Food Chemistry 139 (2013) 663–671

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Food Chemistry

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Distribution of carotenoids in endosperm, germ, and aleurone fractionsof cereal grain kernels

Victoria U. Ndolo a, Trust Beta a,b,⇑a University of Manitoba, Department of Food Science, Winnipeg, Manitoba, Canada R3T 2N2b University of Manitoba, Richardson Centre for Functional Foods & Nutraceuticals, Winnipeg, MB, Canada R3T 2N2

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

Article history:Received 19 July 2012Received in revised form 21 December 2012Accepted 10 January 2013Available online 23 January 2013

Keywords:Non-corn cerealsYellow cornAleuroneCarotenoidsLuteinZeaxanthin

0308-8146/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2013.01.014

⇑ Corresponding author at: University of Manitoba,Winnipeg, Manitoba, Canada R3T 2N2. Tel.: +1 204 474

E-mail address: Trust.Beta@ad.umanitoba.ca (T. Be

To compare the distribution of carotenoids across the grain, non-corn and corn cereals were hand dis-sected into endosperm, germ and aleurone fractions. Total carotenoid content (TCC) and carotenoid com-position were analysed using spectrophotometry and HPLC. Cereal carotenoid composition was similar;however, concentrations varied significantly (p < 0.05). Endosperm fractions had TCC ranging from 0.88to 2.27 and 14.17 to 31.35 mg/kg in non-corn cereals and corn, respectively. TCC, lutein and zeaxanthinin germ fractions were higher in non-corn cereals than in corn. Lutein and zeaxanthin contents werelower in non-corn cereal endosperms. The aleurone layer had zeaxanthin levels 2- to 5-fold higher thanlutein among the cereals. Positive significant correlations (p < 0.05) were found between TCC, carotenoidsanalysed by HPLC and DPPH results. This study is the first to report on carotenoid composition of thealeurone layer. Our findings suggest that the aleurone of wheat, oat, corn and germ of barley have signif-icantly enhanced carotenoid levels.

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1. Introduction

Increased interest in functional foods requires more informa-tion on the phytochemicals including carotenoids in grain cerealsthat have health enhancing properties. Carotenoids are amongthe abundant families of pigments in nature that are responsiblefor the yellow, orange and red colours of fruits, vegetables andgrains. They form part of the antioxidant system in seeds (Howitt& Pogson, 2006). Xanthophyll carotenoids, which include luteinand zeaxanthin are recognised for their antioxidant properties(Gentili & Caretti, 2011; Leenhardt et al., 2006; Miller, Sampson,Candeias, Bramley, & RiceEvans, 1996). Carotenoids act as radicalscavengers and singlet oxygen quenchers (Leenhardt et al., 2006).Epidemiological studies have shown that carotenoid-rich foods re-duce the risk of degenerative diseases, such as cancer, cardiovascu-lar diseases, and age-related macular degeneration and alsomaintain skin health (Burkhardt & Boehm, 2007; Rice-Evans,Sampson, Bramley, & Holloway, 1997; Roberts, Green, & Lewis,2009).

Although a minor component in cereals (Irakli, Samanidou, &Papadoyannis, 2011), some grains contain higher and others lowercontent of carotenoids compared to fruits and vegetables (Abdel-Aal et al., 2002; Humphries & Khachik, 2003). However, carotenoid

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ta).

content is an important characteristic in the utilisation of cerealssuch as durum wheat for pasta production (Hentschel et al.,2002). Several authors studied carotenoid content and compositionin whole grain wheat (Abdel-Aal, Young, Rabalski, Hucl, & Fregeau-Reid, 2007; Adom, Sorrells, & Liu, 2003; Hentschel et al., 2002; Pan-fili, Fratianni, & Irano, 2004) maize or yellow corn (Kimura, Kobori,Rodriguez-Amaya, & Nestel, 2007; Luterotti & Kljak, 2010, and bar-ley (Goupy, Hugues, Boivin, & Amiot, 1999). The main carotenoidsin cereal grains are lutein and zeaxanthin (Hentschel et al., 2002;Panfili et al., 2004). Lutein was the most abundant carotenoid in11 wheat varieties studied by Adom et al. (2003). Okarter, Liu, Sorr-ells, and Liu (2010) found higher levels of lutein and zeaxanthin ineight diverse whole wheat varieties than reported by Adom andothers. Zeaxanthin was the dominant carotenoid in maize whereaslutein was the main component in oat, barley, spelt and durumwheat (Panfili et al., 2004). Studies on carotenoid content and com-position mainly used durum wheat, bread wheat, specialty wheat(Einkorn, Khorasan) (Abdel-Aal et al., 2002, 2007; Hidalgo,Brandolini, Pompei, & Piscozzi, 2006) yellow corn (Hulshof,Kosmeijer-Schuil, West, & Hollman, 2007; Kurilich & Juvik,1999a) while barley and oat were rarely used (Goupy et al.,1999; Panfili et al., 2004). Only a few studied distribution of carote-noids in grain kernels and their fractions (Borrelli, De Leonardis,Platani, & Troccoli, 2008; Hentschel et al., 2002; Panfili et al.,2004). There is limited or no information on carotenoid composi-tion of the aleurone layer.

Although the study used hand dissected fractions, these frac-tions can be obtained mechanically at large scale during milling

Table 1Whole grain weight and percentage proportion of each fraction.

Cereal type Weight (mg) % proportion of whole grain

Whole Bran Germ Endopserm

Purple barley 45.5 15.7 2.6 81.7Non-pigmented barley 47.5 14.4 2 83.6

Mean-barley 46.5 15.1 2.3 82.7Purple wheat 45.7 11.7 1.6 86.8Ambassador wheat 47.6 12 2 86Caledonia wheat 50.8 12.2 2.1 85.8MSU D8006 wheat 48.8 12 1.9 86.1

Mean-wheat 48.2 12.0 1.9 86.2Oat 38.4 8.7 1.8 89.5DASCA corn 378.7 5.7 12.1 82USP1395XR corn 360.6 5.8 11.2 83.0P1508HR corn 394.6 5.2 10.7 84.1

Mean-yellow corn 377.9 5.6 11.3 83.0

Average weight of the various grain cereals (n = 25) and % proportions of the grainfractions (bran, germ and endosperm).

