Part 2.1: Antioxidant Potentials and Phenolic Composition of Tef Varieties: An Indigenous Ethiopian Cereal

2.1.3 Materials and methods

Chemicals and reagents: DPPH, TPTZ (2, 4, 6-tripyridyl-s-triazine), Trolox (6-hydroxyl-2,5,7,8-

tetramethylchroman-2-carboxylic acid), catechin, naringenin, gallic acid, ferulic acid, p-coumaric acid, o-coumaric acid, vanillic acid, caffeic acid, syringic acid, sinapic acid, protocatechuic acid, salicylic acid and trifluoroacetic acid were purchased from Sigma–Aldrich Fine Chemicals (St. Louis, MO). HPLC grade methanol and water were purchased from VWR Chemicals (VWR international S.A.S., France). Analytical grade phenol reagent, aluminum chloride, sodium nitrite,

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methanol, hydrochloric acid, sodium hydroxide, and sodium carbonate were purchased from Chem-Lab (Chem lab NV, Belgium).

Grain sample and preparation: Seven tef varieties i.e. Boset (DZ-Cr-409), Dega (DZ-01-2675),

Quncho (DZ-Cr-387), Simada (DZ-Cr-285), Tsedey (DZ-Cr-37), Zagurey (local) and Zezew (local) were used in this study. All the tef varieties were originating from one location, grown in the same season and were the ones that were available at that region. The names outside the brackets are local names whereas the ones between brackets are the breed name which is specific to each variety. The first five (5) varieties are white whereas the last two (2) are brown. All the tef varieties, grown under similar agricultural conditions, were harvested in the main harvesting season (locally called Meher) in December 2013 and generously obtained (about 3 kg each) from Axum Agricultural Research Center (Tigray, Ethiopia). They were carefully cleaned manually and then milled by a local miller into flour using a disc attrition mill. They were sun dried while standing on the field (before harvest) and milled by disc attrition milling at a local tef miller, in the same way as tef is milled in Ethiopia. Some portions (about 1 kg) of each variety was pre-milled prior to each variety and discarded to prevent cross-contamination among the varieties. The flour passed through a sieve of mesh number 16 (sieve opening 1.19mm, Tylor test sieve, Mentor, OH, USA). Flour samples were packed in polythene pouches and stored at -20oC

until further analysis.

2.1.3.1 Extraction of soluble and bound phenolics

Extraction of soluble phenolics (Fig. 2.1.1) was based on the method described by Gonzales et al. (2014). Briefly, approximately 2 g of flour was placed in a 50 mL falcon tube and homogenized with 15 mL of 100% methanol at 3000 rpm using an Ultra-Turrax (IKA-T18D, Germany) for 45 s. The tubes were then placed on ice for 15 s. The mixture was centrifuged (Z 300 K, Hermle Labortechnik, GmbH, Germany) at 13000 g for 10 min at 4°C. The residue was re-extracted using 10 mL of 80% methanol following the same procedure. The supernatant was further filtered using filter paper with pore size of 5-13 µm (VWR; Leuven, Belgium) and the volume was corrected to 25 mL using 80% methanol. The phenolic content from these extracts will be further referred to as soluble phenolics. After removal of the supernatant, the residues were air dried overnight and stored at −20°C un l further extrac on for bound phenolics.

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Alkaline hydrolysis of bound phenolic content of the residues (Fig. 2.1.1) was done following the optimized method of Gonzales et al. (2014). Briefly, 0.1 g of dried residue obtained after the methanolic extraction was hydrolyzed using 2 mL of 2M NaOH and sonicated (UP 400S, Hielscher, GmbH, Germany) at maximum amplitude (100%) for 30 min at 60°C in a screw-capped test tube previously flushed (dried) with nitrogen. The samples were then neutralized using 2M HCl. Then, 4 mL of methanol (100%) containing 0.1% formic acid was added as an extraction solvent followed by vortex mixing for 2 min. Then the tubes were centrifuged (Z300K, Hermle Labortechnik, GmbH, Germany) for 10 min at 10000 g and 4°C. Extraction was done twice and the supernatants were pooled and standardized to 20 mL using 80% methanol. The phenolic content from these extracts will be further indicated as bound phenolics.

