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Research Article
The antioxidative effect of lipophilized rutin anddihydrocaffeic acid in fish oil enriched milk
Ann-Dorit Moltke Sørensen1, Lone Kirsten Petersen1*, Sara de Diego2**, Nina Skall Nielsen1,
Bena-Marie Lue3***, Zhiyong Yang3, Xubing Xu3 and Charlotte Jacobsen1
1 Division of Industrial Food Research, National Food Institute, Technical University of Denmark, Lyngby,
Denmark2 Department of Biotechnology and Food Science, University of Burgos, Burgos, Spain3 Department of Engineering, Aarhus University, Arhus, Denmark
The antioxidative effect of phenolipids was evaluated in fish oil enriched milk emulsions as a model for a
complex food system. Two different phenolipids modified from dihydrocaffeic acid (with C8 or C18:1) and
rutin (with C12 or C16) were evaluated. Both dihydrocaffeate esters and rutin laurate showed significantly
better antioxidant properties inmilk emulsion compared with the original phenolics. However, rutin palmitate
only performed slightly better as antioxidant than rutin. The results with rutin indicated that a cut-off effect
exists in relation to the alkyl chain length with respect to optimal antioxidant activity in milk emulsions. Thus,
the optimal alkyl chain length is at least below 16 carbon atoms, and maybe even less for rutin esters. For
dihydrocaffeate esters it was not possible to conclude on a cut-off effect in relation to alkyl chain length and
antioxidative effect due to the almost similar antioxidant effect of the two phenolipids. However, there was a
tendency towards octyl dihydrocaffeate being slightly more efficient than oleyl dihydrocaffeate.
Practical application: The finding that phenolipids are better antioxidants in milk emulsions than the
original phenolic acid provides new knowledge that can be used to develop new antioxidant strategies to
protect foods against lipid oxidation. However, the results indicate that both optimization of alkyl chain
length for each type of phenolic, and optimization for each type of emulsion will be necessary in order to get
the best oxidative stability of an emulsion with these phenolipids. Use of efficient antioxidantsmay lower the
amount of antioxidant needed to protect against lipid oxidation and may in addition decrease the costs.
Keywords: Caffeic acid / o/w Emulsion / Polar paradox / Rutin
Received: October 10, 2011 / Revised: January 9, 2012 / Accepted: February 24, 2012
DOI: 10.1002/ejlt.201100354
1 Introduction
The health beneficial effects of n-3 long chain PUFA (LC
PUFAs) such as, e.g. reduced risk of cardiovascular diseases
are well documented. During the last decade substantial
efforts have been put into enriching foods with the healthy
n-3 LC PUFAs as reviewed by Jacobsen et al. [1] These
efforts have been carried out in order to increase the popu-
lations’ intake of especially eicosapentaenoic acid (EPA) and
DHA [2, 3]. Despite the increasing number of n-3 PUFA
enriched foods on the market, consumer acceptance and
shelf-life of such products are still limited by the higher
oxidative susceptibility of unsaturated lipids, which will lead
to an unpleasant fishy off-flavour [4–7]. To retard lipid oxi-
dation, a range of commercial synthetic antioxidants with free
radical scavenging activity and metal chelating properties are
*Current address: CP Kelco ApS, Ved banen 16, DK-4623 Lille Skensved,
Denmark
**Current address: Grupo Siro, Paseo Pintor Rosales 40, Madrid, Spain
***Current address: Novozymes A/S, Krogshoejvej 36, DK-2880
Bagsvaerd, Denmark
Correspondence: Dr. Ann-Dorit Moltke Sørensen, Division of Industrial
Food Research, National Food Institute, Technical University of Denmark,
Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark
E-mail: adms@food.dtu.dk
Fax: þ45 4588 4774
Abbreviations: ATD, automatic thermal desorber; BHT, butylated
hydroxytoluene; DHCA, dihydrocaffeic acid; DHCA C18:1, oleyl
dihydrocaffeate; DHCA C8, octyl dihydrocaffeate; EPA,
eicosapentaenoic acid; LC, long chain; PV, peroxide value; Rutin C12,
rutin laurate; Rutin C16, rutin palmitate
434 Eur. J. Lipid Sci. Technol. 2012, 114, 434–445
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available, e.g. calcium disodium ethylenediaminetetraacetate
(EDTA), butylhydroxytoluene (BHT) and propyl gallate.
However, there is a trend in consumer preference for natural
ingredients such as phenolic compounds rather than syn-
thetic compounds. The major part of natural antioxidants
from plants, are phenolic compounds, e.g. caffeic acid.
Most food products are emulsions, and in these systems
lipid oxidation is suggested to be initiated at the interface
between the oil phase and the aqueous phase or air, and
continued in the oil phase. In emulsions, antioxidants may
mainly partition into three different phases: the aqueous
phase, the oil phase and the interface between oil and water.
Partitioning of antioxidants into the different phases is influ-
enced by their polarity and interactions with other com-
ponents present in the emulsion, e.g. emulsifier [8, 9].
Generally, phenolics are hydrophilic compounds and they
will most likely be located in the aqueous phase.
Furthermore, the polarity of antioxidants in bulk oil and
emulsions has been considered to be decisive for their effi-
ciency. This phenomenon is known as the polar paradox and
states that hydrophilic antioxidants are more efficient in bulk
oils than lipophilic antioxidants. In contrast, lipophilic anti-
oxidants generally function better than hydrophilic anti-
oxidants in emulsions [10]. However, recent studies have
reported results that contradict the polar paradox hypothesis
[11–14]. This suggests that other factors might be equally
important, and more research is urgently required to improve
our understanding about the relationship between the mol-
ecular structure of the antioxidants and their efficacy in
different real food systems.
Recently, several studies have reported the possibility of
changing the polarity of phenolics by lipophilization with fatty
acids of different chain length in order to improve their
antioxidative effect in emulsified media. The current work
in this area has been summarized by Shahidi and Zhong [15].
