Received: 16 February 2016
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Revised: 15 August 2016
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Accepted: 22 August 2016
DOI 10.1111/jfpe.12485
ORIGINAL ARTICLE
Effect of ohmic heating on Polyphenol Oxidase (PPO) inactivation and color change in sugarcane juice Juhi Saxena | Hilal Ahmad Makroo | Brijesh Srivastava Department of Food Engineering and Technology, School of Engineering, Tezpur University, Assam, India Correspondence Dr. Brijesh Srivastava, Department of Food Engineering and Technology, School of Engineering, Tezpur University, Assam 784028, India. Email:
[email protected]
Abstract Sugarcane juice was analyzed for the effect of four different time-temperatures (60–90°C for 5– 20 min) at three electric field strengths (EFS) (24, 32, and 48 V cm-1) on PPO activity in a lab scale ohmic heater. At 608C, the activity decreased with increase in EFS while at 70–908C, increased activity was observed at 32 and 48 V cm21. Up to 97.8% reduction in activity was observed at 32 V cm-1/908C/5 min. The biphasic model was the best to describe PPO inactivation kinetics. The kinetic analysis showed that the decimal reduction time (D-value, minute) decreased with increasing EFS at different temperatures. At 32 V cm-1/908C/5 min, the D-value was 8.25 and 76.55 min for labile and stable PPO fractions, respectively. The electric field sensitivity parameter (ZV-value, V cm-1) was lower at 60 and 708C but increased with treatment temperature. The z-value (z8C) for the labile fraction consistently decreased with increasing EFS and was lower than the stable fraction. Color change was inversely proportional to treatment temperatures at 24V cm-1 and directly proportional at 32 and 48V cm-1. The kinetics for L, a, b values was best described by first order model while colour change was best explained by the combined model.
Practical applications Sugarcane juice is one of the most widely relished beverages of south Asia. However, enzymatic browning is a major factor that limits its storage to only a few hours after extraction. High temperatures and processing times employed during thermal processing cause deleterious changes in the color, flavor and the overall nutritive components of the product. Ohmic heating has been applied to different commercial food products (like strawberries, grapes, orange juice) and has been rendered as a time-efficient process. Therefore, this study was conducted to investigate the effect of ohmic heating on inactivation of the browning enzyme (PPO) in sugarcane juice and its kinetic analysis, so as to suggest an alternative that can be explored further for commercial packaging of sugarcane juice. KEYWORDS
D value, ohmic heating, PPO inactivation kinetics, sugarcane juice, Z value
1 | INTRODUCTION
of the product thereby limiting its storage for a longer time (Bucheli and Robinson, 1994). Polyphenol oxidase (PPO) is one of the major
Sugarcane (Saccharum officinarum) is an exclusively cultivated crop in
enzymes responsible for browning. PPO catalyses the oxidation of
tropical and subtropical regions of the world. Fresh sugarcane juice is a
monophenolic compounds to o-diphenols and o-dihydroxy compounds
popular beverage in many tropical countries and India is its second larg-
to o-quinones which causes browning (Jayaraman et al., 1982). To
est producer next to Brazil. Relished for its sweet taste and flavor, sug-
inhibit enzymatic browning, conventional methods like thermal proc-
arcane juice is a great preventive and healing source for sore throat,
essing and chemical additives (ascorbic acid, sulfites, sodium diethyl
cold, and flu (Khare et al., 2012). However, enzymatic browning is one
dicarbamate) have often been used for inactivation of PPO (Ilhami
of the major causes for deleterious changes in the sensory properties
et al., 2005; Kim et al., 2005) but the nutritive losses caused by
Journal of Food Process Engineering 2016; 00-00
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C 2016 Wiley Periodicals, Inc. V
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exposure to high temperature and processing times have led research-
non-enzymatic browning are also higher during prolonged heat treat-
ers to explore other alternatives for enzyme inactivation. In recent
ment, color kinetics was also conducted to assess the effect of the
years, new techniques like hydrostatic pressure (Rapeanu et al., 2006),
treatment conditions on the sensorial aspect of the sugarcane juice.
