Oxidation kinetics of sardine oil in the presence of commercial immobilized lipases commonly used as biocatalyst

Accep ed Manusc ip Oxida ion kine ics o sa dine oil in he p esence o comme cial immobilized lipases commonly used as bioca alys Ángela Ga cía Solaesa, Ma ía Te esa Sanz, Rod igo Melgosa, Sag a io Bel án PII: S0023-6438(18)30446-8 DOI: 10.1016/j.lw .2018.05.032 Re e ence: YFSTL 7142 To appea in: LWT - Food Science and Technology Recei ed Da e: 22 Janua y 2018 Re ised Da e: 10 May 2018 Accep ed Da e: 11 May 2018 Please ci e his a icle as: Solaesa, Á.Ga cí., Sanz, Ma í.Te esa., Melgosa, R., Bel án, S., Oxida ion kine ics o sa dine oil in he p esence o comme cial immobilized lipases commonly used as bioca alys , LWT - Food Science and Technology (2018), doi: 10.1016/j.lw .2018.05.032. 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. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Oxida ion kine ics o sa dine oil in he p esence o 1 comme cial immobilized lipases commonly used as bioca alys 2 Ángela Ga cía Solaesa, Ma ía Te esa Sanz ∗ , Rod igo Melgosa, Sag a io Bel án 3 Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), 4 Uni e si y o Bu gos, 09001 Bu gos. Spain 5 Abs ac 6 Oxida ion kine ics o sa dine oil ha e been de e mined a 40, 65 and 90ºC by measu ing 7 concen a ion o p ima y and seconda y oxida ion p oduc s in he p esence o 8 comme cial immobilized lipases (Lipozyme 435, Lipozyme RM and Lipozyme TL) 9 commonly used as bioca alys in lipid modi ica ion eac ions. Oxida ion p oduc s 10 concen a ion was ound o be lowe when he immobilized lipases we e added a he 11 highes empe a u es s udied. The lowes oxida ion indices we e obse ed in he 12 p esence o Lipozyme RM. 13 Al hough he mechanism o explain his dec ease in he oxida ion p oduc s is no s ill 14 clea , hese esul s migh indica e ha he use o hese immobilized lipases in lipase- 15 ca alyzed eac ions o ish oils a high empe a u e (90ºC) will yield highe eac ion 16 a es bu also a educ ion o he oxida ion p oduc s o med due o oxida ion o 17 polyunsa u ed a y acids. 18 Keywo ds: ish oil, omega-3, oxida ion p oduc s, comme cial immobilized lipases. 19 ∗Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess e [email protected] MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2 1. In oduc ion 20 Fish oil has high amoun s o omega-3 long-chain polyunsa u a ed a y acids (n-3 21 PUFA), mainly eicosapen aenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22 22:6n-3) which ha e been epo ed o p o ec agains he de elopmen o many diseases 23 (Solaesa, Sanz, Falkebo g, Bel án, & Guo, 2016). Howe e , despi e he high nu i ional 24 alue o hese p oduc s, he high deg ee o unsa u a ion makes ish oils e y p one o 25 au oxida ion. The esul ing b eakdown p oduc s cause o - la o s and ancidi y, loss o 26 nu i ional alue and inally consume ejec ion (Gómez-Alonso, Mancebo-Campos, & 27 Sal ado , 2004). The deg ee and a e o lipid oxida ion is in luenced by he unsa u a ion 28 o a y acids, oxygen concen a ion, empe a u e, su ace a ea, wa e ac i i y and 29 p esence o an i– and p ooxidan s. Tempe a u e has also an impo an impac on lipid 30 oxida ion since an inc ease in empe a u e accele a es oxida ion a es. 31 The p ocess o lipid oxida ion can be desc ibed in h ee gene al s eps: ini ia ion, 32 p opaga ion and e mina ion. The