Genetic Variability in Anthocyanin Composition and Nutritional

Article pubs.acs.org/JAFC

Genetic Variability in Anthocyanin Composition and Nutritional Properties of Blue, Purple, and Red Bread (Triticum aestivum L.) and Durum (Triticum turgidum L. ssp. turgidum convar. durum) Wheats Donatella B. M. Ficco,† Vanessa De Simone,† Salvatore A. Colecchia,† Ivano Pecorella,† Cristiano Platani,† Franca Nigro,† Franca Finocchiaro,§ Roberto Papa,† and Pasquale De Vita*,† †

Consiglio per la Ricerca e la sperimentazione in Agricoltura − Centro di Ricerca per la Cerealicoltura (CRA-CER), S.S. 673, Km 25,200, 71122 Foggia, Italy § Consiglio per la Ricerca e la sperimentazione in Agricoltura − Centro di Ricerca per la Genomica (CRA-GPG), Via S. Protaso, 29017 Fiorenzuola d’Arda (PC), Italy S Supporting Information *

ABSTRACT: Renewed interest in breeding for high anthocyanins in wheat (Triticum ssp.) is due to their antioxidant potential. A collection of different pigmented wheats was used to investigate the stability of anthocyanins over three crop years. The data show higher anthocyanins in blue-aleurone bread wheat (Triticum aestivum L.), followed by purple- and red-pericarp durum wheat (Triticum turgidum L. ssp. turgidum convar. durum), using cyanidin 3-O-glucoside as standard. HPLC of the anthocyanin components shows five to eight major anthocyanins for blue wheat extracts, compared to three anthocyanins for purple and red wheats. Delphinidin 3-O-rutinoside, delphinidin 3-O-glucoside, and malvidin 3-O-glucoside are predominant in blue wheat, with cyanidin 3-O-glucoside, peonidin 3-O-galactoside, and malvidin 3-O-glucoside in purple wheat. Of the total anthocyanins, 40− 70% remain to be structurally identified. The findings confirm the high heritability for anthocyanins, with small genotype × year effects, which will be useful for breeding purposes, to improve the antioxidant potential of cereal-based foods. KEYWORDS: pigmented durum and bread wheats, total anthocyanin content, qualitative parameters, anthocyanin composition, genotype by environment interaction, minerals, total polyphenol, trolox equivalent antioxidant capacity

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INTRODUCTION

the grain pericarp (outer layer), whereas the blue pigment is localized to the grain aleurone layer.11 The anthocyanins were identified and quantified in various blue, purple, and red bread wheats by Abdel-Aal et al.4 The total anthocyanin contents varied from 7.1 to 211.9 μg/g. These pigments normally exist as glycosides (bonded to a sugar moiety), as the anthocyanidin (or aglycon) component alone is extremely unstable. The most common anthocyanin in purple wheat is cyanidin 3-O-glucoside (Cy-3-Glc), followed by peonidin 3-O-glucoside (Pn-3-Glc), whereas delphinidin 3-Oglucoside (Dp-3-Glc) is the most abundant anthocyanin in blue wheat.2,14 Carotenoids are the most important natural pigments, and these compounds have a wide distribution, different structures, and numerous biological functions. In humans, the nutritional importance of carotenoids arises mainly from the pro-vitamin A activity of β-carotene. In addition to their role as precursors of vitamin A, carotenoids have been associated with reduced risk of cancer, decreased cardiovascular disease, protection of the macula region of the retina, prevention of cataracts, and increased iron absorption. The concentration of carotenoids is higher in durum wheat (1.50−4.00 μg/g) than in bread wheat (0.50−2.00 μg/g)15,16 as a result of selection in durum wheat

Wheat (Triticum ssp.) is one of the most common human foods in the world, and recently the focus has been on the possibility of increasing the content of bioactive compounds to improve health and prevent disease.1 Some bioactive compounds can be specific to certain cereals, for example, γ-oryzanol in rice, avenanthramides and saponins in oat, β-glucans in oat and barley, alkylresorcinols in rye, and anthocyanins and carotenoids in pigmented cereal grains.1−3 In particular, there is extensive literature on anthocyanins in colored rice, corn, and to a lesser extent, in barley, buckwheat, sorghum, millet, bread (Triticum aestivum L.) and durum wheat (Triticum turgidum L. ssp. turgidum convar. durum).2,4−9 Anthocyanins are recognized as having the ability to scavenge free radicals that can cause oxidative stress in human cells.10 For a long time, the accumulation of anthocyanins in wheat grain was not considered in breeding programs, except as a genetic marker for specific purposes (feed) to enable their distinction from that used for human consumption or in genetic studies, for example, when carrying out an indirect selection program.11 Indeed, anthocyanin accumulation is an extremely rare trait, which is seen in some forms of tetraploid wheats, with its high frequency in Ethiopian wheat germplasm.11 More recently, a series of blue wheats have been developed through intraspecies crosses from Triticum boeticum, Triticum monococcum, and related wheat species (Agropyron ssp.) with bread wheat.11−13 Early studies confirmed that the purple pigment is localized to © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8686

January 22, 2014 July 24, 2014 July 29, 2014 July 29, 2014 dx.doi.org/10.1021/jf5003683 | J. Agric. Food Chem. 2014, 62, 8686−8695

Journal of Agricultural and Food Chemistry

Article

breeding programs for high yellow pigment concentrations.17 Inheritance of grain or semolina pigment concentrations is complex, although the overall heritability of pigment concentrations is relatively high, ranging from 0.78 to 0.96.18 Recent studies have shown genetic variability for both anthocyanin4,8,19,20 and carotenoid3,17 contents in wheat. For breeding programs, the availability of genetic variability is a key factor to consider, as well as the interaction of genotype × environment (G×E). Few studies have been conducted to evaluate the effects of the environment, defined as a combination of location (L), year (Y), and agronomic management of the crop (M), on the total content of anthocyanins in wheat14 whereas up to now, no studies have been conducted to evaluate the technological performance of these genetic materials. The objectives of the present study were to (i) explore the genetic variability of the total anthocyanin content (TAC) and other qualitative parameters [yellow pigment (YP), thousandkernel weight (TKW), protein content (PC), sodium dodecyl sulfate (SDS) sedimentation volume, and ash content (ASH)] and nutritional parameters [minerals, total polyphenol (TPC), Trolox equivalent antioxidant capacity (TEAC)] in wheat grain; (ii) identify the main anthocyanin compounds in a collection of wheats that includes accessions of tetraploid and hexaploid wheats; and (iii) estimate the effects of G×Y over three growing seasons for all of the traits evaluated. This will thus provide information on the existing variability and genetic resources available to breeders to improve the nutritional value of pasta, bread, and other wheat-based end-products.

