A study by 1H NMR on the influence of some factors affecting lipid in vitro digestion

A s udy by
1
H NMR on he in luence o some ac o s a ec ing lipid
in i o diges ion
Bá ba a Nie a-Eche a ía, Enca nación Goicoechea, Ma ía J. Manzanos, Ma ía D. Guillén
⇑
Food Technology, Facul y o Pha macy, Lasca ay Resea ch Cen e , Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad n°7, 01006 Vi o ia, Spain
a icle in o
A icle his o y:
Recei ed 11 Decembe 2015
Recei ed in e ised o m 11 Ap il 2016
Accep ed 4 May 2016
A ailable online 4 May 2016
Chemical compounds s udied in his a icle:
Ammonium chlo ide (PubChem CID: 25517)
Glucosamine hyd ochlo ide (PubChem CID:
2830832)
Glucose (PubChem CID: 5793)
Glucu onic acid (PubChem CID: 94715)
Glyce ol (PubChem CID: 753)
Monosodium phospha e (PubChem CID:
23672064)
Sodium bica bona e (PubChem CID:
516892)
Sodium sul a e (Pubchem CID: 24436)
U ea (PubChem CID: 1176)
U ic acid (PubChem CID: 1175)
Keywo ds:
1
H NMR
Lipolysis
In i o diges ion
Fish lipids
Bile sal s
abs ac
This a icle ocuses on he impac o se e al expe imen al ac o s, including gas ic acidi ica ion, in es i-
nal ansi ime, p esence o gas ic lipase, sample/diges i e luids a io, concen a ion and na u e o he
enzymes in in es inal juice, and bile concen a ion, on he ex en o in i o lipolysis when using a s a ic
model ha simula es human diges ion p ocesses in mou h, s omach and small in es ine. The s udy was
ca ied ou by P o on Nuclea Magne ic Resonance (
1
H NMR). This echnique p o ides a comple e
molecula pic u e o lipolysis, e idencing o he i s ime, whe he p e e en ial hyd olysis o ce ain
glyce ides o e o he s occu s. A lipolysis deg ee simila o ha epo ed in i o was eached by a ying
ce ain a iables wi hin a physiological ange; among hem, bile concen a ion was ound o be c ucial.
The holis ic iew o his
1
H NMR s udy p o ides in o ma ion o pa amoun impo ance o design sound
in i o diges ion models o de e mine he bioaccessibili y and bioa ailabili y o lipophilic compounds.
Ó2016 The Au ho s. Published by Else ie L d. This is an openaccessa icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. In oduc ion
In ecen yea s, in i o diges ion has a ac ed g ea in e es in
mul iple ields, including ood echnology and nu i ion, and has
become a aluable esea ch ool in s udying he bioaccessibili y
and bioa ailabili y o ele an nu ien s and oxic compounds
(Ga e , Failla, & Sa ama, 1999; Goicoechea, B andon, Blokland,
& Guillén, 2011; Roman, Bu i, & Singh, 2012; Ve san oo ,
Oomen, Van de Kamp, Rompelbe g, & Sips, 2005). Howe e , gas-
oin es inal diges ion is a e y complex and dynamic p ocess
whe e inges ed ood componen s a e submi ed o mechanical
o ces and o diges i e juices un il hey a e ans o med in o small
bioa ailable molecules, some o which can also be me abolized by
he gu mic obio a. Thus, an accu a e e lec ion o he human phys-
iological en i onmen wi hin he diges i e ac in o de o mimic
na u ally occu ing e en s is e y di icul and he pe o mance o
in i o diges ion can be in luenced by se e al expe imen al ac o s.
The in i o diges ion models p oposed in he li e a u e g ea ly
di e in hei complexi y le el, a ying om s a ic o dynamic, and
om one s ep p ocedu es o models ha simula e sequen ially all
o he diges i e p ocess, ha is, hose aking place in he mou h,
s omach and gu , including colonic e men a ion (Kong & Singh,
2010; Li, Hu, & McClemen s, 2011; Minekus, Ma eau, Ha enaa ,
& Huis in ’ Veld, 1995; Molly, Woes yne, & Ve s ae e, 1993;
Ve san oo , Van de Kamp, & Rompelbe g, 2004). Depending on
he esea ch opic and objec i es o he s udy, a wide a ie y o
condi ions has been assayed. The e o e, di e ences can be
h p://dx.doi.o g/10.1016/j. oodchem.2016.05.021
0308-8146/Ó2016 The Au ho s. Published by Else ie L d.
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
⇑
Co esponding au ho .
E-mail add ess: [email p o ec ed] (M.D. Guillén).
Food Chemis y 211 (2016) 17–26
Con en s lis s a ailable a ScienceDi ec
Food Chemis y
jou nal homepage: www.else ie .com/loca e/ oodchem
obse ed be ween he p opo ions o samples/diges i e luids, he
composi ion o diges i e juices, he ansi imes pe o med in each
s ep, o he in ensi y o he mechanical o ces applied (Hu , Lim,
Decke , & McClemen s, 2011). Recen ly, an a emp o homogenize
expe imen al condi ions o in i o diges ion was made (Minekus
e al., 2014). Ne e heless, he i s equi emen o all he in i o
me hodologies should be o mimic in i o mac onu ien diges ion
ex en (Golding & Woos e , 2010; Hu , Decke , & McClemen s,
2009), and o his pu pose i is o pa amoun impo ance o
analyze he in luence o he ac o s a ec ing i .
Rega ding lipid diges ion, he human body shows a high e i-
ciency o his p ocess. A e he enzyma ic hyd olysis ha akes
place in he s omach and mainly in he small in es ine, >95% o
die a y iglyce ides a e abso bed as monoglyce ides o a y acids
(Golding & Woos e , 2010). In i o lipolysis le els epo ed in he
li e a u e a e usually a lowe han hose occu ing in i o, espe-
cially wi h ega d o ish lipids (Ma in, Nie o-Fuen es, Seño áns,
Regle o, & Sole -Ri as, 2010; Ma ze, Meynie , & An on, 2013;
Zhu, Ye, Ve ie , & Singh, 2013). The high esis ance o long chain
polyunsa u a ed acyl g oups o in i o hyd olysis by panc ea ic
lipase could explain he low a es o lipolysis epo ed o ish oils
(Bläckbe g, He nell, Beng sson, & Oli ec ona, 1979). Thus, he
imp o emen o lipolysis unde in i o condi ions is a challenge
ha dese es a deepe knowledge o he ac o s a ec ing lipase
ac i i y. Fu he mo e, his deepe knowledge o lipid diges ion
would help in he u u e o design ood p oduc s wi h speci ic pe -
o mance du ing diges ion; ha is o say, wi h special p ope ies
like a ge ed deli e y.
In his con ex , he e ec o di e en expe imen al ac o s on
lipid in i o diges ion ex en is s udied in his pape , in o de o
ind hose condi ions unde which a lipolysis deg ee simila o ha
occu ed in i o is eached. The s a ing poin me hod was ha
desc ibed by Ve san oo e al. (2004, 2005). Al hough ini ially
designed o assessing bioa ailabili y o ood myco oxins, i has
since been employed o se e al pu poses, mainly ela ed o lipid
esea ch, such as he s udy o mic os uc u al changes in emulsi-
ied lipids (Hu e al., 2009), he a e o oxic compounds esul ing
om lipid oxida ion (Goicoechea e al., 2008, 2011), he in luence
o he cheese ma ix on lipid diges ion (Lamo he, Co beil,
Tu geon, & B i en, 2012), he e ec s o an ioxidan s on lipid oxida-
ion du ing diges ion (Ta ainen, Phuphusi , Suomela, Kuksis, &
Kallio, 2012), he diges ion o ish oil emulsions (Ma ze e al.,
2013), and milk mac onu ien decomposi ion (Kop -Bolanz e al.,
2012). A en ion was paid o di e en expe imen al ac o s,
including gas ic acidi ica ion, in es inal ansi ime, p esence o
gas ic lipase, sample/diges i e luids a io, in es inal enzymes
concen a ion and bile concen a ion. Thei in luence on he lipol-
ysis ad ance was quan i ied by means o P o on Nuclea Magne ic
Resonance (
1
H NMR). This echnique was selec ed because i was
p e iously success ully employed o quan i y iglyce ides, diglyc-
e ides, monoglyce ides, a y acids and glyce ol in lipid mix u es
and o e alua e he ad ance o lipolysis du ing in i o diges ion
(Nie a-Eche a ía, Goicoechea, Manzanos, & Guillén, 2014, 2015).
