abstract
Effects of steeping conditions (time, temperature and soaking solution) and anaerobic storage on the
gamma-aminobutyric acid (GABA) content in waxy hull-less barley grains during germination was
examined. The barley kernel was steeped for 16 h at different temperatures (5, 15 or 35 C) either in
water or in a buffer solution (pH 6.0, 50 mmol/L sodium acetate) and then germinated at 15 C for 72 h.
To reach the optimum water content (36–44 g/100 g) for germination, a longer steeping period was
required when steeping temperature was lower (16 h at 5 C vs. 8 h at 15 C). At 35 C for steeping,
however, the water content in the grains increased excessively, and thus germination percentage became
much less than those at 5 and 15 C. The GABA content increased with increasing germination time and
was higher in the buffer solution than water. These findings indicate that the glutamate decarboxylase
(GAD), which is the rate-limiting enzyme for GABA synthesis, is more activated by extending germination
at controlled pH (6.0). An anaerobic storage with nitrogen in the dark for the germinated barley grains
substantially raised the GABA content: 14.3 mg/100 g after the treatment for 12 h, which was four times
higher than that of control sample (3.7 mg/100 g). Overall results suggest that the steeping prior to
germination greatly affects the GABA production during the germination of barley, and the anoxia
storage with nitrogen after the germination increases the GABA content.
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2. Materials and methods
2.1. Materials
The waxy hull-less barley used in this study was harvested in
2005, and provided by the Honam Agricultural Research Center
(Iksan, Korea).
2.2. Steeping and germination
The barley grains were surface-sterilized by dipping in sodium
hypochlorite solution (0.1 g/100 g) for 30 min and thoroughly
rinsed in deionized water. Steeping was carried out by placing the
barley grains (30 g) in a flask containing 100 ml of sterilized
deionized water or a buffer solution (pH 6.0, 50 mmol/L sodium
acetate) for 8, 16, and 24 h at 5, 15, and 35 C. The optimal pH for
GABA production has been reported to be around 6.0 (Streeter &
Thompson, 1972). Generally, steeping is carried out without cooling
or heating (Bamforth & Barclay, 1993; Rimsten, 2003), but steeping
was done at 5 and 35 C in this study because temperature stress
with cold or heat shocks could enhance the GABA levels (Shelp
et al., 1999). After steeping, the barley grains were subjected to
germination on two layers of moist filter paper in Petri dish (9 cm)
for 24, 48, and 72 h (100% relative humidity) at 15 C, the temperature
recommended for germination of barley (Bamforth & Barclay,
1993; Rimsten, 2003). The germinated barley grains were freezedried
and ground in a laboratory mill (A10 analytical mill, Tekmar
Co., Cincinnati, OH) to a fine powder for analyses.
2.3. Water content during steeping
After steeping, the barley grains were placed on the paper towel
to remove surface water and weighted. The moisture content of the
grains was determined by drying in a convection oven at 100 C for
24 h.
2.4. Germination percentage
The barley grains were considered to be germinated when the
radicle was 1 mm or longer. The germination percentage was
calculated as the number percentage of germinated grains to total
grains tested.
2.5. Anaerobic treatment
The effect of anaerobic treatment was evaluated with the barley
grains germinated for 48 h at 15 C after steeping for 8 h at 15 C in
50 mmol/L sodium acetate buffer solution (pH 6.0). The germinated
barley grains (5 g) were placed in a 250 ml flask, and then purged
with nitrogen gas for 1 min. After covering tightly, the flask was
stored in a dark room at an ambient temperature for 12 h. The
grains were then removed from the containers, immediately
freeze-dried, and ground by a laboratory mill (A10 analytical mill,
Tekmar Co., Cincinnati, OH).
2.6. Analysis of GABA content
The GABA content in the germinated barley grains was determined
by following the procedure of Baum et al. (1996) with
a minor modification. Ground barley flour (200 mg) was added to
a solution (800 ml) of methanol:chloroform:water mixture (12:5:3,
volume basis) in a centrifuge tube. The tube was vortexed and then
centrifuged at 12,000 g at 4 C for 15 min. The supernatant was
collected in a flask, and the residue was extracted again with
a chloroform:water (3:5, v/v) solution (800 ml). The second
supernatant was combined with the first supernatant. The
collected sample was dried and then re-dissolved in water. The
sample was then filtered through a 0.45 mm filter and the GABA
content was measured by an amino acid analysis system (Waters,
Milford, MA) after 6-aminoquioly-N-hydroxysuccinimidyl
carbonate (AQC) derivatization.
2.7. Statistical analysis
All analyses were performed in triplicate. Statistical analyses
were carried out with a Duncan’s multiple test (P < 0.05) using
statistical software SPSS V. 8.2 (SPSS Institute Inc., Cary, NC).