concen a ions o p ima y and seconda y oxida ion 33 p oduc s can be measu ed quan i a i ely and he e o e gi e an indica ion o he 34 oxida i e s a us o he oil. The hyd ope oxide con en , as p ima y oxida ion p oduc s, is 35 usually de e mined by he pe oxide alue (PV) assay. Seconda y oxida ion p oduc s can 36 be de e mined by anisidine alue (AV) and hioba bi u ic acid eac i e subs ances 37 (TBARS) assays. AV measu es mainly 2-alkenals and 2,4-dienals. On he o he hand, 38 he cyclic pe oxides o med by au oxida ion om polyunsa u a ed a y acids wi h h ee 39 o mo e double bonds a e he mos impo an p ecu so s o malonaldehyde and hence 40 sou ce o TBARS (Hoyland & Taylo , 1991). Polyene index (PI) is also used as a good 41 indica o o PUFA de e io a ion in ish oils, de ined as (EPA + DHA): palmi ic acid 42 a io (Pazhouhanmeh , Fa hoosh, Sha i , & Esmaeilzadeh, 2016). 43 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 Omega-3 concen a es can be p oduced by lipase-ca alyzed eac ions since hey can be 44 ca ied ou unde mild condi ions in sho eac ion imes compa ed wi h chemical 45 p ocesses. Some widely used comme cial immobilized lipases used in enzyma ic 46 eac ions a e om Candida an a c ica (No ozym 435 and Lipozyme 435), Rhizomuco 47 miehei (Lipozyme RM IM) and The momyces lanuginose (Lipozyme TL IM). In a 48 p e ious wo k (Solaesa, Sanz, Melgosa, & Bel án, 2017), i was ound ha he 49 oxida ion s a us o he inal eac ion p oduc s o glyce olysis o sa dine oil a 60 and 50 90ºC ca alyzed by he comme cial immobilized lipase, Lipozyme 435, was e en lowe 51 han he oxida ion s a us o he ini ial sa dine oil, de e mined as PV and AV. Especially, 52 hyd ope oxide con en dec eased in a g ea e ex end a he highes eac ion empe a u e 53 assayed. The e o e, an inc ease in eac ion empe a u e, no highe han he maximum 54 dena u a ion empe a u e o he lipase, was posi i e since eac ion a e inc eased and 55 lowe oxida ion s a us o he inal p oduc s was de e mined. 56 Based on hose p e ious esul s, in his wo k, a sys ema ic s udy o he oxida ion 57 p oduc s o ma ion o sa dine oil in he p esence o h ee comme cial immobilized 58 lipases commonly used in lipid modi ica ion eac ions has been ca ied ou in he 59 empe a u e ange whe e lipase ca alyzed eac ions usually ake place. The comme cial 60 lipases used, immobilized on o di e en suppo s, we e Lipozyme 435, Lipozyme RM 61 and Lipozyme TL. Au oxida ion kine ics o sa dine oil, as a con ol, we e i s 62 de e mined a he empe a u es selec ed in his wo k (40, 65 and 90ºC) o de e mine he 63 ne o ma ion a e o he p ima y and seconda y oxida ion p oduc s. A e wa ds, sa dine 64 oil was incuba ed a he same empe a u es in con ac wi h he di e en immobilized 65 lipases o e alua e he educ ion o he oxida ion p oduc s concen a ion in he p esence 66 o hese immobilized lipases. 67 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 4 2. Ma e ials and me hods 68 2.1 Ma e ials 69 Re ined sa dine oil was p o ided by Indus ias A ines S.L. (Spain). Th ee comme cial 70 immobilized lipases we e used in his wo k. Lipozyme 435 om Candida an a c ica B 71 and Lipozyme TL IM om The momyces lanuginosus we e kindly dona ed by 72 No ozymes A/S (Bags ae d, Denma k). Lipozyme RM IM, om Rhizomuco miehei, 73 was pu chased om Sigma Ald ich. The suppo cha ac e is ics o hese immobilized 74 lipases a e summa ized in Table 1. All o he chemicals used in he di e en analyses 75 pe o med in his wo k, we e o analy ical o HPLC g ade. 76 2.2 Oxida ion kine ics o sa dine oil 77 Fi s , au ooxida ion kine ics o sa dine oil we e e alua ed a h ee di e en empe a u es 78 40, 65 and 90 ºC as con ol samples. 