A Review of Pectin Methylesterase Inactivation in Citrus Juice During Pasteurization

Accepted Manuscript
A review of pectin methylesterase inactivation in citrus juice during pasteurization
Sara Aghajanzadeh, Aman Mohammad Ziaiifar
PII: S0924-2244(17)30415-6
DOI: 10.1016/j.tifs.2017.10.013
Reference: TIFS 2100
To appear in: Trends in Food Science & Technology
Received Date: 26 June 2017

Revised Date: 8 October 2017
Accepted Date: 23 October 2017
Please cite this article as: Aghajanzadeh, S., Ziaiifar, A.M., A review of pectin methylesterase
inactivation in citrus juice during pasteurization, Trends in Food Science & Technology (2017), doi:
10.1016/j.tifs.2017.10.013.
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1 Abstract
3 Background
4 Pectin, naturally found in citrus, plays a key role in the quality of the obtained
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5 juices. Pectin methylesterase enzyme (PME) influences the cloud stability,
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6 viscosity, color, mouth feeling and flavor of the juices by de-esterification of
7 pectin. Iinactivation of PME is introduced as a pasteurization index in citrus juices,
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8 due to its higher thermal resistance than the spoilage microorganisms.
9 Scope and approach
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Inactivation of PME using different thermal (conventional, microwave and
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11 ohmic heating) and non-thermal (pulsed electric field, high pressure processing
12 and high pressure carbon dioxide) processes is important in juice production. The
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13 aim of this study was to review the effect of these processing methods on the PME
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14 inactivation in different citrus juices.

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15 Key finding and conclusion
16 Using non-thermal methods in combination with moderate thermal methods can

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17 be more effective in PME inactivation with minimum loss in citrus juice quality.
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19 Keywords: Pectin methylesterase; Citrus juice; Pasteurization; Thermal methods;

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20 Non-thermal methods
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1 A review of pectin methylesterase inactivation in citrus
2 juice during pasteurization
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4 Sara Aghajanzadeh a*, Aman Mohammad Ziaiifar a
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6 Department of Food Process Engineering, Gorgan University of Agricultural Sciences and Natural
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7 Resources, Basij Square, Gorgan, Iran.
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15 Corresponding author email: saraaghajanzadeh@yahoo.com
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25 Abstract
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27 Background
28 Pectin, naturally found in citrus, plays a key role in the quality of the obtained juices.
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29 Pectin methylesterase enzyme (PME) influences the cloud stability, viscosity, color,
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30 mouth feeling and flavor of the juices by de-esterification of pectin. Iinactivation of
31 PME is introduced as a pasteurization index in citrus juices, due to its higher thermal
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32 resistance than the spoilage microorganisms.
33 Scope and approach
34
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Inactivation of PME using different thermal (conventional, microwave and ohmic
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35 heating) and non-thermal (pulsed electric field, high pressure processing and high
36 pressure carbon dioxide) processes is important in juice production. The aim of this
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37 study was to review the effect of these processing methods on the PME inactivation in
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38 different citrus juices.

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39 Key finding and conclusion
40 Using non-thermal methods in combination with moderate thermal methods can be

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41 more effective in PME inactivation with minimum loss in citrus juice quality.
42
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43 Keywords: Pectin methylesterase; Citrus juice; Pasteurization; Thermal methods; Non-

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44 thermal methods
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49 1. Introduction
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51 Citrus fruits are rich sources of vitamins, minerals, fibers and also antioxidants such as
52 phenolic compounds which are effective in prevention of cardiovascular disease, various
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53 cancers and diabetes (Antunes, Dandlen, Cavaco, & Miguel, 2010; Du, Li, Ma, & Liang,
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54 2009). Serving of citrus is therefore recommended in the human diet. Juicing is used to
55 produce a high nutritional product from the citrus which decreases the waste and also
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56 making them available through the year. In addition, drinking juice could be more easily and
57 somewhat more pleasant than fruits and vegetables, especially for children and patients.
58
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Microorganisms are usually considered as an index to control the safety of food.
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59 However, the microbial studies are generally expensive and time-consuming. In
60 pasteurization of the acidic foods such as citrus juice, the inactivation of PME is introduced
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61 as an index, due to its higher thermal resistance than the spoilage microorganisms (Chen &
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62 Wu, 1998; Kimball, 1999; Polydera, Galanou, Stoforos, & Taoukis, 2004; Versteeg,
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63 Rombouts, Spaansen, & Pilnik, 1980). Pectin methylesterase (E.C. 3.1.1.11), also known as
64 pectinase, pectinesterase and pectin methoxylase is naturally found in citrus which is vital in
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65 the fruit ripening (Fayyaz, Asbi, Ghazali, Man, & Jinap, 1995; Oakenfull & Scott, 1984;
66 Schuch, 1994; Tieman & Handa, 1994). PME and high molecular weight compounds
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67 (proteins, hesperidin, cellulose, hemicellulose and pectin) are released and suspended in the
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68 juice during mechanical juice extraction as the endocarp cells are raptured. These colloidal
69 suspensions cause the cloud and turbidity appearance in the juice, being a desirable quality
70 characteristic in citrus juice (Kimball, 1999).
71 Pectin, a small portion of the cloud materials, plays an important role in the cloud
72 stability. It is rich in galacturonic acid units linked together via α (1→ 4) glycoside bonds
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73 with the side chains of sugars. Lots of its carboxyl groups are esterified with methanol to
74 form methoxy groups (Fig. 1. a). These methoxy groups prevent pectin from several
75 reactions including polymerization and gelatinization (Kimball, 1999). De-esterification of
76 the methoxylated pectin occurs by the activity of PME, during which calcium cations can
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77 easily react with demethylated pectin to produce insoluble pectate (Fig. 1.b). This reaction
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78 results in loss of juice cloudy appearance. Inactivation of PME during processing increases
79 the cloud stability and influences the viscosity, color, mouth feeling and flavor of the juices
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80 (Tiwari, Muthukumarappan, O'donnell, & Cullen, 2009).
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82 1. 1. Kinetic analysis
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84 Inactivation of PME is assumed to follow a semi- logarithmic first order kinetic as a

