenols, a cu off effec ha in ol es a oo-sho o a oo-long hyd ophobic chain does no ensu e a p ope in e acial loca ion, which in u n does no esul o an op imal lipase ac i i y, 49 and he cu off effec is simply a na u al consequence o he diffe en ial solubili y o an ioxidan s in he aqueous, in e acial, and oil egions o an emulsion. 50 Fu he mo e, o he phenolic acyl es e s, such as y osol-FAs, ha e shown a sus ained- elease beha io o ee y osol molecules du ing he ime-consuming diges ion p ocess 16 ha could imp o e he bioac i i y scope o polyphenols because o ex ended ac ions. In his sense, he cu en wo k b ings new knowledge and s a egies o he design o slow- elease o mula ions o HT-FAs ha could enhance he phenolic loading cycle ime by p olonging he e minal hal - li e and he eby leng hening hei bioac i i y. This is pa icula ly impo an , as se e al biological p ope ies ha e been ecen ly sugges ed o hese compounds, namely, an idiabe ic, p oli e a i e, and an ioxidan capaci ies. 47,51 In addi ion, i has been demons a ed ha he es e ifica ion o phenolic compounds did no comp omise he bioac i i y o he na i e molecule bu ins ead may enhance i s bioa ailabili y and expand i s applica ion domains. 52 As concluding ema ks, he p esen s udy p o ides new e idence on he bioaccessibili y o lipophenolic de i a i es o HT om di e se oli e oils (EVOO and VOO), showing ha hey a e bioaccessible a e pe o ming a simula ion o GI diges ion unde in i o condi ions, depending la gely on he ype o FA es e i ying HT as well as he ood ma ix. Mo e impo an ly, he gene a ion o HT-FAs du ing in es inal diges ion was epo ed in he p esen wo k, as well as he iden ifica ion o he enzymes esponsible o mos o he syn hesis iden ified (panc ea in). These findings a e o c i ical ele ance om a clinical and echnological poin o iew because o he po en ial biological effec s ha could be expec ed om de no o syn hesized lipophenols, which could ha e a c i ical impac on bo h he hal -li e o plan -based oods and he biological benefi s al eady desc ibed on hose oods and oods uffs con aining he componen s o lipophenols (e.g., HT and FAs). Indeed, he majo ou comes e ie ed sugges ha he use o oily ma ices and hei byp oduc s as po en ial unc ional ing edien s, oods, supplemen s, o cosme ics, o heal h p omo ion and disease isk educ ion, should ake in o conside a ion he o ma ion o new lipophenols du ing diges ion and, consequen ly, he ans o ma ion o he final bioac i e scope and he effec s wi hin he ame o specific pa hophysiological p ocesses. Ul ima ely, u he assays con- ce ning he bioa ailabili y o HT-FAs a e s ill needed, wi h a high impo ance placed on alida ing whe he lipophenol molecules would mig a e ac oss he epi helial ba ie , hei me aboliza ion ci cula ing eac ion, he anspo e s o hese compounds, and he me abolic e en s ha affec hei final shel -li e in he o ganism and bioac i i y in he sepa a e issues and cell ypes. ■AUTHOR INFORMATION Co esponding Au ho s A  ngel Gil-Izquie do −Depa men o Food Science and Technology, Resea ch G oup on Quali y, Sa e y and Bioac i i y o Plan Foods, CEBAS-CSIC, Uni e si y Campus o Espina do, Mu cia 30100, Spain; o cid.o g/0000- 