L-Galactono-γ-lactone dehydrogenase activity and vitamin C content in fresh-cut potatoes stored under controlled atmospheres

Article abstract of DOI:10.1021/jf030071s

L-Galactono-γ-lactone dehydrogenase (GLDH) activity and vitamin C content as ascorbic acid (AA) plus dehydroascorbic acid (DHA) were evaluated in five potato tuber cultivars (Agata, Altesse, Franceline, Manon, and Monalisa). The effect of fresh-cutting and subsequent refrigerated storage of Manon potato under different atmospheres (air, 20% CO₂ + air, 100% N₂, and vacuum packaging) on GLDH activity and vitamin C content was also determined. GLDH from the five potato tuber cultivars showed typical inhibition kinetics by high substrate concentration in the synthesis of AA from its physiological precursor L-galactonic acid-γ-lactone (GL). GLDH activity was not correlated with the corresponding vitamin C content in any potato tuber cultivar. GLDH from all the cultivars presented a major isoform with isoelectric point (IEP) 5, which changed to IEP = 4.3 after minimal processing. In addition, the GLDH-catalyzed synthesis of AA by the new isoform showed typical Michaelis kinetics, in which the enzyme became more efficient to catalyze the reaction. Whether the change in the isoform pattern was due to either post-translational modifications or de novo synthesis of a new isoenzyme remains unanswered. Fresh-cutting increased GLDH activity from 4.7-fold (vacuum packaging) to 11-fold (air) after 6 days. In addition, 100% of vitamin C content was retained in air and decreased in the rest of atmospheres after this storage period, following the sequence vacuum packaging (89%) > 100% N₂ (78%) > 20% CO₂ + air (63%). This tendency was correlated with the corresponding GLDH activity detected in each storage atmosphere, except in the case of 20% CO₂ + air. Vacuum packaging proved to be the best storage condition, because fresh-cut potatoes did not turn brown and retained 89% of initial vitamin C content.

Full text of DOI:10.1021/jf030071s

4296 J. Agric. Food Chem. 2003, 51, 4296−4302
L-Galactono-γ-Lactone Dehydrogenase Activity and Vitamin C
Content in Fresh-Cut Potatoes Stored under Controlled
Atmospheres
JUAN ANTONIO TUDELA,† JOSEÄ ANTONIO HERNAÄ NDEZ,‡ MAR´ıA ISABEL GIL,† AND
JUAN CARLOS ESP´ıN*,†
Research Group on Quality, Safety, and Bioactivity of Plant Foods,
Department of Food Science and Technology and Department of Plant Nutrition and Physiology,
CEBAS-CSIC. P.O. Box 164.30100 Espinardo, Murcia. Spain. Fax: 34-968-39 62 13
L-Galactono-γ-lactone dehydrogenase (GLDH) activity and vitamin C content as ascorbic acid (AA)
plus dehydroascorbic acid (DHA) were evaluated in five potato tuber cultivars (Agata, Altesse,
Franceline, Manon, and Monalisa). The effect of fresh-cutting and subsequent refrigerated storage
of Manon potato under different atmospheres (air, 20% CO2 + air, 100% N2, and vacuum packaging)
on GLDH activity and vitamin C content was also determined. GLDH from the five potato tuber cultivars
showed typical inhibition kinetics by high substrate concentration in the synthesis of AA from its
physiological precursor L-galactonic acid-γ-lactone (GL). GLDH activity was not correlated with the
corresponding vitamin C content in any potato tuber cultivar. GLDH from all the cultivars presented
a major isoform with isoelectric point (IEP) 5, which changed to IEP ) 4.3 after minimal processing.