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85 function of treatment time, which can be written as Eq. 1 (Chen & Wu, 1998; Tajchakavit &
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86 Ramaswamy, 1997b).
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87
A
88 Ln( ) = −k.t Eq. 1
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A0
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90 where, k and t, A0 and A represent the reaction rate constant (min-1) at a specific
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91 treatment condition (time, pressure & etc.), process time (min), the initial and residual
92 activity of PME, respectively. The negative slope of regression of Ln ( ) vs. treatment
93 time denotes the reaction rate constant.
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94 Eq. 2 is used as a nonlinear regression model for inactivation of two isoforms of PME
95 which are inactivated following first-order kinetics under certain conditions (Tribess &
96 Tadini, 2006):
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A
98 = a × exp(−k1 × t ) + (1 − a) × exp(−k2 × t ) Eq. 2
A0
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99
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100 Where “a” and “1-a” represent the activity of resistance and sensitive isoforms,
101 respectively. k1 and k2 are the reaction rate constants (min-1) of resistance and sensitive
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isoforms, respectively. Decimal reduction time (D-value) can be used to quantify the
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103 effect of a specific treatment condition on the PME inactivation during processing. It is
104 defined as the required time to reduce 90% of the PME activity and obtained from Eq. 3:
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105
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106 D − value = Eq. 3
k
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108 D-value of PME varies depending on its isoforms, properties of the juice including a
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109 variety of raw material, pH, total soluble solid, extraction method of the juice and
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110 processing conditions (Kimball, 1999; Rouse, 1953; Snir, Koehler, Sims, & Wicker,
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111 1996). The negative reciprocal slope of the regression of the log of D-value vs.
112 temperature represents the Z-value (Eq. 4), showing the temperature sensitivity of D-
113 values. It is also defined as the temperature difference that causes a ten-fold change in
114 the rate of PME inactivation.
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T2 − T1
116 Z − value = Eq. 4
LogD1 − LogD2
117 Which, T2 and T1 are temperatures corresponding to D2 and D1, respectively. In HP
118 treatment, Z-value (Eq. 5) is similarly derived from the negative slope of the regression
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119 of log D-value vs. applied pressure (Nienaber & Shellhammer, 2001):
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P2 − P1121 Eq.5
Z p − value =
log D1 − log D2
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123 where, P2 and P1 are temperatures corresponding to D2 and D1, respectively.
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The effect of electric field intensity and treatment time on PME inactivation is
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125 however studied with different developed mathematical models. Eq.1 can be used as the
126 first-order fractional conversion model when the “t” is replaced by the frequency (Hz),
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127 the pulse width ( s) or the electrical energy density input (J/m3) (Elez-Martinez,
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128 Suarez-Recio, & Martin-Belloso, 2007). Fermi’s model (Eq. 6) describes the effect of
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129 applied electric intensity on the enzyme activity (Evrendilek, et al., 2012):
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A 1
131 = Eq. 6
A0 1 + exp( E − E h )
a
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132
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133 where, E indicates the electric field intensity (kV/cm), and Eh is a critical level of
134 electric field intensity where the PME activity is 50% (kV/cm) and “a” represents the
135 steepness of the curve around Eh. Weibull distribution model (Eq. 7) reflects the electric
136 field intensity or process time on PME activity (Evrendilek, et al., 2012):
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A x
137 Ln( ) = −( )b Eq. 7
A0 a
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139 where, “x” represents either electric field intensity or process time, “a” is scale
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140 parameter and “b” shows the shape of activity curve as b < 1 indicates a concavity
141 upwards, b = 1 straight line and b > 1 a concavity downwards. Hulsheger’s model (Eq. 8)
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142 presents the relation between PME activity and the applied electric field intensity (E)
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143 and treatment time (t) (Evrendilek, et al., 2012):
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E − Ec
A t −( )
145 =( ) a
Eq. 8
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A0 tc
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147 where, tc, Ec and a are critical treatment time, critical electric field intensity and a
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148 constant, respectively.
149
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150 1. 2. Isoforms of PME