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liquid chromatography (HPLC) grade. Eleven anthocyanin standards were purchased: Cy-3-Glc, cyanidin 3-O-galactoside (Cy-3-Gal), malvidin 3-O-glucoside (Mv-3-Glc), pelargonidin 3-O-glucoside (Pg3-Glc), and Pn-3-Glc from Extrasynthese (Genay, France); and cyanidin 3-O-rutinoside (Cy-3-Rut), Dp-3-Glc, delphinidin 3-Orutinoside (Dp-3-Rut), peonidin 3-O-arabinoside (Pn-3-Ara), peonidin 3-O-galactoside (Pn-3-Gal), and Pt-3-Glc from Polyphenols Laboratory (Sandnes, Norway). Quality Traits Assessment. Thousand-kernel weight was calculated from the mean weight of three sets of 100 grains per plot. PC was assayed using the micro-Kjeldahl method, according to the AACC International,21 which was then multiplied by 5.7. The SDS sedimentation volume was measured on 0.6 g of whole meal flour, according to the UNI 10277 method (Ente Nazionale Italiano di Unif icazione22), expressed as a specific SDS sedimentation volume of whole meal flour (in mL/g). The ash content was determined according to the UNI 2171 method23 for the determination of ash yield by complete combustion. These data are expressed as percentages. Yellow pigments were analyzed according to method 14-50 of AACC International,24 as modified by in ref 25 for microsamples. The data were expressed as micrograms per gram on dry matter. Total Anthocyanin Content Using the pH Differential Method. The extraction and purification of TAC was performed according to the mehtod of Hosseinian,9 with slight modifications. A mixture of methanol acidified with 1 N HCl (85:15; v/v) (8 mL) was added to the whole meal samples (0.5 g) in 50 mL centrifuge tubes and sonicated for 18 min at room temperature in an ultrasonic bath (DT 510 Sonorex, DIGITEC). After centrifugation at 9000g for 15 min at room temperature, the supernatants were recovered into 15 mL centrifuge tubes. The pellets were extracted with a further 4 mL of the acidified methanol, and following the same centrifugation, the second supernatants were added to the first. These total supernatants were incubated at −20 °C in the dark for 48 h to facilitate macromolecule precipitation. After this incubation, the samples were centrifuged at 9000g for 15 min, and the supernatants were filtered using 0.45 μm regenerated cellulose syringe filters (Phenomenex, Torrance, CA, USA). Total anthocyanin content was evaluated using a colorimetric method with different pH solutions. Two aliquots of the supernatants extracted (750 μL) were put into different tubes and diluted (1:2, v/v) with either potassium chloride buffer (0.03 M KCl), for pH 1.00, or sodium acetate buffer (0.4 M CH3CO2Na·3H2O), for pH 4.50. The resulting samples were incubated for 30 min at room temperature in the dark and then filtered with 0.45 μm regenerated cellulose syringe filters. The absorbances of the samples at 520 nm were measured against distilled water as the blank. Total anthocyanin content was corrected for the dry matter and is expressed as Cy-3-Glc equivalents as micrograms per gram dry matter. Anthocyanin Composition Using HPLC Analysis. The anthocyanins were extracted according to the method described by Abdel-Aal and Hucl,14 with slight modifications. Briefly, 500 mg of material was extracted twice by mixing 8 mL of acidified methanol with 1.0 N HCl (85:15; v/v), which was then left in an ultrasonic bath for 30 min, with nitrogen gas introduced into the tubes. The crude extracts were centrifugated at 3000g for 15 min at 10 °C and then stored at −80 °C to precipitate the large molecules. The extracts were recentrifuged at 3000g for 15 min at 10 °C and concentrated in centrifugal evaporators (JOUAN RC 1022, Thermo Electron Corp.). The concentrated extracts (5 mL) were filtered through 0.45 μm regenerated cellulose syringe filters (Phenomenex) before analysis by HPLC. The anthocyanins were separated and quantified with a 1200 series chromatography system (Agilent Technologies, Waldbron, Germany) equipped with a G1379B degasser, G1312B binary pump, G1367D temperature-controlled injector, G1330B temperaturecontrolled column thermostat, G1316B photodiode array detector, and Chemstation Rev. B03.02. A 100 mm × 4.6 mm, 2.6 μm, C18 Kinetex column (Phenomenex) was used for the anthocyanin separation. The column temperature was 40 °C, and the mobile phase consisted of (A) 5% formic acid and (B) methanol 22.5%,

MATERIALS AND METHODS

Genotypes and Field Experiments. In all, 76 pigmented wheat genotypes were analyzed in the present study. These included 70 (27 purple, 43 red) that were durum wheat (T. turgidum L. ssp. turgidum convar. durum Desf; 2n = 4x = 28; tetraploid AABB genome) and three that were blue bread wheat (T. aestivum L.; 2n = 6x = 42; hexaploid AABBDD genome) (Supporting Information Table S1). The collection also included two Italian durum wheat genotypes (Preco, PR22D89) and one Italian bread wheat genotype (Spada), which were used as commercial yellow controls, that is, nonpigmented. The 76 genotypes were grown in field trials during three consecutive growing seasons (2008−2009, 2009−2010, 2010−2011) at the CRACER in Foggia, Italy. The experimental design was a randomized complete block with three replications. Each experimental unit consisted of a 1 m2 plot (two rows of 1 m length, with 0.5 m between rows). During the growing season, standard cultivation practices were used. Table S2 (Supporting Information) reports the monthly precipitation and temperature ranges throughout the entire growth cycle of the durum wheat, from November to June. Whole plots were harvested mechanically on June 13, 2009, June 15, 2010, and June 20, 2011, and the grains were kept at 4 °C until analysis. Approximately 100 g of grain was sampled from each plot shortly before analysis and used for the preparation of whole meal, using an experimental mill (Tecator Cyclotec 1093) with a 0.05 mm sieve. Chemicals and Reagents. Sulfuric acid, hydrogen peroxide, SDS, lactic acid, potassium chloride, sodium acetate (CH3CO2Na·3H2O), formic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-3-carboxylic acid (Trolox), sodium hydroxide, 2,2′-azinobis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS), potassium ferricyanide (K3Fe(CN)6), iron(III) chloride, the gum arabic and catechin standards were purchased from Sigma-Aldrich (Milan, Italy). Methanol, n-butanol, hydrogen chloride, acetonitrile, acetone, and acetic acid were purchased from Carlo Erba (Milan, Italy). All chemicals and solvents used in the present study were of high-pressure 8687

