J. Pac .Sci. Tech.

Vol.19No.5（2010）

Original Paper～～～～～～～～～～～～～～～～～～～～～～～～～～～～

Stimulation of γ-Aminobutyric Acid Production in Vine-ripened
Tomato (Solanum lycopersicum L.) Fruits Under an Adjusted
Aerobic Atmosphere
Nobukazu MAE*, Yoshio MAKINO*†, Seiichi OSHITA*, Yoshinori KAWAGOE*, Atsushi
TANAKA**, Takashi AKIHIRO***, Kazuhito AKAMA***, Satoshi KOIKE****, Chiaki
MATSUKURA****, and Hiroshi EZURA****
Stimulation of γ-aminobutyric acid (GABA) production under low O2 and high CO2 conditions (adjusted
aerobic atmosphere) at two levels of temperature was studied. Vine-ripened tomato fruits were stored under hypoxic
conditions and adjusted aerobic atmospheres as well as in air at 15°C for 12.6 d and at 25°C for 6.6 d. At 15°C, the
GABA concentrations of tomato fruits during storage under one adjusted aerobic atmosphere (O2 14%, CO2 8%) were
not significantly different from that in air. On the other hand, at 25°C, the tomato fruit GABA concentrations under asecond adjusted aerobic atmosphere (O2 11%, CO2 12%) were significantly higher by 72% than that in air 6.6 d fromthe start of storage. Increased accumulation of alanine under this adjusted aerobic atmosphere supports theobservation that these conditions stimulate GABA production. This temperature is 5°C lower than the temperature(30°C) reported to be required to stimulate GABA production. This result is more significant than that reported in thepast because the shelf-life of fresh produce including tomato fruits can be prolonged by storage at low temperatures.
Keywords : Solanum lycopersicum L., γ-aminobutyric acid, modified atmosphere packaging

1. Introduction
The functional compound γ-aminobutyric acid
(GABA)1-3) is widely distributed throughout thebiological world4). Accumulation of GABA in
horticultural products has been well studied.
Akihiro et al.5) found that a high GABA
concentration in a variety of tomato fruits wascaused by low enzymatic activity of
α-ketoglutarate-dependent GABA transaminase.

Previous reports have shown that GABA
accumulates rapidly in plant tissues exposed to avariety of different stresses4). Stress from anoxia
is reported to be effective for stimulating GABA
production4); however, horticultural products
stored under anoxic or hypoxic conditions
generate off-odors due to ethanol fermentationand lose their commercial value6). Makino et al.7)
reported that an adjusted aerobic atmosphere isalso effective for stimulating GABA production*

The University of Tokyo
Sumitomo Bakelite Co., Ltd.
***
Shimane University****
University of Tsukuba†
Corresponding author, Graduste School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657,
Japan, TEL: +81-3-5841-5361, FAX: +81-3-5841-8174, Email:amakino@mail.ecc.u-tokyo.ac.jp**－ 375 －

Stimulation of γ-Aaminobutyric Acid Production in Vine-ripened Tomato (Solanum lycopersicum L.)
Fruits Under an Adjusted Aerobic Atmospher

in tomato fruits at 30°C as well as under hypoxic
conditions. However, this temperature is rather
high for the storage of fresh produce.
Furthermore, Gautier et al.8) reported suppressionof normal ripening when the temperature wasincreased from 27 to 32°C in experiments using
mature green tomato fruits. Therefore, lowstorage temperature and conditions that stimulate
GABA production are required. In the present
study, we attempted to stimulate GABA
production in tomato fruits at 25°C to reduce
storage temperature by 5°C than the previous
study7) and compared this stimulation with that infruits stored at 15°C.