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152 There are several isoforms of PME that representing the various role of this enzyme in
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153 the modification of the cell wall during the plant growth (Jolie, Duvetter, Van Loey, &
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154 Hendrickx, 2010). The number of PME isoforms depends on physicochemical properties of
155 the juice and maturity of the raw material. These are different molar mass (usually ranged
156 25-54kDa), isoelectric point (alkaline, natural or acidic), degree of glycosylation, thermal
157 kinetic properties and catalytic properties (Al-Qsous, et al., 2004; Duvetter, et al., 2009;
158 Jolie, et al., 2010; Kohli, Kalia, & Gupta, 2015). It was shown that the number of PME
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159 isoforms depends on physicochemical properties of the juice and maturity of the raw
160 material.
161 PME is encoded as large pre-pro-proteins that have peptide motifs (Giovane, et al., 2004).
162 All isoforms of this enzyme are catalyzed the same reaction (Jolie, et al., 2010). Active part
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163 of the PME domain is proceeded by an N-terminal extension (PRO region) which differ in
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164 length and it has low level of amino acid identity between the isoforms (Kohli, et al., 2015).
165 Different isoforms can have various mechanisms based on the presence of specific structural
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166 motifs (Pelloux, Rustérucci, & Mellerowicz, 2007).
167 The ratio of the different isoforms can vary considerably based on the development stage
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(like fruit ripening) and organ (in fruit, root or flower) (Jolie, et al., 2010). Different
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169 isoforms have similar inherent activity but different environmental situations are needed for
170 certain substrate and reaction mechanism (Pelloux, et al., 2007). For example, PME is
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171 sensitive to the ionic strength of the environment. As, the enzyme activity increased by
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172 rising in cation concentration up to a certain amount but higher that results in a reduction in
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173 enzyme activity (Duvetter, et al., 2009). Each isoform of the PME has its special pH optima
174 which directly affects its action pattern (Kohli, et al., 2015). Generally, pH optima of the
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175 most of the PME plant sources are ranged from 6 to 8 (Duvetter, et al., 2009).
176 Temperature is another factor affecting the enzyme activity; for example, at the
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177 temperatures above 70 ℃, the heat stable isoform is rapidly inactivated (Duvetter, et al.,
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178 2009). Thermal processing of the juices revealed the existence of several thermal resistance
179 isoforms (Aghajanzadeh, Ziaiifar, Kashaninejad, Maghsoudlou, & Esmailzadeh, 2016a;
180 Cameron, BAKER, & GROHMANN, 1998; Tajchakavit & Ramaswamy, 1997b; Versteeg,
181 et al., 1980). Conventional thermal processing method needs longer time which leads to
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182 nutritional loss and undesirable changes in the color of juice (Ling, Tang, Kong, Mitcham,
183 & Wang, 2015). The ability to steadily meet customer expectations requires alignment of
184 new food processing technologies to reduce or eliminate these unwanted effects. Some of
185 the important novel thermal or non-thermal processing methods consist of microwave (MW)
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186 treatment, ohmic heating (OH), pulsed electric field (PEF), high pressure (HP) and high
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187 pressure carbon dioxide (HPCD) treatment. The objective of this study is therefore to review
188 the effect of these processing methods on the PME inactivation in different citrus juices.
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189
190 2. Juice pasteurization
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192 To produce a juice, different production steps should be planned, including cleaning,
193 sorting, peeling, juice extraction, filtration and thermal or non-thermal treatment
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194 (pasteurization or sterilization), filling, sealing and cooling. Thermal treatment inactivates
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195 the microorganisms and enzymes in order to guarantee the safety and also increase the shelf
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196 life of the juice (Aghajanzadeh, Ziaiifar, et al., 2016a). Generally, low-acid juices (pH > 4.6)
197 are heated at higher temperature than 100℃ (sterilization); while, acidic juices (pH < 4.6)
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198 require the heat treatment below 100 ℃ (pasteurization). The intensity of pasteurization
199 process (temperature and time) is important to guarantee the safety, shelf life and quality of
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200 the juice. Pasteurization process, depending on its intensity, lasts to destroy certain
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201 vegetative microbial species or enzymes up to the desired degree which is performed using
202 several thermal and non-thermal methods.
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206 2. 1. Thermal pasteurization of juice
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208 Conventional, microwave and ohmic heating are well-known thermal pasteurization
209 methods of citrus juices. Depending on the juice, different time-temperature combinations
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210 (process intensity) are applied. As the juice temperature increases, the microorganisms and
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211 enzymes are more deactivated due to reversible or irreversible changes in their structures.
