Intestinal digestion of fish oils and ω-3 concentrates under in vitro conditions

Research Article

Intestinal digestion of fish oils and v-3 concentrates underin vitro conditions

Diana Martin, Juan A. Nieto-Fuentes, Francisco J. Senorans, Guillermo Reglero and

Cristina Soler-Rivas

Facultad de Ciencias, Seccion Departamental de Ciencias de la Alimentacion, Universidad Autonoma de

Madrid, Cantoblanco, Madrid, Spain

A comparative study of the in vitro bioaccesibility of v-3-oils (salmon oil, SO; tuna oil, TO; enriched-v-3

oil as triacylglycerols (TAGs), v-3-TAG; and enriched-v-3 oil as ethyl esters (EEs), v-3-EE) was

performed after treatment with pancreatin (pancreatic lipase as major lipolytic enzyme) at pH 7.5.

Aliquots were taken at different times of digestion for analyzing the evolution of lipid products. The

micellar phase (MP) formed at 120 min of digestion was isolated, its total lipid content was extracted and

its composition in lipid products was analyzed. The rate of hydrolysis of v-3-TAG concentrates was

continuous throughout the time of reaction (51% hydrolysis of TAGs at 120 min), whereas the digestion

of SO andTOwas initially faster but stopped after 10 min of reaction (35 and 38%hydrolysis of TAGs at

120 min of SO and TO, respectively). A poor hydrolysis of EEs took place for the v-3-EE oil (around 7%

hydrolysis of EEs at 120 min). TheMP of v-3-TAG oil, SO, and TOmainly consisted of free fatty acids

(FFAs) and MAGs. The MP from digested v-3-EE oil consisted of FFAs and undigested EEs.

Therefore, the highest degree of hydrolysis and inclusion of lipid products in the micellar structure

was found for the v-3-TAG oil, but compared to fish oils long times of digestion were required. This

experience also shows for the first time the MP composition from v-3-concentrates in the form of EEs.

Practical applications:Commercial v-3 sources can be found as purified fish oil or concentrates in the

form of TAGs, FFAs, and EEs. Despite differences exist regarding their intestinal metabolism, there is

lack of information about the specific composition in lipolytic products of the absorbable fraction (MP)

from v-3-TAG or v-3-EE concentrates. This comparative study showed that (i) the in vitro bioacce-

sibility of v-3-polyunsaturated fatty acid (PUFA) seems to be better as v-3-TAG concentrates than

purified fish oils, but after long times of digestion; and (ii) the in vitro hydrolysis of v-3-PUFA as EEs

seems to be poor, at least after the activity of the major lipolytic enzyme of pancreatin, namely pancreatic

lipase. Furthermore, the inclusion of EEs within micellar structures seems to be limited. These results

contribute to the knowledge of the intestinal lipolysis of v-3 sources by showing the composition of the

MP on lipid products for the first time.

Keywords: Digestion / Fish oil / Mixed micelles / Omega-3 / Pancreatic lipase

Received: April 26, 2010 / Revised: July 29, 2010 / Accepted: September 20, 2010

DOI: 10.1002/ejlt.201000329

1 Introduction

Recommended consumption of fatty acids of the v-3 family,

mainly eicosapentaenoic acid (EPA) and docosahexaenoic

acid (DHA) were advised years ago [1] due to their beneficial

role as anti-thrombotic, anti-inflamatory, and hypolipidemic

fatty acids. Despite the fact that fish is the main source of

these fatty acids, general consumption of fish is quite low for

reaching the minimal intake level of EPA and DHA. An easy

way of increasing long v-3-polyunsaturated fatty acids

(PUFA) intake is by the fish oils supplements oils now

Correspondence: Diana Martin, Facultad de Ciencias, Seccion

Departamental de Ciencias de la Alimentacion, Universidad Autonoma de

Madrid, 28049 Cantoblanco, Madrid, Spain

E-mail: [email protected]

Fax: þ34-914978255

Abbreviations: DAG, diacylglycerol; DHA, docosahexaenoic acid; EE,

ethyl ester; EPA, eicosapentaenoic acid; FFA, free fatty acid; HPLC, high-

performance liquid chromatography; MAG, monoacylglycerol; MP,

micellar phase; OP, oily phase; PUFA, polyunsaturated fatty acid; SO,

salmon oil; TAG, triacylglycerols; TO, tuna oil

Eur. J. Lipid Sci. Technol. 2010, 112, 1315–1322 1315

� 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

available. These sources can be found both as purified fish oil

and as concentrates in the form of triacylglycerols (TAGs),

free fatty acids (FFAs), and ethyl esters (EEs).

