comparative analysis of lipophilic compounds in eggs of organically raised isa brown and araucana...

Chemical Papers 66 (10) 955–963 (2012)DOI: 10.2478/s11696-012-0219-2

ORIGINAL PAPER

Comparative analysis of lipophilic compounds in eggsof organically raised ISA Brown and Araucana hens‡

Adela Pintea, Francisc V. Dulf*, Andrea Bunea, Cristian Matea, Sanda Andrei

University of Agricultural Science and Veterinary Medicine, 3-5 Manastur Street, 400372, Cluj-Napoca, Romania

Received 30 October 2011; Revised 4 May 2012; Accepted 14 May 2012

Hens’ eggs represent a rich source of important nutrients, including lipids and carotenoids. Thelipid composition of hens’ eggs is influenced by genetic factors, age, and diet. The aim of this studywas to compare the fatty acids, cholesterol, and carotenoids content of the egg yolk of ISA Brownand Araucana hens grown in free-range housing systems. Fatty acids and cholesterol were analysedby GC-FID and GC-MS and carotenoids were quantified by RP-HPLC-PDA.The Araucana egg yolk has a higher lipid content and higher egg-to-albumen ratio than the ISABrown yolk, while the total cholesterol, carotenoids content and profile are not significantly different.The lipids of the Araucana egg yolk have a higher content of mono-unsaturated fatty acids (MUFAs),eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and a better n–6/n–3 ratio thanthe ISA Brown egg yolk lipids. The major carotenoids were lutein and zeaxanthin, which accountfor more than 83 % in egg yolk. Eggs of both breeds, when raised organically, represent very goodsources of highly bio-available lutein and zeaxanthin, pigments which are related to lower risk ofage-related macular degeneration. We report for the first time on the fatty acids composition inlipid fractions and the profile and content of carotenoids of the Araucana egg yolk.c© 2012 Institute of Chemistry, Slovak Academy of Sciences

Keywords: egg, lipids, carotenoids, GC-MS, HPLC-PDA

Introduction

Eggs represent one of the commonest foodstuffs inthe human diet. The eggs most widely acceptable toconsumers are hens’ eggs, being a rich source of impor-tant nutrients. Apart from water, egg white containsa large number of proteins. The egg yolk is a valuablesource of proteins, lipids, vitamins (A, D, thiamine,riboflavin, B12), and minerals (iron, potassium, phos-phorus, zinc, selenium) (Cherian et al., 2002; Fredriks-son et al., 2006).

The lipidic fraction of eggs, including the carote-noids, is concentrated in the yolk. Lipids accountfor up to 22 % of the egg yolk and represent animportant source of animal fat for humans. Gas-chromatography with flame ionisation detection (GC-FID) or mass spectrometric detection (MSD) are thepreferred methods for determining the fatty acids

composition of total lipids and lipids fraction in bi-ological samples (Christie, 1989). Fatty acids are usu-ally derivatised to fatty acids methyl esters (FAMEs)by acid- or base-catalysed transesterification withmethanol. FAMEs of egg yolk were previously ob-tained by boron trifluoride–methanol (Cherian etal., 2002; Simčic et al., 2011), hydrochloric acid inmethanol (Ramstedt et al., 1999) and analysed byGC-FID and GC-MS. The major fatty acids in eggyolk lipids are oleic acid C18:1 (40–50 %), palmiticacid C16:0 (20–30 %), and linoleic acid C18:2 (15 %).However, the fatty acids profile and content of individ-ual fatty acids are affected by genetic factors (breed)(Millet et al., 2006; Simčic et al., 2011), age (Nielsen,1998), type of housing (Cerolini et al., 2005; Sammanet al., 2009; Anderson, 2011), and diet (Milinsk etal., 2003; Millet et al., 2006; Cachaldora et al., 2006;Fredriksson et al., 2006; Oliveira et al., 2010). The

*Corresponding author, e-mail: francisc [email protected]‡Presented at the 5th Meeting on Chemistry & Life 2011, Brno, 14–16 September 2011.

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consumption of unsaturated fatty acids, especially n–3 fatty acids, is associated with a reduced risk of car-diovascular diseases (Simopoulos, 2002; De Caterina,2011). Also, n–3 fatty acids display neuroprotectiveproperties and exert beneficial effects on the cognitivefunction associated with ageing (Zhang et al., 2011).Great efforts have been made in recent years to im-prove the fatty acids content and the n–3/n–6 fattyacids ratio in egg yolk by using supplements rich in n–3fatty acids such as fish oil, flaxseed, canola and soy-bean oils, or microalgae (Milinsk et al., 2003; Kivini etal., 2004; Millet et al., 2006; Fredriksson et al., 2006;Cachaldora et al., 2006; Bou et al., 2009).

