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

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LWT - Food Science and Technology

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Use of oregano extract and oregano essential oil as antioxidants in functionaldairy 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,*

aUniversidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR 87020-900, Brazilb Food Research and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Boulevard West, Saint-Hyacinthe, QC J2S 8E3, CanadacDairy 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

Article history:Received 6 July 2011Received in revised form1 November 2011Accepted 5 December 2011

Keywords:Origanum vulgareOriganum minutiflorumLipid oxidationDienesHexanalPropanalDairy beverages

* Corresponding author. Tel.: þ1 450 768 3235; faxE-mail address: michel.britten@agr.gc.ca (M. Britte

0023-6438/$ e see front matter Crown Copyright �doi:10.1016/j.lwt.2011.12.018

a b s t r a c t

To increase health benefits, dairy beverages can be enriched with omega-3 fatty acids, although thesefatty acids are susceptible to oxidation. The use of oregano extract (OE) and oregano essential oil (OEO) asantioxidants in dairy beverages enriched with 2 g/100 g linseed oil was studied. The OE contained269 mg gallic acid equivalents per gram and was shown by the DPPH radical assay to have powerfulantioxidant activity. During 10 days of storage under light or heat conditions, color, conjugated dienehydroperoxides, hexanal, propanal, and dissolved oxygen concentrations were evaluated in dairybeverages containing various concentrations of OE or OEO. It was found that OE was more efficient thanOEO in preventing the formation of conjugated dienes, hexanal, and propanal as well as the depletion ofoxygen induced by heat or light oxidation. The addition of OE or OEO did not affect dairy beveragephysical stability during storage.

Crown Copyright � 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Polyunsaturated fatty acids of the n-3 family (n-3 PUFA) havereceived special attention in recent years because of their potentialhealth benefits for the consumer. Greater intake of n-3 PUFA hasbeen 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-3PUFA 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 beuseful in the prevention and treatment of epilepsy (Scorza et al.,2008). Therefore, the incorporation of n-3 fatty acids into dairyproducts (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,

: þ1 450 773 8461.n).

2011 Published by Elsevier Ltd. All

2007), has been studied with a view to providing consumers withthese health benefits.

One shortcoming of this approach is the high sensitivity of the n-3 PUFA family to oxidation during processing and storage. Oxidationcan change the nutritional and sensory properties of dairy bever-ages. Depending on the storage conditions, two main reactions canaffect lipids. Autoxidation is a free radical chain process occurringspontaneously or at moderate temperatures in the presence ofmolecular oxygen (3O2). Although the reaction between tripletoxygen and fatty acids is thermodynamically unfavorable (Frankel,2005), heat, transition metals, and light can accelerate fatty acidfree radical formation (Choe & Min, 2006). Under light exposure inthe presence of sensitizers such as metals or chlorophylls, tripletoxygen can form singlet oxygen (1O2), a powerful radical generatorthat reacts directly with lipids (Choe & Min, 2006). These reactionscan severely affect dairy product quality.

Polyphenols from plant extracts have been used in differentfood matrices to improve the oxidative stability of food lipids. Atthe time of submitting this work, only a few studies have looked atthe addition of antioxidants to minimize oxidative reactions indairy products (Giroux, Houde, & Britten, 2010; Jacobsen, Let,Nielsen, & Meyer, 2008; Jacobsen, Let, Sørensen, et al., 2008; Let,Jacobsen, & Meyer, 2004).

rights reserved.

M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174168

The aim of this study was to evaluate the efficiency of extractof oregano (Origanum vulgare) and essential oil of oregano(Origanum minutiflorum) in increasing the antioxidant capacity ofdairy beverages enriched with 2 g/100 g linseed oil and reducinglipid oxidation during storage under different heat and lightconditions.

2. Materials and methods

2.1. Material

Dried oregano and oregano essential oil (OEO) were purchasedfrom a local market in Saint-Hyacinthe, QC, Canada. The plantmaterial was milled and stored at �20 �C.

2.2. Oregano extract preparation

For the oregano extract (OE), 50 g oregano powder was infusedfor 10 min in 1 L hot water (80 �C). The infusion was stirred occa-sionally and centrifuged at 3200�g for 6 min. Following filtrationwith Whatman No. 1 filter paper to remove any residuals, theinfusionwas quickly cooled, freeze-dried, and stored at�20 �C. TheOE was prepared in triplicate.

