LWT - Food Science and Technology 47 (2012) 167e174

Contents lists available at SciVerse ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Use of oregano extract and oregano essential oil as antioxidants in functional

dairy beverage formulations
Marcela Boroski a, Hélène J. Giroux b, Hassan Sabik b, Hélène V. Petit c, Jesui V. Visentainer a,
Paula T. Matumoto-Pintro a, Michel Britten b, *
a
Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR 87020-900, Brazil
b
Food Research and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Boulevard West, Saint-Hyacinthe, QC J2S 8E3, Canada
c
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, P.O. Box 90, Sherbrooke, QC J1M 1Z3, Canada

a r t i c l e i n f o a b s t r a c t

Article history: To increase health benefits, dairy beverages can be enriched with omega-3 fatty acids, although these
Received 6 July 2011 fatty acids are susceptible to oxidation. The use of oregano extract (OE) and oregano essential oil (OEO) as
Received in revised form antioxidants in dairy beverages enriched with 2 g/100 g linseed oil was studied. The OE contained
1 November 2011
269 mg gallic acid equivalents per gram and was shown by the DPPH radical assay to have powerful
Accepted 5 December 2011
antioxidant activity. During 10 days of storage under light or heat conditions, color, conjugated diene
hydroperoxides, hexanal, propanal, and dissolved oxygen concentrations were evaluated in dairy
Keywords:
beverages containing various concentrations of OE or OEO. It was found that OE was more efficient than
Origanum vulgare
Origanum minutiflorum
OEO in preventing the formation of conjugated dienes, hexanal, and propanal as well as the depletion of
Lipid oxidation oxygen induced by heat or light oxidation. The addition of OE or OEO did not affect dairy beverage
Dienes physical stability during storage.
Hexanal Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Propanal
Dairy beverages

1. Introduction
Polyunsaturated fatty acids of the n-3 family (n-3 PUFA) have
received special attention in recent years because of their potential
health benefits for the consumer. Greater intake of n-3 PUFA has
been associated with a reduced risk of coronary heart disease (Kris-
Etherton, Harris, & Appel, 2002) and the prevention of prostate,
colorectal, and breast cancer (Shahidi & Miraliakbari, 2004).
Constituents of the membrane phospholipids of human cells, n-3
PUFA have been recommended for preventing and curing inflam-
matory and degenerative diseases (Simopoulos, 1991). Recently,
clinical studies have shown that omega-3 (n-3) fatty acids may be
useful in the prevention and treatment of epilepsy (Scorza et al.,
2008). Therefore, the incorporation of n-3 fatty acids into dairy
products (Bourre, 2005; Giroux, St-Amant, Fustier, Chapuzet, &
Britten, 2008; Goodridge, Ingalls, & Crow, 2001), such as the addi-
tion of fish oil to processed cheese (Ye, Cui, Taneja, Zhu, & Singh,
2009), yogurts, butter, and cream (Kolanowski & Weibbrodt,
* Corresponding author. Tel.: þ1 450 768 3235; fax: þ1 450 773 8461.
E-mail address: michel.britten@agr.gc.ca (M. Britten).
2007), has been studied with a view to providing consumers with
these health benefits.
One shortcoming of this approach is the high sensitivity of the n-
3 PUFA family to oxidation during processing and storage. Oxidation
can change the nutritional and sensory properties of dairy bever-
ages. Depending on the storage conditions, two main reactions can
affect lipids. Autoxidation is a free radical chain process occurring
spontaneously or at moderate temperatures in the presence of
molecular oxygen (3O2). Although the reaction between triplet
oxygen and fatty acids is thermodynamically unfavorable (Frankel,
2005), heat, transition metals, and light can accelerate fatty acid
free radical formation (Choe & Min, 2006). Under light exposure in
the presence of sensitizers such as metals or chlorophylls, triplet
oxygen can form singlet oxygen (1O2), a powerful radical generator
that reacts directly with lipids (Choe & Min, 2006). These reactions
can severely affect dairy product quality.
Polyphenols from plant extracts have been used in different
food matrices to improve the oxidative stability of food lipids. At
the time of submitting this work, only a few studies have looked at
the addition of antioxidants to minimize oxidative reactions in
dairy products (Giroux, Houde, & Britten, 2010; Jacobsen, Let,
Nielsen, & Meyer, 2008; Jacobsen, Let, Sørensen, et al., 2008; Let,
Jacobsen, & Meyer, 2004).

