Chemicals
The necessary chemical standards were obtained from the suppliers shown in parenthesis.
Acetic acid, 99 % (Merck, Darmstadt, Germany); 3-methyl butanal, 98 %; butanoic acid,
97 %; pentan-2-one, 98 %; dihydro-2-methyl-3(2H) furan, 97 %; 2-methylpyrazine, 98
%; 3-furaldehyde, 99 %; 2-furamethanol, 97 %; 2-acetyl furan, 99 %; 2-ethylpyrazine,
98 %; benzaldehyde, 98 %; 5-methyl-2-furfural, 99 %; 2-pentyl furan, 98 %; ethyl
furfural, 98 %; limonene, 99 %; phenyl acetaldehyde, 99 %; 2-acetylpyrrole, 98 %;
2-pyrrole carboxaldehyde, 97 %; 3-methyl phenol, 99 %; 2-ethyl-3,5-dimethylpyrazine,
98 %; linalool, 98 %; 5-hydroxy methyl furfural, 99 %; β-cyclocitral, 97 %; 2,6-dimethoxyphenol,
98 %; β-caryophyllene, 98 %; α-ionone, 98 %; α-copaene, 99 %; β-ionone, 98 % (Aldrich,
Steinheim, Germany)
Preparation of roasted yam tubers
Yam tubers (Discorea rotundata; elongated with thick rough hairy skin) (500 g) each were purchased from Pasar Borong,
Selangor, Malaysia and were identified by Prof. Mohd Yusop of the Faculty of Agriculture,
Malaysia.. The yam samples were stored at 4 ± 1 o C until used. Yam tubers with uniform size, maturity (i.e. yam harvested 220 days
after planting, DAP) and free from defects were selected, washed and hand peeled to
remove the skin and roasted (400 o C for 80 min) in a roaster (Model Duetl-M, Probat and Emmerich, Germany). After roasting,
the yam tubers were removed from the oven and allowed to cool to room temperature
(28 ± 1 o C) prior to solvent extraction.
Yam extract production
A longitudinal section cut (50 g of each roasted yam tubers) obtained from the central
part was pulverized and a portion of the pulverized roasted yam (10 g) was taken for
volatile compounds analysis. Aroma extract was prepared from a 10 g of the pulverized
roasted tubers with a 40 mL of dichloromethane. The suspension was stirred using a
vortex mixer (Heidolph, Rotamax 120, Schwabach, Germany) at 150 rpm and 28 o C for 1 h. The extract was filtered and dried on Na2SO4. The solvent was subsequently removed under a purified nitrogen stream (Turbo Vap
II, Caliper Life Science, Massachusetts, USA) to a volume of 4 mL. The extract was
stored at -20 o C until analysis.
Analysis of volatile compounds
Analysis of volatiles was carried out using the Static headspace-gas chromatography-mass
spectrometry (SH-GC-MS) as reported by Ludwig, et al., [5]. The roasted extract (4
mL) or volatile standards solution (4 mL) was introduced into a 10 mL vial and sealed
with a silicon rubber Teflon cap. The vial was equilibrated (40 o C, 15 min) in the headspace sampler (model 7694 E, Agilent Technologies, Palo Alto,
CA) with pressurized carrier gas for 12 s. Subsequently, 1 mL of the headspace was
injected into a non-polar BPx5 (5 % phenyl polysilylphenylene siloxane) capillary
column (30 m x 0.25 mm i.d., film thickness 0.25 µm; Scientific Instrument Services,
Inc., Ringoes, NJ. USA) in a QP-5050A GC-MS Instrument (Shimadzu, Kyoto, Japan). Helium
at a flow rate of 1.5 mL min -1, injection temperature at 250 o C and a detector temperature of 280 o C were employed. The temperature program commenced at 50 o C and was held for 3 min, raised to 250 o C at the rate of 15 o C min -1, held for 30 min and later increased to 280 o C at a rate of 10 o C min -1, with a final hold time of 5 min.
Mass spectrometry was carried out in electron impact mode using the following
conditions: The source temperature was 250 o C, the quadruple temperature selected was 280 o C and the relative electron multiplier voltage applied was 400 V with a resulting
voltage of 1553 V. The selection ion monitoring mode was employed to improve the detection
limits. Identification of volatile compounds (Table 1) was done comparing their mass
spectra with the spectra of the available pure standards and also by comparing their
retention indices with those of standards and data from literature. The linear retention
indices (RI) of compounds were obtained with a series of alkanes (C6 – C28) injected according to the same chromatographic protocol reported above.
Gas-chromatography-olfactometry analysis
The GC-O system was made up of a Trace Ultra 1300 GC (Thermos Scientific, Waltham,
MA, USA) equipped with an ODP 3 olfactory detector port (Gerstel, Mulheim, Germany).
A BPX5 capillary column (30 m x 0.25 mm i.d., film thickness 0.25 µm; Scientific Instrument
Service, Inc., Ringoes, NJ, USA) was employed. The column temperature program was
similar to that of the GC-MS above. The flow rate was 1 mL min -1 and 1 µL of the yam extract was injected. The split ratio between the FID detector
and the sniffing port was 1:1. Sniffing was conducted through a sniffing cone by three
trained panellists that presented normalized responses.
Aroma extract dilution analysis (AEDA) was employed in establishing the contribution
of each aroma compound to the overall flavour of the roasted yam. The extract was
diluted stepwise with dichloromethane (1:1, v/v) and sniffing of dilutions was continued
until no odour could be detected by sniffers [31]. The last dilution step in which
an aroma compound was sniffed is referred to as the flavour dilution (FD) factor of
the compound [32].
Anti-oxidative activity of volatile compounds
The anti-oxidative activities of the selected aroma compounds were tested according
to the method reported by Yangimoto, Lee, Ochi & Shibamoto [28]. Nine volatile compounds
(4 furans, 2 pyrroles, and 3 pyrazines) with appreciable concentrations in the roasted
yam (Table 2) and previously identified as potential anti-oxidants in roasted coffee
[5] were further quantified by calibration curves to determine their concentrations
in the roasted yam. Standard solutions of each selected volatile compound were analysed
at five levels of concentrations and their chromatographic areas were plotted against
concentration. The coefficients of linearity for the calibration curves were R2 >0.98. Pure reference standards at concentrations equivalent to those of the selected
volatiles found in the roasted yam and at 5, 10, 20, and 50 fold of the initial concentration
found in the roasted yam (Table 2) were prepared. Each reference standard was added
to 2 mL of dichloromethane solution of hexanal (3 mg mL -1) containing 0.2 mg mL -1 of undecane (internal standard) in a 20 mL vial. The sealed vial was heated at 60
o C for 10 min to initiate oxidation reaction of the mixture within the sealed vial.
Analysis of inhibition of aroma compounds toward hexanal was carried out by purging
the headspace of the vial with pure air (1.5 L min -1, 2 s) every 24 h for the first 10 days. The inhibition of aroma compounds toward
hexanal was monitored by gas chromatography (GC) at 5-days interval for 30 days.