Plant Foods Hum Nutr (2014) 69:85–91 DOI 10.1007/s11130-013-0400-y

ORIGINAL PAPER

Effects of Roasting on Phenolic Composition and In vitro Antioxidant Capacity of Australian Grown Faba Beans (Vicia faba L.) Siem Siah & Izabela Konczak & Jennifer A. Wood & Samson Agboola & Christopher L. Blanchard Published online: 12 January 2014 # Springer Science+Business Media New York 2014

Abstract Faba bean phenolic compounds encompassed phenolic acids, flavonols, proanthocyanidins and anthocyanins. Roasting faba beans for 120 min decreased the total phenolic, flavonoid and proanthocyanidin contents by 42, 42 and 30 %, respectively. Roasting beans for 120 min decreased the 2,2diphenyl-1-picrylhydrazyl radical scavenging activity, total equivalent antioxidant capacity and ferric reducing antioxidant power by 48, 15 and 8 %, respectively. High performance liquid chromatography-post column derivatisation revealed the generation of new phenolic compounds as a result of roasting. Antioxidant mechanism of bean less-polar phenolic Electronic supplementary material The online version of this article (doi:10.1007/s11130-013-0400-y) contains supplementary material, which is available to authorized users. S. Siah : J. A. Wood : S. Agboola : C. L. Blanchard Graham Centre for Agricultural Innovation, Wagga Wagga Agricultural Institute, Pine Gully Road Wagga Wagga 2650, Australia S. Siah : I. Konczak Food Futures Flagship, CSIRO Animal, Food and Health Sciences, 11 Julius Avenue, North Ryde 2113, Australia S. Siah : C. L. Blanchard School of Biomedical Sciences, Charles Sturt University, Wagga Wagga 2678, Australia J. A. Wood NSW Department of Primary Industries, Tamworth Agricultural Institute, Calala 2340, Australia S. Agboola School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, Australia

compounds was largely based on free radical scavenging activity. The bean phenolic compounds with reducing capability were heat stable. Roasted faba bean extracts (70 % acetone, v/v) were fractionated into relatively polar and nonpolar fractions; the latter contributed the majority of the antioxidant capacity. The extracts from beans with different seed coat colours differed in their phenolic compositions, which suggest different levels of potential benefits to health. Although roasting initially lowers the bean antioxidant capacity, prolonged roasting at 150 °C for 60 min and longer causes generation of new phenolic compounds and an increased antioxidant capacity. The findings encourage a wider ultilisation of faba beans for human foods particularly in baked/roasted products. Keywords Vicia faba . Roasting . Phenolic compounds . Antioxidant activity/capacity . Hydrolysis . Seed coat colour Abbreviations ABTS C3Geq CHAeq DMAC-HCl DPPH FRAP GAE HPLC HPLC-DAD

S. Siah Grain Growers Ltd, 1 Rivett Road, North Ryde 2113, Australia

HPLC-PCD

Present Address: S. Siah (*) Grain Growers Ltd, PO Box 7, North Ryde 1670, Australia e-mail: [email protected]

LSD ORAC RE

2,2'-azino-bis(3-ethylbenzthiazoline6-sulphonic acid) Cyaniding 3-glucoside equivalents Chlorogenic acid equivalents Hydrochloric acidified 4-dimethylaminocinnamaldehyde 2,2-diphenyl-1-picrylhydrazyl Ferric reducing antioxidant power Gallic acid equivalent High performance liquid chromatography High performance liquid chromatographydiode array detector High performance liquid chromatography-post column derivatisation Least significant difference Oxygen radical absorbance capacity Rutin equivalents

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TEAC TFA TFC TPC Tpro

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Total equivalent antioxidant capacity Trifluoroacetic acid Total flavonoid content Total phenolic content Total proanthocyanidin content

evaluate the effect of dry roasting on phenolic contents and antioxidant capacity of Australian grown faba beans. Acid and alkaline hydrolyses are applied on faba beans phenolic extracts to aid in phenolic compound analyses.

