2. Materials and methods

## 2.1. Polyphenolic profile analysis

## 2.1.1. Samples details

1. Introduction

24 Rediscovery of Landraces as a Resource for the Future

sity is particularly high [4].

tative features [6, 7].

organic production [8–11].

tion methods, able to distinguish landraces [12, 13].

After the massive use of processed acorns, as food source for prehistoric nomadic populations, the most important food discovery was undoubtedly that of cereals. Even now, wheat (Triticum) is one of the main food sources in the world. According to the last FAO report, wheat world production for 2017 was approximately expected in 740 million tons, exceeding the previous last one crop year by 1.2%, and covering about 15% of the world's arable surface [1]. In this scenario, durum wheat production reaches around 30 million tons, accounting approximately for 5–6% of the total world wheat production [2]. Canada, USA, Mexico and Russia are some of the countries around the world where durum wheat is cropped, although the Mediterranean region covers about 60% of world durum wheat production [3], being the EU (Italy, Spain, France and Greece) the leading global producer [2]. South Italy is one of the regions historically most voted to the cereal crops, where the durum wheat varietal biodiver-

For geographical position and ecological condition, Sicily represents the perfect environment for the cultivation of cereals, especially for durum wheat. In addition to the pedo-climatic conditions [5], some historical and socio-cultural aspects had also contributed to enrich the varietal heritage, such as the many invasions that characterized the island during the centuries. All these conditions, together with the historically conducted mass selection and the more recent genetic improvement programs based on the artificial crosses, had contributed to build

In Sicily, old and new durum wheat commercial varieties are currently cropped, but also many ancient landraces or populations characterized by specific bio-morphological traits and quali-

In recent years, all over the world, the attention paid to local and traditional productions is growing, especially in the agro-food sector. Maybe, it is due to the impact of globalization and the social and economic changes, but also to the increased consideration to health and nutritional aspects of food. Also in Sicily, this trend has led to the rediscovery and reuse of landraces both of wheat and other crops, responding to requests for more and more demanding market. The rising price of these local productions are contributing to the farmers' satisfaction, changing an unprofitable job in a renewed professional opportunity also for young businessmen. Furthermore, many recent research studies testify the high healthy and nutraceutical value of landraces, both for high amount of antioxidant compounds and for their natural aptitude to

This growing interest in local landraces has inspired to find effective and objective identifica-

In this chapter a multidisciplinary practical approach based on genotype and phenotype characterization of durum wheat Sicilian landraces is proposed. In particular, the polyphenolic profile of whole wheat grains was analyzed by means of HPLC/DAD and HPLC/ESI-MS.

the extremely wide currently existing varietal panorama [4].

Nine durum wheat (Triticum durum Desf.) landraces ("Margherito," "Manto di Maria," "Ruscia," "Russello SG8," "Scavuzza," "Tumminia SG3," "Trentino," "Tripolino," "Urria") were selected for phenolics profile evaluation.

Grains were cropped, in three plots of 10 m<sup>2</sup> each, using 350 viable seeds/m<sup>2</sup> , during three consecutive years (2012, 2013, 2014), in the fields of the Stazione Sperimentale di Granicoltura per la Sicilia, sited in Santo Pietro - Caltagirone [37�07<sup>0</sup> 1200N; 14�31<sup>0</sup> 1700E; 313 m a.s.l.] (CT, Sicily, Italy). 40 kg N/ha and 90 kg P2O5/ha were supplied at sowing carried out at the beginning of December; nitrogen fertilization with 50 kg N/ha were applied before the beginning of stem elongation stage (20–30 code in the BBCH-scale for cereals). Mechanical weed control methods were carried out in spring time and harvest was performed when physiological maturity of each genotype was reached.

Whole grain samples were milled to a fine powder by a laboratory mill (1093 Cyclotec Sample Mill, Tecator Foss, Hillerød, Denmark) equipped with a 1 mm sieve, immediately cooled to �20�C and kept at this temperature until analysis to protect bioactive components from degradation [14].