0001-7646-0386; Phone: +34968396200; Email: [email p o ec ed] Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle h ps://doi.o g/10.1021/acs.ja c.1c05373 J. Ag ic. Food Chem. 2021, 69, 14165−14175 14172 Sonia Medina −Depa men o Food Science and Technology, Resea ch G oup on Quali y, Sa e y and Bioac i i y o Plan Foods, CEBAS-CSIC, Uni e si y Campus o Espina do, Mu cia 30100, Spain; o cid.o g/0000-0002-7231-6480; Phone: +34968396200; Email: [email p o ec ed] Au ho s Ca olina Alemán-Jiménez −Depa amen o de En e me ía, Uni e sidad Ca ólica de Mu cia, Mu cia 30107, Spain Raul Domínguez-Pe les −Depa men o Food Science and Technology, Resea ch G oup on Quali y, Sa e y and Bioac i i y o Plan Foods, CEBAS-CSIC, Uni e si y Campus o Espina do, Mu cia 30100, Spain; o cid.o g/0000- 0001-6232-712X Juana I. Gallego-Gómez −Depa amen o de En e me ía, Uni e sidad Ca ólica de Mu cia, Mu cia 30107, Spain Agus ín Simonelli-Munoz −Depa amen o de En e me ía, Fisio e apia y Medicina. Uni e sidad de Alme ía, Alme ía 04120, Spain Espé ance Moine −Ins i u des Biomolécules Max Mousse on (IBMM), UMR 5247, CNRS, Uni e si y o Mon pellie , Mon pellie 34093, F ance Thie y Du and −Ins i u des Biomolécules Max Mousse on (IBMM), UMR 5247, CNRS, Uni e si y o Mon pellie , Mon pellie 34093, F ance Céline C aus e −Ins i u des Biomolécules Max Mousse on (IBMM), UMR 5247, CNRS, Uni e si y o Mon pellie , Mon pellie 34093, F ance; o cid.o g/0000-0002-5714- 8749 Fede ico Fe e es −Depa men o Food Technology and Nu i ion, Molecula Recogni ion and Encapsula ion (REM) G oup, Uni e sidad Ca ólica de Mu cia, UCAM, Mu cia 30107, Spain Comple e con ac in o ma ion is a ailable a : h ps://pubs.acs.o g/10.1021/acs.ja c.1c05373 Au ho Con ibu ions # C.A.-J. and R.D.-P. wo au ho s con ibu ed equally o he p esen wo k. Funding This wo k was pa ially unded by he ″FundacionSe neca de la Region de Mu cia″G upo de Excelencia 19900/GERM/15. S.M. was suppo ed by a Pos doc o al Con ac (Saa ed a- Faja do 21078/SF/19) om he Agency o Science and Technology o he Region de Mu cia−″FundacionSe neca″ (Spain). No es The au ho s decla e no compe ing financial in e es . ■ACKNOWLEDGMENTS The au ho s hank he company Olimend os S.L. (Mu cia, Spain) o he supply o ex a i gin oli e oils. ■ABBREVIATIONS BF, bioaccessible ac ion; BHA, bu yla ed hyd oxyanisole; DMSO, dime hyl sul oxide; EVOO, ex a i gin oli e oil; FAs, a y acids; FO, flaxseed oil; HT, hyd oxy y osol; HT-ALA, hyd oxy y osol es e ified wi h α-linolenic acid; HT-FA, hyd oxy y osol es e ified wi h a y acid; HT-LA, hyd oxy y - osol es e ified wi h linoleic acid; HT-OA, hyd oxy y osol es e ified wi h oleic acid; MRM, mul iple eac ion moni o ing; MUFAs, monounsa u a ed a y acids; NMR, nuclea magne ic esonance; PUFAs, polyunsa u a ed a y acids; RF, esidual ac ion; SFAs, sa u a ed a y acids; SGF, simula ed gas ic fluid; SIF, simula ed in es inal fluid; VOO, i gin oli e oil ■REFERENCES (1) Wah bu g, U.; K a z, M.; Cullen, P. Medi e anean Die , Oli e Oil and Heal h. Eu . J. Lipid Sci. Technol. 2002,104, 698−705. (2) Vilaplana-Pé ez, C.; Auón, D.; Ga cía-Flo es, L. A.; Gil- Izquie do, A. 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