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Thermal inactivation kinetic of pectin methylesterase and cloud stability in sour orange juice
Accepted Manuscript Thermal inactivation kinetic of pectin methylesterase and cloud stability in sour orange juice Sara Aghajanzadeh, Aman Mohammad Ziaiifar, Mahdi Kashaninejad, Yahya Maghsoudlou, Ebrahim Esmailzadeh PII:
S0260-8774(16)30119-4
DOI:
10.1016/j.jfoodeng.2016.04.004
Reference:
JFOE 8535
To appear in:
Journal of Food Engineering
Received Date: 13 July 2015 Revised Date:
2 March 2016
Accepted Date: 2 April 2016
Please cite this article as: Aghajanzadeh, S., Ziaiifar, A.M., Kashaninejad, M., Maghsoudlou, Y., Esmailzadeh, E., Thermal inactivation kinetic of pectin methylesterase and cloud stability in sour orange juice, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng.2016.04.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Thermal inactivation kinetic of pectin methylesterase and
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cloud stability in sour orange juice
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a*
a
a
Yahya Maghsoudlou b, Ebrahim Esmailzadeh c a
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Sara Aghajanzadeh , Aman Mohammad Ziaiifar , Mahdi Kashaninejad ,
Department of Food Process Engineering, Gorgan University of Agricultural
Sciences and Natural Resources, Basij Square, Gorgan, Iran.
Department of Food Science and Technology, Gorgan University of Agricultural
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b
Sciences and Natural Resources, Basij Square, Gorgan , Iran.
Biosystem Engineering, Gorgan University of Agricultural Sciences and Natural
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c
Corresponding author email: [email protected]
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*
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Resources, Basij Square, Gorgan , Iran.
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Abstract
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products such as sour orange juice. Inactivation of pectin methylesterase enzyme
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Heat treatment is a common method to improve cloud stability of high acid food
33
In this work, the kinetics of PME thermal inactivation and cloud stability in sour
34
orange juice were investigated. The fresh sour orange juice was heated in a controlled
35
water bath at 60, 70, 80 and 90℃ for different times depending on applied temperature.
36
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(PME), naturally found in citrus, is known as a heat treatment index for these products.
37
inactivation kinetics of PME was calculated in terms of thermal resistance parameters
38
(D & Z values), activation energy, enthalpy, entropy and free energy. The obtained Z-
39
value (36.90℃), activation energy (62.61± 0.84 kJ/mol) and free energy (93.52 - 96.37
40
kJ/mol) revealed the high stability of PME during the heat treatment. Based on thermal
41
resistance of PME, heating times were corrected by considering the come-up time
42
effectiveness. The results showed that cloud value of heated juice increased by PME
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inactivation during heat treatment.
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Time–temperature data was simultaneously recorded using a data-logger. The
Chemical compounds studied in this article
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Sodium chloride (PubChem CID: 5234); Sodium hydroxide (PubChem CID: 14798); Phenolphthalein (PubChem CID: 4764)
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Keywords: Sour orange juice; Heat treatment; Pectin methylesterase; Cloud stability
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1. Introduction
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which are considered to be effective in prevention of cardiovascular disease, various
60
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Citrus fruits contain antioxidants such as ascorbic acid and phenolic compounds
cancers and diabetes (Antunes et al., 2010; Du et al., 2009). Sour orange (Citrus
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aurantium L), also known as bitter orange, is commonly grown in the south-west of
62
Asia, especially in Iran. Despite the bitter and sour taste of this fruit, its juice is used as
63
a seasoning in various food products and salads (He et al., 1997).
64 65
the membrane and high molecular weight compounds are suspended. These
66
compounds consist of proteins, hesperidin, cellulose, hemicellulose and pectin
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As the endocarp cells are ruptured during the citrus juice mechanical extraction,
(Kimball, 1991). These colloidal suspensions are responsible for cloud and turbidity of
68
citrus juice (Kimball, 1991). Cloud stability is a desirable citrus juice quality
69
characteristic which influences the flavor, color and mouth feel of the product (Tiwari
70
et al., 2009).
71 72
cloud stability is undeniable (Kimball, 1991). Pectin is rich in galacturonic acid units
73
linked together via glycoside bonds with side chains of rhamnose, arabinans, galactans,
74
xylose and fucose (Kimball, 1991). Pectin methylesterase (PME), also known as
75
pectinase, pectin esterase and pectin methoxylase, affects the cloud stability and
76
viscosity of citrus juice by de-esterification of the methoxylated pectin and formation
77
of insoluble calcium pectate (Fayyaz et al., 1995; Oakenfull and Scott, 1984).
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Although pectin composes a small portion of the cloud materials, its effect on juice
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Thermal pasteurization is a common method to control the microbial and enzymatic
79
activities in high acid products such as citrus juice (Chen et al., 1993). Table 1 shows
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that there are several isoforms of PME with different thermal resistance which are
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deactivated gradually during citrus juice heat treatment (Cameron et al., 1998;
82
Cinquanta et al., 2010; Tajchakavit and Ramaswamy, 1997; Versteeg et al., 1980;
83
Wicker and Temelli, 1988). Hence there are different PME isoforms depend on citrus
84
variety and heat treatment condition such as used method, time and temperature.
85
Published studies show that thermal resistance of PME is higher than target
86
microorganisms so this enzyme is introduced as heat treatment index for citrus
87
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1980). The residual activity of PME plays an important role in cloud stability of citrus
89
juice (Holland et al., 1976; Rothschild et al., 1975). The inactivation of enzymes and
90
microorganisms mainly occur during holding time and also come-up time (CUT). So it
91
is necessary to estimate the CUT effectiveness to evaluate the exact intensity of heat
92
treatment on PME inactivation. To prevent the overheating of the product and
93
subsequently energy wasting, organoleptic and nutritional losses the effectiveness of
94
CUT should be considered to reduce the heating process time as suggested by Ball
95
(1923) and Tajchakavit & Ramaswamy (1997).
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products (Chen and Wu, 1998; Polydera et al., 2004; Snir et al., 1996; Versteeg et al.,
97
PME inactivation during heat treatment of sour orange juice. Arrhenius plot and
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The objective of this work was to study the kinetic parameters (D and Z-value) of
thermodynamic parameters of PME inactivation such as free energy (∆G), enthalpy
99
(∆H) and entropy (∆S) were also investigated. Due to the effect of PME on pectin de-
100
esterification and sour orange juice turbidity, the cloud value changes were studied by
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considering the CUT effectiveness on PME inactivation during heat treatment.
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2. Materials and methods
2. 1. Sample preparation
103 104 105 106 107 108
Iran) and kept at 4℃ until the experiments were carried out. The juice was extracted
109
from the washed and squeezed fruits using a domestic juicer. It was then filtered by a
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Sour orange (Citrus aurantium L.) was purchased from a local market (Gorgan,
sieve with mesh size 170 to remove large size particles such as pulp, seed, etc.