pressurized carbon dioxide (CO2) containing supercritical CO2 (Chen et al., 1992; Liu et al., 2010); heat treatment by microwaves and elec-
2 | MATERIALS AND METHODS
tric current (Matsui et al., 2007; Xu et al., 2015) have surfaced as PPO inactivation methods. Ohmic heating is an alternative thermal processing technique that allows rapid and volumetric generation of heat within the product as a result of resistance of the food material to the flow of electric current (Sastry and Barach, 2000). Short processing times employed during ohmic heating cause less degradation of color and nutritive composition of the product (Castro et al., 2004; Verghese et al., 2014). Ohmic heating has been studied for a variety of applications like preheating, blanching, pasteurization, and enzyme inactivation in various food products (Li et al., 2013; Lima and Sastry, 1999; Mizrahi, 1996). Castro et al. (2004) established that ohmic heating leads to faster inactivation of enzymes
2.1 | Juice preparation Mature sugarcane stems of “Pharma” variety were sourced from a local farm near Tezpur, Assam. The stems were peeled and cut into smaller cylindrical sections of 1.5–2 cm each. The juice was extracted using Usha Food Processor (make: FP2663, Thane, India). The extracted juice was filtered via fourfolds of the muslin cloth and its physicochemical properties were determined. It was then used for ohmic treatment and analysis. Fresh juice was used for each set of experiments.
2.2 | Ohmic treatment
and similar observations have been reported by Icier et al. (2006) for per-
The ohmic treatment was carried out in a lab scale set up that com-
oxidase in pea puree as well as Demirdoven and Baysal (2014) for pectin
prised of two movable stainless steel (grade 316) electrodes in a hollow
methylesterase inactivation in orange juice. Icier et al. (2008) reported
cylindrical casing as given by Saxena et al. (2016) (Fig. 1), an opening of
that PPO in grape juice was deactivated at a lower critical temperature
5 mm diameter was made on the outer teflon casing which served as
by ohmic heating as compared to conventional heating and the inactiva-
an inlet for feed as well as for temperature measurement using a
tion kinetics followed a one-step first order deactivation.
teflon-coated thermocouple during the experiment. The different EFS
The composition of PPO enzyme varies in different plants, such as
were achieved by adjusting the distance between the electrodes. For
PPO in avocado is composed of six isoenzymes (Kahn, 1976) while that
high EFS, the distance between the electrodes was reduced and hence,
in bean sprouts has two isoenzymes (Nagai and Suzuki, 2003). Saraiva
the volumetric capacity of the equipment varied from 50 mL at 24
et al. (1996) have reported the influence of the composition of food
V cm21 to 34.5 mL at 32 V cm21 and 25 mL at 48 V cm21,
material on the kinetic behavior of peroxidase enzyme. Therefore, there
respectively.
is a possibility that the composition of food material plays an important
Freshly extracted sugarcane juice was fed into the Ohmic heater
role in PPO inactivation too. No published literature is currently avail-
via the inlet and was subjected to four different treatment tempera-
able on the inactivation kinetics of PPO in sugarcane juice. Moreover,
tures (60, 70, 80, and 908C) at three EFS (24, 32, and 48 V cm21) for
only limited reports are available which have studied the combined
four holding times (5, 10, 15, and 20 min). The come up time for differ-
effects of electric field strength (EFS), time and temperature on enzyme
ent temperatures at different EFS are reported in Table 1. A single
inactivation (Icier et al., 2008; Riener et al., 2008). Therefore, the pres-
phase electric supply of 240 V and 50 Hz frequency was used to carry
ent study was conducted to explore the effects of temperature and
out the experiments. The temperature was maintained for different
EFS as well as holding time on PPO inactivation kinetics in sugarcane
holding periods by switching the electric supply on/off with the help of
juice. Because PPO is directly associated with color and chances of
a relay based temperature indicator-cum-controller. The residual PPO
FIGURE 1
Schematic diagram of ohmic heating set up
SAXENA
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T A B LE 1
3
2.4.2 | Model 2: Bi-phasic model
Come-up times for different combinations of ohmic
treatment
This model is used to describe a scheme where the enzyme is a mixture
EFS (V cm21)
Holding temp. (8C)
24
Come-up time (min)
of two isoforms with different thermal and/or electric field sensitivities,
60 6 1
2.12
each following its own first order kinetics (Cruz et al., 2006) and can be
24
70 6 1
2.76
represented as Equation 3.