2.3. FolineCiocalteu assay

Total phenolics in the OE were determined according toSingleton and Rossi (1965) with the following modifications. Thefreeze-dried OE was dispersed in methanol at a concentration of2.50 mg/mL. A 0.25 mL aliquot of extract solution in methanol wasmixed with 0.25 mL Folin-Ciocalteu reagent (previously dilutedwith water; dilution factor ¼ 2), 0.50 mL of a saturated sodiumcarbonate solution, and 4.0 mL water. The mixture was left at roomtemperature in darkness for 30 min and then centrifuged at3200�g for 6 min. The supernatant absorbance was measured at725 nm. A standard curve was prepared using gallic acid, and theresults were expressed as milligrams of gallic acid equivalents pergram 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 byEl-Massry, El-Ghorab, and Farouk (2002) with slight modifications.The OE or OEOwas dispersed in 10 mL methanol at a concentrationof 2.0 mg/mL. Different aliquots of this solution were mixed with2.0 mL DPPH methanolic solution (47 mg/mL). The mixture wasthoroughly vortexed and kept in the dark for 30 min. Absorbancewas then measured at 517 nm against a methanol blank withoutDPPH. The results were expressed as the percentage inhibition ofthe DPPH radical (% Inhibition DPPH�), which was calculatedaccording to the following equation:

% Inhibition DPPH� ¼�AbsDPPH

� � Abssample

�

AbsDPPH�

� 100 (1)

where AbsDPPH� is the absorbance of the DPPH� solutionwithout OEor OEO, and Abssample is the absorbance of the DPPH� solution withOE 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 percentageinhibition.

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 proteinconcentration 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, andBritten (2008). Sodium azide (0.02 g/100 g) was used as a microbialgrowth inhibitor. The OE was added at three concentrations (0.001,0.01, and 0.1 g/100 g), and eachmixturewas stirred for 15min. Afterovernight storage at 4 �C, the pH was adjusted to 6.7. The dairybeverages were enriched with pure linseed oil (Orphée, La MaisonOrphée Inc., Quebec City, QC, Canada) for a final concentration of2 g/100 g. The preparations (40 �C) were pre-emulsified for 3min at8000 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 MPafor the first two passes and to 3.5 MPa for the third pass. Thesamples were batch-pasteurized at 63.5 �C for 30 min in stainlesssteel cups. For the dairy beverages enriched with OEO, the oil wasmixed directly with the linseed oil before homogenization fora 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 beverageswere stored under two different conditions:(1) at an elevated temperature, in a storage cabinet maintained at50 �C; and (2) under light exposure using a fluorescent light (warmwhite, 60 W fluorescent lamps, General Electric, Cleveland, OH) at4 �C. For the light exposure, glass tubes (8.50 � 2.0 cm) containing6.0 mL dairy beverage were capped and laid horizontally at 30 cmfrom the light source. Under both storage conditions, samples weretaken after 0, 1, 2, 3, 4, 6, 8, and 10 days for conjugated dieneanalyses, and after 0, 3, 6, and 10 days for hexanal, propanal, andoxygen analyses.

2.7. Dairy beverage color

Hunter L*, a*, and b* values were measured in a HunterLabspectrocolorimeter (Labscan 2 tristimulus, Hunter AssociatesLaboratory, Inc., Reston, VA) set for illuminant D-65 and an illu-mination area measuring 4.5 cm in diameter. The differencebetween the L*, a*, and b* values of the dairy beverages containingOE or OEO and the control dairy beverage was used to calculate thetotal color difference, according to the following equation:

Total color difference ¼�DL*2 þ Da*2 þ Db*2

�1=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 tubewith a 16-mm internaldiameter and scannedbya light source (880nm) after 0, 7,14, 28, and50 days of storage in darkness at 4 �C. The backscattering intensitywas recorded as a function of sample height. The change in back-scattering intensity at the top (BSmax) and in the middle (BS35mm) ofthe sample during storage was used to monitor phase separation.

2.9. Lipid oxidation

2.9.1. Conjugated dienesConjugated diene (CD) hydroperoxides were evaluated after 0, 1,

2, 3, 4, 6, 8, and 10 days of storage according to Kiokias, Dimakou,

Table 1Hunter L*, a*, and b* values for dairy beverages containing oregano extract (OE) ororegano essential oil (OEO).

L* a* b*

Control 86.4ab �3.48a 6.97aOE 0.001 g/100 g 85.9ab �3.68a 8.74aOE 0.01 g/100 g 85.6b �3.49a 8.40aOE 0.1 g/100 g 81.2c �2.87b 12.77bOEO 0.001 g/100 g 87.0a �3.47a 7.50aOEO 0.01 g/100 g 86.5ab �3.46a 7.07aOEO 0.1 g/100 g 86.4ab �3.41a 7.00a

Values with the same letter within a column are not significantly different atP > 0.05.

