Rapid Screening of Soybean Volatiles
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Head space GC analysis of soybean volatiles. Soybean seeds (0.5 to
0.6 g, corresponding to 2 to 8 grains depending on size) were soaked in
5 ml of distilled water in a headspace vial (22 ml, Perkin Elmer,
Waltham, MA) at room temperature (25 ꢁC) overnight. After removing
the seed coat, the imbibed seeds were homogenized thoroughly with a
Polytron homogenizer (PT10-35, Kinematica, Littau, Switzerland) for
1 min. The homogenate was incubated at 25 ꢁC for 30 min in order to
facilitate the enzyme reaction, and then 5 ml of saturated solution of
CaCl2 was mixed in order to kill the enzymes. The vial was sealed
tightly with a butyl stopper and crimp top seal (National Scientific,
Rockwood, TN). Automated headspace volatile analysis was carried
out with a headspace sampler (HS 40XL, Perkin Elmer) equipped with
a GC (GC-2014, Shimadzu, Kyoto). The vial was incubated at 80 ꢁC
for 30 min, and pressurized for 3 min with N2 gas set at 100 kPa.
Headspace gas was introduced to the GC system for 0.2 min. GC was
performed with a Stabiliwax column (30 m ꢀ 0:25 mm i.d., Restek,
Bellefonte, PA) with 40 ꢁC (1 min) to 180 ꢁC (1 min) at 10 ꢁC/min.
Detection was performed with a FID detector.
SDS–PAGE. Protein profiles were analyzed by a modification of the
procedure of SDS–PAGE developed by Kitamura.5)
Results and Discussion
Headspace volatile screening
A simple and efficient headspace sampling procedure
was established with a headspace sampler coupled to a
GC system. It relies on the formation of volatile
compounds during homogenization and subsequent
incubation of the soaked soybean seeds. The amounts
of volatiles in dry soybean seeds are usually low, but they
increase rapidly during homogenization, which affects
the flavor properties of soybean-derived foods.2) Because
the seed coat hampers efficient homogenization and
flavonoids accumulated in it are known to be inhibitory
to lipoxygenase activity,12) it was removed before
homogenization. With this system, five major volatile
compounds formed in the homogenized soybean seeds,
viz., n-hexanal, 1-penten-3-ol, n-pentanol, n-hexanol,
and 1-octen-3-ol, were analyzed within 12 min (Fig. 1).
The peaks corresponding to these volatile compounds
found with Fukuyutaka (having all three isozymes of
LOX) were much bigger than those found with L-Star
(LOX-less variety), except for 1-octen-3-ol. This is in
good accordance with the volatile compositions exam-
ined in our previous study.2) This indicates that this
system is useful to identify a soybean variety that has
lower (or higher) ability to form volatile compounds.
Almost the same amount of 1-octen-3-ol was found with
Fukuyutaka and L-Star because it was formed inde-
pendently on LOXs.2,13)
Because the homogenate was heated to 80 ꢁC in order
to facilitate vaporization of volatiles during headspace
sampling, it was anticipated that heat degradation of
some nonvolatile compounds (such as lipid hydroper-
oxides) might result in overestimation of the amounts of
volatiles. Because heat degradation of hydroperoxides
can be largely prevented by the addition of lipophilic
antioxidants, we evaluated the effects of the addition of
butylated hydroxytoluene. When headspace GC analysis
was performed in the presence of 1 mM butylated
hydroxytoluene, little difference was found (data not
shown). Hence, we concluded that the volatiles detected
in this system were those mostly formed in the
homogenate but not those formed during headspace
analysis. With this sampling system, about 72 samples
were routinely analyzed per day.