24
80 6 1
3.25
24
90 6 1
3.95
At 5AL exp ð2kL tÞ1 As exp ð2ks tÞ Ao
32
60 6 1
0.98
where AL is the fraction of the thermolabile enzyme and As is the frac-
32
70 6 1
1.17
tion of thermostable enzyme; kL and kS are the inactivation rate con-
32
80 6 1
1.50
stants of labile fraction (min21) and stable fraction (min21) respectively;
32
90 6 1
1.83
and t is time (min).
48
60 6 1
0.20
48
70 6 1
0.32
48
80 6 1
0.45
48
90 6 1
0.55
(3)
2.4.3 | Model 3: Weibull model This model has been developed as an alternative to linear inactivation kinetics (Van Boekel, 2008). In this model, the enzyme inactivation is treated as a function of the residual enzyme activity which is dependent on several factors such as differences in the treatment intensity or
activity was measured for samples treated at all EFS for different tem-
development of resistance by the enzyme. The equation for Weibull
peratures and holding times.
model is given as Equation 4 a5exp ð2btn Þ
2.3 | Determination of residual PPO activity
(4)
where “a” is the decay in enzyme activity measured as the ratio of
The PPO activity was measured by the method described by Ozoglu
activity at any time (t) to the initial enzyme activity at t 5 0; the param-
and Bayindirli (2002). Immediately after the treatment, the juice sample
eter “b” determines the scale of the curve and “n” determines the shape
was chilled in an ice bath maintained at 48C 6 18C. 0.5 mL of treated
of the survival curve where n < 1 denotes upward concavity or increas-
and cooled sugarcane juice was added to a mixture of 1 mL of 0.2 M
ing resistance and n > 1 represents downward concavity or decreasing
catechol solution and 2 mL of phosphate buffer (pH 5 6.5). The absorb-
resistance of the enzyme (n 5 1 would correspond to a linear kinetics).
ance was measured at 420 nm at every 1 min interval till 10 min. The enzyme activity was estimated from the linear portion of the curve of absorbance v/s time. One unit of PPO activity was defined as 0.001D A420/min. The PPO activity of the samples was expressed as %
Current Enzyme Activity 3 100 Initial Enzyme Activity
This model is also a non-linear model which is described by the Equation 5. A5A0 1 ðA0 2A1 Þexp ð2ktÞ
Residual Activity (RA) as given in Equation 1: % RA5
2.4.4 | Model 4: Fractional conversion model
(1)
Enzyme activity analysis was carried out in triplicates for each
(5)
where “A” is the decay in the enzyme activity, A0 is the initial enzyme activity at t 5 0; A1 is the residual activity after treatment time and “k” is the inactivation rate constant.
experimental run.
2.5 | Decimal reduction time and thermal reduction time
2.4 | Kinetic analysis of PPO
The decimal reduction time (D value) is defined as the treatment time Different kinetic models were used to study the kinetic behavior of PPO enzyme during inactivation.
needed for 90% inactivation of initial activity of PPO at a given condition and can be obtained by Equation 6.
2.4.1 | Model 1: First Order
D5
This model is used to describe a one step conversion of the enzyme from its native (active) form to the denatured (inactive) form by a first order irreversible reaction (Equation 2) At a5 5exp ð2ktÞ Ao
2:303 k
(6)
where k is the inactivation rate constant (min21). The inactivation rate constant (k) was determined by modeling the PPO inactivation data using OriginPro 8.5 software. The “k” values of the best fit model were
(2)
selected and subsequently the D values were determined for the given data set using Equation 6.
where “a” is the decay in enzyme activity that is expressed as the ratio
The voltage increase needed for one log reduction in D value at a
of measured activity “At” at time “t” of the treatment to the initial activ-
constant temperature is reflected by ZV value (V cm21) (electric field
ity “Ao” and the coefficient “k” represents the first-order rate constant.
sensitivity parameter). Additionally, the temperature increase required
4
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SAXENA
for a one log reduction in D value at constant EFS is reflected by z value (8C) (thermal sensitivity parameter).
T A B LE 2
Physico-chemical properties of fresh sugarcane juice
Parameters
The ZV value and z value is calculated as the negative reciprocal of the slope of the regression lines of log D versus V and log D versus T, respectively (Liu et al., 2008).
2.6 | Color measurement
ET AL.