664 V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671

and dry fractionation processes (Antoine, Peyron, Lullien-Pellerin,Abecassis, & Rouau, 2004). The main objective of this study wastherefore to investigate the distribution of total and individualcarotenoids in endosperm, germ and aleurone fractions obtainedby hand dissection of diverse cereals grains. A secondary objectivewas to determine the antioxidant activity of carotenoid extracts.

2. Materials and methods

2.1. Chemicals

Acetonitrile, methyl-t-butyl ether (MtBE) 1-butanol and metha-nol were purchased from Fisher Scientific (Whitby, ON, Canada).Carotenoid standards, lutein, zeaxanthin and b-cryptoxanthin werepurchased from ChromaDex Inc. (Santa Ana, CA) and trolox (S)-(�)-6-hydroxy-2,5,7,8-tramethylchroman-2-carboxylic acid and2,2-diphenyl- 1-picrylhydrazyl (95%) was obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).

2.2. Samples

A study was conducted on 7 non-corn (2 barley varieties (purpleand regular), 4 soft wheat varieties (Ambassador, Caledonia, Purplewheat and MSUD8006), 1 oat) and 3 yellow corn (USP1395XR,P1508HR and Dasca-flint corn) cereals. Samples analysed included10 whole grains, 10 endosperm fractions, 10 germ fractions and 10aleurone layer (only for HPLC).

2.3. Sample preparation

2.3.1. Hand dissection/grain fractionatingThe grains were hand dissected to separate the outer pericarp

(bran), inner layer (aleurone layer), germ and endosperm basedon the procedure described by Stewart, Nield, and Lott (1988) withfurther modifications. Briefly, grain brush ends and germs were re-moved by a sharp scalpel under a magnifying glass and the seedscut lengthwise. The grains were soaked in 0.1% sodium hypochlo-rite for 15–20 min to sterilize the surfaces and rinsed using sterile,distilled, deionised water. The seeds were placed in 10 cm petridishes lined with two ashless filters, moistened with 10 mL of ster-ile, distilled, deionised water. The petri dishes were wrapped inaluminium foil and kept at room temperature (20 �C) for 2 days.The pericarp, aleurone layer and endosperm were separated usinga scalpel and stored at �20 �C. Sample were freeze dried, groundusing a multi-use blade grinder, model PCC770 (Loblaws Inc. MO,Canada) to pass through a 0.5 mm sieve screen and no residue>0.5 mm were discarded. The ground samples were stored at�20 �C before extraction and analysis.

2.3.2. Estimation of proportions of seed fractionsTo determine the ratio of the germ, endosperm and bran to

whole grain seed, 25 seeds of each grain sample were randomly se-lected and manually dissected under a magnifying glass. Theweight of germ was calculated by subtracting weight of the endo-sperm with germ from weight of endosperm without germ. Iso-lated germs were randomly weighed to confirm the weight foundby subtraction. The average weight of whole grain and percentageproportions of each fraction are shown in Table 1. The percentageproportions were later used to calculate the distribution of the TCCin the grain fractions.

2.4. Extraction of carotenoids

Carotenoids were extracted according to the method of Abdel-Aal et al. (2007) with some modifications. Briefly, 200 mg of ground

samples (whole grain, endosperm and germ) were mixed with 2 mLof water–saturated butanol in tubes covered with black cap andaluminium foil in a fume hood. The mixtures were vortex for 30 sand carotenoids extracted by shaking for 15 min at speed of 40using a horizontal rotary shaker (RKVSD, ART Inc. Laurel, MD). Aftershaking, the samples were left to stand for 60 min at room temper-ature in the dark and homogenised again before shaking for another15 min. Lastly, the samples were allowed to stand for another60 min. About 1.8 mL of extract were transferred into 2 ml brownmicro-centrifuge tubes and centrifuged at 4000g and 20 �C usingIEC Micromax Microcentrifuge (Thermo Electron Corporations,USA) for 5 min. All the procedures were carried out in the dark.

2.5. Spectrophotometric determination of TCC

Supernatants were transferred from micro-centrifuge tubes intoa semi-micro quartz cuvet and absorbance measured at 450 nm(average absorbance for carotenoids in wheat and corn) using aUltraspec 1100 pro, UV/Visible spectrophotometer (BiomicronLtd., Cambridge, CB4 QFJ, England). All analysis was done in tripli-cate. Total carotenoid content (TCC) was calculated using the fol-lowing equation and expressed as lg lutein equivalent/g sample.

C ¼ ð2� AÞ=S�W½lg=g�

where C = lutein content, lg/g; A = absorbance reading, S = regres-sion coefficient (the number that express the relationship whichis created based on concentration of lutein working standard solu-tions in lg/mL and the absorbance); 2 = dilution factor (the dilutionfactor 2 is based on the total extracted volume of 2 ml) andW = sample weight, g (Abdel-Aal & Young, 2007, 2009).