2. Ma e ials and me hods
2.1. Samples, eagen s and enzymes
Fa med Eu opean sea bass (Dicen a chus lab ax) specimens
we e pu chased om a local supe ma ke . A e cleaning, gu ing,
ille ing and skinning, hey we e submi ed o in i o diges ion.
The a e age weigh o a ille was 252.9 ± 22.0 g and hei a e age
lipid con en was 8.2 ± 1.0% (ww).
Reagen s and enzymes o he p epa a ion o diges i e juices
we e acqui ed om Sigma-Ald ich (S . Louis, MO, USA): Aspe gillus
o yzae
a
-amylase (10065); pepsin om po cine gas ic mucosa
(P7125); lipases om Aspe gillus nige (534781) and Candida ugosa
(62316); panc ea in om po cine panc eas (P1750); lipase ype II
c ude om po cine panc eas (L3126) and bo ine bile ex ac
(B3883).
2.2. In i o diges ion expe imen s
The s a ing poin o his s udy was he in i o diges ion model
de eloped by Ve san oo e al. (2004, 2005) o he ed s a e. The
composi ion o diges i e juices (sali a, gas ic, duodenal and bile)
is gi en in Table 1. Jus be o e he in i o diges ion expe imen s,
he juices we e hea ed o 37 ± 2 °C. The ish sample was p epa ed
by mincing in a g inde , o simula e mechanical disin eg a ion ha
occu s in he mou h. The diges ion expe imen s a ed wi h he
addi ion o 6 ml o sali a o 9 g o minced sea bass sample. A e
5 min o incuba ion, 12 ml o simula ed gas ic juice (GJ) we e
added and he mix u e was o a ed head-o e -heels a 40 pm
o 2 h a 37 ± 2 °C. Thi y minu es a e s a ing he gas ic diges-
ion, pH was se be ween 2 and 3 wi h HCl (37%), simula ing he
g adual acidi ica ion o he chyme ha occu s in i o. A e 2 h o
gas ic diges ion, 2 ml o sodium bica bona e solu ion (1 M),
12 ml o duodenal juice (DJ) and 6 ml o bile juice (BJ) we e added.
Subsequen ly, pH was se be ween 6 and 7, and he mix u e was
o a ed again a 40 pm and incuba ed a 37 ± 2 °C o 2 h.
The in luence o some expe imen al ac o s on he ish lipolysis
was e alua ed. These we e: gas ic pH, in es inal esidence ime,
p esence o lipase in he GJ, sample/diges i e luids a io, enzy-
ma ic composi ion o he DJ and bile concen a ion in he BJ.
Al hough each a iable can be a ec ed by he o he s, he in luence
o each expe imen al ac o on he lipolysis ex en was s udied
sequen ially, keeping he es o he expe imen al condi ions con-
s an bu including he selec ed condi ions o he ac o p e iously
es ed. This selec ion was made conside ing he imp o emen o
lipolysis, he e lec ion o physiological condi ions, as well as p ac-
ical and economical easons. Each diges ion expe imen was ca -
ied ou in iplica e, excep o ha using a lowe amoun o
Table 1
Composi ion o he juices employed in he model desc ibed by Ve san oo e al.
(2004, 2005) used as a s a ing poin in his s udy.
Componen s Sali a Gas ic
Juice (GJ)
Duodenal
Juice (DJ)
Bile
Juice (BJ)
KCl (mmol/l) 12.02 11.06 7.57 5.05
NaCl (mmol/l) 5.10 47.09 119.98 89.99
NaHCO
3
(mmol/l) 20.17 – 40.33 68.86
NaH
2
PO
4
(mmol/l) 7.40 0.22 – –
NH
4
Cl (mmol/l) – 5.72 – –
KH
2
PO
4
(mmol/l) – – 0.59 –
Na
2
SO
4
(mmol/l) 4.79 – – –
KSCN (mmol/l) 2.06 – – –
MgCl
2
(mmol/l) – – 0.53 –
CaCl
2
2H
2
O (mmol/l) – 2.72 1.36 1.51
HCl (37%) (ml/l) – 6.50 0.18 0.15
U ea (mmol/l) 3.33 1.42 1.67 4.16
Glucose (mmol/l) – 3.61 – –
Glucu onic acid (mmol/l) – 0.10 – –
U ic acid (mmol/l) 0.09 – – –
Glucosamine hyd ochlo ide
(mmol/l)
– 1.53 – –
Bo ine se um albumin (g/l) – 1.00 1.00 1.80
Mucin (g/l) 0.025 3.00 – –
a
-amylase (g/l) 0.29 – – –
Pepsin (g/l) – 2.50 – –
Panc ea in (g/l) – – 9.00 –
Panc ea ic lipase (g/l) – – 1.50 –
Bile (g/l) – – – 30.00
pH 6.8 ± 0.2 1.3 ± 0.2 8.1 ± 0.2 8.2 ± 0.2
18 B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26
sample/diges i e luids a io ha was pe o med in duplica e (4.5 g
o ish mea : 6 ml o sali a: 12 ml o GJ wi h lipase o A. nige
added: 12 ml o DJ p oposed by Ve san oo : 6 ml o BJ wi h bile
a 30 g/l).
2.3. Lipid ex ac ion and
1
H NMR spec a acquisi ion
Lipids con ained in minced ish and in diges ed samples we e
ex ac ed using dichlo ome hane as sol en (CH
2
Cl
2
, HPLC g ade,
Sigma-Ald ich, S . Louis, MO, USA), as in a p e ious s udy (Nie a-
Eche a ía e al., 2015). A e wa ds, he
1
H NMR spec a o he lipid
ex ac s we e acqui ed using a B uke A ance 400 spec ome e
ope a ing a 400 MHz. The sample p epa a ion, he acquisi ion
condi ions and he s udy o he spec al da a we e he same as in
p e ious s udies (Guillén & Ruiz, 2004; Nie a-Eche a ía e al.,
2014, 2015). Each spec um was eco ded in duplica e. Table 2
gi es he assignmen o he di e en
1
H NMR signals o he
co esponding p o ons, in ag eemen wi h he abo e-men ioned
s udies. The
1
H NMR spec a shown in he igu es we e plo ed
a a ixed alue o absolu e in ensi y o be alid o compa a i e
pu poses using he Mes eNo a p og am (Mes elab Resea ch,
San iago de Compos ela, Spain).
2.4. Equa ions de i ed om
1
H NMR spec al da a employed o he
quan i ica ion o he se e al lipoly ic p oduc s in he diges a es and he
ex en o lipid diges ion
Bea ing in mind ha he signal a eas in he spec a a e
p opo ional o he numbe o p o ons ha gene a e hem
and he p opo ionali y cons an is he same o all kinds o p o-
ons, he numbe o moles (N) o 2-monoglyce ides (2-MG),
1-monoglyce ides (1-MG), 1,2-diglyce ides (1,2-DG), iglyce ides
(TG), a y acids (FA) and glyce ol (Gol) in each sample can be
exp essed as ollows (Nie a-Eche a ía e al., 2014):
N
2-MG
¼Pc

A
K
=4ð1Þ
N
1-MG
¼Pc

A
L
ð2Þ
N
1;2-DG
¼Pc

A
IþJ
2N
1-MG

=2ð3Þ
Table 2
Chemical shi assignmen s and mul iplici ies o he
1
H NMR signals in CDCl
3
o he main p o ons o glyce ides and a y acids p esen in ish lipid samples be o e and a e in i o
diges ion. The signal le e s ag ee wi h hose gi en in Fig. 1.