0023-6438/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2011.12.018
168 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174
The aim of this study was to evaluate the efficiency of extract
of oregano (Origanum vulgare) and essential oil of oregano
(Origanum minutiflorum) in increasing the antioxidant capacity of
dairy beverages enriched with 2 g/100 g linseed oil and reducing
lipid oxidation during storage under different heat and light
conditions.
2. Materials and methods
2.1. Material
Dried oregano and oregano essential oil (OEO) were purchased
from a local market in Saint-Hyacinthe, QC, Canada. The plant
material was milled and stored at 20  C.
2.2. Oregano extract preparation
For the oregano extract (OE), 50 g oregano powder was infused
for 10 min in 1 L hot water (80  C). The infusion was stirred occa-
sionally and centrifuged at 3200g for 6 min. Following filtration
with Whatman No. 1 filter paper to remove any residuals, the
infusion was quickly cooled, freeze-dried, and stored at 20  C. The
OE was prepared in triplicate.
2.3. FolineCiocalteu assay
Total phenolics in the OE were determined according to
Singleton and Rossi (1965) with the following modifications. The
freeze-dried OE was dispersed in methanol at a concentration of
2.50 mg/mL. A 0.25 mL aliquot of extract solution in methanol was
mixed with 0.25 mL Folin-Ciocalteu reagent (previously diluted
with water; dilution factor ¼ 2), 0.50 mL of a saturated sodium
carbonate solution, and 4.0 mL water. The mixture was left at room
temperature in darkness for 30 min and then centrifuged at
3200g for 6 min. The supernatant absorbance was measured at
725 nm. A standard curve was prepared using gallic acid, and the
results were expressed as milligrams of gallic acid equivalents per
gram of extract (mg GAE/g).
2.4. DPPH assay
The antioxidant activity of OE and OEO was studied by evalu-
ating their free-radical-scavenging effect on the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical based on the method proposed by
El-Massry, El-Ghorab, and Farouk (2002) with slight modifications.
The OE or OEO was dispersed in 10 mL methanol at a concentration
of 2.0 mg/mL. Different aliquots of this solution were mixed with
2.0 mL DPPH methanolic solution (47 mg/mL). The mixture was
thoroughly vortexed and kept in the dark for 30 min. Absorbance
was then measured at 517 nm against a methanol blank without
DPPH. The results were expressed as the percentage inhibition of
the DPPH radical (% Inhibition DPPH), which was calculated
according to the following equation:
 
