Kinetic study and kinetic parameters of lipase‐catalyzed glycerolysis of sardine oil in a homogeneous medium

1 DOI: 10.1016/S1872-2067(15)61040-3 CJC-2015-11-042 编辑润色稿 Kine ic s udy and kine ic pa ame e s o lipase-ca alyzed glyce olysis o sa dine oil in a homogeneous medium. Ángela Ga cía Solaesa, Ma ía Te esa Sanz*, Sag a io Bel án, Rod igo Melgosa 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 The p oduc ion o polyunsa u a ed a y acids (PUFAs) concen a es by enzyma ic ca alysis has gained in e es due o hei s e eospeci ici y and he milde condi ions needed compa ed o he use o ino ganic ca alys s. The enzyma ic glyce olysis o sa dine oil by Lipozyme 435 o ge PUFA concen a es in he o ms o di- and monoacylglyce ols (DAGs, MAGs) in an op imized amoun o e -bu anol as he o ganic sol en was s udied. Fi s , mass ans e limi a ion o he eac ion sys em was analyzed. The e ec o di e en ope a ing a iables such as lipase loading, empe a u e and eed composi ion was in es iga ed. A semi-empi ical kine ic model based on he e e sible elemen a y eac ions o glyce olysis and hyd olysis o he glyce ides was employed o co ela e he expe imen al kine ic da a. A mole a io glyce ol:oil o 3:1 was he op imum, which p oduced mo e han 84 w % o MAG a 50ºC. A compa ison wi h o he glyce olysis sys ems was pe o med using MAG yield, eac ion a e and signi icance o kine ic pa ame e s. Keywo ds: lipase-ca alyzed; glyce olysis; e -bu anol; mass ans e ; kine ic model. Recei ed 18 No embe 2015. Accep ed 4 Janua y 2016. ∗ Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess: e [email protected] 1. In oduc ion Fish oil is ich in omega-3 (n-3) polyunsa u a ed a y acids (PUFAs) such as eicosapen aenoic acid (EPA) and docosahexaenoic acid. The heal h bene i s o n-3 a y acids ha e been widely es ablished in he li e a u e [1-3]. Among he di e en ypes o lipid de i a i es con aining PUFA concen a es, MAG and DAG has good bioa ailabili y [4, 5]. In addi ion, MAG o i s mix u es wi h DAG accoun o 75 % o wo ldwide emulsi ie p oduc ion [6]. The p ocess cu en ly used in indus y o ob ain MAG is glyce olysis using an ino ganic alkaline ca alys a high empe a u e (220 - 260ºC). This me hod has se e al disad an ages such as i gi es a da k colo and bu n as e as well as high ene gy consump ion. Fu he mo e, chemical glyce olysis is no sui able o p oducing MAG ich in PUFA due o oxidiza ion p oblems. Enzyma ic glyce olysis is an a ac i e al e na i e o he p oduc ion o MAG ich in PUFA since he eac ion can be ca ied ou unde mild condi ions [7] and s uc u ed p oduc s a e ob ained. The immiscibili y o he eac an s, glyce ol and oil leads o mass ans e limi a ion in he glyce olysis o oils. 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 [8], comp essed luids [9], and ionic liquids [10] in o de o imp o e he mass ans e . 2 Recen ly he use o di e en su ac an s o inc ease he in e acial a ea [11] and ul asound i adia ion [12] ha e also been p oposed o educe mass ans e limi a ion. This pape is pa o a wide p ojec o he op imiza ion o MAG p oduc ion by enzyma ic glyce olysis o sa dine oil. Fi s , di e en e -alcohols we e e alua ed as he sol en used o c ea e a homogeneous phase [13]. Te ia y alcohols enhance he enzyme ac i i y and accele a e he eac ion a e as compa ed o he sol en - ee sys em [14]. In a p e ious wo k, e -pen anol was selec ed as he sol en and he e ec o he glyce ol:oil mole a io was e alua ed o i s e ec on kine ic beha io and MAG yield. The glyce olysis p oduc was subsequen ly ac iona ed by a wo-s ep molecula dis illa ion o ob ain a concen a ed p oduc o MAG and DAG ich in PUFA [15]. In his wo k, a di e en e ia y alcohol, e -bu anol was used as he sol en . Te - bu anol has been used in di e en glyce olysis sys ems o ege able oils such as oli e oil [16, 17], palm oil [18], camellia oil [19] and sun lowe oil [8, 20]. The main objec i e o his wo k is o p esen a de ailed kine ic s udy o enzyma ic glyce olysis o e ined sa dine oil in e -bu anol as he sol en ca alyzed by a comme cial lipase Lipozyme 435. The amoun o e - bu anol added o c ea e a monophasic sys em has been op