111 112
2.2. Heat treatment
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Heat treatments were carried out in a water bath (WNB-22, Memmert, Germany) at
115
different temperatures (60, 70, 80 and 90℃) for various times. The juice sample (15
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ml) was sealed in a test tube (15 mm outer diameter, 160 mm length and 1 mm
117
thickness). Time–temperature data of sample during heating was recorded using a 1mm
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diameter copper–constantan thermocouple (T-type) and a data logger (TC-08,
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Pichotechnology Co, UK). The sample was then rapidly cooled using ice-water bath to
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minimize the effect of cooling time on the enzyme inactivation.
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2. 3. Methods of analysis
2.3.1. Fresh sour orange juice analyses
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Density, total acidity, pH, total soluble solids, moisture content and total ash of fresh sour orange juice were determined (AOAC, 2012). A glass pycnometer was used 3
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to measure the density of sample (g/cm ) at 25°C. Total acidity was determined by
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expressed as citric acid percentage. pH was measured using pH-meter (W3B, BEL,
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Italy) at 25°C. Total soluble solids (°Brix) were determined by a refractometer
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(UV/VIS80 +T, PG Instrument, US) at 25°C. Moisture content of sour orange juice was
133
measured by drying the sample to a constant weight at 105 ± 1°C in a hot-air oven
134
(FD53, Binder, Germany). The ash content was measured by burning the juice in a
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titration the juice with NaOH 0.1 N solution using phenolphthalein as indicator and
136
the pulp volume of centrifuged juice at 1500 rpm for 10 min was measured to
137
determine the pulp content of the sample.
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silica crucible at 525°C up to a constant weight. According to Kimball (1999) method,
2. 3. 2. PME Activity
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To measure the PME activity, 5 ml of sour orange juice was mixed with 20 ml of
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1% pectin-salt solution (10 g pectin and 15.3 g NaCI diluted in 1 L distilled water) and
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incubated at 30℃. The pH of the solution was adjusted to 7 using NaOH (2 N). Then,
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the pH of the solution was readjusted to 7.7 with NaOH (0.05 N). Finally, 0.1ml of
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NaOH (0.05 N) was added and the time to regain pH 7.7 was recorded (Kimball,
146
1991). The enzyme activity unit (PEU) was calculated according to Eq. 1:
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PEU (unit/ml) =
. .
(1)
149 150
2. 3. 3. Cloud value
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152 153
heated for 76, 66, 48, 35 and 21 min at 70, 75, 80, 85 and 90℃, respectively.
154
According to method of Versteeg et al. (1980), 5 ml of sour orange juice was
155
centrifuged at 3000 rpm for 10 min at room temperature. Cloud value was measured as
156
supernatant absorbance at 660 nm (T-80, UV/VIS Double Beam Spectrophotometer).
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In this method absorbance of distilled water was considered as a blank.
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In order to study the kinetic of cloud stability, the 15 ml of sour orange juice was
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2. 3. 4. Kinetic parameters analysis
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The thermal inactivation of PME is often described by first order kinetic model described by the following equation:
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Log = −k . t
162 163 164
(2)
165 166
Where A and A0 are residual and initial enzyme activity (PEU), respectively. k is -1
168
to reduce the PME by 90% is called decimal reduction time (D-value) which is
169
inversely related to the reaction rate constant (Eq. 3):
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the inactivation rate constant (min ) and t represents the time (min). The time required
167
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D = 2.303/ k
(3)
172 173
The negative slope of D-value on a log scale against the corresponding temperature
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is termed the Z-value which represents the increase in temperature to attain 10 folds
175
decrement in D-value (Eq. 4):
176 177
Z=
$% &$'
(4)
( )' &( )%
6
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on time–temperature profiles during the CUT, D and Z values were computed from the
181
uncorrected heating times. The effective heating times (te) were calculated using Eq. 5
182
in which the lethality (L) was computed using the uncorrected Z-value.
183
te =* L dt = * 10
./.012 3
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Where T2 and T1 are temperatures corresponding to D2 and D1, respectively. Based
dt
(5)
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each heat treatment). Using the corrected thermal times, D and Z values were
188
recalculated. The more accurate thermal times were recalculated using the Eq. 5 with
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the new Z-value. This procedure was repeated until difference between two sequential
190
Z-values were within 5 %. Finally, the CUT effectiveness was calculated at each heat
191
treatment temperature by the calculated lethality or thermal time (Eq. 5) divided by the
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CUT (Tajchakavit and Ramaswamy, 1997).
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Where Tref is the reference temperature (in this study, the water bath temperature in
194
microorganisms so it should be considered in calculation of the process time (Ball,
195
1923). When the heating time is longer than CUT, a correction factor equal to effective
196
portion of CUT is required. In the present study, this factor is obtained from time-
197
temperature and inactivation profile of PME during heat treatment (Tajchakavit and
198
Ramaswamy, 1997).
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Ball (1923) mentioned that 42% of CUT influences the inactivation of enzymes and
200
Arrhenius model (Eq. 6). Thermal inactivation of PME is related to breaking up the
201
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Temperature dependency of a reaction constant is usually described by the
hydrogen bonds, unfolding of tertiary protein structure and amino acid thermal
202
deamidation of the enzyme (Tanaka and Hoshino, 2003). According to Eq. 6, the slope
203
of the semi-logarithm of reaction rate constant (k) vs. reciprocal of absolute
204
temperature is equal to the activation energy (Ea).
205 206
/45
k = k 6.
(6)
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Where Ea is activation energy (J/mol), K0 is frequency factor or the Arrhenius
209
constant (min-1), R is the universal gas constant (8.3144 J/mol K), and T is absolute
210
temperature (K).
211
Enthalpy, entropy and free energy were described by Eyring models (Eq. 7 to 9).
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Enthalpy at each temperature was calculated according to Eq. 7: ∆H = ∆E − RT
(7)
Where ∆H represents enthalpy (J.mol ) and ∆E is activation energy (J/mol). Free
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∆G = −RTlnKh/k B T
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energy (J.mol-1) at different temperature was calculated by Eq. 8:
212 213 214 215 216 217 218 219
(8)
220 221 222
J) and k B is Boltzmann Constant (1.3806×10-23 J.K-1). Entropy (∆S, J.mol-1.K-1) for
223
PME inactivation was calculated using Eq. 9:
224
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Where K is inactivation constant rate of PME, h is Planck constant (6.6262× 10-34
∆S = ∆H − ∆G /T
225
(9)
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2.4. Statistical analysis
226 227 228 229 230 231
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All experiments were performed in triplicate and the presented results are the mean of the obtained value ± standard deviation. Results were submitted to analysis of
232
variance (ANOVA) with significance level of P < 0.05. All statistical analyses were
233
conducted using the SAS software (version 9.1).
234
3. Results and Discussion
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235
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3. 1. Physicochemical properties of sour orange juice
238 239
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sour orange juice can be categorized in high acid food products (pH < 3.7); therefore
241
the heat treatment below 100℃ is adequate to inactivate the enzyme in order to
242
guarantee the cloud stability of the product.
243
3. 2. Thermal inactivation kinetics of PME
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Table 2 shows the physicochemical properties of fresh sour orange juice. As shown,
244 245 246 247
temperatures. The high sensitive fraction of PME was rapidly inactivated during CUT.