Units
Values
Water content
(%)
81.05 6 0.30
Total solids
(%)
18.95 6 0.42
Acidity
(%)
0.16 6 0.02
TSS
(8B)
19.6 6 0.10
pH
–
5.42 6 0.07
Color changes in the sugarcane juice were analyzed using a Hunter Lab
Vitamin C
(mg/100 mL)
4.57 6 0.13
color, USA. The equipment was calibrated by placing the standard black
Reducing sugars
(%)
0.66 6 0.02
and white tile in the cell transmittance section. A glass cuvette (3.5 3 4
PPO activity
Units
69.40 6 0.47
3 1.5 cm3) containing ohmic-treated juice was placed in the cell transmittance specimen compartment. The lid of the compartment was closed and the analysis was then conducted. Three Hunter parameters,
EFS for different holding times has been plotted in Fig. 2. The degree
namely “L” (lightness), “a” (redness to greenness) and “b” (yellowness to
of inactivation of PPO followed a different trend at different treatment
blueness) were measured and total color differences were calculated
conditions. The effect of temperature, holding time, EFS and their
by Equation 7.
interaction on PPO inactivation was found to be significant (p < 0.01). The maximum inactivation was observed at 32 V cm21 at 90 8C held
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h i 2 ðL0 2 LÞ2 1 ða0 2 aÞ2 1 ðb0 2 bÞ2 DE 5
(7)
for 5 min. Overall, at 60 8C there was a decrease in residual PPO activity with increasing EFS; at 70 and 80 8C, a significant increase in the
where L0, a0, b0 are the values of fresh juice sample and L, a, b are the
residual PPO activity was observed from 24 to 32 V cm21 following a
values of the treated samples.
decreased PPO activity at 48 V cm21 and at 90 8C, the residual PPO
To explain the color change phenomenon in sugarcane juice, the
activity observed an increase at 48 V cm21. These observations were
data were fitted to first order model (Model 1) and combined model
found to be significant (p < 0.01). A significant (p < 0.01) increase in the
(combination of zero and first order) (Ibarz et al., 1999) as given in
PPO activity was observed during initial subjection at 24 V cm21 at
Equations 2 and 8, respectively. The combined model follows a two-
60 8C held for 5 min which may be due to experimental error. There-
stage mechanism, where the maillard reaction (stage 1) follows a zero-
after, the residual PPO activity decreased with holding time. No such
order kinetics and destruction of natural fruit pigments (stage 2) fol-
increase was visible in the samples upon initial subjection to 32 and 48
lows a first order kinetics.
V cm21 at 60 8C for 5 min.
DE5kc ð12exp ð2k1 ÞtÞ
(8)
where kc 5 k0/k1. “k0” is the zeroth order rate constant and k1 is the first order rate constant.
In addition, the effect of holding time on the residual PPO activity was also observed. Although greater inactivation was visible at 48 V cm21 from 60 to 80 8C in comparison to 24 and 32 V cm21, the residual PPO activity increased with holding time. For 32 V cm21, this increase in residual PPO activity with holding time was visible at 90 8C
2.7 | Statistical analysis
only. The increase in the enzyme activity with holding time at constant
All the data were statistically analyzed using SPSS software 16.0. Anal-
temperatures can be attributed to the pulsating ohmic treatment that
ysis of Variance (ANOVA) and Duncan’s multiple range tests were car-
can influence biochemical reactions by changing the molecular spacing
ried out on the observed data at 99% confidence level. Kinetic
and increasing interchain reactions to give a better enzyme–substrate
modeling was performed for PPO inactivation at different processing
interaction (Castro et al., 2004). Similar results have been reported for
conditions using OriginPro software 8.5. The statistical criteria used for
the deactivation of trypsin at 55–70 8C (Margot et al., 1997) and for
discrimination among the kinetic models were R value and v value.
inactivation of peroxidase enzyme in the range of 40–80 8C by thermo-
Best fit model was obtained on the basis of R2 value, v2 value and the
sonication (Cruz et al., 2006). Giner et al. (2005) have suggested that
residual plot. The experiments were run in triplicates.