The fish oil-derived concentrates have the advantage of

containing a higher level of EPA and DHA than fish oil.

However, diverse studies have evidenced that the metabolism

of fish oils and v-3-concentrates fish oils under the different

structural forms, including both digestion, absorption, and

transport steps, is different and contradictory. Nordoy et al.

[2] reported a similar absorption of v-3 fatty acids in fish oil

given as EEs or TAGs. On the contrary, Yang et al. [3] found

a slower hydrolysis by pancreatic lipase of fish oil as EEs

compared to the same oil as TAGs. Regarding digestion,

differences have been attributed to the location of the double

bonds, but not to the chain length and degree of unsaturation

[3]. Moreover, the location of fatty acids in the glycerol

moiety has also a role in the digestion process. Thus, low

hydrolysis rate by pancreatic lipase for EPA andDHA located

at sn-1 and sn-3 positions of fish oil TAG have been stated,

but not definitively evidenced [4].

After degradation by pancreatic lipase, intestinal lipid

digestion requires the solubilization of the hydrolysis prod-

ucts in mixed bile salt–lipid micelles [5]. The presence of

lipids in the duodenum stimulates the secretion of bile salts,

phosphatidylcholine, and cholesterol from the gall bladder

and pancreatic fluids (containing pancreatic lipase/co-lipase,

etc.) from the pancreas. Lipases binds at the surface of the oil

droplets formed by the gall bladder emulsifiers hydrolysing

lipids into their digestion products generating a series of

colloidal species including micelles, mixed micelles, vesicles,

and emulsion droplets, named as micellar phase (MP) from

now on. Such hydrolysis products included in the MP are

derived firstly from degradation of TAGs, which yields a FFA

and a diacylglycerol (DAG) and secondly DAG is degraded

to another FFA and a monoacylglycerol (MAG). Mixed

micelles containing digestion products (FFAs and MAG)

is the common way of absorption of lipids by enterocites for

subsequent resynthesis to TAGs by the 2-MAG pathway [6].

Despite the diverse studies that have reported the con-

troversial about digestion, absorption, and transport of v-3

concentrates under different forms, the study of the lipid

content and composition of the MP formed during luminal

metabolism of these lipids is scarce. Several studies have been

carried out regarding composition of the whole digestion

medium by in vitro assays [3, 7–9] but most of them did

not deal with the potentially absorbable fraction, whichmight

be the hydrolysis products included in the MP.

In the present study, the intestinal digestion of differentv-

3-rich oils (EPA- and DHA-rich oils) was carried out under

in vitro conditions in order to study the change in the gener-

ation of lipid products during the reaction and to isolate the

MP formed at the end of the process. The total hydrolytic

products included in the MP as well as the composition of

such MP in hydrolytic products were also analyzed. In order

to cover diverse parameters related to the oil source that

might influence the digestion process, the assay included fish

oils with different EPA/DHA ratio (tuna oil (TO) as a rich

DHA fish oil and salmon oil (SO) as a rich EPA fish oil), as

well as fish oil-derived concentrates, both in the form of TAG

and in the form of EE.

2 Materials and methods

2.1 Reagents and materials

Sodium sulfate anhydrous, Trizma-maleate, NaCl, CaCl2,

pancreatin, bile salts, and phosphatidyl choline from hen egg

were purchased from Sigma–Aldrich Chemie GmbH

(Steinheim, Germany). Sodium hydroxide, hydrochloric acid

from Panreac (Barcelona, Spain). All solvents used were of

high-performance liquid chromatography (HPLC) grade

from Lab-Scan (Dublin, Ireland). The SO, TO, and the

v-3-concentrate oil as EE 60% DHA/20% EPA (v-3-EE)

were purchased from Jedwards International Inc. (Quincy,

MA; respectively). The v-3-concentrate oil as TAG 60%

DHA/20% EPA (v-3-TAG) was obtained by random inter-

esterification of the commercial v-3-EE oil according to

Haraldsson et al. [10]. The ‘‘v-3’’ term will refer to EPA

plus DHA fatty acids from now on. The lipid composition of

oils is shown in Table 1.