dx.doi.org/10.1021/jf5003683 | J. Agric. Food Chem. 2014, 62, 8686−8695


Article

Table 1. Combined Analysis of Variance (ANOVA), Genetic (σ2G) and Genotype × Year Interaction (σ2GY) Variances (±SE) and Narrow-Sense Heritability Estimates on a Genotype-Mean Basis for Qualitative Traits Evaluated of 76 Pigmented Wheat Genotypes over Three Growing Seasons at Foggia, Italya year genotype G×Y residual total

DF

TAC

2 75 150 228 455

4.450 × 10 2.553 × 105§ 1.276 × 104§ 2.618 × 101 2.685 × 105

63.72 408.19§ 98.98§ 7.79 578.69

8479.515 8823.235§ 5135.975§ 269.954 22708.678

9.267 × 10 7.824 × 102§ 6.473 × 102§ 8.368 × 10−1 2.357 × 103

11.52 4.55§ 5.69§ 0.31 22.08

10.28§ 79.79§ 66.62§ 3.18 159.86

553.12 ± 92.6 42.49 ± 4.9 92.8

0.79 ± 0.15 0.31 ± 0.04 69.7

13.90 ± 3.3 16.52 ± 2.0 43.9

1.02 ± 0.3 2.15 ± 0.2 32.1

0.10 ± 0.03 0.21 ± 0.02 31.1

0.0037 ± 0.002 0.0183 ± 0.002 16.1

σ2G (σ2GY) heritability (H2B, %)

YP 2§b

TKW §

PC §

SDS 2§

ASH §

a TAC, total anthocyanin content (μg/g); YP, yellow pigment (μg/g); TKW, thousand-kernel weight (g); PC, protein content (%); SDS, sodium dodecyl sulfate sedimentation volume (mL/g); ASH, ash content (%). b§, significant at p ≤ 0.001.

acetonitrile 22.5%, and ultrapure water, at a flow rate of 0.5 mL/min. The gradient was programmed as follows: 0 → 12 min, 9% B; 12 → 25 min, 35% B; 25 → 30 min, 50% B; 30 → 35 min, 9% B. The anthocyanins were detected at 520 nm and identified according to their retention times, using UV−vis spectra authenticated standards. The stock standard solutions were prepared by weighing 1 mg of each standard and dissolving each in 1 mL of acidified methanol. The working standard solutions were prepared by diluting the stock solution, and then they were injected. The range of injected standard was from 0 to 40 ng. All of the coefficients (R2) ranged from 0.99981 to 0.99998. Data are expressed as micrograms per gram dry matter. Mineral Element Analysis. The mineral element concentrations (Fe, Mn, Cu, Zn) of the whole meal were determined using a Varian Vista-MPX simultaneous inductively coupled plasma optical emission spectrometer, with CCD detection, as described in detail by Ficco et al.26 All of the mineral determinations were compared to certified reference materials (NIST SRM 8436: durum wheat flour on dry weight). The moisture content was determined by oven-drying samples of each material at 60 °C for 24 h and then allowing the samples to cool in a desiccator for 4 h prior to reweighing. The data are expressed as milligrams per kilogram. Total Polyphenol Content. Total polyphenol content was quantified using the Prussian blue assay,27 as modified and described in detail by Finocchiaro et al.5 The absorbance was measured at 700 nm using a DU-64 spectrophotometer (Beckman Instruments, Brea, CA, USA). Increasing concentrations of catechin standard (from 0.3 to 1.0 mmol/L, initial concentrations) were used to construct the calibration curve. To verify the linearity of the curve in the range in these samples, the assay was standardized against 0.001 M catechin, and the polyphenols were expressed as catechin equivalents as grams per kilogram dry matter. Trolox Equivalent Antioxidant Capacity. Total antioxidant capacity was evaluated according to the Trolox equivalent antioxidant capacity method,28 as described in detail by Finocchiaro et al.5 The TEAC method is based on decoloration of the ABTS radical cation at 734 nm in the presence of antioxidants. The data are expressed in millimoles of Trolox (used as the antioxidant reference) per kilogram dry matter. Statistical Analysis. The results of TAC, YP, TKW, PC, SDS, and ASH were subjected to a combined analysis of variance (ANOVA) using Statistica software (StatSoft version 7.1 StatSoft, Inc., Tulsa, OK, USA) (1), whereas the analysis of minerals, TPC, and TEAC were subject to one-way ANOVA (2), according to the following equations:

yijk = μ + αi + βj + (αβ)ij + eijk

(1)

yijk = μ + αi + eik

(2)

represents the effect of interaction of genotype i with year j, whereas e represents the residual effect. Means were identified as being significantly different on the basis of Fischer’s protected least significant differences (LSD) at a probability level of 5%. The variance components were calculated from a mixed model (genotype and genotype by year random effects; year and block within year fixed effects), as analyzed using the restricted maximum likelihood procedure. Narrow-sense heritability (H2B) was estimated from the resulting variance components on a genotype-mean basis. Pearson correlation coefficients (r) were used to study the relationship among the qualitative traits evaluated.