248
After that, the slope of the PME inactivation curve decreased due to presence of heat
249
resistance fraction; so the extension of the heat treatment had no especial effect on
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Fig. 1 shows the nonlinear inactivation of PME during heat treatment at applied
251
thermal resistances were recognized in the sour orange juice treated at 60℃ while at
252
70, 80 and 90℃ only two isoforms were revealed (Fig 1a). Inactivation kinetics of heat
253
resistant PME fractions is shown in Fig. 1b. Significant difference in PME inactivation
254
behavior during heat treatment of the samples at different temperatures was observed
255
(P < 0.05). In this study the thermal inactivation of the heat resistant fraction of PME
256
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PME inactivation of heat resistance fraction. Three isoforms of PME with different
257
was completely inactivated up to 70℃ and also isoform with higher thermal resistance
258
influences considerably on the turbidity or cloud stability of citrus juice (Cameron et
259
al., 1998). The D-values were obtained from Fig.1b as summarized in Table 3. The
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was investigated during the holding time because the heat sensitive fraction of PME
261
(Ingallinera et al., 2005; Sio et al., 2001; Tajchakavit and Ramaswamy, 1997).
262
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results show that D-value decreased with increase in temperature during heat treatment
263
PME (R > 0.98); that indicates the high resistance of this enzyme to the heat
264
treatment. Ingallinera et al. (2005) studied the thermal resistance of PME in different
265
cultivars of orange juice. They reported the Z-value for this enzyme in Navel,
266
Sanguinello, Tarocco and Moro were 21.5, 16.7, 34.7 and 17.2℃, respectively.
267
Tajchakavit & Ramaswamy (1997) reported the Z-values 17.6 and 31.1°C respectively
268
for the heat sensitive and resistant fractions of PME during heat treatment of orange
269
juice in water bath; while only one fraction of PME with Z-value of 13.4°C was found
270
Fig. 2 shows the Z-value of 36.9 °C was obtained for the heat resistant fraction of 2
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isoforms in orange juice was between 9.2 and 16.4℃ during heat treatment from 75 to
272
95℃. These results show that thermal resistance of PME is dependent on fruit cultivar
273
(Kimball, 1991; Rouse, 1953), applied treatment method and different isoforms of the
274
enzyme (Cameron and Grohmann, 1996).
275
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during heat treatment in microwave. Sio et al. (2001) reported that the Z-value of PME
276
considerable degradation in a specific compound (Vikram et al., 2005). Ea, required
277
energy for inactivation of PME in sour orange juice, was 62.61± 0.84 kJ/mol (Fig. 3).
278
Ingallinera et al. (2005) reported that activation energy of different cultivars of orange
279
juice ranging from 65 - 135 kJ/mol during conventional heating at 70 - 85℃. These
280
high Ea revealed the high thermal resistance of PME. Generally, activation energy is
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High activation energy indicates that a slight change in temperature results in
influenced by used heat treatment conditions, type of fruit and its cultivar.
282 283
are shown in Table 4. Enthalpy (∆H) decreased insignificantly (P > 0.05) by increasing
284
the temperature. As enthalpy is related to the bonds strength and is the measure of
285
energy obstacle that must be overcome by reacting molecules (Vikram et al., 2005), the
286
small variations in ∆H represent the insignificance changes in PME structure during
287
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The thermodynamic parameters for thermal inactivation of sour orange juice PME
288
denaturation of heat resistance PME isoform is an endothermic reaction (D'Amico et
289
al., 2003; Nielsen et al., 2003). Entropy (∆S) is associated with the number of
290
molecules with sufficient energy to react (Vikram et al., 2005). The negative obtained
291
entropy that didn’t change during heat treatment (P > 0.05) represents the chemical
292
reaction to hydrogen and disulfide bonds forming in enzyme structure during heat
293
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sour orange juice heat treatment. The positive value of enthalpy also indicates that the
treatment (Anema and McKenna, 1996; Dannenberg and Kessler, 1988; Owusu et al.,
294
1992). Free energy (∆G) measures the spontaneity of a reaction (D'Amico et al., 2003).
295
∆G increased by rising in temperature (P < 0.05). The high amount of ∆G approved the
296
high thermal resistance of PME in sour orange juice (Kouadio et al., 2013).
297 298
3. 3. Temperature-time history in heat treatment of sour orange juice
299 300
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During the CUT, the juice temperature reaches to the target temperature. As shown,
301
an increase in process temperature leads to an insignificant decrease in CUT (P >
302
0.05).
303 304
61.30% at 60, 70, 80 and 90℃, respectively. At higher process temperature, the effect
305
of CUT on PME inactivation increased. These results could be used in control and
306
optimization of heat treatment of sour orange juice in water bath at different
307
temperatures.
308
3. 4. Process time calculations and cloud value
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The CUT effectiveness on the PME inactivation was 45.97%, 52.79%, 57.62% and
309 310 311 312
60, 70, 80 and 90℃, PME activity residuals were 1.18 ×10-4, 4.13×10-5, 3.54×10-5 and
313
2.48×10-5, respectively. Rothschild et al. (1975) and Holland et al. (1976) mentioned
314
that citrus juice is considered commercially stable when the PME activity is less than
315
10-4 unit/ml. Subsequently in all applied temperatures, except 60℃, the cloud stability
316
of sour orange juice was guaranteed by 1D (90%) reduction in initial activity of PME
317
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PME activity of fresh sour orange juice was 4.37×10-4 unit/ml. After treatment at
occurred. Using the Eq 5, considering 80℃ as Tr, and also the CUT effectiveness, heat
318
treatment time is computed 76, 66, 48, 35 and 21 min at 70, 75, 80, 85 and 90℃,
319
respectively.
320 321
corrected time. Absorbance of fresh juice was 0.444 ± 0.08 at 660 nm which increased
322
insignificantly (P > 0.05) 0.482 ± 0.04, 0.511 ± 0.07, 0.524 ± 0.05, 0.535 ± 0.06 and
323
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Fig. 4 shows changes in cloud value during sour orange juice heat processing in
0.542 ± 0.08 after heat treatment at 70, 75, 80, 85 and 90℃, respectively. The results
324
also showed that the processing time influences on the cloud value significantly (P <
325
0.05).
326 327
4. Conclusions
328 329
In this study, the inactivation of PME and some thermodynamic parameters of sour
330
orange juice were described by calculating the D and Z values. Different fractions of
331
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332
CUT, based on the estimated Z-value of PME, was used to estimate the required time
333
for sour orange juice heat treatment at 70, 75, 80, 85 and 90℃. Thermal treatment
334
results in an increase in cloud value by reduction in PME activity. In conclusion, the
335
optimal thermal treatment based on PME inactivation improves the desirable turbid
336
appearance of the product.
337
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PME with different thermal resistance were found during heat treatment. Corrected
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298.
399
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Polydera, A., Galanou, E., Stoforos, N., Taoukis, P., (2004). Inactivation kinetics of
400
juices and comminuted products of Israeli citrus fruits*. International Journal of Food
401
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Rothschild, G., Vliet, C.V., Karsenty, A., (1975). Pasteurization conditions for
Science & Technology 10(1), 29-38.
