Journal of Food Engineering 66 (2005) 187–192
www.elsevier.com/locate/jfoodeng
Study of lipoxygenase and peroxidase as indicator enzymes
in green beans: change of enzyme activity, ascorbic acid
and chlorophylls during frozen storage
K. Savasß Bahcßeci, Arda Serpen, Vural G€
okmen *, Jale Acar
Department of Food Engineering, Hacettepe University, 06532 Beytepe, Ankara, Turkey
Received 24 October 2003; accepted 1 March 2004
Abstract
Effects of two blanching conditions using peroxidase (POD) and lipoxygenase (LOX) as indicator enzyme on residual enzyme
activities, ascorbic acid and chlorophyll content in green beans during frozen storage were studied. No reactivation of both LOX
and POD enzyme was observed during storage. The losses of ascorbic acid and chlorophylls during storage followed first order
kinetics. Half-life of ascorbic acid in unblanched green beans was determined to be 1.89 months. It increased to 2.15 and 3.48
months by blanching at 70 C for 2 min (for >90% LOX inactivation) and 90 C for 3 min (for >90% POD inactivation),
respectively. Half-lifes of chlorophyll a (Chl a) and chlorophyll b (Chl b) were determined to be 7.32 and 13.11 months in unblanched green beans. Blanching green beans at 70 C for 2 min decreased the half-lifes of Chl a and Chl b to 5.05 and 10.09 months
while blanching at 90 C for 3 min increased to 8.26 and 16.70 months, respectively. The results clearly showed that a blanching
treatment to inactivate POD retains the quality attributes of green bean better during frozen storage.
2004 Elsevier Ltd. All rights reserved.
Keywords: Peroxidase; Lipoxygenase; Blanching indicator; Frozen storage; Ascorbic acid; Chlorophylls
1. Introduction
Freezing is used to maintain product quality over
long storage and results in a slower rate of most deteriorative reactions such as senescence, enzymatic decay,
chemical decay and microbial growth. However, freezing does not prevent off-flavor development, color and
texture deterioration in frozen vegetables because enzyme systems remain active even at sub-zero temperatures (Rodriguez-Saona, Barrett, & Selivonchick, 1995).
In order to prevent enzymatic reactions during processing, most vegetables must first be blanched.
Blanching can be carried out by different methods, but
water blanching is most widely used techniques for this
purpose.
Blanching is a thermal process designed to inactivate
the enzymes responsible for generating the off-flavors
and off-odors. Apart from enzyme inactivation,
blanching of vegetables prior to freezing has several
advantages, but also a number of disadvantages. The
*
Corresponding author. Fax: +90-312-2992-123.
E-mail address: [email protected] (V. G€
okmen).
0260-8774/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2004.03.004
advantages include stabilization of texture, flavor and
nutritional quality, destruction of microorganisms and
wilting of leafy vegetables, which assists in packaging
(Cano, 1996). However, since blanching is a heat treatment, changes associated with mild thermal processing
can be expected. These include loss of turgor in cells, due
to thermal destruction of membrane integrity, and partial degradation of cell wall polymers. A further effect of
thermal processing is the degradation of chromophores
such as chlorophyll, resulting in color change. Pigment
degradation will continue to take place through frozen
storage. The extent of the color change and the rate of
the reactions could be affected by the severity of heat
treatment. For example, the greater the extent of conversion of chlorophyll in the initial product, the more
rapid will be deterioration during the frozen storage.
Blanching can lead to thermally induced degradation of
nutrients such as ascorbic acid. It can also lead to
leaching of nutrients. Hence, a determination must be
made as to whether or not the unblanched product has
adequate stability to retain structural integrity and
quality attributes upon freezing and during frozen
storage, and a decision must be reached as to the extent
188
K.S. Bahcßeci et al. / Journal of Food Engineering 66 (2005) 187–192
of blanching needed to ensure optimum product quality
(Reid, 1998).