an increase in EFS can yield considerable improvement in inactivation
2
2
of enzymes. However, in the present study, although the residual PPO
3 | RESULTS AND DISCUSSION 3.1 | Effect of ohmic treatment on residual PPO activity
activity in sugarcane juice after a treatment time of 15 min at 70 8C decreased with increasing EFS but at 80 8C after 15 min treatment time, a significant increase (p < 0.01) in the residual PPO activity was observed at 48 V cm21. Terefe et al. (2010) have reported a temperature range of 80–1008C to cause resistance in PPO enzyme to inactiva-
The physicochemical properties of fresh sugarcane juice were esti-
tion during thermal treatment. Therefore, it is suggested that in
mated and are reported in Table 2. The PPO activity in the fresh juice
addition to the possible changes in the molecular spacing at higher EFS
was observed to be 69.4 6 0.47 U mL21. The effect of ohmic treat-
of 48 V cm21, simultaneous subjection to high temperature (80 8C)
ment on residual PPO activity at different levels of temperature and
confers resistance to PPO for inactivation. Sankhla et al. (2012) have
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FIGURE 2
5
Change in % Residual PPO activity with time at (a) 608C (b) 708C (c) 808C and (d) 908C
reported a traditional thermal treatment of 80 8C for 20 min or 80 8C
refrigerated conditions. In this study, a combination of 80 8C for 5 min
for 10 min with addition of chemical treatments (like KMS and citric
at all EFS resulted in residual PPO activity in the range suggested by
acid) to give satisfactory results for sugarcane juice preservation. In
Mao et al. (2007). The non-thermal effect exerted on PPO during
another study, Mao et al. (2007) have suggested a residual PPO activity
ohmic heating has been clearly illustrated which makes this process
of 7–11% to prevent browning in sugarcane juice during storage under
time-efficient. Further studies on quality evaluation may be conducted
Summary of the performance of selected models fitted to residual PPO activity at different EFS for different treatment temperaturesa
T A B LE 3
EFS (V cm21) 24
v2
0.566–0.970
0.004–0.031
Patterned
Biphasic
0.898–0.973
0.000–0.005
Random
Weibull distribution
0.928–0.959
0.002–0.013
Patterned
Fractional conversion
0.880–0.923
0.004–0.011
Random
First order
0.870–0.971
0.008–0.011
Patterned
Biphasic
0.928–0.973
0.002–0.008
Random
Weibull distribution
0.796–0.971
0.001–0.004
Patterned
Fractional conversion
0.913–0.942
0.002–0.007
Random
First order
0.834–0.952
0.002–0.010
Patterned
Biphasic
0.942–0.988
0.003–0.008
Random
Weibull distribution
0.778–0.853
0.002–0.015
Patterned
Fractional conversion
0.910–0.944
0.007–0.018
Random
First order
32
48
a
R2
Models
2
Good models have higher R (coefficient of determination), lower chi-square values and random residuals.
Residual plot
6
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ET AL.
to compare the sensorial and nutritive attributes of the processed juice. No published literature is available to support the effect of EFS at high holding temperatures on enzyme activity.
3.2 | PPO inactivation kinetics 3.2.1 | Mathematical modeling The first order, biphasic, Weibull, and fractional conversion models were attempted to model PPO inactivation data whose regression coefficient (R2) and chi square (v2) values are listed in Table 3. The inactivation rate constants (k, min21) were determined by fitting the residual PPO activity data to the four mathematical models. The model with the highest range of R2value and lowest range of v2 value was selected. Consequently, the biphasic model was found to be the best fit for all the treatment conditions with the R2 between 0.898 and 0.988 (Figure 3). Thus, according to this model, PPO of sugarcane juice consists of the labile and the stable fractions. Similar results have been reported by Jakob et al. (2010) for inactivation of peroxidase in carrots subjected to ohmic heating at different voltage gradients. In addition, Li et al. (2013) have also suggested the first order biphasic model for inactivation of urease in soymilk by ohmic heating. However, different treatments like high pressure processing have suggested a zero, first as well as second order model for PPO inactivation in Fuji apples (Falguera et al., 2013). This discrepancy may have resulted from the difference in the composition of the food materials chosen for PPO inactivation studies as pH, enzyme and ion concentration as well as the treatment method employed tend to affect the kinetic behavior of the enzyme (Saraiva et al., 1996).