The cholesterol content in eggs is commonly de-termined by GC-FID after saponification of the to-tal lipid extract (Millet et al., 2006) or by enzymatic-spectrophotometric methods (Simčic et al., 2009; Gul-temirian et al., 2009). Due to epidemiological stud-ies showing a positive correlation between plasmacholesterol concentration and the incidence of coro-nary diseases, the high cholesterol content of eggs isregarded as an undesirable characteristic. However,it was shown that most people are able to consumeeggs without any significant increase in total and LDLcholesterol (Lewis et al., 2000). Millet et al. (2006)demonstrated that Araucana hen eggs contained morecholesterol than commercial breeds but this was notinfluenced by diet.

Carotenoids are the pigments of egg yolk, and theirconcentration is an important attribute, since con-sumers associate an intense colour with eggs that areboth healthier and of higher quality. Since carotenoidsare not produced by animals, their level in animalproducts, including egg yolk, is related strictly to diet.Egg yolk contains xanthophylls (oxygenated deriva-tives) like lutein and zeaxanthin as major pigments.Due to their extended polyene chain, carotenoids aresusceptible to oxidation, the addition of electrophiles,and E/Z isomerisation caused by heat, light, or chem-icals (Britton et al., 2004). Also, the relatively lowconcentration of carotenoids in most biological sam-ples requires highly sensitive analytical methods. Themost useful method for carotenoids analysis is high-performance liquid chromatography (HPLC) with aUV-VIS diode array detector (DAD) or MSD. Becausecarotenoids are lipophilic compounds, the reversed-phase HPLC on C18 and C30 stationary phases isused, mostly in a gradient system. C30 columns arepreferred due to their capacity to resolve positionaland geometric isomers (Oliver & Palou, 2000; Řezankaet al., 2009). The egg yolk carotenoids were deter-mined in organic or conventional eggs using a C30stationary phase, a gradient system based on tert-butyl methyl ether, methanol, and water, and DADor APCI-MS detection (Schlatterer & Breithaupt,2006; Perry et al., 2009). RP-HPLC-DAD separationson different C18 stationary phases were also usedfor quantification of total and major carotenoids in

eggs (Karadas et al., 2005; van Ruth et al., 2011).Carotenoids are regarded as beneficial in the preven-tion of major diseases, including certain cancers, car-diovascular, and eye diseases (Krinsky & Johnson,2005). A high intake of lutein and zeaxanthin by hu-mans is related to a decreased risk of age-related mac-ular degeneration, the leading cause of blindness inWestern countries (Landrum & Bone, 2001). It wasdemonstrated that the bio-availability of lutein andzeaxanthin from egg yolk was higher than from supple-ments and from green vegetables like spinach (Handel-man et al., 1999; Chung et al., 2004; Burns-Whitmoreet al., 2010). Egg yolk can be significantly enrichedwith lutein (Tyczkowski & Hamilton, 1986; Leeson &Caston, 2004) and other carotenoids through nutri-tional manipulation (Hamilton et al., 1990; Breithauptet al., 2003; Fredriksson et al., 2006).

The aim of this study was to compare the fattyacids, cholesterol, and carotenoids composition of theegg yolk of ISA Brown and Araucana hens raised or-ganically in a free-range housing system. We also re-port for the first time on the fatty acids distribution inphospholipids and cholesterol ester fraction in Arau-cana hens.

Experimental

ISA Brown and Araucana hens were raised in afree-range housing system and received the same or-ganic food comprising a mixture of corn, wheat, andbarley, with no supplements and water ad libitum.

Lipid and carotenoids extraction

Total lipids extraction was performed using a mix-ture of chloroform/methanol (ϕr = 2 : 1) as de-scribed by Folch et al. (1957). Carotenoids were ex-tracted following the procedure of Schlatterer and Bre-ithaupt (2006). Egg yolks of both breeds were pooledand homogenised. An aliquot (5 g) was extractedthree times with a mixture of light petroleum/ethylacetate/methanol (ϕr = 1 : 1 : 1). The extractswere transferred into a separation funnel and wa-ter was added to allow phase separation. The upperphase was evaporated to dryness in the presence ofethanol. The oily phase was quantitatively transferredwith TBME/methanol into a volumetric flask, filtered(PTFE, 0.45 µm filters) and subjected to HPLC-PDAanalysis.

Chromatographic separation of carotenoids

HPLC-PDA separation of carotenoids was per-formed using a Shimadzu LC20 AT high perfor-mance liquid chromatograph (HPLC) with a SPD-M20A diode array detector. A YMC C30 column(24 cm × 4.6 mm; particle size: 5 µm) and agradient consisting of two solvents were used: sol-

