Substrates emulsification process to improve lipase-catalyzed sardine oil glycerolysis in different systems. Evaluation of lipid oxidation of the reaction products

Accep ed Manusc ip Subs a es emulsi ica ion p ocess o imp o e lipase-ca alyzed sa dine oil glyce olysis in di e en sys ems. E alua ion o lipid oxida ion o he eac ion p oduc s Ángela Ga cía Solaesa, Ma ía Te esa Sanz, Rod igo Melgosa, Sag a io Bel án PII: S0963-9969(17)30370-8 DOI: doi: 10.1016/j. ood es.2017.07.048 Re e ence: FRIN 6845 To appea in: Food Resea ch In e na ional Recei ed da e: 18 May 2017 Re ised da e: 18 July 2017 Accep ed da e: 19 July 2017 Please ci e his a icle as: Ángela Ga cía Solaesa, Ma ía Te esa Sanz, Rod igo Melgosa, Sag a io Bel án , Subs a es emulsi ica ion p ocess o imp o e lipase-ca alyzed sa dine oil glyce olysis in di e en sys ems. E alua ion o lipid oxida ion o he eac ion p oduc s, Food Resea ch In e na ional (2017), doi: 10.1016/j. ood es.2017.07.048 This is a PDF ile o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. ACCEPTED MANUSCRIPT 1 Subs a es emulsi ica ion p ocess o imp o e lipase-ca alyzed sa dine oil glyce olysis in di e en sys ems. E alua ion o lipid oxida ion o he eac ion p oduc s Ángela Ga cía Solaesa, Ma ía Te esa Sanz  , Rod igo Melgosa, Sag a io Bel án Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), Uni e si y o Bu gos, 09001 Bu gos. Spain Abs ac Mono- and diacylglyce ols ich in omega-3 ha e a g ea in e es due o hei good bioa ailabili y and oxida ion s abili y compa ed wi h o he kind o omega-3 concen a es. The main d awback in mono- and diacylglyce ols p oduc ion by glyce olysis is he immiscibili y o he subs a es, oil and glyce ol. To imp o e mass ans e a es, a oiding he use o o ganic sol en s, emulsi ica ion o bo h eac an s as e e se micelles (glyce ol-in-oil) was ca ied ou p e ious o lipase-ca alyzed sa dine oil glyce olysis. Subs a e emulsi ica ion yielded highe eac ion a es compa ed o kine ics wi h no p e ious emulsi ica ion, bu s ill lowe han in o ganic sol en s. To a oid he use o o ganic sol en , SC-CO2 was used as eac ion medium bu no kine ic ad an ages we e demons a ed in he p essu e ange om 15 o 25 MPa. By inc easing empe a u e, om 40 o 90 ºC, eac ion a es inc eased bo h in a sol en - ee sys em and in SC-CO2 medium. I was also ound ha an inc ease in empe a u e does no lead o an inc ease in he inal oxida ion s a us o he eac ion p oduc s. This beha io was due o he so p ion capaci y o he Lipozyme 435 suppo , gi ing lowe oxida ion s a us a he highes empe a u e, 80-90 ºC. Keywo ds: ish oil, glyce olysis, mic oemulsion, SC-CO2, pe oxides adso p ion. Chemical compounds s udied in his a icle: Glyce ol (PubChem CID: 753); Eicosapen aenoic acid (PubChem CID: 446284); Docosahexaenoic acid (PubChem CID: 445580); Ae osol OT (PubChem CID: 23673837); Tween 80 (PubChem CID: 5281955).  Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess e [email protected] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 1. In oduc ion The impo ance o omega-3 polyunsa u a ed a y acids (n-3 PUFA), especially eicosapen aenoic acid (EPA) and docosahexaenoic acid (DHA), in human nu i ion and disease p e en ion is ully ecognized scien i ically (K is-E he on, Ha is, & Appel, 2002; Riedige , O hman, Suh, & Moghadasian, 2009). n-3 PUFA supplemen s a e a ailable in di e en chemical o ms. Among he di e en ypes o lipid de i a i es con aining n-3 PUFA concen a es, monoacylglyce ols (MAG) and diacylglyce ols (DAG) ha