3. Results and discussion
3.1. Water uptake
Fig. 1 represents the water content of the barley grains steeped
in water at different temperatures. The grains steeped in the buffer
displayed similar results (data not shown). The water content was
increased by steeping, and the increment was significant at the
early stage of steeping (up to 8 h). It was reported that at the
beginning of steeping, the embryo and hull absorbed water more
rapidly than did the starchy endosperm (Bamforth & Barclay, 1993).
0
10
20
30
40
50
60
0 5 10 15 20 25
Steeping time (h)
Water content (g/100g)
Fig. 1. Moisture content of barley grains steeped in water for 0, 8, 16, and 24 h at 5
(–$A$–$), 15 (d) and 35 (..) C. Data reported are the mean SD of triplicate
determinations.
H.-J. Chung et al. / LWT - Food Science and Technology 42 (2009) 1712–1716 1713
The rapid increase in water content at the early stage was attributed
to the water uptake by embryo since the barley used was hull-less.
The water content was greater as the steeping temperature was
increased (Fig. 1). For instance, the water content after steeping for
24 h reached 38, 43, and 51 g/100 g at 5, 15, and 35 C, respectively.
Dewar et al. (1997) reported that steeping temperature had
a significant effect on water uptake of sorghum grains. Shimoda,
Ogawa, Takashita, and Omori (1998) observed that increased
steeping temperature enhanced the water uptake of barley grains.
However, it was found that the barley grains steeped at 35 C in this
study promoted mold growth on surface, resulting in an undesired
flavor. With rice grains, soaking in warm water (30 C) for several
hours caused microorganisms to grow rapidly (Bandara, Vithanege,
& Bean, 1991). Similar result has also been reported by Dewar et al.
(1997) with sorghum. They claimed that the steeping for an
extended period with excess water might lead to anoxic conditions,
which might be compounded by microbial proliferation because
the oxygen requirements by the grains could not be satisfied.
3.2. Germination percentage
The germination percentage was significantly affected by
steeping temperature (Fig. 2). When the steeping was done in water
(Fig. 2a), the barley grains germinated rapidly within 24 h at all
steeping temperatures tested. The germination percentages after
steeping at 5 and 15 C were 80.9 and 85.5%, respectively, whereas
that after steeping at 35 C was much lower (48.0%). The water
content after steeping is one of the critical factors in determining
the germination percentage (Davidson, Eastman, & Thomas, 1976;
Rimsten, 2003). Rimsten (2003) suggested that steeping was
important for controlling the development of enzymes needed for
germination because the grains absorbed water during steeping.
The optimum water content for germination in barley grains has
been reported to be from 39 to 44 g/100 g (Haraldsson et al., 2004),
and from 38 to 48 g/100 g (Son et al., 2002). The water content of
the barley in this study ranged between 36 and 44 g/100 g by
steeping at 5 and 15 C, respectively, which was near optimal for
germination. However, the water content of the barley steeped at
35 C was 51 g/100 g, which was too high. Therefore, steeping at the
low temperatures resulted in higher germination percentages than
that at 35 C. Bamforth and Barclay (1993) reported that steeping at
a high temperature could lead to the inhibition in growth of rootlet
and damage to the embryo, which resulted in reduced germination.
In this study, the chit length after germination was much shorter in
barley grain steeped at 35 C than that at 5 or 15 C (data not
shown). Therefore, it could be concluded that the germination
percentage was highly dependent on the steeping temperature, and
if the steeping temperature was too high the germination was
impaired.
When the steeping of barley grains was carried out in the buffer
solution (pH 6.0), the germination percentage increased continually
with germination time regardless of steeping temperature
(Fig. 2b). At the early stage of germination (24 h), the germination
percentage of barley grains steeped in a buffer solution (20–40%)
was lower than that in water (45–65%). This result was in agreement
with the studies that have reported retardations in germination
and growth of seedlings under saline conditions (Al-Karaki,
2001; Othman, Al-Karaki, Al-Tawaha, & Al-Horani, 2006). Ions in
the buffer solution might affect the germination ratio, but the
germination percentages at 72 h after steeping in the buffer solution
at 5 and 15 C were similar to that after steeping in water
(Fig. 2a vs. b). Like the case of water steeping, the higher temperature
(35 C) resulted in lower germination percentages. However,
the germination percentage after steeping at 35 C in buffer was
higher than that in water (63% vs. 48% at 72 h). In addition, the
steeping in buffer produced much less amount of off-flavors than
that in water. This could possibly suggest that the buffer solution
inhibited microorganism growth. Se