M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174 169

Tsaprouni, and Oreopoulou (2006) with slight modifications.Samples (25 mL) of the dairy beverages were diluted in 2.5 mLisooctane: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 mmPVDFfilter. The absorbance of the filtrate was measured at 232 nmagainst an isooctane:2-propanol blank. The CD concentration wascalculated 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 analysesPropanal and hexanal formation in the dairy beverages

was evaluated after 0, 3, 6, and 10 days of storage using headspacesolid-phase microextraction (HS-SPME) combined with gaschromatographyemass spectrometry (GCeMS). Samples (3 g) ofthe dairy beverages were sealed in 10 mL amber vials. The SPMEfiber (85 mm Carboxen/PDMS, Supelco, Oakville, ON, Canada) wasinserted into the headspace of the vial and left there for 44 min at40 �C. Volatile compoundswere desorbed by inserting the fiber intothe injection port of a Varian CP-3800 gas chromatograph (PaloAlto, CA) operated in splitless mode for 3min at 300 �C. Heliumwasused as carrier gas with a constant flow rate of 1 mL min�1. Thecompounds were separated on a VF-5ms column (equivalent to DB-5ms; Varian, Mississauga, ON, Canada) measuring 30 � 0.25 mmand with a 25-mm film thickness. The oven temperature programbegan with 3 min at 35 �C, followed by a 6 �C min�1 increase to80 �C, a 20 �C min�1 increase to 280 �C, and 2 min at 280 �C. ASaturn 2000 mass spectrometry detector (Varian) was used anddetection was carried out on the total ion current obtained byelectron impact at 70 eV. The selected ions were 55, 57, and 58 forpropanal and 99 for hexanal. The mass range acquisition was m/z30e250. Retention time and mass spectra ions were obtained foreach pure commercial standard (SigmaeAldrich, Oakville, ON,Canada). Calibration curves were prepared using hexanal andpropanal standards (SigmaeAldrich).

2.10. Dissolved oxygen

During storage, dissolved oxygen concentration (mg/L) in thedairy 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 toa split-plot factorial designwith concentration in the main plot andexposure time to heat or light in the subplot. Variance analysis wasused to determine whether the factors and their interactions hada 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 usingwater 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 dispersionand incubation for 2 h in an ultrasonic bath. The phenolic content ofthe extract was 269 mg GAE/g, which agrees with the values foundby Sahin et al. (2004) and �Skerget et al. (2005) (220 and 186 mgGAE/g, respectively) but is higher than the 39.4 mg GAE/g obtainedby Chun, Vattem, Lin, and Shetty (2005).

The antioxidant activity of oregano was evaluated against theDPPH radical to measure the ability of the OE to donate hydrogento stabilize the radical, as assessed spectrophotometrically. The OE

showed a high potential to inhibit the DPPH radical, with IC50values of 26.7 � 0.9 mg/mL. This result is in agreement with that ofBoroski et al. (2011) although a potential less than 9.9 mg/mL wasreported by Sahin et al. (2004). Rosmarinic acid, caffeic acid,coumaric acid, quercetin, and carvacrol are the compoundsresponsible for the antioxidant activity of oregano (Exarchou et al.,2002).

The IC50 value for the OEO was higher than 500 mg/mL. A similarresult was previously reported (Sahin et al., 2004). The non-polarcharacter of the antioxidants present in OEO may explain thepoor 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 colorparameters gradually changed as the OE concentration increased.The lightness of the dairy beverages (L* value) decreased from 86 to81 with increasing OE concentration from 0 to 0.1 g/100 g. Thisresult was expected, considering the dark color of the extract. Boththe a* and b* parameters increased in parallel with OE concen-tration. Schamberger and Labuza (2007) reported that the additionof green tea polyphenols to milk increased redness (a* values).Similar trends were observed in the present experiment withincreased OEO concentration, although the differences were notstatistically significant (P > 0.05).