Table 1. Lipid composition of oils before in vitro intestinal digestion

(%)a)

SO TO v-3-TAG v-3-EE

TAGs 100.0 100.0 60.2 n.d.

DAGs n.d. n.d. 23.8 n.d.

MAGs n.d. n.d. 10.9 22.8

FFAs n.d. n.d. 5.2 n.d.

EEs n.d. n.d. n.d. 77.2

Fatty acid profile

C14:0 8.7 3.7 n.d. n.d.

C16:0 19.3 17.7 n.d. n.d.

C16:1 10.2 6.4 n.d. n.d.

C16:2 n.d. 2.4 n.d. n.d.

C16:3 n.d. 1.3 n.d. n.d.

C18:0 4.6 4.6 1.1 1.1

C18:1 14.5 19.1 1.3 1.3

C18:2 1.2 3.4 n.d. n.d.

C18:3 1.4 1.0 0.4 0.4

C20:1 3.9 4.5 1.0 1.0

C18:4 n.d. 0.5 n.d. n.d.

C20:4 n.d. 1.9 3.1 3.1

C22:1 n.d. 2.1 1.0 1.0

C20:5 18.4 8.2 23.9 23.9

C24:1 n.d. n.d. 4.2 4.2

C22:5 1.8 1.5 2.1 2.1

C22:6 10.9 21.7 62.0 62.0

n.d. – not detected.a) v-3 refers to C20:5 and C22:6 fatty acids.

1316 D. Martin et al. Eur. J. Lipid Sci. Technol. 2010, 112, 1315–1322


2.2 In vitro digestion

The in vitro digestion model was carried out following a

combination of methods proposed by Sek et al. [8] and

Christensen et al. [11]. Briefly, the sample (1 g) was mixed

with 54 mL of 50 mM Trizma-maleate buffer pH 7.5 in a

thermostatically controlled vessel (378C) under continuous

stirring. The pH was adjusted and maintained to 7.5 (with

1 M NaOH) using a viscotrode (Metrohm, Herisau,

Switzerland) electrode and a titrator device (Titrino plus

Metrohm 877, Switzerland). Simulation of intestinal diges-

tion was started by addition of 5 mMCaCl2, 150 mMNaCl,

and 6 mL of a pancreatin solution (1000 IUB/mL, 11.8 mM

bile salts, and 1.3 mM phosphatidyl choline in 50 mM

Trizma-maleate buffer pH 7.5). In vitro digestion of each

sample was performed in triplicate.

In order to study the evolution of lipid products through-

out the hydrolytic process, additional digestions of each

sample were performed following the same process, but

aliquots (700 mL) were collected at 0, 5, 10, 20, 30, 45,

60, 90, and 120 min of digestion for total lipids extraction.

2.3 Micellar phase isolation

After 120 min of in vitro digestion, the MP was isolated

according to Soler-Rivas et al. [12]. Digested samples were

submitted to centrifugation (4000 rpm 40 min 208C)

(5810R Eppendorf Iberica, Madrid, Spain) which separated

digested samples into a poorly emulsified oil phase, a highly

emulsified aqueous phase, and a precipitated pellet phase.

The oily phase (OP) typically contains undispersed oil. This

fractionmight correspond to the fraction that in vivo would be

wasted in faeces or transformed by colonic flora. The aqueous

phase or MP includes micellar and vesicular structures and

should correspond to the potentially bioaccesible fraction.

The pellet contains precipitated insoluble (calcium) soaps

of fatty acids liberated during the pancreatic digestion [13].

2.4 Lipid extraction

The isolated MP was mixed with 100 mL of hexane/methyl-

tert-butyl ether (50:50 v/v) in a separatory funnel. Mixture

was shaken vigorously during 5 min and allowed to separate

the two phases. Organic phase was collected and placed in a

separate vessel. Afterwards, sodium sulfate anhydrous (10 g)

was added and stirred for 30 min. In the mean time, a second

extraction was performed on the remaining aqueous phase in

the separatory funnel and treated as explained for the first

extraction. Organic phases obtained after both extractions

were pooled together and concentrated using a rotary evap-

orator at 308C.Concentrated extracts were dissolved in 5 mL

of methyl-tert-butyl ether, centrifuged (15 000 rpm 5 min),

and the supernatants were dried under nitrogen stream. The

total lipids extracted were estimated and stored at 48C until

further analysis.