imized based on liquid-liquid equilib ium (LLE) da a p e iously de e mined [13]. This alue was compa ed wi h he amoun o e -bu anol added o o he glyce olysis sys ems. The esul s in e ms o MAG and DAG yields we e compa ed wi h li e a u e da a epo ed o di e en ype o oils and ela ed o he high ac i i y o he lipase o sho and medium chain leng h a y acids. Fi s , he ex e nal and in e nal mass ans e esis ances we e analyzed in he he e ogeneous sys em o he immobilized lipase. Mass ans e limi a ion can play an impo an ole in he eac ion. Howe e , in mos glyce olysis s udies epo ed in he li e a u e, no mass ans e s udies we e pe o med. Ma hema ical models a e needed o p edic and op imize he indus ial p ocess. Howe e , no many wo ks in he li e a u e deal wi h he kine ic modeling o glyce olysis. One o he i s wo ks was ca ied ou by Moquin e al. [9]. In ha wo k, he kine ics o he non-ca alyzed glyce olysis o soybean oil in SCCO2 medium we e co ela ed by a sequence o e e sible eac ions o ake in o accoun he pa allel hyd olysis eac ion. The same model was used by Vale io e al. [11] in he kine ic s udy o sol en - ee lipase-ca alyzed glyce olysis o oli e oil by No ozym 435 wi h T i on X-100 as su ac an . Al hough glyce olysis and hyd olysis eac ions we e p oposed, no in o ma ion on he expe imen al FFA p oduc ion and a e o change o glyce ol we e p o ided and only he TAG, MAG and DAG concen a ions we e used in he i ing p ocedu e o ob ain he kine ic pa ame e s. The mechanism o glyce olysis and hyd olysis o pu e POP (1,3-palmi in-2-olein) by Rhizopus a hizus lipase was s udied by Tan and Yin [21] by including hyd olysis, es e i ica ion and isome iza ion o MAG and DAG. Chei silp e al. [22] p oposed a Ping-Pong Bi Bi model ha ocused on he kine ics o he hyd olysis and es e i ica ion s eps in ol ed in he glyce olysis o palm oil in an ace one/isooc ane mix u e (3:1 / ). Wa e was dissol ed in glyce ol (10 % w/ o wa e added o glyce ol) and he e o e a la ge amoun o 3 wa e was p esen in he eac ion medium. Recen ly, Voll e al. [17] p oposed a kine ic model based on he o de ed-sequen ial Bi Bi mechanism o a lipase-ca alyzed glyce olysis sys em o oli e oil in e -bu anol as he sol en . In ha wo k, he eac ion p oduc s we e exp essed as o al amoun o MAG, DAG, TAG and FFA by weigh pe cen age on a sol en - ee basis composi ion. No expe imen al in o ma ion on he glyce ol concen a ion a e o change was p o ided. Fiame i e al. [12] used a simila model o he one p oposed by Voll e al. [17] in he glyce olysis o oli e oil by ul asound i adia ion. Howe e , he pa ame e s we e no p o ided in he open li e a u e al hough hey could be a ailable upon eques o he au ho s. In his wo k, a simila app oach o ha p e iously p oposed by Moquin e al. [9] was used. The kine ic pa ame e s we e compa ed when possible wi h p e ious alues epo ed in he li e a u e. This model was able o conside he concen a ion o all he compounds in ol ed in he glyce olysis sys em: TAG, DAG, MAG, FFA, glyce ol and wa e . 2. Expe imen al 2.1 Ma e ials Re ined sa dine oil was p o ided by Indus ias A ines S.L. (Spain) wi h a wa e con en o 0.19 ± 0.03%. 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 ± 0.04%. Te -bu anol (TB) was pu chased om Me ck wi h a pu i y o ≥ 99% and a wa e con en o 0.20 ± 0.03%. The p oduc s we e s o ed o e ac i a ed 3 Å molecula sie e o keep hem d y. The ood g ade lipase Lipozyme 435 om Candida an a c ica (immobilized on a mac opo ous hyd ophobic ac ylic esin) was dona ed by No ozymes A/S (Bags ae d, Denma k). The wa e con en o his lipase was 3.5 ± 0.3% as de e mined in iplica e by Ka l-Fishe i a ion wi h a Mi subishi CA-20 mois u e me e . Acco ding o No ozymes A/S, he speci ic ac i i y o he lipase is ≥ 8000 p opyl lau a e uni s/g. No addi ional wa e was added o he sys em. The e o e, wa e p esen in he eac ion medium came only om he eac an s. 2.2 Enzyma ic Glyce olysis o Sa dine Oil Di e en ials con aining a mix u e o sa dine oil, glyce ol and TB we e incuba ed a di e en empe a u es om 303 o 333 K in a wa e ba h wi h s i ing. Di e en mole a ios o subs a e and enzyme dosage we e also