Color parameters were monitored during storage, and the totalcolor difference was calculated. The total color difference is a singlevalue that takes into account the difference between the L*, a*, andb* values of the sample and thus assesses global changes in thesamples. Storage of the control dairy beverage at 50 �C increasedthe total color difference, with a value of 9.4 after 10 days (Fig. 1).Maillard reactions between milk proteins and lactose were likelyresponsible for the color change in the dairy beverages. Accordingto Fig. 1a, OE was efficient in preventing color change at 50 �C,especially at high concentrations (P < 0.05). Supplementing dairybeverages with 0.1 g/100 g OE reduced the total color difference bya factor of 4.8 after 10 days when compared to the control.Schamberger and Labuza (2007) showed that the addition of greentea flavonoids to milk reduced Maillard browning, and a similareffect was expected from the phenolic compounds containedin OE.

The OEO also prevented color changes in the beverages duringstorage at 50 �C (Fig. 1b). When OEO was used at 0.1 g/100 g, thetotal color difference in the dairy beverages was reduced by halfafter 10 days. Under light exposure storage, neither OE nor OEO hadan effect on beverage total color change (P > 0.05).

40

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Col

or d

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a

100

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10

Col

or d

iffer

ence

106Time (days)

b

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.

M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174170

3.3. Physical stability

Dairy beverages enriched with linseed oil consist of a complexdispersion of fat globules, casein micelles, and minerals. Duringstorage, changes in homogeneity, particle size, and interactionsmight induce phase separation (Durand, Franks, & Hosken, 2003).Those changes can be monitored by means of a backscatteringprofile as a function of time. In the dairy beverages (Fig. 2), a slightincrease in the backscattering intensity at the top of the sample(BSmax), corresponding to the formation of a cream layer, wasobserved during the first week of storage. The backscatteringintensity in the middle of the sample (BS35mm) decreased slightlyduring storage as a result of fat droplets moving to the top of thesample. The addition of OE or OEO at the highest concentrations didnot 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 showeda phase separation pattern similar to that of the dairy beveragesenriched with 2 g/100 g linseed oil. The BSmax of the commercialmilk increased by 21% compared to 15% for the dairy beveragesduring 14 days of storage.

3.4. Conjugated dienes

Dairy beverages enriched with linseed oil (2 g/100 g) containabout 1.06 g/100 g linolenic acid (18:3 n-3), 0.56 g/100 g linoleic

0 10 20 30 40 500

20

40

60

80

100

Back

scat

terin

g (%

)

Time (days)

Fig. 2. Backscattering of dairy beverages (with 2 g/100 g linseed oil) containing oreganoextract (OE) or oregano essential oil (OEO) at the top (BSmax) of the sample: (-) control,(C) OE 0.1 g/100 g, (:) OEO 0.1 g/100 g; and in the middle (BS35mm) of the sample: (;)control, (A) OE 0.1 g/100 g, (=) OEO 0.1 g/100 g, as a function of storage time.

acid (18:2 n-6), and 0.30 g/100 g oleic acid (18:1 n-9). The highPUFA content makes these beverages susceptible to oxidation.

Conjugated diene hydroperoxides are produced during the firstphase of lipid oxidation by the reaction between peroxyl radicalsand oxygen (3O2). The autoxidation of linoleic and linolenic acidsproduces conjugated products (Choe et al., 2006), which absorb UVlight at 232 nm, and their second oxidation products, such asketones, which absorb at 272 nm (Luzia & Jorge, 2009). In thecontrol dairy beverage, CDswere formed after light exposure or heatduring storage. Both light and elevated temperature induced CDformation in the control dairy beverage, but the concentration wasmuch higher for the samples exposed to light than for those storedat 50 �C (Fig. 3). During storage at elevated temperatures, Maillardreactions occur, and the resulting products act as antioxidants andincrease the stability of lipids (Frankel, 2005; Giroux et al., 2010).Furthermore, at high temperatures or in the presence of metal, CDsare readily broken into smaller compounds such as aldehydes,ketones, acids, esters, and alcohols (Choe & Min, 2006).

In the experiment using OE, antioxidant concentration, storagetime, and storage conditions significantly affected (P < 0.05) CDformation (Fig. 3). In the light-induced oxidation experiment,a gradual rise in CD concentration was observed over the 10 daystorage period (Fig. 3a). The formation of CDs was slightly reducedat 0.001 g/100 g OE and strongly inhibited at concentrations greaterthan or equal to 0.01 g/100 g. Compared to the control, the use of0.01 g/100 g OE reduced CD formation after 6 days by 88%. Underlight exposure, the presence of sensitizers and transition catalyticmetals 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 theprevention of these reactions, antioxidants from OE might act assinglet oxygen quenchers or metal chelators.

