Agronomic Characterization and α- and β-ODAP ... - ACS Publications

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Agronomic Characterization and α- and β‑ODAP Determination through the Adoption of New Analytical Strategies (HPLC-ELSD and NMR) of Ten Sicilian Accessions of Grass Pea Fabio Gresta,† Concetta Rocco,‡ Grazia M. Lombardo,§ Giovanni Avola,∥ and Giuseppe Ruberto*,‡ †

Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Loc. Feo di Vito, 89122 Reggio Calabria, Italy Istituto del CNR di Chimica Biomolecolare, Via Paolo Gaifami 18, 95126 Catania, Italy § Dipartimento di Scienze Agrarie e Alimentari, Università di Catania, Via Valdisavoia 5, 95123 Catania, Italy ∥ Istituto del CNR per la Valorizzazione del Legno e delle Specie Arboree, Via Paolo Gaifami 18, 95126 Catania, Italy ‡

S Supporting Information *

ABSTRACT: Ten accessions of grass pea (Lathyrus sativus L.) from different Sicilian sites, cultivated in the same environmental conditions, were analyzed for their morphological and productive parameters and for the content of two non-protein amino acids: α- and β-ODAP (α- and β-N-oxalyl-L-α,β-diaminopropionic acid). The β-isomer is the neurotoxin responsible for the neuron disease known as lathyrism. This analysis was carried out using two common analytical methodologies never applied in their determination, an HPLC separation with evaporative light scattering (ELS) as detector, and nuclear magnetic resonance (NMR). The content of the two isomers falls in the range reported for these compounds: 0.42−0.74 and 2.69−4.59 g/kg for αand β-ODAP, respectively; and the two methods yield comparable results. High productivity and a high protein content were detected in three Sicilian accessions. Low β-ODAP content was found to be linked to accessions with heavier seeds and those originating at lower altitudes. KEYWORDS: Lathyrus sativus L., α- and β-ODAP, NMR, HPLC-ELSD, seed yield, protein content

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INTRODUCTION Grain legumes are an essential component of crop systems and human diet all over the world. The importance of grain legumes as food, as animal fodder, and as soil fertility improvement tools is widely recognized, and some of these, such as broad beans, peas, and chick peas, have been widely studied from both agronomic and chemical perspectives.1−3 Some others, commonly referred to as minor grain legumes,4 have been almost completely neglected by the research community. However, this group of plants, which includes legumes of minor economic significance such as grass pea, cowpea, lupin, etc., shows a very limited input requirement in terms of fertilization and irrigation. In the past few years these plants have received increasing attention because of their potential as rotation crops in semiarid regions5 and as functional food in the human diet thanks to their valuable protein content and fatty acid profile.6−8 Among these, grass pea (Lathyrus sativus L.) is a legume crop which has been grown since ancient times.9 Its origin is localized in southwest and central Asia, from where it spread into the Eastern Mediterranean. Today it is still widely used in India, Bangladesh, Nepal, Afghanistan, Ethiopia, and Syria.10 Grass pea has been one of the main sources of protein for the people and animals of the Mediterranean basin for millennia,11,12 but it is now cultivated in a considerably reduced area. In Italy, it is cultivated in the center-south part of the peninsula ranging from Tuscany to Sicily, with very limited surface and average farm land size ranging from 0.1 to 0.4 ha,13 but it still grows in marginal environments free from herbicides, © 2014 American Chemical Society