e good bioa ailabili y and oxida ion s abili y (He nandez, 2014; Lawson & Hughes, 1988). Addi ionally, i mus be also conside ed ha die a y TAG a e hyd olyzed in he small in es ine o sn-2-MAG being he mos a o able s uc u e o n- 3 PUFA o be adso bed by in es inal mucosa (Banda a e al. 2012). In addi ion, MAG o i s mix u es wi h DAG accoun o 75% o he wo ldwide emulsi ie p oduc ion (Zhong e al., 2009). The well-known d awbacks o he con en ional chemical glyce olysis echnique (ene gy in ensi e, low yields (30–40%), oxidized p oduc s) ha e p omp ed a g owing in e es in he de elopmen o al e na i e p ocesses o he p oduc ion o MAG and DAG ich in n-3 PUFA. Enzyme-ca alyzed eac ion is an a ac i e al e na i e since he eac ion can be ca ied ou unde mild condi ions (Bo nscheue , 1995; Fel es, de Oli ei a, Block, & Ninow, 2013). To o e come he p oblem o he immiscibili y o glyce ol and oil, di e en app oaches ha e been used in he li e a u e o imp o e he con ac be ween he eac an s and hence educe mass ans e limi a ion. Lipase-ca alyzed glyce olysis has been ca ied ou in di e en eac ion media such as o ganic sol en s (Dams up e al., 2006), comp essed luids (Moquin, Temelli, King, & Palcic, 2005) and ionic liquids (Guo & Xu, 2006), in o de o imp o e he mass ans e . The cos , oxici y and ene gy equi ed o sol en emo al om he p oduc mix u e, a e impo an aspec s o be conside ed when dealing wi h con en ional sol en sys ems (P a , Hayle , & Wells, 2014). Recen ly, he uses o di e en su ac an s o inc ease he in e acial a ea, and ul asound i adia ion ha e been also p oposed o educe mass ans e limi a ion (Fiame i e al., 2012; Valé io, Ro ani, T eichel, De Oli ei a, & Oli ei a, 2010). Bioca aly ic p ocessing in mic oemulsion sys em has ecei ed a en ion in o de o inc ease con ac be ween subs a es. The o ma ion o a mic oemulsion o he eac an s (glyce ol-in-oil) as e e se micelles can help o imp o e mass ans e a es. Fu he mo e, lipases demons a e high in e acial ac i i y in micelle sys ems because he o ma ion o he ac i e si e du ing he eac ion ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3 occu s a he in e ace be ween he subs a es and he enzyme. Se e al ood g ade su ac an s a e able o s abilize he micella sys em imp o ing sys em homogenei y (Ca alho & Cab al, 2000; S ama is, Xenakis, & Kolisis, 1999). Ne e heless, i mus be aken in o accoun ha some ood g ade su ac an s ha e chemical unc ions ha could be modi ied by lipases. Fo ins ance, he lipase No ozym 435 p esen ed ac i i y a pa icula condi ions owa ds some su ac an s as soy leci hin and Tween in glyce olysis eac ions (Camino Fel es, Villeneu e, Ba éa, de Oli ei a, & Ninow, 2012). To a oid his p oblem, o he syn he ic su ac an s, such as sodium (bis-2-e hyl-hexyl) sul osuccina e (ae osol-OT o AOT), ha e been used. AOT has been epo ed o o m micelles in a g ea numbe o nonpola subs ances and se e al o he pola sol en s such as glyce ol (Fiame i e al., 2009). In his case, good esul s ha e been ob ained in glyce olysis sys ems when adding mo e ha 7.5% o AOT (Fiame i e al., 2009). Howe e , he high amoun o his su ac an may gene a e p oblems du ing emo al p ocesses (S ama is, Xenakis, & Kolisis, 1994). Ano he al e na i e o o ganic sol en s is he use o he supe c i ical luids (SCFs) as eac ion medium. Supe c i ical ca bon dioxide (SC-CO2) is p obably he mos used SCF due o i s addi ional bene i s (non- oxic, non- lammable, eadily a ailable a high pu i ies and low cos s, and ela i ely mild