Enzyme assay. n-Hexanal formation from linoleic acid (99% pure,
Sigma, St. Louis, MO) or from linoleic acid 13-hydroperoxide
(prepared with soybean LOX1 as reported previously2)) was deter-
mined essentially as described previously.10,11) In brief, soybean seeds
were imbibed overnight at 25 ꢁC and homogenized with 98 vol (v/w)
of distilled water with a Polytron mixer on ice. After centrifugation at
6,000 rpm (T15A36 rotor, Hitachi, Tokyo) for 10 min at 4 ꢁC, the
supernatant was taken as crude enzyme solution. The enzymatic
reaction was carried out with the enzyme solution (equivalent to 2.5 mg
of seeds) in the presence of 1 mM linoleic acid (prepared as 50 mM
solution in 0.2% Tween 20, emulsified before use with a tip-type
sonicator) or 0.4 mM linoleic acid 13-hydroperoxide (prepared as
50 mM solution in ethanol) in 0.1 M Na phosphate (pH 7.0, with 1 ml of
total volume of the reaction mixture) at 25 ꢁC for 10 min. After the
reaction, 2 ml of 0.1% 2,4-dinitrophenylhydrazine in ethanol contain-
ing 0.5 M acetic acid and 0.1% butylated hydroxytoluene were added
with 100 nmol of n-heptanal as an internal standard, and this was
incubated for 30 min at 25 ꢁC. The hydrozone derivatives were
extracted with 2 ml of hexane, and the hexane extract was washed
once with brine. After drying in vacuo, the residue was dissolved with
100 ml of CH3CN. A portion (2 ml) of the solution was separated with a
HPLC system (L-2130, Hitachi) equipped with a Mightysil RP-18
column (250 ꢀ 4:6 mm, Kanto Chemicals, Tokyo). The solvent was
comprised of CH3CN:H2O:tetrahydrofurane (80:19:1, v/v) at a flow
rate of 1 ml/min. Detection was performed with absorbance at 350 nm.
n-Hexanal was quantified using a calibration curve constructed with
n-heptanal as an internal standard.
1-Octen-3-ol forming activity. For photosensitized oxygenation,
linoleic acid (20 mg) was dissolved in 2 ml of methanol containing
75 mg mlꢂ1 of methylene blue (Wako Pure Chemicals, Osaka). The
solution was cooled to 4 ꢁC and irradiated for 3 h with a 250 W sodium
lamp (Panasonic, Osaka) with continuous introduction of oxygen gas to
the solution. The formation of linoleic acid hydroperoxides was
monitored by TLC (Silica gel 60, Merck, Whitehouse Station, NJ) with
a developing solvent consisting of hexane:2-propanol:acetic acid
(110:10:10, v/v). The hydroperoxides were detected by spraying N,N0-
dimethyl-p-phenylene diaminedihydrochloride solution (10 mg/ml in
methanol:water:acetic acid, 28:25:1, v/v). After photosensitization
reaction, unreacted linoleic acid was removed using a silica gel (Wakogel
C-300, Wako Pure Chemicals) column with hexane:ether (9:1, v/v).
The crude hydroperoxide was eluted with hexane:ether (6:4, v/v). The
solvent was removed in vacuo, and the hydroperoxides were emulsified
Selection of soybean varieties having modified vola-
tile formation
We evaluated ability to form volatile compounds after
homogenizing soybean seeds of 626 varieties collected
worldwide. The first screening resulted in 29 candidates,
then repeated analyses of these candidates confirmed
four varieties, Laredo, Daizu B, BRS213, and Santa
Maria, as the ones having unusual volatile-forming
properties (Fig. 2).
The amounts of volatile compounds formed from
homogenized soybean seeds of BRS213 were much
lower than those found with a normal soybean variety,
Fukuyutaka (Fig. 3). The profile was quite similar to that
of L-Star, which had no LOX. Santa Maria formed C6
and C5 volatiles in amounts approximately half of those
in 0.2% (w/v) Tween 20 in 0.1
M Na phosphate (pH 6.5) to a final concen-
tration of 10 mM. The concentration of hydroperoxides was calculated
from conjugated diene absorption at 234 nm (" ¼ 25;000 Mꢂ1 cmꢂ1).
The activity to form 1-octen-3-ol was determined at 25 ꢁC in a
reaction mixture containing 0.1 ml of the hydroperoxides and 2 ml of
crude enzyme solution. The reaction mixture was incubated for 15 min;
thereafter, 0.2 ml of 0.1 N NaOH was added to stop the reaction.
Nonenzymatic activity was estimated by using boiled crude enzyme
instead. The amount of 1-octen-3-ol was determined by headspace GC
analysis, essentially as described above. One unit of enzyme activity
was defined as the ability to form 1 mmole of 1-octen-3-ol per min.
Matsui, Kenji