In addition, the GLDH-catalyzed synthesis of AA by the new isoform showed typical Michaelis kinetics,
in which the enzyme became more efficient to catalyze the reaction. Whether the change in the
isoform pattern was due to either post-translational modifications or de novo synthesis of a new
isoenzyme remains unanswered. Fresh-cutting increased GLDH activity from 4.7-fold (vacuum
packaging) to 11-fold (air) after 6 days. In addition, 100% of vitamin C content was retained in air
and decreased in the rest of atmospheres after this storage period, following the sequence vacuum
packaging (89%) > 100% N2 (78%) > 20% CO2 + air (63%). This tendency was correlated with the
corresponding GLDH activity detected in each storage atmosphere, except in the case of 20% CO2
+ air. Vacuum packaging proved to be the best storage condition, because fresh-cut potatoes did
not turn brown and retained 89% of initial vitamin C content.
KEYWORDS: L-Galactono-γ-lactone dehydrogenase (GLDH); fresh-cut potato; ascorbic acid; vitamin C;
enzyme kinetics; controlled atmosphere; minimal processing; vacuum packaging
INTRODUCTION
important role in growth and metabolism and acts as a free
radical scavenger (3-5). Ascorbate is oxidized by ROS to yield
monodehydroascorbate, which forms dehydroascorbic acid
(DHA). DHA also exhibits biological activity, because it can
be readily converted into AA in the human body (6).
Reactive oxygen species (ROS) cause oxidative stress, which
alters cellular structures and cell death processes in humans,
leading to aging and a number of pathologies, including cancer
as well as cardiovascular and neurodegenerative diseases (1).
Therefore, there is great interest in the health-promoting effects
of antioxidants that prevent oxidative stress by scavenging ROS
(2).
The main human dietary antioxidants are vitamins E and C,
phenolic compounds, and carotenoids. Ascorbic acid (AA) is
the most active form of vitamin C, and it is quantitatively the
predominant antioxidant in plant cells, where it plays an
Potatoes play an important role in the diet, because it is the
most consumed vegetable in many countries (7). Despite their
moderate AA content (10-30 mg/100 g fresh weight), potatoes
are a major source of AA in the Western diet, because of the
large amounts consumed (8).
There is increasing consumer demand for fresh-cut fruits and
vegetables, including fresh-cut potatoes, as a source of vitamins
and antioxidants. However, controls during fresh-cut processing
are essential, because antioxidants such as phenolics and AA
are affected when bruising, trimming, and cutting of fruits and
vegetables occurs (9, 10). Therefore, research should focus on
† Department of Food Science and Technology.
* To whom correspondence should be addressed. E-mail: jcespin@
cebas.csic.es.
‡ Department of Plant Nutrition and Physiology.
10.1021/jf030071s CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/13/2003

GLDH Activity and Vitamin C in Potatoes
J. Agric. Food Chem., Vol. 51, No. 15, 2003 4297
Processing and Storage Atmospheres. Potatoes were hand-peeled
and washed with running water. After peeling, the tubers were sorted
for absence of visual defects and uniform color. Potatoes were cut in
8 × 8 mm2 strips with a manual potato cutter (Sammic CF-4, Azpeitia,
Spain) at room temperature. The strips were washed again with running
water and then strained in a draining board to remove surface water.
Uniform strips were selected and broken pieces discarded. No post-
harvest chemical washing treatment was applied. One hundred grams
of strips were selected at random from the whole bunch of fresh-cut
potato strips as one replicate. Three replicates were used for each
treatment and sampling date. Four different storage atmospheres were
studied: storage in air (AIR), controlled atmosphere of 20% CO2 +
air (CA 20% CO2), controlled atmosphere of 100% N2 (CA 100% N2)
and vacuum packaging (VAC).
the optimal maintenance of both nutritional and sensorial
qualities of fresh-cut products. In this context, a recent study
has reported the retention of vitamin C in fresh-cut potatoes
stored in air, whereas vitamin C content decreased in fresh-cut
potatoes stored under modified atmosphere packaging (10). This
work demonstrated the biosynthesis and accumulation of vitamin
C when fresh-cut potatoes were stored in various atmospheres,
and the authors suggested that this phenomena could be related
to changes in the enzyme activity of L-galactono-γ-lactone
dehydrogenase (GLDH, EC 1.3.2.3), which catalyzes the final
step of AA biosynthesis (11, 12).