Materials and Methods Introduction

Samples

Faba beans (Vicia faba L.), commonly known as broad beans, are cultivated as human food and animal feed in temperate and subtropical regions of the world. Faba beans are good source of starch, protein and fiber offering low fat content. They are harvested at vegetative stage when the pods and seeds are fresh and green, and used as a vegetable. Alternatively, they can be harvested at maturity stage after the pods and beans dry out, in which the resulting dry seeds are termed pulses. In Australia, faba beans are grown as a break crop and mostly harvested at maturity. Greater than 80 % of Australian grown faba beans are exported to more than 40 different countries which accounts for an export value of US$68 million per annum (5 years averaged, 2007–11) [1]. The majority of the Australian faba beans crop is exported to the Middle East and Asia. Commercial faba bean varieties are buffcoloured whereas the red-coloured faba beans are not grown commercially. Con sumption o f faba b ean s h as a fav oura ble hypocholesterolaemic effect that may reduce the risk of cardiovascular diseases in vivo [2]. Faba beans also contain substantial minor constituents such as L-3,4dihydrophenylalanine (L-dopa), phytic acids and phenolic compounds that are associated with health benefits [3–6]. For instance, phenolic compounds, natural antioxidants, exhibit antihypertensive, anticancer and antimutagenic activities [5, 7], L-dopa and phytic acids may have anti-cancer potentials and provide treatments to Parkinson’s Disease [3, 6, 8]. The effects of various processing methods such as soaking, sprouting, freezing, boiling, pressure cooking and steaming on phenolic contents and antioxidant capacity of faba beans have been investigated [9–11]. While a number of reports deal with the impact of dry heating on phenolic contents and antioxidant capacity of pulses [12–14], similar studies on faba beans are scarce. Furthermore, the effects of dry heat treatment on phenolic contents and antioxidant capacity of pulses are dependent on bean types, phenolic extraction methods and dry heating conditions [12, 15]. For instance, dry toasting increases the total phenolic contents of black-eyed peas, kidney and pinto beans but not that of soybeans [12]; roasted black bean, yellow soybean and giant lentils at 150 °C for 10 and 30 min have a similar Trolox equivalent antioxidant capacity (TEAC) as their unprocessed beans, while further roasting for 60 min increases their TEAC [15]. The present study aims to

Two faba bean cultivars: Nura (buff-coloured) and Rossa (redcoloured) were grown to maturity at Wagga Wagga Agricultural Institute, Australia, in 2009. The bean samples were dried at 50 °C for 3 days before storing at −18 °C. The bean samples were ground into flours using an IKAUniversalmühle M20 Grinder (Janke and Kunkel, Staufen, Germany) and stored in screw cap plastic containers at 4 °C. Dry Roasting Seeds were placed on an open tray and roasted using dry heat in an oven (Premium Laboratory Oven, Thermoline Scientific, Australia) at 150 °C for 60 min [15]. The roasted beans were cooled at room temperature, ground into flour and stored in screw cap plastic containers at 4 °C. Preparation of Crude Phenolic Extract Extraction of phenolic compounds was carried out on the raw and roasted faba bean flours by dispersing them in 70 % (v/v) acetone at a solid to solvent ratio of 1:10. The acetone was removed using a rotary vacuum evaporator (Rotavapor R-205; Buchi, Switzerland) and the extracts were lyophilized [5, 10, 16]. Distilled water was used to dissolve the dried extract and filtered (0.45 μm) before analyses. Some non-dissolvable material was removed during the filtration process. Preparation of Purified Phenolic Extract The crude extracts were purified using an open chromatography columns filled with Amberlite XAD7 mesh 20–60 acrylic resin (Sigma-Aldrich). The crude extracts were added to resins kept in milliQ water. The columns were washed with 1 % acetic acid (v/v) (2 L) solution. Phenolic compounds were eluted with pure acetone. The eluted extract was concentrated using a rotary evaporator at 40 °C under reduced pressure and freeze-dried. Total Phenolic Content, Total Flavonoid Content and Antioxidant Capacity Assays The total phenolic content (TPC), total flavoid content (TFC), diphenylpicrylhydrazyl (DPPH) radical scavenging capacity,

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Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays were conducted as previously described [5]. The proanthocyanidin content (TPro) assay was carried out according to the hydrochloric acidified 4-dimethylaminocinnamaldehyde (DMAC-HCL) protocol as described by Li et al. [17] with a reduced volume to suit a 96-well microplate format.

High Performance Liquid Chromatography (HPLC) with Post Column Derivatisation (PCD) Online The HPLC-PCD system for phenolic compound profiling was set up as previously described [5, 10] where antioxidant activity of phenolic compounds was measured on-line using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) cation radical.