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vent A: methanol/tert-butyl methyl ether/water (ϕr= 81 : 15 : 4) and solvent B: tert-butyl methylether/methanol/water (ϕr = 90 : 7 : 3). The gradientwas: 0 min, 1 % of solvent B; 50 min, 55 % of B; 51min, 60 % of B; 52 min, 60 % of B; 54 min, 1 % of B inA, followed by equilibration of the column for 15 min.The flow-rate was adjusted to 1.0 mL min−1. The sam-ple or standard (20 µL) was injected into the HPLCsystem. The DAD detector was set at 450 nm; chro-matographic data and UV-VIS spectra were recorded,stored, integrated, and compared using Shimadzu LCsolution 1.11.SP1 software. Lutein, zeaxanthin, andβ-carotene standards were provided by LGC Stan-dards, UK; β-cryptoxanthin was purified in our labo-ratory (from Physalis alkekengi L.). HPLC-grade sol-vents were from Merck (Darmstadt, Germany). Eachstandard was injected separately and separated usingthe same chromatographic conditions as the analysedsamples. The retention time and absorption spectrumwere recorded in the range of 300–550 nm. The pu-rity of individual peaks was checked by evaluatingthe matching of the absorption spectra at the ups-lope, apex, and downslope of the peaks. The intra-dayrelative standard deviation (RSD) of retention times(Rt) determined for six replicates of standards solu-tion ranged from 0.09 % to 0.23 %. Identification ofcarotenoids in samples was carried out by comparingthe retention time and UV-VIS absorption spectrumof each compound with those of the above standardcompounds and by co-chromatography with authen-tic standards (Schiedt & Liaaen-Jensen, 1995). Co-chromatography was performed by spiking sampleswith carotenoids standards and HPLC separation un-der the same conditions. The absorption spectra ofindividual carotenoids were compared with those ofthe standards by their overlapping, using DAD detec-tor software. A calibration curve was established witheach carotenoid by plotting peak area against con-centration for five concentrations ranging from 0.5–30 µg mL−1. The coefficients of determination R2 forlutein, zeaxanthin, β-cryptoxanthin, and β-carotenewere 0.9914, 0.9945, 0.9983, and 0.9913, respectively.All analyses were performed in triplicate.

GC-FID analysis of cholesterol

After saponification of the total lipid extract,cholesterol was derivatised with N,O-bis-(trimethyl-silyl)-trifluoroacetamide (BSTFA) containing1 mass % of trimethylchlorosilane (TMCS) in pyridine.Trimethyl silyl ether (TMS) derivatives of cholesterolwere immediately injected into a Shimadzu GC-2010system equipped with a FID and a Varian CP-Sil 5CBcapillary column, (25 m × 0.25 mm, film thickness0.12 µm). The injector temperature was 260◦C andthe detector temperature was 290◦C. The tempera-ture programme was: 3 min at 130◦C, 10◦C min−1 upto 300◦C, and 5 min isothermal. A calibration curve

with cholesterol (0.05–5 mg mL−1) was constructedfor quantitative analysis by plotting peak area againstconcentration. The coefficient of determination was R2

= 0.9947.

HPTLC separation of lipid classes

The total lipid extract was subsequently sep-arated by HPTLC on Silica gel 60 F254 plates(10 cm × 20 cm). The chromatographic chamberswere saturated with the solvent system: propan-1-ol/chloroform/methanol/methyl acetate/0.25mass % aq. KCl (ϕr = 8.3 : 8.3 : 2.7 : 10 : 3)1 h prior to development (Heape et al., 1985).This method enabled the separation of: cholesterolesters (CE), phosphatidylethanolamine (PE), phos-phatidylcholine (PC), phosphatidylinositol and phos-phatidylserine (PI + PS), and sphingomyelin (SPH).The lipid bands of standard compounds were visu-alised using a mixture of copper acetate (3 mass %)and phosphoric acid (8 mass %) and heating to 130◦Cfor 10 min. The lipids were identified by comparingthe Rf with retention times of standard compoundspurchased from Sigma–Aldrich (Munich, Germany).

GC-MS analysis of FAMEs

Fatty acid methyl esters (FAMEs) of total lipidextracts and of the fractions obtained by HPTLC sep-aration (CE, PE, PC, PI + PS, SPH) were preparedusing a boron trifluoride–methanol complex (Morrison& Smith, 1964). A Perkin–Elmer Clarus 600T GC-MS(Perkin–Elmer, Shelton, USA) equipped with a BPx-70 column (60 m × 0.25 mm i.d., 0.25 µm film thick-ness; Ringwood, Australia) was used for the FAMEsanalysis. The initial oven temperature of 140◦C wasincreased to 220◦C at a rate of 2◦C min−1 and heldfor 25 min. Flow-rate of the carrier gas He and thesplit ratio were 0.8 mL min−1 and 1 : 24, respectively.The injector temperature was 210◦C. The positive ionelectron impact (EI) mass spectra were recorded ationisation energy of 70 eV and a trap current of 100 µAwith a source temperature of 150◦C. The mass scanswere performed within the range of m/z 22–395 at arate of 0.14 scan per second with an intermediate timeof 0.02 s between the scans. The injection volume was1 µL. FAMEs were identified by comparison of reten-tion times with those of known standards (Supelco�

37 component FAME Mix, 47885-U) and comparisonof the resulting mass spectra with the spectra in ourdatabase (NIST MS Search 2.0).