Despite the importance of AA in both plant and human
physiology, the metabolic pathway leading to AA biosynthesis
is not yet fully understood. In fact, the last step in the
biosynthesis pathway has been recently elucidated in plants,
involving the conversion of L-galactonic acid-γ-lactone (GL)
to ascorbic acid in a GLDH-catalyzed reaction (4, 13). GLDH
is bound to the inner mitochondrial membrane (11, 12, 14) and
is highly specific for the AA precursor GL. This enzyme has
been previously studied in a few sources, such as white and
sweet potato (11, 15), spinach (12), cauliflower (16), sweet
pepper (17), and kidney bean (14). However, there are no
previous studies concerning the effect of minimal processing
(cutting and cold storage in different atmospheres) on this
enzyme and the possible relationship with AA content.
For AIR, CA 20% CO2, and CA 100% N2 atmospheres, fresh-cut
potato strips were placed in 250-mL jars as one replicate. A continuous
humidified flow at a rate of 15 mL/min was obtained by using flow
boards with needle valves. Changes in O2 and CO2 concentrations in
AIR, CA 20% CO2, and CA 100% N2 were monitored daily, using a
gas chromatograph (Perkin-Elmer autosystem, Connecticut) equipped
with a thermal conductivity detector.
For VAC, multilayer film bags (BB4L Cryovac, Sealed Air S. L.,
Sant Boi de Llobregat, Spain) were used. The film characteristics were
as follows: 59-µm thickness and permeance at 23 °C and 0% RH of
1.4 × 10-13 mol ‚ s-1 ‚ m-2 ‚ Pa-1 for O2 and 7.1 × 10-13 mol ‚ s-1
‚ m-2 ‚ Pa-1 for CO2. The film physical properties are similar to the
polymers used in the fresh-cut potato industry. A gas exchange device
with a vacuum packaging machine (Zermat, Carburos Meta´licos S. A.,
Madrid, Spain) was utilized. Vacuum packaging was carried out by
exclusion of air from the bags.
Therefore, the aim of the present work is to study the kinetic
characterization of the enzyme GLDH in five potato cultivars
(Agata, Altesse, Franceline, Manon, and Monalisa) and its
correlation with initial AA content in potato tuber. In addition,
GLDH activity, vitamin C content, and browning severity were
evaluated in the cultivar Manon upon cutting and cold storage
in different controlled atmospheres (i.e., air (AIR), high CO2
concentration (20% CO2 + AIR), 100% N2, and vacuum
packaging (VAC)).
Sensory Evaluation. Browning of fresh-cut potatoes was evaluated
immediately after cutting and after 1, 2, 3, 4, 5, and 6 day intervals by
a panel of five judges. Evaluation was scored on a 3-point scale (1 )
no browning; 2 ) moderate; 3 ) severe).
Ascorbate Extracts and HPLC Analysis. AA and DHA contents
were determined by the method of Zapata and Dufour (18), with some
modifications previously reported by Tudela et al. (10).
Ascorbate content was determined in all the potato tuber cultivars
(hereafter termed as “initial AA”). In addition, ascorbate was also
evaluated daily, during six storage days, for Manon cultivar. Ascorbate
analysis was performed in triplicate. Mean values ( standard deviation
are shown.
MATERIALS AND METHODS
Reagents. AA, bovine serum albumin (BSA), citric acid, L-cysteine,
cytochrome C (Cyt c), dehydroascorbic acid (DHA), GL, 1,2-phe-
nylenediamine dihydrochloride (OPDA), ethylenediaminetetraacetic
disodium salt (EDTA), mannitol, 3-[N-morpholino] propanesulfonic
acid (MOPS), sodium cyanide (NaCN), sodium fluoride (NaF), nitro
blue tetrazolium chloride (NBT; 3,3′-(3,3′-dimethoxy-4,4′-biphenylene)-
bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride]), Triton X-100
(TX-100) were purchased from Sigma-Aldrich (St. Louis, MO). All
other reagents were of analytical grade and supplied by Merck
(Darmstadt, Germany). Milli-Q system ultrapure water (Millipore Corp.,
USA) was used throughout this research.