During storage at a high temperature, the CD concentration inthe control dairy beverage increased from 24 to 79 mmol/kg after10 days (Fig. 3b). Increasing the OE concentration gradually reducedCD formation during storage. At 0.1 g/100 g OE, CD formation wastotally inhibited.

In the dairy beverages prepared with OEO, antioxidantconcentration, storage time, and storage conditions significantlyaffected (P < 0.05) CD formation (Fig. 4). At the highest concen-tration tested (0.1 g/100 g), OEO reduced light-induced CD forma-tion by about 50% after 6 days of storage (Fig. 4a). After 10 days,however, CD concentration was close to that in the control dairybeverage. When used at lower concentrations, OEO had very littleeffect on light-induced CD formation. The OEO reduced CDformation during storage at 50 �C but only at the highest concen-tration (Fig. 4b). After 6 days, CD concentrationwas reduced by 65%when compared to the control dairy beverage. However, as

0 2 4 6 8 100

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Time (days)

[CD

] mm

ol/k

gfa

t

0 2 4 6 8 100

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400

[CD

] mm

ol/k

gfa

t

Time (days)

ba

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).

M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174 171

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 protectinglinseed oil than compounds from OEO, a finding that can beexplained by antioxidant chemistry. The polar characteristic ofphenolic compounds allows them to better act as antioxidants inthe 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 inapolar systems. Thus, OE may act as an antioxidant in dairybeverages enriched with omega-3 fatty acids.

3.5. Dissolved oxygen

The initial oxygen concentration in the control dairy beverageswas 6.4 � 0.6 mg/L. Rapid oxygen depletion was observed duringthe first 3 days of storage at 50 �C or of exposure to light. Similarresults were obtained previously (Giroux, Acteau, et al., 2008;Giroux, St-Amant, et al., 2008).

Fig. 5 shows the changes in dissolved oxygen during the storageof dairy beverages prepared with OE. Antioxidant concentrationand storage time significantly affected dissolved oxygen concen-tration (P < 0.05) (Fig. 5). Under light or heat exposure, an increasein antioxidant concentration slowed down oxygen depletionduring storage. At a 0.1 g/100 g concentration, OE totally preventedoxygen depletion for 6 days in dairy beverages exposed to light(Fig. 5a), and the oxygen concentration remained higher than in thecontrol dairy beverage at the end of the storage period. The oxygendepletion 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

a

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] mm

ol/k

gfa

t

Fig. 4. Changes in the conjugated diene (CD) concentration during storage of dairy bevera(C) 0.001 g/100 g, (:) 0.01 g/100 g, (;) 0.1 g/100 g. Beverage samples were exposed to

slightly retard oxygen depletion in the beverages exposed to heat.After 6 days of storage at 50 �C, the oxygen concentration wasindependent of OE concentration and similar to that in the control(Fig. 5b).

The OEO concentration, storage time, and storage conditionshad a significant effect (P < 0.05) on dissolved oxygen concentra-tion in the dairy beverages (Fig. 6). Similar to OE, OEO was moreeffective in preventing oxygen depletion in the beverages storedunder light than in the beverages stored at 50 �C. Compared to theresult achieved with OE, a much smaller effect on the oxygendepletion pattern of dairy beverageswas observedwith OEO, whichcould only retard oxygen depletion in the beverages exposed tolight (Fig. 6a) and had almost no effect on the beverages stored at50 �C (Fig. 6b).

Under both storage conditions, OE was more efficient than OEOin preventing oxygen depletion. The phenolic compounds(from OE) played an important role in the oxidative stability of thedairy beverages, preventing oxygen uptake in the early stages ofradical reactions. Different mechanisms might explain thisprotection. Polyphenolic compounds might chelate Fe2þ ions andprevent the generation of singlet oxygen. Kim et al. (2009) foundthat the synthetic aromatic antioxidant TBHQ has a strong singletoxygen quenching property that occurs based on the chargetransfer mechanism. Kristinová, Mozuraityte, Storrø, and Rustad(2009) reported an efficiency feature of propyl gallate thatprevents 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 orheat) of dairy beverages similar to those used in the presentexperiment resulted in no change in dissolved oxygen concentra-tion as a function of time.

0 2 4 6 8 10Time (days)

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ges (with 2 g/100 g linseed oil) containing oregano essential oil (OEO): (-) control,light (a) or stored at 50 �C (b).

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Time (days)0 2 4 6 8 10

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a b

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).

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g/L)

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ba

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).