c i ical condi ions) ha a e appealing when choosing en i onmen al eplacemen o o ganic sol en s (Ma suda, 2013; Rezaei, Temelli, & Jenab, 2007). SC-CO2 has liquid-like densi y bu gas-like iscosi y esul ing in high mass ans e being a clean al e na i e o eplace o ganic sol en s. Enzyma ic concen a ion o n-3 PUFA in supe c i ical luids (SCFs) is an in e es ing op ion o he p e en ion o oxida ion du ing p ocessing o ish oil (Lin, Chen, & Chang, 2006; Roh, Kim, & Choi, 2015). Besides, SC-CO2 can be easily sepa a ed om he eac ion p oduc s by simple dep essu iza ion and allows ac iona ion o he eac ion p oduc s. Some p e ious s udies o enzyma ic eac ions o di e en lipid sou ces in SC-CO2 ha e been epo ed in he li e a u e. Howe e , in case o enzyma ic glyce olysis, o he comp essed luids such as p opane, n-bu ane, and ace one, ha e been used (Esmelind o e al., 2008; Tai & B unne , 2011; Valé io e al., 2010). Some s udies o glyce olysis o ege able oils in SC-CO2 a high empe a u es can be ound bu wi h no enzyma ic ca alys (Moquin e al., 2005; Temelli, King, & Lis , 1996). In a p e ious wo k, a de ail kine ic s udy o glyce olysis o sa dine oil using Lipozyme 435 o m Candida an a c ica B as bioca alys in an op imized amoun o e -bu anol was pe o med (Solaesa, Sanz, Bel án, e al., 2016). Te .bu anol helped o c ea e a ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 homogeneous phase and o educe mass ans e limi a ions. Howe e , o ganic sol en s p esen di e en en i onmen al conce ns. In his wo k, o imp o e con ac be ween subs a es, a oiding he use o o ganic sol en s, emulsi ica ion o glyce ol and oil be o e glyce olysis eac ion was conside ed. Glyce olysis eac ion has been pe o med in a sol en ee sys em a a mosphe ic p essu e and in SC-CO2 as eac ion medium wi h p e ious susbs a es emulsi ica ion. The e ec o adding a su ac an , AOT o Tween 80, o s abilize he emulsion, on glyce olysis pe o mance has been also s udied. Glyce olysis has been de e mined a di e en ope a ing empe a u esa a mosphe e p essu e, 0.1 MPa, and in SC-CO2 medium in he p essu e ange om 15 o 25 MPa. Since n-3 PUFA a e highly suscep ible o oxida ion; he oxida i e s a us o he inal eac ion p oduc s was e alua ed h ough he pe oxide and anisidine alues. Reac ion yields and he oxida ion alues o he eac ion p oduc s we e compa ed o bo h sys ems. 2. Ma e ials and me hods 2.1 Ma e ials Re ined sa dine oil was p o ided by Indus ias A ines S.L. (Spain) wi h 18.3% o EPA and 7% o DHA and a wa e con en o 0.2% (Solaesa, Bucio, Sanz, Bel án, & Rebolleda, 2014). Glyce ol was pu chased om Sigma Ald ich wi h a pu i y o ≥ 99.5% and a wa e con en o 0.18%. The ood g ade lipase Lipozyme 435 om Candida an a c ica B (immobilized on a mac opo ous hyd ophobic ac ylic esin), was dona ed by No ozymes A/S (Bags ae d, Denma k). Ca bon dioxide (99.9%) was supplied by Ai Liquide S.A. (Spain). Polyoxye hylene so bi an monoolea e (Tween 80) and sodium bis (2-e hylhexyl) sul osuccina e (Ae osol AOT o AOT), used as ood g ade su ac an s, we e pu chased by Sigma Ald ich. All o he chemicals used in di e en analyses we e o analy ical o HPLC g ade. 2.2 Emulsi ica ion p ocess Mic oemulsions o he glyce olysis sys em o sa dine oil we e p epa ed a a ixed mole a io o 3:1 (glyce ol:oil) since his mole a io was ound as he op imum in a p e ious kine ic s udy (Solaesa, Sanz, Bel án, e al., 2016). A high-speed blende (Micc a D9 equipped wi h a DS-20/PF EMR o o –s a o ) a di e en speeds, om 16000 o 35000 