Signal Chemical shi (ppm) Mul iplici y Func ional g oup
Type o p o ons Compound
A0.88 –CH
3
Sa u a ed, monounsa u a ed
x
-9 and/o
x
-7 acyl g oups and FA
0.89 –CH
3
Unsa u a ed
x
-6 acyl g oups and FA
B0.97 –CH
3
Unsa u a ed
x
-3 acyl g oups and FA
C1.19–1.42 m
*
–(CH
2
)
n
–Acyl g oups and FA
D1 1.61 m –OCO–CH
2
–CH
2
–Acyl g oups in TG, excep o DHA, EPA and ARA acyl g oups
1.62 m –OCO–CH
2
–CH
2
–Acyl g oups in 1,2-DG, excep o DHA, EPA and ARA acyl g oups
1.63 m –OCO–CH
2
–CH
2
–,
COOH–CH
2
–CH
2
–
Acyl g oups in 1-MG and FA, excep o DHA, EPA and ARA acyl g oups
1.64 m –OCO–CH
2
–CH
2
–Acyl g oups in 2-MG, excep o DHA, EPA and ARA acyl g oups
D2 1.69 m –OCO–CH
2
–CH
2
–EPA and ARA acyl g oups in TG
1.72 m COOH–CH
2
–CH
2
–EPA and ARA acids
E1.92–2.15 m
**
–CH
2
–CH@CH– Acyl g oups and FA, excep o –CH
2
– o DHA acyl g oup in b-posi ion
in ela ion o ca bonyl g oup
F1 2.26–2.36 d –OCO–CH
2
–Acyl g oups in TG, excep o DHA acyl g oups
2.33 m –OCO–CH
2
–Acyl g oups in 1,2-DG, excep o DHA acyl g oups
2.35 –OCO–CH
2
–
COOH–CH
2
–
Acyl g oups in 1-MG and FA, excep o DHA acyl g oups
2.38 –OCO–CH
2
–Acyl g oups in 2-MG, excep o DHA acyl g oups
F2 2.37–2.41 m –OCO–CH
2
–CH
2
–DHA acyl g oups in TG
2.39–2.44 m COOH–CH
2
–CH
2
–DHA acid
G2.77 @HC–CH
2
–CH@Diunsa u a ed
x
-6 acyl g oups and FA
H2.77–2.90 m @HC–CH
2
–CH@Polyunsa u a ed
x
-6 and
x
-3 acyl g oups and FA
I3.65 ddd ROCH
2
–CHOH–CH
2
OH Glyce yl g oup in 1-MG
J3.73 m
***
ROCH
2
–CH(OR
0
)–CH
2
OH Glyce yl g oup in 1,2-DG
K3.84 m
***
HOCH
2
–CH(OR)–CH
2
OH Glyce yl g oup in 2-MG
L3.94 m ROCH
2
–CHOH–CH
2
OH Glyce yl g oup in 1-MG
N4.18 ddd ROCH
2
–CHOH–CH
2
OH Glyce yl g oup in 1-MG
O4.22 dd,dd ROCH
2
–CH(OR
0
)–CH
2
OR
00
Glyce yl g oup in TG
P4.28 ddd ROCH
2
–CH(OR
0
)–CH
2
OH Glyce yl g oup in 1,2-DG
Q4.93 m HOCH
2
–CH(OR)–CH
2
OH Glyce yl g oup in 2-MG
R5.08 m ROCH
2
–CH(OR
0
)–CH
2
OH Glyce yl g oup in 1,2-DG
S5.27 m ROCH
2
–CH(OR
0
)–CH
2
OR
00
Glyce yl g oup in TG
T5.28–5.46 m –CH@CH–Acyl g oups and FA
Abb e ia ions: : iple ; m: mul iple ; TG: iglyce ides; DHA: docosahexaenoa e; EPA: eicosapen aenoa e; ARA: a achidona e; 1-MG: 1-monoglyce ide; FA: a y acid;
1,2-DG: 1,2-diglyce ide; 2-MG: 2-monoglyce ide; d: double .
*
O e lapping o mul iple s o me hylenic p o ons in he di e en acyl g oups o a y acids ei he in b-posi ion, o u he , in ela ion o double bonds, o in
c
-posi ion, o
u he , in ela ion o he ca bonyl g oup.
**
O e lapping o mul iple s o he
a
-me hylenic p o ons in ela ion o a single double bond o he di e en unsa u a ed acyl g oups o a y acids.
***
This signal shows di e en mul iplici y i he spec um is acqui ed om he pu e compound o aking pa in he mix u e.
B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26 19
N
TG
¼Pc

A
NþOþP
2N
1;2DG
2N
1-MG
ðÞ=4ð4Þ
N
FA
¼Pc

10A
2:26—2:37
þPc

5A
2:37—2:44
60N
TG
40N
1;2-DG
ð
18N
1-MG
13N
2-MG
Þ=20 ð5Þ
N
Gol
¼N
FA
N
1;2-DG
2N
2-MG
2N
1-MG
ðÞ=3ð6Þ
whe e Pc is he p opo ionali y cons an ela ing he numbe o
p o ons ha gene a e a signal, Ais he a ea o he signal in ol ed
(see Table 2) and A
2.26–2.37
and A
2.37–2.44
a e he a eas o signals
a 2.26–2.37 and 2.37–2.44 ppm espec i ely. These equa ions we e
used in o de o quan i y he se e al p oduc s gene a ed du ing
lipolysis and he ex en o lipid diges ion in he diges a es, ollow-
ing he app oaches ou lined in he Sec ions 3.2.1–3.2.3 o he
Resul s and Discussion sec ion.
2.5. S a is ical analysis
The signi icance o he di e ences on he mola pe cen ages o
he di e en kinds o lipoly ic p oduc s p esen in he diges a es,
and on he hyd olysis le el, iglyce ide ans o ma ion and lipid
bioaccessibili y we e de e mined by one-way a iance analysis
(ANOVA) ollowed by Tukey b es a p< 0.05, using SPSS .19
(IBM, NY, USA).
3. Resul s and discussion
3.1. Gene al
Due o he b oad ange o applica ions o in i o diges ion
me hodology, e y di e en models ha e been p oposed o mimic
his complex physiological p ocess. The selec ed model should no
only be able o e lec as closely as possible he human physiology
in o de o gene a e consis en and ealis ic esul s, bu should also
be ela i ely simple, cos e ec i e, easily applicable and ep o-
ducible. Likewise, he me hod employed o de e mine he ex en
o lipolysis mus be sound, and he de ini ion o he la e clea .
The model de eloped by Ve san oo e al. (2004, 2005) was
used as he s a ing poin o de elopmen o a new me hod. To
e alua e he changes in he in i o diges ion lipolysis as a conse-
quence o he a ia ions in se e al expe imen al ac o s, he de e -
mina ion o he ex en o lipolysis i s ly in he s a ing me hod,
and hen in all o he subsequen in i o diges ion expe imen s,
is equi ed.
3.2. De e mina ion o he ex en o lipolysis in he diges a es o sea
bass mea ob ained using Ve san oo condi ions
Fig. 1 shows he
1
H NMR spec a o he ish lipids be o e in i o
diges ion (BD) and a e in i o diges ion using he Ve san oo
condi ions (DV). Some egions a e su icien ly enla ged o show
he mos signi ican di e ences in he in ensi y o he signals,
whose assignmen is gi en in Table 2. The BD spec um con ains
he ypical signals (A,B,C,D1,D2,E,F1,F2,G,H,T) co esponding
o he p o ons o he main acyl g oups o ish lipids and o hose
speci ic o iglyce ides (TG) (O,S).
Ne e heless, when compa ing DV and BD spec a, no iceable
di e ences a e obse ed. Al hough ce ain signals (A,B,C,E,G,
H,T) emain almos unchanged a e diges ion, hose ela ed o
he glyce ol backbone o TG (O,S) show lowe in ensi ies.
Mo eo e , in he DV spec um signals ela ed o he p o ons in
he glyce ol backbone o newly o med glyce ides appea ; hese
a e p edominan ly 1,2-diglyce ides (1,2-DG) (J,P,R) and 2-
monoglyce ides (2-MG) (K,Q), which is in ag eemen wi h lipid
hyd olysis ha occu s wi hin he human ac and he egiospeci-
ici y o he lipases added o he duodenal juice. Al hough in e y
low in ensi y, signals Iand L, co esponding o 1-monoglyce ides
(1-MG), a e also p esen in he DV spec um. The occu ence o
1-MG in he diges a es can be explained by he isome iza ion o
2-MG, as occu s in i o (Ma son & Volpenhein, 1964).