Laguerre et al. [11, 16] have recently reported on the anti-
oxidative effect of lipohilized chlorogenic and rosmarinic
acids. For chlorogenic acid the antioxidant capacity increased
as the alkyl chain length was increased from 1 to 12 carbon
atoms, whereas further increase of the chain length resulted in
a drastic decrease in the antioxidant capacity. On the basis of
these results the authors suggested a so-called cut-off effect
related to the length of the lipid chain attached to chlorogenic
acid, which they explained as follows: When, the hydropho-
bicity of the lipophilized compound increases to above a
certain level, the lipophilized compound is suggested to form
micelles in the aqueous phase. Thereby, they will not be
available as antioxidants at the interface and in turn their
efficacy will be reduced [11]. For rosmarinate esters, the octyl
rosmarinate improved the antioxidative effect eight times
compared to rosmarinic acid. Thus, the results lead to the
conclusion that lipophilization with medium chain fatty acids
is a promising way to increase the antioxidant activity [16].
However, the results obtained with chlorogenate and ros-
marinate esters have also led to the suggestion that the exact
location of antioxidants in the discontinuous phase, inter-
facial layer or oil droplets, is important for the activity of the
antioxidants [11, 16]. In addition, a study on lipophilized
dihydrocaffeic acids and their antioxidative effect in o/w
emulsions reported by Sørensen et al. [17] suggested that
lipophilized dihydrocaffeic acid tended to follow the newly
suggested cut-off effect in relation to the alkyl chain length
attached although only two chain lengths were evaluated. In
contrast, lipophilized rutin added to o/w emulsions did not
show a cut-off effect, since the esters, rutin laurate and rutin
palmitate, were consistently less effective compared with the
rutin [13]. However, only two chain lengths (C12 and C16)
were evaluated along with rutin, hence alkyl chain shorter
than C12 lipophilized on rutin may indicate a cut-off effect.
From these studies it may be concluded that the cut-off effect
is specific for the individual lipophilized phenolic com-
pounds, i.e. the optimal chain length may vary between
different phenolics.
Recent research on lipophilized phenolic compounds has
mostly paid attention to their production, in vitro antioxidant
activity and their effect in simplified model systems, whereas
studies on their effect in real food systems are lacking. Inmore
complex systems such as real food the antioxidant behaviour
might be influenced by interaction with other components
present in the emulsions, e.g. emulsifier and iron [15, 18].
However, on the basis of studies in simple o/w emulsions we
hypothesize that lipophilization of phenolics will increase
their antioxidant efficacy in emulsified food enriched with
n-3 PUFAs. Moreover, we hypothesize that the alkyl chain
length will affect the antioxidative effect of these lipophilized
phenolic compounds as was observed in a recent study with
simple o/w emulsions [17], and that the effect of the alkyl
chain length may be different from that observed in simple
model systems due to the presence of other potentially inter-
acting compounds. Therefore, the aim was to evaluate the
antioxidative effect of dihydrocaffeic acid lipophilized with
either octyl or oleyl alcohol and rutin lipophilized with lauric
acid or palmitic acid in fish oil enriched milk. Fish oil
enriched milk was chosen as previous studies have shown
that it is highly susceptible to lipid oxidation [1]. Moreover, it
is a complex food system in which antioxidants may interact
with different compounds, e.g. proteins. Lipophilized rutin
and dihydrocaffeic acid were chosen as antioxidants since this
study is a continuation of recent studies performed in our lab
in simplified o/w emulsions [12, 13, 17] and results could
show different effects of lipophilization in simple versus com-
plex emulsions.
2 Material and methods
2.1 Materials
Fresh milk (0.5 and 1.5% fat content) was purchased in a
local supermarket. Fish oil without antioxidant added was
supplied by Maritex Norway (subsidiary of TINE BA,
Eur. J. Lipid Sci. Technol. 2012, 114, 434–445 Antioxidant efficacy of lipophilized phenolics 435
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Norway). This oil had an initial PV of 0.1 meq peroxides/kg
oil, tocopherol content of 204 mg a-tocopherol, 102 mg g-
tocopherol and 42 mg d-tocopherol/kg oil, and the fatty acid
composition was as follows: 14:0, 3.0%; 16:0, 8.7%; 16:1,
8.2%; 18:0, 1.9%; 18:1, 20.9%; 18:2, 1.8%; 18:4, 2.6%;
20:1, 12.5%; 20:5 (EPA), 9.4%; 22:1, 5.9%; 22:5, 1.1%
and 22:6 (DHA), 11.6%. The total percentages of n-3 and
n-6 PUFA in the oil were 24.7 and 2.7%, respectively.
Rutin (purity �98%), caffeic acid (purity �98%),
dihydrocaffeic acid (purity �98%) and oleyl alcohol (purity
85%) were from Sigma–Aldrich (Steinheim, Germany).
Lipophilized rutin with lauric (C12) or palmitic (C16) acids,
both with a purity of 98%, were synthesized at the National
Food Institute, Division of Industrial Food Research
(Technical University of Denmark, Lyngby Denmark). For
further details about the lipophilization process refer to Lue
et al. [19]. Lipophilized dihydrocaffeic acid with octyl (C8)
with a purity of 80% or oleyl alcohol (C18:1) with a purity of
60% were synthesized at the Department of Engineering,
Faculty of Science (Aarhus University, Arhus, Denmark).
Chemicals were from Merck (Darmstadt, Germany) and
external standards for identification and quantification of
secondary volatile oxidation products were all from Sigma–
Aldrich (Steinheim, Germany). All solvents were of HPLC
grade and purchased from Lab-Scan (Dublin, Ireland).
2.2 Production of fish oil enriched milk
Milk with 0.5% fat and with 1.5% fat was mixed (1:1 v/v) to
obtain a total fat content of 1%. Subsequently, the milk was
pasteurized at 728C for 15 s and the fish oil (0.5% v/v) and
antioxidant were added (for specification on antioxidant
addition, see Section 2.3). Emulsions were prepared in two
steps: pre-emulsification and homogenization. During pre-
emulsification, the heated milk with fish oil and antioxidant
added was stirred with an Ultra-Turrax (Step 7, 1 min, Janke
& Kunkel IKA-Labortechnik, Staufen, Germany). The pre-
emulsion was then homogenized at a pressure of 25 and
250 bar with four circulations of the emulsion at RT using
a table homogenizer from GEA Niro Soavi Spa (Parma,
Italy). The emulsions (100 g) were stored in 100 mL blue
cap bottles at 58C. Samples, one flask pr. code, were taken at
Day 0, 3, 6, 9 and 12 and divided in brown glass bottles for
different analysis and stored at �408C until analyses of per-
oxides, volatiles, tocopherols and fatty acids were performed.