Fractionation of Crude Phenolic Extracts Using a Preparative HPLC Fractionation of crude phenolic extracts was carried out using a preparative scale HPLC system (ProStar Prep HPLC system, Varian) consisting of a Varian ProStar ultraviolet–visible detector (Model 320), Varian PrepStar Solvent Delivery Module pumps (Model SD-1) and a Varian ProStar fraction collector (Model 701). An aliquot (2 mL) of crude extract from the dry roasted Nura for 60 min at 150 °C (0.25 g/mL) in phase A was injected for separation using a Varian Dynamax Microsorb 100-5 C18 reverse phase column (250×21.4 mm). The gradient elution was the same as the HPLC-PCD system as previously described [5, 10] with a flow rate of 15 mL/min. Two fractions: fraction A and B, were collected which represented the elution from 1–10 min to 10–30 min respectively from the analytical HPLC system. The collected fractions were concentrated and lyophilised.

Hydrolysis

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Alkaline Hydrolysis The alkaline hydrolysis method was adapted from Luthria and Pastor-Corrales [19]. The purified phenolic extract (10 mg) was hydrolysed using 2 M sodium hydroxide (5 mL) for 30 min at 45 °C (laboratory oven, Memmert, Germany). The mixture was then acidified by adding 7.2 M hydrochloride acid (1.4 mL). The conjugated phenolic acids were released by adding ethyl acetate (6.4 mL) and vortexed for 20 s. The extraction was repeated three times and the combined ethyl acetate fraction was evaporated to a complete dryness. The dried remains were dissolved in 80 % (v/v) methanol (5 mL) and vortexed for 30 s (three times). The solution was filtered (0.45 μm) before analysis. Identification and Quantification of Phenolic Compounds by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) The HPLC system (Shimadzu, Japan) consisted of an auto injector (SIL-10 AD VP), a degasser (DGU-20A, pumps (LC10 AD)), a diode array detector (SPD-M10A), a column oven (CTO-10AS) and a system controller (SCL-10A). Phases A and B were 0.5 % TFA (v/v) in distilled water and acetonitrile:TFA:water (95:0.5:4.5, v/v/v), respectively. The column oven was maintained at 25 °C and the solvent elution rate was 1 mL/min. The gradient elution method used to separate phenolic compounds was outlined by Konczak et al. [20]. The hydroxybenzoic acids/flavanols were detected at 280 nm and quantified as gallic acid equivalents (GAE/gDW); hydroxycinnamic acids were measured at 326 nm and expressed as chlorogenic acid equivalents (CHAeq/gDW); flavonols were quantified at 370 nm and expressed as rutin equivalents (RE/ gDW), and the anthocyanins were assessed at 520 nm and expressed as cyanidin 3-glucoside equivalents (C3Geq/gDW). Statistical Analysis The data were analysed using GenStat 13th edition http://www. vsn-intl.com (VSN International Ltd, UK). All data was analysed by ANOVA or student’s t-test (two tailed). The data with least significant difference (LSD) calculated at the 5 % level.

Acid Hydrolysis The acid hydrolysis of the purified phenolic extract was performed as described in Daniel et al. [18]. Dried sample extract (10 mg) was hydrolysed using 2 mL of 2 M trifluoroacetic acid (TFA) (Sigma-Aldrich) solution for 2 h at 120 °C in a heating block (Reacti-Therm III Heating/ Stirring Module, Thermo Scientific, USA). Subsequently, the acid solution was made up to 5 mL in a volumetric flask with aqueous 80 % methanol (v/v) and stored at −18 °C. The solution was filtered (0.45 μm) before analysis.

Results Effects of Roasting on Phenolic Contents of Faba Beans The effects of dry roasting on extraction yields, total phenolic content (TPC), total flavonoid content (TFC) and total proanthocyanidin content (TPro) of faba bean cultivar Nura at different time points is shown in Table 1. Roasting the faba beans for 10 to 120 min resulted in a significantly higher

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Table 1 Effect of dry roasting on material yields, phenolic contents and antioxidant capacity of faba beans Roasting time (minute)