2.6. Determination of carotenoid composition by HPLC

Fresh extractions were done to determine carotenoid composi-tion using the same procedure described above up to centrifuga-tion of the extracts. After centrifugation, the supernatantobtained was filtered through a 0.45 lm nylon disc filter intobrown HPLC vials and stored at �20 �C overnight before analysis.HPLC analysis of carotenoid composition included the aleuronelayer fraction and analysis was done in duplicate. The determina-tion of carotenoid composition was done according to the methoddescribed by Abdel-Aal et al. (2007) with some modifications.Briefly, the chromatographic separation and quantification ofcarotenoids was carried out on an HPLC (Waters 2695) equippedwith a photodiode array detector (PAD) (Waters 996) and

V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671 665

autosampler (Waters 717 plus) (Waters, Milford, MA) using YMCTM

carotenoid S-3, 3 lm packing, 4.6 � 100 mm column (Waters, Mil-ford, MA). The column was operated at 35 �C, 20 lL of sample wasinjected by the auto sampler and eluted with a gradient systemconsisting of (A) methanol/methyl tert-butyl ether/Milli-Q water(81:15:4, v/v/v) and (B) methyl tert-butyl ether/methanol (90:10,v/v). The flow rate was set at 1 mL/min. The gradient wasprogrammed as follows: 0–9 min, 100–75% A; 10–12 min 0% A;12–13 min, 0–100% A; and 13–15 min, 100% A. The separatedcarotenoids were detected and measured at 450 nm. The elutedcarotenoids were identified based on similarity of retention time,elution sequence and UV/Vis spectra with those of standards andthe ones reported in literature. Lutein and zeaxanthin standardswere used for identification and quantification whereasb-cryptoxanthin was only used for identification as specified bymanufacturer. Five concentrations in the range of 0.05–0.5 and0.25–2.5 lg/ml per injection of 20 lL were prepared for luteinand zeaxanthin to generate regression equations for quantification.The regression equations (y = 4983.3x + 7.8715 and y = 3182.6x �80.362) showed a linear relationship with R2 of 0.9999 and0.9983 for lutein and zeaxanthin, respectively.

2.7. Determination of DPPH radical scavenging activity

The DPPH radical scavenging activity of carotenoids extractswas determined according to the method described by Brand-Wil-liams (1995) and as modified by Li, Pickard, and Beta (2007) withfurther modifications. Aliquots of 3.9 ml of 60 lM DPPH in metha-nol were mixed with 0.1 mL of the carotenoid extracts from wholegrain, endosperm, germ and aleurone layer. The mixtures wereheld for 30 min under subdued light. The absorbance of DPPH rad-icals was read at 515 nm against methanol as a blank using a Ultra-spec 1100 pro, UV/Visible spectrophotometer (Biomicron Ltd.Cambridge, CB4 QFJ, England). The analysis was done in duplicate.The standard curve was constructed using trolox concentrationsranging from 100 to 700 lmol. The percentage scavenging of DPPHradical was calculated according to the following equation:

DPPH radical scavenging activity (%) = [(1 � Asample,t)/(Acontrol)]� 100, where Acontrol is the absorbance of DPPH radical in methanolat 0 min, Asample is the absorbance of DPPH radical and sample ex-tract or standard at 30 min.

2.8. Statistical analysis

The analytical data were reported as mean ± standard deviation(SD) of triplicate analysis for TCC and duplicate analysis for HPLC

Table 2Total carotenoid content (mg/kg) in whole grain, endosperm, germ and bran fractions.

Cereal ID Whole graina Endosper

Purple barley 4.54 ± 0.12d 1.14 ± 0.0Non-pigmented barley 2.25 ± 0.08 g 0.88 ± 0.0

Mean-barley 3.40 1.01Purple wheat 2.62 ± 0.03f 1.71 ± 0.0Ambassador wheat 2.11 ± 0.04 g 1.73 ± 0.0Caledonia wheat 2.73 ± 0.05ef 1.98 ± 0.0MSU D8006 wheat 2.84 ± 0.06e 2.27 ± 0.0

Mean-wheat 2.57 1.92Oat 1.8 ± 0.05 h 1.18 ± 0.0Dasca corn 26.46 ± 0.18a 31.35 ± 0USP1395XR corn 12.86 ± 0.03c 14.17 ± 0P1508HR corn 15.24 ± 0.02b 18.79 ± 0

Mean-yellow corn 18.19 21.44

a Values are mean ± standard deviation (n = 3). Values with a different letter in each cb Calculated by subtraction: TCC in whole grain-(TCC in endosperm – TCC in germ) bc Figures in parentheses are percentage contribution of fraction to TCC in whole grain

assay of independent extractions. One- way analysis of variance(ANOVA) of results was performed using SAS statistical softwareversion 9.2 (SAS Institute Inc., Cary, NC). Significant differencesamong samples for TCC and carotenoid composition were assessedusing Duncan multiple range test at p < 0.05. Correlations betweenparameters were examined by Pearson’s correlation test.

3. Results and discussion

3.1. Variation in total carotenoid content in whole grain, endospermand germ

3.1.1. Whole grainTable 2. shows total carotenoid content (TCC) expressed as lu-

tein equivalent (mg/kg) in whole grain. Levels of TCC varied signif-icantly different (p < 0.05) among different cereal types and withincereal varieties. Non-corn cereals had lower TCC compared to corn.The average TCC was 3.40, 2.57 and 18.19 mg/kg for barley, wheatand yellow corn, respectively. Oat had the lowest TCC (1.8 mg/kg).Among the non-corn cereals, purple barley had highest levels(4.54 mg/kg). TCC of wheat varieties (range 2.11–2.84 mg/kg)was similar to those reported by other authors (Hentschel et al.,2002; Konopka, Czaplicki, & Rotkiewicz, 2006; Panfili et al.,2004). However, Luterotti & Kljak, 2010 reported a lower rangeof TCC (1.1–1.3 mg/kg) in wheat flour. Compared to values in lowyellow pigmented durum wheat reported by Ramachandran,Pozniak, Clarke, and Singh (2010), TCC of wheat varieties in thepresent study were 2.2- to 2.5-fold lower. Purple barley had 2-foldhigher TCC compared to regular barley Table 2. However, use ofcolorimetric methods to determine TCC may overestimate theamounts. For instance, TCC was over estimated by 20% in wheat(Abdel-Aal et al., 2007) and only 30–50% of the yellow pigmentswere carotenoids in durum wheat (Hentschel et al., 2002). Hent-schel and others suggested the presence of other unknown pig-ments contributing to the yellow colour in grains. This may havebeen the case with purple barley, as not all pigments were carote-noids according to HPLC results to be discussed in Section 3.5. TCCof yellow corn in the present study were higher than values re-ported recently for yellow corn (11–23 mg/kg) (Luterotti & Kljak,2010) although they were within the same range of 19.3–26.4 mg/kg reported for ten maize varieties (Rios et al., 2009).