In sho , om he simple obse a ion o he BD and DV spec a
i can be deduced ha he ex en o lipolysis eached in he DV
sample is a he limi ed, and ha an impo an amoun o TG
emains unhyd olyzed.
All o he abo e, di ec ly obse able in he spec a, can also be
quan i ied. Thus, wo app oaches can be used, ei he based on
he glyce yl s uc u es o on he a y acids and acyl g oups
p esen .
3.2.1. App oach based on he glyce yl s uc u es p esen in he
diges a es
Using he equa ions p o ided in he Ma e ials and me hods Sec-
ion 2.4, he mola pe cen ages o he di e en kinds o glyce ides
in ela ion o he o al numbe o moles o glyce yl s uc u es
(NT
GS
) can be de e mined:
TG%¼100N
TG
=NT
GS
ð7Þ
1;2-DG%¼100N
1;2-DG
=NT
GS
ð8Þ
2-MG%¼100N
2-MG
=NT
GS
ð9Þ
1-MG%¼100N
1-MG
=NT
GS
ð10Þ
Gol%¼100N
Gol
=NT
GS
ð11Þ
NT
GS
¼N
TG
þN
1;2-DG
þN
2-MG
þN
1-MG
þN
Gol
ð12Þ
The esul s ob ained a e gi en in Table 3. Using Ve san oo
condi ions (DV) a conside able p opo ion o TG emained in ac
(69.2 ± 3.3%), a below he hyd olysis pe o mance epo ed
in i o (95% o TG ans o med in o FA and MG) (Golding &
Fig. 1.
1
H NMR spec a o lipid ex ac s o sea bass be o e (BD) and a e in i o
diges ion ollowing he condi ions desc ibed by Ve san oo e al. (2004, 2005)
(DV) and he ones p oposed a e s udying he in luence o ce ain ac o s (DO).
Some spec al egions ha e been p ope ly enla ged and he signal le e s ag ee wi h
hose in Table 2.
20 B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26
Woos e , 2010), hus p o iding e idence o limi ed lipolysis in he
s a ing me hod. I is also no ewo hy ha 1,2-DG was he mos
abundan glyce ide among he lipoly ic p oduc s and ha com-
ple e hyd olysis o TG in o FA and Gol occu ed o a sligh ex en .
Mo eo e , aluable in o ma ion conce ning he p e e en ial
hyd olysis o he glyce yl s uc u es du ing diges ion is ob ained
om he compa a i e analysis o hese mola pe cen ages; g ea e
hyd olysis o he es e bonds occu ed in TG han i did in DG and
in MG. Indeed, he posi ional speci ici y o panc ea ic lipase would
explain his dec ease o he hyd olysis a e in pa ial glyce ides (TG
>1,2-DG >MG), because lipase inds wo ou e chains in TG, only
one in 1,2-DG (and 1-MG) and none in 2-MG (Desnuelle &
Sa a y, 1963).
3.2.2. App oach based on he a y acids and acyl g oups p esen in he
diges a es
Lipolysis can also be analyzed conside ing he acyl g oups (AG)
bound o he di e en kinds o glyce ides and he FA p esen . The
mola pe cen age o FA (FA%) and hose o he AG bounded o TG
(AG
TG
%), 1,2-DG (AG
1,2-DG
%), 2-MG (AG
2-MG
%) and 1-MG (AG
1-MG
%)
in ela ion o he o al numbe o moles o hese AG plus FA
(NT
AG+FA
) can be de e mined:
AG
TG
%¼100ð3N
TG
Þ=NT
AGþFA
ð13Þ
AG
1;2-DG
%¼100ð2N
1;2-DG
Þ=NT
AGþFA
ð14Þ
AG
2-MG
%¼100N
2-MG
=NT
AGþFA
ð15Þ
AG
1-MG
%¼100N
1-MG
=NT
AGþFA
ð16Þ
FA%¼100N
FA
=NT
AGþFA
ð17Þ
NT
AGþFA
¼3N
TG
þ2N
1;2-DG
þN
2-MG
þN
1-MG
þN
FA
ð18Þ
The esul s ob ained using his second app oach a e also gi en
in Table 3. The p opo ion o po en ially non-abso bable s uc u es
(AG
TG
%+AG
1,2-DG
%) emained e y high using Ve san oo condi-
ions, his being app oxima ely 4- old highe han ha o bioacces-
sible molecules (MG and FA). Mo eo e , hese la e we e mainly
FA (see AG
2-MG
%, AG
1-MG
%, FA%). Howe e , i mus be no ed ha
excep o TG, di e en alues a e ob ained due o he di e en sig-
ni icance o each quan i ica ion me hod. Indeed, a mass balance
could be success ully applied wi h only he i s app oach. Despi e
his, all o he de e mina ions ob ained om
1
H NMR spec al da a
p o ide a comple e molecula pic u e o lipolysis (CMP
L
), which is
a holis ic iew o he lipolysis a a molecula le el, allowing a
deepe s udy o lipid diges ion.
3.2.3. O he pa ame e s usually employed o desc ibe he ex en o
lipolysis
The ex en o ish lipid in i o diges ion can also be es ima ed
using di e en pa ame e s employed by o he au ho s. One o
hem is he so-called hyd olysis le el (H
L
%)(Zhu e al., 2013),
which can be calcula ed using his equa ion:
H
L
%¼100N
FA
=NT
AGþFA
ð19Þ
Ano he p oposed pa ame e (A mand e al., 1999; Vina o
e al., 2012a, 2012b) is he deg ee o ans o ma ion o TG
(T
TG
%). This only conside s he hyd olysis ha occu ed in TG
and can be de e mined using he ollowing equa ion:
T
TG
%¼100ðN
TGi
N
TG
Þ=N
TGi
ð20Þ
whe e N
TGi
is he numbe o moles o TG ini ially p esen in he
sample, which can be es ima ed in each sample as NT
GS
, assuming
ha he lipids be o e diges ion consis exclusi ely o TG
Table 3
Comple e molecula pic u e o he lipolysis (CMP
L
) unde gone in he diges a es o sea bass mea unde di e en expe imen al condi ions. Di e en le e s wi hin each column indica e a signi ican di e ence (p< 0.05).