pm, was used by pulses du ing 3 minu es. To p epa e he su ac an - ee emulsion as e e se micelles, he app op ia e amoun o glyce ol (10 g) was added d op by d op o ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 he sui able amoun o oil (30 g) while being comple ely mixed a high speed. Dispe sed (glyce ol) and con inuous (sa dine oil) phases we e iden i ied by he dilu ion es (Mize e al., 2013). Fu he mo e, di e en concen a ions (0.5, 1 and 1.5% in glyce ol o oil as indica ed in Table 2) o wo ood g ade su ac an s, AOT and Tween 80, we e es ed in o de o imp o e he s abili y o he emulsion. A de ined quan i y o each su ac an was dissol ed in oil o in glyce ol, depending on i s solubili y. The cha ac e iza ion o he emulsions was pe o med 10 min a e emulsi ica ion o a oid any c eaming o coalescence e ec . Pa icle size dis ibu ion (PSD), mean d ople diame e and polydispe si y index (PDI) o samples we e measu ed by dynamic ligh sca e ing (DLS), using a Ze asize Nano ZS appa a us (Mal e n Ins umen s L d., UK) o e alua e he bes condi ions o p oduce a s able emulsion wi h small (o he smalles ) d ople size. 2.3 Lipase-ca alyzed glyce olysis o sa dine oil in di e en sys ems A compa a i e s udy o lipase-ca alyzed glyce olysis in di e en sys ems was ca ied ou . All he expe imen s we e conduc ed in a ba ch mode keeping cons an he enzyme concen a ion a 5 w % (by weigh o subs a es) and he subs a e mole a io (3:1, glyce ol o oil) acco ding o p e ious wo k (Solaesa, Sanz, Bel án, e al., 2016). Table 1 summa izes all glyce olysis eac ions ha ha e been done in his wo k. Expe imen s 1 - 6 ha e been ca ied ou a a mosphe ic p essu e in a sol en ee sys em in a 100 mL jacke ed ba ch eac o . Fi s o all, expe imen s 1 and 2 we e ca ied ou o e alua e he e ec o p e ious subs a es emulsi ica ion on eac ion a e. Expe imen s 3 and 4 we e pe o med wi h emulsi ied subs a es s abilized by adding a ood g ade su ac an , AOT and Tween 80 espec i ely, a he op imum concen a ion p e iously de e mine in sec ion 2.2. Expe imen s 2, 5 and 6 we e pe o med o e alua e he e ec o eac ion empe a u e, 50, 80 and 90ºC espec i ely. Glyce olysis eac ion was ca ied ou as ollows. Once emulsion was p epa ed, i was cha ged in o he eac o . La e , he lipase was added and a ni ogen s eam was applied. The eac o was hen closed and he s i ing sys em by impelle s was connec ed. A he mos a ic wa e ba h allows wo king a he desi ed empe a u e. The eac o was co e ed wi h oil pape o a oid he ligh exposu e. On he o he hand, expe imen s 7-13 ha e been ca ied ou in SC-CO2 as eac ion medium. They we e pe o med in a high p essu e ba ch s i ed ank eac o made o s ainless s eel, ha ing an in e nal olume o 100 mL (Melgosa e al., 2017). A eshly ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 p epa ed emulsion and he lipase we e cha ged in o he eac o p o ided wi h magne ic agi a ion and hen i was closed, placed in a he mos a ic wa e ba h and connec ed o he p essu e ci cui . Subsequen ly, SC-CO2 was ed in o he eac o by means o a high p essu e pump (ISCO 260 D) up o he desi ed p essu e, which was main ained by a digi al p essu e con olle . Ope a ing p essu e and empe a u e ha e been a ied in he ange be ween 15-25 MPa (Exp.7-9) and 40-90ºC (Exp. 7 and 10-13). In bo h sys ems, samples we e aken pe iodically du ing 8 h, il e ed and s o ed a -18ºC up o analysis. 