3.1.2. EndospermNon-corn cereal endosperms had significantly lower TCC

(p < 0.05) which ranged from 0.88 to 2.27 mg/kg compared to14.17–31.35 (mg/kg) in yellow corn Table 2. MSUD8006 wheat

ma Germa Branb

2 g (20.5)c 12.68 ± 0.16b (7.2) 3.28 (72.3)2 h (32.8) 14.77 ± 0.23a (3.3) 1.22 (54.2)

13.73 2.256 f (56.5) 8.45 ± 0.39e (5.3) 1.00 (38.2)4f (70.6) 9.87 ± 0.24c (9.5) 0.42 (19.9)2e (62.3) 9.42 ± 0.1d (7.3) 0.83 (30.4)5d (68.7) 8.71 ± 0.12e (5.9) 0.72 (25.4)

9.11 0.743 g (58.9) 6.08 ± 0.25f (6.1) 0.63 (35.0).22a (97.2) 3.19 ± 0.02 h (1.4) 0.37 (1.4).16c (91.6) 4.81 ± 0.14 g (4.2) 0.55 (4.3).02d (106) 3.33 ± 0.03 h (2.4) None

3.78 0.46

olumn are statistically different at p < 0.05 (Duncan’s multiple range test).ased on seed fraction weight proportions to whole grain..

666 V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671

and regular barley had the highest and least TCC, respectively.Within yellow corn varieties, Dasca had the highest TCC. On aver-age, TCC of barley, wheat and oat endosperm were 29.7%, 74.7%and 65.5% of total carotenoid content of the corresponding wholegrain implying concentration of carotenoids in the germ of barley.

3.1.3. GermIn contrast, TCC were high in germ fraction of non-corn cereals

(6.08–14.77 mg/kg) and low in yellow corn (3.19 – 4.81 mg/kg) Ta-ble 2. The concentrations were significantly different (p < 0.05)among all the cereal types. The ratios of TCC in the germ of non-corn cereals to germ of corn cereals were 3.6:1, 2.4:1 and 1.6:1for barley, wheat and oat respectively. The highest TCC were ob-served in regular barley germ followed by purple barley, Ambassa-dor wheat, MSUD8006 wheat, purple wheat and oat among thenon-corn cereals. The higher content of TCC in germ would beattributed to presence of yellow carotenoid pigments which werefound in trace amounts in endosperm of non-corn cereals. Onestudy reported 1.8-fold higher TCC in the germ of durum wheatthan in whole grain content (Panfili et al., 2004). In this study, germcarotenoids were 4.0-, 3.5- and 3.3-fold higher in barley, wheat andoat, respectively compared to TCC of their whole grains. These re-sults show that barley germ is a more concentrated source of carot-enoid than wheat germ. Oat germ had about 66.7% of the TCC ofwheat germ. These results confirm that in wheat, carotenoids areconcentrated in the germ, and suggest that the same is applicablein barley and oat. Barley, wheat and oat germ fractions may there-fore be targeted for use as food ingredients with enhanced caroten-oid content.

3.2. Contribution of each grain fraction to TCC in whole grain

In non-corn cereals, TCC contribution to whole grain variedwidely while in yellow corn it was within the same range Table 2.TCC contribution ranged from 20.5–70.6% in the endosperm; 3.3–9.5% in germ and 19.9–72% in bran within non-corn cereals. In yel-low corn it was >91.6–106% from the endosperm only and between1.4% and 4.3% from the germ and bran. Similarly, Weber (1987) re-ported 90–107% and 1.3–3.6% TCC contributions from horny andfloury endosperm and, germ and tip cap of corn inbreds,respectively.

3.2.1. EndospermAmong the non-corn cereals, wheat and oat endosperm contrib-

uted above 50% of TCC to whole grain Table 2. Ambassador wheatcontributed the highest TCC (70.6%) despite having the lowest TCCin whole grain among the wheat varieties. Worth noting was thelow contribution (20.5%) of purple barley endosperm to wholegrain. In corn, the endosperm contributed the highest, which isattributed to the yellow pigment responsible for its colour (Coul-tate, 2009). TCC contributions to whole grain were highest forthe endosperm since it is the largest fraction.

3.2.2. GermAlthough TCC of non-corn cereals was the highest in the germ

fraction, its contribution to TCC in whole grain was the lowestcompared to contributions from endosperm and bran fractions(by difference). Therefore it may be inferred that, although TCCconcentrations in the germ fractions of wheat (7.26–13.33%), bar-ley (5.34–9.48%), and oat (6.11%) were the highest, their contribu-tion to TCC in whole grain are minimal because the germ onlyconstitutes about 1.9–2.0% of whole grain. Yellow corn germ con-tributed the lowest TCC (1.47–4.20%) to its whole grain. Slightlylower ranges of TCC contribution (1.3–3.6%) of corn germ to wholegrain were reported by Weber (1987).

3.2.3. BranTCC in bran fraction were calculated by subtraction based on

bran percentage composition and total carotenoid content in germ,endosperm and whole grain. The percentage contributions were be-tween 19.9–72.3% (63.3% average) in non-corn cereals and 1.4–4.3%(2.9% average) in yellow corn Table 2. Although this study foundnegligible amounts of TCC, Kean, Hamaker, and Ferruzzi (2008) re-ported 1.77–6.50 mg/kg of carotenoid in yellow corn bran. The lat-ter used mechanically separated bran fraction while our study usedhand dissected fractions which were pure with zero to little adulte-ration of endosperm fraction. TCC from purple barley (72.3%) andpurple wheat (38.2%) bran fraction were higher than contributionsfrom the bran of non-pigmented barley and wheat varieties. Thehigh TCC contribution from bran of purple barley and purple wheatmay be attributed to pigmentation in the seed coat of these grains.According to Fratianni, Irano, Panfili, and Acquistucci (2005), theseed coat contains interfering pigments which would lead to overestimation of the total carotenoid content.