212 Thermal inactivation of PME begins above 40℃ as a result of breaking up the hydrogen
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213 bonds, unfolding of tertiary protein structure and amino acid thermal deamidation (Tanaka
214 & Hoshino, 2003). The plot of PME inactivation vs. thermal processing time is often curve-
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linear (Fig. 2). This fact is related to the existence of various isoforms of this enzyme with
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216 different thermal resistances, which are deactivated gradually during the process. These
isoforms are categorized to heat sensitive and heat resistance by breaking the curve to
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218 several linear ones or first order rate curves (Cinquanta, Albanese, Cuccurullo, & Di Matteo,
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219 2010; Espachs-Barroso, Van Loey, Hendrickx, & Martín-Belloso, 2006; Tajchakavit &
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220 Ramaswamy, 1997b). A different isoform of PME can be also recognized depending on the
221 used method, processing time and juice temperature variation during heat treatment (Table
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222 1).
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226 Conventional thermal pasteurization refers to heating the juice up to the desired
227 temperature and holding it for a specific time, based on the thermal resistance of target
228 microorganism or enzyme. In a batch water bath, the simplest pasteurization equipment, the
229 packed juice is maintained at a high enough temperature for the needed time. While in a
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230 continuous water bath pasteurizer, the product is subjected to continuous hot water sprays
231 while moving through a tunnel. In this method, steam at atmospheric pressure (100°C) is
232 occasionally injected to increase the rate of heating (Ramaswamy & Marcotte, 2005). Heat
233 exchangers, applying widely in juice processing, are classified according to transfer process
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234 (direct and indirect), construction (tubular, plate type, extended surface and regenerative)
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235 and flow arrangement (single and multi-pass) (Shah & Sekulic, 2003). Based on the type of
236 product, availability of equipment, cost and energy consumption, batch or continuous
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237 conventional treatment devices can be used.
238 Two isoforms of PME were recognized in Valencia orange juice pulp during heating in a
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water-bath at 60 to 90℃ with Z-values of 10.8℃ for heat resistance and 6.5℃ for sensitive
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240 isoforms (Wicker & Temelli, 1988). In the other study, when the orange juice (Old South
Brand) was heated in a well-agitated water-bath at the same temperature two isoforms with
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242 higher Z-values (31.1°C and 17.6 °C) were also found (Tajchakavit & Ramaswamy, 1997b).
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243 As well, the presence of two isoforms of PME was reported during heating the mixed orange
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244 juices (Pera and Lime varieties) with controlled pH (3.6 - 4.0) in a plate heat exchanger at
245 82.5, 85 and 87.5℃. A non-linear regression model was used to describe the kinetics of
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246 enzyme inactivation. The quicker inactivation of the enzyme was reported at lower pH and
247 higher temperature (Tribess & Tadini, 2006). These differences in the obtained results were
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248 related to using various varieties of the orange and treatment conditions such as applied
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249 temperature. As, Z-value of PME during heating of Sanguinello orange juice at 75 to 95℃,
250 75 to 85℃ and 85 to 95℃ were reported 10.5, 9.2 and 16.4℃, respectively (Sio, Palmieri,
251 Servillo, Giovane, & Castaldo, 2001). In the other study, when sour orange (Citrus
252 aurentium L.) juice treated at 60℃, three isoforms of PME were recognized while at higher
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253 temperatures of 70, 80 and 90 ℃ two isoforms were only revealed (Aghajanzadeh et al.,
254 2016). It was found two isoforms of this enzyme during key lime (Citrus aurantiifolia) juice
255 thermal treatment at the same conditions (Aghajanzadeh, Kashaninejad, & Ziaiifar, 2016).
256 PME was totally inactivated during pasteurization of concentrated orange juices (100℃
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257 for 7 min) and no enzyme activity was found in stored juice for 240 days, resulting in no
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258 variation in cloud stability. The PME activity of the commercially pasteurized Spanish
259 orange juice (77℃ for 20 s) decreased during 2 weeks storage at 4 and 10℃. An increase in
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260 the enzyme activity was however observed between week 3 to 6 (Esteve, Frigola, Rodrigo,
261 & Rodrigo, 2005). This can be related to re-activity of thermal resistance isoform
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(presenting about 0 to 33% of the total PME, (Snir, et al., 1996)). Therefore, process
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263 conditions (time and temperature) should be planned based on the inactivation of this
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264 isoform to achieve the higher cloud stability and shelf life.
265 In general, the conventional thermal processing needs long heating time that leads to
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266 quality deteriorations in vitamin content, color and rheological characteristics (Ling, et al.,
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267 2015). Over the years, new thermal processing technologies have emerged to reduce or
268 eliminate these unwanted effects.