3.2.2 | Effect of ohmic heating on decimal reduction time (D-value) The inactivation rate constant (k) values for the best fit model (biphasic model) were obtained and decimal reduction time (min) was calculated by Equation 6 values of kL (labile), kS (stable), and DL (labile) and DS (stable), respectively for all the treatment conditions are provided in Table 4. At a particular temperature, DL values were found to be smaller than their corresponding DS values at different EFS suggesting that the labile fraction could be inactivated easily than the stable one on increasing the EFS. Also, at particular EFS, the DL and DS values decreased with increasing temperature. Similar results have been
Biphasic model for ppo inactivation in sugarcane juice at (A) 24 V cm21, (B) 32 V cm21, and (C) 48 V cm21
FIGURE 3
reported by Zhi et al. (2008) where the D values of PPO enzyme decreased steeply as the EFS increased. Similarly, the D values have also been reported to decrease with increase in temperature and pressure in watermelon juice; apple juice and red beet extract (Gui et al., 2007; Liu et al., 2008, 2013).
stable fraction of the PPO enzyme. Yang et al. (2000) have reported that one of the possible reasons for this behavior of PPO enzyme can be attributed to its tetramer structure and a high molecular weight which make it more susceptible to high temperature of the order of
At 608C, DL (33.86 min) and DS (329 min) at 24 V cm21 were
80–100 8C in the presence of electric field. Therefore, proper optimiza-
almost 3.48 and 2.85 times the DL (9.71 min) and DS (115.1 min) at 48
tion in terms of EFS and temperature is required to result in maximum
21
V cm
, respectively; while on comparing the effect of temperature, it
inactivation of PPO in sugarcane juice.
was observed that at 24 V cm21, DL(33.86 min) and DS (329 min) at 60 8C were 2.83 and 3.16 times the DL(11.93 min) and DS (103.8 min) at 90 8C, respectively. This suggests that increasing the EFS at a partic-
3.3 | Effect of EFS on PPO
ular temperature causes higher destruction of the labile fraction while
The sensitivity of D values to the EFS could be expressed by the
increasing temperature at particular EFS causes a greater effect on the
parameter ZV value, which is defined as the EFS change that result in a
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7
Kinetic parameters for inactivation of polyphenol oxidase (PPO) from sugarcane juice by ohmic treatment based on the bi-phasic model
T A B LE 4
Temp (8C)
EFS (V cm21)
AL
AS
KL (min21)
KS (min21)
DL (min)
DS (min)
60
24
0.524
0.524
0.068
0.007
33.86
32
0.559
0.559
0.108
0.010
48
0.551
0.551
0.237
24
0.558
0.558
32
0.528
48
0.527
24 32
70
80
90
ZV,L (V cm21)
ZV,S (V cm21)
ZT,L (8C)
ZT,S (8C)
329
64.51 (R2 5 0.98)
57.80 (R2 5 0.98)
21.21
227.9
69.93 (R2 5 0.95)
59.88 (R2 5 0.97)
0.020
9.71
115.1
44.64 (R2 5 0.97)
52.91 (R2 5 0.99)
78.74 (R2 5 0.97)
55.55 (R2 5 0.96)
0.085
0.009
27.05
255.1
36.76 (R2 5 0.99)
43.29 (R2 5 0.98)
0.528
0.121
0.013
18.96
174.7
0.527
0.371
0.032
6.208
71.96
0.645
0.654
0.132
0.015
17.38
150.06
46.08 (R2 5 0.99)
42.73 (R2 5 0.98)
0.599
0.400
0.190
0.023
12.1
97.97
50.25 (R2 5 0.99)
49.01 (R2 5 0.97)
48
0.524
0.524
0.435
0.056
5.29
41.12
24
0.647
0.643
0.193
0.022
11.93
103.8
32
0.545
0.543
0.279
0.030
8.25
76.55
48
0.556
0.534
0.596
0.065
3.86
34.91
10-fold decrease in the D values and is provided in Table 4. At 60 and
the thermal sensitivity of the stable fraction at that EFS. The explana-
70 8C, the labile fraction exhibited a lower ZV value (ZV,L5 44.64 and
tion for such an observation is that inactivation occurs through several
36.76 V cm21) than the stable fraction (ZV,S5 52.91 and 43.29
mechanisms and each has its own temperature dependence. Protein
21
), respectively, indicating a possible tendency that the labile
unfolding is one of the major reasons for inactivation by thermal treat-
fraction was more susceptible to the treatment conditions. However,
ment. However, in Ohmic treatment, other factors such as loss of
at 80 and 908C, the labile fraction was observed to have a slightly
metallic ions, changes in molecular spacing, substrate-enzyme interac-
V cm
21
higher ZV value (46.08 and 50.25 V cm
) than the stable fraction
tion are related to enzyme inactivation (Castro et al., 2004) and thus
(42.73 and 49.01 V cm21), respectively, suggesting that the stable frac-
can be responsible for decrease in ZT,S at 48 V cm21. This explains the
tion may have become more susceptible to inactivation as the temper-
possible reason for the odd behavior of the enzyme at 48 V cm21 at
ature increased. Although no published literature is available for kinetic
different temperatures for different holding times.