Statistics

All analyses were done in triplicate. The resultsare presented as the means of measurements of threesamples analysed individually in triplicate. Statisti-cal differences between samples were tested using Stu-


Table 1. Egg parameters and total lipid, cholesterol, and carotenoids content

SampleParameter p-values

Araucana ISA Brown

Egg mass/g 44.67 (2.10)b 63.91 (3.20)a < 0.001Yolk mass/g 14.82 (1.10)a 15.03 (1.30)a 0.2279Yolk/egg ratio/% 33.17 (0.91)a 23.51 (0.85)b < 0.0001Total lipid content per g of egg yolk/g 0.27 (0.02)a 0.20 (0.01)b < 0.0001Total cholesterol content per g of egg yolk/mg 14.80 (0.98)a 14.20 (0.93)a 0.3578Total carotenoids content per 100 g of egg yolk/mg 3.17 (0.29)a 3.32 (0.31)a 0.6884

The above are mean values from three samples (n = 3, standard deviation in parentheses) analysed individually in triplicate. Meanvalues in the same row with different superscript letters are significantly (p < 0.001) different (Student’s t-test).

dent’s t-test and ANOVA (Tukey’s Multiple Compar-ison Test; GraphPad Prism Version 4.0, GraphPadSoftware Inc., San Diego, CA, USA). The level of sig-nificance was set at p < 0.05.

Results and discussion

In the present study, the lipophilic compounds inegg yolks of two breeds (Araucana and ISA Brown)were investigated. The egg mass of Araucana hens(44.67 g) was significantly lower than that of ISABrown eggs (63.91 g), as also was the yolk/egg ratio(Table 1). The total egg yolk lipid content was alsosignificantly higher in the Araucana (0.27 g per g ofegg yolk) than in the ISA Brown eggs (0.20 g per gof egg yolk). The level of cholesterol was higher, al-beit not significantly, in the Araucana eggs (14.80 mgper g of egg yolk) than in ISA Brown eggs (14.20 mgper g of egg yolk) from hens fed on the same corn-based organic feed (Table 1). Both breeds showed asimilar carotenoid content (3.17 mg per 100 g of eggyolk vs. 3.32 mg per 100 g of egg yolk) (Table 1) andprofile (Table 2)). Considering the average yolk mass,eggs from Araucana hens would provide 4 g of lipids,219 mg of cholesterol, and 0.47 mg of carotenoids perportion (one egg), while ISA Brown hens eggs wouldprovide 3 g of lipids, 213 mg of cholesterol, and 0.50mg of carotenoids per portion (one egg).

Carotenoids from egg yolk were extracted andseparated by HPLC-PDA (Fig. 1). The same majorcarotenoids were identified in the eggs: lutein, zea-xanthin, β-apo-8′-carotenal, β-cryptoxanthin, and β-carotene. UV-VIS absorption spectra of carotenoidsprovided information on the chromophore in molecu-les, position of absorption maxima and the fine struc-ture, as useful tools for the identification of these com-pounds (Britton et al., 2004). The absorption maximaof major carotenoids identified in egg samples are sim-ilar to the maxima given in the literature (Britton etal., 2004; Schlatterer & Breithaupt, 2006). The com-pound with retention time of 12.5 min showed an ad-ditional maximum in the UV region (332 nm). Accord-ing to the position of the maximum and the retentiontime, the compound could be a cis-isomer of lutein but

Fig. 1. HPLC chromatogram of carotenoids isolated fromAraucana hen egg yolk. Peak identification: 1 – un-known, 2 – lutein, 3 – zeaxanthin, 4 – β-apo-8′-carotenal, 5 – β-cryptoxanthin, 6 – β-carotene.

this was not confirmed under these experimental con-ditions. The presence of cis-isomers of lutein and zeax-anthin in raw eggs was previously reported by Perryet al. (2009). The profile of carotenoids was very sim-ilar in the egg yolk of the two breeds. Together, luteinand zeaxanthin represent more than 83 % of the to-tal carotenoids content, while β-apo-8′-carotenal, β-cryptoxanthin, and β-carotene are present in very lowamounts (Table 2). No synthetic carotenoids such ascanthaxanthin or citranaxanthin were detected in theegg samples.

Hens’ eggs are a significant source of fatty acids inthe human diet and consumers show increasing inter-est in functional foods containing n–3 polyunsaturatedfatty acids (PUFAs). The major fatty acids in the to-tal lipids extracted from ISA Brown and Araucanaegg yolks were oleic (39. 8 % and 43.1 %), palmitic(28.1 % and 23.6 %), linoleic (13,26 % and 11.87 %),and stearic acids (9.33 % and 10.19 %) (Fig. 2, Ta-bles 3 and 4). However, statistical analysis of the fattyacids of the total lipids extract of both types of eggsshowed some differences with regard to the composi-tion and ratio of n–6 PUFAs to n–3 PUFAs. Arau-cana eggs have a significantly higher (p < 0.05) con-tent of monounsaturated fatty acids (49.05 %) thanISA Brown eggs (44.99 %). Also, the total content of