Preparation of Mitochondria-Enriched Potato Extracts. The
extracts were obtained according to the method of Struglics et al. (19),
with some modifications. Fifty grams of potato were added to 50 mL
of extraction medium (0.9 M mannitol, 30 mM MOPS, 3mM EDTA,
25 mM L-cysteine, and 0.3% (w/v) BSA). The pH was adjusted to 7.3
by dropwise addition of 8 M KOH. The mixture was homogenized
with five consecutive pulses for 3 s in an Osterizer blender (Sunbeam,
Delray Beach, FL) and further filtered through nylon gauze (120-µm
mesh). The homogenate was left standing for 5 min, allowing the starch
to sediment, and then centrifuged at 1600g for 15 min in a Beckman
J2-21 centrifuge (Altberville, MN), using a Beckman JA-14 rotor. The
supernatant was decanted and centrifuged at 12800g for 25 min. The
pellets were redissolved in 30 mL of washing medium (0.3 M mannitol,
10 mM MOPS, 1mM EDTA, and 0.1% (w/v) BSA, pH 7.2) and
centrifuged at 17400g for 10 min, using a Beckman JA-20 rotor. Every
final pellet (8 pellets) enriched in mitochondria was redissolved in 300
µL of washing medium without BSA (0.3 M mannitol, 10 mM MOPS,
and 1 mM EDTA). This means that every extraction assay yielded a
total volume of 2.4 mL of mitochondrial-enriched extract.
Plant Material. Agata, Altesse, Franceline, Manon, and Monalisa
potatoes were selected because of their availability throughout the
present experiment. These potatoes were used for the screening of
cultivars (initial GLDH activity and AA content). The Manon cultivar
was selected to evaluate the effect of fresh-cutting and storage under
controlled atmospheres on both GLDH activity and vitamin C content.
Altesse tubers were harvested at the middle of August, while Agata,
Franceline, Manon, and Monalisa were harvested in September-October.
After harvest, all potatoes were stored at 7-8 °C and 90% relative
humidity (RH). Tubers were supplied by Agroinnova S. L. (Tordesillas,
Spain) and transported by car to the laboratory in less than 12 h, where
those with defects (cuts and bruises) were discarded. At the laboratory,
sound tubers were kept at 4 °C and 70% RH in darkness for 2 days
before processing. Each potato cultivar assayed here included a complete
certificate of “traceability” which specified all the aspects and factors
concerning the cultivar: agronomic and climatic conditions (soil, type
and doses of fertilization, irrigation, pesticides, herbicides, temperature,
etc.), analysis at harvest (starch, dry matter, nitrates, etc.), culinary tests
(frying, cooking, etc.), size, etc.
The extraction protocol was repeated three times for each cultivar.
In addition, the protocol was also repeated three times for each treatment
and storage day, in the case of the Manon cultivar, to evaluate the
effect of cutting and storage conditions on GLDH activity.
GLDH Assay. GLDH activity was measured at 25 °C, according to
the method of Imai et al. (20), with minor modifications. NaCN was
included to prevent reoxidation of reduced Cyt c by the Cyt c oxidase
present in the extract. Cyt c is used as a coupled reagent to determine

4298 J. Agric. Food Chem., Vol. 51, No. 15, 2003
Tudela et al.
GLDH activity. The precursor GL is converted to ascorbic acid, which
reduces Cyt c (20), with the subsequent change in Cyt c spectrum and
corresponding absorbance (reduced Cyt c has a maximum at 550 nm).