AbsDPPH  Abssample


% Inhibition DPPH ¼  100
AbsDPPH 
where AbsDPPH is the absorbance of the DPPH solution without OE
or OEO, and Abssample is the absorbance of the DPPH solution with
OE or OEO after 30 min. The analyses were carried out in triplicate,
and the OE or OEO concentration providing 50% inhibition (IC50)
was obtained by plotting concentrations versus percentage
inhibition.
2.5. Dairy beverage preparation
Low-heat skim milk powder (35.3 g/100 g protein; Agropur,
Granby, QC, Canada) was dispersed in deionized water to a protein
concentration of 3.5 g/100 g and supplemented with Fe (as FeSO4)
at a level of 0.001 g/100 g as described by Giroux, Acteau, Sabik, and
Britten (2008). Sodium azide (0.02 g/100 g) was used as a microbial
growth inhibitor. The OE was added at three concentrations (0.001,
0.01, and 0.1 g/100 g), and each mixture was stirred for 15 min. After
overnight storage at 4  C, the pH was adjusted to 6.7. The dairy
beverages were enriched with pure linseed oil (Orphée, La Maison
Orphée Inc., Quebec City, QC, Canada) for a final concentration of
2 g/100 g. The preparations (40  C) were pre-emulsified for 3 min at
8000 rpm using an Ultra-Turrax T25 homogenizer (IKA, Staufen,
Germany) fitted with an S25KV-25F dispersing tool and homoge-
nized with a single-stage Emulsiflex-C5 homogenizer (Avestin,
Ottawa, ON, Canada). Homogenization pressure was set to 20 MPa
for the first two passes and to 3.5 MPa for the third pass. The
samples were batch-pasteurized at 63.5  C for 30 min in stainless
steel cups. For the dairy beverages enriched with OEO, the oil was
mixed directly with the linseed oil before homogenization for
a final concentration of 0.001, 0.01, or 0.1 g/100 g in the beverages.
A control dairy beverage (without OE or OEO) was also prepared.
2.6. Storage conditions
The dairy beverages were stored under two different conditions:
(1) at an elevated temperature, in a storage cabinet maintained at
50  C; and (2) under light exposure using a fluorescent light (warm
white, 60 W fluorescent lamps, General Electric, Cleveland, OH) at
4  C. For the light exposure, glass tubes (8.50  2.0 cm) containing
6.0 mL dairy beverage were capped and laid horizontally at 30 cm
from the light source. Under both storage conditions, samples were
taken after 0, 1, 2, 3, 4, 6, 8, and 10 days for conjugated diene
analyses, and after 0, 3, 6, and 10 days for hexanal, propanal, and
oxygen analyses.
2.7. Dairy beverage color
Hunter L*, a*, and b* values were measured in a HunterLab
spectrocolorimeter (Labscan 2 tristimulus, Hunter Associates
Laboratory, Inc., Reston, VA) set for illuminant D-65 and an illu-
mination area measuring 4.5 cm in diameter. The difference
between the L*, a*, and b* values of the dairy beverages containing
OE or OEO and the control dairy beverage was used to calculate the
total color difference, according to the following equation:
 1=2
Total color difference ¼ DL*2 þ Da*2 þ Db*2 (2)
2.8. Physical stability
The stability of the dairy beverages was analyzed using a Beck-
man Coulter QuickSCAN analyzer (Coulter Corporation, Miami, FL).
The sample (7 mL) was placed in a glass tube with a 16-mm internal
diameter and scanned by a light source (880 nm) after 0, 7, 14, 28, and
50 days of storage in darkness at 4  C. The backscattering intensity
was recorded as a function of sample height. The change in back-
(1)
scattering intensity at the top (BSmax) and in the middle (BS35mm) of
the sample during storage was used to monitor phase separation.
2.9. Lipid oxidation
2.9.1. Conjugated dienes
Conjugated diene (CD) hydroperoxides were evaluated after 0, 1,
2, 3, 4, 6, 8, and 10 days of storage according to Kiokias, Dimakou,
 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174
Tsaprouni, and Oreopoulou (2006) with slight modifications.
Samples (25 mL) of the dairy beverages were diluted in 2.5 mL
isooctane:2-propanol (2:1 mL/mL). After mixing for 30 s in a cap-
ped plastic tube, the contents were filtered through a 0.45 mm PVDF
filter. The absorbance of the filtrate was measured at 232 nm
against an isooctane:2-propanol blank. The CD concentration was
calculated using the molar absorptivity of linoleic acid (ε ¼ 26 000)
and expressed as millimoles per kilogram of fat.
2.9.2. HS-SPME-GC-MS analyses
Propanal and hexanal formation in the dairy beverages
was evaluated after 0, 3, 6, and 10 days of storage using headspace
solid-phase microextraction (HS-SPME) combined with gas
chromatographyemass spectrometry (GCeMS). Samples (3 g) of
the dairy beverages were sealed in 10 mL amber vials. The SPME
fiber (85 mm Carboxen/PDMS, Supelco, Oakville, ON, Canada) was
inserted into the headspace of the vial and left there for 44 min at
40  C. Volatile compounds were desorbed by inserting the fiber into
the injection port of a Varian CP-3800 gas chromatograph (Palo
Alto, CA) operated in splitless mode for 3 min at 300  C. Helium was
used as carrier gas with a constant flow rate of 1 mL min1. The
compounds were separated on a VF-5ms column (equivalent to DB-
5ms; Varian, Mississauga, ON, Canada) measuring 30  0.25 mm
and with a 25-mm film thickness. The oven temperature program
began with 3 min at 35  C, followed by a 6  C min1 increase to
80  C, a 20  C min1 increase to 280  C, and 2 min at 280  C. A
Saturn 2000 mass spectrometry detector (Varian) was used and
detection was carried out on the total ion current obtained by
electron impact at 70 eV. The selected ions were 55, 57, and 58 for
propanal and 99 for hexanal. The mass range acquisition was m/z
30e250. Retention time and mass spectra ions were obtained for
each pure commercial standard (SigmaeAldrich, Oakville, ON,
Canada). Calibration curves were prepared using hexanal and
propanal standards (SigmaeAldrich).
2.10. Dissolved oxygen
During storage, dissolved oxygen concentration (mg/L) in the
dairy beverages was determined using an Orion 850 Aplus dis-
solved oxygen meter (Thermo Electron Corp., Beverly, MA).
2.11. Statistical analysis
The dairy beverages were prepared in triplicate according to
a split-plot factorial design with concentration in the main plot and
exposure time to heat or light in the subplot. Variance analysis was
used to determine whether the factors and their interactions had
a significant effect on the measured parameters (SAS Institute Inc.,
Cary, NC).
3. Results and discussion
3.1. Antioxidant activity
The average extraction yield from dried oregano leaves using
water at 80  C was 25.4  0.6 g/100 g. A similar yield (26.2 g/100 g)

was reported by Skerget et al. (2005) using methanol dispersion
and incubation for 2 h in an ultrasonic bath. The phenolic content of
the extract was 269 mg GAE/g, which agrees with the values found