2.4 Analysis o he eac ion p oduc s The neu al lipid p o ile (TAG, DAG, MAG and FFA) was analyzed by a no mal phase high pe o mance liquid ch oma og aphy (NP-HPLC). The ch oma og aphic appa a us consis ed o a HPLC sys em (Agilen 1200) o med by a qua e na y pump and an au o- injec o . The ch oma og aphic sepa a ion o he compounds was ca ied ou a oom empe a u e wi h a Lich osphe Diol column (5 μm, 4 mm × 250 mm) and de ec ion was pe o med by an e apo a i e ligh sca e ing de ec o (Agilen 1200 se ies) a 35ºC and 0.35 MPa. G adien elu ion was achie ed by mobile phases A (isooc ane) and B (me hyl e -bu yl e he :ace ic acid = 99.9:0.1, / ). The me hod and calib a ion p ocedu e ha e been p e iously epo ed (Solaesa, Sanz, Falkebo g, e al., 2016). The egioisome s o DAG and MAG could no be dis inguished by he applied analy ical p ocedu e, so he o al amoun o MAG and DAG was epo ed o he kine ic expe imen s. The lipid p o ile esul s we e exp essed in glyce ol ee basis. 2.5 Lipid oxida ion analysis The oxida ion s a us has been de e mined using wo assays: pe oxide alue (PV) and anisidine alue (AV). The PV measu es he concen a ion o hyd ope oxides o med in he ini ial s ages o lipid oxida ion (p ima y oxida ion). PV was de e mined ollowing he AOAC O icial Me hod 965.33 by an au oma ic i a o Me h om 905 Ti ando (AOAC O icial Me hod 965.33, 2000). The AV is an es ima ion o he concen a ion o non- ola ile seconda y oxida ion p oduc s (mainly 2-alkenals and 2,4-dienals). The AV was measu ed acco ding o AOCS o icial me hod (Cd 18–90), using a UV-Visible spec opho ome e (AOCS O icial Me hod Cd 18-90, 2017). PV and AV allow calcula ing o al oxida ion (TOTOX) by he o mula: TOTOX = 2PV + AV [1] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 PV and AV ha e been de e mined o he supplied e ined sa dine oil and he inal eac ion mix u es ob ained a e 8 h a he di e en empe a u es The lipid phase was sepa a ed o analysis om he lipase and he emained glyce ol by cen i uga ion a 5000 pm and 35ºC du ing 10 minu es. The uppe phase, ee o glyce ol, o med by he lipid ac ion (TAG, DAG, MAG and FFA) was collec ed unde N2 a mosphe e and s o ed a -18ºC up o analysis. 2.6 S a is ical analysis All analyses we e conduc ed using so wa e S a g aphics X64. The esul s a e p esen ed as a mean  s anda d de ia ion o a leas h ee eplica es. The signi icance o he di e ences was de e mined based on an analysis o he a iance wi h he Tukey’s hones ly signi ican di e ence (HSF) me hod a p- alue ≤ 0.05. 3. Resul s and discussion 3.1 Op imiza ion o he emulsi ica ion p ocess and cha ac e iza ion o he emulsion 3.1.1 Su ac an - ee emulsions The e ec o emulsi ica ion speed on emulsion s abili y wi hou he addi ion o su ac an s has been e alua ed by measu ing he polydispe si y index (PDI) and he d ople diame e o he emulsion ob ained in he ange om 16000 o 35000 pm. A any o he emulsi ica ion speeds essayed, d ople diame e was lowe han 2 µm bu 29000 pm we e needed o ob ain a PDI below 1. The lowes polydispe si y index was ob ained a he highes speed assayed in his wo k, 35000 pm; howe e , oaming was obse ed. The e o e 29000 pm was selec ed o u he subs a e emulsi ica ions. A his speed he mean d ople diame e o he emulsion was 301 ± 34 nm and he PDI a ound 0.4. The su ac an - ee emulsion p esen ed a PDI lowe han 1 only up o 20 minu es, al hough a longe imes, s ill a anslucen and homogeneous sys em was isually obse ed. In any case, he emulsion was p epa ed and immedia ely used as eac ion media. 