Extraction yield (g/100gDW)†

TPC (mgGAE/ gDW)‡

TFC (mgCE/gDW)§

TPro (mgCE/gDW)∫

DPPH (μmolTE/ gDW)ß

TEAC (μmolTE/ gDW)Δ

FRAP (μmolFe2+eq/ gDW)Ω

0 10 30 60 120 5 % LSD

7.71b 8.62a 8.63a 8.39a 8.77a 0.50

10.7a 8.3b 5.5c 5.2c 6.2c 1.2

2.8a 2.3b 1.6c 1.8c 1.6c 0.3

0.23a 0.16b 0.14bc 0.13c 0.16b 0.03

53.4a 34.0b 24.1c 34.3b 27.8c 4.7

72.7a 66.2b 57.4c 49.8d 61.8bc 4.6

18.2b 19.3ab 19.2ab 19.5ab 19.7a 1.5

The data marked with same superscripts in the respective analysis/assay (column) was not significant different (p>0.05); † extraction yields were expressed as g of extract per 100 g of dry beans; ‡ total phenolic content (TPC) was expressed as mg gallic acid equivalents per g of dry beans; § total flavonoid content (TFC) was expressed as mg of catechin equivalents per g of dry beans; ∫ total proanthocyanidins (TPro) was expressed as mg of catechin equivalents per g of dry beans; ß 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was expressed as μmol Trolox equivalents per g of dry beans; Δ Trolox equivalent antioxidant capacity (TEAC) was expressed as μmol Trolox equivalents per g of dry beans; Ω ferric reducing antioxidant power (FRAP) was expressed as μmol Fe2+ equivalents per g of dry beans

extraction yield in comparison to that of raw beans by about 10 %. The extraction yield of phenolic extracts from the roasted beans was similar regardless of the roasting duration. The TPC and TFC of the raw beans were significantly higher than those of roasted beans, regardless of roasting duration. Roasting for 10 min resulted in a 20 % decrease in TPC and TFC compared to that of raw beans, while a decrease by 40– 50 % in TPC and TFC was observed in beans roasted for 30– 120 min. Roasting beans for 10 min lowered the TPro by 30– 40 % with no further reduction at longer roasting times.

These peaks were more pronounced in the extract obtained from the beans roasted for 120 min.

Effect of Roasting on Faba Bean Antioxidant Capacity The effect of dry roasting on antioxidant capacity of faba bean extracts was evaluated using the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity, Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP) assays (Table 1). In comparison to the raw beans, roasting for up to 120 min reduced the DPPH radical scavenging activity in the beans by 40–50 %. A similar tendency was observed in the TEAC assay, where the TEAC values of the roasted beans were 70–90 % that of raw beans. In contrast, roasting for 10, 30 and 60 min did not alter the FRAP values, while roasting for 120 min increased these values compared to the raw faba beans. Effect of Roasting on HPLC-PCD Profiles of Faba Bean Phenolic Extracts Figure 1 shows the HPLC-PCD profiles of extracts from raw and roasted beans. The compounds eluted in two separate regions: retention time 0–10 min and 10–30 min. Additional new or altered HPLC peaks were observed in the chromatograms of roasted bean extracts at retention time 10–30 min in correspondence to that of raw bean extracts (marked by *).

Fig. 1 High performance liquid chromatography-post column derivatisation (HPLC-PCD) profiles of crude phenolic extracts from a raw faba beans, b roasted faba beans for 60 min and c 120 min at 150 °C (asterisk, new/altered peaks)

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Table 2 Phenolic contents and antioxidant capacity of Fraction A and B obtained from the crude extract of roasted faba beans (roasting at 150 °C for 60 min) Extraction yield (g/100gDW)† TPC (mgGAE/gDW)‡ TPro (mgCE/gDW)§ FRAP (μmolFe2+eq/gDW)∫ ORAC (μmolTE/gDW)Φ Crude extract 8.39±0.32a Fraction A 2.43±0.07b Fraction B 0.50±0.01c

5.24±0.63a 0.34±0.04c 2.31±0.09b

0.13±0.01a 0.001±0.000c 0.09±0.01b

19.46±0.68a 1.09±0.17c 8.14±0.35b

94.43±16.30a 18.36±1.60b 19.81±5.60b

The results marked with different superscripts in the individual column were significantly different from each other (p

Effects of roasting on phenolic composition and in vitro antioxidant capacity of Australian grown faba beans (Vicia faba L.).

Faba bean phenolic compounds encompassed phenolic acids, flavonols, proanthocyanidins and anthocyanins. Roasting faba beans for 120 min decreased the ...
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