by Sahin et al. (2004) and Skerget et al. (2005) (220 and 186 mg
GAE/g, respectively) but is higher than the 39.4 mg GAE/g obtained
by Chun, Vattem, Lin, and Shetty (2005).
The antioxidant activity of oregano was evaluated against the
DPPH radical to measure the ability of the OE to donate hydrogen
to stabilize the radical, as assessed spectrophotometrically. The OE
169
showed a high potential to inhibit the DPPH radical, with IC50
values of 26.7  0.9 mg/mL. This result is in agreement with that of
Boroski et al. (2011) although a potential less than 9.9 mg/mL was
reported by Sahin et al. (2004). Rosmarinic acid, caffeic acid,
coumaric acid, quercetin, and carvacrol are the compounds
responsible for the antioxidant activity of oregano (Exarchou et al.,
2002).
The IC50 value for the OEO was higher than 500 mg/mL. A similar
result was previously reported (Sahin et al., 2004). The non-polar
character of the antioxidants present in OEO may explain the
poor efficiency in stabilizing the hydrophilic DPPH radical. Carva-
crol, thymol, and other monoterpene hydrocarbons were previ-
ously identified as the major antioxidants in OEO (Kulisic, Radonic,
Katalinic, & Milos, 2004).
3.2. Dairy beverage color
The initial Hunter L*, a*, and b* parameters in the dairy bever-
ages containing OE or OEO were measured (Table 1). The color
parameters gradually changed as the OE concentration increased.
The lightness of the dairy beverages (L* value) decreased from 86 to
81 with increasing OE concentration from 0 to 0.1 g/100 g. This
result was expected, considering the dark color of the extract. Both
the a* and b* parameters increased in parallel with OE concen-
tration. Schamberger and Labuza (2007) reported that the addition
of green tea polyphenols to milk increased redness (a* values).
Similar trends were observed in the present experiment with
increased OEO concentration, although the differences were not
statistically significant (P > 0.05).
Color parameters were monitored during storage, and the total
color difference was calculated. The total color difference is a single
value that takes into account the difference between the L*, a*, and
b* values of the sample and thus assesses global changes in the
samples. Storage of the control dairy beverage at 50  C increased
the total color difference, with a value of 9.4 after 10 days (Fig. 1).
Maillard reactions between milk proteins and lactose were likely
responsible for the color change in the dairy beverages. According
to Fig. 1a, OE was efficient in preventing color change at 50  C,
especially at high concentrations (P < 0.05). Supplementing dairy
beverages with 0.1 g/100 g OE reduced the total color difference by
a factor of 4.8 after 10 days when compared to the control.
Schamberger and Labuza (2007) showed that the addition of green
tea flavonoids to milk reduced Maillard browning, and a similar
effect was expected from the phenolic compounds contained
in OE.
The OEO also prevented color changes in the beverages during
storage at 50  C (Fig. 1b). When OEO was used at 0.1 g/100 g, the
total color difference in the dairy beverages was reduced by half
after 10 days. Under light exposure storage, neither OE nor OEO had
an effect on beverage total color change (P > 0.05).
Table 1
Hunter L*, a*, and b* values for dairy beverages containing oregano extract (OE) or
oregano essential oil (OEO).
L* a* b*
Control 86.4ab 3.48a 6.97a
OE 0.001 g/100 g 85.9ab 3.68a 8.74a
OE 0.01 g/100 g 85.6b 3.49a 8.40a
OE 0.1 g/100 g 81.2c 2.87b 12.77b
OEO 0.001 g/100 g 87.0a 3.47a 7.50a
OEO 0.01 g/100 g 86.5ab 3.46a 7.07a
OEO 0.1 g/100 g 86.4ab 3.41a 7.00a
Values with the same letter within a column are not significantly different at
P > 0.05.
170 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174

10 a 10 b

Color difference

Color difference
8 8

6 6

4 4

2 2

0 0
4 10
6 10 6 10
Time (days) Time (days)

Fig. 1. Effect of the concentration of oregano extract (a) and oregano essential oil (b) on total color difference in dairy beverages during storage at 50  C. Control,
0.001 g/100 g, 0.01 g/100 g, 0.1 g/100 g.

3.3. Physical stability
Dairy beverages enriched with linseed oil consist of a complex
dispersion of fat globules, casein micelles, and minerals. During
storage, changes in homogeneity, particle size, and interactions
might induce phase separation (Durand, Franks, & Hosken, 2003).
Those changes can be monitored by means of a backscattering
profile as a function of time. In the dairy beverages (Fig. 2), a slight
increase in the backscattering intensity at the top of the sample
(BSmax), corresponding to the formation of a cream layer, was
observed during the first week of storage. The backscattering
intensity in the middle of the sample (BS35mm) decreased slightly
during storage as a result of fat droplets moving to the top of the
sample. The addition of OE or OEO at the highest concentrations did
not affect dairy beverage stability (Fig. 2). For comparison purposes,
the physical stability of commercial homogenized milk with 2 g/
100 g fat was evaluated after 14 days of storage under the condi-
tions examined in the present study. The commercial milk showed
a phase separation pattern similar to that of the dairy beverages
enriched with 2 g/100 g linseed oil. The BSmax of the commercial
milk increased by 21% compared to 15% for the dairy beverages
during 14 days of storage.
3.4. Conjugated dienes
Dairy beverages enriched with linseed oil (2 g/100 g) contain
about 1.06 g/100 g linolenic acid (18:3 n-3), 0.56 g/100 g linoleic
100
80
Backscattering (%)
acid (18:2 n-6), and 0.30 g/100 g oleic acid (18:1 n-9). The high
PUFA content makes these beverages susceptible to oxidation.
Conjugated diene hydroperoxides are produced during the first
phase of lipid oxidation by the reaction between peroxyl radicals
and oxygen (3O2). The autoxidation of linoleic and linolenic acids
produces conjugated products (Choe et al., 2006), which absorb UV
light at 232 nm, and their second oxidation products, such as
ketones, which absorb at 272 nm (Luzia & Jorge, 2009). In the
control dairy beverage, CDs were formed after light exposure or heat
during storage. Both light and elevated temperature induced CD
formation in the control dairy beverage, but the concentration was
much higher for the samples exposed to light than for those stored
at 50  C (Fig. 3). During storage at elevated temperatures, Maillard
reactions occur, and the resulting products act as antioxidants and
increase the stability of lipids (Frankel, 2005; Giroux et al., 2010).
Furthermore, at high temperatures or in the presence of metal, CDs
are readily broken into smaller compounds such as aldehydes,
ketones, acids, esters, and alcohols (Choe & Min, 2006).
In the experiment using OE, antioxidant concentration, storage
time, and storage conditions significantly affected (P < 0.05) CD
formation (Fig. 3). In the light-induced oxidation experiment,
a gradual rise in CD concentration was observed over the 10 day
storage period (Fig. 3a). The formation of CDs was slightly reduced
at 0.001 g/100 g OE and strongly inhibited at concentrations greater
than or equal to 0.01 g/100 g. Compared to the control, the use of
0.01 g/100 g OE reduced CD formation after 6 days by 88%. Under
light exposure, the presence of sensitizers and transition catalytic
metals favored the reaction of 3O2 to form singlet oxygen (1O2)
(Frankel, 2005), which reacts directly with the high-electron-
density double bonds of PUFA (Choe & Min, 2006). For the
prevention of these reactions, antioxidants from OE might act as
singlet oxygen quenchers or metal chelators.