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270 2. 1. 2. Microwave heating

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271
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272 Microwave is a part of the electromagnetic (EM) spectrum waves with a frequency range
273 between 300 MHz and 300 GHz (Benlloch-Tinoco, Igual, Rodrigo, & Martínez-Navarrete,
274 2013). Two frequencies of 915 and 2450 MHz are specified by the Federal Communications
275 Commission in food industrial applications. In pasteurization process, the choice of MW
276 frequency is important in uniform and efficient heating of the product, which is related to
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277 microwave system (electric field intensity and food positioning in cavity) and food
278 characteristics (dielectric properties, homogeneity and quantity) (Ahmed, Ramaswamy,
279 Kasapis, & Boye, 2009; Cinquanta, et al., 2010). As the food is directly heated, MW
280 pasteurization is faster, more effective and economical than the conventional heat treatments
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281 (Cinquanta, et al., 2010; Villamiel, Castillo, Martín, & Corzo, 1998).
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282 Two principal mechanisms involve in MW heating consist of dipole rotation and ionic
283 polarization; which is the related to the alternate of the MW electric field between the two
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284 electrodes (Ahmed, et al., 2009; Benlloch-Tinoco, et al., 2013). During MW pasteurization,
285 when the juice is placed between the electrodes the polar molecules (like water) and ionic
286
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compounds (such as salts) are continuously reoriented to align themselves with MW electric
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287 field (Fig. 3). The heat is generated in the sample as result of pushing, pulling and colliding
288 between these molecules or ions during this rearrangement.

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289 In comparison to the conventional treatment, come up time (CUT) is shorter in MW

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290 treatment that resulted in lower food nutritional loss (Tajchakavit & Ramaswamy, 1997b).
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291 PME, having protein structure with numerous polar and charged sites, could be influenced
292 by an electrical field of MW (Tajchakavit & Ramaswamy, 1997a). MW energy may be

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293 insufficient to break up the covalent bonds of PME but some non-covalent bonds such as
294 hydrophobic, electrostatic and hydrogen bonds may be disrupted.

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295 It was found only one isoform of PME with Z-value of 13.4℃ during MW heating (700
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296 W, 2450 MHz) of orange juice (Old South Brand); the higher microwave power absorption
297 was also observed when the larger volume of the juice was treated (Tajchakavit &
298 Ramaswamy, 1997b). They reported that in comparison to the conventional heating in a
299 well-agitated water bath, MW heating was more effective in PME inactivation and
300 calculated smaller D-values. This observation can be directly related to the time–temperature
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301 profile of a linear CUT that indicated a more rapid heating than the conventional one
302 (Tajchakavit & Ramaswamy, 1997b). 97.5% and 98.8% reduction in PME activity were
303 observed during orange (Navel variety) juice heating using continuous-flow MW system
304 (525 W, 2450 MHz) at 89.7 ℃ and 96.4 ℃ , respectively. The higher PME inactivation
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305 (98.90%) was found when the juice was heated in a tubular conventional heating system at
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306 89.8℃ and either 96.5℃, which can be related to the shorter process time in MW heating
307 method (Villamiel, et al., 1998). PME activity in grapefruit (Star Raby variety) juice
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308 reduced up to 90% during MW treatment (900 W for 30 s); it was approximately equal to
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309 PME reduction (12%) during conventional heating in water bath at 80 ℃ for 11s (Igual,
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310 García-Martínez, Camacho, & Martínez-Navarrete, 2010). During MW heating (3000W,
311 2.45 GHz) of sweet orange (Citrus sinensis Osbeck) juice two different isoforms of the
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312 enzyme were recognized; as the heat sensitive one was inactivated after 1 min treatment at
313 70℃ with Z-value of 22.1℃ (Cinquanta et al., 2010).

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314 During batch MW heating, the major factor limiting the PME inactivation is formation of
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315 hotspots which is related to the non-uniform heating; which brings inappropriate lethality
316 calculation and decrease in efficiency of the pasteurization (Salazar-González, San Martín-
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317 González, Vergara-Balderas, López-Malo, & Sosa-Morales, 2014; Zhao, Huang, & Yan,
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318 2011). However, this is not the case in MW juice pasteurization, due to the liquid nature and
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319 uniform heating of this kind of products (lumped system of heat transfer). During juice
320 treatment using MW, continuous stirring of the sample overcomes this problem and results
321 in a uniform heating (Cinquanta, et al., 2010). Additionally, applying a continuous MW
322 system could be more effective way to increase the power absorption by the juice (Stratakos,
323 Delgado-Pando, Linton, Patterson, & Koidis, 2016). In this condition, the temperature of the
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324 sample and also uniformity heating could be easily controlled by changing the flow rate of
325 the juice. Continuous MW heating system (900 W, 2450 MHz) was used in Navel orange
326 juice processing. 95.36% and 93.04% reduction in PME activity were observed in moderate
327 temperature using 40 ml/min-83 ℃ and 50 ml/min-75 ℃ heating conditions, respectively.
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328 However, 86.08% PME inactivation was only achieved when the juice was heated
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329 conventionally in a water bath at 95℃ for 1 min. The rate of PME inactivation increased
330 during MW heating in comparison to the conventional heating of the juice in water bath; as,
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331 D-value was reduced from 70 s for conventional treatment to 39.42 s and 38.76 s when the
332 MW processing method with 40 and 50 ml/min flow rate were applied, respectively
333
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(Demirdöven & Baysal, 2016). In literature, no study was performed about the variation of
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334 PME activity during storage of citrus juices. However, no regeneration of PME activity in
thermally pasteurized Guava juice (Psidium guajava L.) was found during storage (Salazar-
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336 González, et al., 2014).