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Rouse, A., (1953). Distribution of pectinesterase and total pectin in component parts
402 403
of citrus fruits, Food Technology. INST FOOD TECHNOLOGISTS SUITE 300 221 N
404
LASALLE ST, CHICAGO, IL 60601-1291, pp. 19-19.
405
Sio, F., Palmieri, A., Servillo, L., Giovane, A., Castaldo, D., (2001).
406
Thermoresistance of pectin methylesterase in Sanguinello orange juice. Journal of food
407
biochemistry 25(2), 105-115.
408
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Snir, R., Koehler, P., Sims, K., Wicker, L., (1996). Total and thermostable pectinesterases in citrus juices. Journal of Food Science 61(2), 379-382.
409 410 411
Kinetics of Pectin Methylesterase in Orange Juice Under Batch Mode Heating
412
Conditions. LWT-Food Science and Technology 30(1), 85-93.
413
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Tajchakavit, S., Ramaswamy, H., (1997). Thermalvs. Microwave Inactivation
414
kinetics of Bacillus amyloliquefaciensα‐amylase in an aqueous solution of sodium
415
dodecyl sulphate and the kinetics in the solution of anionic‐phospholipid vesicles. Biotechnology and applied biochemistry 38(2), 175-181.
417
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Tanaka, A., Hoshino, E., (2003). Similarities between the thermal inactivation
416
Tiwari, B., Muthukumarappan, K., O'donnell, C., Cullen, P., (2009). Inactivation
418
kinetics of pectin methylesterase and cloud retention in sonicated orange juice.
419
Innovative Food Science & Emerging Technologies 10(2), 166-171.
420
Versteeg, C., Rombouts, F., Spaansen, C., Pilnik, W., (1980). Thermostability and
421
orange juice cloud destabilizing properties of multiple pectinesterases from orange.
422
Journal of Food Science 45(4), 969-971.
423
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Vikram, V., Ramesh, M., Prapulla, S., (2005). Thermal degradation kinetics of
424
nutrients in orange juice heated by electromagnetic and conventional methods. Journal
425
of Food Engineering 69(1), 31-40.
426
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pulp. Journal of Food Science 53(1), 162-164.
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Wicker, L., Temelli, F., (1988). Heat inactivation of pectinesterase in orange juice
427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445
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Figure Captions:
455 456 457
various temperatures and b. expanded view of thermal inactivation of heat resistance
458
fraction.
459
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Fig. 1. a. Residual PME activity in sour orange juice as a function of heating time at
Fig. 2. Temperature sensitivity of heat resistance isoform of PME in sour orange juice.
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Fig. 3. Arrhenius plot of thermal inactivation of PME in sour orange juice.
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Fig. 4. Cloud value changes during heat treatment of sour orange juice at different
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temperatures.
460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476
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477 478 479 480 481 482 483 484 485
16
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Table Captions:
486 487
Table 1. Published data for thermal inactivation of PME of citrus juice.
488 489
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Table 2. Some physicochemical properties of fresh sour orange juice.
490 491 492
processing at various temperatures.
493
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Table 3. Constant rate and D-value for PME in sour orange juice during thermal
494 495
60-90℃.
496
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Table 4. Thermodynamic parameters of PME during treatment of sour orange juice at
17
497
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Table 1. Published data for thermal inactivation of PME of citrus juice. Product
Temperature range (℃)
Equipment
sweet orange juice
60-85
microwave two
82.5 – 87.5
plate heat exchanger
75 - 95
oil bath
Valencia orange juice
80
water bath
orange juice
60-90
Valencia orange juice
50-65
orange juice
60 - 90
water bath
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Reference
Cinquanta et al. (2010)
Tribess & Tadini (2006)
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Two
four two
microwave one water bath
two
water bath
three
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60 - 90
Two
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Mixed juice of two orange varieties (Pera & Lime) Sanguinello orange juice
Number of isoforms
Sio et al. (2001)
Cameron et al. (1998) Tajchakavit & Ramaswamy (1997) Wicker & Temelli (1988) Versteeg et al. (1980)
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Properties Value pH 3.01 ±0.02 Acidity (%citric acid) 5.32 ±0.04 Density (g/cm3) 1.045 ±0.01 Total soluble solids (°Brix) 10.66 ±0.04 Moisture content (%) 87.03 ±0.06 Pulp content (%) 2 ±0.14 Ash (dry basis) (%) 0.31 ±0.03 mean ± Standard Deviation (n=3).
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Table 2. Some physicochemical properties of fresh sour orange juice.
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Table 3. Constant rate and D-value for PME in sour orange juice during thermal processing at various temperatures. Constant rate (min -1)
D-value (min)
R2
60
0.0151 ± 0.001
152.5 ± 2.86
0.92
70
0.0289 ± 0.001
77.3 ± 2.20
0.94
80
0.0465 ± 0.002
49.5 ± 2.41
0.96
90
0.104 ± 0.008
22.1 ± 1.78
0.94
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TE D
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mean ± Standard Deviation (n=3).
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Temperature (℃)
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Table 4. Thermodynamic parameters of PME during treatment of sour orange juice at 6090℃.
60 70 80 90
Enthalpy (kJ/mol)
Entropy (kJ/mol)
Free energy (kJ/mol.K)
59.84 ± 0.85 59.76 ± 0.83 59.67 ± 0.84 59.59 ± 0.85
-0.101 ± 0.02 -0.101 ± 0.01 -0.102 ± 0.01 -0.101 ± 0.02
93.52 ± 0.01 94.47 ± 0.03 96.00 ± 0.02 96.37 ± 0.01
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mean ± Standard Deviation (n=3).
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Temperature (℃)
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2.5
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1.5
90℃
1.0
60℃
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Log ((PEU/PEU0) × 100)
2.0
0.0 0
1
2
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0.5
3
4 5 Time (min)
6
70℃ 80℃
7
8
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(a)
1.6
1.4
1.2 1.1 1.0
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1.3
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Log ((PEU/PEU0) × 100)
1.5
60℃ 70℃ 80℃
0.9
90℃
0.8 0.7 0.6
0
2
4 Time (min) 6
8
10
(b) Fig. 1. a. Residual PME activity in sour orange juice as a function of heating time at various temperatures and b. expanded view of thermal inactivation of heat resistance fraction.
9
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4 3.5 3
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R² = 0.989
2 1.5
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1 0.5 0 55
60
65
70 75 Temperature (℃)
80
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50
85
90
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Fig. 2. Temperature sensitivity of heat resistance isoform of PME in sour orange juice.
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Log (D)
2.5
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1/Temperature × 103 (K-1) 2.8
2.9
3
-1.5
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y = -7.5301x + 18.392 R² = 0.9862 -3.5
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Ln (k)
-2.5
3.1
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2.7
-4.5
-5.5
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Fig. 3. Arrhenius plot of thermal inactivation of PME in sour orange juice.
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0.25
70℃
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75℃ 80℃ 85℃
0.15
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90℃
0.05
0.00 0
10
20
30
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0.10
40
50
60
70
80
TE D
Time (min)
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Fig. 4. Cloud value changes during heat treatment of sour orange juice at different temperatures.
AC C
Ln (treted juice abs/fresh juice abs)
0.20
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Highlights -
PME is known as a pasteurization index in sour orange juice.