During storage at a high temperature, the CD concentration in

the control dairy beverage increased from 24 to 79 mmol/kg after
60 10 days (Fig. 3b). Increasing the OE concentration gradually reduced
CD formation during storage. At 0.1 g/100 g OE, CD formation was
40 totally inhibited.
In the dairy beverages prepared with OEO, antioxidant
concentration, storage time, and storage conditions significantly
20
affected (P < 0.05) CD formation (Fig. 4). At the highest concen-
tration tested (0.1 g/100 g), OEO reduced light-induced CD forma-
0 tion by about 50% after 6 days of storage (Fig. 4a). After 10 days,
0 10 20 30 40 50 however, CD concentration was close to that in the control dairy
Time (days) beverage. When used at lower concentrations, OEO had very little
effect on light-induced CD formation. The OEO reduced CD
Fig. 2. Backscattering of dairy beverages (with 2 g/100 g linseed oil) containing oregano
extract (OE) or oregano essential oil (OEO) at the top (BSmax) of the sample: (-) control,
formation during storage at 50  C but only at the highest concen-
(C) OE 0.1 g/100 g, (:) OEO 0.1 g/100 g; and in the middle (BS35mm) of the sample: (;) tration (Fig. 4b). After 6 days, CD concentration was reduced by 65%
control, (A) OE 0.1 g/100 g, (=) OEO 0.1 g/100 g, as a function of storage time. when compared to the control dairy beverage. However, as
 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174 171

400
a 400
b
350 350
300 300

[CD] mmol/kg fat

[CD] mmol/kg fat

250 250
200 200
150 150
100 100
50 50
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Fig. 3. Changes in the conjugated diene (CD) concentration during storage of dairy beverages (with 2 g/100 g linseed oil) containing oregano extract (OE): (-) control,
(C) 0.001 g/100 g, (:) 0.01 g/100 g, (;) 0.1 g/100 g. Beverage samples were exposed to light (a) or stored at 50  C (b).

observed for storage under light exposure (Fig. 4a), CD concentra-
tion increased by 10 days of storage.
Phenolic compounds from OE were more effective in protecting
linseed oil than compounds from OEO, a finding that can be
explained by antioxidant chemistry. The polar characteristic of
phenolic compounds allows them to better act as antioxidants in
the aqueous phase of the emulsion compared to OEO compounds.

Skerget et al. (2005) studied the behavior of OE in oils and emul-
sions and observed a more protective effect in emulsions than in
apolar systems. Thus, OE may act as an antioxidant in dairy
beverages enriched with omega-3 fatty acids.
3.5. Dissolved oxygen
The initial oxygen concentration in the control dairy beverages
was 6.4  0.6 mg/L. Rapid oxygen depletion was observed during
the first 3 days of storage at 50  C or of exposure to light. Similar
results were obtained previously (Giroux, Acteau, et al., 2008;
Giroux, St-Amant, et al., 2008).
Fig. 5 shows the changes in dissolved oxygen during the storage
of dairy beverages prepared with OE. Antioxidant concentration
and storage time significantly affected dissolved oxygen concen-
tration (P < 0.05) (Fig. 5). Under light or heat exposure, an increase
in antioxidant concentration slowed down oxygen depletion
during storage. At a 0.1 g/100 g concentration, OE totally prevented
oxygen depletion for 6 days in dairy beverages exposed to light
(Fig. 5a), and the oxygen concentration remained higher than in the
control dairy beverage at the end of the storage period. The oxygen
depletion pattern in the control dairy beverage stored at 50  C
(Fig. 5b) was similar to that observed under light exposure (Fig. 5a).
Concentrations of OE greater than 0.01 g/100 g were required to
slightly retard oxygen depletion in the beverages exposed to heat.
After 6 days of storage at 50  C, the oxygen concentration was
independent of OE concentration and similar to that in the control
(Fig. 5b).
The OEO concentration, storage time, and storage conditions
had a significant effect (P < 0.05) on dissolved oxygen concentra-
tion in the dairy beverages (Fig. 6). Similar to OE, OEO was more
effective in preventing oxygen depletion in the beverages stored
under light than in the beverages stored at 50  C. Compared to the
result achieved with OE, a much smaller effect on the oxygen
depletion pattern of dairy beverages was observed with OEO, which
could only retard oxygen depletion in the beverages exposed to
light (Fig. 6a) and had almost no effect on the beverages stored at
50  C (Fig. 6b).
Under both storage conditions, OE was more efficient than OEO
in preventing oxygen depletion. The phenolic compounds
(from OE) played an important role in the oxidative stability of the
dairy beverages, preventing oxygen uptake in the early stages of
radical reactions. Different mechanisms might explain this
protection. Polyphenolic compounds might chelate Fe2þ ions and
prevent the generation of singlet oxygen. Kim et al. (2009) found
that the synthetic aromatic antioxidant TBHQ has a strong singlet
oxygen quenching property that occurs based on the charge
transfer mechanism. Kristinová, Mozuraityte, Storrø, and Rustad
(2009) reported an efficiency feature of propyl gallate that
prevents background oxygen uptake in liposome systems.
In a previous study (Giroux, Acteau, et al., 2008; Giroux,
St-Amant, et al., 2008), storage in the dark at 4  C (i.e. no light or
heat) of dairy beverages similar to those used in the present
experiment resulted in no change in dissolved oxygen concentra-
tion as a function of time.