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RESULTS ANOVA and Year Effect. The results of combined analysis of variance (ANOVA) made on data obtained from three growing seasons are given in Table 1. Highly significant year (Y), genotype (G), and G×Y effects were detected for each of the investigated traits. Component of variance (σ2G and σ2GY) and narrow-sense heritability estimates on a genotype-mean basis were also evaluated (Table 1). Total anthocyanin content showed the highest H2B (92.8%) value followed by YP (69.7%). The narrow-sense heritability estimates were small for all other traits, ranging from 16.1% (ASH) to 43.9% (TKW). The three experimental years showed similar annual temperature ranges, whereas the variations in the total rainfall levels were more significant (Supporting Information Table S2). In particular, the growing seasons of 2009−2010 and 2010−2011 had analogous rainfall, as 435.8 mm and 470.2 mm, respectively. This rainfall was more concentrated in the winter months with respect to the 2008−2009 growing season, during which it was distributed over the entire cycle of the plants, with about 2-fold the rainfall considered as the seasonal average over the past 52 years. These data significantly influenced all of the parameters analyzed, and in particular the PC, SDS sedimentation volume, and ASH, for which the highest values were recorded in the third year (16.90%, 3.03 mL/g, and 2.39%, respectively; Table 2). The behavior observed for TAC, however, was completely the opposite. In this case, the values were higher for those recorded in the first two growing seasons (19.0 and 18.8 μg/g, in 2008−2009 and 2009−2010, respectively), whereas the lowest TAC (17.1 μg/g) was recorded in the third study year (2010−2011). The levels of YP did not follow any clear trend, with an increased accumulation of carotenoids observed in the second growing season (2009−2010), when the minimum value of TKW was also recorded (35.1 g).

μ represents the overall mean of all plots in all years, αi represents the effect of the ith genotype, βj represents the effect of jth year, and αβij 8688



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tetraploid wheat, with particular reference to the durum wheat varieties used as references in the present study, showed higher mean TKW (49.18 g) as compared to pigmented blue wheat (35.46 g). Considering the genotypic variability within each group (Supporting Information Table S1), among the blue wheat group, Sebesta Blue2 was the genotype with the highest TAC (174.27 μg/g), YP (7.01 μg/g), SDS sedimentation volume (4.90 mL/g), and ash (2.45%) but also the lowest TKW (32.05 g). Compared to the commercial bread wheat (Spada), the blue wheat showed good qualitative traits, in terms of both pigment content (anthocyanins and carotenoids) and technological traits (PC and SDS sedimentation volume). The purple durum wheats were characterized by a wide range of values for all of the traits evaluated. Wide variations were seen for TAC, which ranged from 8.12 μg/g (Mog) to 50.22 μg/g (378b), for PC, which ranged from 12.47% (MP3) to 17.88% (ELS 6304-56), and for TKW, which ranged from 34.53 g (286b) to 49.70 g (ELS 6404-130-3). The 378b genotype showed the best performance in terms of TAC, PC, and SDS sedimentation volume. The group of red durum wheats followed the same trend as the purple wheat, with the major differences being for TAC, which ranged from 1.20 μg/g (ELS 6404-139-3) to 24.57 μg/g (ELS 6304-71), for TKW, which ranged from 27.91 g (ELS 6404-114-2) to 49.75 g (ELS 6404-139-3), and for PC, which ranged from 13.02% (ELS 6404-116-3) to 18.38% (ELS 6404159-2). Interestingly, the lower ash values ranged from 1.98% (337) to 2.06% (ELS 6404-85-2). Both of the durum pigmented groups are characterized by relatively variable YP and SDS sedimentation volume (from 5.26 to 7.34 μg/g and from 2.2 to 3.9 mL/g, respectively). Anthocyanin Composition of Pigmented Wheats. The anthocyanin composition according to the HPLC analysis for the three different pigmented groups collected in the year 2010−2011 are reported in Table 4 and the Supporting Information (Table S3). These show different profiles for each of the groups in terms of the 11 standard compounds used as references for their identification (see Materials and Methods; Figure 1A). No anthocyanins were detected in the commercial wheats used as references (Figure 1C,F). In the bread wheat, the blue aleurone was most represented by Dp-3-Rut (33.44 μg/g), Dp-3-Glc (13.68 μg/g), and Mv-3Glc (12.04 μg/g), followed, with lower levels, by Cy-3-Rut (8.42 μg/g), Cy-3-Glu (3.07 μg/g), Pn-3-Ara (2.22 μg/g), Pn3-Gal (1.94 μg/g), and Pn-3-Glc (0.88 μg/g) (Figure 1B). Among the genotypes belonging to this group, Sebesta Blue2

Table 2. Mean Values of TAC and Qualitative Traits of 76 Pigmented Wheat Genotypes Evaluated over Three Growing Seasons at Foggia, Italya year

TAC

YP

TKW

PC

SDS

ASH

2008−2009 2009−2010 2010−2011 LSD(0.05)

18.98 18.82 17.14 0.07

5.90 6.75 6.02 0.04

45.48 35.08 41.88 0.24

13.93 13.83 16.90 0.01

2.69 2.99 3.03 0.03

2.21 2.03 2.39 0.01

a TAC, total anthocyanin content (μg/g); YP, yellow pigment (μg/g); TKW, thousand-kernel weight (g); PC, protein content (%); SDS, sodium dodecyl sulfate sedimentation volume (mL/g); ASH, ash content (%).

Qualitative Parameters among and within the Blue, Purple, and Red Wheat Groups. The data reported in Table 3 subdivide the genotypes according to the color of the pericarp and their ploidy level, and these show higher values of TAC in the pigmented wheat than in the commercial varieties, with values close to zero. In particular, the blue wheat had a higher mean TAC (118.37 μg/g) than the purple (22.60 μg/g) and red (9.91 μg/g). This means that TAC in the blue wheat genotypes was 5-fold greater than for the purple and 12-fold greater than for the red tetraploid wheat. The blue wheat was also characterized by the highest mean SDS sedimentation volume (4.40 mL/g). Compared to the bread and durum commercial varieties, the pigmented wheat also showed greater accumulation of ash in the grain than the commercial varieties (2.38, 2.21, and 2.22%, for blue, purple, and red wheats, respectively). Different behavior was seen for YP of the grain. In this case, the commercial varieties of durum wheat, which were genetically improved for this YP trait,17,29 showed the highest YP (9.84 μg/g) compared to both the commercial control bread wheat variety Spada (4.49 μg/g) and the pigmented durum wheat (about 6 μg/g). A good level of variability was also seen for PC, for which the means were included in the range from 11.90 to 13.72% in the commercial wheat; among the pigmented wheats, the red had the highest PC (15.10%), followed by the purple (14.89%) and blue (13.54%). Contrary to what was observed for PC, the qualitative responses of the genetic materials in terms of SDS sedimentation volume were higher for the blue wheat (4.40 mL/g) with respect to the wheat with purple and red pericarp grain (2.80 mL/g each). Thousand-kernel weight was significantly affected by the level of ploidy and the genetic improvements carried out with these two wheat species over the past decades.30,31 Indeed, the