Process optimization involves measuring the rate of
enzyme destruction, so the blanch time is just long enough to destroy the indicator enzyme (Cano, 1996).
Many processors utilize a heat treatment sufficient to
inactivate peroxidase, one of the more stable enzymes
present, and not incidentally, one of the enzymes whose
activity is relatively easy to measure. Some researchers
suggested that targeting peroxidase leads to a more severe
heat treatment than is required for many vegetables and
that the enzymes responsible for quality loss, which have
been identified, have a lower stability than peroxidase
(Reid, 1998). Recently, use of LOX as an indicator of
proper blanching has been recommended as more significant in determining storage stability in frozen vegetables (Barrett & Theerakulkait, 1995; Nagy-Gasztonyi,
Kardos-Neumann, & Biacs, 2000; Sheu & Chen, 1991;
Williams, Lim, Chen, Pangborn, & Whitaker, 1986).
The objective of this study was to optimize blanching
operation for green beans in terms of enzyme reactivation and losses of ascorbic acid and chlorophylls during
frozen storage using LOX and POD enzymes as
blanching indicators, separately.
2. Materials and methods
2.1. Materials
Linoleic acid (99% pure), chlorophylls a and b were
purchased from Sigma and other chemicals from Merck.
Green beans were obtained from a local market in
Ankara.
2.2. Blanching
An ordinary bench top constant temperature water
bath was used as the water blanching equipment. Green
beans were size graded, washed, drained and blanched in
distilled water at 60–96 C for different times to determine thermal inactivation kinetics of POD and LOX.
From thermal inactivation data, blanching treatments at
70 C for 2 min and 90 C for 3 min were selected to
inactivate >90% of initial LOX and POD activity in
green beans, respectively. Following blanching, green
beans were plunged into an ice bath and stored in
polyethylene bag at )18 C for 12 months. POD and
LOX activities, ascorbic acid and chlorophyll contents
were measured monthly during frozen storage.
2.3. Enzyme assays
2.3.1. Preparation of crude enzyme extract
One hundred grams of green beans were homogenized in 150 ml of water at 4 C in a blender for 3 min.
The slurry was filtered through three layers of cheesecloth and centrifuged at 25,000g for 20 min. The
supernatant was used as the crude enzyme extract.
2.3.2. POD and LOX assays
POD activity was measured as the change in absorbance at 420 nm using guaiacol and H2 O2 as substrates
based on the method of Sheu and Chen (1991). LOX
activity was measured at 234 nm with a linoleic acid
substrate solution according to G€
okmen, Bahcßeci, and
Acar (2002b). A double beam spectrophotometer (Shimadzu, 2101 PC) and a 1 cm path-length cuvette were
used for enzyme measurement.
2.4. Ascorbic acid analyses
Suitable amounts of green beans and metaphosphoric
acid solution were homogenized using a Virtis homogenizer at medium speed for 2 min. The homogenized
sample was filtered through a black band filter paper
and then 0.45 lm Millipore disposable filter. The clarified sample was divided into two parts: One part was
directly analysed for ascorbic acid (AA) content.
Dithiothreitol was added into the other part at a ratio of
1 mg/ml and it was kept at dark for 90 min to convert
any dehydroascorbic acid (DHAA) to AA. After complete conversion of DHAA was achieved, the sample
was analysed for its total AA content. HPLC analyses
were performed according to G€
okmen, Kahraman,
Demir, and Acar (2000).
2.5. Chlorophyll analyses
Ten grams of sample was weight into a homogenizer
cup that contained 0.1 g CaCO3 and 1 ml of 1% BHT to
stabilize pigments against tissue acids and oxidation,
respectively. 50 ml of methanol was added and the
mixture was simultaneously homogenized and extracted
in a Virtis homogenizer at medium speed for 2 min.
After tissue is thoroughly disintegrated, the extract was
filtered through a black band filter paper into a 100-ml
volumetric flask. The residue and filter paper were returned to the homogenizer cup with additional 50 ml
portion of methanol, and the extraction was repeated.