s udied. The amoun o TB added was ixed a a mass a io o 1.5:1 (TB:subs a es) on he basis o p e ious s udies on LLE [13]. A selec ed ime in e als ( om i e minu es up o eigh hou s), a sample o he eac ion mix u e was wi hd awn and il e ed h ough a mic o il e (0.45 µm, Sa o ius RC) o s op he eac ion by emo ing he lipase. All samples we e s o ed a −18 ᵒC p io o analysis. The eusabili y o Lipozyme 435 in his p ocess was es ed by ecycling he immobilized enzyme in six ba ches. A e each un, he lipase was washed once wi h TB, and hen wice wi h hexane in o de o elimina e he emaining compounds. A e wa ds, he lipase was d ied a 303 K and s o ed in a desicca o unde acuum. No signi ican educ ion in enzyme ac i i y was ound. In any e en , a esh bioca alys was used in each un. Te -bu anol was e apo a ed unde acuum using a o a y e apo a o (Heibolph VV2000) a 333 K. In his way, 4 TB can be eused by using he molecula sie e o elimina e he wa e con en . 2.3 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 35 ºC 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 we e p e iously epo ed [23]. The egioisome s o DAG and MAG could no be dis inguished by he applied analy ical p ocedu e. The e o e he o al amoun o MAG and DAG was epo ed o he kine ic expe imen s. The analysis o he emaining glyce ol was pe o med by a high empe a u e gas ch oma og aph (HT-GC) sys em (HP 6890 Se ies GC Sys em) equipped wi h a lame ioniza ion de ec o (FID), a used silica capilla y column o 30 m × 0.25 mm i.d. coa ed wi h a 0.25 µm ilm hickness o 65% phenyl me hylpolisiloxane (65HT) as he s a iona y phase and an Agilen Technologies 7683B Se ies au oma ic injec o . The me hod and calib a ion p ocedu e we e p e iously epo ed [13]. 2.4 Kine ic modeling The o e all glyce olysis eac ion can be desc ibed by: TAG + 2 Gly 3 MAG [1] Howe e , glyce olysis is belie ed o ollow a wo-s ep eac ion. Fi s , one molecule o glyce ol eac s wi h one molecule o TAG o yield one molecule o DAG and ano he molecule o MAG. The eac ion o one molecule o DAG wi h one molecule o glyce ol can also ake place o yield wo molecules o MAG: TAG + Gly DAG + MAG [2] DAG + Gly 2 MAG [3] The b eakdown o TAG due o eac ion wi h MAG can also occu o p oduce wo molecules o DAG [9]: TAG + MAG 2 DAG [4] E en in he p esence o small amoun s o wa e in he glyce olysis eac ion medium, unwan ed hyd olysis eac ions mus be conside ed: TAG + H2O DAG + FFA [5] DAG + H2O MAG + FFA [6] k 1 k5 k3 k 2 k4 k 6 k 7 k9 k 8 k 10 k 1 k7 5 MAG + H2O GLY + FFA [7] Kine ic models a e needed o p edic and simula e he eac ion. By o mula ing he mass balance equa ion o all he species o he eac ion sys em, he concen a ion p o ile e sus ime can be ob ained. In his way, he p ocess can be op imized. The a e o change in concen a ion o each o he eac ion componen s a e desc ibed by he ollowing di e en ial equa ions: 𝑑𝑑𝑛𝑛𝑇𝑇𝑇𝑇𝑇𝑇 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑑𝑑 =−𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺+𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−𝑘𝑘5𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇+ 𝑘𝑘6(𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇)2−𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [8] 𝑑𝑑𝑛𝑛𝐷𝐷𝐷𝐷𝐷𝐷 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 =𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺-𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−𝑘𝑘3𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺+𝑘𝑘4(𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇)2+ 2𝑘𝑘5𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−2𝑘𝑘6(𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇)2+𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [9] 𝑑𝑑𝑛𝑛𝑀𝑀𝐷𝐷𝐷𝐷 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 =𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺-𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇+2𝑘𝑘3𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺−2𝑘𝑘4(𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇)2− 