The total lipids from aliquots taken at different times of

digestion were extracted by adding 1100 mL of hexane/

methyl-tert-butyl ether (50:50 v/v) in polypropylene tubes

of 2000 mL. Mixture was centrifuged for 5 min at

15 000 rpm. Organic phase containing separated lipids was

collected and placed in a separate tube. Afterwards, sodium

sulfate anhydrous was added and centrifuged for 5 min at

15 000 rpm. In the mean time, a second extraction was

performed on the remaining aqueous phase in the polypro-

pylene tube and treated as explained for the first extraction.

Organic phases obtained after both extractions were pooled

together and dried under nitrogen stream and stored at 48Cuntil further analysis.

2.5 High-performance liquid chromatographyanalysis

Hydrolysis products (TAGs, DAGs, MAGs, and FFAs) were

determined by HPLC on a Kromasil silica 60 column

(250 mm � 4.6 mm, Analisis Vinicos, Tomelloso, Spain)

coupled to a CTO 10A VP 2 oven, a LC-10AD VP pump,

a gradient module FCV-10AL VP, a DGU-14A degasser,

and an evaporative light scattering detector (ELSD-LT) from

Shimadzu (IZASA, Spain). The column temperature was

maintained at 358C. Twenty microliter of the diluted samples

(4 mg/mL) were injected. Details of the HPLC methodology

and the mobile phase utilized have been previously reported

[14]. Quantification was based on calibration curves per-

formed with appropriate standards (Sigma–Aldrich Chemie

GmbH).

2.6 Gas chromatography analysis

For the analysis of EEs, 1 mL of the sample diluted in hexane

was injected into an Agilent (Avondale, PA) GC (6890N

Network GC System) coupled to an autosampler (Agilent

7683B). The capillary column was a 30 mHP-88 (Avondale)

(0.25 mm i.d.). The temperatures of the injector and the FID

were 220 and 2508C, respectively. The temperature program

was as follows: starting at 1008C and then heating to 1808C at

208C/min; followed by heating from 180 to 2208C at 158C/

min. The final temperature (2208C) was held for 30 min.

Identification of the EEs was based on the retention times and

relative area percentages of a PUFA no 3 standard (#4-7085)

obtained from Supelco.

3 Results and discussion

3.1 Lipid products during in vitro intestinal digestionof v-3 oils

The evolution of intestinal digestion of SO, TO, v-3-TAG,

and v-3-EE was compared under in vitro conditions by

monitoring the change in the level of TAGs, DAGs,

MAGs and FFAs throughout 120 min (Fig. 1). Such reaction

Eur. J. Lipid Sci. Technol. 2010, 112, 1315–1322 Comparative study of the digestion of v-3-oils 1317


time was chosen for comparative vreasons and according to

physiological times [12, 15]. In the case of purified fish oils

(SO and TO), there was a decrease in the TAG level and a

parallel increase in DAG, MAG, and FFAs as lipolysis pro-

ceeded. These changes took place faster during the initial

minutes of hydrolysis (from 0 to 30 min for SO and to 20 min

for TO). After these times of digestion, the rate of TAG

hydrolysis kept constant and no further lipolysis of TAGs,

DAGs, or MAGs seemed to occur, neither in SO nor TO.

Thus, the remaining TAG at 120 min was around 64.6 g/

100 g of lipids for SO (35.4% of hydrolysis) and 61.6 g/100 g

of lipids for TO (38.4% of hydrolysis). Sek et al. [16] also

reported a decrease in the rate of TAGs hydrolysis after

10 min of in vitro digestion of long-chain TAGs and no

complete hydrolysis of TAGs after 30 min of digestion

monitoring. Similarly, Ikeda et al. [4] did not reach the

complete in vitro digestion of TAGs rich in EPA or DHA

at the second position after 60 min of reaction time, the

reaction keeping constant from 10 min of digestion. The

early decreased degradation of lipids might suggest that

hydrolysis of fish oils under in vitro conditions performed

seems to be so fast to saturate micellar solubilization of

lipolytic products, which might hinder the interaction of

anchored pancreatic enzymes and substrates, this phenom-

ena being slightly faster for TO compared to SO. This was in

accordance with the slightly higher levels of final lipid prod-

ucts (FFAs and MAGs) reached for TO compared to SO,

especially regarding to theMAGs ratio (7.3 g ofMAGs/100 g

of lipids for TO and 4.7 g of MAGs/100 g of lipids for SO).