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337 As a final point, in calculation of thermal resistance of PME in juice some parameters
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338 should be also considered to define the thermal condition such as heating mode (continuous
339 or batch), heating power, mass or flow rate of the sample, fruit cultivar and different
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340 isoforms of the enzyme (Aghajanzadeh, Ziaiifar, et al., 2016a; Cinquanta, et al., 2010).
341
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342 2. 1. 3. Ohmic heating

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344 Ohmic heating also is known as Joule heating, electrical resistance heating, direct
345 electrical resistance heating, electro-heating and electroconductive heating (N. Kaur & Singh,
346 2015). In this method, food is placed or passed through the electrodes during which its
347 temperature increases due to electrical current exposure (N. Kaur & Singh, 2015). The
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348 advantages of OH are related to the uniform heating of the food products, compact design of
349 the system and high efficiency in energy transfer (Leizerson & Shimoni, 2005; Somavat,
350 2011). The major limitation of OH in industrial scale is related to high-cost installation and
351 operation (N. Kaur & Singh, 2015).
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352 The intensity of ohmic heating treatment of the juice depends on the product electrical
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353 conductivity, particle size, ionic concentration, field strength and properties of the electrodes
354 (N. Kaur & Singh, 2015). Foods containing abundant ionic concentration can be easily
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355 heated due to a reduction in electrical conductivity (Demirdöven & Baysal, 2014). Besides
356 thermal effect, electro-plasmolysis (electropermeabilization) as a non-thermal phenomenon
357
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can rupture of the food cells, inactivate the microorganisms and enzymes (G. Kaur &
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358 Aggarwal, 2015). Cell membrane shows a particular dielectric strength depending on its
359 lipid content (acting as an insulator). Applying low frequency (50 to 60 Hz) and high electric
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360 field intensity results in exceeding the natural dielectric strength; these changes allow the
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361 cell walls to build up charges and form disruptive pores with different sizes (Evrendilek, et
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362 al., 2012). Depending on the strength of the applied electric field, the formed pores could be
363 differ in size and it can also reseal after a short period of treatment time (N. Kaur & Singh,
EP
364 2015). By this way, destruction of the cellular membranes happens and the extraction of the
365 cell content and inactivation of microorganisms will occur (Bazhal, Lebovka, & Vorobiev,
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366 2003). On the other hand, electro-plasmolysis forces the PME to be released from the pulp.
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367 When the OH is applied, the enzyme surface and its environment are modified by ionization
368 and distribution of solution components. It can be concluded that the combination of thermal
369 and non-thermal effects of OH processing are responsible for PME inactivation.
370 Similar to the conventional method using plate heat exchanger (90℃ for 50 s), 90–98%
371 reduction in PME activity was observed during Shamuti orange juice ohmic heating (50 Hz,
16
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372 20cm electrode gap, 8 kV maximum voltage). The PME activity did not significantly alter
373 during storage of the juice for 100 days, reflecting the irreversible inactivation of the
374 enzyme during both applied thermal methods (Leizerson & Shimoni, 2005). In the other
375 study, 96% reduction in PME activity was found after OH treatment (69°C, 50 Hz, 42 V/cm)
PT
376 of the Navel orange juice; however, 88.3% reduction was only observed after conventional
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377 heating (95℃, 1 min) of the sample (Demirdöven & Baysal, 2014). These results revealed
378 the high efficiency of OH treatment on PME inactivation during juice processing, even at
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379 equal or lower temperatures when compared with conventional heating.
380 D-values of PME inactivation in orange juice were calculated 0.71 and 1.07 min during
381
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OH (42 V/cm, 50 Hz and 69 °C) and conventional thermal treatment (95 °C, 1 min),
AN
382 respectively (Demirdöven & Baysal, 2014)..
In general, inactivation of the PME can be increased and the required holding time will
M
383
384 be reduced by applying the higher electric field strength (Uemura, Kobayashi, & Takahashi,
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385 2015; Yıldız & Baysal, 2006). Conditions of OH heating treatments such as time, the final
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386 temperature of the juice and intensity of the process (electric field and frequency) are the
387 key factors affecting the PME inactivation.