pesticides, or chemical fertilizers. Grass pea is able to grow on poor soils and in dry climates, showing high abiotic and biotic stress resistance,11,14 and it has valuable nitrogen fixation greater than other commercial legumes, a good adaptation to low input environments, and a high seed protein content.5,15−17 Grass pea has been used very little for two reasons, first, because of its low seed production, and second, because of the presence of the neurotoxic components: β-diaminopropionic acids (β-ODAP), identified as the cause of neurolathyrism.18 This pathology, which implies an irreversible paralysis of the legs, appears in people who use grass pea as their only food source with high consumption over a long period.19,20 A similar pathology, named konzo (tied legs), is caused by a large consumption of cassava, Manihot esculenta Crantz (Euphorbiaceae). Konzo and neurolathyrism give similar pathological effects with no differences in clinical diagnosis, though in cassava the toxins have been identified as cyanogenic glucosides producing HCN.21 A further risk factor due to the consumption of grass pea and cassava is the scarce presence in both plants of sulfur amino acids, namely, methionine and cysteine.22 Data shows, for example, that if the consumption of L. sativus is combined with cereals, rich in sulfur amino acids, the toxicity of ODAP is drastically reduced and the disease (neurolathyrism) is rarely a problem.10,23 Received: Revised: Accepted: Published: 2436

September 16, 2013 February 19, 2014 February 22, 2014 February 22, 2014 dx.doi.org/10.1021/jf500149n | J. Agric. Food Chem. 2014, 62, 2436−2442

Journal of Agricultural and Food Chemistry

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Qualitative Analyses and Isolation of α- and β-ODAP. 10 g of dried seeds was milled and extracted with water (50 mL) for 12 h at room temperature and under continuous stirring. The mixture was centrifuged (20 min, 4000 rpm) and the supernatant charged on a column filled with Dowex-50W-8% cross-linked, dry mesh 200−400 (20 mL) in acid (H+) form. The elution was carried out using water, and the fractions were analyzed by thin layer chromatography (TLC, cellulose, acetone:butanol:water:formic acid 7:7:4:4), spraying with ninhydrin (isomer α reacts in pale rose, isomer β in pale blue). All positive fractions were pooled, and the solvent was removed at reduced pressure. The mixture was analyzed by HPLC-ELSD and NMR HPLC Analyses. The previous mixture was analyzed by HPLC in the following conditions: HPLC model P580 (Thermo Fisher Scientific, Waltham, MA, USA), reverse phase column Luna C18 250 mm × 4.6 mm i.d. × 5 μm (Phenomenex, Torrance, CA, USA), kept at 20 °C in a model TCC-100 thermostated oven (Thermo Fisher Scientific, Waltham, MA, USA). The elution was performed using a mixture of 0.13% formic acid in water; flow 0.5 mL/min; detector, model Alltech 3300 evaporative light scattering detector (ELSD) (Grace, Deerfield, IL, USA); drift tube temperature 52 °C, nebulizer gas nitrogen, gas flow 1.5 L/min. Retention times of α-ODAP and βODAP: 3.40 and 3.63 min, respectively. 1 H NMR Analyses. 5 mL of the previous mixture was evaporated to dryness and the residue dissolved in 2 mL of a 0.1 N solution of DCl in D2O. The mixture was submitted to nuclear magnetic resonance (1H NMR) in the following conditions: spectrometer model Avance 400 (Bruker Corp., Billerica, MA, USA) with 5 mm probe and Z gradient at 400.13 MHz (1H), pulse power for 90°, −1.1 db; 90° pulse length, 8.0 μs; measurement temperature, 299 K; pulse angle, 90°; preacquisition delay, 6.5 μs; acquisition time, 7 min; relaxation delay, 50 s; scan number, 8; sweep width, 12 ppm; number of FID points, 32K; number of frequency points, 64K; line broadening, 0.3 Hz. α-ODAP: 1H NMR, CH2, 3.47 Hα dd (13.4, 5.2 Hz), 3.28 Hβ dd (13.4, 8.4 Hz); CH, 4.60 dd (8.4, 5.2 Hz).31,32 β-ODAP: 1H NMR, CH2, 3.76 Hα dd (14.8, 3.8 Hz), 3.67 Hβ dd (14.8, 6.8 Hz); CH, 4.03 dd (6.8, 3.8 Hz).31,32 Isolation of β-ODAP. 10 mL of the previous mixture of α- and βODAP coming from the chromatographic separation was dried and the residue dissolved in distilled water and left at room temperature for 24 h. A white precipitate was separated by filtration and analyzed with the HPLC-ELSD and NMR procedures previously described. The resulting compound was pure β-ODAP, which was used to build the calibration curves for the quantitative analyses of the two amino acids in the HPLC analyses, and for the calibration methods in the NMR analyses. Quantitative Analyses. Sample Treatments. 1 g of plant material (seeds of grass pea) of the ten samples in this study was extracted according to the procedure previously described. A 260 mL fraction coming from the Dowex column, containing the two neurotoxins, was