Table 2. Carotenoid composition of egg yolk determined by HPLC-PDA

Carotenoid content per g of egg yolk/µga

Peak Compound Rt/min λmax/nmAraucana ISA Brown p-values

1 Unknown 12.53 419, 443, 470 2.43 (0.22)b 2.62 (0.24)a 0.00372 Lutein 14.41 420, 445, 473 16.26 (0.38)b 16.96 (0.42)a 0.00113 Zeaxanthin 16.37 426, 452, 476 10.21 (0.59)a 10.01 (0.50)a 0.06174 β-Apo-8′-carotenal 17.54 462 0.97 (0.04)a 0.95 (0.03)a 0.07425 β-Cryptoxanthin 26.91 427, 450, 478 0.71 (0.03)b 0.81 (0.04)a 0.04296 β-Carotene 40.14 426, 452, 476 0.18 (0.02)a 0.14 (0.01)b 0.0202

a) Values are mean of three samples (n = 3, standard deviation in parentheses), analysed individually in triplicate. Mean valuesin the same row with different superscript letters indicate: very significant (0.001 < p < 0.01) and significant (0.01 < p < 0.05)differences (Student’s t-test); Rt – retention time.

Table 3. Fatty acids composition of total lipids and lipid classes in ISA Brown hen egg yolk determined by GC-MS

Fatty acid/%a

Fatty acidTL CE PE PI + PS PC SPH

Lauric 12:0 1.24 (0.11) 2.09 (0.05) 5.60 (0.20) 5.41 (0.25) 0.57 (0.10) 4.20 (0.18)Myristoleic 14:1 n–5 0.05 (0.02) nd nd 1.62 (0.10) nd 2.69 (0.12)Myristic 14:0 0.38 (0.10) 3.84 (0.15) 3.72 (0.16) 5.87 (0.23) 0.88 (0.13) 3.07 (0.15)Pentadecanoic 15:0 0.03 (0.01) 1.67 (0.10) nd tr nd 0.67 (0.12)7-Hexadecenoic 16:1 n–9 0.66 (0.09) 3.33 (0.14) nd nd nd ndPalmitoleic 16:1 n–7 2.96 (0.12) nd 1.74 (0.11) nd nd ndPalmitic 16:0 28.10 (1.10) 30.11 (1.18) 24.30 (0.98) 23.86 (0.90) 28.68 (1.20) 27.15 (1.15)Margaric 17:0 0.11 (0.03) 0.89 (0.04) nd nd nd ndα-Linolenic 18:3 n–3 0.07 (0.02) 0.65 (0.10) 3.14 (0.13) 11.52 (0.38) 10.55 (0.35) 5.47 (0.20)Linoleic 18:2 n–6 13.26 (0.63) 5.53 (0.22) 7.59 (0.21) 6.33 (0.200) 15.86 (0.54) 5.23 (0.18)Oleic 18:1 n–9(Z) 39.80 (1.11) 22.42 (0.70) 24.24 (0.56) 10.23 (0.20) 24.09 (0.61) 14.56 (0.34)Elaidic 18:1 n–9(E) 1.44 (0.10) 0.31 (0.09) 2.27 (0.11) 1.10 (0.10) 0.85 (0.08) 2.43 (0.12)Stearic 18:0 9.33 (0.32) 24.87 (0.90) 21.30 (0.60) 19.76 (0.54) 15.22 (0.48) 31.47 (1.23)Arachidonic 20:4 n–6 1.89 (0.11) nd 3.13 (0.17) 1.68 (0.11) 3.30 (0.12) ndEicosapentaenoic (EPA) 20:5 n–3 0.04 (0.01) nd nd tr nd 0.48 (0.10)11,14,17-Eicosatrienoic 20:3 n–3 0.15 (0.05) nd nd nd nd nd11,14-Eicosadienoic 20:2 n–6 0.07 (0.03) 0.38 (0.08) nd 4.25 (0.15) nd nd11-Eicosenoic 20:1 n–9 0.08 (0.02) nd nd 5.59 (0.21) nd 2.04 (0.12)Arachidic 20:0 0.02 (0.01) 3.91 (0.15) 2.00 (0.11) 2.78 (0.14) nd ndDocosahexaenoic (DHA) 22:6 n–3 0.32 (0.08) tr 0.97 (0.10) nd nd 0.54 (0.09)

a) Mass % of total fatty acids; values are mean (standard deviation in parentheses) of three samples of each hen egg yolk analysedindividually in triplicate; TL – total lipids, CE – cholesterol esters, PE – phosphatidylethanolamine, PC – phosphatidylcholine, PI+ PS – phosphatidylinositol and phosphatidylserine, SPH – sphingomyelin; tr – traces, nd – not detected.

EPA and DHA acids, as well as of arachidonic acid,is higher in Araucana eggs than in ISA Brown. As aconsequence, a very significant difference (p < 0.001)was observed for the n–6/n–3 ratio (26.24 vs. 17.0),with a higher content of n–3 acids in Araucana eggs(Table 5).