#### 2.1.2. Chemicals

All solvents and reagents used in this study were high purity laboratory solvents by Carlo Erba (Milano, Italy); HPLC grade water and acetonitrile were obtained from VWR (Milano, Italy). Pure vitexin (apigenin 8-C-glucoside) and orientin (luteolin 8-C-glucoside) were provided by Extrasynthese (Lyon, France) whilst vanillic acid, ferulic acid, p-coumaric acid and caffeic acid were purchased from Sigma (Sigma-Aldrich s.r.l., Milano, Italy).

#### 2.1.3. Extraction of free and bound phenolic compounds

Phenolic acids and flavonoids represent the most common form of phenolic compounds found in whole grains, existing as soluble free compounds, soluble conjugates esterified to sugars and other low molecular mass components, and insoluble bound forms either encapsulated in the cell-wall structures or chemically bound at molecular level [15].

According to Lo Bianco et al. [11], free phenolics were recovered by applying the method proposed by Dinelli et al. [14] with few changes. In brief, 1 g of whole wheat flour was mixed under vigorous stirring for 10 min with 20 mL of an acidic aqueous methanol solution (80% methanol, 19% water, 1% formic acid). The resulting heterogeneous mixture was transferred into standard glass sample tubes and centrifuged at 2500 g/min for 10 min. After that, the supernatant was removed and the extraction was repeated. Collected supernatants were pooled, evaporated to dryness, and then stored at 20C until use.

range 120–1800 at a resolving power of 25,000 (full-width-at-half-maximum, at m/z 200, RFWHM), resulting in a scan rate of >1.5 scans/s when using automatic gain control target of 1.0 106 and a C-trap inject time of 250 ms, under the following conditions: capillary temperature 300C, nebulizer gas (nitrogen) with a flow rate of 60 arbitrary units; auxiliary gas flow rate of 10 arbitrary units; source voltage 3 kV; capillary voltage 82.5 V; tube lens voltage 85 V. The Orbitrap MS system was tuned and calibrated in positive modes, by infusion of solutions of a standard mixture of sodium dodecyl sulfate (Mr 265.17 Da), sodium taurocholate (Mr 514.42 Da) and Ultramark (Mr 1621 Da). Data acquisition and analyses were performed using the Xcalibur

Phenolic Fingerprinting and Glumes Image Analysis as an Effective Approach for Durum Wheat Landraces…

http://dx.doi.org/10.5772/intechopen.79595

27

For the glumes image analysis, ears of the same ten wheat landraces were reaped, at the time of maximum ripening, in order to include a widest morphological and environmental variabil-

From three to six ears were sampled and from two to four glumes were removed from the spikelets of the ear middle section and from the both sides of each ear. The glumes were stored

Digital images of glumes samples were acquired using a flatbed scanner (ScanMaker 9800 XL, Microtek Denver, CO), applying the same resolution and scanning area conditions reported in Grillo et al. [4]. As suggested by Venora et al. [17], before digital image capture, the scanner was standardized according to the calibration protocol proposed by Shahin and Symons [18]. Morpho-colorimetric features were only measured for sound intact glumes, rejecting that ones with broken beak or shoulder, distinguishing in right and left side of the ear. A total of 902

Code Variety/landrace Sample amount

mar6 Margherito 06 97 mm1 Manto di Maria 01 95 rsc9 Ruscia 09 97 russg8 Russello 13 SG8 97 sca1 Scavuzza 01 95 tre2 Trentino 02 119 tri2 Tripolino 02 80 tumsg3 Tumminia SG3 94 urr1 Urrìa 01 88

ity, the wheat ears were collected during three consecutive years (2012, 2013, 2014).

at room temperature under controlled conditions (20C and 50% RH).

software.

2.2. Glume image analysis

2.2.1. Samples details

2.2.2. Images acquisition

wheat glumes were analyzed (Table 1).

Table 1. List of the ten different wheat local varieties studied.