3.1.2 Su ac an s abilized emulsions The use o a su ac an was also es ed in his wo k o imp o e he emulsion s abili y and eac ion a es. Two ood g ade su ac an s, AOT and Tween 80, wi h hyd ophilic ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 lipophilic balance (HLB) alues o 10 and 15 espec i ely, we e used a di e en amoun s (0.5, 1 and 1.5%). PDI o he emulsions p epa ed adding hese su ac an s we e measu ed a speci ic imes o e alua e hei s abili y (Table 2). Al hough a su ac an was added o s abilize he emulsions, PDI in he di e en emulsions inc eased wi h ime in all cases (Table 2). The highe s abili y was obse ed when 0.5% o Tween 80 and 1.5% o AOT we e p e iously dissol ed in glyce ol. In hese cases he emulsion was ound o be s able o a leas 1 h. PSD was e alua ed o emulsions wi h he highes s abili y o med by adding 0.5 % o Tween 80 and 1.5 % o AOT in glyce ol and compa ed wi h hose ob ained in su ac an - eeemulsion. Smalle micelles we e ob ained when a su ac an was added o he sys em wi h medium pa icle sizes alues o 67 ± 5 nm, 94 ± 4 nm and 301 ± 34 nm o AOT 1.5 % and 0.5 % o Tween 80 dissol ed in glyce ol and su ac an - ee emulsion, espec i ely. 3.2 Glyce olysis eac ion o sa dine oil by Lipozyme 435 3.2.1 E ec o subs a es emulsi ica ion on he eac ion a e Fig. 1 compa es he kine ics o he glyce olysis eac ion in a sol en ee medium a a mosphe ic p essu e wi h and wi hou p e ious emulsi ica ion o he subs a es (Exp 1 and 2 espec i ely). As i can be obse ed, when no p e ious emulsi ica ion o he eac an s was ca ied ou , mass ans e limi a ions lead o lowe ini ial eac ion a e. These limi a ions a e e lec ed in he alues o he ini ial slope o TAG composi ion as unc ion o ime being 0.15  0.01 (mol TAG %·min-1) wi hou subs a es emulsi ica ion and 0.279  0.008 (mol TAG %·min-1) o subs a e emulsi ica ion. Fo a e e se micelle sys em, highe in e acial a ea is p o ided, which a o s lipase- ca alyzed eac ions. A longe eac ion imes, eac ion a es become simila due o he MAG and DAG o ma ion as emulsi ie s. The low HLB alues o MAG and DAG mean ha hey end o s abilize e e se micelles sys ems (O’B ien, 2004). Simila esul s we e obse ed by Awadallak e al. (Awadallak, Voll, Ribas, Ca dozo, & Edson, 2013) in he enzyma ic palm oil hyd olysis unde ul asound i adia ion o p oduce DAG. They also pe o med a con ol eac ion (wi hou ul asound in luence) o compa e he deg ee o hyd olysis in bo h sys ems, being a ound 20% a e 12 h in he con ol eac ion and almos 40% when ul asound was used be o e he eac ion. Bu a longe eac ion imes (24 h) he deg ee o hyd olysis becomes simila . The e o e, hey also ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 15 Oil Chemis s’ Socie y. h p://doi.o g/10.1007/s11746-006-1171-5 Esmelind o, Â. F. A., Fiame i, K. G., Ceni, G., Co azza, M. L., T eichel, H., de Oli ei a, D., & Oli ei a, J. V. (2008). Lipase-ca alyzed p oduc ion o monoglyce ides in comp essed p opane and AOT su ac an . Jou nal o Supe c i ical Fluids. h p://doi.o g/10.1016/j.sup lu.2008.05.004 Eu opean Pha macopoeia 5.0 (2005). Omega-3-acid T iglyce ides. Fel es, M. M. C., de Oli ei a, D., Block, J. M., & Ninow, J. L. (2013). The P oduc ion, Bene i s, and Applica ions o Monoacylglyce ols and Diacylglyce ols o Nu i ional In e es . Food and Biop ocess Technology. h p://doi.o g/10.1007/s11947-012-0836-3 Fiame i, K. G., Us a, M. K., De Oli ei a, D., Co azza, M. L., Fu igo, A., & Vladimi Oli ei a, J. (2012). Kine ics o ul asound-assis ed lipase-ca alyzed glyce olysis o oli e oil in sol en - ee sys em. 