The total lipid extract was fractionated usingHPTLC and the fatty acids profile was determinedin major phospholipids and in cholesterol ester frac-tions. The cholesterol ester (CE) fraction was charac-terised by the highest degree of saturation, saturatedfatty acids (SFAs) representing 67 % and 73 % in ISABrown and Araucana eggs, respectively (Table 5). Thepercentages of palmitic, stearic, and arachidic acids inCE are the highest among the lipid classes investi-gated and also higher than in the total lipid extracts

(Tables 3–5). The CE fraction had the lowest levelof PUFAs, especially EPA and DHA, in both breeds.Sphingomyelins are characterised by a high content ofSFAs (66 % in ISA Brown and 68 % in Araucana), alow total content of PUFAs and MUFAs, and a verylow n–6/n–3 ratio (Table 5). The presence of long-chain fatty acids EPA and DHA in the sphingomyelinfraction is of interest. The glycerophospholipids PC,PE, PI + PS have comparable contents of SFAs, buthigher levels of PUFAs and MUFAs than the CE andSPH fractions. They also showed some differences re-garding the MUFAs and PUFAs distribution. The PEfraction of Araucana eggs have the highest level ofPUFAs (20.82 %), while in ISA Brown eggs PUFAswere found in a greater proportion in the PC fraction(29.29 %).


Fig. 2. GC-MS chromatogram of the FAMEs from total lipids of Araucana egg yolks analysed with a BPx-70 column. Peaks of fattyacids: (1) lauric (12:0), (2) myristoleic (14:1 n–5), (3) myristic (14:0), (4) pentadecanoic (15:0), (5) 7-hexadecenoic (16:1n–9), (6) palmitoleic (16:1 n–7), (7) palmitic (16:0), (8) margaric (17:0), (9) α-linolenic (18:3 n–3), (10) linoleic (18:2 n–6),(11) oleic (18:1 n–9(Z)), (12) elaidic (18:1 n–9(E)), (13) stearic (18:0), (14) arachidonic (20:4 n–6), (15) eicosapentaenoic(20:5 n–3), (16) 11,14,17-eicosatrienoic (20:3 n–3), (17) 11,14-eicosadienoic (20:2 n–6), (18) 11-eicosenoic (20:1 n–9), (19)arachidic (20:0), (20) docosahexaenoic (22:6 n–3).

Table 4. Fatty acids composition of total lipids and lipid classes in Araucana hen egg yolk determined by GC-MS

Fatty acid/%a

Fatty acidTL CE PE PI + PS PC SPH

Lauric 12:0 0.90 (0.10) 3.75 (0.13) 0.79 (0.15) 3.40 (0.12) 0.66 (0.11) 16.88 (0.40)Myristoleic 14:1 n–5 0.03 (0.01) nd nd 2.26 (0.11) nd 1.44 (0.10)Myristic 14:0 0.28 (0.08) 4.26 (0.18) 1.43 (0.12) 3.15 (0.17) 0.53 (0.05) 0.18 (0.06)Pentadecanoic 15:0 0.05 (0.02) 1.99 (1.15) nd 1.57 (0.09) nd 0.34 (0.10)7-Hexadecenoic 16:1 n–9 0.87 (0.10) 4.92 (0.22) nd nd nd ndPalmitoleic 16:1 n–7 2.46 (0.13) nd nd nd 0.64 (0.06) 0.95 (0.11)Palmitic 16:0 23.63 (1.11) 32.22 (1.21) 27.24 (0.98) 27.05 (1.12) 32.84 (1.16) 24.37 (1.10)Margaric 17:0 0.20 (0.05) 0.85 (0.10) nd 3.28 (0.15) 0.68 (0.10) 1.32 (0.08)α-Linolenic 18:3 n–3 0.09 (0.02) 1.52 (0.11) 4.58 (0.12) 9.77 (0.21) 1.86 (0.12) 8.56 (0.20)Linoleic 18:2 n–6 11.87 (0.28) 4.15 (0.16) 10.75 (0.24) 4.44 (0.14) 10.33 (0.27) 4.69 (0.14)Oleic 18:1 n–9(Z) 43.10 (1.28) 15.13 (0.40) 16.85 (0.35) 17.30 (0.42) 27.01 (0.55) 11.83 (0.21)Elaidic 18:1 n–9(E) 2.41 (0.12) 0.32 (0.10) 1.36 (0.14) 1.87 (0.10) 1.64 (0.12) 0.37 (0.11)Stearic 18:0 10.19 (0.32) 26.62 (0.60) 31.51 (0.72) 25.91 (0.59) 18.83 (0.40) 23.24 (0.46)Arachidonic 20:4 n–6 2.81 (0.15) nd 5.11 (0.18) nd 4.98 (0.16) ndEicosapentaenoic (EPA) 20:5 n–3 0.06 (0.01) nd nd nd nd 0.62 (0.10)11,14,17-Eicosatrienoic 20:3 n–3 0.16 (0.05) nd nd nd nd 0.71 (0.12)11,14-Eicosadienoic 20:2 n–6 0.12 (0.03) 0.98 (0.15) nd nd nd 0.73 (0.12)11-Eicosenoic 20:1 n–9 0.18 (0.04) nd nd nd nd 1.30 (0.15)Arachidic 20:0 0.03 (0.01) 3.29 (0.11) nd nd nd 1.68 (0.10)Docosahexaenoic (DHA) 22:6 n–3 0.56 (0.10) tr 0.38 (0.09) nd nd 0.79 (0.11)

