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
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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]
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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
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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
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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
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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
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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]
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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
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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
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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)
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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)
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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)
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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)
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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)
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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)
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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
-
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