79 Fo each expe imen , s oppe ed e lenmeye glass lasks we e illed wi h 70 g o e ined 80 sa dine oil and immedia ely applied a ni ogen s eam in he lask. A e wa ds hey we e 81 pe ec ly closed and co e ed wi h oil pape o a oid he ligh exposu e. Sa dine oil 82 samples we e incuba ed in a wa e ba h wi h o bi al agi a ion a he co esponding 83 empe a u e. A di e en ime in e als, du ing10 h, aliquo s we e wi hd awn o ollow 84 he oxida ion kine ics. 85 When he oxida ion kine ics we e de e mined in he p esence o he immobilized 86 lipases, he sa dine oil was pu in con ac wi h he di e en immobilized comme cial 87 lipases, Lipozyme 435, Lipozyme RM and Lipozyme TL, a 3.5% w/w concen a ion. 88 This concen a ion was ound o be sui able o lipid modi ica ion eac ions (Solaesa e 89 al., 2016). As o he au ooxida ion kine ics, samples we e wi hd awal a di e en ime 90 in e als du ing 10 h. 91 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 All samples we e s o ed a -18ºC up o oxida ion p oduc s analysis. Duplica e 92 expe imen s we e ca ied ou a each empe a u e. 93 2.3 De e mina ion o p ima y oxida ion p oduc s: pe oxide alue (PV) 94 PV was de e mined by iodome ic i a ion ollowing he AOAC O icial Me hod 95 (AOAC O icial Me hod 965.33, 2000) by an au oma ic i a o Me h om 905 Ti ando. 96 PV was exp essed as milliequi alen s o O 2 pe kilog am o sample. All samples we e 97 analyzed in iplica e. 98 2.4 De e mina ion o seconda y oxida ion p oduc s 99 2.4.1 Anisidine alue (AV) 100 The AV was measu ed acco ding o AOCS O icial Me hod (AOCS O icial Me hod Cd 101 18-90, 2017), using a UV-Visible spec opho ome e a 350nm. All samples we e 102 analyzed in iplica e. 103 2.4.2 Thioba bi u ic acid eac i e subs ances (TBARS) assay 104 TBARS we e de e mined acco ding o he spec opho ome ic me hod desc ibed by 105 No eel Semb in he Mas e 's Thesis (No eel Semb, 2012). The me hod is based on he 106 o ma ion o a pink complex wi h s ong abso bance a 532-535nm due o he p esence 107 o hioba bi u ic acid eac i e subs ances (TBARS). TBARS is exp essed as mg o 108 malondialdehyde (MDA) pe kilog am o oil. All samples we e analyzed in iplica e. 109 2.5 Fa y acid analysis 110 The ini ial sa dine oil and he oil samples a e 10 h o incuba ion a 40, 65 and 90ºC 111 we e analyzed by he AOAC me hod (AOAC O icial Me hod 991.39, 2000) o e alua e 112 he a y acid p o ile and he polyene index (EPA+DHA/16:0). An Agilen gas 113 ch oma og aph (6890N Ne wo k GC Sys em) equipped wi h a lame ioniza ion de ec o 114 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 6 (FID) and a used silica capilla y column (OmegawaxTM-320, 30 m x 0.32 mm i.d.) 115 was used. The me hod and he calib a ion p ocedu e was de eloped p e iously (Solaesa 116 e al., 2016). The samples we e analyzed in iplica e. 117 2.6 S a is ical analysis 118 S a is ical analyses we e conduc ed using so wa e S a g aphics X64. Con ol 119 expe imen s and oxida ion s a us o sa dine oil in con ac wi h he immobilized lipases 120 we e ca ied ou in duplica e. Fo each oxida ion p oduc , analysis