M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174172

3.6. GCeMS analyses

Aldehydes are lipid oxidation products that can significantlyaffect food sensory properties at very low concentrations, namely1.6 and 0.15 mg/L for propanal and hexanal, respectively (Frankel,2005). In the dairy beverages, propanal concentration was higherthan hexanal concentration. Propanal is the result of the oxidationof linolenic acid, which is a major fatty acid in linseed oil. Hexanaloriginates from linoleic acid oxidation.

The dairy beverage preparations were pasteurized at 63.5 �C for30 min. After pasteurization, propanal and hexanal concentrationsin 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/

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anal

(mg/

L)

Time (days)

a

Fig. 7. Changes in the propanal (a) and hexanal (b) concentrations in control dairy beverag(-) or stored at 50 �C (C). Dashed lines correspond to the threshold values for the senso

100 g showed lower concentrations of propanal (w0.20 mg/L forbothOE andOEO) andhexanal (0.028� 0.005 and0.015� 0.003mg/L for OE and OEO, respectively) immediately after pasteurization.Antioxidants from OE and OEO reduced hexanal and propanalproduction 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 tolight or heat. Increased propanal and hexanal concentrations wereobserved during the first 6 days under light exposure, and concen-trations remained stable until 10 days. During storage at 50 �C, thehighest concentrations of propanal andhexanalwere reached after 3days. Although elevated temperature seemed to accelerate initialaldehyde formation, the hexanal and propanal levels at the end of

0 2 4 6 8 10

0,0

0,1

0,2

0,3

0,40.4

0.1

0.2

0.3

0.0

Hex

anal

(mg/

L)

Time (days)

b

e (with 2 g/100 g linseed oil) during storage. Beverage samples were exposed to lightry perception of oxidation (Frankel, 2005).

42 420

20

40

60

80

100

Prop

anal

inhi

bitio

n (%

)

3 4 5 60

20

40

60

80

100

Hex

anal

inhi

bitio

n (%

)

ba

OE – 50 °C OE – Light OEO – 50 °C OEO - Light OE – 50 °C OE – Light OEO – 50 °C OEO - 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). Beveragesamples 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.

M. Boroski et al. / LWT - Food Science and Technology 47 (2012) 167e174 173

the storage period were similar under both conditions. Final alde-hyde concentrations were above the threshold values for thesensory perception of oxidation (Frankel, 2005).

Adding OE or OEO to the beverage formulations reducedpropanal and hexanal production during storage. The inhibition ofaldehyde production during the last 7 days of storage was calcu-lated from the concentration difference with the control andexpressed as the percentage of inhibition (Fig. 8). Propanalproduction was strongly inhibited as the concentration of OE orOEO increased (Fig. 8a). On average, inhibition was 2.4 timesgreater with OE than with OEO (P < 0.05). At low concentrations(0.001 g/100 g and 0.01 g/100 g), OE showed better inhibition ofpropanal production in the beverages exposed to light than in thoseexposed to heat. At the highest OE concentration (0.1 g/100 g),propanal inhibition was similar for both storage conditions andaveraged 92%. At the lowest concentration tested (0.001 g/100 g),OEO showed no effect on propanal production in the beveragesexposed to light or heat. At the highest concentration, the inhibi-tion of propanal production was only approximately 60%.

The inhibition of hexanal production was also measured, andthe results are presented in Fig. 8b. The effect of OE and OEO on theinhibition of hexanal production was quite similar to the observedeffect on propanal inhibition (Fig. 8a). As a general trend, higherinhibition was observed when OE was used compared to OEO.However, the difference was smaller than that for propanal inhi-bition (Fig. 8a) and is not statistically significant (P > 0.05).

Interestingly, the incorporation of 0.1 g/100 g OE or OEOmaintained propanal and hexanal concentrations below thethreshold values for sensory perception (Frankel, 2005) after 10days of exposure to light or heat.

4. Conclusion

OE and OEO reduced light- and heat-induced oxidation ofomega-3 fatty acids and change in color during storage of dairybeverages enriched with linseed oil. OE showed better antioxidantproperties than OEO. Physical stability of dairy beverages was notaffected by the addition of OE or OEO. These natural antioxidantscan be added to dairy beverages enriched with omega-3 fatty acidto effectively inhibit oxidation during storage. However, beforethese ingredients are used in dairy beverage formulations theirimpact on sensory properties and consumer acceptance will needto be evaluated.

Acknowledgments

We thank the Coordination for the Improvement of HighEducation Personnel (CAPES) Foundation and Agriculture and Agri-Food Canada for their financial support.

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