3.3. Separation and identification of carotenoids in whole grains andtheir grain fraction

The short method was chosen assuming that water-saturatedbutanol extracted mainly polar carotenoids unlike non-polar ones,which are found in minimal amounts in cereal grains. Lutein and lu-tein esters amounted to about >90% of the yellow pigment com-pared to approximately 1% of b-carotene in wheat (Lepage & Sims,1968). Identification of carotenoids was accomplished by compar-ing the retention times (tR) in the samples with those of the externalstandards and the UV–Visible absorption spectra in published liter-ature. In non-corn cereals and corn, two major peaks were identi-fied. A third major peak was found in corn. Minor peaks were alsoobserved in some cereals and their fractions. Compared to peaksin the standard mixture (Fig. 1A), the two major peaks were identi-fied as lutein and zeaxanthin, the primary carotenoids found in cer-eal grains and products (Fratianni et al., 2005; Panfili et al., 2004).

Lutein and zeaxanthin were identified in all whole grain sam-ples. Barley grains had an additional minor peak at tR 5.9 min (y)(Fig. 1B). In yellow corn, the peak at tR 8.1 min was identified asb-cryptoxanthin. Other unknown minor peaks were at tR 3.3 min(x), 5.9 min (y) and 6.6 min (z) (Fig. 1C). In contrast, only luteinwas identified in the endosperm fraction of non-corn cereals(Fig. 1D) while corn endosperm had similar peaks to the ones iden-tified in the whole grain and one additional unknown peak at tR

7.1 min (w).Lutein and zeaxathin were detected in the aleurone layers of

wheat and oats while zeaxanthin was only detected in barley;however, zeaxanthin was not detected in the endosperm of othernon-corn cereals. This finding suggests that the aleurone layer innon-corn cereal may be closely adhering to the bran than the endo-sperm. In addition to the lutein and zeaxanthin peaks, Caledoniawheat had an unknown peak at tR 3.3 min (x) (Fig. 1E). Apart fromlutein, zeaxanthin and b-cryptoxanthin, the aleurone layer of yel-low corn also had an unknown peak at tR 5.9 min (y) (Fig. 1E). Lu-tein and zeaxanthin were identified in all the germ fractions. Inaddition to these two, non-pigmented barley and Caledonia germalso had minor peaks at tR 3.3 min (x) and 5.9 min (y) (Fig. 1F),while purple barley germ only had the latter peak (Fig. 1H).

These unknown peaks (x, y, z and w) were proposed to be cis-isomers of lutein on the basis of UV/Vis spectra and absorptionmaxima (kmax) with ones reported in literature. The elution se-quence of the unknown peaks in the present study was similar tothe ones reported by Abdel-Aal et al. (2007) in wheat and corn.Based on this literature, the unknown peaks were suggested tobe 13-cis lutein, 9-cis lutein and 90-cis lutein, respectively. Inaddition, the observed spectra absorption maxima of 440, 442,

Fig. 1. LC–UV/Vis chromatograms of carotenoids separated from standard mixture (A); whole grain: purple barley (B), and DASCA corn (C); endosperm: Caledonia wheat (D);Aleurone layer: Caledonia wheat (E) and Dasca corn (F) and germ: Caledonia wheat (G) and purple barley (H); 1 = lutein, 2 = zeaxanthin, 3 = b-cryptoxanthin, x = 13-cis lutein,y = 90-cis lutein, z = 9-cis lutein.

V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671 667

430 and 442 (Fig. 2A and B) of the unknown peaks were compara-ble to ones reported by Gentili and Caretti (2011). Gentili and Car-etti (2011) identified 13-cis lutein and 9-cis lutein at 420/443/471and 444/471 nm, respectively, in maize flour. Similarly cis-luteinshave been reported in wheat, fruits and vegetables (Humphries &Khachik, 2003) and in the present study, they were also found inyellow corn and barley.

3.4. Quantification of lutein and zeaxanthin in whole grains

Quantitative data were calculated from the linear calibrationcurves. The concentration of lutein and zeaxanthin in non-corncereals and yellow corn varied widely and were significantly differ-ent (p < 0.05).

Lutein ranged from 101–1034 lg/kg in non-corn cereals and3414–3891 lg/kg in yellow corn (Table 3). Lutein content was sig-nificantly different (p < 0.05) among the different cereal types andwithin the cereal varieties. Lutein was the primary and major com-ponent in non-corn cereals. Among the non-corn cereals, wheatvarieties had the highest lutein levels on average. Oat had the low-est levels of lutein. Purple barley had two times more lutein com-pared to non-pigmented barley. The ranges of lutein content inwheat varieties are comparable to 841–1340 lg/kg of lutein con-tent in einkon wheat (Hidalgo et al., 2006). In wheat, barley andoat grains, lutein was reported as the major xanthophyll and zea-xanthin as the minor (Luterotti & Kljak, 2010; Okarter et al.,2010; Panfili et al., 2004). Panfili and others reported higheramounts of lutein in soft and durum wheat (1310 and 2650 lg/

Fig. 2. LC chromatograms and absorption spectra for non-pigmented barley germ (A) and DASCA corn endosperm (B), showing maximum wavelength absorption spectra at3.3 min (X), 5.8 min (Y); 6.6 min (Z) and 7.1 min (W).

Table 3Carotenoid composition in whole grain and its distribution in grain fractions (lg/kg)a.