No effect of cyanide was observed on GLDH activity, in accordance
with previous reports (15). The enzyme preparation with GLDH activity
was constituted by 200 µL of the mitochondria-enriched extract and
200 µL of a solution containing 0.2% TX-100 and 50 mM sodium
phosphate buffer (PB) pH 8 (solution A). After vigorous shaking, the
enzyme preparation was left for 2 h at 4 °C until its assay, to release
the enzyme from the mitochondrial inner membrane. The standard
reaction mixture for determining GLDH activity contained 60 µM Cyt
c, 25 mM PB pH 8.0, 1 mM GL, 60 µM NaCN, and 20 µL of enzyme
preparation (6 µg protein) in a total volume of 1 mL. GLDH activity
was quantified as the Cyt c reduction rate at 550 nm (ꢀ550 nm ) 29 500
M-1cm-1) (21). The linear increase of absorbance at 550 nm was
recorded immediately after the addition of the substrate GL. The linear
regression of the spectrophotometric recording rendered GLDH activity.
No activity was detected in the absence of GL as well as without
previous incubation with solution (A). One unit of enzyme activity was
defined as the amount of extract required to oxidize 1 µmol GL
(equivalent to the formation of 2 µmol reduced Cyt c) per min. GLDH
activity was measured in freshly prepared extracts. It should be stressed
that GLDH activity was lost after 3 days in both fresh (4 °C) and frozen
(-20 °C) extracts (with or without TX-100 treatment), which should
be taken into account in the characterization of this enzyme.
Figure 1. High substrate inhibition kinetics of GLDH from potato tuber.
The reaction mixture contained 60 µM Cyt c, 25 mM PB pH 8.0, 60 µM
NaCN, 20 µL of enzyme preparation (6 µg protein), and increasing GL
concentrations from 0.12 to 6 mM at 25 °C. Total assay volume was 1
mL. The vertical lane indicates the average value of standard deviations
(SD). Symbols designate the experimental data and solid lines the
nonlinear regression fitting of experimental data to eq 1 (see Materials
and Methods for details).
GLDH activity from potato tuber (i.e., before cutting) is hereafter
termed as “initial” GLDH activity.
The spectrophotometric assays for determining GLDH activity were
recorded in a UV-1603 Shimadzu spectrophotometer (Tokyo, Japan).
Temperature was controlled at 25 °C with a temperature controller (CPS
240A Shimadzu), checked using a precision of (0.1 °C.
RESULTS AND DISCUSSION
Kinetic Data Analysis. The apparent Vm (maximum rate), Km
(Michaelis constant), and KSI (substrate inhibition constant) values were
calculated from triplicate measurements of the rate for each initial
substrate (GL) concentration, using three different extracts of each
potato cultivar. The mean value of each kinetic constant is shown.
GLDH Activity and Vitamin C Content in Potato Tubers.
GLDH activity on increasing GL concentrations revealed typical
inhibition kinetics by high substrate concentration in the five
potato cultivars assayed, which was in accordance with previous
reports (Figure 1) (11, 16). However, inhibition by high
substrate concentration has been reported to greatly depend on
the plant source. For instance, GLDH from cauliflower required
32 mM GL to show substrate inhibition (16), whereas GLDH
from potato required much lower GL concentrations, as shown
in the present study and other reports as well (11). Nonlinear
regression analysis of experimental data from Figure 1 to eq 1
revealed that kinetic constants of the GLDH-catalyzed reaction
also varied depending on the potato cultivar (Table 1). The
greatest variation was observed for KSI values, which ranged
from 0.95 (Franceline potato) to 6.81 mM (Altesse potato). Km
values ranged from 0.23 (Monalisa potato) to 1.09 mM
(Franceline potato). These values are in the range of those
previously reported for GLDH from white potato (11).
In addition, it should be noted that GLDH activity from potato
was lost after 3 days in both fresh (4 °C) and frozen (-20 °C)
extracts. This behavior was in accordance with that reported
for the stability of GLDH from spinach mitochondria, which
became inactive after 15 h at 4 °C in 100 mM Tris-HCl (pH
8.0) containing 0.25 M sucrose (12).