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388 Pectin, a critical compound in the cloudy appearance, is released into the juice due to
389 electroporation effect. It was reported that pectin content of orange juice was increased up to
C
390 2% after OH (Demirdöven & Baysal, 2014). 18.4% rise in pectin content of orange juice
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391 was also found after electro-plasmolysis application at 27 V/cm (Demirdöven, 2009;
392 Demirdöven & Baysal, 2009). During OH treatment, the juice cloud stability hence
393 increases due to inactivation of PME in addition to the release of pectin into the juice.
394
395
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396 2. 2. Non- thermal pasteurization of juice
397
398 Thermal processing of the juice, most common pasteurization method, presents some
399 disadvantages such as loss of nutritional and organoleptic quality which are directly related
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400 to the degradation of chemical compounds. In addition, changes in physical aspects of the
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401 juice can be occurred as the color of the juice is affected by the non-enzymatic browning
402 occurring via thermal degradation of ascorbic acid as a heat sensitive vitamin (Burdurlu,
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403 Koca, & Karadeniz, 2006). Hence, deterioration of physicochemical properties reduces the
404 acceptance of the food products. Nowadays, to produce a juice with better quality,
405
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researchers and food producers attend to apply the non-thermal methods such as PEF, HP
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406 and HPCD (Table 2).
407
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408 2. 2. 1. Pulsed electric field

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409
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410 Pulsed electric field, a fast and non-thermal technology, is applied in the processing of
411 various foods, especially in juice production. In batch and continuous PEF systems, food is
EP
412 placed between or passed through electrodes, respectively. Food is subjected to certain and
413 controllable high voltage electric pulses (up to 70 kV/cm) for few microseconds in order to
C
414 inactivate target microorganisms and enzymes (Buckow, Baumann, Schroeder, & Knoerzer,
AC
415 2011).
416 The major limitations of PEF treatment in industrial scale are non-uniformity in food,
417 metal emission of the electrode into the food, high cost in electrical energy consumption and
418 high initial investment in equipment (Buckow, et al., 2011; Mastwijk & Bartels, 2005). This
419 non-uniformity is resulted from the electric field and even temperature distributions relating
18
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420 to juice flow rate, pulse shape, electrode insulator configuration, the design of the electrode
421 and chamber (Buckow, et al., 2011; Mastwijk & Bartels, 2005). Food direct directly contacts
422 with electrodes and emission of electrode metal leading to the formation of electrolytic
423 products (Mastwijk & Bartels, 2005). PEF influences only the vegetative forms of bacteria
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424 and being incapable in spore inactivation; it is therefore only used in pasteurization of acidic
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425 food products (Mastwijk & Bartels, 2005).
426 Like OH treatment, the effect of PEF on microbial cell destruction is related to the
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427 electroporation (N. Kaur & Singh, 2015). In general, a difference between PEF and OH is
428 related to the intensity of the treatment. As in PEF usually hundred fold or higher field
429
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strengths than OH are used (kV/cm versus tens of V/cm). The needed treatment time for
AN
430 PEF is usually based on s or ms but OH treatment is based on seconds to minutes
(Kulshrestha & Sastry, 2006).

M
431
432 Effect of PEF on enzyme inactivation depends on the intensity of treatment (such as field
D
433 strength, temperature and time), food ingredients and its physical properties (Buckow, et al.,
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434 2011). Enzymes like other protein structures have net charges and dipole movements in an
435 aqueous medium such as juice. The presence of ions in the juice increases its electrical
EP
436 conductivity (Zhang, Barbosa-Cánovas, & Swanson, 1995). By this way, electrical current is
437 established in the juice when subjected to an electrical field. Due to the changes in the
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438 conformational structure, PME is inactivated during PEF process (Samaranayake & Sastry,
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439 2016).
440 Without any important change in final temperature, 80% reduction in PME activity was
441 observed in Navelina orange juice treated using PEF system at 35 kV/cm for 1500 s. Three
442 models (first-order fractional conversion, Fermi’s and Hulsheger’s) were accurately used to
443 describe the PME activity as function of the treatment time, the electric field intensity and
19
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444 the combination effect of these PEF processing parameters (Elez-Martinez, et al., 2007).
445 During this rapid treatment, the temperature of the juice doesn’t significantly change (Yeom,
446 Streaker, Zhang, & Min, 2000). Hence, PEF treated juice presents sensorial and nutritional
447 qualities close to the fresh one, even with longer shelf life (Mastwijk & Bartels, 2005). In
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448 another work, it was reported that 79% reduction in PME activity during PEF treatment
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449 (25kV/cm, 340 , 63 ℃ ) of orange - carrot juice, with no reversible activation of the
450 enzyme during storage for 10 and 8.5 weeks at 2 ℃ and 12 ℃ , respectively (Rodrigo,
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451 Barbosa-Cánovas, Martinez, & Rodrigo, 2003). During PEF treatment of the Kozan-specific
452 orange juice, 35.1% (13.8 kV/cm, 1033.9 μs) and 93.8% (25.3 kV/cm, 1206 μs) reduction
453
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in PME activity were observed with no recovery in PME activity during 180 days storage at
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454 4 ℃ (Agcam, Akyıldız, & Evrendilek, 2014). About 90% reduction in PME activity was
M
455 reported when the Valencia orange juice was processed using electric field intensity of 35
456 kV/cm for 59 s, similarly no restoration during storage for 112 days at 22 ℃ was found
D
457 (Yeom, et al., 2000). PME activity in Valencia orange juice treated by PEF (at 35kV/cm for
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458 184 ms, 2.2 ms pulse duration) decreased about 83.2% with final juice temperature of 61.9℃.
459 Just 24.1% of PME was inactivated when this juice was lonely heated in oil bath at 62.9℃
EP
460 (Yeom, Zhang, & Chism, 2002). These represent the remarkable synergistic effects of
combination of thermal and non-thermal treatments on PME inactivation during juice