Table 3. Descriptive Statistics of TAC and Qualitative Traits Evaluated in a Collection of 76 Pigmented Wheat Groups over Three Growing Seasons (2009−2011) at Foggia, Italya Triticum aestivum blue (n = 3) TAC YP TKW PC SDS ASH

Triticum durum control (n = 1)

purple (n = 27)

red (n = 43)

control (n = 2)

mean

range

mean

mean

range

mean

range

mean

LSD(0.05)

118.37 6.13 35.46 13.54 4.40 2.38

82.75−174.27 5.01−7.01 32.05−38.59 12.75−14.64 4.10−4.90 2.28−2.45

0.75 4.49 43.28 11.90 3.10 1.91

22.60 6.20 40.45 14.89 2.80 2.21

8.12−50.22 5.26−7.10 34.53−49.70 12.47−17.88 2.20−3.40 2.05−2.31

9.91 6.12 40.97 15.10 2.80 2.22

1.20−24.57 5.28−7.34 27.91−49.75 13.02−18.38 2.20−3.90 1.98−2.39

0.00 9.84 49.18 13.72 3.20 1.99

0.37 0.21 1.23 0.07 0.13 0.04

a

TAC, total anthocyanin content (μg/g); YP, yellow pigment (μg/g); TKW, thousand-kernel weight (g); PC, protein content (%); SDS, sodium dodecyl sulfate sedimentation volume (mL/g); ASH, ash content (%). 8689



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Table 4. Anthocyanin Components (μg/g) of 76 Pigmented Wheat Groups Evaluated over the 2010−2011 Growing Season at Foggia, Italya Triticum aestivum

Triticum durum

blue (n = 3)

purple (n = 27)

red (n = 43)

anthocyanin component

mean

range

mean

range

mean

range

Cy-3-Glc Cy-3-Rut Dp-3-Glc Dp-3-Rut Pn-3-Glc Pn-3-Gal Pn-3-Ara Mv-3-Glc

3.07 8.42 13.68 33.44 0.88 1.94 2.22 12.04

1.17−5.57 7.12−10.97 9.88−18.81 31.77−35.86 0.76−0.99 1.94 0.95−3.49 10.26−13.81

10.34 − − − − 0.58 − 0.48

0.63−29.00

4.02 − − − − 0.33 − 0.22

0.59−10.23

0.06−3.11 0.17−2.35

0.06−3.87 0.06−0.43

a

Cy-3-Glc, cyanidin 3-O-glucoside; Mv-3-Glc, malvidin 3-O-glucoside; Pg-3-Glc, pelargonidin 3-O-glucoside; Cy-3-Gal, cyanidin 3-O-galactoside; Pn-3-Glc, peonidin 3-O-glucoside; Cy-3-Rut, cyanidin 3-O-rutinoside; Dp-3-Glc, delphinidin 3-O-glucoside; Dp-3-Rut, delphinidin 3-O-rutinoside; Pn-3-Gal, peonidin 3-O-galactoside; Pn-3-Ara, peonidin 3-O-arabinoside; Pt-3-Glc, petunidin 3-O-glucoside; −, not detectable.

Figure 1. Typical HPLC chromatogram for standard mixture (A), blue bread wheat (B) versus yellow commercial bread (C), purple durum wheat (D), and red durum wheat (E) versus commercial durum wheat (F).

followed by Pn-3-Gal (0.33 μg/g) and Mv-3-Glc (0.22 μg/g). ELS 6404-117-4 was the genotype with the highest levels of anthocyanin compounds (11.39 μg/g), in agreement with its TAC (20.78 μg/g) detected with the spectrophotometric system. For 10 of the 43 wheat genotypes with red pericarp, these anthocyanins were not detected, probably because the amount is below the threshold of detectability of the instrument used. To quantitatively analyze and confirm the relationships among anthocyanin components, TAC, YP, TKW, PC, SDS, and ASH, the Pearson correlation analysis was applied to the obtained data (Table 5). Higher TAC, but not TKW, was significantly associated with an increase in the concentration of almost all individual anthocyanin components, a finding suggesting that among the tested genotypes there was not a direct influence of the seed weight on the anthocyanin concentration. Positive associations were also recorded between SDS and the anthocyanin content in the grain. In contrast, YP and PC were correlated negatively with each other, but in relation to other characters, they did not show any association.

also showed the highest levels of anthocyanin compounds (85.36 μg/g) by HPLC, as compared to the spectrophotometric analysis. The anthocyanin profile of the durum wheat genotypes with purple pericarp was simpler than that for the blue wheat. For the purple, HPLC analysis showed a total anthocyanin compound of 11.81 μg/g, with the most representative compound being Cy-3-Glc (10.34 μg/g), followed by Pn-3Gal (0.58 μg/g) and Mv-3-Glc (0.48 μg/g) (Figure 1D). Within this group, the behavior of the individual genotypes was relatively diverse, especially for the content of Cy-3-Glc, which oscillated between 0.63 μg/g (ELS 6304-69) and 29.00 μg/g (T-1303). T-1303 is the genotype that also showed the highest Mv-3-Glc (2.35 μg/g). For all of the other compounds, extremely small quantities were detected. A profile similar to that of the previous group was also seen for the red wheat (Figure 1E). In this case, the data confirm the presence of the same anthocyanin compounds, although in smaller quantities. Cy-3-Glc was also confirmed as the most representative compound for this group of wheat (4.02 μg/g), 8690


1.000 0.974 0.866 0.996 0.183 0.911 0.578 0.075 0.915 0.005 −0.270 −0.201 0.722 0.240