Figure 2.1. 1 Extraction process of soluble and bound phenolic compounds About 2 g (dm) Flour

Centrifugation 1st extraction with 15 mL 100% methanol

Ultra-Turrax assisted extraction

Filtrate Residue Residue Soluble phenolic

compounds

Ultrasonic assisted alkali extraction Centrifugation

Filtration

2nd extraction with 10 mL of 80% methanol

Filtration

Filtrate Residue

Bound phenolic compounds

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2.1.3.2 Determination of total phenolic and flavonoid contents

The TPC of each extract was determined using the method described by Singleton et al. (1999). Briefly, 1 mL of each of bound and soluble extracts was mixed with 0.5 mL of 10 times diluted Folin-Ciocalteu reagent in a test tube, vortex mixed and were allowed to stand for 6 min. The reaction was neutralized by adding 1.5 mL of saturated sodium carbonate (20%), followed by the addition of 1 mL double distilled water and then thoroughly mixed. The contents were allowed to stand for 2h in dark at room temperature. The absorbance of the resulting blue color supernatant was measured at 760 nm using a spectrophotometer (Model 4001/4, Thermo Spectronic, USA) using methanol as a blank. Total phenolic content in each extract was determined using a standard curve prepared from gallic acid and the results were expressed as mg GAE/100 g flour dm).

Total flavonoid content (TFC) was determined according to the method described by Dewanto et al. (2002). Briefly, 75 µL of 5% NaNO2 was mixed with 1 mL of the extracts and 1 mL of water, and

thoroughly vortex mixed. After 6 min, 150 µL of a 10% AlCl3 solution was added, and the mixture

was allowed to stand for another 5 min. Then, 0.5 mL of 1M NaOH was added and the contents were allowed to stand for 15 min in dark at room temperature. The absorbance was measured at 510 nm using a spectrophotometer (Model 4001/4, Thermo Spectronic, USA) and methanol was used as a blank. The flavonoid content was determined using a standard curve prepared from catechin and the results were expressed as mg catechin equivalent (CE)/100 g flour dm).

2.1.3.3 Determination of antioxidant capacity

DPPH free-radical scavenging capacity: The method described by Kumaran and Karunakaran

(2006) was used to measure the antiradical activity against the DPPH radical. The DPPH (0.1mM, 2 mL) solution in methanol was added to 100 µL extracts, vortex mixed for 10 s and left in the dark for 30 min at room temperature. The absorbance of the solution was measured at 517nm using a spectrophotometer (Model 4001/4, Thermo Spectronic, USA). The scavenging capacity of DPPH radical was calculated with respect to the Trolox standard curve and the results were expressed as µmol TE/100 g flour dm.

Ferric reducing antioxidant power (FRAP): The ferric reducing antioxidant power (FRAP) was

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acetate buffer (300 mM, pH 3), 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl3.6H2O

solutions were prepared. The fresh working solution (FRAP) was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3.6H2O solution. Bound and soluble flour

extracts (100 µL) were allowed to react with 3 mL of the FRAP solution for 30 min in the dark at room temperature. The absorbance of the colored complex was measured at 593 nm using a spectrophotometer (Model 4001/4, Thermo Spectronic, USA). Methanolic solutions of known Fe2+ concentrations, ranging from 200 to 1000 µmol/L FeSO

4.7H2O, were used for the preparation

of a calibration curve. The FRAP antioxidant capacity was expressed in µmol Fe2+/g flour dm.

2.1.3.4 Phenolic profiling

A method as outlined by Wen et al. (2005) was implemented for the determination of PCs. The separation of PCs was performed with an Agilent 1100 series HPLC system equipped with on-line degasser (Model 590, Alltech elite degassing system, USA), quatpump (G 1311A), AlltimaTM -

Colomn18 5u (4.6 mm × 150 mm; GRACE, Deerfield, USA), photodiode array detector (DAD) (G 1315B, Agilent 1100 series). Instrument control and data analysis was carried out using Agilent HPLC Chemstation 10.1 edition through Windows 2000. The flow rate of the mobile phase was kept at 0.5 mL/min. Mobile phase A was HPLC grade water containing 0.02% trifluoroacetic acid (TFA), and phase B was HPLC grade methanol containing 0.02% TFA. The gradient conditions were: 0-5 min, 25% B; 5-10 min, 25-30% B; 10-16 min, 30-45% B; 16-18 min, 45% B; 18-25 min, 45-80% B; 25-30 min, 80% B; 30-40 min, 80-25% B. The temperature of the column was controlled at 25 °C. Injection volume was 10 µL. The detection wavelengths of DAD were set at four selected positions: 254, 275, 305, and 320 nm. Identification of the PCs was done by comparing retention times and spectra from the DAD detector with those of pure standards. o-Coumaric acid (5mg/L) was used as internal standard, and quantification was performed by external calibration curves for each identified phenolic compound.

2.1.3.5 Statistical analysis

All extracts were made in triplicate. Results are reported as mean ± standard deviation on a dry matter basis (dm). The differences of mean values among tef varieties were determined using one-way analysis of variance (ANOVA) followed by Tukey’s Honest Significant Differences (HSD)

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multiple rank test at P < 0.05 significance level. All statistical analyses were performed using SPSS version 22 (SPSS Inc., Chicago, IL, USA).

In document Compositional and nutritional properties of tef and tef-based food products (Page 54-59)