The final residue was washed with methanol to remove
the traces of pigment. The filtrates were combined and
completed to volume with methanol. The extraction
and filtration steps were carried under low light intensity
and at low temperature (4 C), respectively, to avoid
pigment degradations. The extract was filtered through
a 0.45 lm membrane filter and 10 ll was injected into
the HPLC column. HPLC analyses were performed
using the conditions described by G€
okmen, Bahcßeci, and
Acar (2002a).
K.S. Bahcßeci et al. / Journal of Food Engineering 66 (2005) 187–192
3. Results and discussion
3.1. Determination of blanching conditions
Reduction of >90 % activity of indicator enzyme was
aimed for determination of blanching conditions since it
is recommended for optimum quality of vegetable during frozen storage. Complete inactivation of enzymes
can be easily achieved either using higher temperatures
or increasing the time of thermal process. But in this
case, properties of the products such as color, texture,
flavor, aroma and nutritive quality can be adversely
affected. For maximum retention of quality, clearly the
need is for sufficient heat treatment to stabilize the
product against quality deterioration but at the same
time, to minimize quality loss as a direct result of
heating.
In our study, two enzymes (POD and LOX) were
considered as the indicator of blanching adequacy.
Fig. 1 shows the residual enzyme activities during water
blanching of green beans at different temperatures. At
60 C, 90% of LOX activity was lost within the first
10 min, but the residual activity remained relatively
stable during further 20 min of blanching. Rapid inactivation was observed in green bean LOX at 70 C and
higher temperatures. A blanching treatment at 70 C for
Residual LOX Activity (%)
100
60˚C
70˚C
80
80˚C
60
40
20
0
0
5
10
15
20
Blanching time (min)
(a)
Residual POD Activity (%)
100
80
2 min was found sufficient to inactivate 90% of LOX’s
initial activity. POD was determined to be more resistant to heat treatment than LOX in green beans. 90% of
inactivation of green bean POD could be achieved by a
blanching treatment at 90 C for 3 min.
Lee, Smith, and Hawbecker (1988) noted that
blanching of beans at 82 C for 3.5 min is necessary for
reduction of 90% POD activity and also retaining
quality during frozen storage. In another research, it
was reported that POD is completely inactivated with
the blanching of green beans at 93.3 C for 2 min
(Barrett & Theerakulkait, 1995).
3.2. Changes in POD and LOX activities during frozen
storage
The thermal inactivation of enzymes is reversible and
the enzymes can recover their activity under certain
conditions. It is reported that the reactivation of enzyme
activity after inactivation by heat is one of the properties
of POD. While POD reactivation was observed by many
researchers in model systems (Adams, Harvey, &
Dempsey, 1996; Halpin, Pressey, Jen, & Mondy, 1989;
Powers, Costello, & Leung, 1984; Rodrigo, Rodrigo,
Alvarruiz, & Frigola, 1997), no reactivation was reported during frozen storage of vegetables (Barrett &
Theerakulkait, 1995; McDaniel, Montgomery, Latham,
& Lundahl, 1988; Rodriguez-Saona et al., 1995; Sheu &
Chen, 1991). Also there is no information about the
LOX reactivation in literature.
Fig. 2 shows the changes of POD and LOX activities
in blanched and unblanched green beans during frozen
storage. There was no reactivation in both POD and
LOX enzymes during frozen storage. Blanching at 90 C
for 3 min completely inactivated green bean LOX and
any residual activity was determined in these samples
during frozen storage. LOX activities in blanched (LOX
indicator) and unblanched green beans were relatively
stable (Fig. 2a). Blanching at 70 C for 2 min resulted in
a 90% and 30% inactivation of LOX and POD enzymes,
respectively. POD activities of both unblanched and
blanched (LOX and POD indicator) green beans were
relatively stable during 6 months of frozen storage, but
tended to decrease afterward especially for unblanched
and blanched (LOX indicator) green bean samples.