Dp-3-Glc

1.000 0.783 0.987 0.127 0.819 0.698 0.060 0.872 −0.013 −0.251 −0.211 0.711 0.194

Dp-3-Rut

1.000 0.844 0.395 0.864 0.350 0.467 0.895 0.048 −0.252 −0.206 0.562 0.245

Cy-3-Glc

1.000 0.171 0.873 0.644 0.066 0.908 0.007 −0.264 −0.211 0.724 0.224

Cy-3-Rut

1.000 0.207 0.025 0.359 0.271 −0.043 −0.099 0.040 0.032 0.042

Pn-3-Gal

1.000 0.189 0.116 0.846 −0.015 −0.265 −0.130 0.636 0.286

Pn-3-Glc

1.000 −0.051 0.504 0.042 −0.117 −0.220 0.460 0.004

Pn-3-Ara

1.000 0.284 0.021 −0.043 −0.128 −0.065 0.037

Mv-3-Glc

1.000 0.025 −0.327 −0.176 0.637 0.282

TAC

1.000 −0.035 −0.328 −0.030 0.093

YP

1.000 0.134 −0.099 −0.233

TKW

1.000 0.000 0.231

PC

1.000 0.158

SDS

1.000

ASH

Cy-3-Glc, cyanidin 3-O-glucoside; Mv-3-Glc, malvidin 3-O-glucoside; Pg-3-Glc, pelargonidin 3-O-glucoside; Cy-3-Gal, cyanidin 3-O-galactoside; Pn-3-Glc, peonidin 3-O-glucoside; Cy-3-Rut, cyanidin 3O-rutinoside; Dp-3-Glc, delphinidin 3-O-glucoside; Dp-3-Rut, delphinidin 3-O-rutinoside; Pn-3-Gal, peonidin 3-O-galactoside; Pn-3-Ara, peonidin 3-O-arabinoside; Pt-3-Glc, petunidin 3-O-glucoside; TAC, total anthocyanin content; YP, yellow pigment; TKW, thousand-kernel weight; PC, protein content; SDS, sodium dodecyl sulfate sedimentation volume; ASH, ash content. The coefficients in bold are significant at p < 0.05.

a

Dp-3-Glc Dp-3-Rut Cy-3-Glc Cy-3-Rut Pn-3-Gal Pn-3-Glc Pn-3-Ara Mv-3-Glc TAC YP TKW PC SDS ASH

Table 5. Pearson Correlation Coefficients among Anthocyanin Components, TAC, YP, TKW, PC, SDS, and ASH of 76 Pigmented Wheat Genotypes Evaluated over Three Growing Seasons at Foggia, Italya

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Table 6. Grain Minerals Concentration for Pigmented Wheat Groups Evaluated during the 2010−2011 Growing Season at Foggia, Italy Triticum aestivum blue (n = 3)

Triticum durum

control (n = 1)

purple (n = 27)

red (n = 43)

control (n = 2)

mineral

mean

range

mean

mean

range

mean

range

mean

LSD(0.05)

Fe (mg/kg) Mn (mg/kg) Cu (mg/kg) Zn (mg/kg)

46.13 40.90 6.00 33.13

43.90−47.30 37.80−46.50 4.80−7.70 31.30−36.30

30.30 37.20 5.70 21.60

36.71 41.07 6.87 31.28

30.10−42.00 17.60−51.50 4.30−9.60 24.10−40.40

36.71 41.25 6.71 31.54

30.80−46.80 30.80−51.70 3.80−9.20 22.90−40.60

32.85 38.05 7.40 25.55

5.61 7.75 1.64 4.87

Other Qualitative Parameters and Nutritional Properties of Pigmented Wheat. To complete the characterization of the pigmented wheat, the 76 genotypes collected in the crop year 2010−2011 were also explored for the mineral content of their grain (Table 6 and Supporting Information Table S4). The highest levels of Fe were in blue wheat (46.13 mg/kg), whereas for the other pigmented groups, the means for Fe were lower. A similar trend was seen for Zn in the grain. The blue wheat showed the highest Zn (33.13 mg/kg), followed by the red and purple wheats (31.54 and 31.28 mg/kg, respectively). The other two elements analyzed, Mn and Cu, do not show a definite trend, and the commercial variety PR22D89 showed the highest concentrations of Mn (42.40 mg/kg) and Cu (9.50 mg/kg). Considering the genotypic variability within each group, the blue bread wheat did not show as wide a range as for the purple wheat. Indeed, the data show variations for Fe, from 30.10 mg/kg (286b) to 42.00 mg/kg (ELS 6304-56), for Mn, from 17.60 mg/kg (226) to 51.50 mg/kg (ELS 6304-56), for Cu, from 4.30 (286b) to 9.60 mg/kg (ELS 6404-75-3, ELS 6304-56), and for Zn, from 24.10 mg/kg (9503) to 40.40 mg/ kg (ELS 6404-75-3). Within the red group, for Fe, the range was from 30.80 mg/ kg (377b) to 46.80 mg/kg (ELS 6404-159-4), for Mn, from 30.80 mg/kg (377b) to 51.70 mg/kg (ELS 6404-92-4), for Cu, from 3.80 mg/kg (ELS 6404-139-3) to 9.20 mg/kg (1842), and for Zn, from 22.90 mg/kg (377b) to 40.60 mg/kg (ELS 640492-4). Table 7 shows the means for TPC and TEAC in relation to a selected number of genotypes, as representative of each group considered in the present study. These data show a significant correlation between the means of TPC and TEAC (0.97; p ≤ 0.001). In particular, there was greater accumulation of TPC in

the blue-pigmented wheat, as > purple, > red, which is reflected in the performance of the TEAC. The commercial variety Preco appeared to show abnormal behavior, where a high TPC was seen (4.79 g/kg), and its TEAC was comparable to that for purple durum wheat (19.44 vs 20.08 mmol Trolox/kg).