The droplet size was measured at Day 1, 6 and 12 without
pre-freezing.
2.3 Experimental design
The experimental design for Experiment 1 and 2 is described
in details below and summarized in Table 1.
Experiment 1: Five different antioxidants were evaluated,
rutin, rutin laurate, rutin palmitate, dihydrocaffeic acid and
oleyl dihydrocaffeate in a concentration of 100 mM in fish oil
enriched milk. Dihydrocaffeic acid and oleyl dihydrocaffeate
were added directly to the milk, whereas rutin, rutin laurate
and rutin palmitate were first dissolved or suspended in
1.5 mL acetone due to dissolving problems in the milk emul-
sion. The acetone with antioxidant was then added to the
heated milk. To obtain the same condition for all emulsions,
1.5 mL acetone was added to the milk emulsions with dihy-
drocaffeic acid, oleyl dihydrocaffeate and the control (no
antioxidant added).
Experiment 2: Four different antioxidants were evaluated,
caffeic acid, dihydrocaffeic acid, octyl dihydrocaffeate and
oleyl dihydrocaffeate. Antioxidant concentration tested was
similar to that in Experiment 1 (100 mM). The synthesized
Table 1. Experimental design
Antioxidant applied Sample code
Concentration of antioxidant
mM mg/kg
Experiment 1: Acetone addition (1.5 mL)
Control Control – –
Rutin Rutin 100 61.1
Rutin laurate (C12) Rutin C12 100 79.3
Rutin palmitate (C16) Rutin C16 100 84.9
Dihydrocaffeic acid DHCA 100 18.2
Oleyl dihydrocaffeate (C18:1) DHCA C18:1 100 72.1
Experiment 2: No acetone addition
Control Control – –
Dihydrocaffeic acid DHCA 100 18.2
Octyl dihydrocaffeate (C8) DHCA C8 100 36.8
Oleyl dihydrocaffeate (C18:1) DHCA C18:1 100 72.1
Oleyl alcohol Oleyl alcohol 107 28.8
Caffeic acid Caffeic acid 100 18.0
436 A.-D. M. Sørensen et al. Eur. J. Lipid Sci. Technol. 2012, 114, 434–445
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oleyl dihydrocaffeate was only 60% pure, and contained
�40% free oleyl alcohol. Therefore, an emulsion with oleyl
alcohol with the same amount of oleyl alcohol (29 mg/kg) as
in the emulsion with oleyl dihydrocaffeate was included to
evaluate the effect of oleyl alcohol on lipid oxidation in the
milk emulsion. The antioxidants were dissolved directly in
the heated milk before homogenization.
2.4 Droplet size determination
The size of the fat droplets in milk emulsions was determined
by laser diffraction with a Mastersizer 2000 (Malvern
Instruments Ltd., Worcestershire, UK). A few droplets of
the milk emulsion were suspended directly in re-circulating
water (2800 rpm, obscuration 14–16%). The set-up used
was the Fraunhofer method, which assumes that all sizes
of particles scatter with equal efficiencies and that the
particles is opaque and transmits no light [20]. The results
were reported as surface mean diameter,
D3;2 ¼P
nid3iP
nid2i
where d is the diameter of individual droplets.
2.5 Measuring lipid oxidation
2.5.1 Extraction of lipids
Lipids were extracted from fish oil enriched milk (15 g)
according to the method described by Bligh and Dyer [21]
with reduced amount of solvent applied [22]. The analysis
was done in duplicate and further used for determination of
peroxide value, fatty acid composition and tocopherol
concentration.
2.5.2 Primary oxidation products, peroxide value (PV)
Peroxide value in the lipid extracts were determined by a
colorimetric method based on formation of an iron–thiocya-
nate complex measured according to the method described
by Shanta and Decker [23], n ¼ 2.
2.5.3 Secondary volatile oxidation products –dynamic headspace
Volatiles were collected on TenaxTM tubes (Perkin Elmer,
Norwalk, CT, USA) by purging the fish oil enriched milk
(8 g) with nitrogen (150 mL/min, 30 min) at 458C. An
ATD-400 automatic thermal desorber was used for thermally
desorbing the collected volatiles. The transfer line of the
ATD was connected to a 5890 IIA gas chromatograph
(Agilent Technologies, Palo Alto, CA, USA) equipped with
a DB wax column (length 30 m � I.D. 0.25 mm � 0.5 mm
film thickness, J&W Scientific, CA, USA) coupled to a HP
5972A mass selective detector. Temperature program was as
follows: 5 min at 458C, 1.58C/min from 45 to 558C,
2.58C/min from 55 to 908C, 128C/min from 90 to 2208Cand hold for 4 min at 2008C. The MS was operating in the
electron ionization mode at 70 eV and mass to charge ratios
between 29 and 200 were scanned. For quantification of the
different volatiles, solutions with external standards at differ-
ent concentrations were prepared and analysed from milk
with no fish oil added. The results are given in ng/g milk
(n ¼ 3).
2.5.4 Tocopherol concentration
Lipid extract was evaporated under nitrogen, re-dissolved in
heptane and analysed byHPLC (Agilent 1100 Series, Agilent
Technologies, Palo Alto, CA, USA) according to the AOCS
method [24] to determine tocopherol concentration in the
different milk samples (n ¼ 4).
2.5.5 Fatty acid composition
Lipid extract was evaporated under nitrogen. First, the glyc-
erol bound fatty acids were transesterified with methanolic
NaOH (0.5 M). Then, hydrolytic released and free fatty acids
were methylated by a boron trifluoride reagent (20%) cata-
lysed process. Methyl esters were extracted with heptane
followed by separation on GC (HP 5890A, Agilent
Technologies, Palo Alto, CA, USA). The procedure was
according to the AOCS methods [25, 26], n ¼ 2.
2.5.6 Sensory
Preliminary sensory evaluation was performed by an expert
panel composed of two persons. Each assessor evaluated one
milk sample at a time. After each milk sample the assessors
discussed the odour of the sample (only Experiment 2).