𝑘𝑘5𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇+𝑘𝑘6(𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇)2+𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘12𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [10] 𝑑𝑑𝑛𝑛𝐷𝐷𝑡𝑡𝐺𝐺 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 = −𝑘𝑘1𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺 +𝑘𝑘2𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇−𝑘𝑘3𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺+𝑘𝑘4(𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇)2+𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘12𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [11] 𝑑𝑑𝑛𝑛𝐹𝐹𝐹𝐹𝐷𝐷 𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 = 𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇+𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇+𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂−𝑘𝑘12𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [12] 𝑑𝑑𝑛𝑛𝐻𝐻2𝑂𝑂𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ⁄ 𝑑𝑑𝑡𝑡 =−𝑘𝑘7𝑥𝑥𝑇𝑇𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘8𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘9𝑥𝑥𝐷𝐷𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘10𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇−𝑘𝑘11𝑥𝑥𝑀𝑀𝑇𝑇𝑇𝑇𝑥𝑥𝐻𝐻2𝑂𝑂+𝑘𝑘12𝑥𝑥𝑇𝑇𝑡𝑡𝐺𝐺𝑥𝑥𝐹𝐹𝐹𝐹𝑇𝑇 [13] As explained abo e in he analy ical p ocedu e, he s e eoisome s o DAG and MAG could no be dis inguished and no di e ence was made be ween hem in he model. The concen a ions o he eac ion p oduc s we e exp essed on a sol en - ee basis. TAG, DAG, MAG, FFA and glyce ol concen a ions we e expe imen ally de e mined. The wa e concen a ion could no be measu ed e sus eac ion ime. Acco ding o Moquin e al. [9], i is possible o es ima e he change in wa e concen a ion by sub ac ing he expe imen al FFA concen a ion om he ini ial wa e concen a ion since he o ma ion o one mole FFA equi es one mole o k11 k 12 6 wa e (Equa ions 5-7). The a e cons an s o he six kine ic equa ions we e ob ained by sol ing he se o di e en ial equa ions simul aneously. The di e en ial equa ions we e sol ed nume ically wi h a ou h o de Runge-Ku a me hod and he pa ame e s we e op imized by minimizing he ollowing objec i e unc ion (O.F.): 𝑂𝑂. F. = ∑ ∑ �xi,exp−xi,calc�2 n i=1 all samplesnsamples ·100 [14] using he simplex Nelde -Mead me hod. The subsc ip “i” e e s o he di e en componen s in he glyce olysis sys em: TAG, DAG, MAG, FFA, glyce ol and wa e . The subsc ip s “exp” and “calc” e e o he expe imen al and calcula ed mole ac ion o he di e en componen s o each expe imen al kine ic da a poin (nsamples) The oo mean squa e de ia ion ( msd) was calcula ed o e alua e he quali y o he i ing: 𝑟𝑟𝑟𝑟𝑟𝑟𝑑𝑑=�∑�𝑤𝑤𝑖𝑖𝑒𝑒𝑒𝑒𝑒𝑒−𝑤𝑤𝑖𝑖𝑐𝑐𝑡𝑡𝑡𝑡𝑐𝑐�2 𝑁𝑁𝑂𝑂𝑁𝑁𝑁𝑁 𝑖𝑖=1 𝑁𝑁𝑂𝑂𝑁𝑁𝑁𝑁 [15] whe e NOBS is he o al numbe o kine ic da a poin s o all he kine ic expe imen s and 𝑤𝑤𝑖𝑖𝑒𝑒𝑒𝑒𝑒𝑒 and 𝑤𝑤𝑖𝑖𝑐𝑐𝑡𝑡𝑡𝑡𝑐𝑐 a e he expe imen al and calcula ed weigh ac ions o he eac ion compounds. 3. Resul s and discussion 3.1 Mass ans e analysis Ex e nal and in apa icle mass ans e esis ance can in luence he obse ed eac ion a e in he e ogeneous ca aly ic p ocesses such as immobilized lipase bioca alysis. Be o e he s udy o he e ec o he kine ic a iables, he mass ans e a e was analyzed. Te -bu anol was used as he o ganic sol en o p o ide an en i onmen whe e oil and glyce ol can in e ac since bo h eac an s a e comple ely immiscible. Te -bu anol helps o c ea e a homogeneous phase and also dec eases he iscosi y o he eac ion medium since bo h eac an s a e highly iscous, especially glyce ol (Table 1). To e alua e he ex e nal mass ans e esis ance, he glyce olysis eac ion was ca ied ou a di e en s i ing speeds, om 120 o 200 pm, while keeping cons an he es o he eac ion condi ions. The esul s a e p esen ed in Table 1. F om hese esul s, i can be concluded ha he e was no inc ease in he ini ial eac ion a e o MAG o ma ion in he speed ange s udied. This esul was expec ed since ex e nal di usion does no usually con ol he o e all a e unless he s i ing speed is e y low o he eac ion mix u e is e y iscous [24]. Te -bu anol helps o dec ease he iscosi y o he eac ion medium since i s iscosi y is 100 ime smalle han he iscosi y o glyce ol (Table 1), esul ing in a low ex