The evolution of in vitro digestion of the v-3-TAG fish oil

concentrate was different from that observed for fish oils

(Fig. 1). The initial rate of hydrolysis of fish oils seemed to

be faster compared to v-3-TAG fish oil concentrate. Thus, at

30 min of digestion, there was 38.3% hydrolysis of TAGs for

SO, 38.2% hydrolysis of TAGs for TO, and 14.0% hydrolysis

of TAGs for v-3-TAG fish oil. The different results on

extension of hydrolysis of fish oils respect to v-3-TAG oil

might be related to the distribution of long-chain PUFA on

the glycerol moiety. It is known that PUFA containing double

bonds close to the carboxyl group are resistant to attack by

pancreatic lipase [9]. Taking into account that the major

lipolytic enzyme of the pancreatin, namely pancreatic lipase,

is a sn-1,3 specific enzyme, the higher the distribution of

resistant long-chain PUFA on the sn-1,3 location, the lower

0102030405060708090

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TAGs

FFA

DAGs

MAGs

EEs

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MAGs

EEs

Figure 1. Evolution of lipid products (TAGs, FFAs, DAGs, MAGs, and EEs) (g/100 g of lipids) during in vitro intestinal digestion of v-3 oils

(EPA and DHA rich oils). SO, TO, v-3-TAG (v-3 concentrate from fish oil in form of TAGs), and v-3-EE (v-3 concentrate from fish oil in form

of EEs).



the expected extension of hydrolysis. In the case of SO and

TO, previous studies have shown that most EPA is located

both at sn-1,3 positions or randomly distributed on the glyc-

erol backbone, whereas DHA seems to be largely at sn-2

position [17–21]. Thus, the similar course of hydrolysis of

SO and TO during the in vitro process might be related to the

similar distribution of resistant v-3 fatty acids within glycer-

ides of these oils. Contrarily, the analyzed v-3-TAG oil in the

present assay was assumed to show a random distribution of

DHA and EPA among the three positions of the glycerol

backbone, since it originated from a random enzymatic proc-

ess of synthesis. This different regio-distribution of v-3 fatty

acids on the glycerol moiety respect to fish oils might be

related to the different course of hydrolysis reaction of the

v-3-TAG oil compared to SO or TO. Furthermore, regard-

less of the location of v-3 fatty acids, the higher total level of

v-3 fatty acids (EPA plus DHA) of the v-3-TAG oil respect

to fish oils (29% for SO, 30% for TO, and 86% for v-3-TAG

oil) might also explain the differences on the rate of hydroly-

sis. Thus, Yang et al. [3] found evidences that fish oil TAGs

containing 2–3 long-chain PUFA per molecule were more

resistant to hydrolysis by pancreatic lipase.

Contrary to fish oils, the rate of in vitro digestion of v-3-

TAG concentrates did not reach a plateau and, despite the

slower rate of digestion, the hydrolysis of v-3-TAG concen-

trates continuously progressed and seemed to extend beyond

120 min. Thus, the level of TAGs changed from 60.2 g/

100 g of lipids at 0 min to 29.6 g/100 g of lipids at

120 min (50.8% of hydrolysis). Moreover, the hydrolysis

of DAGs was also evidenced. Thus, the content of DAGs

changed from 23.8 g/100 g of lipids at 0 min to 10.4 g/100 g

of lipids at 120 min (56.2% of hydrolysis). These decreases

in TAGs andDAGs corresponded to a continuous increase in

the ratio of FFAs, changing from 5.2 g/100 g of lipids at

0 min to 43.0 g/100 g of lipids at 120 min of reaction.

Moreover, after 20 min of reaction, it seemed that the

MAG level tended to increase (from 8.7 g/100 g of lipids

at 20 min to 16.9 g/100 g of lipids at 120 min) likely due to

the degradation of DAGs. Contrary to fish oils, it is possible

that the slower hydrolysis of v-3-TAG oil would not saturate

the micellar solubilization of lipid products as they were

produced, allowing the activity of the anchored pancreatic

lipase for longer times.