was pe o med in 121 iplica e. The esul s a e p esen ed as a mean ± s anda d de ia ion. The signi icance o 122 he di e ences was de e mined based on an analysis o he a iance wi h he Tukey's 123 hones ly signi ican di e ence (HSD) me hod a p- alue ≤ 0.05. Fo each oxida ion 124 p oduc , signi icance di e ence has been de e mined among he h ee lipases a he 125 h ee ope a ing empe a u es a a speci ic con ac ime. ANOVA was also pe o med o 126 analyze he s a is ical signi icance o ime by compa ing oxida ion p oduc 127 concen a ion o each enzyme and empe a u e along ime. 128 The es ima ion o he pa ame e s o he models es ed in his wo k was pe o med by 129 using he Ma qua d algo i hm (S a g aphics X64). 130 3. Resul s and discussion 131 3.1 Chemical quali y o ini ial sa dine oil 132 The a y acid p o ile and some quali y pa ame e s o he supplied e ined sa dine oil 133 used in his wo k a e p esen ed in Table 2. The a y acid p o ile was simila o hose 134 p e iously epo ed o sa dine oil (Homayooni, Saha i, & Ba zega , 2014; No iega- 135 Rod íguez e al., 2009; Okada & Mo isse , 2007; Solaesa, Bucio, Sanz, Bel án, & 136 Rebolleda, 2014). The polyene index (PI) o he supplied e ined sa dine oil used in his 137 wo k was 1.39 (Table 2) being simila o he PI alue epo ed o sa dine oil 138 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 7 (Homayooni e al., 2014). O he impo an quali y pa ame e s such as acid alue, PV 139 and AV o he supplied e ined sa dine oil we e 0.45 ± 0.04 mg KOH/g, 4.8 ± 0.6 meq 140 O 2 /kg and 19.7 ± 0.4, espec i ely. These alues we e e y close o he limi s allowed 141 by GOED Volun a y Monog aph (GOED, 2015), which a e 0.5 mg KOH/g o acid 142 alue, 5 meq O 2 /kg oil o PV and 20 o AV. The e o e, he supplied e ined sa dine oil 143 used in his wo k was pa ially oxidized and oxida ion eac ion a es could be as e due 144 o he p esence in he medium o oxida ion p oduc s. Acco ding o To o-Vázquez e al. 145 (1993) he induc ion pe iod was educed as ini ial pe oxide alue inc eased, based on 146 hei s udy o co n oil oxida ion. The e ined sa dine oil used in his wo k p esen ed a 147 alue o 46 mg o MDA/kg. Rega ding TBARS con en , he maximum is no clea ly 148 speci ied. A documen by FAO es ablishes o esh ish oil a TBARS alue o 50 mg o 149 MDA/kg ish oil, al hough i is e e ed o ishes eed (Masson S, 1994). The induc ion 150 pe iod (h) by Rancima , de e mined a 70ºC and 20 L/h o ai low a e was 7.2 hou s, 151 simila o ha ob ained by No iega e al. (No iega-Rod íguez e al., 2009) o c ude and 152 deodo ized sa dine oil (10.4 and 17.7 h, espec i ely) using Rancima a 60ºC and 7 L/h 153 o ai low a e. 154 3.2 Oxida ion kine ics o sa dine oil a di e en empe a u es 155 3.2.1 Kine ic aspec s o oxida ion p oduc s 156 Pe oxide alues we e de e mined o e ime du ing 10 h a 40, 65 and 90ºC (Fig. 1a). A 157 any incuba ion ime, hyd ope oxide con en inc eased wi h incuba ion empe a u e. Fig. 158 1a also shows ha pe oxide alue con inuously inc eased wi h ime du ing 10 h. This 159 means ha hyd ope oxide a e o ma ion was s ill highe han hyd ope oxide a e 160 decomposi ion in he pe iod o ime co e ed in his wo k o all he empe a u es. 161 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 8 The kine ics o lipid oxida ion is no an easy ask. Reac ions a es a e usually desc ibed 162 by a pseudo-ze o, pseudo- i