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DISCUSSION Increasing attention has been paid to anthocyanins, due to their frequent presence in plants, particularly berry fruits, vegetables, grapes, and more recently in pigmented (black/blue/purple) cereal grains, for their health benefits as dietary antioxidants.4,19 Most of the studies in the literature have been related to pigmented bread wheat and field trials conducted in a single growing season, and only rarely these investigations were carried out on tetraploid wheat genotypes. In the present study, the results are reported for a large collection of wheat genotypes. These are primarily represented by durum wheat genotypes, but also by bread wheat, with the aim being not only to evaluate the anthocyanin levels and composition in the grain but also to characterize other qualitative traits, which have not been investigated to date. The agronomic evaluation conducted for 3 years has also allowed estimation of the effect of growing season (Y) on the qualitative performance of these genetic materials. Total Anthocyanin Content, Qualitative Traits, and Nutritional Properties of Pigmented Wheats. Our results confirm that blue wheat has, among wheat genetic resources, the highest TAC in the grain.32−34 Recently, Syed Jaafar et al.35 analyzed the TAC levels in a series of genotypes of wheat, including the same three Sebesta Blue genotypes analyzed in the present study. Although their mean TACs were relatively higher (159.8, 251.8, and 130.2 μg/g, for Sebesta Blue1, Sebesta Blue2, and Sebesta Blue3, respectively), the authors also confirm the graded list in the present study in which the Sebesta Blue2 genotype shows the highest TAC. In particular, the TAC range observed in the present study, for the three Sebesta Blue genotypes throughout the entire study period (82.7−174.3 μg/g), was quite similar to that found in blue wheat by Abdel-Aal and Hucl32 and Zofajova et al.33 (157 and 193.3 μg/g, respectively). However, sligthly lower were the values reported by Eticha et al.34 and Knievel et al.,19 between 39.9 and 106 μg/g. By contrast, higher values ranging from 76 to 286 μg/g were reported in refs 4, 7, and 19, although these other studies investigated different genotypes with respect to those considered in the present study. Comparison with the literature of TAC in purple and red durum wheats is difficult, as most of the studies reported were conducted using hexaploid wheat varieties. The means recorded in the present study for purple durum wheat are comparable to

Table 7. Mean Values of TPC and TEAC for a Subset of Pigmented Wheat Genotypes Collected during the 2010− 2011 Growing Season at Foggia, Italya TPC

TEAC

Triticum aestivum

species

blue

color

Sebesta Blue1

6.82

24.11

Triticum durum

purple

World Seeds3 MP3 ELS 6404-115-5 ELS 6404-140-2 ELS 6404-116-1 Preco

6.26 5.67 3.16 3.34 3.23 4.79

24.61 20.53 15.11 14.23 15.57 19.44

0.96

0.74

red control LSD(0.05)

genotype

a

TPC, total polyphenol content (g/kg); TEAC, Trolox equivalent antioxidant capacity in wheat grain (mmol Trolox/kg). 8692



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those reported in numerous other studies.2,35 In these cases, TACs ranged from 27.0 μg/g34 for the variety BVAL 258034 to 476.9 and 522.7 μg/g, recorded by Knievel et al.19 for the breeding of a purple wheat line and by Hossenian et al.9 in a stressed field trial, respectively. In the case of red wheat, the mean TACs in the present study were lower than for purple wheat, and in agreement with the means of 5, 5.2, and 5.76− 7.97 μg/g observed by Escribano-Bailòn et al., Abdel-Aal et al., and Syed Jaafar et al.,2,32,35 respectively. The carotenoid pigments were lower than the anthocyanin pigments, which is in agreement with Abdel-Aal et al.,32 who reported lower YP than TAC. Moreover, Eticha et al.34 reported a range of YP from 2.6 to 7.6 μg/g for 13 blue and purple wheat genotypes. Here, no significant differences were observed among the three pigmented wheat groups, which suggests that for this character, there has been no selection, in contrast to the commercial varieties of durum wheat. For these latter, the present study indeed shows very high YP, which confirms the strong selective pressure that has been exerted by breeders to increase the content of the carotenoid pigments in the durum wheat grain.17 The highest PC recorded in the present study is also comparable to the studies of Abdel-Aal et al.32 and Eticha et al.34 for both bread and durum wheats and the blue and purple wheats, whereas the red durum wheat shows values similar to those of the two commercial varieties used as controls. Total polyphenol and TAC are also good indicators of antioxidant capacity, and some studies have reported high correlation between antioxidant capacity and TPC.36,37 On a selected number of the genotypes chosen within each of the groups of the pigmented wheat in the present study, TPC and TEAC were determined. The positive association (0.64; p < 0.001) between TPC and the ability of these compounds to capture reactive oxygen species confirms previous studies on wheat38−40 and on other crop species.41 Our data confirm in part also the results obtained by Eticha et al.34 relating to the antioxidant effects produced by TAC. Indeed, although it is less than that for TPC, the positive association between TAC and TEAC is significant (data not shown). However, the mean TEAC for durum wheat varieties (e.g., Preco) that are characterized by the absence of TAC and a high content of YP suggests that TEAC for some genotypes might be due to one or more other groups of pigments. This confirms the significant, but not exclusive, role that the anthocyanins appear to have in the control and capture of reactive oxygen species.42 For the mineral concentrations across the entire collection, the data in the present study are within the ranges defined previously by Ficco et al., Liu et al., and Ozkan et al.26,43,44 on nonpigmented tetraploid wheat. With the exception of Cu, the concentrations of Mn, Zn, and Fe in purple wheat were higher than those of the commercial varieties (3.0, 5.8, and 3.9%, respectively). Interestingly, the mineral contents in the blue wheat analyzed in the present study are characterized by the highest Fe content (46.13 mg/kg), with a limited range of variation, whereas the red wheat is characterized by the highest Zn content, which is more variable. These data indicate that these variations can be exploited for breeding activities to improve the mineral availability in the final products. Effects of Year on Total Anthocyanin Content and Qualitative Parameters of Pigmented Wheats. In our work we did not observe a strong effect of the growing season and of the G×Y effect on the TAC. Indeed, our results are similar to those obtained by Bustos et al.,45 who analyzed the