The initial GLDH activity was compared with the corre-
sponding initial AA content in each variety (Table 1) in order
to investigate a possible simple correlation. However, a non-
simple relationship was found between initial AA content and
any kinetic constant that characterized GLDH activity from
potato tuber (i.e., AA vs Vm, AA vs Km, AA vs KSI, and AA vs
Vm/Km) (results not shown). Therefore, initial AA content of
potato tuber cannot be simply correlated to initial GLDH
activity; other enzymes (phosphoglucose isomerase, phospho-
mannose isomerase, galactose dehydrogenase, etc.), and/or
substrate precursors (fructose-6-phosphate, galactose, mannose-
6-phosphate, GL) belonging to the AA biosynthesis pathway
Substrate inhibition constants (KSI) were calculated by using the
equation
Vm [S]
V )
(1)
S
KSI
Km + [S] 1 +
(
)
(22), where [S] is the GLDH substrate (GL) concentration.
Nonlinear regression fittings were carried out by using the Mar-
quardt-Levenberg algorithm (23), implemented in the Sigma Plot 6.0
program for Windows (SPSS Science, Chicago, USA). Coefficient of
variation (CV ) (mean/SD) * 100) was always less than 10% in the
determination of the above kinetic values.
Protein Assay. Protein concentrations were determined by the
method of Bradford (24) using bovine serum albumin as a standard.
Isoelectric Focusing (IEF) Experiments. Enzymatic extracts were
obtained as described above. A sample of 1 mL of extract was mixed
with 1 mL of solution A and left for 2 h at 4 °C, to release the enzyme
from the membrane. Afterward, the mixture was concentrated by using
Microcon YM-10 filter membrane (Millipore Corporation, USA), and
4 µL of the concentrated sample were applied to the gel. Isoelectric
points (IEP) of GLDH were determined in the electrophoresis unit Phast
System (Pharmacia, Sweden). Isoelectric focusing gels (PhastGel from
Pharmacia) with IEP range of 3-9 were used. Conditions for running
IEF experiments were those suggested by the manufacturer. Broad IEP
(3-10) kit markers from Pharmacia were used.
The gels were rinsed with 15 mM PB pH 8 and further incubated
with a mixture containing 150 µM NaCN, 15 mM PB pH 8, 1 NBT
mM, and 1 mM GL, to develop enzyme activity. GHLD activity was
visualized by the appearance of blue bands in the gel. No bands were
detected in the gels in the absence of the substrate GL. Stained gels
were immediately scanned. Some faint bands were lost during image
processing.

GLDH Activity and Vitamin C in Potatoes
J. Agric. Food Chem., Vol. 51, No. 15, 2003 4299
a
b
Table 1. Kinetic Constants of GLDH and Vitamin C Content (AA+DHA) in Five Potato Tuber Cultivars (before cutting)
AA
DHA
(mg/100 g fw)
vitamin C
(mg/100 g fw)
cultivar
Vm (µM/min)
Km (mM)
KSI (mM)
Vm/Km (min-1
)
(mg/100 g fw)
Manon
Agata
Monalisa
Franceline
Altesse
1.50 ± 0.08
1.85 ± 0.12
1.71 ± 0.11
3.86 ± 0.25
0.57 ± 0.03
0.41 ± 0.03
0.60 ± 0.04
0.23 ± 0.01
1.09 ± 0.05
0.42 ± 0.03
1.57 ± 0.13
1.63 ± 0.15
4.47 ± 0.41
0.95 ± 0.08
6.81 ± 0.51
3.6 × 10-3 ± 3.5 × 10-4
3.1 × 10-3 ± 2.9 × 10-4
7.4 × 10-3 ± 6.8 × 10-4
3.5 × 10-3 ± 2.8 × 10-4
1.3 × 10-3 ± 1.1 × 10-4
16.4 ± 0.7
6.1 ± 0.3
9.4 ± 0.3
8.1 ± 0.3
7.5 ± 0.2
3.2 ± 0.4
1.6 ± 0.2
3.2 ± 0.2
2.2 ± 0.2
1.8 ± 021
19.6 ± 2.5
7.7 ± 1.1
12.6 ± 0.9
10.3 ± 1.1
9.3 ± 1.1
a GLDH assay conditions are specified in Materials and Methods. Assay mixture included 60 µM Cyt c, 25 mM PB pH 8.0, 1 mM GL, 60 µM NaCN, and 20 µL of
enzyme preparation (6 µg protein). b AA and DHA contents were determined by the method of Zapata and Dufour (18), with some modifications previously reported by
Tudela et al. (10).
together with enzymes that regenerate ascorbic acid, such as
dehydroascorbate reductase (DHAR) and/or monodehydroascor-
bate reductase (MDHAR), must be critically involved.