e nal mass ans e esis ance and i ac s as an ine ca ie o he eac an s o he ac i e si e o he enzyme. Hence, 170 pm was chosen o all he glyce olysis eac ions. Slow in apa icle di usion can educe he o e all eac ion a e, especially i he eac an molecules a e la ge [25] and ha e a low mobili y in he lipase suppo . Ches e ield e al. [26] analyzed he ela i e magni ude o he 7 ex e nal liquid mass ans e esis ance o he combined in e nal esis ances (in apa icle di usion and eac ion esis ances) in he e hanolysis o was e cooking oil using No ozym 435 by plo ing he ecip ocal ini ial eac ion a e (1/ o) as a unc ion o in e se lipase loading (1/m). This plo should be a s aigh line, wi h a slope p opo ional o he combined in e nal esis ances, and he in e cep is p opo ional o he in e phase mass ans e esis ance. Figu e 1 illus a es his linea dependence in he glyce olysis o sa dine oil. The linea i p o ed ha he a e con olling s ep is he combined in e nal esis ances since he in e cep can be conside ed negligible. To e alua e he in apa icle di usion e ec , he lipase Lipozyme 435 was sepa a ed in o wo ac ions by a 400 µm sie e (46 w % o Lipozyme 435 pa icles wi h ϕp > 400 µm). Kine ic expe imen s we e ca ied ou wi h each o he ac ions ob ained and compa ed wi h he esul s ob ained wi h unsie ed lipase. Figu e 2 shows ha he ini ial eac ion a e o MAG o ma ion was inc eased by dec easing he pa icle size o Lipozyme 435. This may indica e in e nal mass ans e limi a ion o he la ge pa icles, al hough he same MAG yield was achie ed a long eac ion ime. A signi ican po e di usion esis ance was also ound by Ches e ield e al. [26] in he e hanolysis s udy wi h No ozym 435 ( echnical g ade o Candida an a ica). The expe imen al Thiele modulus, φexp, was calcula ed o e alua e he in apa icle esis ance [27]: 𝜙𝜙𝑒𝑒𝑒𝑒𝑒𝑒=�𝑑𝑑𝑒𝑒 6�2𝑟𝑟𝑒𝑒𝑒𝑒𝑒𝑒,𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒 𝐷𝐷𝑒𝑒𝑒𝑒𝑒𝑒𝐶𝐶𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒,𝑡𝑡 [16] dp is he mean pa icle diame e o Lipozyme 435 (dp = 383 µm, [26]). The e ec i e di usi i y, De , was e alua ed using [28]: 𝐷𝐷𝑒𝑒𝑒𝑒𝑒𝑒=𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒−𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑠𝑠𝑡𝑡𝜀𝜀𝑒𝑒𝜎𝜎 𝜏𝜏 [17] whe e εp, τ and σ a e Lipozyme 435 po osi y, o uosi y and cons ic ion ac o . These alues we e aken om Ches e ield [26] o No ozym 435 (εp = 0.5, τ = 6 and σ = 1). Dsubs a e-sol en is he molecula di usi i y o he eac an s (glyce ol and ish oil) in he eac ion medium ( e -bu anol in his wo k). I was es ima ed using he Wilke-Chang equa ion [29]: 𝐷𝐷𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑟𝑟𝑡𝑡𝑡𝑡𝑒𝑒−𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑛𝑛𝑡𝑡=7.4·10−8𝑇𝑇(𝑀𝑀𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒𝜓𝜓𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑠𝑠𝑡𝑡) 𝜂𝜂𝑠𝑠𝑡𝑡𝑡𝑡𝑠𝑠𝑒𝑒𝑠𝑠𝑡𝑡𝑉𝑉𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑡𝑡𝑠𝑠𝑡𝑡𝑡𝑡𝑒𝑒 0.6 [18] whe e Dsubs a e-sol en is he di usion coe icien o he subs a e in he sol en (cm2·s-1), Msubs a e is he molecula weigh o he sol en (g/mol), T is he empe a u e (K), ηsol en is he iscosi y o he sol en , cP, Vsus a e is he mola olume o he subs a e a i s no mal boiling empe a u e, cm3/mol and ψ he associa ion ac o o he sol en (dimensionless, ψ = 1 o non-associa ed compounds). The pa ame e s alues used in he calcula ion o φ a e lis ed in Table 2. Mola olumes a he no mal boiling poin we e es ima ed by he Tyn and Calus me hod [29]: 8 𝑉𝑉= 0.285𝑉𝑉𝑐𝑐1.048 [19] whe e Vc is he c i ical olume in cm3/mol. Vc o glyce ol was 255 cm3/mol [29]. No da a o Vc o ish oil was ound in he li e a u e. The co esponding es ima ed alue o iolein (Vc = 3235.65 cm3/mol) was used [30]. Φ was e alua ed o bo h subs a es, glyce ol and sa dine oil, a 323 K o exp,glyce ol = 0.0173 mmol·L-1·s-1, Cglyce ol,o = 47.5 mmol·L-1 exp, ish oil = 0.023 mmol·L-1·s-1 C ish oil,o = 47.5 mmol·L-1. Acco ding o Bailey [31], when Φ is su icien ly la ge (Φ ≥ 3), di usion o subs a e is slow ela i e o i s consump ion. When Φ < 0.3 he limi ing a e p ocess is he chemical eac ion. Φ o di usion o glyce ol in he eac ion medium was ound