2.6 Data analyses
2.6.1 Statistics
The obtained results were analysed by two way ANOVA
(GraphPad Prism, Version 4.03, GraphPad Software,
Inc.). The Bonferroni multiple comparison was used to test
differences between samples or storage time (significance
level p<0.05).
2.6.2 Inhibition percentages
To compare the efficacy of the antioxidants in the two differ-
ent emulsion systems, inhibition percentages were calculated.
Inhibition ½%� ¼ 1� SampleAntioxidantSampleControl
� �� 100
Eur. J. Lipid Sci. Technol. 2012, 114, 434–445 Antioxidant efficacy of lipophilized phenolics 437
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3 Results
3.1 Characteristics of the fish oil enriched milkemulsions
The characteristics, i.e. droplet size and content of EPA and
DHA of the different fish oil enriched milk emulsions are
summarized in Table 2. Average sizes of the lipid droplet in
the milk were between 0.45 and 0.74 mm during the storage
period. In Experiment 1, the emulsion with antioxidants had
a significantly larger droplet size, whereas a similar effect was
not observed in the second experiment. The droplet sizes
measured in the different milk emulsion were unchanged
during storage. Content of EPA and DHA in the different
milk emulsions at day 0 indicated similar levels of 3.26–3.47
and 4.03–4.44% EPA and DHA of total fatty acids, respect-
ively. At Day 12, oleyl dihydrocaffeate (Experiment 2) was
the only milk emulsion with a decrease in EPA and DHA
>0.1%. However, the EPA and DHA contents increased to
the same extent during storage in several emulsions. Thus,
the decreases in EPA and DHA in oleyl dihydrocafeate
(Experiment 2) may be due to day to day variation for the
measurements rather than an actual decrease. This finding
suggests that lipid oxidation did not significantly affect EPA
and DHA contents in any of the milk emulsions.
3.2 Peroxide values in fish oil enriched milkemulsions
Peroxide values obtained in Experiment 1 and 2 during
storage are shown in Fig. 1. For Experiment 1 a lag phase
Table 2. Droplet size D3,2 [mm] for lipid droplets in fish oil enriched milk given as an average during storage (average W SD) and content of
EPA and DHA (wt% of total lipids) at day 0 and 12 in the different milk emulsions
Sample code Droplet size (mm)
EPA content (%wt of total lipids) DHA content (%wt of total lipids)
Day 0 Day 12 Day 0 Day 12
Experiment 1
Control 0.45 � 0.01 3.47 � 0.08 3.51 � 0.09 4.35 � 0.24 4.47 � 0.21
Rutin 0.70 � 0.03 3.26 � <0.01 3.51 � 0.15 4.03 � 0.07 4.43 � 0.28
Rutin C12 0.74 � 0.01 3.42 � 0.04 3.48 � <0.01 4.26 � 0.20 4.30 � 0.06
Rutin C16 0.59 � 0.01 3.43 � 0.08 3.44 � 0.01 4.26 � 0.21 4.27 � 0.01
DHCA 0.70 � 0.03 3.47 � 0.05 3.58 � 0.01 4.35 � 0.15 4.57 � <0.01
DHCA C18:1 0.72 � 0.02 3.51 � 0.08 3.48 � 0.01 4.44 � 0.16 4.36 � 0.01
Experiment 2
Control 0.73 � 0.02 3.41 � 0.01 3.75 � 0.07 4.15 � 0.03 4.53 � 0.02
DHCA 0.65 � 0.07 3.37 � 0.02 3.31 � 0.04 4.10 � 0.04 4.04 � 0.08
DHCA C8 0.70 � 0.03 3.31 � 0.01 3.31 � 0.07 4.05 � 0.04 4.02 � 0.16
DHCA C18:1 0.74 � 0.04 3.43 � 0.08 3.26 � 0.06 4.09 � 0.01 3.99 � 0.02
Oleyl alcohol 0.73 � 0.02 3.46 � 0.01 3.58 � 0.01 4.22 � 0.04 4.45 � 0.03
Caffeic acid 0.61 � 0.09 3.37 � 0.07 3.37 � 0.02 4.13 � 0.09 4.07 � 0.04
Sample codes refer to Experimental design Table 1.
0 5 10 150
5
10
15
Storage time [Days]
PV [m
eq p
erox
ides
/ kg
oil]
0 5 10 150
1
2
3
4
5
6
7
PV [m
eq p
erox
ides
/ kg
oil]
(A)
(B)
ControlRutin palmitate
Rutin laurate
RutinOleyl dihydrocaffeateDihydrocaffeic acid
Oleyl alcoholControl
Dihydrocaffeic acidCaffeic acid
Oleyl dihydrocaffeateOctyl dihydrocaffeate
Figure 1. Concentration of peroxides measured as PV [meq.
peroxides/kg oil] in the different fish oil enriched emulsions during
storage. Error bars indicate SD of the measurements (n ¼ 2). (A)
Experiment 1: control (&), rutin (D), rutin laurate (~), rutin palmitate
(!), dihydrocaffeic acid (*) and oleyl dihydrocaffeate (*). (B)
Experiment 2: control (&), dihydrocaffeic acid (*), octyl dihydro-
caffeate (^), oleyl dihydrocaffeate (*), oleyl alcohol (X) and caffeic
acid (5).
438 A.-D. M. Sørensen et al. Eur. J. Lipid Sci. Technol. 2012, 114, 434–445
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was observed until day 3 for the emulsion without antioxidant
added (Fig. 1A) as the difference between day 0 and 3 was
insignificant. In contrast, the lag phase for emulsion with
antioxidant added was longer than 3 days and for milk emul-
sion with rutin laurate the lag phase could not be determined
within the storage period (PV never increased). This indi-
cated that rutin laurate efficiently inhibited the development
of peroxides. However, the data could also indicate that
peroxides were developed and decomposed at equal rates.
Investigation of secondary oxidation products (volatiles),
described later, resolves this. After the end of the lag phase,
the concentration of peroxides increased with different rates
in the different emulsions. At day 12 the ranking of the different
milk emulsions with respect to PV level was as follows: controla
(highest level) � rutin palmitateab � rutinb ¼ oleyl dihydro-
caffeateb ¼ dihydrocaffeic acidb>rutin lauratec.