s and pseudo second o de eac ions. In any case, he o de 163 o he eac ion a e does no comply wi h he s oichiome y o he eac ion (Kamal-Eldin 164 & Yanishlie a, 2005). Labuza and Be gquis (Labuza & Be gquis , 1983) ound ha 165 lipid oxida ion we e hal -o de wi h espec o pu e lipids in model sys ems. Howe e , 166 in complex ood sys ems he da a some imes i ze o o de as well (Labuza & Be gquis , 167 1983). In his wo k, o ma ion o p ima y oxida ion p oduc s ollowed a pseudo-ze o 168 o de kine ic model: 169 PV =PV o + k· [1] 170 whe e PV o deno es he ini ial pe oxide alue and k is he eac ion a e cons an o a 171 ze o o de eac ion (meq O 2 ·kg oil -1 ·h -1 ). Reac ion a e cons an s, k, we e de e mined 172 om he slope o plo ing he PV as a unc ion o incuba ion ime. Table 3 lis s he 173 eac ion a e cons an o he h ee empe a u es assayed in his wo k, oge he wi h he 174 quali y o he i ing. Pseudo- i s and pseudo-second o de kine ics we e also ied, bu 175 he i ing was wo se han o pseudo-ze o o de eac ion. Gomez-Alonso e al. (Gómez- 176 Alonso e al., 2004) also ound ha o ma ion o p ima y oxida ion p oduc s o oli e oil 177 in he empe a u e ange om 25 o 75ºC ollowed a pseudo-ze o o de kine ics. 178 Fig. 1b and 1c show he a ia ion o he AV and he TBARS con en , espec i ely, o e 179 ime a he h ee empe a u es s udied in his wo k. I can be obse ed ha , bo h AV and 180 TBARS con en , con inuously inc eased wi h ime and empe a u e in he i s 10 h o 181 incuba ion ime, mainly a he highes empe a u es, 90ºC and 65ºC. A 40ºC he 182 oxida ion a es we e slowe and he seconda y oxida ion p oduc s concen a ion did no 183 inc ease in a g ea ex en . Compa ing Fig. 1a, 1b and 1c i can be obse ed ha 184 gene a ion o seconda y oxida ion p oduc s was aking place simul aneously wi h 185 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 15 ega d, Wang e al. (1991) in he s udy o anses e i ica ion eac ion o iolein wi h 328 lau ic acid ca alyzed by Lipozyme IM 20 obse ed ha , when es ing he ope a ion 329 s abili y o he immobilized lipase a 65 ºC o e 10 numbe o eac ion ba ches, 10 h 330 each ba ch, hyd ope oxide con en dec eased h ough he epea ed ba ches, bu so did 331 he lipase ac i i y. These au ho s p oposed ha hyd ope oxide decomposi ion p oduc s 332 could cause inac i a ion o he enzyme. 333 Al hough, mechanism is no s ill clea , among he di e en phenomenon ha could be 334 aking place, adso p ion o he oxida ion p oduc s on he lipase suppo could play an 335 impo an ole. Lipozyme RM is immobilized on o a weak-base anion exchange esin, 336 ha ing good capabili y o adso p ion p ocess. I s hyd ophilic na u e could esul in a 337 highe a ini y o hyd ophilic oxida ion p oduc s. On he con a y, Lipozyme 435 was 338 immobilized on o a hyd ophobic suppo (Chen e al., 2008). The seconda y oxida ion 339 p oduc s, as well as hyd ope oxides, a e conside ed pola molecules and, i is well 340 known ha he highe he deg ee o oxida ion o an oil, he mo e pola he oxida ion 341 p oduc s usually a e (Kamal-Eldin & Yanishlie a, 2005). This could pa ially explain 342 he di e ence obse ed among bo h immobilized lipases. In case o Lipozyme TL, 343 al hough i was immobilized on silica gel, a hyd ophilic ma e ial, i s pa icle size ange 344 was bigge (250-1000 µm) as well as he ue densi y o he suppo (1830 kg/m 3 , 345 Zhang, 2007). The e o e, i s su ace a ea was conside ably lowe . In Table 1 i can be 346 obse ed ha he a e age alue o Lipozyme RM pa icle size was smalle (200~600 347 µm) han he pa icle size o he o he s wo immobilized lipases (300-1000 µm). 