Fraction Whole grain Endosperm Aleurone Germ Whole grain Endosperm Aleurone Germ

Cereal type Lutein Zeaxanthin

Purple barley 699 ± 0.02e 45 ± 0.00f 190 ± 0.00d 2517 ± 0.21b 624 ± 0.02d nd nd 3015 ± 0.11dRegular barley 295 ± 0.02f 66 ± 0.01f 151 ± 0.06d 2630 ± 0.0ab 651 ± 0.03d nd nd 5855 ± 0.00a

Mean-barley 497 56 171 2573 637 – – 4435Purple wheat 596 ± 0.00e 554 ± 0.01de 534 ± 0.08d 1714 ± 0.01c 543 ± 0.02de nd 1217 ± 0.04d 3233 ± 0.01cAmbassador wheat 741 ± 0.01ed 493 ± 0.03e 370 ± 0.03d 1566 ± 0.08c 359 ± 0.01e nd 629 ± 0.05f 2491 ± 0.06eCaledonia wheat 905 ± 0.01cd 559 ± 0.03de 350 ± 0.02d 2531 ± 0.15b 411 ± 0.01de nd 680 ± 0.05ef 3726 ± 0.01bMSUD8006 wheat 1034 ± 0.02c 624 ± 0.04d 446 ± 0.05d 2816 ± 0.17a 439 ± 0.03de nd 580 ± 0.04f 2927 ± 0.00d

Mean-wheat 819 557 425 2157 438 – 776 3094Oat 101 ± 0.00f 43 ± 0.00f 181 ± 0.02d 1340 ± 0.00d 356 ± 0.0e nd 943 ± 0.11de 1960 ± 0.13fDASCA corn 3891 ± 0.21a 3365 ± 0.00c 3171 ± 0.01c 236 ± 0.00e 18369 ± 0.19a 13210 ± 0.32a 13730 ± 0.13a 873 ± 0.04ghUS P139SXR corn 3762 ± 0.16a 3672 ± 0.14b 7631 ± 1.14a 307 ± 0.01e 4593 ± 0.11c 7294 ± 0.18b 7993 ± 0.31b 969 ± 0.01 gP1508HR corn 3414 ± 0.07b 3881 ± 0.03a 5277 ± 0.04b 261 ± 0.01e 6674 ± 0.23b 7710 ± 0.05b 7232 ± 0.19c 818 ± 0.00 h

Mean-yellow corn 3689 3639 5360 268 9879 9404 9652 886

Nd – Not detected.a Values are mean ± standard deviation (n = 2). Values with a different letter in each column are statistically different at p < 0.05 (Duncan’s multiple range test).

668 V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671

kg), barley (860 lg/kg) and oat (230 lg/kg). Lutein was on average7.4 times and 4.5 times higher in corn than in barley and wheat,respectively. In non-corn cereals, lutein accounted for 22.2–70.2%of the total carotenoid content. Among wheat varieties, lutein con-tributed about 52–70%, which was lower than 78–85% reported inMindum and Thatcher wheat and 80–90% in einkorn wheat (Abdel-Aal et al., 2002).

Zeaxanthin levels ranged from 356 to 650 lg/kg and 4593–18,369 lg/kg in non-corn cereals and yellow corn, respectively.The zeaxanthin content in yellow corn are similar to those reportedin maize (Kurilich & Juvik, 1999b). In the non-corn cereals, zeaxan-

thin content were significantly different (p < 0.05) among cerealtypes but similar within cereal varieties. On average, barley varie-ties had the highest levels of 637 lg/kg, followed by wheat varie-ties (438 lg/kg) and oat had the least (356 lg/kg). Zeaxanthincontent were significantly different (p < 0.05) within the corn vari-eties, with Dasca having 3.9 times and 2.7 times the amounts inUSP1395XR and P1508HR corn. Zeaxanthin content of 6430 and120 lg/kg, in corn and wheat, respectively were previously re-ported (Panfili et al., 2004). On average zeaxanthin content in bar-ley and wheat were only 4.4% and 6.4% of the amounts found inyellow corn. Furthermore, zeaxanthin accounted for 29.8–77.8%

V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671 669

and 55.0–82.5% in non-corn cereals and corn varieties, respec-tively. Lutein contributed 10–40% (Kimura et al., 2007) and zeaxan-thin 30–60% of the total carotenoid in corn (Hulshof et al., 2007).

Differences in lutein and zeaxanthin content in wheat grainsand others cereals observed in this study would be attributed tovarietal and genetic differences and growing location which affectchemical composition of the cereal grains (Kurilich & Juvik, 1999a;Panfili et al., 2004).

3.5. Quantification and distribution of lutein and zeaxanthin in grainfractions

Table 3 reports carotenoid composition in grain fractions of di-verse cereal grains and of particular interest is the aleurone layerwhich has very limited literature.

3.5.1. Aleurone layerThe range of lutein content was lower in non-corn cereals 151–

534 lg/kg compared to 3171–7631 lg/kg in yellow corn. On aver-age wheat varieties had 2.3–2.4 times higher lutein content thanoat and barley. The amount of lutein in barley varieties was not sig-nificantly different, presumably agreeing with Fratianni et al.(2005) that pigmentation in seed coat leads to overestimation ofTCC. Among corn (average 5360 lg/kg), highest levels of luteinwere observed in USP1395XR (7631 lg/kg) and the least in DASCA(3171 lg/kg). However lutein content was not significantly differ-ent among the non-corn cereals (p < 0.05). The amounts of lutein inoat and barley aleurone layers were 4- and 3 times higher than inendosperm fractions. Similarly in corn, lutein content was higherin aleurone than in the endosperm.

Zeaxanthin levels varied significantly (p < 0.05) both in non-corn cereals and corn varieties. It ranged from 580–1217 lg/kgamong the non-corn cereals with higher levels (7232–13730 lg/kg) in yellow corn. Purple wheat had the highest zeaxanthinamong non-corn cereals, followed by oat, Caledonia wheat, Ambas-sador wheat and MSUD8006 wheat. DASCA corn had the highestlevels of zeaxanthin. Zeaxanthin has been reported to be concen-trated in the germ fraction of wheat (Panfili et al., 2004) and cornendosperm (Kean et al., 2008). In the present study, zeaxanthinwas also the dominant carotenoid in the aleurone layers. In wheatand yellow corn varieties, zeaxanthin content was 1.8-fold higherthan lutein content while in oats it was 5.2-fold higher than lutein.