400
a 400
b
350 350
300 300
[CD] mmol/kg fat

[CD] mmol/kg fat

250 250
200 200
150 150
100 100
50 50
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Fig. 4. Changes in the conjugated diene (CD) concentration during storage of dairy beverages (with 2 g/100 g linseed oil) containing oregano essential oil (OEO): (-) control,
(C) 0.001 g/100 g, (:) 0.01 g/100 g, (;) 0.1 g/100 g. Beverage samples were exposed to light (a) or stored at 50  C (b).
172 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174

8 8
a b

Dissolved oxygen (mg/L)

Dissolved oxygen (mg/L)
6 6

4 4

2 2

0 0

0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Fig. 5. Changes in the dissolved oxygen concentration during storage of dairy beverages (with 2 g/100 g linseed oil) containing oregano extract (OE): (-) control, (C) 0.001 g/100 g,
(:) 0.01 g/100 g, (;) 0.1 g/100 g. Beverage samples were exposed to light (a) or stored at 50  C (b).

a b 8
8

Dissolved oxygen (mg/L)

Dissolved oxygen (mg/L)

6
6

4
4

2
2

0
0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Fig. 6. Changes in the dissolved oxygen concentration during storage of dairy beverages (with 2 g/100 g linseed oil) containing oregano essential oil (OEO): (-) control,
(C) 0.001 g/100 g, (:) 0.01 g/100 g, (;) 0.1 g/100 g. Beverage samples were exposed to light (a) or stored at 50  C (b).

3.6. GCeMS analyses
Aldehydes are lipid oxidation products that can significantly
affect food sensory properties at very low concentrations, namely
1.6 and 0.15 mg/L for propanal and hexanal, respectively (Frankel,
2005). In the dairy beverages, propanal concentration was higher
than hexanal concentration. Propanal is the result of the oxidation
of linolenic acid, which is a major fatty acid in linseed oil. Hexanal
originates from linoleic acid oxidation.
The dairy beverage preparations were pasteurized at 63.5  C for
30 min. After pasteurization, propanal and hexanal concentrations
in the control beverage were 0.87  0.17 and 0.050  0.011 mg/L,
respectively. The dairy beverages prepared with OE or OEO at 0.1 g/
100 g showed lower concentrations of propanal (w0.20 mg/L for
both OE and OEO) and hexanal (0.028  0.005 and 0.015  0.003 mg/
L for OE and OEO, respectively) immediately after pasteurization.
Antioxidants from OE and OEO reduced hexanal and propanal
production during the pasteurization of the dairy beverages.
Fig. 7 presents the changes in hexanal and propanal concentra-
tions in the control dairy beverage during 10 days of exposure to
light or heat. Increased propanal and hexanal concentrations were
observed during the first 6 days under light exposure, and concen-
trations remained stable until 10 days. During storage at 50  C, the
highest concentrations of propanal and hexanal were reached after 3
days. Although elevated temperature seemed to accelerate initial
aldehyde formation, the hexanal and propanal levels at the end of

5
a 0.4
0,4 b
4
0.3
0,3
Propanal (mg/L)

Hexanal (mg/L)

3
0,2
0.2
2
0,1
0.1
1

0.0
0,0
0
0 2 4 6 8 10 0 2 4 6 8 10
Time (days) Time (days)

Fig. 7. Changes in the propanal (a) and hexanal (b) concentrations in control dairy beverage (with 2 g/100 g linseed oil) during storage. Beverage samples were exposed to light
(-) or stored at 50  C (C). Dashed lines correspond to the threshold values for the sensory perception of oxidation (Frankel, 2005).
 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174 173