Compared to Experiment 1, the off set for peroxide devel-
opment was faster in Experiment 2 (Fig. 1B). Moreover, the
PV level in the milk emulsion without antioxidant and
the one with dihydrocaffeic acid added was higher than in
the samemilk emulsion in Experiment 1. Only two emulsions
in Experiment 2 had a lag phase, and these were the milk
emulsions with oleyl dihydrocaffeate and octyl dihydrocaf-
feate added. For all other milk emulsions the development of
PVs was triggered in different rates already at the beginning of
storage depending on the antioxidant added. At day 12 the
ranking ofmilk emulsions according to PV level was as follows:
oleyl alcohola>controlb>dihydrocaffeic acidc ¼ caffeic
acidc>oleyl dihydrocaffeated ¼ octyl dihydrocaffeated.
Thus, in contrast to the findings for rutin esters in
Experiment 1, the chain length of the fatty acid esterified
to dihydrocaffeic acid did not influence the development of
peroxides in the emulsions differently.
The inhibition percentage calculated for Experiment 1
based on PV development (Table 3), clearly showed that
rutin laurate was the most efficient antioxidant in inhibiting
the formation of peroxides when added to milk emulsion.
However, oleyl dihydrocaffeate and rutin palmitate was as
efficient as rutin laurate at Day 6, but their antioxidative
efficiency decreased after Day 6. For Experiment 2 it is clear
from the inhibition percentage that both dihydrocaffeate
esters added were very effective in inhibiting the peroxides
formation. Inhibition percentages for those milk emulsions
with the same antioxidants applied in both experiments;
dihydrocaffeic acid and oleyl dihydrocaffeate, indicated bet-
ter inhibition of PV development in Experiment 1 with
acetone added for dihydrocaffeic acid emulsion after Day 3
and in Experiment 2 without acetone for oleyl dihydrocaf-
feate emulsion. Thus, the addition of acetone seemed to have
no significant impact on the development of peroxides. In
spite of the higher PVs in Experiment 2, the efficacy of
antioxidants in fish oil enriched milk seemed to be better
for octyl and oleyl dihydrocaffeate without acetone than
oleyl dihydrocaffeate, rutin laurate and rutin palmitate with
acetone added at least before Day 9. However, before a
clear conclusion can be drawn the data on volatile oxidation
products must be considered.
3.3 Development of volatile oxidation products
Five different volatiles were measured in the stored milk
emulsions and three of them is shown in Fig. 2: 1-penten-
3-one, 1-penten-3-ol and 2,6-nonadienal. These volatiles are
shown since they illustrate the general trend in the develop-
ment of volatiles during storage in the two experiments.
Furthermore, they represent decomposition of n-3 fatty acids
and are known to have impact on the development of fishy
off-flavour [27].
For Experiment 1, the volatiles are shown in Fig. 2A, C
and E, respectively. Concentration of 1-penten-3-one in milk
Table 3. Calculated inhibition percentages of PV level in the different milk emulsions in both storage experiments
Milk emulsions
Inhibition of PV during storage (%)
Day 0 Day 3 Day 6 Day 9 Day 12
Experiment 1
Rutin �6 18 42 34 21
Rutin laurate (C12) 27 32 59 77 78
Rutin palmitate (C16) 37 �18 57 32 11
Dihydrocaffeic acid 18 �15 31 52 29
Oleyl dihydrocaffeate (C18:1) 34 21 58 31 26
Experiment 2
Dihydrocaffeic acid 35 29 11 15 23
Octyl dihydrocaffeate (C8) 45 63 83 57 57
Oleyl dihydrocaffeate (C18:1) 47 59 70 59 49
Oleyl alcohol 63 3 �95 26 �15
Caffeic acid 57 51 33 34 31
The values are calculated according to the control emulsions from the respective experiment.
Eur. J. Lipid Sci. Technol. 2012, 114, 434–445 Antioxidant efficacy of lipophilized phenolics 439
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emulsions increased until Day 9, whereafter the concen-
tration decreased at different rates in the different emulsions.
Milk emulsions with no antioxidant and dihydrocaffeic acid
added had no lag phase, whereas milk emulsions with rutin,
rutin palmitate and oleyl dihydrocaffeate added had a lag
phase of 3 days before these emulsions started developing 1-
penten-3-one. The concentration of 1-penten-3-one in milk
emulsion with rutin laurate added did not increase during
storage. The ranking of the emulsions according to concen-
tration of 1-penten-3-one before the decrease in concentration
(Day 9) was as follows: rutin lauratea<oleyl dihydrocaffea-
teb<rutin palmitatec � dihydrocaffeic acidc<rutind<controle.
The observed decline in the concentration of 1-penten-3-one at
the end of the storage period might be due to a reduction of
this volatile to 1-penten-3-ol either by the antioxidant or
other components in the milk emulsion. For the other vol-
atiles measured in this experiment, the concentration
increased after a shorter or longer lag phase (Fig. 2C
and E). Similar for these volatiles were that the control milk
emulsion had the shortest lag phase and the highest concen-
tration of the volatiles measured at the end of the storage. The
lag phase for the development of 1-penten-3-ol in milk emul-
sion was 3 days for control emulsion and emulsions with
dihydrocaffeic acid, and 6 days for oleyl dihydrocaffeate,
rutin and rutin palmitate added, whereas milk emulsion with
rutin laurate had a lag phase of 9 days (Fig. 2C). The duration
of the lag phase for development of 2,6-nonadienal was
slightly different from that of 1-penten-3-ol. Here, the milk
emulsion with rutin only had 3 days lag phase together with
dihydrocaffeic acid and control emulsions, andmilk emulsion
with oleyl dihydrocaffeate added had an infinite lag phase
(2,6-nonadienal never increased, Fig. 2E). After the end of
the lag phase the concentration of volatiles increased in the
different milk emulsions, except for 2,6-nonadienal in the
Figure 2. Concentration of three volatiles [ng/g] in the different fish oil enriched milk emulsions during storage. (A,B) 1-penten-3-one
Experiment 1 and 2, (C,D) 1-penten-3-ol Experiment 1 and 2, (E,F) 2,6-nonadienal Experiment 1 and 2, respectively. Error bars indicate
SD of the measurements (n ¼ 3). Symbols: control (&), rutin (D), rutin laurate (~), rutin palmitate (!), dihydrocaffeic acid (DHCA*), oleyl
dihydrocaffeate (DHCA C18:1 *), octyl dihydrocaffeate (DHCA C8 ^), oleyl alcohol (X) and caffeic acid (5).