Fac o s Mola pe cen ages o glyce ides in ela ion o he o al numbe
o glyce yl s uc u es p esen
Mola pe cen ages o acyl g oups (AG) and a y acids (FA) in
ela ion o he o al numbe o AG + FA p esen
TG% 1,2-DG% 2-MG% 1-MG% Gol% AG
TG
%AG
1,2-DG
%AG
2-MG
%AG
1-MG
% FA%
S a ing poin (Sample DV) (Ve san oo condi ions) 69.2 ± 3.3a 17.6 ± 1.0a 6.4 ± 2.0a 1.3 ± 0.4a 5.5 ± 0.2a 69.2 ± 3.3a 11.7 ± 0.7a 2.1 ± 0.7a 0.4 ± 0.1a 16.5 ± 1.9a
Delay o pH shi ing in he gas ic s ep (60 min) 69.0 ± 2.6a 18.1 ± 1.2a 6.0 ± 1.4a 1.2 ± 0.2a 5.7 ± 0.6a 69.0 ± 2.6a 12.1 ± 0.8a 2.0 ± 0.5a 0.4 ± 0.1a 16.5 ± 1.5a
Inc ease o in es inal esidence ime (4 h) 63.0 ± 3.1a 19.1 ± 1.6a 6.7 ± 1.3a 2.5 ± 0.6a 8.7 ± 1.6a 63.0 ± 3.1a 12.8 ± 1.1a 2.2 ± 0.4a 0.8 ± 0.2a 21.2 ± 2.2a
Addi ion o lipase in he GJ (100 U/ml o A. nige lipase) 48.9 ± 4.3b 23.5 ± 0.7a 12.0 ± 1.7b 2.2 ± 0.2a 13.4 ± 2.2b 48.9 ± 4.3b 15.7 ± 0.4a 4.0 ± 0.6b 0.7 ± 0.1a 30.7 ± 3.4b
Dec ease o a io ood sample/diges i e luids olume (4.5 g ood: 6 ml S: 12 ml GJ:
12 ml DJ: 6 ml BJ)
39.6 ± 5.3c 18.2 ± 2.9a 9.4 ± 1.5ab 2.0 ± 0.5a 30.8 ± 1.3c 39.6 ± 5.3c 12.1 ± 1.9a 3.1 ± 0.5ab 0.7 ± 0.2a 44.5 ± 3.0c
Inc ease o lipase concen a ion in he DJ (DJ2: 9 g/l o panc ea in and 9.6 g/l lipase) 39.9 ± 3.5c 18.7 ± 1.4a 8.7 ± 2.3ab 1.5 ± 0.6a 31.1 ± 0.9c 39.9 ± 3.5c 12.5 ± 1.0a 2.9 ± 0.8ab 0.5 ± 0.2a 44.2 ± 1.5c
Dec ease o bile concen a ion in he BJ (Sample DO) (18.75 g/l) 4.6 ± 2.1d 23.0 ± 5.2a 23.2 ± 1.9c 5.4 ± 1.1b 43.8 ± 5.0d 4.6 ± 2.1d 15.3 ± 3.5a 7.7 ± 0.6c 1.8 ± 0.4b 70.5 ± 4.8d
Abb e ia ions: TG: iglyce ides; 1,2-DG: 1,2-diglyce ide; 2-MG: 2-monoglyce ide; 1-MG: 1-monoglyce ide; Gol: glyce ol; DV: diges ed ish lipid ex ac s ollowing Ve san oo condi ions; S: sali a; GJ: gas ic juice; DJ: duodenal
juice; BJ: bile juice; DO: diges ed ish lipid ex ac s ollowing he op imized condi ions p oposed in his s udy.
B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26 21

(99.2 ± 0.2%), ha a o al ex ac ion o lipids is pe o med, and ha
he possible des uc ion o FA du ing diges ion is negligible.
Finally, o es ima e he ex en o lipolysis in diges ion om a
physiological poin o iew, some au ho s p oposed he pa ame e
lipid bioaccessibili y (L
BA
%)(Kenmogne-Domguia, Meynie , Viau,
Llamas, & Geno , 2012):
L
BA
%¼100ðN
1-MG
þN
2-MG
þN
FA
Þ=NT
AGþFA
ð21Þ
As can be obse ed in Table 4, he H
L
% eached wi h he s a ing
me hod is e y low, eleasing less han 20% o acyl g oups. How-
e e , he p opo ion o iglyce ide ans o med in o DG, MG o
Gol (T
TG
%) was app oxima ely 30%. Finally, he L
BA
% eached when
using he s a ing me hod was close o 20%. The small di e ence
be ween H
L
% and L
BA
% indica es ha he mola pe cen age o AG
suppo ed on MG was much smalle han ha o FA (see Table 3).
H
L
% coincides exac ly wi h he FA% gi en in Table 3 and ha
T
TG
% is exac ly (100-TG%) o (100-AG
TG
%), also gi en in Table 3.
These wo pa ame e s a e included in he abo e-de ined comple e
molecula pic u e o lipolysis (CMP
L
), p o iding a de ailed iew o
he molecula composi ion o he diges a e.
3.3. S udy o he in luence o se e al ac o s on he ex en o in i o
lipolysis
3.3.1. E ec o gas ic pH acidi ica ion
Following he diges ion model desc ibed abo e, he addi ion o
12 ml o gas ic juice (GJ) o 9 g o minced sea bass led o an ini ial
gas ic pH o 4.7 ± 0.2 and 30 min la e an acidi ica ion o he
chyme o 2.5 ± 0.5, wi h HCl (37%), was ca ied ou . Howe e ,
in i o, gas ic pH was e y low in basal condi ions (1.8–1.9) and
a e 30 min o ood inges ion i ma kedly inc eased up o 6.1,
mainly due o he bu e capaci y o die a y p o eins. Then, a e
3 h i p og essi ely e u ned o basal alues (A mand e al., 1999).
Wi h his in mind, in he cu en s udy he acidi ica ion o he
chyme o 2.5 ± 0.5 was pe o med a e 60 min o he addi ion o
GJ, ins ead o 30 min, wi h he aim o mimicking human condi-
ions. This change did no cause any signi ican di e ence o he
ex en o lipolysis (Tables 3 and 4). Ne e heless, as i is close o
physiological condi ions, his modi ica ion was main ained in he
subsequen diges ion expe imen s.
Simila esul s we e ob ained when apeseed oil in wa e emul-
sion was in i o diges ed and no signi ican di e ence ela ing o
o al lipolysis was obse ed when gas ic pH was main ained
ei he a 2.5 o 4.0 (Kenmogne-Domguia e al., 2012). The limi ed
impac o gas ic pH was a ibu ed o he lack o gas ic lipase.
3.3.2. E ec o in es inal ansi ime
The s a ing me hod p oposes an in es inal esidence ime o
2 h; howe e , in i o i is o he o de o 3 h, anging om 1 o 6
(Ve san oo e al., 2004). Mo eo e , when in i o diges ion mod-
els a e used o ma ine oods, longe in es inal esidence imes a e
employed, usually up o 4 h (Hu e al., 2011). In line wi h his, an
inc ease o in es inal esidence ime o 4 h was es ed in o de o
ensu e a g ea e deg ee o lipolysis.
As can be obse ed in Table 3, a sligh inc ease (p> 0.05) in he
ex en o lipid in i o diges ion was ob ained. As o mola pe -
cen ages in ela ion o he o al numbe glyce yl s uc u es p e-
sen , app oxima ely 6% mo e TG we e hyd olyzed mainly
yielding MG and Gol, he eby eleasing po en ially abso bable lipid
s uc u es (MG, FA). I is also no iceable ha he inc ease o in es i-
nal esidence ime a ou s he isome iza ion o 2-MG o 1-MG.
Indeed, 2-MG a e e y uns able in aqueous solu ion and a alkaline
pH (Desnuelle & Sa a y, 1963), as hey a e a he in es inal s ep.
Simila obse a ions can be deduced by compa ing mola pe cen -
ages in ela ion o he o al numbe o AG + FA. Al hough he pe -
cen age o non-abso bable AG (AG
TG
%+AG
1,2-DG
%) was sligh ly
educed, i s ill emained e y high because app oxima ely 75%
o ini ial AG we e no bioaccessible. Compa ing he alues o H
L
%,
T
TG
% and L
BA
% be o e and a e inc easing in es inal esidence ime
(Table 4), a simila inc ease is obse ed, indica ing ha a longe
esidence ime mainly led o he hyd olysis o TG in o MG, Gol
and FA. Mo eo e , he small di e ence be ween H
L
% and L
BA
% al-
ues shows ha AG
MG
% was much lowe han FA%. These deduc-
ions can also be di ec ly deduced om he CMP
L
(Table 3).
Conside ing all o he abo e, a 4 h-in es inal ansi ime was pe -
o med in he subsequen diges ions expe imen s.
3.3.3. E ec o he addi ion o lipase o he gas ic juice (GJ)
The model de eloped by Ve san oo e al. (2004, 2005)
ocuses on in es inal lipolysis and does no include any lipase in
he gas ic s ep. Ne e heless, se e al au ho s epo ed a no able
con ibu ion o gas ic lipase o he ex en o lipolysis in heal hy
adul s. These au ho s indica e ha al hough panc ea ic lipase is
mainly esponsible o a diges ion, lipolysis can s a in he s om-
ach ca alyzed by an acid-s able gas ic lipase, which hyd olyzes 5–
35% o TG (A mand e al., 1999; Ca ie e, Ba owman, Ve ge , &
Laugie , 1993; Pa umi e al., 2002). This pa ial hyd olysis is
belie ed o be o pa amoun impo ance o igge ing he subse-
quen diges ion in he small in es ine, because gas ic lipase p o-
mo es he p e-emulsi ica ion o lipids by al e ing he in e acial
composi ion o lipid d ople s h ough newly o med p oduc s,
mainly DG and FA. Ta ainen e al. (2012), ollowing he model
p oposed by Ve san oo e al. (2004, 2005), simula ed his p e-
emulsi ica ion e ec by adding s anda d compounds, such as FA
and DG, o he samples p io o diges ion.