The evolution in the ratio of lipid products during in vitro

digestion of v-3-EE oil was quite different compared to fish

oils and v-3-TAG fish oil concentrates (Fig. 1). The level of

EEs only decreased from an initial value of 77 g/100 g of

lipids to 71.8 g/100 g of lipids (93% of remained EE) after

120 min of reaction. Therefore, a poor hydrolysis of EEs took

place under the in vitro digestion conditions performed. As

previously explained, pancreatic lipase is considered the main

lipolytic enzyme of the pancreatin extract and it is usually

assumed that porcine pancreatin generally lacks carboxyl

ester lipase for hydrolyzing EEs [22–24]. Therefore, the

obtained results were mainly related to the action of

pancreatic lipase on EEs. The resistance of EEs against

pancreatic lipolysis has been evidenced in diverse studies

[3, 9, 25]. As suggested, the double bond close to the ester

bond might lead to steric hindrance effect for enzymatic

hydrolysis [9, 26]. Differences in substrate emulsification

have been also proposed for explaining the lower rate of

hydrolysis of EEs [3].

In the case of the v-3-EE oil, it should be also pointed out

that this oil initially contained MAGs together with EEs

(Table 1), being in the form of 1-MAG. Despite the low

proportion of this glyceride structure, its hydrolysis by 1,3-

specific pancreatic lipasemight also contribute to the released

fatty acids during in vitro digestion. In fact, a progressive

decrease in the level ofMAGswas observed during the course

of the reaction.

3.2 Lipid composition of theMPobtained after in vitrointestinal digestion of v-3 oils

The total lipid content of the MP in most cases was around

30% of total lipid before digestion (29.5% � 2.0 for SO,

29.4% � 1.1 for TO, and 28.4% � 1.4 for v-3-TAG oil).

Therefore, regardless the different degree of hydrolysis

occurred during the digestion of fish oils and v-3-TAG oil

(Fig. 1), it seems that the rate of inclusion of lipid products

from degradation of v-3-TAG fish oil concentrate under

in vitro conditions was similar to that for purified fish oils.

In the case of the formed MP of the digested v-3-EE oil, the

total lipid content was much lower compared to the rest of

digested oils (10.2% � 1.7). This was in agreement with the

lower rate of hydrolysis of EEs measured during digestion

reaction (Fig. 1).

Previous studies have not been found regarding the total

lipid content of the MP formed after digestion of v-3 con-

centrates fish oils as TAGs or EEs, but the lipid contents of

the MPs obtained for SO, TO, and v-3-TAG oil were within

the range reported by Hofmann and Borgstrom [5] in in vivo

human assays during corn oil digestion. These authors were

the first that evidenced the formation of two mainly phases

(oily and aqueous) after centrifugation of the intestinal con-

tent from digested fat frommen. In such study, they reported

variable results from 16 to 45% for the lipid content of the

formedMP respect to the total lipid intestinal content during

corn oil digestion. Under in vitro conditions of lipolysis,

Patton and Carey [27] reported a 13.1% of total lipids in

the aqueous phase formed from digested olive oil. Factors

such as pH, temperature, and enzyme concentrations have

been considered for explaining different results between stud-

ies [16].

As previously reported, the in vitro digestion of v-3-TAG

oil yielded a higher ratio of lipid products compared to fish

oils after 120 min of reaction (Fig. 1). However, as reported

above, the total lipid content of the MP of fish oils and

v-3-TAG oil was practically the same. Those lipid products

non-included in the MP of digested v-3-TAG oil were lost in



the precipitated pellet (data not shown), probably as insolu-

ble calcium soaps, because after centrifuging the digestion

medium, a lack of OP was obtained and only the MP and the

precipitated pellet separated. Therefore, it seems that despite

the higher hydrolysis of v-3-TAG oil compared to fish oils, it

would not result in a higher inclusion of digested products in

the MP. Any kind of limitation in the level of dispersion of

lipid products in the MP might take place. Whether this is a

limitation of the in vitro model or this would also occur under

in vivo conditions would need further investigations. The

dispersion of hydrolysis products in the MP is based on

micellar solubility, which depends on the chemical form of

the lipid, the degree of unsaturation of the fatty acid, and the

presence of other lipids [6]. On the other hand, Sek et al. [16]

evidenced that higher proportions of FFAs and MAGs dis-

persed into the aqueous phase as the concentration of bile

salts increased in the digestion medium.

The lipid composition of the separated MPs from

digested oils are shown in Fig. 2. In vitro digestion of fish

oils led to the formation of a MP consisted of FFAs (53.9 g/

100 g of lipids for SO and 55.9 g/100 g of lipids for TO) and

MAGs (37 g/100 g of lipids for both oils). Minor proportions

of TAGs andDAGswere also detected in theMP of both oils.