0.094 m2) were used to create hypoxic conditions
with high CO2 levels around the tomato fruits.
Micro-perforated PET/low-density polyethylene(LDPE) film pouches (O2 permeance: 0.16
mmol·m−2·h−1·kPa−1, film thickness: 47 µm;
surface area: 0.085 m2) were used to contain acontrolled aerobic atmosphere at 15 and 25°C,
respectively. The atmospheric conditions werecreated as previously described10). Macroperforated pouches (the same PET/LDPE film as
the micro-perforated pouch with 5 mm∅ × 8
holes) were used as a control in which thein-package atmosphere was expected to be thesame as that of ambient air.

2. Materials and Methods

2.2 Storage of Tomato Fruits
Two tomato fruits were sealed in a pouchcontaining 10 g C2H4 absorbent (Picón et al.,1993) using an impulse sealer. A total of 72
pouches with C2H4 absorbent were prepared andstored at 15°C for 12.6 d (harvested in
November) and at 25°C for 6.6 d (harvested in
December) in dark constant temperature units.
The temperature of 15°C was the storage
temperature effective for maintaining the qualityof tomato fruits according to Nakhasi et al.11). In
contrast, the storage temperature (25°C) was
selected in an attempt to reduce the temperature
at which GABA production is stimulatedcompared to that of 30°C selected in the previous
study7). Storage periods at each temperature were
subjectively determined and were terminatedwhen the fruit lost its visual commercial value
due to softening12) or growth of fungi13).

2.1 Materials
Vine-ripened
tomatofruits(Solanumlycopersicum L.9), House Momotaro cultivar)
were harvested on November 15 and December
13, 2007, at a farm in Hiratsuka (Kanagawa
Prefecture, Japan). After harvesting, the samples
were maintained at 10–16°C, transported to thelaboratory within 1 d, and stored in a constanttemperature unit (15°C) for another 1 d before
use. The average mass of the 78 pieces of fruit
harvested on November 15 was 0.198 kg (S.D. ±0.012; range 0.175–0.225 kg). The average massof the 78 pieces of fruit harvested on December
13 was 0.196 kg (S.D. ± 0.012; range
0.171–0.226 kg). Intact fruits without any
pretreatments were used for the following
experiments.
Three-layer
laminated(polyethylene
terephthalate (PET)/aluminum/polyethlene) highbarrier pouches Lamizip® (AS ONE Corporation,
Tokyo) (O2 permeance: 1.7 × 10−5 mmol·m−2·h−1·
kPa−1, film thickness: 110 µm; surface area:

2.3 Data Analysis
Changes in the in-package O2 and CO2
concentrations were monitored using the method－ 376 －

J. Pac .Sci. Tech.

No.5（2010）

Vol.19of Makino et al.14).
Changes in amino acid concentrations of all
tomato samples were monitored using the methodof Makino et al.7).
JMP® 8.0 software (SAS Institute Inc., Cary,
NC, USA) was used for statistical analysis. Whenbetween-class variation was significant at P <0.05 using one-way analysis of variance(ANOVA), mean values were compared by theleast significant difference (LSD) test (P < 0.05).

3. Results and Discussion
Gas composition in the macro-perforated
pouch was the same as that in the surrounding air
regardless of temperature. Changes in O2 andCO2 concentrations in the high barrier andmicro-perforated pouches over time are shown in
Fig.1.

In the high barrier pouch at 15°C, the

mean

O2

concentration

decreasedfromapproximately 21% to 5% after 4-d storage, andremained at that level until the end of storage(12.6 d). The mean CO2 concentration increased
from 0% to 13% after 2.2-d storage, andgradually increased to 16% at the end of storage.
At 25°C, mean O2 concentration decreased to 4%

Fig.1 In-package atmosphere changes inpouches at 15 and 25℃ (n = 3). Open andclosed symbols denote micro-perforated film
and three-layer laminated high barrier
pouches, respectively. Circles and triangles
denote in-package O2 and CO2 concentrations,
respectively. Mean ± SE of three observations
are plotted.

