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.
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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]
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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
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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
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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

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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
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(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
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(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
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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
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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

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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
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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
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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
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Oil Me hyl and E hyl Es e s.
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Chen, B., Hu, J., Mille , E. M., Xie, W., Cai, M., & G oss, R. A. (2008). Candida
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No iega-Rod íguez, J. A., O ega-Ga cía, J., Angulo-Gue e o, O., Ga cía, H. S.,
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Pazhouhanmeh , S., Fa hoosh, R., Sha i , A., & Esmaeilzadeh, R. (2016). Oxida ion
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Rod igues, R. C., & Fe nandez-La uen e, R. (2010). Lipase om Rhizomuco miehei as
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Sa hi el, S., Huang, J., & P inyawiwa kul, W. (2008). The mal p ope ies and
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applica ions o he A henius equa ion o e alua ing iscosi y and oxida ion a es
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456
Sil a Rod igues, J., Peixo o do Valle, C., Pinhei o Gue a, P. D. A. G., De Sousa Rios,
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M. A., Quei oz Mal ei a, J. De, & Rica do, N. M. P. S. (2017). S udy o kine ics
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om i sh isce a oil. Fuel P ocessing Technology, 161, 95–100.
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Solaesa, Á. G., Bucio, S. L., Sanz, M. T., Bel án, S., & Rebolleda, S. (2014).
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Cha ac e iza ion o T iacylglyce ol Composi ion o Fish Oils by Using
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Ch oma og aphic Techniques. Jou nal o Oleo Science, 63(5), 449–460.
463
Solaesa, Á. G., Sanz, M. T., Falkebo g, M., Bel án, S., & Guo, Z. (2016). P oduc ion
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Solaesa, Á. G., Sanz, M. T., Melgosa, R., & Bel án, S. (2017). Subs a es
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emulsi ica ion p ocess o imp o e lipase-ca alyzed sa dine oil glyce olysis in
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Resea ch In e na ional, 100(May), 572–578.
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S iede , M. M., Pinhei o, C. P., Bo ba, V. S., Pohndo , R. S., Cada al, T. R. S., &
472
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Pin o, L. A. A. (2017). Bleaching op imiza ion and win e iza ion s ep e alua ion in
473
he e inemen o ice b an oil. Sepa a ion and Pu i ica ion Technology, 175, 72–
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78.
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Sulli an, J. C., Suzanne, R., Jo ica, F., & Jean, A. (2015). Oxida ion Ra es o
476
T iacylglyce ol and E hyl Es e Fish Oils. Jou nal o he Ame ican Oil Chemis s’
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Socie y, 92, 561–569.
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To o-Vazquez, J.F., Cas illo-M., A.A., & He nández-C., R. (1993). A mul iple- a iable
479
app oach o s udy co n oil oxida ion. Jou nal o he Ame ican Oil Chemis 's
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Socie y, 70(3), 261-267.
481
Wang, Y., & Go don, M.H. (1991). E ec o lipid oxida ion p oduc s on he
482
anses e i ica ion ac i i y o an immobilized lipase. Jou nal o Ag icul u ad and
483
Food Chemis y, 39, 1693-1695.
484
Xu, X., Timm-H., M., Skall Nielsen, N., Po sgaa d, T., & Jacobsen, Ch. (2005) E ec s
485
o an ioxidan s on he lipase-ca alyzed acidolysis du ing p oduc ion o s uc u ed
486
lipids. Eu opean Jou nal o Lipid Science and Technology, 107, 464-468.
487
Yin, H., & Sa hi el, S. (2010). Physical P ope ies and Oxida ion Ra es o Un e ined
488
Menhaden Oil ( B e oo ia pa onus ). Jou nal o Food Science, 75(3), 163–168.
489
Zhang, H. (2007). E alua ion o P ac ical P ocess Aspec s o Lipozyme TL IM
490
Ca alyzed Bulk Fa Modi ica ion in a Ba ch Reac o . The Open Bio echnology
491
Jou nal, 1, 72–80.
492
493
494
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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)
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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)
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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

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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.
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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
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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.