These results indicate that the aleurone layer of wheat, oat andcorn cereals have significant levels of lutein and zeaxanthin, whichhave antioxidant properties in addition to ferulic acid. Aleuronegrains in the aleurone layer are surrounded by lipid droplets andcarotenoids in this layer may be protecting the membranes fromlipid peroxidation. Konopka et al. (2006) suggested that the lipidfractions of grains contain carotenoids which may enhance theantioxidant activity of the aleurone layer.

3.5.2. EndospermOnly lutein was detected in the endosperm fraction of non-corn

cereals ranging from 43 lg/kg (oat) to 624 lg/kg (MSUD8006) and3171–7631 lg/kg in yellow corn. The average lutein content was9.9 times higher in wheat than in barley. Although, wheat hadthe highest lutein content among the non-corn cereals, its levelswere 6.5 times lower compared to levels in corn. In corn, lutein lev-els varied significantly among the varieties. Similarly, zeaxanthinlevels were significantly (p < 0.05) high in DASCA corn(13210 lg/kg).

3.5.3. GermLutein and zeaxanthin content in the germ fraction had an

opposite trend to what was observed in whole grain, endospermand aleurone layer.

Lutein levels ranged from 1340 to 2816 lg/kg in non-corn cere-als and 236–307 lg/kg in yellow corn (Table 3). On average, barleyvarieties had the highest levels of lutein (2573 lg/kg) and oat hadthe least (1340 lg/kg) among the non-corn cereals. Lutein contentwas much lower in corn germ (268 lg/kg). The proportion of luteinto total carotenoid in the germ was 30.9–49.0% and 21.3–24.2% innon-corn cereals and corn, respectively.

Zeaxanthin content ranged from 1960 to 5855 lg/kg in non-corncereals and 818–969 lg/kg in yellow corn. In barley and wheatgerm, the average zeaxanthin content was 5.6 and 3.5 times higherthan in yellow corn. The levels of zeaxanthin were lower in yellowcorn; however, the proportions of zeaxanthin to lutein were high incorn (75.8–78.7%) and low in non-corn cereals (51.0–68.1%). Thehigh concentrations of lutein and zeaxanthin in the germ fractionwould be attributed to their role as antioxidants and in promotingseed germination (Rogozhin, Verkhoturov, & Kuriliuk, 2001).

Within the germ fraction, the ratio of zeaxathin to lutein variedamong the types of cereal grains. In purple barley and MSUD8006wheat, the ratio was 1:1 whereas as in oat, Caledonia, Ambassadorwheat, purple wheat and non-pigmented barley, the ratio wasfrom 1.5:1 to 2.2:1. The ratio was much higher in yellow corn rang-ing from 3.1–3.7:1 despite the low levels. Comparing the levels oflutein and zeaxanthin in the germ fraction to those in the wholegrain, results clearly showed that the germ is a more concentratedsource of these carotenoids especially zeaxanthin. The ratio of lu-tein in germ to whole grain ranged between 2 and 3 times in wheatand purple barley. In oat and non-pigmented barley germs, the ra-tio was very high, 13 and 9 times respectively. The levels of zea-xanthin content in germ were higher than in whole grains acrossthe cereals and varied widely. It was 9-fold in Caledonia andnon-pigmented barley, 7-fold in Ambassador and MSUD8006wheat, 5.5- to 5.9-fold in oat and purple wheat and 4.8-fold in pur-ple barley. A higher ratio (21 times) of zeaxanthin in germ to zea-xanthin in whole grain was found in soft wheat (Panfili et al.,2004). However, for both lutein and zeaxanthin, respectively, theratios in corn were low, 0.06 and 0.04 in Dasca corn, 0.08 and0.13 in USP1395XR and 0.07 and 0.10 in P1508HR corn. These re-sults suggest that zeaxanthin is mainly localised and highly con-centrated in the germ fraction and aleurone layer in non-corncereals, and in the endosperm and aleurone layer of corn varieties.

Concurring with Panfili et al. (2004), lutein was found in all thegrain fractions however; in the present study it was unevenly dis-tributed. Zeaxanthin was not detected in endosperm of non-corncereals and barley aleurone layer, but it was more concentratedin germ as previously reported with some considerable amountsin the aleurone layer. In addition, this study is the first, to theauthors’ knowledge, to report on lutein and zeaxanthin contentin aleurone layer fractions of different cereals. Comparing theamounts of zeaxanthin in grain fractions, the levels in the aleuronelayer ranked second from the germ.

3.6. Antioxidant activity of carotenoids extracts from whole grain andits fractions

Lutein and zeaxanthin have been reported as important antiox-idants (Gentili & Caretti, 2011; Leenhardt et al., 2006) and hencethe extracts used for HPLC analysis were also examined for theirantioxidant activity using DPPH method. DPPH assay measuresthe reducing ability of antioxidants towards DPPH radical throughdiscolouration, showing the % of DPPH that has been quenched.The results presented are for the scavenging activity at 30 min.There was a wide and significant variation in %DPPH scavengingactivity among the different cereal types and their grain fractions(p < 0.05) (Fig. 3).

The %DPPH scavenging activity in whole grains was generallylow (616.4%) in non-corn cereal and slightly higher

670 V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671

(P20 6 23.9%) in yellow corn. The %DPPH scavenging activity inMSUD8006 and Caledonia wheats were below the lowest scaveng-ing activity (11.9%) of minimum trolox concentration (100 lM).However, a positive correlation was found between scavengingactivity and total carotenoid (lutein and zeaxanthin) content(r = 0.6945, p < 0.05). Similarly, %DPPH scavenging activity in theendosperm was low in non-corn cereals (<5%) cereal and slightlyhigher in corn (<10%). In the non-corn cereals the scavenging activ-ity decreased in the following order: oat > Ambassador wheat > -non-pigmented barley, purple barley > purple wheat > MSUD8006wheat > Caledonia wheat (Fig. 3). Despite this observation, a signif-icant and strong correlation was found between DPPH scavengingactivity and carotenoid content analysed by HPLC in the endo-sperm fraction (r = 0.9647, p < 0.05).