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JAOCS, Jou nal o he Ame ican Oil Chemis s’ Socie y, 86, 783–789. h p://doi.o g/10.1007/s11746-009-1402-7 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 18 Fig. 1 .TAG composi ion as a unc ion o ime in he glyce olysis o sa dine oil in sol en ee sys em wi h () and wi hou (◊) subs a es emulsi ica ion a a mosphe ic p essu e (0.1 MPa). Reac ions we e pe o med a MR = 3:1 (glyce ol:oil), T = 50ºC, enzyme loading 5 % w . o subs a es. 0 20 40 60 80 100 0 100 200 300 400 500 TAG (mol %) Time (min) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 19 Fig. 2. TAG composi ion as a unc ion o ime in he glyce olysis o sa dine oil p e ious subs a es emulsi ica ion wi h 1.5% o AOT in glyce ol (□), 0.5% o Tween 80 in glyce ol (○) and su ac an ee () a a mosphe ic p essu e (0.1 MPa).. Reac ions we e pe o med a MR = 3:1 (glyce ol:oil), T = 50ºC, enzyme loading 5 % w . o subs a es. 0 20 40 60 80 100 0 100 200 300 400 500 TAG (mol %) Time (min) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 20 Fig. 3. Time cou se o lipase-ca alyzed glyce olysis eac ion o sa dine oil in e -bu anol medium (hollow symbols) and in sol en ee (solid symbols) wi h p e ious emulsi ica ion o he subs a es a a mosphe ic p essu e (0.1 MPa). Legend: TAG (, ▲), DAG (□, ■), MAG (◇, ◆) and FFA (○, ●). Reac ions we e pe o med a MR = 3:1 (glyce ol:oil), T = 50ºC, enzyme loading 5 % w . o subs a es. 0 20 40 60 80 100 0 100 200 300 400 500 Lipid composi ion (mol %) Time (min) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 21 Fig. 4. E ec o p essu e in SC-CO2 as eac ion medium on glyce olysis con e sion o sa dine oil and p oduc s yield wi h p e ious subs a es emulsi ica ion . (∆) con e sion o TAG, (□, ◊, and ○) yields o DAG, MAG and FFA, espec i ely a e 7 h o eac ion ime. Reac ions we e pe o med a MR = 3:1 (glyce ol:oil), T = 50ºC, enzyme loading 5 % w . o subs a es. 0 20 40 60 80 100 0 5 10 15 20 25 Con e sion o Yield (mol %) P essu e (MPa) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 22 Fig. 5. E ec o empe a u e on MAG + DAG composi ion as unc ion o ime in he glyce olysis o sa dine oil wi h p e ious subs a es emulsi ica ion in SC-CO2 a 15 MPa: 40ºC (◊), 50ºC (□), 65ºC (∆), 80ºC (○) and 90ºC (x). Reac ions we e pe o med a MR = 3:1 (glyce ol:oil) and enzyme loading 5 % w . o subs a es. 0 20 40 60 80 100 0 100 200 300 400 500 600 MAG + DAG (mol %) Time (min) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 23 Fig. 6. In luence o eac ion empe a u e on PV (whi e ba s) and AV (g ey ba s) in he inal eac ion mix u e a e 7 h a 15 MPa in SC-CO2 as eac ion medium wi h p e ious subs a es emuls icia ion. Measu emen s gi en a e mean alues based on ou de e mina ions. Limi allowed is he maximum o each axis. Values wi h di e en le e s in each ype o analysis (PV o AV) a e signi ican ly di e en when applying he Tukey's hones ly signi ican di e ence (HSD) me hod a p- alue ≤ 0.05. ab c b b a ab ab a ab b b b 10 15 20 25 30 0 2 4 6 8 10 Ini ial 40 50 65 80 90 AV PV (mEq pe oxide kg-1 oil) Reac ion empe a u e (ºC) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 24 Table 1. Summa y o he eac ion condi ions o lipase-ca alyzed sa dine oil glyce olysis eac ions ca ied ou in his wo k. Exp. Reac ion medium P essu e (MPa) Tempe a u e (ºC) Emulsi ica ion Su ac an 1 Sol en ee 0.1 50 No - 2 Yes - 3 Yes AOT 4 Yes Tween 80 5 80 Yes - 6 90 Yes - 7 SC-CO2 as sol en 15 50 Yes - 8 20 Yes - 9 25 Yes - 10 15 40 Yes - 11 65 Yes - 12 80 Yes - 13 90 Yes - ACCEPTED MANUSCRIPT