348 The e o e, he adso p ion could be inc ease due o a highe equency o collisions 349 be ween adso ba e and adso ben . In any case, i mus be highligh ed ha he a e age 350 pa icle o hese immobilized lipases was much highe han he pa icle size o 351 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 16 comme cial abso ben s such as Tonsil ® bleaching ea hs which ange om 25 o 150 352 µm (S iede e al., 2017). 353 The oxida ion s a us o sa dine oil in con ac wi h he immobilized lipases (C immob-lipase ) 354 has been ela ed o he co esponding da a o he con ol sa dine oil p e iously 355 de e mined in sec ion 3.2 a he same empe a u e and incuba ion imes (C con ol ), C immob- 356 lipase /C con ol , o each ype o oxida ion p oduc . Figu es 3a, 3b, 4a, 4b, 5a and 5b show 357 he a io C immob-lipase /C con ol o he PV, AV and TBARS le el change a 90 and 65ºC, 358 espec i ely o he h ee immobilized lipases. The oxida ion s a us o sa dine oil in 359 con ac wi h he immobilized lipases a 40ºC showed no di e ence wi h ha o he 360 sa dine oil in he absence o immobilized lipases, o e en sligh ly highe oxida ion 361 s a us. The e o e, he esul s ha e no been plo ed. Fo he h ee immobilized lipases 362 es ed in his wo k a 65 and 90ºC, he a io C immob-lipase /C con ol dec eased o e ime. 363 Based on he shape o he Fig. 3-5 he ollowing equa ion has been used o co ela e he 364 da a o e ime: 365 C  !"#$%&',) C *+),#,) -=exp.−k + 0 [5] 366 whe e C immob-lipase, is he concen a ion o oxida ion compounds in sa dine oil in con ac 367 wi h immobilized lipases a a ce ain con ac ime, , C con ol, is he concen a ion o 368 oxida ion compounds in con ol sa dine oil (in he absence o immobilized lipases) a 369 he same incuba ion ime, , n is an adjus men pa ame e and k is an empi ical cons an . 370 This equa ion was based on he modi ied model o B imbe g p oposed by Mon e e al. 371 (Mon e e al., 2015) in he s udy o educ ion o colo and oxida ion p oduc s o ca p oil 372 wi h blends o bleaching ea h and ac i a ed ca bon. The dependence o k pa ame e on 373 empe a u e was assumed o ollow an A henius ype ela ionship (Eq. 2), al hough 374 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 17 only he highes empe a u es, 90 and 65º C we e conside ed in he i ing p ocedu e. 375 Table 6 lis s he pa ame e s ob ained o equa ion 5 o ela e he oxida ion p oduc s 376 concen a ion in con ac wi h he h ee immobilized lipases and he con ol oil o he 377 h ee measu ed pa ame e s, PV, AV and TBARS con en . Fo all he oxida ion p oduc s, 378 he highes E a was ound o Lipozyme TL p o ing ha his immobilized lipase is mo e 379 empe a u e-dependen on he educ ion o oxida ion p oduc s in he bulk oil. The 380 con inuous lines in Fig. 3-5 ep esen he equa ion 5, showing good i ing. 381 4. Conclusions 382 The oxida ion kine ics o sa dine oil e ealed ha hyd ope oxide and TBARS o ma ion 383 was highe han ha o seconda y oxida ion p oduc s de e mined as AV, due o he high 384 PUFA con en . Oxida ion s a us was lowe in he p esence o h ee comme cial 385 immobilized lipases, in he same empe a u e ange, especially a high empe a u es, 65 386 and 90ºC. Howe e , a 40ºC, oxida ion p oduc s o ma ion seemed o be s ill p omo ed. 387 Lipozyme RM yielded he lowe oxida ion indices, qui e below o hose o he ini ial 388 ish oil. 389 Al hough exac mechanism o educ ion o oxida ion p oduc s in he p esence o 390 comme cial immobilized lipases is no ye clea . I is an impo an inding since high 391 empe a u es a e usually a oided when dealing wi h lipid modi ica ion o ish oil due o 392 i s high con en o PUFA, e y p one o oxida ion. By using hese ype o lipases, 393 empe a u es, a leas , up o 90ºC can be used ha ing a double bene i o empe a u e; 394 on one hand highe eac ion a es and, on he o he hand lowe oxida ion s a us. 395 Howe e , eusabili y o he enzyme should be u he conside ed. 396 Acknowledgemen s 397 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 18 The au ho s hank he Eu opean Regional De elopmen Fund (ERDF) and Jun a de 398 Cas illa y León [g an numbe BU055U16] o inancial suppo . AGS acknowledges 399 Uni e si y o Bu gos and RM MINECO [g an numbe BES-2013-063937] o hei 400 p e-doc o al con ac s. 401 402 403 404 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 19 Re e ences 405 AOAC O icial Me hod 965.33. (2000). Pe oxide Value o Oils and Fa s. 406 AOAC O icial Me hod 991.39. (2000). Fa y Acids in Encapsula ed Fish Oils and Fish 407 Oil Me hyl and E hyl Es e s. 408 AOCS O icial Me hod Cd 18-90. (2017). p-Anisidine Value. 409 Chen, B., Hu, J., Mille , E. M., Xie, W., Cai, M., & G oss, R. A. (2008). 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The Open Bio echnology 491 Jou nal, 1, 72–80. 492 493 494 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 23 495 496 497 Fig. 1. (a) PV, (b) AV and (c) TBARS o sa dine oil du ing incuba ion ime a 40ºC (∆), 498 65ºC (□) and 90ºC (◊). Con inuous lines ep esen he pseudo-ze o o de kine ic model (Table 499 3). 500 0 10 20 30 40 50 60 0 2 4 6 8 10 12 PV, meqO2/kg oil ime, h 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 AV ime, h 0 25 50 75 100 125 150 175 200 0 2 4 6 8 10 12 TBARS (mg MDA/kg oil) ime, h (a) (b) (c) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 24 501 502 503 504 Fig. 2. (a) A henius plo and (b) Ey ing plo o he oxida ion o sa dine oil, PV (○), AV (□), 505 TBARS (∆). Uni s o k a e lis ed in Table 3. Time uni s in Ey ing plo o k alues ha e been 506 exp essed as s. 507 508 509 -2 -1 0 1 2 3 0.0027 0.0028 0.0029 0.0030 0.0031 0.0032 0.0033 ln k 1/T (K-1) (a) -16.0 -15.2 -14.4 -13.6 -12.8 -12.0 -11.2 0.0027 0.0028 0.0029 0.0030 0.0031 0.0032 0.0033 ln (k/T) 1/T (K-1) (b) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 31 Table 4. Ac i a ion ene gy om he A henius equa ion o he o ma ion o hyd ope oxides in di e en kind o oils ound in li e a u e. Type o oil T ange (ºC) E a , kJ/mol Re e ence Re ined sa dine oil 40 - 90 37.1 This wo k Un e ined menhaden oil 45 - 85 30.9 (Yin & Sa hi el, 2010) Un e ined pollock oil 24 - 90 33.2 (Sa hi el e al., 2008) Re ined soybean oil 25 - 80 73.6 (Lee e al., 2007) Re ined sun lowe oil 79.5 Vi gin oli e oil 52.3 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 32 Table 5. Oxida ion p oduc s concen a ion o sa dine oil samples in con ac wi h Lipozyme 435, Lipozyme RM and Lipozyme TL a 40, 65 and 90ºC a di e en incuba ion imes. Oxida ion Index Immobilized lipase T (ºC) Con ac ime (h) 1 2.5 5 7.5 10 PV Lipozyme 435 90 B 