effect of crop management on TAC. Different from when a wider range of environmental conditions were explored, a larger effect associated with a significant G×E was observed. Moreover, the results of the G×E response on the accumulation of anthocyanins in wheat grain and their results appear to conflict. For instance, whereas water stress, as argued by Eticha et al.,34 results in lower accumulation of TAC, Hossenian et al. 9 reported that this stress promotes accumulation of anthocyanin pigments in grain. Moreover, Abdel-Aal et al.7 argued that in blue and purple wheats, the anthocyanin concentration is significantly influenced by growing conditions and that the environmental effect is much stronger in purple wheat, due to the pigment location in the outer pericarp. Probably, the intensity and duration of the stress may also produce substantialy different outcomes associated with the differences in environmental conditions not different from what is also occurring for complex traits such as grain yield.29 Considering our results, the behavior of anthocyanin pigments appears similar to that of carotenoid pigments, for which there is a wide literature available that supports for this trait a strong genotypic component and a high heritability.18,46,47 For the other traits evaluated (TKW, PC, ASH, and SDS), the data reveal that these are more influenced by genotype and G×Y, confirming a greater difficulty in obtaining a genetic progress as reported in refs 29 and 48. Anthocyanin Composition of Pigmented Wheat. Compared to other cereals, such as maize and rice, the anthocyanin composition of pigmented wheat is not well characterized.2,8 Moreover, the studies reported in the literature have almost always referred to a few selected genotypes of durum wheat. For these reasons, the present study appears to be the first that has examined a large number of purple/red tetraploid wheats. Our analysis was conducted using 11 standard compounds of the anthocyanins, with reference to bread wheat. Our data confirm delphinidin (Dp-3-Rut and Dp3-Glc) as the main aglycone in blue wheat, as it represents 68.9% of the total anthocyanins,4,7,8,19,49 followed by cyanidin, in the form of Cy-3-Glu and Cy-3-Rut. However, compared to previous studies, in all three Sebesta genotypes evaluated, relatively large levels of Mv-3-Glc were also found, whereas the peonidin aglycone is always present, and it is glycosylated with various sugars (glucoside, arabinoside, and galactoside) for each of the three genotypes. To date, more than 15 types of anthocyanins have been identified in purple wheat,35 which suggests greater complexity in the genetic control of purple durum wheat compared to blue bread wheat.20 However, with the exception of the study of Hossenian et al.,9 in which they identified and quantified 13 anthocyanin compounds using HPLC analysis, in all of the other studies, the number of compounds identified was limited to cyanidin in the form of Cy-3-Glc and Cy-3-Rut.4,35 Although the chromatographic profiles of purple wheat appear more simple than those generated by blue wheat, the retention times overlapped only in part. In the purple and red tetraploid wheats in the present study, Cy-3-Glc was confirmed as the predominant anthocyanin, at 87.5 and 91.4% of TAC in purple and red wheats, respectively, followed by Pn-3-Gal (4.91− 7.50%) and Mv-3-Glc (4.06−5.00%), which is consistent with previous results.9,50 It must be emphasized, however, that with respect to blue wheat, purple and red wheats express lower levels of TAC, and although the chromatograms of wheat with purple and red pericarp appear simpler, this does not mean that other compounds are not present. It could be that the 8693


Journal of Agricultural and Food Chemistry extraction, identification, and quantification methods used have not allowed for the identification of all of the compounds examined. When the relationship between TAC determined by colorimetry and the sum of the individual anthocyanins determined by HPLC was examined, a significant positive correlation was obtained, with a correlation coefficient of 0.90 (p < 0.05). The difference might be due to the contribution of other pigments present in the grain that have an absorbance at 520 nm, and to be sure of this, there is the need to know more through more sensitive detection systems. In conclusion, pigmented wheat genotypes exhibited large variations in quality and composition of anthocyanins as well as antioxidant activity. Cyanidin and delphinidin are the main anthocyanidins identified in the present collection of pigmented wheat and, although there is a need for more reliable and sensitive methods for their identification and quantification, the knowledge gained from the present study represents a very important aspect toward our better understanding of the genetic mechanisms that regulate the pathways of synthesis of anthocyanins in wheat. For wheat, these genetic materials represent a source of genes/traits that will be very interesting for the improvement and development of new genotypes with enhanced nutritional properties. The high heritability values recorded for TAC and YP in wheat grain suggest that both traits can be combined, through specific crossing programs, in commercial wheat varieties with high technological grain quality to improve their nutritional value. The characterization of these materials was also extended to the minerals content and, the higher contents of Fe and Zn make it even more attractive for the production of functional foods and also as added ingredients. In this way, the antioxidant activity of the wheat-derived products can be improved significantly. However, to ensure a real effect of wheat grain pigments and minerals on human health, it is necessary to undertake further studies to evaluate the stability of these compounds during the manufacture of the final products (bread, pasta, and bakery products), milling, bread- and pasta-making, and cooking and to estimate the effects on human health through the evaluation of biomarkers of target functions and biological response.

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ACKNOWLEDGMENTS

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ABBREVIATIONS USED

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REFERENCES

Original seeds of pigmented wheats were kindly provided by Dr. Harold Bockelaman, (USDA-ARS, National Small Grain Collection, Aberdeen, ID, USA). We thank Cecilia di Paola for technical assistance and Dr. Antonio Troccoli for meteorological data support. We are grateful to Dr. Christopher Berrie for scientific English language editorial assistance.

Cy-3-Gal, cyanidin 3-O-galactoside; Cy-3-Glc, cyanidin 3-Oglucoside; Cy-3-Rut, cyanidin 3-O-rutinoside; Dp-3-Glc, delphinidin 3-O-glucoside; Dp-3-Rut, delphinidin 3-O-rutinoside; G×E, genotype × environment; G×Y, genotype × year; HPLC, high-performance liquid chromatography; Mv-3-Glc, malvidin 3-O-glucoside; PC, protein content; Pg-3-Glc, pelargonidin 3-O-glucoside; Pn-3-Ara, peonidin 3-O-arabinoside; Pn-3-Gal, peonidin 3-O-galactoside; Pn-3-Glc, peonidin 3O-glucoside; Pt-3-Glc, petunidin 3-O-glucoside; SDS, sodium dodecyl sulfate; TAC, total anthocyanin content; TEAC, Trolox equivalent antioxidant capacity; TKW, thousand-kernel weight; TPC, total polyphenol; YP, yellow pigment

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(P.D.V.) Phone: +39-0881-742972. Fax +39-0881-713150. Email: [email protected]. Funding

This study was supported by the Italian Ministry of Agriculture (MiPAAF), with the special grant RGV/Trattato FAO (Risorse Genetiche Vegetali), and by the Italian Ministry of Economic Development, with the special grant PAQ (Pasta e Nuovi Prodotti Alimentari ad Alta Qualità da Cereali Italiani). Notes

The authors declare no competing financial interest. 8694



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Genetic Variability in Anthocyanin Composition and Nutritional

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