-
Beside destruction of target microorganisms, the optimal thermal treatment based on PME inactivation improves the turbid appearance of the juice. Different fractions of PME with different thermal resistance were found during thermal
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-
treatment. -
Corrected come up time, based on the estimated Z-value of PME, was used to estimate the time of sour orange juice pasteurization.
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Thermal treatment led to increment in cloud value by reduction in PME activity.
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S0260-8774(16)30119-4
DOI:
10.1016/j.jfoodeng.2016.04.004
Reference:
JFOE 8535
To appear in:
Journal of Food Engineering
Received Date: 13 July 2015 Revised Date:
2 March 2016
Accepted Date: 2 April 2016
Please cite this article as: Aghajanzadeh, S., Ziaiifar, A.M., Kashaninejad, M., Maghsoudlou, Y., Esmailzadeh, E., Thermal inactivation kinetic of pectin methylesterase and cloud stability in sour orange juice, Journal of Food Engineering (2016), doi: 10.1016/j.jfoodeng.2016.04.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Thermal inactivation kinetic of pectin methylesterase and
1
cloud stability in sour orange juice
2 3
a*
a
a
Yahya Maghsoudlou b, Ebrahim Esmailzadeh c a
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Sara Aghajanzadeh , Aman Mohammad Ziaiifar , Mahdi Kashaninejad ,
Department of Food Process Engineering, Gorgan University of Agricultural
Sciences and Natural Resources, Basij Square, Gorgan, Iran.
Department of Food Science and Technology, Gorgan University of Agricultural
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b
Sciences and Natural Resources, Basij Square, Gorgan , Iran.
Biosystem Engineering, Gorgan University of Agricultural Sciences and Natural
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c
Corresponding author email: [email protected]
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*
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Resources, Basij Square, Gorgan , Iran.
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Abstract
29 30 31
products such as sour orange juice. Inactivation of pectin methylesterase enzyme
32
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Heat treatment is a common method to improve cloud stability of high acid food
33
In this work, the kinetics of PME thermal inactivation and cloud stability in sour
34
orange juice were investigated. The fresh sour orange juice was heated in a controlled
35
water bath at 60, 70, 80 and 90℃ for different times depending on applied temperature.
36
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(PME), naturally found in citrus, is known as a heat treatment index for these products.
37
inactivation kinetics of PME was calculated in terms of thermal resistance parameters
38
(D & Z values), activation energy, enthalpy, entropy and free energy. The obtained Z-
39
value (36.90℃), activation energy (62.61± 0.84 kJ/mol) and free energy (93.52 - 96.37
40
kJ/mol) revealed the high stability of PME during the heat treatment. Based on thermal
41
resistance of PME, heating times were corrected by considering the come-up time
42
effectiveness. The results showed that cloud value of heated juice increased by PME
43
inactivation during heat treatment.
44
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Time–temperature data was simultaneously recorded using a data-logger. The
Chemical compounds studied in this article
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Sodium chloride (PubChem CID: 5234); Sodium hydroxide (PubChem CID: 14798); Phenolphthalein (PubChem CID: 4764)
45 46 47 48 49 50
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Keywords: Sour orange juice; Heat treatment; Pectin methylesterase; Cloud stability
51 52 53 54 55 56
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1. Introduction
57 58 59
which are considered to be effective in prevention of cardiovascular disease, various
60
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Citrus fruits contain antioxidants such as ascorbic acid and phenolic compounds
cancers and diabetes (Antunes et al., 2010; Du et al., 2009). Sour orange (Citrus
61
aurantium L), also known as bitter orange, is commonly grown in the south-west of
62
Asia, especially in Iran. Despite the bitter and sour taste of this fruit, its juice is used as
63
a seasoning in various food products and salads (He et al., 1997).
64 65
the membrane and high molecular weight compounds are suspended. These
66
compounds consist of proteins, hesperidin, cellulose, hemicellulose and pectin
67
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As the endocarp cells are ruptured during the citrus juice mechanical extraction,
(Kimball, 1991). These colloidal suspensions are responsible for cloud and turbidity of
68
citrus juice (Kimball, 1991). Cloud stability is a desirable citrus juice quality
69
characteristic which influences the flavor, color and mouth feel of the product (Tiwari
70
et al., 2009).
71 72
cloud stability is undeniable (Kimball, 1991). Pectin is rich in galacturonic acid units
73
linked together via glycoside bonds with side chains of rhamnose, arabinans, galactans,
74
xylose and fucose (Kimball, 1991). Pectin methylesterase (PME), also known as
75
pectinase, pectin esterase and pectin methoxylase, affects the cloud stability and
76
viscosity of citrus juice by de-esterification of the methoxylated pectin and formation
77
of insoluble calcium pectate (Fayyaz et al., 1995; Oakenfull and Scott, 1984).
78
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Although pectin composes a small portion of the cloud materials, its effect on juice
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Thermal pasteurization is a common method to control the microbial and enzymatic
79
activities in high acid products such as citrus juice (Chen et al., 1993). Table 1 shows
80
that there are several isoforms of PME with different thermal resistance which are
81
deactivated gradually during citrus juice heat treatment (Cameron et al., 1998;
82
Cinquanta et al., 2010; Tajchakavit and Ramaswamy, 1997; Versteeg et al., 1980;
83
Wicker and Temelli, 1988). Hence there are different PME isoforms depend on citrus
84
variety and heat treatment condition such as used method, time and temperature.
85
Published studies show that thermal resistance of PME is higher than target
86
microorganisms so this enzyme is introduced as heat treatment index for citrus
87
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88
1980). The residual activity of PME plays an important role in cloud stability of citrus
89
juice (Holland et al., 1976; Rothschild et al., 1975). The inactivation of enzymes and
90
microorganisms mainly occur during holding time and also come-up time (CUT). So it
91
is necessary to estimate the CUT effectiveness to evaluate the exact intensity of heat
92
treatment on PME inactivation. To prevent the overheating of the product and
93
subsequently energy wasting, organoleptic and nutritional losses the effectiveness of
94
CUT should be considered to reduce the heating process time as suggested by Ball
95
(1923) and Tajchakavit & Ramaswamy (1997).
96
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products (Chen and Wu, 1998; Polydera et al., 2004; Snir et al., 1996; Versteeg et al.,
97
PME inactivation during heat treatment of sour orange juice. Arrhenius plot and
98
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The objective of this work was to study the kinetic parameters (D and Z-value) of
thermodynamic parameters of PME inactivation such as free energy (∆G), enthalpy
99
(∆H) and entropy (∆S) were also investigated. Due to the effect of PME on pectin de-
100
esterification and sour orange juice turbidity, the cloud value changes were studied by
101
considering the CUT effectiveness on PME inactivation during heat treatment.
102
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2. Materials and methods
2. 1. Sample preparation
103 104 105 106 107 108
Iran) and kept at 4℃ until the experiments were carried out. The juice was extracted
109
from the washed and squeezed fruits using a domestic juicer. It was then filtered by a
110
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Sour orange (Citrus aurantium L.) was purchased from a local market (Gorgan,
sieve with mesh size 170 to remove large size particles such as pulp, seed, etc.