100
a 100
b

Propanal inhibition (%)

Hexanal inhibition (%)

80 80

60 60

40 40

20 20

0 0
2
OE – 50 °C OE – Light OEO 4– 50 °C OEO - Light 3 °C OE – 4Light
OE – 50 OEO 5– 50 °C OEO
6 - Light

Fig. 8. Inhibition of the production of propanal (a) and hexanal (b) during the storage of dairy beverages enriched with oregano extract (OE) or oregano essential oil (OEO). Beverage
samples were exposed to light or stored at 50  C. Concentrations of OE or OEO were: 0.001 g/100 g, 0.01 g/100 g, 0.1 g/100 g.

the storage period were similar under both conditions. Final alde- References
hyde concentrations were above the threshold values for the
sensory perception of oxidation (Frankel, 2005). Boroski, M., de Aguiar, A. C., Boeing, J. S., Rotta, E. M., Wibby, C. L., Bonafé, E. G., et al.
(2011). Enhancement of pasta antioxidant activity with oregano and carrot leaf.
Adding OE or OEO to the beverage formulations reduced Food Chemistry, 125, 696e700.
propanal and hexanal production during storage. The inhibition of Bourre, J. M. (2005). Where to find omega-3 fatty acids and how feeding animals
aldehyde production during the last 7 days of storage was calcu- with diet enriched in omega-3 fatty acids to increase nutritional value of
derived products for human: what is actually useful? Journal of Nutrition, Health
lated from the concentration difference with the control and and Aging, 9, 232e242.
expressed as the percentage of inhibition (Fig. 8). Propanal Choe, E., & Min, D. B. (2006). Mechanisms and factors for edible oil oxidation.
production was strongly inhibited as the concentration of OE or Comprehensive Reviews in Food Science and Food Safety, 5, 169e186.
Chun, S. S., Vattem, D. A., Lin, Y. T., & Shetty, K. (2005). Phenolic antioxidants from
OEO increased (Fig. 8a). On average, inhibition was 2.4 times clonal oregano (Origanum vulgare) with antimicrobial activity against Heli-
greater with OE than with OEO (P < 0.05). At low concentrations cobacter pylori. Process Biochemistry, 40, 809e816.
(0.001 g/100 g and 0.01 g/100 g), OE showed better inhibition of Durand, A., Franks, G. V., & Hosken, R. W. (2003). Particle sizes and stability of UHT
bovine, cereal and grain milks. Food Hydrocolloids, 17, 671e678.
propanal production in the beverages exposed to light than in those
El-Massry, K. F., El-Ghorab, A. H., & Farouk, A. (2002). Antioxidant activity
exposed to heat. At the highest OE concentration (0.1 g/100 g), and volatile components of Egyptian Artemisia judaica L. Food Chemistry, 79,
propanal inhibition was similar for both storage conditions and 331e336.
Exarchou, V., Nenadis, N., Tsimidou, M., Gerothanassis, I. P., Troganis, A., &
averaged 92%. At the lowest concentration tested (0.001 g/100 g),
Boskou, D. (2002). Antioxidant activities and phenolic composition of extracts
OEO showed no effect on propanal production in the beverages from Greek oregano, Greek sage, and summer savory. Journal of Agricultural and
exposed to light or heat. At the highest concentration, the inhibi- Food Chemistry, 50, 5294e5299.
tion of propanal production was only approximately 60%. Frankel, E. N. (2005). Lipid oxidation (2nd ed.). Bridgwater, England: The Oily Press.
Giroux, H. J., Acteau, G., Sabik, H., & Britten, M. (2008). Influence of dissolved gases
The inhibition of hexanal production was also measured, and and heat treatments on the oxidative degradation of polyunsaturated fatty
the results are presented in Fig. 8b. The effect of OE and OEO on the acids enriched dairy beverage. Journal of Agricultural and Food Chemistry, 56,
inhibition of hexanal production was quite similar to the observed 5710e5716.
Giroux, H. J., Houde, J., & Britten, M. (2010). Use of heated milk protein-sugar blends
effect on propanal inhibition (Fig. 8a). As a general trend, higher as antioxidant in dairy beverages enriched with linseed oil. LWT-Food Science
inhibition was observed when OE was used compared to OEO. and Technology, 43, 1373e1378.
However, the difference was smaller than that for propanal inhi- Giroux, H. J., St-Amant, J. B., Fustier, P., Chapuzet, J. M., & Britten, M. (2008). Effect of
electroreduction and heat treatments on oxidative degradation of a dairy
bition (Fig. 8a) and is not statistically significant (P > 0.05). beverage enriched with polyunsaturated fatty acids. Food Research International,
Interestingly, the incorporation of 0.1 g/100 g OE or OEO 41, 145e153.
maintained propanal and hexanal concentrations below the Goodridge, J., Ingalls, J. R., & Crow, G. H. (2001). Transfer of omega-3 linolenic acid
and linoleic acid to milk fat from flaxseed or linola protected with formalde-