Over the last 20 years, these concerns and the discovery of variability in the level of ODAP concentration have led to the setting up of germplasm and breeding programs which aim to select those genotypes with the lower ODAP levels.10−12,24,25 In fact, the most common quantitative ranges for the two isomers are 0.07−7.52 and 0.05−0.79 g/dry kg for β and α isomers (Figure 1), respectively.26−30

Figure 1. The two non-protein amino acids, α- and β-ODAP, of L. sativus.

With this in mind exploring agronomic, Sicilian accessions of varieties which merge concentration.

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the present paper has the purpose of qualitative, and chemical traits of ten grass pea in order to identify those high productive traits with low ODAP

MATERIALS AND METHODS

Field Trial and Plant Material. The trial was carried out in the experimental field of the University of Catania (Sicily, Southern Italy, 10 m a.s.l.) in 2006−2007 using 10 accessions of grass pea taken from farmers’ collections (Table 1). The seeds were sown on the 30th November into ploughed soil fertilized with 100 kg/ha of P2O5. The experiment was arranged in a complete randomized block design with plots of 4 × 3 m, replicated three times. A sowing rate of 60 plant/m2 with rows 1.0 m far was adopted. Seeds were collected at the end of June 2007 and immediately processed for the analyses. No irrigation was used, and weeds were managed mechanically. The soil was a clay-loam soil with pH 8.1 and medium nutrient status. During the trial rainfall and temperature were recorded. Average temperature ranged from 10 °C in January to 25 °C in June while the minimum never went below 5 °C and maximum reached 32 °C at the end of the crop cycle. Total rainfall was 495 mm, 50% of which fell in December. Temperature and rainfall data did not differ significantly from the thirty-year values. In the field the following parameters were measured: plant height at flowering, pods per plant, seeds per pod, 1000 seed weight, seed yield, seed dimension (three-dimensional parameters: longitudinal, transversal, and thickness). Seed protein content was calculated as: (Kjeldahl nitrogen content × 6.25).

Table 1. Main Parameters of the Studied Grass Pea Accessions (± Standard Error) code

provenance

altitude (m a.s.l.)

height at flowering (cm)

pod/plant (n)

seed/pod (n)

1 2 3 4 5 6 7 8 9 10 M CV (%)

Aidone 1 Aidone 2 Aidone 3 Troina Licodia Eubea Bronte Corleone Piana degli Albanesi Maletto Mineo

550 550 550 970 480 750 530 650 1000 450

31 (±1.9) 31 (±2.3) 31 (±2.0) 31 (±1.9) 31 (±1.8) 31 (±2.1) 26 (±1.6) 31 (±2.2) 31 (±1.9) 26 (±1.7) 30 2.22

17.1 (±2.55) 11.8 (±2.18) 13.3 (±2.64) 8.6 (±1.59) 5.9 (±1.76) 13.1 (±2.79) 7.1 (±1.94) 12.2 (±2.34) 13.9 (±2.13) 10.4 (±2.47) 10.5 20.93

2.4 (±0.17) 2.1 (±0.11) 1.9 (±0.11) 2.2 (±0.13) 2.6 (±0.15) 2.1 (±0.12) 2.3 (±0.12) 2.1 (±0.10) 2.4 (±0.15) 2.1 (±0.13) 2.3 9.44

2437

1000 seed wt (g)

seed yield (t/ ha)

242.7 250.2 235.5 288.5 252.6 220.7 345.1 181.4 196.6 307.5 247.1 19.83

0.82 (±0.16) 1.48 (±0.19) 1.40 (±0.17) 1.86 (±0.21) 0.86 (±0.11) 0.98 (±0.12) 0.69 (±0.10) 0.81 (±0.11) 0.81 (±0.12) 0.96 (±0.18) 1.07 35.69