The solid residue from the free phenolic extraction was subjected to alkaline hydrolysis to recover the bound phenolic compounds, according to Mattila et al. [16]. Distilled water (12 mL) and 5 mL of 10 M NaOH were added to the residue and stirred overnight at room temperature. The mixture was acidified to pH = 2 and then extracted three times with 15 mL of a 1:1 (v/v) mixture of cold diethyl ether and ethyl acetate by manually shaking and centrifuging. Organic layers were combined, evaporated to dryness, and dissolved into 2 mL of the aqueous methanol solution to analytical determinations.

#### 2.1.4. HPLC/DAD quantitative analyses

For HPLC/DAD analyses dry extracts were reconstituted in 3 mL of the extracting solvent and immediately analyzed. Quantitative analyses were carried out on a UltiMate3000 "UHPLC focused" instrument equipped with a binary high pressure pump, a Photodiode Array detector, a Thermostatted Column Compartment and an Automated Sample Injector (Thermo Scientific, Italy). Collected data were processed through a Chromeleon Chromatography Information Management System v. 6.80. Chromatographic runs were all performed using a reverse-phase column (Gemini C18, 250 4.6 mm, 5 μm particle size, Phenomenex, Italy) equipped with a guard column (Gemini C18 4 3.0 mm, 5 μm particle size, Phenomenex, Italy). Wheat polyphenols were eluted with the following gradient of B (formic acid, 2.5% solution in acetonitrile) in A (2.5% solution of formic acid in water): 0 min: 5% B; 10 min: 15% B; 30 min: 25% B; 35 min: 30% B; 50 min: 90% B; then kept for 7 min at 100% B. The solvent flow rate was 1 mL/min and. Quantifications were carried out at 350 nm using orientin (R2 = 0.9999) as external standard; the detector was set at 280 nm to build the calibration curve for vanillic acid (R2 = 0.9997), whilst vitexin (R2 = 0.9999), caffeic acid and ferulic acid were quantified at 330 nm using the corresponding reference substances (R2 = 0.9999 and R2 = 0.9998, respectively). The same reference wavelength was used for the quantification of coumarins against p-coumaric acid (R2 = 0.9998). All analyses were carried out in triplicate.

#### 2.1.5. Identification of main components via HPLC/ESI-MS

In order to unambiguously identify the chromatographic signals and/or to confirm peak assignments, a series of HPLC/ESI/MS analyses were performed on wheat samples. In this case, variable aliquots (1.0–1.5 mL) of the above mentioned hydro-alcoholic solutions coming from quantitative analyses (see previous paragraph) were transferred into standard laboratory vials and brought to dryness in vacuo with a rotary evaporator (Heidolph Laborota 400). The resulting yellowish residues were then re-dissolved in 500 μL of the original hydroalcoholic solution and submitted to qualitative analyses. The HPLC apparatus used was the same described above, whilst ESI mass spectra were acquired by a Thermo Scientific Exactive Plus Orbitrap MS (Thermo Fisher Scientific, Inc., Milan, Italy), using a heated electrospray ionization (HESI II) interface. Mass spectra were recorded operating in negative ion mode in the m/z range 120–1800 at a resolving power of 25,000 (full-width-at-half-maximum, at m/z 200, RFWHM), resulting in a scan rate of >1.5 scans/s when using automatic gain control target of 1.0 106 and a C-trap inject time of 250 ms, under the following conditions: capillary temperature 300C, nebulizer gas (nitrogen) with a flow rate of 60 arbitrary units; auxiliary gas flow rate of 10 arbitrary units; source voltage 3 kV; capillary voltage 82.5 V; tube lens voltage 85 V. The Orbitrap MS system was tuned and calibrated in positive modes, by infusion of solutions of a standard mixture of sodium dodecyl sulfate (Mr 265.17 Da), sodium taurocholate (Mr 514.42 Da) and Ultramark (Mr 1621 Da). Data acquisition and analyses were performed using the Xcalibur software.