440 A.-D. M. Sørensen et al. Eur. J. Lipid Sci. Technol. 2012, 114, 434–445
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emulsion with oleyl dihydrocaffeate added. At day 12 the
ranking according to concentration of 1-penten-3-ol and 2,6
nonadienal were as follows: rutin lauratea<oleyl dihydrocaf-
feate and dihydrocaffeic acidb<rutin and rutin palmita-
tec<controld and oleyl dihydrocaffeatea<rutin laurate and
dihydrocaffeic acidb<rutin palmitate and rutinc<controld,
respectively. Overall, the concentrations of volatiles were
lower in emulsions containing the lipophilized compounds
compared with the original phenolic. However, rutin palmi-
tate sometimes led to higher concentration of volatiles than
rutin in the milk emulsions.
The development of volatiles in emulsions from
Experiment 2 is shown in Fig. 2B, D and F. As observed
in Experiment 1, the concentration of 1-penten-3-one first
increased and then decreased. However, in Experiment 2 this
only happened in three emulsions: oleyl alcohol, dihydrocaf-
feic acid and control emulsion. Moreover, these emulsions
had a faster increase in the concentration of 1-penten-3-one
than the other emulsions. For milk emulsions with octyl and
oleyl dihydrocaffeate added, the development of 1-penten-3-
one did not increase the first 3 days. The concentration of 1-
penten-3-one in the emulsions before the decrease (Day 6)
was ranked as follows: octyl dihydrocaffeatea<oleyl dihydro-
caffeatea,b<caffeic acidb<dihydrocaffeic acidc<controld<oleyl
alcohole (Fig. 2B). The same milk emulsions, as observed for
development of 1-penten-3-one, also had a faster increase for
the other measured volatiles, except for 2,6-nonadienal. In
these emulsions (control, dihydrocaffeic acid and oleyl alco-
hol) no lag phase was observed, whereas the other 3 emul-
sions with caffeic acid, oleyl dihydrocaffeate and octyl
dihydrocaffeate added, had a lag phase. For the development
of 1-penten-3-ol, the lag phase was 3 days for milk emulsion
with caffeic acid and oleyl dihydrocaffeate added, whereas it
was 9 days with octyl dihydrocaffeate added. At the end of the
storage period the concentration of 1-penten-3-ol in the
different emulsions was ranked as follows: octyl dihydrocaf-
featea<oleyl dihydrocaffeateb<caffeic acidc<dihydrocaffeic
acidd<controle<oleyl alcohold. Similar to Experiment 1,
the development of 2,6-nonadienal had a longer lag phase
than observed for the other volatiles measured. Emulsions
with oleyl alcohol and dihydrocaffeic acid had together with
the control emulsion, the shortest lag phases of 3 days fol-
lowed by the emulsion with caffeic acid (6 days). The longest
lag phase was observed in emulsions with octyl and oleyl
dihydrocaffeate, which continued during the entire storage
period (Fig. 2F). According to concentration of 2,6-nona-
dienal at Day 12, the order of emulsions was as follows: oleyl
and octyl dihydrocaffeatea<caffeic and dihydrocaffeic acid-
sb<controlc<oleyl alcohold. In summary, the difference for
the different volatiles was the length of the lag phase, whereas
the ranking of the emulsions according to volatile concen-
trations was more or less similar for the different volatiles.
Similar to the PV, the inhibition percentages were calcu-
lated, however only at the end of the storage and for the
concentration of 1-penten-3-one when it was highest at day 9
and 6 for Experiment 1 and 2, respectively. The inhibition
percentage from Experiment 1 (Table 4) clearly showed that
rutin laurate followed by oleyl dihydrocaffeate were the most
efficient antioxidants in inhibiting the formation of volatiles
compared to other antioxidants. Depending on the volatile, it
was different whether it was rutin or rutin palmitate that was
more efficient. For 1-penten-3-ol and 2,4-heptadienal rutin
was more efficient compared to rutin palmitate, whereas
for 1-penten-3-one, 2-hexenal and 2,6-nonadienal it was
Table 4. Calculated inhibition percentages of volatile concentration in the different milk emulsions in both storage experiments
Milk emulsions
Inhibition of volatiles at day 12 (%)
1-Penten-3-onea) 1-Penten-3-ol 2-Hexenal 2,4-Heptadienal 2,6-Nonadienal
Experiment 1
Rutin 17 48 22 25 25
Rutin laurate 76 78 48 63 65
Rutin palmitate 39 41 26 14 34
Dihydrocaffeic acid 32 59 38 56 55
Oleyl dihydrocaffeate 52 66 41 60 b)
Experiment 2
Caffeic acid 69 45 31 32 47
Dihydrocaffeic acid 18 8 8 20 37
Oleyl dihydrocaffeate 75 66 43 54 b)
Octyl dihydrocaffeate 80 77 54 46 b)
Oleyl alcohol �13 �26 �25 �7 �55
The values were calculated according to the control emulsions from the respective experiment.a) For development of 1-penten-3-one the inhibition percentages were calculated at the day with highest concentration before decreasing, that
means that it was calculated at day 9 for experiment 1 and day 6 for experiment 2.b) This indicate that the specific volatile was not detected in this emulsion and therefore the inhibition percentage was not calculated.
Eur. J. Lipid Sci. Technol. 2012, 114, 434–445 Antioxidant efficacy of lipophilized phenolics 441
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opposite. For Experiment 2 it is clear from the inhibition
percentages that both dihydrocaffeate esters were very effec-
tive in inhibiting the formation of volatiles. However, octyl
dihydrocaffeate was more efficient regarding the inhibition of
1-penten-3-one, 1-penten-3-ol and 2-hexenal, whereas oleyl
dihydrocaffeate was more efficient in inhibiting the formation
of 2,4-heptadienal. When comparing the efficiency of the
phenolics, caffeic acid was more efficient as antioxidant than
dihydrocaffeic acid. The impurity, oleyl alcohol, clearly
showed prooxidative behaviour in the milk emulsion.