o be 1.9·10-2. Howe e , a alue o 0.36 was ob ained o he di usion o ish oil in e -bu anol, p obably due o he bigge oil molecules ha can lead o mo e di usional limi a ion (Table 2). In any case, he alue o Φ was close o he limi o 0.3 and he obse ed a e can be conside ed kine ically con olled. Based on he Φ alues, he lipase was used in i s comme cially a ailable size wi hou sie ing o u he kine ic expe imen s. Yang e al. [20] s udied he e ec o he loading o No ozym 435 on he glyce olysis o sun lowe oil. They ound ha an enzyme loading o mo e han 10% esul ed in only a small inc ease in MAG yield. The e o e hey sugges ed ha 10-15% o enzyme loading was enough o ob ain he maximum eac ion pe o mance. Mo eo e , o he au ho s as Vale io e al. and Fiame i e al. [11, 12] ha e shown ha high enzyme concen a ions can lead o he o ma ion o agg ega es, making he enzyme ac i e si e una ailable o he subs a es. Based on his and he esul s shown in Figu e 1, u he glyce olysis kine ics we e pe o med wi h 10 w % o Lipozyme 435 based on eac an weigh . 3.2 Glyce olysis eac ion sys em The p esence o a ca alys is necessa y since i has been shown in he li e a u e [16] ha unde 70ºC he obse ed eac ion a e wi hou a ca alys is nea ly ze o. Figu e 3c shows a ypical glyce olysis p o ile o ish oil a he mole a io o glyce ol:sa dine oil o 3:1 a 323 K wi h 10% o lipase loading in e -bu anol (68 % o e - bu anol). The main eac ion p oduc a he abo e condi ions was MAG (a ound 51 % mole pe cen age), bu DAG and FFA p oduc ion we e also obse ed al hough he mole pe cen was a ound 3 % o bo h compounds. The ini ial wa e con en in he eac ion medium was less han 1 % by weigh bu i was nea ly 10 % o he mole con en o wa e in he eac ion medium. The e o e FFA p oduc ion can be obse ed. TAG consump ion was nea ly comple e wi h a mole pe cen a equilib ium condi ions lowe han 2 %. 3.2.1 E ec o eac an mole a io The ini ial mole eac an a io (MR) was a ied be ween 1 and 9. Figu es 3a-3d show he glyce olysis p oduc p o ile exp essed in mole ac ion on a sol en - ee basis. The eac ion a e o o ma ion o MAG was always highe han ha o DAG and FFA. The p esence o a sol en , e -bu anol, helped bo h eac an s o di use o he ac i e si es o he enzyme and MAG o ma ion was a o ed. Vale io e al. [11] s udied he kine ics o 9 glyce olysis o oli e oil in a su ac an sys em (wi h T i on X-100 as su ac an ) as an al e na i e o he use o o ganic sol en s, and ound ha he DAG ini ial eac ion a e was highe han ha o MAG e en wi h an excess o glyce ol (MR=9:1). This beha io could be due o mass ans e limi a ion and can be compa ed o a si ua ion o low glyce ol concen a ion in he eac ion medium. The op imal MR glyce ol:oil mus conside he MAG yield as well as he excess o glyce ol employed in he glyce olysis eac ion. The equilib ium yield o MAG was calcula ed as: 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝑟𝑟𝐸𝐸𝐸𝐸𝑟𝑟 𝑀𝑀𝑀𝑀𝑀𝑀 𝑦𝑦𝐸𝐸𝑦𝑦𝐸𝐸𝑑𝑑 (%)=𝑀𝑀𝑡𝑡𝑡𝑡𝑒𝑒𝑠𝑠 𝑡𝑡𝑒𝑒 𝑀𝑀𝑇𝑇𝑇𝑇 𝑖𝑖𝑛𝑛 𝑡𝑡ℎ𝑒𝑒 𝑒𝑒𝑒𝑒𝑠𝑠𝑖𝑖𝑡𝑡𝑖𝑖𝑠𝑠𝑟𝑟𝑖𝑖𝑠𝑠𝑒𝑒 𝐼𝐼𝑛𝑛𝑖𝑖𝑡𝑡𝑖𝑖𝑡𝑡𝑡𝑡 𝑒𝑒𝑡𝑡𝑡𝑡𝑒𝑒𝑠𝑠 𝑡𝑡𝑒𝑒 𝑇𝑇𝑇𝑇𝑇𝑇·3·100 [20] Figu e 4 shows ha he MAG equilib ium yield emained p ac ically cons an a a MR highe han 5:1. A simila beha io was obse ed by Ches e ield e al [26] in he e hanolysis o was e co onseed cooking oil by No ozym 435. These au ho s p oposed he ollowing ela ionship o he equilib ium yield: 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝑟𝑟𝐸𝐸𝐸𝐸𝑟𝑟 𝑀𝑀𝑀𝑀𝑀𝑀 𝑦𝑦𝐸𝐸𝑦𝑦𝐸𝐸𝑑𝑑 (%)=𝑡𝑡 1+𝑒𝑒𝑒𝑒𝑒𝑒�𝑅𝑅𝑀𝑀𝑡𝑡−𝑅𝑅𝑀𝑀 𝑠𝑠� [21] Non-linea eg ession was pe o med by using he Ma qua d algo i hm (S a g aphics) gi ing a = 89.285 de ined as he limi ing no malized MAG equilib ium [26], b = 0.922 and RMo = 1.35 wi h 2 = 0.999. McNeil (1990) also ound ha he MAG equilib ium yield was independen o he glyce ol:oil mole a io om mole a io highe han 5:1. To ake in o accoun he excess o glyce ol employed, Figu e 4 also shows he MAG composi ion (exp essed as mole pe cen age) on a sol en - ee basis and on a sol en and glyce ol- ee basis. In he lipid basis (no glyce ol), on inc easing he MR, he MAG con en inc eased sha ply om a MR o 1:0 o 3:1 and hen he MAG con en sligh ly inc eased in he lipid ac ion. On a sol en - ee