(±5.44) (±4.81) (±5.33) (±5.27) (±5.15) (±4.94) (±5.19) (±5.86) (±5.67) (±5.24)

protein content (%) 26.0 26.2 25.4 27.4 25.8 26.8 25.4 25.7 26.9 27.4 26.6 2.92

(±0.29) (±0.35) (±0.31) (±0.45) (±0.41) (±0.41) (±0.38) (±0.31) (±0.33) (±0.40)

dx.doi.org/10.1021/jf500149n | J. Agric. Food Chem. 2014, 62, 2436−2442

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Figure 2. 1H NMR quantitative determination of β- and α-ODAP, with the use of maleic acid and alanine as standards (* solvent impurity). dried out. The final residue was dissolved in 25 mL of distilled water, and the solution was analyzed for its content of α- and β-ODAP adopting the two analytical procedures previously described, namely, HPLC-ELSD and NMR. HPLC-ELSD Analyses. 5 mL of the previous solution was diluted (9:1) with 0.1 N HCl. 10 μL was analyzed by HPLC-ELSD in the conditions previously described. Each analysis was carried out in triplicate. For the quantitation of both amino acids the pure β-ODAP, previously purified, was used to build two calibration curves in the amount comprising between 2140 and 778 ng (y = 0.5493x + 486.43, R2 = 0.9997) for β-ODAP and between 476 and 214 ng (y = 0.9639x + 164.51, R2 = 1) for α-ODAP. NMR Analyses. 20 mL of the same solution was concentrated, and each residue was dissolved in 2 mL of 0.1 N DCl in D2O. 500 μL of the resulting solution was then submitted to nuclear magnetic resonance (1H NMR) in the previously described conditions, performing four measurements every 10 min. For the calculations alanine (56 μL of a solution 0.068 M in D2O) and maleic acid (56 μL of a solution 0.069 M in D2O) were used as standards. Phase and baseline corrections were performed manually, as were the signal integrations. In the case of improper baseline corrections the BIAS and SLOPE functions were activated for the integral calculation. The 1H NMR signals selected for the quantification of α-ODAP and β-ODAP were at 3.28 and 4.03 ppm, respectively.31,32 The reference signals of the standard compounds were the methyl group of alanine at 1.55 ppm and the vinyl protons of maleic acid at 6.31 ppm (Figure 2). Validation and Calibration Procedures. For the validation and intercomparisons three model mixtures were prepared gravimetrically. Mixture NMR-1 with the following molar ratio: β-ODAP (85.42 mol/ mol %), alanine (8.50 mol/mol %), maleic acid (6.08 mol/mol %). Mixture NMR-2: β-ODAP (1.97 mol/mol %), alanine (95.73 mol/mol %), maleic acid (2.30 mol/mol %). Mixture NMR-3: β-ODAP (21.44 mol/mol %), alanine (7.94 mol/mol %), maleic acid (71.62 mol/mol %). In order to check the linearity of the method, 13 model solutions containing β-ODAP were prepared in different molar ratios (from 2.02 to 99.04 mol/mol) solved in DCl/D2O. Figure 3 shows the experimentally determined molar ratios for β-ODAP and experimental value versus estimated experimental value.

Figure 3. Tests of linearity. Theoretical and experimental molar ratios of the 13 model mixtures, calculated for β-ODAP. Correlation coefficient: 0.99996 (A). Difference between gravimetric and experimental value vs estimated experimental value yHat (with yHat =1.003x, where x represents the gravimetric value) (B).