Inhibition percentages in milk emulsions with the same anti-
oxidants applied in both experiments; dihydrocaffeic acid,
oleyl dihydrocaffeate, indicated better inhibition of volatile
development in Experiment 1 with acetone added for dihy-
drocaffeic acid emulsion, and in general no difference in the
inhibition percentages by oleyl dihydrocaffeate in the two
experiments. Hence, the addition of acetone seemed to have
no significant impact on the development of volatiles, which
was also concluded from the formation of peroxides. The
inhibition percentages indicate that three of the lipophilized
antioxidants were better than the phenolic they originated
from.
3.3.1 Tocopherol in the fish oil enriched emulsions
Three of the tocopherol homologues were detected (a-, g-
and d-tocopherol), but only the concentration of a-toco-
pherol changed significantly during the storage period
(Fig. 3). This might be due to the higher amount of this
homologue compared with the others. The concentration of
a-tocopherol was reduced most in the milk emulsion without
antioxidant added (control emulsion) and least in emulsion
with rutin laurate followed by oleyl dihydrocaffeate in
Experiment 1 and caffeic acid and oleyl dihydrocaffeate fol-
lowed by octyl dihydrocaffeate in Experiment 2. Hence,
tocopherol was better preserved in these emulsions maybe
due to less lipid oxidation.
3.3.2 Sensory evaluation
As volatiles cause off-flavour, the preliminary sensory evalu-
ation of emulsions was expected to reflect the result of volatile
development in the emulsions. Only milk emulsions in
Experiment 2 was evaluated by their odour by a sensory
expert panel at storage day 9 and 12 (data not shown).
Experiment 1 was not possible to evaluate due to addition
of the acetone, which resulted in a strong solvent odour.
Clearly, the most oxidized emulsion according to the sensory
evaluation was the emulsion with oleyl alcohol due to a clear
off-odour of oxidized fish oil at Day 9 which developed to
lacquer/painty at day 12.Moreover, the control emulsion had
a clear fishy off-odour as well as metallic odour at day 9. The
most oxidatively stable emulsions, as determined by odour
evaluation, were emulsions with oleyl and octyl dihydrocaf-
feate. These two emulsions were evaluated to have ocean and
chemical/flower odour, respectively. The expert panel could,
however, not discriminate between these emulsions with
respect to their level of fishy off-odour.
4 Discussion
4.1 Physical stability and lipid oxidation
All the milk emulsions were physically stable and no changes
in the droplet size during storage were observed. In
Experiment 1 there was a significant difference in size of
the lipid droplets in the control emulsion (0.45 mm) com-
pared to the other emulsions (0.59–0.74 mm). This could be
due to either less lipid incorporated in the emulsion or an
unintended slightly different homogenization than for the
other emulsions in this experiment. However, it is clear from
Table 2 that the emulsion had the same amount of fish oil as
the other emulsions. Thus, the smaller droplets may indicate
a slightly longer homogenization of this emulsion. Lipid
oxidation is initiated at the interface of the droplet, therefore
the size of total interfacial area is hypothesized to influence
the lipid oxidation. Thus, increased droplet size may result in
decreased lipid oxidation [9]. However, the literature in this
Figure 3. Concentration of a-tocopherol in the different fish oil
enriched emulsions at day 0 and 12 (A) Experiment 1 and (B)
Experiment 2. Error bars indicate SD of the measurements
(n ¼ 4). Different letterswithin same storage day indicate significant
differences in concentration betweenemulsions. Abbreviation: Rutin
C12, rutin laurate; Rutin C16, rutin palmitate; DHCA, dihydrocaffeic
acid; DHCA C18:1, oleyl dihydrocaffeate; DHCA C8, octyl
dihydrocaffeate.
442 A.-D. M. Sørensen et al. Eur. J. Lipid Sci. Technol. 2012, 114, 434–445
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area is conflicting, and some studies support the hypothesis
[4, 28], whereas in milk emulsions the opposite has been
observed [29, 30]. Therefore, the increased oxidation in the
control emulsion was most likely due to no protection of the
lipids by antioxidants and not due to the different droplet size.
Furthermore, the control emulsion in Experiment 2 had the
same droplet size as the other emulsions, but was still the
most oxidized emulsion together with emulsion with oleyl
alcohol. Hence, the results indicated limited effect of inter-
face area on lipid oxidation in complex emulsion systems.
4.2 Oxidative stability of the milk emulsions and theinfluence of alkyl chain length
The results indicated thatmilk emulsions without antioxidant
or with oleyl alcohol added generally oxidized faster than
emulsions with either phenolics or phenolipids added.
Interestingly, rutin laurate was a more efficient antioxidant
compared to rutin palmitate and rutin it-self, which indicated
a cut-off effect related to the alkyl chain length. However, for
lipophilized dihydrocaffeic acid it was less clear whether such
cut-off effect existed, as differences in oxidation parameters
not always was significantly different for octyl and oleyl
dihydrocaffeate emulsions.
In emulsion with rutin laurate, PV did not increase during
storage, which could indicate efficient inhibition by rutin
laurate or fast decomposition of peroxides to volatiles. The
findings that milk emulsion with rutin laurate added had low
concentrations of volatiles showed that, rutin laurate effec-
tively inhibited formation of both primary and secondary
oxidation products. Interestingly, the findings regarding
the efficacy of rutin esters as antioxidants were different in
milk emulsions compared with earlier findings in simple o/w
emulsion [13]. Thus, in this study on fish oil enriched milk,
both rutin esters had an antioxidative effect, but rutin laurate
was a more efficient antioxidant than rutin palmitate and
rutin it-self. In contrast, rutin laurate and rutin palmitate
were less effective antioxidants when compared with rutin
in simple o/w emulsion [13]. However, rutin laurate also
exerted stronger antioxidative activity than rutin and rutin
palmitate in a LDL assay, which is a more complex system
than an o/w emulsion [13]. Thus, these findings indicate that
the cut-off effect is influenced by the system, i.e. simple o/w
emulsion or more complex emulsion systems such as LDL
and milk. In addition, the fish oil enriched milk emulsion
contains proteins and otherminor components, which are not
present in a simple o/w emulsion. These components might
have interacted and influenced the efficacy or location of the
phenolipids. Shahidi and Zhong [15] suggested that not only
the partitioning of the antioxidant influence the efficacy of the
antioxidant, but also the emulsifier in emulsified medium.