analysis of PPO enzyme by ohmic heating, Liu et al. (2013) have
In addition, the ZT,L was higher than ZT,S at all EFS suggesting that
reported the increase in the susceptibility of the stable fraction of PPO
the labile fraction was less sensitive to temperature as compared to the
enzyme in water melon juice at high pressures.
stable fraction on exposure to different EFS. Studies on inactivation of PPO by high pressure have also reported a decreased thermal sensitiv-
3.4 | Effect of temperature on PPO
ity of the labile fraction and an increased thermal sensitivity of the sta-
Thermal sensitivity of the PPO enzyme at particular EFS can also be expressed in terms of z value which may be defined as the change in temperature required to cause 90% change in D value (Table 4). The z value for labile fraction (ZT,L,8C) was found to increase with EFS, rang-
ble fraction after the pressure pretreatments (Gui et al., 2007; Liu et al., 2013).
3.5 | Effect of ohmic heating on color change
ing from 64.5 to 78.7 8C suggesting the decrease in thermal sensitivity
The color degradation of sugarcane juice during ohmic treatment was
of the fraction at higher EFS. The z-value for stable fraction (ZT,S) was
assumed to be a partial contribution of PPO, nonenzymatic browning,
found to be in a narrow range of 55.5–59.8 8C but no trend could be
pigment destruction, and electrochemical degradation of the electro-
21
established because of the slight increase at 48 V cm
. The results
des. The results are presented as L/L0, a/a0, b/b0, DE in the plots shown
indicate that there is a strong probability of the stable fraction of the
in Figures 4–7. Generally the sample lightness decreases with increas-
enzyme to be more sensitive to temperature under the influence of dif-
ing temperature during thermal treatment of juice sample (Ibarz et al.,
ferent EFS in comparison to the labile fraction. The increase in the z
1999; Rattanathanalerk et al., 2005). However, in the present study
value indicates a possibility that increase in EFS may decrease the ther-
the lightness was found to increase with increasing temperature at 24
mal sensitivity of the labile fraction as well as the stable fraction but a
V cm21. This may be due to increased destruction of sensitive pig-
slight decrease in ZT,S at 48 V cm21 suggests that high EFS increases
ments (like chlorophyll) of sugarcane juice. The lightness decreased
8
|
SAXENA
ET AL.
The change of redness (a/a0) of sugarcane juice at (A) 24 V cm21, (B) 32 V cm21, and (C) 48 V cm21
FIGURE 5
The change of lightness (L/L0) of sugarcane juice at (A) 24 V cm21, (B) 32 V cm21, and (C) 48 V cm21
FIGURE 4
change (DE) observed an unusual trend at 24 V cm21, where DE 21
(Figure 4). The redness
decreased with increasing temperature from 60 to 90 8C (Figure 7).
increased with temperature increase (Figure 5) and the yellowness
This may be explained by a small but significant increase in the residual
decreased with temperature increase (Figure 6) at every EFS. The color
PPO activity on initial exposure of sugarcane juice to 60 8C at 24
with temperature increase at 32 and 48 V cm
SAXENA
|
ET AL.
9
The change of yellowness (B/B0) of sugarcane juice at (A) 24 V cm21, (B) 32 V cm21, and (C) 48 V cm21
FIGURE 7
V cm21. Because the activity decreased with time as well as increasing
of the ratio of k0 and k1, a higher DE suggests a greater role of maillard
temperature, the color change also decreased. The DE increased with
reaction and electrochemical degradation of electrodes than residual
FIGURE 6
21
increasing temperature at 32 and 48 V cm
. Because DE is function
The change of total color difference (DE) of sugarcane juice at (A) 24 V cm21, (B) 32 V cm21, and (C) 48 V cm21
PPO activity.
10
|
T A B LE 5
SAXENA
ET AL.