The low percentage of DPPH scavenging activity in the endo-sperm of the non-corn cereals may be attributed to the low totalcarotenoid content and absence of zeaxanthin. Mortensen andSkibsted (1997) studied the role of carotenoid structure in radicalscavenging reactions and reported that zeaxanthin is more reactivethan lutein although their order of reactivity only shows slight dif-ferences. However, we could not explain why corn endosperm de-spite its high lutein and zeaxanthin content, had low DPPHscavenging activity compared to the other fractions.

In contrast, %DPPH scavenging activity in the germ fractionsshowed different trends, as was observed in TCC and HPLC analysisresults. Compared to the other fractions, scavenging activity wasthe highest in the germ ranging from 26.2% to 45.5% in non-corncereal and 22.8–25.8% in yellow corn. Within the germ fractions,scavenging activity was the highest in wheat varieties (MSUD8006and Caledonia) and lowest in corn. The germ of barley varieties, de-spite having higher amounts of total carotenoid (Table 3) com-pared to wheat varieties, had lower DPPH scavenging activity.This discrepancy may have contributed to the weak but significantcorrelation observed between %DPPH scavenging activity and lu-tein and zeaxanthin content (r = 0.48023, p = 0.03).

%DPPH scavenging activity in the aleurone layer of wheat vari-eties was about a third (31.4%) to half (46.9%) the activity observedin their germ fractions. Barley aleurone layer had the lowest scav-enging activity. The high scavenging activity of wheat aleuronemay be attributed to approximately 1.2–2 times more zeaxanthincompared to lutein while the low scavenging activity in barleywould be due to lack of zeaxanthin and low levels of lutein inthe aleurone layer. A positive significant correlation was observedbetween DPPH scavenging activity and lutein + zeaxanthin contentin aleurone layers (r = 0.9604, p < 0.05). Zhou, Su, and Yu (2004) re-ported highest concentration of 2980 lg/kg (lutein + zeaxanthin)in Australian wheat bran, which also includes the aleurone layer.

01020304050607080

PB NB PW AW CW MW OA DC USC PC

Cereal type

%D

PPH

sca

veng

ing

activ

ity

Whole Endo Germ Aleurone

Fig. 3. %DPPH scavenging activity of WSB extracts from whole grain, endospermand germ of different cereals. PB, purple barley; NB-regular barley; PW, purplewheat; AW, ambassador wheat; CW, Caledonia wheat, MW, MSUD8006 wheat; OA,Oat; DC, Dasca corn; USC,USP1395XR corn; PC, P1508HR corn. The vertical barsrepresents the standard deviations (n = 2).

These results suggest that carotenoids may be localised in aleuronelayer rather than the other bran layers (pericarp, seed coat). Thereis need therefore for further research to analyse carotenoid contentin the other layers of wheat and oat bran. The highest scavengingactivity was observed in yellow corm aleurone ranging from 60.0to 73.8%. This may be attributed to the high levels of lutein in aleu-rone layer compared to endosperm since average zeaxanthin levelswere similar in the two fractions.

3.7. Relationship between carotenoid content and %DPPH scavengingactivity

Positive and strong correlations were found between carotenoidcontent and %DPPH scavenging activity as discussed above. In con-trast, no correlation was found between antioxidant activity andcarotenoid content in methanolic extracts of cereal grains (Choi,Jeong, & Lee, 2007). Similarly, Thaipong, Boonprakob, Crosby, Cisn-eros-Zevallos, and Byrne (2006) reported zero and negative corre-lations between carotenoid content in different fruit juice extractsand their antioxidant activity using DPPH. The positive correlationobserved in this study would be attributed to lutein and zeaxan-thin contents extracted using the water saturated-butanol. Accord-ing to Baublis, Decker, and Clydesdale (2000) aqueous extractsfrom cereal grain products showed significant antioxidant activity.In addition, lutein and zeaxanthin have hydroxyl groups on each b-ring (Miller et al., 1996) and these polar functional groups renderthem more accessible to aqueous environments (Rice-Evanset al., 1997). Furthermore strong correlations were also wereobserved between TCC and carotenoid content determined byHPLC (Fig. 4). A similar trend in correlation coefficients was alsoobserved in germ, whole grain and endosperm. The correlation

Fig. 4. Correlation between lutein and zeaxanthin content determined by liquidchromatography and total carotenoid content determined by spectrophotometry.

V.U. Ndolo, T. Beta / Food Chemistry 139 (2013) 663–671 671

was slightly lower in germ fractions (R2 = 0.8591) compared to thatof endosperm (R2 = 0.9833) and whole grain (R2 = 0.9656).

4. Conclusion

Carotenoid composition was similar except in yellow cornwhich also had b-cryptoxanthin. Generally, lutein and zeaxanthinand TCC were all unevenly distributed across the grain kernel. Innon-corn cereals, lutein and zeaxanthin were concentrated in thegerm unlike in yellow corn where they were concentrated in theendosperm and aleurone layer. This study is the first to report oncarotenoid composition of the aleurone layer. Oat, wheat and yel-low corn aleurone layer exhibited higher levels of zeaxanthin com-pared to lutein content. Antioxidant activity of carotenoid inaleurone layer of non-corn cereals was about 50% of the germ,the most concentrated source of carotenoid in cereals. A high cor-relation between %DPPH scavenging and total lutein and zeaxan-thin content was found. Apart from yellow corn endosperm andaleurone layers, germ and aleurone layers of oat and wheat, andgerm of barley may be utilised as food ingredients with enhancedcarotenoid content in development of functional foods for healthyconscious consumers.

Acknowledgements

The infrastructure used for the research was generously fundedby the Canada Foundation for Innovation (New Opportunities Fundand Leaders Opportunities Fund). This project was partially sup-ported by the Agri-Food Research and Development Initiative(ARDI) under the Canada-Manitoba Growing Forward initiative:Growing Forward – Working together to build an innovative andprofitable agriculture and agri-food sector. We are thankful forthe technical support from Alison Ser, Yang Qiu, and Pat Kenyonof the Department of Food Science, University of Manitoba.

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