Effect of Fresh-Cutting and Storage Atmospheres on
Vitamin C Content, GLDH Activity, and Browning. The
potato cultivar Manon was chosen to investigate the effect of
minimal processing and subsequent storage of potato strips in
different controlled atmospheres (AIR, CA 20% CO2, CA 100%
N2, and VAC) on vitamin C content and GLDH activity together
with the influence on browning degree. This cultivar was chosen
due to the combination of its intermediate GLDH activity and
higher vitamin C content than the other cultivars (Table 1).
Vitamin C Content and Browning. Potato strips retained their
vitamin C content after 6 days of storage in AIR at 4 °C (Figure
2), in accordance with a recent report (10). Although AIR
storage is an interesting approach to monitor the overall process
and even to preserve vitamin C content, obviously it is not a
feasible way to keep potato strip quality, due to the severe
browning development (Figure 3). Browning did not have
relevant influence on vitamin C content in the present study, in
agreement with a recent report which stressed the lack of
correlation between browning development and vitamin C
content (25). After 6 days of storage, vitamin C decreased in
the rest of atmospheres following the sequence VAC (89%
retention) > CA 100% N2 (78%) > CA 20% CO2 (63%)
(Figure 2). The vitamin C content was approximately similar
to that of AA in all treatments, because the quantitative variation
of DHA after 6 days was minimal (Figure 2). According to
our results, VAC packaging could be recommended for storing
fresh-cut potatoes in the attempt to preserve both vitamin C
content and browning degree (Figures 2 and 3). On the other
hand, the storage of fresh-cut potatoes at high CO2 atmosphere
(CA 20% CO2) yielded both the highest loss of vitamin C
content and severe browning degree after 4 days (Figures 2
and 3).
GLDH ActiVity. GLDH activity of fresh-cut potatoes stored
in different atmospheres was evaluated during the 6 day period
to give a likely explanation to the observed vitamin C biosyn-
thesis. Fresh-cutting provoked an increase of GLDH activity in
potato strips in all the atmospheres reaching the maximum
approximately at the fourth day of storage (Figure 4). The
highest induction was observed in the strips stored in AIR (15.5-
fold induction) and the lowest increase when stored in CA 20%
CO2 (6.7-fold induction). This meant that the overall tendency
concerning the variation of vitamin C content during the 6 days
could be explained, at least partially, by the corresponding
GLDH activity (Figures 2 and 4).
Figure 2. Influence of the atmosphere composition on the ascorbic acid
(AA), dehydroascorbic acid (DHA) and vitamin C content during 6 days
of storage at 4 °C of fresh-cut Manon potato strips.
activity was the same in the endosperm and only slightly
increased in the embryos. These results suggested that the
increase of AA was mainly due to the novo synthesis, rather
than to the recycling of the oxidized ascorbate (26). Therefore,
these observations agree with our results and seems that the
observed vitamin C retention after storage could be related with
the induction of GLDH activity, and the increase in this enzyme
could indicate high AA requirements during the storage period
of fresh-cut potatoes. However, the full corroboration of this
hypothesis should involve the determination of both DHAR and
It has been previously described that GLDH activity and AA
content increased in both embryos and endosperm during the
germination of Pinus pinea seeds. However, DHAR activity
progressively decreased during germination, whereas MDHAR

4300 J. Agric. Food Chem., Vol. 51, No. 15, 2003
Tudela et al.
Figure 3. Effect of atmosphere composition on the degree of browning
of fresh-cut Manon potato strips stored during 6 days at 4 °C. The scale
ranged from 1 (no browning) to 3 (severe browning).