4.3 ± 0.4 a AB 3.8 ± 0.1 ab A 3.1 ± 0.1 ab A 3.1 ± 0.1 ab A 3.3 ± 0.3 b 65 A 4.6 ± 0.1 a A 4.6 ± 0.1 bc A 4.3 ± 0.5 cd A 3.9 ± 0.1 b A 3.9 ± 0.1 c 40 A 4.6 ± 0.2 a B 5.2 ± 0.0 cd C 6.5 ± 0.0 e B 5.3 ± 0.5 c AB 5.0 ± 0.1 d Lipozyme RM 90 B 4.8 ± 0.3 a AB 3.6 ± 0.3 a A 2.7 ± 0.2 a A 2.2 ± 0.1 a A 1.9 ± 0.1 a 65 C 4.4 ± 0.3 a B 3.6 ±0.2 a A 2.9 ± 0.1 ab A 2.5 ± 0.2 a A 2.4 ± 0.1 a 40 A 4.6 ± 0.1 a A 5.4 ± 0.2 cd B 8.3 ± 0.3 B 7.4 ± 1.0 d A 5.1 ± 0.4 d Lipozyme TL 90 C 5.0 ± 0.0 a C 5.3 ± 0.2 cd B 3.6 ± 0.0 bc A 2.9 ± 0.1 ab B 4.0 ± 0.3 c 65 C 5.4 ± 0.2 a C 5.6 ± 0.2 d B 4.6 ± 0.2 d A 3.9 ± 0.1 bc A 3.5 ± 0.2 bc 40 A 5.2 ± 0.0 a C 11.9 ± 0.7 e D 15.0 ± 0.1 g C 11.9 ± 0.5 e B 9.1 ± 0.0 e AV Lipozyme 435 90 A 22.0 ± 0.0 de BC 26.0 ± 0.0 e B 25.1 ± 0.4 d D 28.2 ± 0.2 e C 26.5 ± 0.5 c 65 A 22.1 ± 0.5 de A 22.3 ± 1.2 cd A 22.2 ± 0.2 c AB 23.4 ± 0.6 bc B 25.9 ± 0.8 c 40 A 21.0 ± 0.4 cd AB 21.5 ± 0.3 bc AB 21.6 ± 0.2 c AB 21.5 ± 0.7 b B 22.1 ± 0.9 b Lipozyme RM 90 B 19.5 ± 0.4 ab AB 19.2 ± 0.7 a AB 19.0 ± 0.3 ab AB 17.9 ± 0.7 a A 17.4 ± 0.2 a 65 B 20.0 ± 0.3 bc B 19.8 ± 0.3 ab A 18.2 ± 0.1 a A 17.7 ± 0.7 a AB 18.9 ± 0.5 a 40 A 18.8 ± 0.1 a A 19.4 ± 0.2 a A 19.7 ± 0.1 b A 18.2 ± 1.5 a A 19.2 ± 0.0 a Lipozyme TL 90 A 22.8 ± 0.1 e A 24.1 ± 0.1 de AB 24.8 ± 0.1 d B 26.8 ± 0.7 de C 33.1 ± 1.2 d 65 A 21.4 ± 0.3 d A 21.7 ± 0.2 bc B 24.8 ± 0.5 d B 24.5 ± 0.6 cd B 25.1 ± 0.5 c 40 A 21.4 ± 0.5 d A 22.0 ± 0.3 c A 23.1 ± 0.8 c A 22.3 ± 0.5 bc A 22.4 ± 0.2 b TBARS Lipozyme 435 90 B 60 ± 4 cd B 58 ± 4 cd A 37 ± 3 ab A 34 ± 6 b A 42 ± 2 b 65 A 55 ± 4 abcd A 48 ± 4 abc A 43 ± 2 bc A 49 ± 5 c A 54 ± 4 c 40 AB 57 ± 3 bcd AB 56 ± 3 bcd A 54 ± 0 c AB 64 ± 0 d B 70 ± 2 d Lipozyme RM 90 D 42 ± 4 a CD 39 ± 4 a BC 28 ± 1 a AB 20 ± 0 a A 15 ± 1 a 65 C 51 ± 4 abc C 45 ± 4 ab B 33 ± 2 ab A 22 ± 3 ab A 14 ± 0 a 40 A 43 ± 3 ab B 59 ± 3 cd CD 73 ± 4 d D 85 ± 2 e BC 69 ± 3 d Lipozyme TL 90 B 66 ± 3 d B 61 ± 4 d A 30 ± 1 a A 24 ± 1 ab A 22 ± 2 a 65 C 64 ± 3 cd BC 61 ± 1 d BC 54 ± 2 c B 50 ± 4 cd A 37 ± 5 b 40 A 52 ± 9 abcd BC 110 ± 11 e D 143 ± 7 e C 120 ± 5 B 105 ± 2 e Values wi h di e en small le e s in he same column o each oxida ion p oduc 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. Values wi h di e en capi al le e s in he same ow o each lipase a a ce ain incuba ion empe a u e 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. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 33 Table 6. Pa ame e s o equa ion 5 wi h an A ehnius ype dependence on empe a u e o k pa ame e o oxida ion p oduc s quan i ied by PV, AV and TBARS o he samples in con ac wi h Lipozyme 435, Lipozyme RM and Lipozyme TL. Immobilized lipase PV AV TBARS k o E a , kJ/mol n R 2 k o E a , kJ/mol n R 2 k o E a , kJ/mol n R 2 Lipozyme 435 5.9·10 3 26.5 0.59 0.998 2.3·10 5 45.2 1.03 0.968 2.2·10 2 19.9 0.78 0.973 Lipozyme RM 9.6·10 2 21.2 0.68 0.999 8.9·10 4 32.03 0.76 0.989 3.2·10 2 19.6 0.78 0.993 Lipozyme TL 9.0·10 3 28.7 0.76 0.992 5.3·10 5 46.7 0.84 0.919 1.0·10 4 33.5 1.28 0.995 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Highligh s • PV and TBARS inc ease as e han AV in he au oxida ion o sa dine oil. • Oxida ion p oduc s concen a ion dec eased in he p esence o immobilized lipases. • The dec ease was highe o PV and TBARS han anisidine eac ed compounds. • Lipozyme RM p esen ed he bes esul s o ob ain lowe oxida ion indices.