In order to explore the relationships between the parameters, a Pearson correlation was performed. 2438

dx.doi.org/10.1021/jf500149n | J. Agric. Food Chem. 2014, 62, 2436−2442

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RESULTS AND DISCUSSION

Morphological, Productive, and Qualitative Traits. Studied accessions did not differ in relation to phenological stages. For all the accessions seed emergence occurred 24 days after sowing, flowering began in the second week of February, pod set in the second week of March, and pod maturity at the end of June, period in which seeds were harvested. Plant height, detected at the beginning of flowering, was about 31 cm for all the studied accessions, with the exception of ‘Corleone’ and ‘Mineo’, where heights were 26 cm (Table 1). This was lower than values reported by other authors,33,34 but it can be explained by the wide inter-row distance adopted to facilitate measurements and harvest. Grain yield was 1.07 t/ha on average; the best results were achieved by accessions ‘Troina’, ‘Aidone 2’, and ‘Aidone 3’ (1.86, 1.48, and 1.40 t/ha, respectively). The lowest yield was recorded in ‘Corleone’ with 0.69 t/ha. The remaining accessions showed values between 0.81 and 0.98 t/ha. The production of the best accessions is in agreement with those reported previously as 1.7−2.6 t/ha,35 1.2−1.6 t/ha,11 and 1.6− 2.0 t/ha.5 As regards yield components, on average the number of pods per plant was 10.5, with a coefficient of variation of 20.9%. The highest number of pods per plant was found on ‘Aidone 1’ (17.1), while the lowest was recorded in ‘Licodia Eubea’ (5.9). The number of seeds per pod was of 2.3 on average with a limited range of variability (9.44%). On the contrary, weight of the 1000 seeds was quite variable (19.83%) with an average value of 247.1 g ranging from 181.4 to 345 g. For this parameter the accessions ‘Corleone’ and ‘Mineo’ (345.1 and 307.5 g, respectively) significantly differ from the others (Table 1). The boxplot in Figure 4 summarizes results for seed morphological data. With regard to longitudinal section, the accession ‘Aidone 3’, ‘Corleone’, and ‘Mineo’ revealed the highest median values equal to 11 mm with data skewed to the right, although the skew is more prominent in the last. The accessions ‘Aidone 3’ and ‘Mineo’ showed 50% of the readings within 10 and 11 mm, whereas ‘Corleone’ showed the highest interquartile extent, ranging from 10 to 12 mm. When the transversal section was taken into account, accession ‘Corleone’, in accordance with the longitudinal data, showed the median value (11 mm) higher than the 90th percentile of all the other accessions. No valuable differences were observed in thickness. The protein content of grass pea accessions was on average of 26.6% with a CV of 2.9. It ranged from 25.4 of genotypes ‘Aidone 3’ and ‘Corleone’ to 27.4% of genotypes ‘Troina’ and ‘Mineo’ (Table 1). These values are in agreement with those reported in the literature,12,36 comparing several samples from eleven different accessions, which found an average value of 27.2%, and with that described by Yan et al.,28 which reports values between 25.6 and 27.3%. It must be emphasized that this value of grass pea protein content is in general higher than those of field pea, chick pea, and fava bean, which are, instead, much more commonly consumed. Chemical Analyses. The contents of α- and β-ODAP in the ten accessions of Sicilian grass pea were listed in Table 2. It must be emphasized that the two analytical methodologies here used were applied for the first time in the determination of αand β-ODAP in L. sativus. Compared to the usual methods used for the determination of the two ODAP isomers, the instrumental tools adopted here do not require any

Figure 4. Distribution of the seed dimensions. In the boxes plot, each representing the distribution of 25 data, the lowest and highest boundaries indicate the 25th and 75th percentile, respectively, and the black line within the box marks the median. Whiskers on the right and left of the box indicate the 90th and 10th percentiles, respectively. Dark circles represent the 95th and the 5th percentiles.

derivatization procedure,37−39 so they are relatively much more simple. The two procedures gave comparable results, as shown in Table 2, with some differences observed in the determination of the minor component, α-ODAP. The content of the two isomers falls in the range normally reported for these compounds.26−30 In particular, the accessions ‘Aidone 3’ and ‘Corleone’ show the lower amount 2439

dx.doi.org/10.1021/jf500149n | J. Agric. Food Chem. 2014, 62, 2436−2442

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Table 2. Content of β- and α-ODAP (g/kg) of Ten Sicilian Accessionsa NMR

a

code

provenance

1 2 3 4 5 6 7 8 9 10

Aidone 1 Aidone 2 Aidone 3 Troina Licodia Eubea Bronte Corleone Piana degli Albanesi Maletto Mineo