It is interesting to point out that regardless the slightly higher

hydrolysis of TO compared to SO (Fig. 1), no differences

were reflected in the composition of the isolated MP. This

was especially surprising for the level of MAGs, since as

previously discussed, digested TO showed a final MAG con-

tent higher than that of SO at 120 min of digestion in the

whole medium of reaction (Fig. 1).

The MP from digested v-3-TAG concentrate mainly

consisted of FFAs (56.6 g/100 g of lipids) and MAGs

(42.1 g/100 g of lipids) but TAGs were not detected. This

result shows that the composition of the MP formed after

digestion of v-3-TAG fish oil concentrates might be different

to that of fish oils, the presence of lipid products dominating

in the case of concentrates. Therefore, it seems that for a same

level of inclusion of lipids in the MP of digested fish oils and

v-3-TAG oil, the proportion of lipid products would be

higher in the case of digested v-3-TAG oil.

The formed MP from digested v-3-EE fish oil concen-

trate was quite different to that obtained for the rest of

assayed oils (Fig. 2). This MP mainly consisted of FFAs

(57.5 g/100 g of lipids) and undigested EEs from the initial

oil (38.4 g/100 g of lipids) as well as a slightMAGs ratio. The

obtained results show that regardless the scarce hydrolysis of

v-3-EE fish oil (Fig. 1), a low percentage of non-digested EEs

are effectively included in the MP within the rest of digestion

products (FFAs). Regarding the rest of non-included lipids in

the MP, they were separated in the OP (data not shown),

because after centrifuging the digestion medium a lack of

precipitated pellet was obtained and only the MP and the OP

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pids

MP of digested ω-3 EE

Figure 2. Lipid composition (g/100 g of lipids) of the isolated MPafter in vitro intestinal digestion of v-3 oils (EPA and DHA rich oils). SO, TO,

v-3-TAG (v-3 concentrate from fish oil in form of TAGs), v-3-EE (v-3 concentrate from fish oil in form of EEs).



separated. Several authors have reported that absorption of

EPA and DHA is equal both from EEs and from TAGs in

humans [2, 3], regardless the lower hydrolytic rate of EEs by

pancreatic lipase. However, the exact mechanisms of absorp-

tion of v-3 fatty acids as EEs, as well as the lipid composition

of the micellar structures during their intestinal hydrolysis,

have not been clarified. The present study showed that only

low levels of non-hydrolyzed EEs seemed to include in micel-

lar structures (10.2% � 1.7) after in vitro intestinal digestion

of EEs by pancreatic lipase. Nevertheless, in vivo studies for

confirming these findings would be of interest, as well as the

potential contribution of carboxyl ester lipase on the hydroly-

sis of EEs, as a natural enzyme of the human pancreatic juice.

4 Conclusions

This comparative study showed that the in vitro intestinal

digestion ofv-3 (EPA andDHA) sources as fish oil, TAGs, or

EEs concentrates was different, regarding both the evolution

of lipid products during digestion, the rate of digestion and

the inclusion of hydrolysis products in the potential absorb-

able fraction as mixed micelles. The v-3-TAG concentrates

progressed slowly but continuously throughout the whole

time of digestion, whereas the hydrolysis of fish oils stopped

at initial times and was more incomplete. Moreover, the

MP from v-3-TAG concentrates showed a slightly higher

proportion of v-3 rich lipid products. In the case of the v-3-

EE concentrate, most EEs remained undigested and scarce

lipids were included in the MP. The findings of the present

assay contributed to the knowledge of the intestinal lipolysis

of v-3 sources, especially those results concerning the

inclusion of lipid products on micellar structures under

in vitro conditions, as the first study in depth on this subject.

The authors would like to thank the Ministerio de Ciencia e

Innovacion, Spain (AGL 2008-05655) and the Community of

Madrid, Spain (ALIBIRD-CM S-2009/AGR-1469) for

supporting this research. Diana Martin thanks the Ministerio de

Ciencia e Innovacion and Fondo Social Europeo for funding her

postdoctoral ‘‘Juan de la Cierva’’ contract.

The authors have declared no conflict of interest.

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Intestinal digestion of fish oils and ω-3 concentrates under in vitro conditions