threshold values for sensory perception (Frankel, 2005) after 10
hyde. Canadian Journal of Animal Science, 81, 525e532.
days of exposure to light or heat. Jacobsen, C., Let, M. B., Nielsen, N. S., & Meyer, A. S. (2008). Antioxidant strategies for
preventing oxidative flavour deterioration of foods enriched with n-3 poly-
unsaturated lipids: a comparative evaluation. Trends in Food Science and Tech-
4. Conclusion nology, 19, 76e93.
Jacobsen, C., Let, M. B., Sørensen, A. D. M., Horn, A. F., Timm-Heinrich, M., &
OE and OEO reduced light- and heat-induced oxidation of Nielsen, N. S. (2008). Applications of natural antioxidants in omega-3 enriched
foods. Electronic Journal of Environmental, Agricultural and Food Chemistry, 7,
omega-3 fatty acids and change in color during storage of dairy
3288e3295.
beverages enriched with linseed oil. OE showed better antioxidant Kim, J. I., Lee, J. H., Choi, D. S., Won, B. M., Jung, M. Y., & Park, J. (2009). Kinetic
properties than OEO. Physical stability of dairy beverages was not study of the quenching reaction of singlet oxygen by common synthetic anti-
oxidants (tert-Butylhydroxyanisol, tert-di-Butylhydroxytoluene, and tert-
affected by the addition of OE or OEO. These natural antioxidants
Butylhydroquinone) as compared with a-Tocopherol. Journal of Food Science,
can be added to dairy beverages enriched with omega-3 fatty acid 74, C362eC369.
to effectively inhibit oxidation during storage. However, before Kiokias, S. N., Dimakou, C. P., Tsaprouni, I. V., & Oreopoulou, V. (2006). Effect of
these ingredients are used in dairy beverage formulations their compositional factors against the thermal oxidative deterioration of novel food
emulsions. Food Biophysics, 1, 115e123.
impact on sensory properties and consumer acceptance will need Kolanowski, W., & Weißbrodt, J. (2007). Sensory quality of dairy products fortified
to be evaluated. with fish oil. International Dairy Journal, 17, 1248e1253.
Kris-Etherton, P. M., Harris, W. S., & Appel, L. J. (2002). Fish consumption, fish oil,
omega-3 fatty acids, and cardiovascular disease. Circulation, 106, 2747e2757.
Acknowledgments Kristinová, V., Mozuraityte, R., Storrø, I., & Rustad, T. (2009). Antioxidant activity of
phenolic acids in lipid oxidation catalyzed by different prooxidants. Journal of
We thank the Coordination for the Improvement of High Agricultural and Food Chemistry, 57, 10377e10385.
Kulisic, T., Radonic, A., Katalinic, V., & Milos, M. (2004). Use of different methods for
Education Personnel (CAPES) Foundation and Agriculture and Agri- testing antioxidative activity of oregano essential oil. Food Chemistry, 85,
Food Canada for their financial support. 633e640.
174 M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174
Let, M. B., Jacobsen, C., & Meyer, A. S. (2004). Effects of fish oil type, lipid
antioxidants and presence of rapeseed oil on oxidative flavour stability of
fish oil enriched milk. European Journal of Lipid Science and Technology, 106,
170e182.
Luzia, D. M. M., & Jorge, N. (2009). Antioxidant activity of lemon seed extract
(Citrus limon) added to soybean oil in accelerated incubator-storage test.
Química Nova, 32, 946e949.
Sahin, F., Güllüce, M., Daferera, D., Sökmen, A., Sökmen, M., Polissiou, M., et al.
(2004). Biological activities of the essential oils and methanol extract of Orig-
anum vulgare ssp. vulgare in the Eastern Anatolia region of Turkey. Food Control,
15, 549e557.
Schamberger, G. P., & Labuza, T. P. (2007). Effect of green tea flavonoids on Maillard
browning in UHT milk. LWT-Food Science and Technology, 40, 1410e1417.
Scorza, F. A., Cysneiros, R. M., Arida, R. M., Terra-Bustamante, V. C., de
Albuquerque, M., & Cavalheiro, E. A. (2008). The other side of the coin:
beneficiary effect of omega-3 fatty acids in sudden unexpected death in
epilepsy. Epilepsy and Behavior, 13, 279e283.
Shahidi, F., & Miraliakbari, H. (2004). Omega-3 (n-3) fatty acids in health and
disease: Part 1-Cardiovascular disease and cancer. Journal of Medicinal Food, 7,
387e401.
Simopoulos, A. P. (1991). Omega-3 fatty acids in health and disease and in growth
and development. American Journal of Clinical Nutrition, 54, 438e463.
Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144e158.

Skerget, M., Kotnik, P., Hadolin, M., Hras, A. R., Simoni c, M., & Knez, Z. (2005).
Phenols, proanthocyanidins, flavones and flavonols in some plant materials and
their antioxidant activities. Food Chemistry, 89, 191e198.
Ye, A., Cui, J., Taneja, A., Zhu, X., & Singh, H. (2009). Evaluation of processed cheese
fortified with fish oil emulsion. Food Research International, 42, 1093e1098.