HPLC-ELSD

β 3.05 3.69 2.96 3.46 3.21 3.47 2.69 3.56 4.59 3.00

(±0.09) (±0.12) (±0.15) (±0.10) (±0.04) (±0.05) (±0.05) (±0.08) (±0.05) (±0.20)

α d b d bc cd bc e b a d

0.51 0.48 0.42 0.50 0.53 0.74 0.44 0.50 0.61 0.47

β

(±0.02) (±0.04) (±0.04) (±0.12) (±0.03) (±0.04) (±0.03) (±0.08) (±0.19) (±0.01)

b b b b ab a b b ab b

3.12 3.68 2.93 3.45 3.21 3.43 2.61 3.57 4.45 3.04

(±0.12) (±0.08) (±0.07) (±0.07) (±0.08) (±0.01) (±0.05) (±0.08) (±0.06) (±0.08)

α de b e c d c f bc a de

0.60 0.72 0.52 0.56 0.60 0.57 0.60 0.60 0.60 0.55

(±0.03) (±0.04) (±0.08) (±0.12) (±0.03) (±0.10) (±0.07) (±0.05) (±0.11) (±0.01)

±SD reported in parentheses. Different letters in each column indicate significant difference for p ≤ 0.05.

Table 3. Pearson Correlation Analysis of Productive and Chemical Parametersa seed/ pod pod/plant seed/pod 1000 seed wt seed yield protein β-ODAP (NMR) α-ODAP (NMR) β-ODAP (HPLC) α-ODAP (HPLC) a

−0.278

1000 seed wt

seed yield

protein

−0.586* 0.0692

−0.0825 −0.453 0.111

0.0361 −0.0352 0.0818 0.335

β-ODAP (NMR)

α-ODAP (NMR)

β-ODAP (HPLC)

α-ODAP (HPLC)

0.297 0.151 −0.651* 0.0571 0.402

0.252 0.181 −0.468 −0.209 0.407 0.514

0.33 0.149 −0.677* 0.0739 0.426 0.995*** 0.509

0.00136 0.222 −0.0919 −0.00463 −0.147 0.328 −0.00226 0.344

altitude 0.166 0.0564 −0.344 0.307 0.523 0.733* 0.485 0.709* −0.106

*, **, and *** indicate significance for 0.05, 0.01, and 0.001, respectively.

of β isomer, whereas the samples ‘Aidone 2’ and ‘Maletto’ had a higher content of the same isomer, as shown by the NMR and HPLC-ELSD evaluations (Table 2). For the α isomer a slightly more complex situation is observed: from the NMR analyses the content of this isomer is lower in the accessions ‘Aidone 3’ and ‘Corleone’, as for the β isomer, instead the higher amount is reported for the samples ‘Bronte’ and ‘Maletto’; by the HPLC analyses the accessions ‘Aidone 3’ and ‘Mineo’ show the lower amount, whereas six samples, namely, ‘Aidone 2’, ‘Aidone 1’, ‘Licodia Eubea’, ‘Corleone’, ‘Maletto’, and ‘Piana degli Albanesi’, show a comparable and higher amount of α isomer. These results are confirmed by the ANOVA analysis of the aforesaid quantitative results, which shows significant differences for the β isomer in the two analytical approaches (NMR and HPLC), whereas for the α isomer a less significant difference by NMR analyses and no differentiation by HPLC are observed (Table 2). A remarkable chemical aspect of β-ODAP, which could affect the quantitative determination of this compound and its isomer in grass pea, is its spontaneous isomerization into the nontoxic isomer. This reaction is strongly influenced by the pH of the aqueous solution and, mainly, by heating, giving an equilibrium with about 40% of α-form.28,31,40−42 In order to verify if the aforesaid isomerization took place during the experimental procedures (extraction, analytical determinations, etc.) used in this study, pure β-ODAP was left in water at neutral pH and running 1H NMR spectra at the initial time (t0), after 24 h (t1), four days (t2), one week (t3), two weeks (t4), six weeks (t5), and sixteen weeks (t6). Only after one week an appreciable amount of α isomer was recorded (ca. 5% with respect to β isomer), whereas in the last experiment (t6) a 6:4 ratio between β and α isomers was