In o de o e alua e he e ec o gas ic lipase, diges ion expe -
imen s we e ca ied ou simul aneously using GJ wi hou and wi h
lipase. Taking in o accoun ha human gas ic lipase (HGL)
emains ac i e in he duodenum, whe e i s ill shows a conside -
able hyd oly ic ac i i y (Ca ie e e al., 1993), he pe o mance o
gas ic lipase on ish lipolysis was e alua ed a he end o he
whole diges ion. Based on p e ious s udies (Roman e al., 2012;
an Aken, Bomho , Zoe , Ve beek, & Oos e eld, 2011), lipase om
Aspe gillus nige was selec ed o simula e HGL, because his ungal
Table 4
Di e en pa ame e s desc ibing he lipolysis ex en unde gone in he in i o diges ion o sea bass mea unde di e en expe imen al condi ions. Di e en le e s wi hin each
column indica e a signi ican di e ence (a p< 0.05).
Fac o s H
L
%T
TG
%L
BA
%
S a ing poin (Sample DV) (Ve san oo condi ions) 16.5 ± 1.9a 30.8 ± 3.3a 19.0 ± 2.7a
Delay o pH shi ing in he gas ic s ep (60 min) 16.5 ± 1.5a 31.0 ± 2.6a 18.9 ± 1.9a
Inc ease o in es inal esidence ime (4 h) 21.2 ± 2.2a 37.0 ± 3.1a 24.3 ± 2.5a
Addi ion o lipase in he GJ (100 U/ml o A. nige lipase) 30.7 ± 3.4b 51.1 ± 4.3b 35.4 ± 3.9b
Dec ease o a io ood sample/diges i e luids olume (4.5 g ood: 6 ml S: 12 ml GJ: 12 ml DJ: 6 ml BJ) 44.5 ± 3.0c 60.4 ± 5.3bc 48.3 ± 3.3c
Inc ease o lipase concen a ion in he DJ (DJ2: 9 g/l o panc ea in and 9.6 g/l lipase) 44.2 ± 1.5c 60.1 ± 3.4c 47.6 ± 2.5c
Dec ease o bile concen a ion in he BJ (Sample DO) (18.75 g/l) 70.5 ± 4.8d 95.4 ± 2.1d 80.1 ± 5.6d
Abb e ia ions: H
L
: Hyd olysis le el; T
TG
: T iglyce ide ans o ma ion; L
BA
: Lipid bioaccessibili y; DV: diges ed ish lipid ex ac s ollowing Ve san oo condi ions; S: sali a,
GJ: gas ic juice; DJ: duodenal juice; BJ: bile juice; DO: diges ed ish lipid ex ac s ollowing he op imized condi ions p oposed in his s udy.
22 B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26
lipase shows a simila egiospeci ici y, a wide op imum pH ange
(2.5–5.5) and esis ance agains p o eases.
As shown by he CMP
L
(Table 3), he addi ion o 100 U/ml o A.
nige lipase o he GJ p o okes no iceably inc eased lipolysis,
dec easing TG% signi ican ly om 63.0 o 48.9%, in ag eemen wi h
in i o hyd oly ic e iciency a ibu ed o HGL (5–35% o TG). Con-
sequen ly, 1,2-DG%, 2-MG% and Gol% inc eased in a simila p opo -
ion. I can also be obse ed in Table 3 ha only one hi d o AG
+ FA p esen we e po en ially abso bable a e his diges ion expe -
imen (see AG
2-MG
%, AG
1-MG
% and FA%). The pa ame e s employed
o desc ibe lipolysis (Table 4) also show a signi ican inc ease in
ela ion o he expe imen s ca ied ou in he absence o gas ic
lipase. This inc ease is highe in T
TG
% han in H
L
% and L
BA
%, sug-
ges ing again ha he hyd olysis o he es e bond occu ed o a
g ea e ex en in TG han in pa ial glyce ides. Thus, like panc ea ic
lipase, his ungal lipase p e e en ially a acks he es e bonds o
TG a he han hose o pa ial glyce ides. Mo eo e , i should be
aken in o accoun ha TG a e he main glyce yl s uc u es p esen
and hus migh ha e he bes chance o in e ac ing wi h lipase.
Since he in i o gas oduodenal lipolysis he e pe o med by
he GJ con aining lipase, occu ed o a lesse ex en (FA% om
21.2 o 30.7, see Table 3) han ha o an Aken e al. (2011),
who epo ed 30% o FA% a e in i o gas ic diges ion o se e al
ood emulsions, a highe concen a ion o A. nige lipase (200 U/
ml) was also assayed. Howe e , no signi ican di e ences we e
obse ed (da a no shown). This limi ed inc ease in he ex en o
lipolysis ega dless o he amoun o gas ic lipase used could be
explained by hind ance caused by p o ona ed long chain FA accu-
mula ed a he su ace o lipid d ople s, which could inhibi u he
lipolysis (Pa umi e al., 2002).
Mo eo e , assays wi h unspeci ic Candida ugosa lipase we e
also pe o med, bu wi h less success ul esul s (da a no shown).
Thus, a gas ic juice ha included A. nige lipase a 100 U/ml was
used o he ollowing diges ion expe imen s.
3.3.4. E ec o ood/diges i e luids a io
In he s a ing me hod, a p opo ion o 9 g ( ood): 6 ml (sali a):
12 ml (GJ): 12 ml (DJ): 6 ml (BJ) is p oposed. Ne e heless, lowe
ood/diges i e luids a ios ha e been employed in la e s udies
o di e en ood ma ixes (Hu e al., 2009; Kop -Bolanz e al.,
2012; Lamo he e al., 2012; Ma ze e al., 2013).
Taking all he abo e conside a ions in o accoun , in i o diges-
ion expe imen s we e pe o med employing he condi ions
selec ed in he p e ious sec ion, and using 9 g and 4.5 g o minced
ish. Table 3 shows he CMP
L
a e diges ion in bo h cases. The
educ ion o he sample amoun p o oked a signi ican dec ease
in TG% (10%). Likewise, 1,2-DG%, 2-MG% and 1-MG% we e
educed due o comple e hyd olysis o glyce ides; in ac , i is
wo h no ing he inc ease (p< 0.05) o Gol% and FA%; howe e ,
app oxima ely hal o he AG + FA p esen emained as non-
abso bable (AG
TG
%+AG
1,2-DG
%). These obse a ions a e in ag ee-
men wi h he pa ame e s o Table 4;H
L
% and L
BA
% unde wen a
highe inc ease han T
TG
% did, which sugges s ha he use o
4.5 g o sample led o g ea e hyd olysis in DG and MG han when
9 g we e used. Simila esul s we e also obse ed by Li e al. (2011)
using he pH-s a i a ion me hod. These au ho s submi ed h ee
di e en amoun s o a co n oil emulsion o in i o diges ion and
epo ed he lowes a e and ex en o lipid hyd olysis when
diges ing he highes amoun . Hence, he lowes ood/diges i e lu-
ids a io assayed was employed in he subsequen expe imen s.
3.3.5. E ec o he enzyma ic composi ion o he duodenal juice (DJ)
Duodenal juice is a complex mix u e in which a leas h ee
di e en ypes o panc ea ic lipase enzymes a e sec e ed,
colipase-dependen lipase being he one mainly esponsible o a
diges ion. Ca boxyl es e hyd olase (bile sal s imula ed lipase) and
phospholipase A2 a e conside ed mino componen s o panc ea ic
juice (Reis, Holmbe g, Wa zke, Lese , & Mille , 2009). Mo eo e ,
wide composi ional a ia ions can be obse ed due o he amoun
and ype o ood inges ed, he indi idual cha ac e is ics and he
ime o day (Cla ysse e al., 2009; Kalan zi e al., 2006). Despi e his
a iable composi ion, he concen a ion o panc ea ic lipases
in i o is epo ed o always be in la ge excess o e subs a e,
ensu ing a comple e lipid diges ion (Reis e al., 2009).