Figure 5. (A) Evolution of GLDH kinetics upon storing fresh-cut Manon
potato in AIR during 6 days at 4 °C. (9) Initial GLDH activity in Manon
potato tuber (before processing, day 0). Assay conditions were the same
as those detailed in Figure 4. Symbols designate experimental data and
solid lines the nonlinear regression fitting of experimental data to eq 1 (in
the case of potato tuber, 9) and to Michaelis equation (v ) Vm [S]/(Km
+ [S]) in Manon potato strips. (B) Evolution of Vm and Km values (obtained
from nonlinear regression analysis of Figure 5A) upon storing Manon
potato strips in AIR during 6 days at 4 °C. (C) Evolution of GLDH catalytic
power (Vm / Km) (obtained from nonlinear regression analysis of Figure
5A) upon storing Manon potato strips in AIR during 6 days at 4 °C.
Figure 4. GLDH activity evolution of Manon potato strips stored during 6
days at 4 °C under different atmospheres. The reaction mixture contained
60 µM Cyt c, 25 mM PB pH 8.0, 60 µM NaCN, 1 mM GL, and 20 µL of
enzyme preparation (6 µg protein) at 25 °C. Total assay volume was 1
mL.
MDHAR activities, which was not approached in the present
study.
The highest AA retention (AA content at day 6 minus initial
AA content) corresponded to the highest GLDH activity in AIR
(GLDH activity at day 6 minus initial GLDH activity). On the
other hand, the highest loss of AA was observed in the potato
strips stored in CA 20% CO2, which presented low GLDH
activity (Figures 2 and 4). However, the daily variation of
GLDH activity was not simply correlated with the daily
evolution of AA, as demonstrated by the low correlation values
(R) and high P values obtained (results not shown).
To deepen in the effect of fresh-cutting on GLDH, the kinetic
behavior of this enzyme was evaluated every day in those
potatoes strips stored in AIR. Storage in AIR was chosen
because the GLDH activity was higher than that in the other
atmospheres. Figure 5A shows the variation of GLDH kinetics
versus days of storage. It should be stressed that the substrate
inhibition kinetics observed in potato tubers (Figures 1 and 5A,
day 0) changed to typical Michaelis kinetics, in which the
inhibition by high substrate concentration disappeared (Figure
5A). To our knowledge, the change of GLDH kinetics as
strategy to face wounding stress has not been previously
published. In addition, apparent Vm values increased more than
7-fold after 6 days of storage, whereas apparent Km values
decreased 2-fold the first day of storage after cutting and
remained constant until day 6 (Figure 5B). The overall
conclusion of this change is that the enzyme GLDH became
more efficient to catalyze the conversion of GL to AA thanks
to the 14-fold increase of the catalytic power (Vm/Km) after 6
days of storage and the absence of inhibition by high substrate
concentration from the first day after cutting (Figure 5). This
could be related to the requirement of more antioxidant power
by plant cells (by synthesizing AA) to face the wounding-
generated stress by minimal processing (17, 27). In addition,
although other factors seem to be involved, this induction
process was enhanced by the presence of oxygen and inhibited
by high CO2 concentration (Figure 4), which in turn, could be
related to the lowest vitamin C retention observed in the strips
stored under this atmosphere (Figure 2).
IEF experiments revealed that GLDH presented one major
isoenzyme or isoform with isoelectric point 5 (IEP ) 5) in all
potato tuber cultivars before the processing (results not shown
for all the cultivars, Figure 6 A,B in Manon cultivar). The IEP

GLDH Activity and Vitamin C in Potatoes
J. Agric. Food Chem., Vol. 51, No. 15, 2003 4301
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ACKNOWLEDGMENT
The authors are grateful to Agroinnova S.L. for providing the
potato tubers.

4302 J. Agric. Food Chem., Vol. 51, No. 15, 2003
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Received for review January 29, 2003. Accepted May 1, 2003. This work
has been partially supported by a grant from CICYT, Spain, project
PTR 1995-0625OP. J. A. T. has a fellowship from Ministerio de
Educacio´n, Cultura y Deporte (Spain), reference AP2001-2493.
JF030071S