a) Mass % of total fatty acids; values are mean (standard deviation in parentheses) of three samples of each hen egg yolk analysedindividually in triplicate; TL – total lipids, CE – cholesterol esters, PE – phosphatidylethanolamine, PC – phosphatidylcholine, PI+ PS – phosphatidylinositol and phosphatidylserine, SPH – sphingomyelin; tr – traces, nd – not detected.

Araucana hens originate from South America buthave recently been bred by some fanciers in Europe.The Araucana eggs are smaller than those of commer-cial breeds and have a blue colour due to biliverdin(Gultemirian et al., 2009). Consumers show increas-

ing interest in eggs from organically raised hens andeggs produced by “fancy” breeds, which are consid-ered a better alternative to conventional eggs.

The general quantitative data for Araucana andISA Brown eggs (mass, yolk/albumen ratio, total


Table 5. Statistical analysis of fatty acids distribution in total lipids and lipid classes

Fatty acid/%a

Sample ∑SFAs

∑MUFAs

∑PUFAs n–3 PUFAs n–6 PUFAs EPA + DHA n–6/n–3

TL a39.21 (1.68)c b44.99 (1.46)a a15.80 (0.93)c a0.58 (0.16)e a15.22 (0.77)b b0.36 (0.09)b a26.24aCE 67.38 (2.57)a 26.06 (0.93)bc 6.56 (0.40)e 0.65 (0.10)e 5.91 (0.30)e tr 9.00b

ISA BrownPE 56.92 (2.05)b 28.25 (0.78)b 14.83 (0.61)c 4.11 (0.23)d 10.72 (0.38)d 0.97 (0.10)a 2.60c

PI + PS 57.68 (2.06)b 18.54 (0.61)e 23.78 (0.84)b 11.52 (0.38)a 12.26 (0.46)c tr 1.00ePC 45.35 (1.91)c 24.94 (0.69)c 29.71 (1.01)a 10.55 (0.35)b 19.16 (0.66)a nd 1.80d

SPH 66.56 (2.83)a 21.72 (0.70)d 11.72 (0.57)d 6.49 (0.39)c 5.23 (0.18e 1.02 (0.19)a 0.80e

TL b35.28 (1.69)d a49.05 (1.68)a a15.67 (0.64)c a0.87 (0.18)e a14.80 (0.46)a a0.62 (0.11)b b17.00aCE 72.98 (3.48)a 20.37 (0.72)cd 6.65 (0.42)e 1.52 (0.11)d 5.13 (0.31)b tr 3.40c

AraucanaPE 60.97 (1.97)b 18.21 (0.49)de 20.82 (0.63)a 4.96 (0.21)c 15.86 (0.42)a 0.38 (0.09)b 3.20c

PI + PS 64.36 (2.24)b 21.43 (0.63)c 14.21 (0.35)d 9.77 (0.21)b 4.44 (0.14)b nd 0.45dPC 53.54 (1.82)c 29.29 (0.73)b 17.17 (0.55)b 1.86 (0.12)d 15.31 (0.43)a nd 8.23b

SPH 68.01 (2.30)ab 15.89 (0.68)e 16.10 (0.79)bc 10.68 (0.53)a 5.42 (0.26)b 1.41 (0.21)a 0.50d

a) Mass % of total fatty acids; values are mean (standard deviation in parentheses) of three samples of each hen egg yolk analysedindividually in triplicate; means in the same column followed by different subscript letters indicate significant differences (p <0.05) within egg yolks lipid classes; different superscript letters in front of the mean values in the same column mean significantdifferences between total lipid of the two egg yolks (p < 0.05, p < 0.001, Student’s t-test); TL – total lipids, CE – cholesterolesters, PE – phosphatidylethanolamine, PC – phosphatidylcholine, PI + PS – phosphatidylinositol and phosphatidylserine, SPH –sphingomyelin, SFAs – saturated fatty acids, MUFAs – monounsaturated fatty acids, PUFAs – polyunsaturated fatty acids, EPA+ DHA – eicosapentaenoic and docosahexaenoic acid; tr – traces; nd – not detected.

lipid, and cholesterol content; Table 1) obtained in thisstudy are in agreement with the data reported by Mil-let et al. (2006) and Gultemirian et al. (2009). In ad-dition, new data are given on the carotenoids contentand the fatty acids distribution in lipid classes, ob-tained by chromatographic separations (HPLC-DAD,HPTLC, GC-FID, and GC-MS). Despite the generalbelief that Araucana eggs contain only a small amountof cholesterol, more recent studies have demonstratedthe cholesterol concentration to be higher in Arau-cana than in commercial breeds such as ISA Brown orLohman Selected Leghorn (Millet et al., 2006). Takinginto account the total mass, the high yolk/egg ratio,and the high lipid content of the egg yolk, the choles-terol intake per serving will be higher for Araucanaeggs than for commercial breeds.