70˚C
60
80˚C
3.3. Changes in ascorbic acid during frozen storage
90˚C
40
96˚C
20
0
0
(b)
189
5
10
15
20
Blanching time (min)
Fig. 1. Residual (a) LOX, and (b) POD activities of green beans
blanching after different time and temperature.
The initial AA content of unblanched fresh beans was
found to be 222.42 mg/kg and it decreased to 153.60 mg/kg
after blanching at 70 C for 2 min and 133.34 mg/kg
after blanching at 90 C for 3 min, respectively. AA content of unblanched green beans decreased significantly
during frozen storage following first order kinetics. AA
losses in unblanched, 70 C · 2.0 min blanched and
90 C · 3.0 min blanched green beans were found to be
190
K.S. Bahcßeci et al. / Journal of Food Engineering 66 (2005) 187–192
240
80
unblanched
blanched (ind. LOX)
60
40
20
0
0
1
2
3
4
5
6
7
8
180
120
60
0
9
0
Storage time (month)
(a)
1
2
3
4
5
6
7
100
increase for 3 months then to decrease through storage.
Blanching treatments prior to frozen storage resulted in
an increase on DHAA contents of green beans as a results of AA oxidation. Similar to AA, DHAA contents
of blanched green beans also tended to decrease through
frozen storage (Fig. 3). Only green beans in which
blanched at 90 C for 3 min to inactivate POD contained
AA and DHAA while unblanched and blanched at
70 C for 2 min green beans contained any AA and
DHAA at the end of 9 months of frozen storage.
80
60
unblanched
blanched (ind LOX)
blanched (ind POD)
40
0
0
1
2
3
4
5
6
7
8
9
Storage time (month)
(b)
Fig. 2. Changes in (a) LOX, and (b) POD activities in green beans
during frozen storage at )18 C.
3.4. Changes in chlorophylls during frozen storage
93.65%, 86.21% and 68.64% after 6 months of storage,
respectively. During frozen storage of unblanched green
beans at )18 C, the half-life of AA was calculated as
1.89 months. It increased to 2.15 and 3.48 months by
blanching at 70 C for 2 min and 90 C for 3 min,
respectively (Table 1).
The first step of AA degradation is oxidation to
DHAA and in the second step, it hydrolyses to diketogluconic acid. While AA and DHAA have equal antiscorbutic activity, diketogluconic acid has no biological
activity. So AA together with DHAA constitute of total
vitamin C effect. Unblanched green beans were found to
contain 25.69 mg/kg of DHAA prior to frozen storage.
DHAA content of unblanched green beans tended to
Color is the primary quality attribute by which the
consumer assesses natural and processed foods. Frozen
vegetables are subjected to color modifications which
take place during blanching and/or during frozen storage. Chlorophylls are mainly responsible for the color in
green beans. Chl a and Chl b contents of fresh green
beans were determined to be 131.97 and 69.28 mg/kg,
respectively, with an initial Chl a/Chl b ratio of 1.90.
Chl a and Chl b contents of green beans were affected by
blanching time and temperature with the conversion of
chlorophylls into corresponding epimers and pheophytins. 1.14 mg/kg of the epimer of Chl a and 0.51 mg/kg
of pheophytin a formed in green beans by blanching at
70 C for 2 min. 7.89 mg/kg of the epimer of Chl a, 7.22
Table 1
Degradation rate constants and half-lifes of AA, chlorophylls a and b in green beans during frozen storage at )18 C
Rate constant (k), 1/month
a
AA
Chlorophyll a
Chlorophyll b
b
Half-life (t1=2 ) month
UB
LOX
POD
UBa
LOXb
PODc
0.3671
0.0947
0.0529
0.3229
0.1373
0.0687
0.1989
0.0839
0.0415
1.89
7.32
13.11
2.15
5.05
10.9
3.48
8.26
16.70
Unblanched.