111 112
2.2. Heat treatment
113 114
Heat treatments were carried out in a water bath (WNB-22, Memmert, Germany) at
115
different temperatures (60, 70, 80 and 90℃) for various times. The juice sample (15
116
ml) was sealed in a test tube (15 mm outer diameter, 160 mm length and 1 mm
117
thickness). Time–temperature data of sample during heating was recorded using a 1mm
118
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diameter copper–constantan thermocouple (T-type) and a data logger (TC-08,
119
Pichotechnology Co, UK). The sample was then rapidly cooled using ice-water bath to
120
minimize the effect of cooling time on the enzyme inactivation.
121 122
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2. 3. Methods of analysis
2.3.1. Fresh sour orange juice analyses
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Density, total acidity, pH, total soluble solids, moisture content and total ash of fresh sour orange juice were determined (AOAC, 2012). A glass pycnometer was used 3
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to measure the density of sample (g/cm ) at 25°C. Total acidity was determined by
123 124 125 126 127 128 129 130
expressed as citric acid percentage. pH was measured using pH-meter (W3B, BEL,
131
Italy) at 25°C. Total soluble solids (°Brix) were determined by a refractometer
132
(UV/VIS80 +T, PG Instrument, US) at 25°C. Moisture content of sour orange juice was
133
measured by drying the sample to a constant weight at 105 ± 1°C in a hot-air oven
134
(FD53, Binder, Germany). The ash content was measured by burning the juice in a
135
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titration the juice with NaOH 0.1 N solution using phenolphthalein as indicator and
136
the pulp volume of centrifuged juice at 1500 rpm for 10 min was measured to
137
determine the pulp content of the sample.
138
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silica crucible at 525°C up to a constant weight. According to Kimball (1999) method,
2. 3. 2. PME Activity
139 140
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To measure the PME activity, 5 ml of sour orange juice was mixed with 20 ml of
142
1% pectin-salt solution (10 g pectin and 15.3 g NaCI diluted in 1 L distilled water) and
143
incubated at 30℃. The pH of the solution was adjusted to 7 using NaOH (2 N). Then,
144
the pH of the solution was readjusted to 7.7 with NaOH (0.05 N). Finally, 0.1ml of
145
NaOH (0.05 N) was added and the time to regain pH 7.7 was recorded (Kimball,
146
1991). The enzyme activity unit (PEU) was calculated according to Eq. 1:
147 148
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PEU (unit/ml) =
. .
(1)
149 150
2. 3. 3. Cloud value
151
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152 153
heated for 76, 66, 48, 35 and 21 min at 70, 75, 80, 85 and 90℃, respectively.
154
According to method of Versteeg et al. (1980), 5 ml of sour orange juice was
155
centrifuged at 3000 rpm for 10 min at room temperature. Cloud value was measured as
156
supernatant absorbance at 660 nm (T-80, UV/VIS Double Beam Spectrophotometer).
157
In this method absorbance of distilled water was considered as a blank.
158
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In order to study the kinetic of cloud stability, the 15 ml of sour orange juice was
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2. 3. 4. Kinetic parameters analysis
160 161
The thermal inactivation of PME is often described by first order kinetic model described by the following equation:
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Log = −k . t
162 163 164
(2)
165 166
Where A and A0 are residual and initial enzyme activity (PEU), respectively. k is -1
168
to reduce the PME by 90% is called decimal reduction time (D-value) which is
169
inversely related to the reaction rate constant (Eq. 3):
170
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the inactivation rate constant (min ) and t represents the time (min). The time required
167
171
D = 2.303/ k
(3)
172 173
The negative slope of D-value on a log scale against the corresponding temperature
174
is termed the Z-value which represents the increase in temperature to attain 10 folds
175
decrement in D-value (Eq. 4):
176 177
Z=
$% &$'
(4)
( )' &( )%
6
178
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179 180
on time–temperature profiles during the CUT, D and Z values were computed from the
181
uncorrected heating times. The effective heating times (te) were calculated using Eq. 5
182
in which the lethality (L) was computed using the uncorrected Z-value.
183
te =* L dt = * 10
./.012 3
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Where T2 and T1 are temperatures corresponding to D2 and D1, respectively. Based
dt
(5)
184 185 186 187
each heat treatment). Using the corrected thermal times, D and Z values were
188
recalculated. The more accurate thermal times were recalculated using the Eq. 5 with
189
the new Z-value. This procedure was repeated until difference between two sequential
190
Z-values were within 5 %. Finally, the CUT effectiveness was calculated at each heat
191
treatment temperature by the calculated lethality or thermal time (Eq. 5) divided by the
192
CUT (Tajchakavit and Ramaswamy, 1997).
193
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Where Tref is the reference temperature (in this study, the water bath temperature in
194
microorganisms so it should be considered in calculation of the process time (Ball,
195
1923). When the heating time is longer than CUT, a correction factor equal to effective
196
portion of CUT is required. In the present study, this factor is obtained from time-
197
temperature and inactivation profile of PME during heat treatment (Tajchakavit and
198
Ramaswamy, 1997).
199
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Ball (1923) mentioned that 42% of CUT influences the inactivation of enzymes and
200
Arrhenius model (Eq. 6). Thermal inactivation of PME is related to breaking up the
201
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Temperature dependency of a reaction constant is usually described by the
hydrogen bonds, unfolding of tertiary protein structure and amino acid thermal
202
deamidation of the enzyme (Tanaka and Hoshino, 2003). According to Eq. 6, the slope
203
of the semi-logarithm of reaction rate constant (k) vs. reciprocal of absolute
204
temperature is equal to the activation energy (Ea).
205 206
/45
k = k 6.
(6)
207 208
7
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Where Ea is activation energy (J/mol), K0 is frequency factor or the Arrhenius
209
constant (min-1), R is the universal gas constant (8.3144 J/mol K), and T is absolute
210
temperature (K).
211
Enthalpy, entropy and free energy were described by Eyring models (Eq. 7 to 9).
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Enthalpy at each temperature was calculated according to Eq. 7: ∆H = ∆E − RT
(7)
Where ∆H represents enthalpy (J.mol ) and ∆E is activation energy (J/mol). Free
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∆G = −RTlnKh/k B T
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energy (J.mol-1) at different temperature was calculated by Eq. 8:
212 213 214 215 216 217 218 219
(8)
220 221 222
J) and k B is Boltzmann Constant (1.3806×10-23 J.K-1). Entropy (∆S, J.mol-1.K-1) for
223
PME inactivation was calculated using Eq. 9:
224
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Where K is inactivation constant rate of PME, h is Planck constant (6.6262× 10-34
∆S = ∆H − ∆G /T
225
(9)
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2.4. Statistical analysis
226 227 228 229 230 231
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All experiments were performed in triplicate and the presented results are the mean of the obtained value ± standard deviation. Results were submitted to analysis of
232
variance (ANOVA) with significance level of P < 0.05. All statistical analyses were
233
conducted using the SAS software (version 9.1).
234
3. Results and Discussion
236
235
237
3. 1. Physicochemical properties of sour orange juice
238 239
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240
sour orange juice can be categorized in high acid food products (pH < 3.7); therefore
241
the heat treatment below 100℃ is adequate to inactivate the enzyme in order to
242
guarantee the cloud stability of the product.