Fo in i o s udies, di e en ypes o DJ ha e been p oposed.
These a y widely in ionic composi ion and ype and amoun o
enzymes added (Kop -Bolanz e al., 2012; Li e al., 2011;
Ve san oo e al., 2004). Due o di icul ies in ob aining human
enzymes, in mos s udies pu i ied comme cial enzymes om
mammal panc eas o ungi a e used. The DJ p oposed by
Ve san oo e al. (2004, 2005) includes po cine panc ea in
ex ac , oge he wi h addi ional po cine lipase o coun e ac he
lowe ac i i y o lipase wi h espec o amylase in he comme cial
ex ac compa ed o ha epo ed in i o.
To in es iga e he e ec o he na u e and concen a ion o he
enzymes o DJ on lipid diges ion, ou in i o diges ion expe i-
men s o 4.5 g o minced ish we e ca ied ou in pa allel, employ-
ing ei he he DJ de ailed in Table 1 (DJ1) o 3 di e en app oaches
(DJ2-4). These di e only in he con en o panc ea in and po cine
panc ea ic lipase. Thus, DJ2 con ains 9 g/l o panc ea in and 9.6 g/l
o lipase, based on Li e al. (2011),DJ3 includes 18 g/l o panc ea in
bu no addi ional lipase, acco ding o Kop -Bolanz e al. (2012), and
DJ4, 18 g/l and 9.6 g/l o panc ea in and lipase espec i ely.
Fig. 2a shows he mola pe cen ages o he glyce yl s uc u es
p esen in he diges a es a e he use o DJ1-4. Quan i a i e da a
epo ed on Tables 3 and 4 co espond o he diges a es ob ained
using DJ2. Thus, he inc ease in he concen a ion o duodenal
enzymes did no lead o any signi ican inc ease o TG hyd olysis
du ing in i o diges ion and he pe cen ages o hyd oly ic p oduc s
ob ained using DJ2-4 we e e y simila o hose ob ained using
DJ1. This limi ed impac on ex en o lipolysis could be e idence
ha he amoun and ype o enzymes p esen in DJ1 a e al eady
in excess o e subs a e. Simila esul s we e ob ained by Li e al.
(2011) when examining he e ec o lipase concen a ion (0–
4.8 g/l) on he o al amoun o FA eleased du ing he in i o diges-
ion o a co n oil emulsion. The ini ial a e o lipolysis clea ly
inc eased as he amoun o lipase inc eased, bu in e ms o he
inal amoun o FA eleased, ha is he ex en o lipid diges ion,
simila esul s we e ob ained o lipase concen a ions be ween
0.4 and 2.4 g/l. Since he enzyma ic composi ion o DJ is no a
key ac o limi ing he hyd olysis unde he condi ions o his
s udy, no modi ica ion o duodenal juice composi ion was included
in he la e expe imen s and ini ial DJ1 was con inued o be
employed.
3.3.6. E ec o he bile concen a ion in he bile juice (BJ)
Bile sal s a e na u al biosu ac an s p esen in he gu lumen
ha play an impo an ole in lipolysis by egula ing he composi-
ion o lipid-wa e in e ace, whe e hyd olysis occu s (Golding &
Woos e , 2010). Depending on bile concen a ion in ela ion o
he c i ical micelle concen a ion, panc ea ic lipase ac i i y is
enhanced o inhibi ed (Bo gs öm & E lanson, 1973). On he one
hand, bile sal s solubilize newly o med lipoly ic p oduc s in o
bile-phospholipid mixed micelles, hus emo ing hem om he
in e ace oge he wi h o he su ac an s (p o eins, phospholipids)
ha may also be p esen in he adso p ion laye , displacing bound
lipases. On he o he hand, he highly su ac an na u e o bile sal s
can es ic lipase adso p ion a he in e ace, limi ing a diges ion
by s e ical hind ance. This inhibi o y e ec can be elie ed by he
p esence o colipase, a non-enzyma ic p o ein co ac o ha
helps lipase o ancho a he in e ace and s abilizes lipase
B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26 23
con o ma ional a angemen , allowing lipolysis o con inue
(Bläckbe g e al., 1979; Reis e al., 2009).
A bile concen a ion o 30 g/l is employed in he s a ing model
(Table 1); his co esponds o 8 mM in he chyme, which ep o-
duces he biochemical en i onmen o he small in es ine du ing
he ed s a e (Ve san oo e al., 2004). Howe e , he amoun o
bile p esen in he small in es ine luc ua es o e diges ion ime,
and his alue is also in luenced by ood composi ion and by indi-
idual cha ac e is ics; in human pos pand ial aspi a es alues
anging om 5 o 15 mM o bile ha e been obse ed (Cla ysse
e al., 2009; Kalan zi e al., 2006).
Bea ing in mind he abo e men ioned, he impac o di e en
bile concen a ions (0, 7.5, 15.0, 18.75, 30.0 and 60.0 g/l in he BJ)
on he ex en o in i o lipolysis was also in es iga ed. Fig. 2b
shows he mola pe cen ages o he glyce yl s uc u es p esen in
he diges a es ob ained. Quan i a i e da a epo ed in Table 3
and 4 co espond o he diges a es ob ained using a bile concen a-
ion o 18.75 g/l. As can be obse ed in he igu e, he use o low
bile concen a ions (0–18.75 g/l) led o signi ican ly lowe TG%
han he use o high ones (30, 60 g/l). This dec ease o TG yielded
mainly 2-MG and Gol, indica ing ha lowe bile concen a ion
g ea ly a ou ed he hyd olysis no only in TG, bu also in pa ial
glyce ides, especially in DG. This can also be in e ed om he
CMP
L
(Table 3) and he h ee pa ame e s shown in Table 4.
The lesse ex en o lipolysis obse ed a high bile concen a-
ions is in ag eemen wi h p e ious s udies (Bo gs öm &
E lanson, 1973; Li e al., 2011). These esul s con i m he key ole
o bile sal s in con olling lipase ac i i y. I should be poin ed ou
ha his clea e ec o bile concen a ion on lipolysis can be
enhanced by he ac ha as i is a s a ic model, he diges ion p od-
uc s gene a ed a e no emo ed om he media, in con as o
wha occu s in i o. When using bile a 30 and 60 g/l, c i ical micel-
la concen a ion could be eached ha would limi he hyd olysis
eac ion d ama ically because, al hough su ac an s a e emo ed
om he in e ace, i is domina ed by bile molecules. Vina o
e al., (2012a, 2012b) p oposed a de ailed inhibi ion mechanism
o panc ea ic lipase and e ealed he oles played by emulsi ie s
and bile. Fu he mo e, he DJ used in his s udy lacks addi ional
colipase ( o ha con ained in panc ea in), which could explain in
pa he majo impac o bile on he lipolysis (Bläckbe g e al.,
1979). A high bile concen a ions, he absence o su icien coli-
pase mean ha he elec os a ic epulsion o lipases a bile sal
co e ed in e aces could no be o e come, g ea ly in luencing he
hyd olysis eac ion du ing he in es inal s ep.
Rega ding he low bile concen a ions es ed, Fig. 2b shows ha
he use o bile a 0 and 7.5 g/l led o highe TG% and lowe MG%
han a e using 15 and 18.75 g/l, al hough di e ences a e no s a-
is ically signi ican . In ac , due o a wo se dispe sion o lipid d o-
ple s in he absence o bile, a smalle su ace a ea would be
a ailable o lipase ac i i y a he in e ace (Bo gs öm &
E lanson, 1973). Mo eo e , wi hou a bile dispe sing e ec , he
p esence o su ace ac i e molecules gene a ed by lipases and p o-
eases a he in e ace could hinde lipase-subs a e di ec con ac
(Mun, Decke , & McClemen s, 2007).