observed. It was noted that in this last experiment also an appreciable amount of 2,3-diaminopropionic acid (DAPRO) was recorded (see Supporting Information); the latter is the hydrolysis product of β- and α-ODAP, confirming that a competition between the isomerization and hydrolysis occurs in these conditions.31 Therefore, if on one hand the β isomer isomerizes to the α isomer, on the other hand the process is very slow and cannot occur during the experimental procedures (mainly extraction step) adopted in this study, thus confirming that those detected are the real amounts of the two isomers in the Sicilian grass pea. Pearson Correlation. Interesting relationships were found between agronomic and chemical parameters (Table 3). One expected negative correlation was found between the number of pods per plant and the 1000 seed weight (−0.586, p < 0.045). This is commonly measured in grain crops since increasing number of pods means that the weight of seeds decreases. Another interesting significant correlation is the one between 1000 seed weight and β-ODAP determined both with the NMR method and with the HPLC method (−0.651, p < 0.041, and −0.677, p < 0.032, respectively). In this case the βODAP seems negatively related with the seed weight. This observation is in agreement with Tadesse and Bekele,43 who found negative association between seed size and ODAP content on fifty grass pea land race accessions. However, it is in contrast with the findings reported by Dahiya,44 who, when analyzing eighteen genotypes of grass pea with a paperchromatography technique, found that smaller seeds contain lower concentrations of ODAP. Another valuable correlation was found between the βODAP content determined with HPLC and NMR and the altitude from where the seeds were collected (0.709, p < 0.021, 2440

dx.doi.org/10.1021/jf500149n | J. Agric. Food Chem. 2014, 62, 2436−2442

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and 0.733, p < 0.015, respectively). From this relation, it emerges that seeds from environments with higher altitudes show a higher content of β-ODAP. On the contrary, a recent study carried out on nine grass pea genotypes in five different environments in Ethiopia showed a negative correlation between altitude and β-ODAP content;45 therefore, the mechanism of this relation is not yet clear and needs to be fully elucidated. In agreement with Tadesse and Bekele,43 no association was found between ODAP and protein content, indicating that ODAP is synthesized independently of protein content. The last significant correlation refers to chemical parameters: β-ODAP HPLC is highly positively correlated to β-ODAP NMR (0.995, p < 0.001), testifying that the two methods are comparable and equally usable. The application of two well consolidated analytical methodologies, namely, NMR and HPLC-ELSD, gives comparable results in the quantitative determination of the two amino acids, showing at the same time some operative advantages with respect to the more complex and time-consuming analytical procedures adopted in this field up to now. Accessions ‘Aidone 2’, ‘Aidone 3’, and ‘Troina’ showed a high seed yield and a valuable protein content; namely, ‘Aidone 3’ showed also a low β-ODAP content and so represents good material for a future breeding program. Moreover, correlations revealed that accessions with heavier seeds and those coming from low altitude environments have a lower β-ODAP content. In conclusion, the results of this study show that the Sicilian grass pea accessions do not present very high amounts of the neurotoxic β-ODAP. In any case, taking into account the very low consumption of this species and relatively low level of the neurotoxins, its sporadic presence in the human diet does not represent a real health risk. In this view, Sicilian grass pea can be considered a niche product strongly characterizing local cultural and gastronomic traditions.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S6 including the structures and 1H NMR spectra of β-ODAP, α-ODAP, and DAPRO and the HPLC-ELSD profile of a mixture of α-ODAP and β-ODAP. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 095 7338347. Fax: +39 095 7338310. E-mail: [email protected]. Funding

This research work was financially supported by Consiglio Nazionale delle Ricerche (CNRRome). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Dario D’Angelo [SOPAT 47, Valguarnera (EN), Assessorato Risorse Agricole ed AlimentariRegione Siciliana].

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