after 1.1-d storage and remained at approximately
2% until the end of storage (6.6 d). The CO2
concentration increased to 18% after 1.1-dstorage and continued to increase up to 41% at
the end of storage. In the micro-perforated film
pouches at 15°C, a steady state was reached 2.2 dfrom the start of storage. Mean O2 and CO2
concentrations of 14% and 8%, respectively, weremaintained until the end of storage (12.6 d). At
25°C, a steady state was reached after 1.1-dstorage, and mean O2 and CO2 concentrationsof 11% and 12%, respectively, weremaintained until the end of storage (6.6 d). The
gas composition in the storage packaging was,regardless of storage temperatures, hypoxic in
the high barrier pouch and aerobic in the
micro-perforated film pouch.
Changes in GABA concentrations over time
are shown in Fig.2. At 15°C, the GABA

－ 377 －

Stimulation of γ-Aaminobutyric Acid Production in Vine-ripened Tomato (Solanum lycopersicum L.)
Fruits Under an Adjusted Aerobic Atmospher

between the two studies may be due to adifference in the variety of fruits. However, theobservation that GABA in fruits stored undermodified atmospheres was not significantly
stimulated at 15°C was reproduced in the present
study. These results suggest that hypoxic
conditions are needed to stimulate GABA
production at 15°C. However, hypoxic conditions
causing off-odors due to fermentation are notdesirable for the storage of fresh products. On theother hand, after 6.6-d storage at 25°C, the
GABA concentrations were significantly higher
in the micro-perforated pouches and in the highbarrier pouches than that in the macro-perforated
pouches. Decrease in storage O2 and increase instorage CO2 concentrations are considered topromote GABA production at 25°C and our
results indicate that the effect is influenced bystorage temperature. Makino et al.7) reported thatconcentration was significantly higher in thean adjusted aerobic atmosphere is effective for

Fig.2 Changes in γ-aminobutyric acid in tomato
fruits at 15 and 25℃ (n = 6). Circles, triangles,
and squares denote values under ambient air,hypoxic conditions with high CO2 levels, andadjusted aerobic (low O2 and high CO2
conditions) atmospheres, respectively. Mean ±SE of six observations are plotted. Within thesame time interval, symbols followed by thesame letter are not significantly different (P <0.05; one-way ANOVA with LSD test).

stimulating GABA production in tomato fruits at
30°C. However, this temperature is rather highfor storing fresh produce. Futhermore, Gautier etal.8) reported that suppression of normal ripening
was observed when temperatures increased from
27°C to 32°C in experiments using green mature
tomatofruits.

In

thepresent

study,

wedemonstrated that GABA production in tomato
fruits can be promoted at 25°C. Among theatmospheres tested in the current study, GABA

high barrier pouch than in the micro-and

production in tomato fruits was found to be

macro-perforated pouches after 9.6-d storage.

stimulated the most under hypoxic conditions

7)

According to a previous investigation , nocreated by the high barrier pouches (Fig.2); this issignificant differences were observed among thesimilar to the observations made in a study on

three packaging methods at 15°C. The difference

tea15). However, this atmosphere is not suitable－ 378 －

J. Pac .Sci. Tech.

for storing fresh produce, as mentioned above.

Vol.19No.5（2010）

significantly lower in the high barrier pouch than

Glutamate concentrations were significantlyin the other pouches at 6.6-d storage at 25°C. If

higher in fruits stored in macro-perforated

glutamate, a main substrate in the glutamic acidpouches than in the other pouches after 12.6-ddecarboxylase (GAD) reaction, is consumed tostorage at 15°C and after 3.9-d storage at 25°C

produce GABA, the glutamate concentration(Fig.3).

would decrease with GABA accumulation, as has

Notably,theconcentration

wasbeen demonstrated in radish leaves16). e
The glutamate concentrations after 6.6-dstorage at 25°C were inversely proportional to the
GABA concentrations. This reaction may be
associated

with

the

difference

in

GABA

concentrations among fruits contained in thethree kinds of pouches. However, an increase inglutamate molecules did not correspond with areduction in GABA molecules. Glutamate is
produced from α-ketoglutarate by glutamate andα-ketoglutarate-dependent GABA transaminase
reactions, and is being consumed by the GAD
reaction17).