Taking in o accoun he esul s ob ained, high bile concen a-
ions migh explain he low in i o lipolysis a es p e iously
Fig. 2a. Mola pe cen ages o he di e en glyce yl s uc u es p esen in he lipid ex ac s o in i o diges ed sea bass when a ying he na u e and concen a ion o enzymes
in he duodenal juice (DJ1: 9 g/l o panc ea in and 1.5 g/l o lipase; DJ2: 9 g/l o panc ea in and 9.6 g/l lipase; DJ3: 18 g/l o panc ea in; DJ4: 18 g/l panc ea in and 9.6 g/l
lipase). Di e en le e s wi hin each lipoly ic p oduc ep esen a signi ican di e ence (p< 0.05).
Fig. 2b. Mola pe cen ages o he di e en glyce yl s uc u es p esen in he lipid ex ac s o in i o diges ed sea bass when a ying bile concen a ion in he bile juice (0, 7.5,
15, 18.75, 30 and 60 g/l). Di e en le e s wi hin each lipoly ic p oduc ep esen a signi ican di e ence (p< 0.05).
24 B. Nie a-Eche a ía e al. / Food Chemis y 211 (2016) 17–26
epo ed (Kop -Bolanz e al., 2012; Ma ze e al., 2013). By con as ,
an in i o diges ion s udy on oil-in-wa e emulsions obse ed ha
by inc easing bile concen a ion, he lipolysis a e is also inc eased
(Mun e al., 2007). As o he imp o emen o he ex en o ish
lipolysis, a bile concen a ion o 18.75 g/l in he BJ (5 mM in he
chyme acco ding o Ve san oo e al. (2004)) would be sui able.
E en i his concen a ion s ill emains lowe han ha used in
in i o p o ocols simula ing ed s a e, i can be conside ed wi hin
he physiological ange, since his concen a ion was epo ed in
some in i o duodenal aspi a es (Cla ysse e al., 2009; Lindahl,
Ungell, Knu son, & Lenne näs, 1997).
3.4. P oposed condi ions o a highe in i o ex en o lipolysis
Conside ing all he abo e s udies, an op imized in i o diges-
ion me hod ha allows one o ob ain a TG hyd olysis le el close
o ha occu ing in i o, can be p oposed o ish lipid diges ion.
The modi ica ions o ca y ou ega ding he s a ing me hod
include: delaying gas ic pH acidi ica ion ( om 30 o 60 min),
inc easing in es inal ansi ime ( om 2 o 4 h), adding A. nige
lipase o he gas ic juice (100 U/ml), dec easing ood/diges i e lu-
ids a io (using 4.5 ins ead o 9 g o ood), and dec easing bile con-
cen a ion ( om 30 o 18.75 g/l).
The imp o emen s made can be obse ed in he lowe pa o
Fig. 1, by compa ing he spec a o diges ed ish lipid ex ac s ol-
lowing he s a ing p o ocol (DV) and he op imized one p oposed
(DO). Ma ked di e ences can be obse ed, especially in he in en-
si y o speci ic signals o TG (O,S), almos impe cep ible in DO,
which indica es ha hey ha e unde gone ex ensi e hyd olysis.
Likewise, he highe in ensi ies o signals ela ed o 1-MG (L,I),
2-MG (K,Q) and 1,2-DG (J,P,R) a e u he e idence o ad anced
lipolysis in DO. Di e ences in he chemical shi and mul iplici y o
signals D1,D2,F1 and F2 a e also no iceable; in he DO spec um
highe chemical shi s (1.63, 1.71, 2.34 and 2.41 ppm) can be
obse ed han in he DV spec um (1.61, 1.69, 2.30 and
2.38 ppm). In ac , as he hyd olysis ad ances, signals D1,D2,F1
and F2 co esponding o TG g adually dec ease whe eas hose co -
esponding o he same kinds o p o ons suppo ed on 1,2-DG, 2-
MG, 1-MG and FA appea cen e ed a highe chemical shi s
(Table 2).
This quali a i e in o ma ion ag ees wi h he quan i a i e da a
epo ed. As shown by he CMP
L
(Table 3), TG% in sample DO is
e y low (4.6 ± 2.1%), indica ing ha app oxima ely 95% o TG ini-
ially p esen we e hyd olyzed, close o in i o pe o mance. No
signi ican modi ica ion o 1,2-DG% was no iced in ela ion o he
s a ing me hod, whe eas he occu ence o po en ially abso bable
glyce ides (2-MG, 1-MG), inc eased signi ican ly. Rega ding his
la e , i should be no ed ha 1-MG% emained almos cons an
in he p e ious expe imen s; he e o e, i s inc ease when using
he new condi ions migh be mainly explained by he highe occu -
ence o 2-MG (23.2 ± 1.9%). Mo eo e , app oxima ely 44% o he
ini ial TG we e comple ely hyd olyzed in sample DO (see Gol%),
in ag eemen wi h le els epo ed in i o (Bo gs öm, T yding, &
Wes öö, 1957). Hence, he p esen s udy highligh s ha he TG
hyd olysis in o Gol and 3 FA is no only es ic ed o in i o condi-
ions and canno be dismissed in in i o s udies, as i has been p e-
iously (Lamo he e al., 2012; Li e al., 2011). Rega ding mola
pe cen ages exp essed in ela ion o he o al numbe o FA + AG,
mo e han wo hi ds o he o al AG ini ially p esen we e eleased
as FA (70.5 ± 4.8%), which we e he main o m o abso bable lipids,
conside ing he alues ob ained o AG
2-MG
% and AG
1-MG
%.
The pa ame e s shown in Table 4 also e lec he abo e-
commen ed in Table 3. Thus, H
L
%, T
TG
% and L
BA
% in sample DO
a e h ee- o ou old highe han ha eached wi h he s a ing
model (DV), highligh ing he signi ican imp o emen o ish lipid
diges ion caused by a ying, wi hin a physiological ange, he
abo e-men ioned expe imen al ac o s. The di e ences obse ed
be ween T
TG
% and L
BA
%(15%), and L
BA
% and H
L
%(10%), sugges
ha sample DO con ains simila p opo ions o DG and MG, as
shown by CMP
L
(see 1,2-DG%, 2-MG% and 1-MG% in Table 3).
4. Conclusions
The p esen s udy p o ides u he knowledge on he impac o
expe imen al ac o s on lipid in i o diges ion, which is o pa a-
moun impo ance in gaining a be e insigh in o he limi a ions
o he p o ocols cu en ly employed in in i o s udies, and hus
unde s anding he esul s ob ained. The addi ion o gas ic lipase,
he dec ease o ood/diges i e luids a io and he dec ease o bile
concen a ion signi ican ly imp o ed lipolysis. Howe e , o all he
expe imen al a iables s udied, he bile concen a ion was ound
o be he key ac o o con olling in i o lipolysis in he s a ic
model employed. Wi h he modi ica ions p oposed, app oxima ely
95% o iglyce ides unde wen hyd olysis and 80% o ish lipids
acyl g oups we e bioaccessible. Since an accu a e ma ch o na u-
ally occu ing e en s is necessa y o consis en s a emen s and
p edic ions, he op imiza ion o in i o diges ion expe imen al
condi ions should be ca ied ou o each kind o sample when
equi ed, especially in bioaccessibili y and bioa ailabili y s udies.
1
H NMR was e idenced o be a sound echnique in he lipid diges-
ion ield, p o iding a holis ic molecula iew o lipolysis, gi ing a
g ea deal o in o ma ion in a as single- un and wi hou any
chemical modi ica ion o he sample. To he bes o he au ho s’
knowledge, his is he i s ime ha he me hodology p oposed
o he e alua ion o he in luence o ce ain ac o s on he lipolysis
ad ance is able o p o ide in o ma ion on he speci ic es e bond
a ec ed by each ac o in each case.
Acknowledgemen s
This wo k has been suppo ed by he Spanish Minis y o
Economy and Compe i i eness (MINECO, AGL2012-36466,
AGL2015-65450-R), by he Basque Go e nmen (EJ-GV, GIC10/
85-IT-463-10) and by he Uni o Educa ion and Resea ch ‘‘Food
Quali y and Sa e y” (UPV/EHU-UFI-11/21). All au ho s pa icipa e
in he COST Ac ion FA1005-INFOGEST. B.N-E hanks UPV/EHU o
a p edoc o al con ac .
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