The carotenoids content and fatty acids profile inAraucana egg yolks in comparison with ISA brown eggyolks is reported for the first time in this paper. Thesimilar values obtained for both breeds show that thecarotenoids content appears to be influenced by dietrather than by genetic factors. Schlatterer and Brei-thaupt (2006) found a comparable amount and a sim-ilar pattern of lutein and zeaxanthin in egg yolks pro-duced in accordance with ecological husbandry. Thezeaxanthin content is usually much lower than luteincontent and this is more evident for barn and cagetype husbandry. Also, hens raised under intensive hus-bandry contain only traces of or no β-cryptoxanthin(Schlatterer & Breithaupt, 2006). The eggs of bothbreeds investigated in this study showed a relativelyhigh content of zeaxanthin and quantifiable amountsof β-cryptoxanthin. This can be explained by thehens being fed on corn, rich in zeaxanthin and con-

taining β-cryptoxanthin. Egg yolks from free rangehens were found to have a higher carotenoids levelthan intensively housed hens (33.93 µg vs. 14.77 µgper g of egg yolk), but lower levels than in quail andpheasant (Karadas et al., 2005). It was shown thatcarotenoids profiling by HPLC combined with chemo-metrics can be useful for the authentication of eggsfrom organically-raised hens from those raised con-ventionally (van Ruth et al., 2011).

The intake of animal products with a higher con-tent of unsaturated fatty acids, especially n–3 fattyacids, is recommended because these lipids can pre-vent the development of cardiovascular and inflam-matory pathologies and can reduce the risk of manychronic diseases (Simopoulos, 2002; Bou et al., 2009).Recent studies have demonstrated the key role of n–3 fatty acids. Human diets have suffered significantchanges, evolving from a ratio n–6/n–3 of 1 : 1 to15 : 1–16.7 : 1 in the Western diet (Simopoulos, 2002).A high level of n–6 fatty acids and a very high ratioof n–6/n–3 fatty acids promote the pathogenesis ofmany severe diseases like cancer, inflammatory, andauto-immune diseases (Simopoulos, 2002). As can beseen in Table 5, Araucana eggs have a more favourablen–6/n–3 ratio than ISA Brown eggs. Millet et al.(2006) and Gultemirian et al. (2009) also observeda higher amount of MUFAs and a more favourablen–6/n–3 ratio in Araucana eggs compared with com-mercial breeds, even if the breeding conditions anddiet were not identical. Since, in our experiment, thehens of both breeds were fed on the same diet, the ge-netic factor appears to be determinant for the differ-ences observed in the fatty acids composition in eggs.In consequence, strategies to improve this ratio in food


are extremely important for a healthy human diet.Ramstedt et al. (1999) reported 95 % of SFAs in

the sphingomyelin fraction of hen egg yolk; more re-cently, Kivini et al. (2004) found 73 % of SFAs and25 % of oleic acid. We found a lower content of SFAsand a higher level of unsaturated fatty acids, includ-ing EPA and DHA in the SPH fraction. The high-est degree of unsaturation (MUFAs + PUFAs) wasfound in phosphatidylcholine (54.5 % in ISA Brownand 46.46 % in Araucana eggs). There are differencesin the fatty acids profile of lipid classes between thetwo breeds investigated. It was shown that the fattyacid profile of phospholipids in egg yolk could be in-fluenced by diet (G�ladkowski et al., 2011); this couldexplain the differences between different studies car-ried out under various experimental conditions.

Conclusions

Araucana egg yolk has higher lipid content andhigher egg/albumen ratio compared with ISA Brownhens fed on the same diet, while the cholesterol andthe carotenoids contents are not significantly differ-ent. The lipids of Araucana egg yolk have a lower con-tent of SFAs, a higher content of MUFAs, EPA, andDHA, and a better n–6/n–3 ratio than ISA Brown eggyolk lipids. The carotenoids content and profile reflectthe high content of xanthophylls in the organic foodfor both breeds. Eggs of both breeds raised organi-cally represent very good sources of highly bioavailablelutein and zeaxanthin, pigments which are related to areduced risk of age-related macular degeneration. Wereport for the first time on the fatty acids composi-tion in lipid fractions and the profile and content ofcarotenoids in Araucana egg yolk.

Acknowledgements. This work was financially supported bya grant from the Romanian National Authority for ScientificResearch, CNCS – UEFISCDI, project no. PN-II-ID-PCE-2011-3-072. The authors wish to thank Dr. Marius Savu forproviding us with the egg samples.

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comparative analysis of lipophilic compounds in eggs of organically raised isa brown and araucana...

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