This is due to saturation of the interfacial area by emulsifier,
which leaves less interfacial area available for antioxidant
location. Hence, emulsifiers may compete with antioxidants
for localization at the interface, where oxidation is initiated.
Moreover, recent experiments with phenolic compounds and
two different emulsifiers in simple o/w emulsion have shown
interaction of the antioxidants with emulsifier and iron [18].
The phenolic compounds investigated were caffeic acid,
coumaric acid, naringenin and rutin, which were evaluated
for interactions with Citrem and Tween with or without iron
present. Interactions in this study may have influenced the
efficacy and location of the antioxidant.
Both octyl dihydrocaffeate and oleyl dihydrocaffeate
exerted stronger antioxidative effects than dihydrocaffeic acid
in fish oil enriched milk. The efficacy of octyl dihydrocaffeate
was slightly better than oleyl dihydrocaffeate in inhibiting the
formation of some volatiles however for other volatiles it was
not significantly better. These findings are different from the
findings obtained with these phenolipids in a simple o/w
emulsion [17]. In the o/w emulsion system the difference
between octyl and oleyl dihydrocaffeate as antioxidants
was clearer than in the milk emulsions, where octyl dihydro-
caffeate was a significantly better antioxidant than oleyl
dihydrocaffeate. Importantly, in both studies oleyl dihydro-
caffeate contained impurities such as oleyl alcohol. When
added in the milk emulsion oleyl alcohol resulted in a proox-
idative effect in contrast to its addition in the simple o/w
emulsion. Since oleyl alcohol acted as a prooxidant in fish oil
enrichedmilk it might have reduced the antioxidative effect of
oleyl dihydrocaffeate as antioxidant. Thus, the antioxidative
effect may have been even better for oleyl dihydrocaffeate
than octyl dihydrocaffeate if a more purified compound was
used, but this needs to be further investigated. The slight
difference in results obtained from the o/w emulsion and milk
emulsion may, similar to effects of rutin esters, be explained
by the different systems tested. For example the effect of
emulsifier and its position on the interface may have led to
interactions with the antioxidant [15].
In vitro antioxidant assays preformed in our previous
studies have generally showed better antioxidant activity
for the original phenolic than for the lipophilized phenolic
compound [12, 17]. However, in emulsions the dihydracaf-
feates were better antioxidants than dihydrocaffeic acid in
both simple o/w emulsions and milk emulsions, and lipophi-
lized rutin was better than rutin in milk emulsions. Thus, the
results from in vitro antioxidant assays cannot solely predict
the antioxidant properties in emulsion systems, and other
factors such as interactions and partitioning may also influ-
ence antioxidant activity. The partitioning of lipophilized
dihydrocaffeic acid and rutin in the different phases has
previously been evaluated [13, 17]. Only rutin laurate could
be measured in the water phase of a simple o/w emulsion and
only in small amounts (3.8%). The finding that lipophilized
rutin did not have any antioxidative effect in o/w emulsions
compared to rutin, whereas lipophilization with C12
improved its efficacy in milk indicates that interactions with,
e.g. proteins may influence the location of the lipophilized
rutin in milk emulsions. Thus, rutin laurate may be located
more favourably in the milk emulsion to act as antioxidant,
Eur. J. Lipid Sci. Technol. 2012, 114, 434–445 Antioxidant efficacy of lipophilized phenolics 443
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i.e. close to the interface instead of as a micelle in the water
phase or in the core of the oil droplets. A similar explanation
could be given for the differences observed with oleyl dihy-
drocaffeate. However, further research is needed in order to
be able to conclude on possible interaction in the milk and
changed partitioning of the phenolipids as a consequence of
the interaction.
4.3 Fish oil enriched milk and human health
Overall it was possible to stabilize fish oil enriched milk by
adding phenolipids. The current amount of fish oil in this
milk will only be a supplement of n-3, since 500 mL milk
daily will result in an intake of 50 mg EPA and DHA. EU
recommendation is currently at an intake of 250 mg daily due
to proved health beneficial effects, thus 500 mL milk daily
will cover 20% of the recommendeted. Consumption of n-3
enriched milk solely may not increase the human health, but
together with other fish or fish oil enriched products.
5 Conclusions
In conclusion, both phenols and phenolipids acted as anti-
oxidants in milk emulsions enriched with fish oil. However,
the phenolipids were more efficient antioxidants especially
rutin laurate, octyl and oleyl dihydrocaffeate. Despite the fact
that only two chain lengths were evaluated, the results tend to
follow the cut-off effect in relation to alkyl chain length and
antioxidative effect for rutin esters. The optimal alkyl chain
length for a rutin ester in fish oil enrichedmilk is at least below
a chain length of 16 carbon atoms. For dihydrocaffeate esters
it was not possible to conclude on a specific cut-off effect in
relation to alkyl chain length and antioxidative effect. To be
able to conclude on the optimal lipid chain length attached to
the phenolic compounds in relation to their strongest anti-
oxidant protection, more studies on the effect of rutin and
dihydrocaffeic acid with different lipid chain lengths are
needed. The antioxidative effect of phenolipids seems quite
complex and it would be of great value to be able to under-
stand the effects of both the lipid chain length and the type of
emulsion system on the antioxidative effect of the lipophilized
compounds. Taken together, these results clearly show that the
polar paradox hypothesis is too simple and must be reconsid-
ered. Moreover, the almost untouched area of phenolipids as
antioxidants in real food systems deserves more attention as the
results have indicated very promising effects of these com-
pounds that could be utilized by the industry in the future.
We thank Maritex Norway (subsidiary of TINE BA, Norway)
for providing the fish oil to our research. The study was financed by
the Danish Council for Strategic Research (Programme committee
for food, nutrition and health) and the Directorate for food,
Fisheries and Agri Business.
The authors have declared no conflict of interest.
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