Blanched (ind. LOX).
c
Blanched (ind. POD).
b
9
Fig. 3. Rates of changes of AA and DHAA contents of unblanched
and blanched green beans during frozen storage at )18 C.
20
a
8
Storage time, month (-18˚C)
120
POD activity (%)
AA (unblanched)
AA (blanched, LOX indicator)
AA (blanched, POD indicator)
DHAA (unblanched)
DHAA (blanched, LOX indicator)
DHAA (blanched, POD indicator)
100
AA or DHAA, mg/kg
LOX activity (%)
120
c
K.S. Bahcßeci et al. / Journal of Food Engineering 66 (2005) 187–192
highest ratio of Chl a/Chl b at the end of 9 months at
)18 C, while the green beans blanched at 70 C for
2 min had the lowest among all (Fig. 4b).
140
Unblanched / Chl a
Unblanched / Chl b
Blanched (ind. LOX) / Chl a
Blanched (ind. LOX) / Chl b
Blanched (ind. POD) / Chl a
Blanched (ind. POD) / Chl b
Chlorophylls, mg/kg
120
100
4. Conclusion
80
60
40
0
1
2
(a)
3
4
5
6
7
8
9
Storage time, month (-18˚C)
2.2
Unblanched
Blanched (ind. LOX)
Blanched (ind. POD)
2.0
Chl a / Chl b Ratio
191
1.8
1.6
1.4
Here, POD and LOX were tested as the indicator
enzymes for the adequacy of blanching treatment in
green beans, separately. The green bean LOX was found
to be more sensitive to heat and easily inactivated by a
mild blanching treatment such as 70 C for 2 min. Even
no LOX reactivation was observed after such a
blanching treatment, some important quality attributes
including ascorbic acid and chlorophylls became more
susceptible to break down during frozen storage. However, increasing blanching time and temperature to
inactivate green bean POD (90 C for 3 min) ensured the
retention of these quality attributes at the highest extend. Overall results suggest using POD as the indicator
of blanching adequacy in green beans to be frozen.
1.2
1.0
(b)
Acknowledgements
0
1
2
3
4
5
6
7
8
9
Storage time, month (-18˚C)
Fig. 4. Rates of changes of (a) Chl a and Chl b contents, (b) Chl a/Chl b
ratios of unblanched and blanched green beans during frozen storage
at )18 C.
mg/kg of the epimer of Chl b and 9.97 mg/kg of pheophytin b formed by blanching at 90 C for 3 min.
Blanching was found to influence the degradation
rate constants of both Chl a and Chl b during storage,
expectedly. Half-lifes of Chl a and Chl b in green beans
were calculated to be 7.32 and 13.11 months, respectively. Blanching at 70 C for 2 min resulted in a decrease, while blanching at 90 C for 3 min an increase in
the half-lifes of Chl a and Chl b during frozen storage
(Table 1). Chl a appeared to be more sensitive than Chl
b in all unblanched and blanched samples (Fig. 4a).
Similar to AA, degradation of chlorophylls during frozen storage also followed first-order kinetics.
Formation of pheophytins from chlorophylls was
progressive during frozen storage in addition to its formation during blanching. 5.38, 28.34 and 56.16 mg/kg of
pheophytin a were determined in unblanched green
beans, blanched green beans at 70 C for 2 min and at
90 C for 3 min, respectively, at the end of 9 months of
frozen storage. Increasing amounts of pheophytins,
especially in green beans blanched at 70 C for 2 min
resulted in a conversion of bright green color into dull
olive color. The rates of change of initial Chl a/Chl b
ratio of unblanched and blanched green beans differed.
The green beans blanched at 90 C for 3 min had the
The authors would like to thank The Scientific and
€ ITAK),
_
Technical Research Council of Turkey (TUB
Agriculture, Forestry and Food Technologies Research
Grant Committee (TOGTAG) for financial support to
this research project (Project no. TOGTAG 2633).
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