Kinetic parameters for color change of sugarcane juice by ohmic treatment L/L0
a/a0
b/b0
DE
Temp (8C)
EFS (V cm21)
k1 (min21)
R2
k1 (min21)
R2
k1 (min21)
R2
kc (min21)
R2
60
24
1.21 E-03
0.83
23.25E-03
0.98
1.35E-03
0.96
1.052
0.84
32
7.07 E-04
0.85
22.45E-02
0.99
1.37E-03
0.96
0.665
0.75
48
2.75E-04
0.90
22.15E-02
0.99
6.41E-03
0.99
0.358
0.77
24
1.01 E-03
0.87
21.68E-02
0.96
1.02E-02
0.99
0.893
0.74
32
7.77 E-04
0.82
22.94E-02
0.99
8.79E-03
0.96
0.773
0.75
70
80
90
48
3.34 E-04
0.80
22.65E-02
0.99
1.40E-02
0.99
0.488
0.87
24
6.37E-04
0.77
22.98E-02
0.92
2.08E-02
0.98
0.751
0.75
32
9.47 E-04
0.78
23.40E-02
0.95
1.69E-02
0.98
0.971
0.76
48
4.28 E-04
0.86
23.05E-02
0.99
2.23E-02
0.99
0.640
0.88
24
5.11E-04
0.84
23.45E-02
0.78
2.90E-02
0.99
0.779
0.71
32
1.12 E-03
0.72
23.72E-02
0.86
2.43E-02
0.99
1.197
0.88
48
7.06 E-04
0.76
23.29E-02
0.96
4.44E-02
0.99
0.961
0.80
The data were fitted to the kinetic models and the parameters are
higher color change. The increasing EFS gave more prominent color
reported in Table 5. It was found that the L, a, and b values followed
change than temperature increase at a particular EFS which suggests
the first order reaction kinetics while the DE could be explained best
the use of lower EFS to reduce the color change of treated juice to a
by the combined model. These observations are in accordance with
minimum.
those reported for the DE due to thermal treatment (Ibarz et al., 1999;
Ohmic heating causes effective reduction of residual PPO activity
Rattanathanalerk et al., 2005), however, a recent study on ohmic heat-
in short processing times, however, holding times at specific tempera-
ing of tomato juice has reported D E to follow first order kinetics (Mak-
tures may increase the residual PPO activity. This field offers scope for
roo et al., 2016). The rate of change (k1) in lightness decreased with
tremendous research especially for application to sugarcane juice as
increasing EFS at 60 and 70 8C whereas it increased at 70 and 80 8C.
lower holding times may allow the retention of flavor components to a
The rate of change in redness increased with increasing EFS at all treat-
large extent while simultaneously preserving color and other organo-
ment temperatures whereas the rate of change of yellowness increased
leptic properties. Therefore, ohmic heating is an energy efficient
with EFS at 60 8C only. The rate of change of color showed both zero
method that can be thought of as an alternative for preservation of
and first order mechanism and was found to decrease with increasing
sugarcane juice as it has opened up new opportunities for research and
EFS at 60 and 70 8C suggesting that the destruction of natural pig-
development in this field.
ments was higher than the maillard reaction which is the opposite in case of thermal processing of pear puree and peach puree as reported
R EF ER E N CE S
by Ibarz et al. (1999) and Garza et al. (1999). No definite trend could be
Bucheli, C. S. & Robinson, S. P. (1994). Contribution of enzymic browning to color in sugarcane juice. Journal of Agricultural Food Chemistry, 42, 257–261.
established at 80 and 90 8C.
4 | CONCLUSION The maximum inactivation of PPO was 97.8% at a holding time of 5 min at 90 8C at 32 V cm21. The kinetic analysis indicated that the biphasic model was best fit to depict the inactivation of PPO in sugarcane juice suggesting the presence of two isoforms of the PPO enzyme. The trend for the decimal reduction time (D value) for both labile and stable fraction, suggests that the efficiency of PPO inactivation increases with both EFS and temperature. The ZV values (V cm21) indicate that the labile fractions were more easily inactivated at 60– 70 8C while the stable fractions were found to become susceptible to inactivation at 80–90 8C. The thermal sensitivity parameter (z value, 8C) suggested that the heat sensitivity of the labile fractions was less than the stable fraction under different EFS. The increase in the degree of redness and yellowness at high temperatures and EFS results in a
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