basis, when glyce ol was conside ed in he global composi ion, a maximum was obse ed in he MAG con en a a MR o 3:1, due o he excess o glyce ol employed ha was no consumed. Table 3 summa izes he glyce ide equilib ium composi ion ound in his wo k, as well as o o he glyce olysis sys ems in he li e a u e ha use e -bu anol as sol en and immobilized Candida an a c ica as he bioca alys . The esul s a e exp essed in weigh pe cen age on a lipid basis since in mos s udies, he composi ion was usually exp essed his way. Al hough di e en lipase loadings we e used in Table 3, he da a lis ed in his able co esponded in mos cases o equilib ium condi ions and he compa ison o he MAG yield can be es ablished as alid. Table 3 shows he di e en esul s in e ms o he MAG and DAG yields a he same ini ial MR (as will be explained in Sec ion 3.2.2, he e ec o empe a u e on he MAG equilib ium yield was no impo an ). Fo ins ance, a he MR glyce ol:oil o 4:1, he MAG pe cen age on a lipid basis anged om 70% o sun lowe oil o 91% o una oil. Rega ding he ype o oil, ish oils ga e a highe MAG yield han ege able oils. Acco ding o he shape and p ope ies o he scissile a y acid binding si es o Candida an a c ica lipase, in he li e a u e, i has been epo ed ha his lipase has high ac i i y o sho and medium chain leng h a y acids [32]. Table 4 p esen s he a y acid composi ion o he oils lis ed in Table 3. I can be obse ed ha ish oils 16 Table 3. Equilib ium composi ion o glyce olysis eac ion ound in his wo k and o o he glyce olysis sys ems ound in he li e a u e ha use e -bu anol as sol en and immobilized Candida an a c ica as bioca alys . Oil T, K % E MR % TB % MAG % DAG % TAG % FFA Re e ence Sa dine 323 10 1:1 3:1 5:1 9:1 63 68 68 74 43.0 ± 1.5 83.3 ± 2.1 89.1 ± 1.8 92.9 ± 1.5 25.8 ± 1.9 6.9 ± 1.1 3.7 ± 0.8 2.0 ± 0.7 24.6 ± 1.5 5.9 ± 1.0 3.0 ± 1.0 2.4 ± 0.8 6.6 ± 1.1 3.8 ± 1.1 4.0 ± 1.3 2.8 ± 1.1 This wo k Sun lowe 323 21 4:1 73 71.3 22.1 0.6 5.2 [8] Sun lowe a 313 15 4.5:1 60 70 25 1 4 [20] Tuna 318 15 4:1 58.6 90.8 2.5 5.5 1.2 [37] Camelia 323 5 4:1 66 74.1 ± 2.7 24.6 ± 0.1 1.3 ± 0.1 - -b [19] Oli e a, c 328 328 328 343 343 10 2.5 2.5 2.5 2.5 6:1 3:1 3:1 9:1 9:1 45 45 80 45 80 67 34 42 53 60 17 15 19 11 14 12 50 36 33 23 4 1 3 3 3 [16] Oli e a 328 10 6:1 45 ∼62 ∼19 ∼15 ∼4 [17] (a) G aphical lec u e (b) No e e ence o FFA o ma ion (c) Da a a 720 min o eac ion ime. 17 Table 4. Composi ion o medium chain leng h a y acids in he oils used in he glyce olysis sys ems lis ed in Table 3. Oil Medium chain leng h a y acids (%) Re e ence C14:0 C16:0 C16:1 Sa dine 12.4 ± 0.4 22.8 ± 0.2 12.5 ± 0.1 This wo k Tuna 4.2 30.6 4.7 [38] Sun lowe 0.1 6.7 0.2 [8] Oli e 0.1 – 1.2 7.0 – 16.0 - [16, 17] Camellia - 8.2 - [19] 18 Table 5. Calcula ed kine ic pa ame e s a di e en glyce ol:oil mole a ios (T = 323.15 K, 10 w % Lipozyme 435 based on subs a e weigh ). Objec i e unc ion and oo mean squa e de ia ion (w %) o he glyce olysis p oduc s. Model pa ame e Mole a io 1:1 3:1 5:1 9:1 k 1 0.0350 0.0264 0.0263 0.0285 k 2 0.0023 0.0248 0.0292 0.0276 k 3 0.7638 0.9167 0.9411 0.9411 k 4 0.0749 0.0400 0.0348 0.0338 k 5 0.0108 0.0096 0.0047 -- k 6 -- -- -- -- k 7 -- -- -- -- k 8 0.0711 0.0046 -- -- k 9 1.8168 1.9017 1.9022 1.9019 k 10 0.4052 0.0234 0.0202 0.0209 k 11 0.5853 0.8911 0.8942 0.8983 k 12 1.9714 1.8052 1.8033 1.8008 O.F. 0.0019 0.0013 0.0009 0.0012 Roo mean squa ed de ia ion (w %) TAG 4.7 3.4 5.6 4.6 DAG 1.0 0.9 0.4 0.2 MAG 3.8 2.7 5.8 5.6 FFA 1.1 0.7 0.5 0.3 Glyce ol 0.4 1.3 1.7 2.2 Wa e 0.2 0.3 0.1 0.3 Table 6. Calcula ed kine ic pa ame e s a di e en eac ion empe a u es (MR = 3:1, 10 w % Lipozyme 435 based on subs a e weigh ). Objec i e unc ion and oo mean squa e de ia ion (w %) o he glyce olysis p oduc s. Model pa ame e Reac ion Tempe a u e, K 303 313 323 333 k 1 0.0104 0.0164 0.0264 0.0357 k 2 0.0106 0.0201 0.0248 0.0494 19 k 3 0.9132 0.9132 0.9167 0.9194 k 4 0.0380 0.0395 0.0400 0.0436 k 5 0.0072 0.0114 0.0096 0.0174 k 6 -- -- -- -- k 7 -- -- -- -- k 8 0.0041 0.0049 0.0046 0.0049 k 9 1.9020 1.9017 1.9017 1.9021 k 10 0.0207 0.0223 0.0234 0.0241 k 11 0.8967 0.8923 0.8911 0.8970 k 12 