243
3. 2. Thermal inactivation kinetics of PME
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Table 2 shows the physicochemical properties of fresh sour orange juice. As shown,
244 245 246 247
temperatures. The high sensitive fraction of PME was rapidly inactivated during CUT.
248
After that, the slope of the PME inactivation curve decreased due to presence of heat
249
resistance fraction; so the extension of the heat treatment had no especial effect on
250
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Fig. 1 shows the nonlinear inactivation of PME during heat treatment at applied
251
thermal resistances were recognized in the sour orange juice treated at 60℃ while at
252
70, 80 and 90℃ only two isoforms were revealed (Fig 1a). Inactivation kinetics of heat
253
resistant PME fractions is shown in Fig. 1b. Significant difference in PME inactivation
254
behavior during heat treatment of the samples at different temperatures was observed
255
(P < 0.05). In this study the thermal inactivation of the heat resistant fraction of PME
256
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PME inactivation of heat resistance fraction. Three isoforms of PME with different
257
was completely inactivated up to 70℃ and also isoform with higher thermal resistance
258
influences considerably on the turbidity or cloud stability of citrus juice (Cameron et
259
al., 1998). The D-values were obtained from Fig.1b as summarized in Table 3. The
260
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was investigated during the holding time because the heat sensitive fraction of PME
261
(Ingallinera et al., 2005; Sio et al., 2001; Tajchakavit and Ramaswamy, 1997).
262
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results show that D-value decreased with increase in temperature during heat treatment
263
PME (R > 0.98); that indicates the high resistance of this enzyme to the heat
264
treatment. Ingallinera et al. (2005) studied the thermal resistance of PME in different
265
cultivars of orange juice. They reported the Z-value for this enzyme in Navel,
266
Sanguinello, Tarocco and Moro were 21.5, 16.7, 34.7 and 17.2℃, respectively.
267
Tajchakavit & Ramaswamy (1997) reported the Z-values 17.6 and 31.1°C respectively
268
for the heat sensitive and resistant fractions of PME during heat treatment of orange
269
juice in water bath; while only one fraction of PME with Z-value of 13.4°C was found
270
Fig. 2 shows the Z-value of 36.9 °C was obtained for the heat resistant fraction of 2
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271
isoforms in orange juice was between 9.2 and 16.4℃ during heat treatment from 75 to
272
95℃. These results show that thermal resistance of PME is dependent on fruit cultivar
273
(Kimball, 1991; Rouse, 1953), applied treatment method and different isoforms of the
274
enzyme (Cameron and Grohmann, 1996).
275
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during heat treatment in microwave. Sio et al. (2001) reported that the Z-value of PME
276
considerable degradation in a specific compound (Vikram et al., 2005). Ea, required
277
energy for inactivation of PME in sour orange juice, was 62.61± 0.84 kJ/mol (Fig. 3).
278
Ingallinera et al. (2005) reported that activation energy of different cultivars of orange
279
juice ranging from 65 - 135 kJ/mol during conventional heating at 70 - 85℃. These
280
high Ea revealed the high thermal resistance of PME. Generally, activation energy is
281
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High activation energy indicates that a slight change in temperature results in
influenced by used heat treatment conditions, type of fruit and its cultivar.
282 283
are shown in Table 4. Enthalpy (∆H) decreased insignificantly (P > 0.05) by increasing
284
the temperature. As enthalpy is related to the bonds strength and is the measure of
285
energy obstacle that must be overcome by reacting molecules (Vikram et al., 2005), the
286
small variations in ∆H represent the insignificance changes in PME structure during
287
TE D
The thermodynamic parameters for thermal inactivation of sour orange juice PME
288
denaturation of heat resistance PME isoform is an endothermic reaction (D'Amico et
289
al., 2003; Nielsen et al., 2003). Entropy (∆S) is associated with the number of
290
molecules with sufficient energy to react (Vikram et al., 2005). The negative obtained
291
entropy that didn’t change during heat treatment (P > 0.05) represents the chemical
292
reaction to hydrogen and disulfide bonds forming in enzyme structure during heat
293
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sour orange juice heat treatment. The positive value of enthalpy also indicates that the
treatment (Anema and McKenna, 1996; Dannenberg and Kessler, 1988; Owusu et al.,
294
1992). Free energy (∆G) measures the spontaneity of a reaction (D'Amico et al., 2003).
295
∆G increased by rising in temperature (P < 0.05). The high amount of ∆G approved the
296
high thermal resistance of PME in sour orange juice (Kouadio et al., 2013).
297 298
3. 3. Temperature-time history in heat treatment of sour orange juice
299 300
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During the CUT, the juice temperature reaches to the target temperature. As shown,
301
an increase in process temperature leads to an insignificant decrease in CUT (P >
302
0.05).
303 304
61.30% at 60, 70, 80 and 90℃, respectively. At higher process temperature, the effect
305
of CUT on PME inactivation increased. These results could be used in control and
306
optimization of heat treatment of sour orange juice in water bath at different
307
temperatures.
308
3. 4. Process time calculations and cloud value
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The CUT effectiveness on the PME inactivation was 45.97%, 52.79%, 57.62% and
309 310 311 312
60, 70, 80 and 90℃, PME activity residuals were 1.18 ×10-4, 4.13×10-5, 3.54×10-5 and
313
2.48×10-5, respectively. Rothschild et al. (1975) and Holland et al. (1976) mentioned
314
that citrus juice is considered commercially stable when the PME activity is less than
315
10-4 unit/ml. Subsequently in all applied temperatures, except 60℃, the cloud stability
316
of sour orange juice was guaranteed by 1D (90%) reduction in initial activity of PME
317
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PME activity of fresh sour orange juice was 4.37×10-4 unit/ml. After treatment at
occurred. Using the Eq 5, considering 80℃ as Tr, and also the CUT effectiveness, heat
318
treatment time is computed 76, 66, 48, 35 and 21 min at 70, 75, 80, 85 and 90℃,
319
respectively.
320 321
corrected time. Absorbance of fresh juice was 0.444 ± 0.08 at 660 nm which increased
322
insignificantly (P > 0.05) 0.482 ± 0.04, 0.511 ± 0.07, 0.524 ± 0.05, 0.535 ± 0.06 and
323
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Fig. 4 shows changes in cloud value during sour orange juice heat processing in
0.542 ± 0.08 after heat treatment at 70, 75, 80, 85 and 90℃, respectively. The results
324
also showed that the processing time influences on the cloud value significantly (P <
325
0.05).
326 327
4. Conclusions
328 329
In this study, the inactivation of PME and some thermodynamic parameters of sour
330
orange juice were described by calculating the D and Z values. Different fractions of
331
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332
CUT, based on the estimated Z-value of PME, was used to estimate the required time
333
for sour orange juice heat treatment at 70, 75, 80, 85 and 90℃. Thermal treatment
334
results in an increase in cloud value by reduction in PME activity. In conclusion, the
335
optimal thermal treatment based on PME inactivation improves the desirable turbid
336
appearance of the product.