This

relationship

is

not

fully

understood because of the complex metabolic
pathways involved. Streeter and Thompson16)
suggested that glutamate may be generated by thedecompositionofproteins

inlong-term

incubation. Therefore, they incubated trimmed
radish leaves for 6 h at room temperature anddetected a decrease in glutamate with an increase
Fig.3 Changes in glutamate in tomato fruits at
15 and 25℃ (n = 6). Circles, triangles, andsquares denote values under ambient air,hypoxic conditions with high CO2 levels, andadjusted aerobic (low O2 and high CO2
conditions) atmospheres, respectively. Mean ±SE of six observations are plotted. Within thesame time interval, symbols followed by thesame letter are not significantly different (P >0.05; one-way ANOVA with LSD test).

in GABA. In the current study, intact tomato
fruits were stored for 6.6 d at 25°C and for 12.6 d
at

15°C.

Liu

and

Luh18)

reportedthat

concentrations of GABA and glutamate in tomato
paste from harvest-delayed fruit were elevated
due to delayed harvest. Differences in sample
storage conditions appear to be the cause ofdifferences in the glutamate data described above.

－ 379 －

Alanine concentration was significantly lower

Stimulation of γ-Aaminobutyric Acid Production in Vine-ripened Tomato (Solanum lycopersicum L.)
Fruits Under an Adjusted Aerobic Atmospher

in the macro-perforated pouch than in the otherpouches at 9.6-d storage at 15°C (Fig.4). This
result reflects those of Streeter and Thompson16)
who

reported

accumulated

that
in

alanineradishand

GABA

leavesunder

anoxic/hypoxic conditions. At 25°C, alanine
concentration was significantly higher in the
micro-perforated pouch than that in the otherpouches after 3.9-d storage (Fig.4). Makino etal.7) reported that alanine production is morestimulated under an adjusted aerobic atmospherethan that under hypoxic conditions at 30°C andthis was shown at 25°C in the present study.
Miyashita et al.19) reported that O2 stresspromoted the decomposition of accumulated
alanine-by-alanine aminotransferase (AlaAT) (EC2.6.1.2) in Arabidopsis (Arabidopsis thaliana (L.)
Heynh.).

This

suggests

that

alaninemay

decompose under hypoxic conditions throughstrong O2 stress and may be maintained underFig.4 Changes in alanine in tomato fruits at 15
and 25 ℃ (n = 6). Circles, triangles, andsquares denote values under ambient air,hypoxic conditions with high CO2 levels, andadjusted aerobic (low O2 and high CO2
conditions) atmospheres, respectively. Mean ±SE of six observations are plotted. Within thesame time interval, symbols followed by thesame letter are not significantly different (P >0.05; one-way ANOVA with LSD test).

adjusted aerobic atmospheres through weak O2stress. The accumulation of alanine in tomato
fruits under the adjusted aerobic atmosphere
supports

theobservationthatatmosphericconditions stimulate GABA production16). At
15°C,

the

alanineconcentration

was

not

significantly different among three atmospheres,apart from at 9.6 d. This suggests that dynamicchanges in alanine concentration are affected by
the storage temperature as well as GABA
concentration.
At 25°C, GABA production in vine-ripened
tomato fruits under low O2 and high CO2
conditions (adjusted aerobic atmosphere) was
significantly more stimulated than that in air. The

accumulation of alanine under the adjusted
aerobic atmosphere supports the observationthat this atmosphere type is effective forstimulating GABA production. The atmosphereand temperature conditions may be applied tothe storage and distribution of tomato fruits to－ 380 －

J. Pac .Sci. Tech.

produce high GABA concentrations,enhancing the value of the fruit.

thus

4. Acknowledgment
This study was financially supported by aGrant-in-Aid
for
Scientific
Research(No.19380140 and 21380155) from the Japan
Society for the Promotion of Science and the
Matsushima
Horticultural
Development
Foundation.

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（Received 1 June 2010）（Accepted 12 August 2010）