1.8017 1.8041 1.8052 1.8069 O.F. 0.0027 0.0021 0.0013 0.0030 Roo mean squa ed de ia ion (w %) TAG 4.7 4.7 3.4 5.5 DAG 0.6 0.7 0.9 0.6 MAG 2.8 3.1 2.7 4.7 FFA 0.4 0.6 0.7 0.7 Glyce ol 1.9 2.3 1.3 1.7 Wa e 0.4 0.2 0.3 0.3 Table 7. Ac i a ion ene gy, kJ/mol, o he di e en s eps in some glyce olysis sys ems S ep Ea,i ( his wo k) Ea,i [17]* Ea,i [11]** 1 35.18 18.30 27.91 2 40.12 5.36·10-5 0.06 3 0.20 -- 47.08 4 3.52 1.097·10-4 2.68·10-12 5 20.64 8.397·10-4 208.17 6 -- 0.35 11.46 7 -- 15.55 62.83 8 4.10 -- 13.33 9 0.001 2.33 1.11·10-13 10 4.20 -- 69.46 11 0.009 45.46 71.48 20 12 0.08 15.77 81.77 (*) Sol en = e -bu anol (**) Su ac an sys em Table 8. Roo mean squa e de ia ion (w %) o glyce olysis p oduc s ob ained wi h ou model equa ions (1-6) TAG DAG MAG FFA Glyce ol Wa e Re e ence 4.74 0.63 4.02 0.60 1.65 0.25 This wo k 4.19 2.73 3.58 1.04 -- -- [17] (--) da a no epo ed Figu e 1. E ec o ca alys loading on ini ial eac ion a e o MAG o ma ion (T = 323 K, MR = 3:1). y = 4.1704x - 0.0277 R² = 0.9878 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.00 0.05 0.10 0.15 0.20 0.25 1/ o (L·min·mmol-1) 1/m (L·genzyme-1) 21 Figu e 2. E ec o pa icle size (○) dp < 400 µm; (◇) unsie ed lipase; (△) dp > 400 µm on MAG o ma ion eac ion: T = 323 K, 5 w % Lipozyme 435 loading, MR =3:1. S anda d unce ain y u (mole ac ion) = 0.02. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 100 200 300 400 MAG mole ac ion, lipid base ime, min a) (b) (a) 22 Figu e 3. Time cou se o he glyce olysis eac ion a di e en mole a ios (MR): (a) 9:1, (b) 5:1, (c) 3:1 (d) 1:1; 323 K, 10 w % Lipozyme  435 loading; □ MAG ○ FFA △ TAG ◇ glyce ol × DAG. Con inuous lines a e o he model in his wo k. S anda d unce ain y u (mole ac ion) = 0.02. 0 10 20 30 40 50 60 70 80 90 100 012345678910 MAG equilib ium yield, MAG composi ion Mola a io (MR) glyce ol:sa dine oil (c) (d) 23 Figu e 4. MAG equilib ium yield (□) as a unc ion o ini ial mole a io (MR) glyce ol:oil. The con inuous line is o Eq. 20. MAG composi ion as mole pe cen age on a sol en and glyce ol ee-basis (●) and on a sol en ee-basis (○). Con inuous lines a e he equilib ium composi ion ob ained wi h he model in his wo k. Figu e 5. Binodal cu e o he e na y sys em glyce ol + ish oil + e -bu anol a 303.15 K (−) and 323.15 K (-- -). Ini ial composi ion o glyce olysis eac ion in e -bu anol medium: ● his wo k, ○ Sun lowe oil [8], ◇ Sun lowe oil [20], Δ Tuna oil [37], + Camellia oil [19], □ Oli e oil [17], × Oli e oil [16]. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Weigh ac ion o e -bu anol Weigh ac ion o glyce ol One phase- egion Two phases- egion 24 Figu e 6. Ini ial eac ion a e as a unc ion o ini ial mole a io glyce ol:oil (323 K, 10 w % Lipozyme 435 loading): □ MAG ○ FFA △ TAG ◇ glyce ol × DAG. 0 1 2 3 4 5 6 7 8 9 10 0246810 o, mmoles·min-1·L-1 Mole a io (MR) glyce ol:sa dine oil (a) (b) 25 Figu e 7. Time cou se o he glyce olysis eac ion a di e en empe a u es: ( a) 303 K, (b) 313 K, (c) 323 K, (d) 333 K; 10 w % Lipozyme  435 loading, MR =3:1; □ MAG ○ FFA △ TAG ◇ glyce ol × DAG. Con inuous lines a e o he model in his wo k. S anda d unce ain y u (mole ac ion) = 0.02. Kine ic s udy o lipase-ca alyzed glyce olysis o sa dine oil in a homogeneous media. Compa ison o glyce olysis kine ic pa ame e s. SOLAESA Ángela G., SANZ M. Te esa*, BELTRÁN Sag a io, MELGOSA Rod igo Uni e si y o Bu gos, Spain This wo k p esen s a de ailed kine ic s udy o enzyma ic glyce olysis o sa dine oil ca alyzed by he comme cial lipase Lipozyme 435. Glyce olysis is ca ied ou in e -bu anol o c ea e a homogeneous sys em a oiding mass ans e limi a ions. Kine ic s udy o lipase-ca alyzed glyce olysis o sa dine oil in a homogeneous media.Compa ison o 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0100 200 300 400 500 Composi ion, mole ac ion ime, min MAG TAG DAG FFA GLY Time cou se o glyce olysis eac ion a 3:1 mole a io, 323 K and 10 w % Lipozyme  435 loading Lipozyme  435 CH 2 –O –CO – R 1 CH –O –CO – R 2 CH 2 –O –CO – R 3 CH 2 –O –CO – R 1 CH –OH (R 2 ) CH 2 –OH Te -bu anol media (DAG FFA) 2 CH 2 –OH CH –OH CH 2 –OH MAG 3 Glyce olTAG (c) (d)