337
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PME with different thermal resistance were found during heat treatment. Corrected
SC
References
338 339 340 341 342
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Cameron, R.G., Baker, R.A., Grohmann, K., (1998). Multiple forms of
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Ingallinera, B., Barbagallo, R.N., Spagna, G., Palmeri, R., Todaro, A., (2005).
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AC C
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Thermoresistance of pectin methylesterase in Sanguinello orange juice. Journal of food
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Snir, R., Koehler, P., Sims, K., Wicker, L., (1996). Total and thermostable pectinesterases in citrus juices. Journal of Food Science 61(2), 379-382.
409 410 411
Kinetics of Pectin Methylesterase in Orange Juice Under Batch Mode Heating
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Tajchakavit, S., Ramaswamy, H., (1997). Thermalvs. Microwave Inactivation
414
kinetics of Bacillus amyloliquefaciensα‐amylase in an aqueous solution of sodium
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Tanaka, A., Hoshino, E., (2003). Similarities between the thermal inactivation
416
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418
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Innovative Food Science & Emerging Technologies 10(2), 166-171.
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Versteeg, C., Rombouts, F., Spaansen, C., Pilnik, W., (1980). Thermostability and
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orange juice cloud destabilizing properties of multiple pectinesterases from orange.
422
Journal of Food Science 45(4), 969-971.
423
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Vikram, V., Ramesh, M., Prapulla, S., (2005). Thermal degradation kinetics of
424
nutrients in orange juice heated by electromagnetic and conventional methods. Journal
425
of Food Engineering 69(1), 31-40.
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pulp. Journal of Food Science 53(1), 162-164.
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Wicker, L., Temelli, F., (1988). Heat inactivation of pectinesterase in orange juice
427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445
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Figure Captions:
455 456 457
various temperatures and b. expanded view of thermal inactivation of heat resistance
458
fraction.
459
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Fig. 1. a. Residual PME activity in sour orange juice as a function of heating time at
Fig. 2. Temperature sensitivity of heat resistance isoform of PME in sour orange juice.
SC
Fig. 3. Arrhenius plot of thermal inactivation of PME in sour orange juice.
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Fig. 4. Cloud value changes during heat treatment of sour orange juice at different
EP
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temperatures.
460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476
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477 478 479 480 481 482 483 484 485
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Table Captions:
486 487
Table 1. Published data for thermal inactivation of PME of citrus juice.
488 489
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Table 2. Some physicochemical properties of fresh sour orange juice.
490 491 492
processing at various temperatures.
493
SC
Table 3. Constant rate and D-value for PME in sour orange juice during thermal
494 495
60-90℃.
496
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Table 4. Thermodynamic parameters of PME during treatment of sour orange juice at
17
497
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Table 1. Published data for thermal inactivation of PME of citrus juice. Product
Temperature range (℃)
Equipment
sweet orange juice
60-85
microwave two
82.5 – 87.5
plate heat exchanger
75 - 95
oil bath
Valencia orange juice
80
water bath
orange juice
60-90
Valencia orange juice
50-65
orange juice
60 - 90
water bath
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Reference
Cinquanta et al. (2010)
Tribess & Tadini (2006)
SC
Two
four two
microwave one water bath
two
water bath
three
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60 - 90
Two
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Mixed juice of two orange varieties (Pera & Lime) Sanguinello orange juice
Number of isoforms
Sio et al. (2001)
Cameron et al. (1998) Tajchakavit & Ramaswamy (1997) Wicker & Temelli (1988) Versteeg et al. (1980)
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Properties Value pH 3.01 ±0.02 Acidity (%citric acid) 5.32 ±0.04 Density (g/cm3) 1.045 ±0.01 Total soluble solids (°Brix) 10.66 ±0.04 Moisture content (%) 87.03 ±0.06 Pulp content (%) 2 ±0.14 Ash (dry basis) (%) 0.31 ±0.03 mean ± Standard Deviation (n=3).
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Table 2. Some physicochemical properties of fresh sour orange juice.
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Table 3. Constant rate and D-value for PME in sour orange juice during thermal processing at various temperatures. Constant rate (min -1)
D-value (min)
R2
60
0.0151 ± 0.001
152.5 ± 2.86
0.92
70
0.0289 ± 0.001
77.3 ± 2.20
0.94
80
0.0465 ± 0.002
49.5 ± 2.41
0.96
90
0.104 ± 0.008
22.1 ± 1.78
0.94
AC C
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TE D
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mean ± Standard Deviation (n=3).
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Temperature (℃)
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Table 4. Thermodynamic parameters of PME during treatment of sour orange juice at 6090℃.
60 70 80 90
Enthalpy (kJ/mol)
Entropy (kJ/mol)
Free energy (kJ/mol.K)
59.84 ± 0.85 59.76 ± 0.83 59.67 ± 0.84 59.59 ± 0.85
-0.101 ± 0.02 -0.101 ± 0.01 -0.102 ± 0.01 -0.101 ± 0.02
93.52 ± 0.01 94.47 ± 0.03 96.00 ± 0.02 96.37 ± 0.01
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mean ± Standard Deviation (n=3).
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Temperature (℃)
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2.5
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1.5
90℃
1.0
60℃
SC
Log ((PEU/PEU0) × 100)
2.0
0.0 0
1
2
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0.5
3
4 5 Time (min)
6
70℃ 80℃
7
8
TE D
(a)
1.6
1.4
1.2 1.1 1.0
EP
1.3
AC C
Log ((PEU/PEU0) × 100)
1.5
60℃ 70℃ 80℃
0.9
90℃
0.8 0.7 0.6
0
2
4 Time (min) 6
8
10
(b) Fig. 1. a. Residual PME activity in sour orange juice as a function of heating time at various temperatures and b. expanded view of thermal inactivation of heat resistance fraction.
9
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4 3.5 3
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R² = 0.989
2 1.5
SC
1 0.5 0 55
60
65
70 75 Temperature (℃)
80
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50
85
90
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Fig. 2. Temperature sensitivity of heat resistance isoform of PME in sour orange juice.
AC C
Log (D)
2.5
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1/Temperature × 103 (K-1) 2.8
2.9
3
-1.5
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y = -7.5301x + 18.392 R² = 0.9862 -3.5
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Ln (k)
-2.5
3.1
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2.7
-4.5
-5.5
AC C
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Fig. 3. Arrhenius plot of thermal inactivation of PME in sour orange juice.
ACCEPTED MANUSCRIPT
0.25
70℃
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75℃ 80℃ 85℃
0.15
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90℃
0.05
0.00 0
10
20
30
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0.10
40
50
60
70
80
TE D
Time (min)
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Fig. 4. Cloud value changes during heat treatment of sour orange juice at different temperatures.
AC C
Ln (treted juice abs/fresh juice abs)
0.20
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Highlights -
PME is known as a pasteurization index in sour orange juice.
-
Beside destruction of target microorganisms, the optimal thermal treatment based on PME inactivation improves the turbid appearance of the juice. Different fractions of PME with different thermal resistance were found during thermal
RI PT
-
treatment. -
Corrected come up time, based on the estimated Z-value of PME, was used to estimate the time of sour orange juice pasteurization.
EP
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Thermal treatment led to increment in cloud value by reduction in PME activity.
AC C
-
1