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461
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462 pasteurization.
463
464
465
466
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467 2. 2. 2. High pressure processing
468
469 High pressure processing is one of the alternative methods mainly used in inactivation of
470 microorganisms and enzymes in juice production. Dynamic high pressure (DHP) refers to
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471 homogenization process that a liquid product is forced through a very narrow and variable
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472 orifice affecting the physicochemical properties of the juice (at high pressure and high
473 velocity). This causes changes in the treated food. DHP treatment differs from static high
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474 pressure due to rheological phenomena such as cavitation, friction, shear and turbulence
475 (Lacroix, Fliss, & Makhlouf, 2005). During this non-thermal processing, the food is
476
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subjected to uniform pressures in the range of 100 – 700 MPa at around room temperature
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477 (Raso, Calderon, Gongora, Barbosa‐Canovas, & Swanson, 1998). In HP, depending on the
M
478 liquid component, the temperature increases approximately 3 ℃ per 100 MPa (Rastogi,
479 Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007). The main advantage of this
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480 method is related to the low thermal treatment condition that the temperature of the food
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481 does not exceed more than 40℃; it is therefore categorized as a non-thermal technology
482 (Welti-Chanes, Ochoa-Velasco, & Guerrero-Beltrán, 2009). This fact beside the stability of
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483 covalent bonds to high pressure brings the better preservation of the nutritional values (like
C
484 vitamins and pigments), sensorial and quality aspects of the food products (Polydera, et al.,
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485 2004; Welti-Chanes, et al., 2009). The major limitation of the application of HP in industrial
486 scale rather than the conventional one is related to the high cost of installation and operation
487 (N. Kaur & Singh, 2015).
488 Enzyme inactivation under high pressure treatment is a very complicated phenomenon
489 including several events causing PME inactivation by folding or unfolding its native
21
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490 structure (Chakraborty, Kaushik, Rao, & Mishra, 2014). The changes in active site and
491 protein denaturation lead to inactivation and also change the functionality of the enzyme.
492 The application of pressure may have a reversible or irreversible and partial or complete
493 unfolding effect on the enzyme structure (Fig. 4). During high pressure processing, the
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494 volume of the juice decreases upon unfolding that is related to the existence of voids and
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495 internal cavities in the folded state, these phenomena change the structure of the enzyme
496 (Alexandrakis, et al., 2014). Destabilization of hydrophobic aggregation takes also place
SC
497 initiating water molecules to be forced into the protein interior. Hence, the decrease in
498 enzyme activity using high pressure treatment may be related to the denaturation of the
499
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globular proteins, the tertiary structures that are preserved in large part by hydrophobic and
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500 electrostatic interactions (Lacroix, et al., 2005). High pressure impacts on changes in the
501 tertiary structure of the enzyme to partial unfolding and molecular rearrangement; it
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502 therefore reduces the PME activity by affecting the substrate – enzyme binding that results
D
503 in less functional or non-functional active sites (Lacroix, et al., 2005). It supposed that the
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504 pressure amplifies the hydrogen bonds formation that composes the secondary structure.
505 This would be the reason of less impact of high pressure on secondary structure than the
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506 tertiary one (Alexandrakis, et al., 2014).
507 It was also found this enzyme was inactivated during treatment at 300 or 400 MPa for 10
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508 min in the formulated juice containing concentrated Satsuma mandarin, water and purified
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509 PME from orange peel. It was discovered that soluble solids protect the enzyme from
510 inactivation. No reversible activity of PME was found in the stored juice at 0 to 37℃ for 3
511 months (Ogawa, Fukuhisa, Kubo, & Fukumoto, 1990).
512 D-value of PME inactivation in orange juice with pH equal 3.7 during treatment at 100 to
513 400 MPa varied from 8250 to 260 min. Also, the calculated D-value during HP processing
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514 (270 to 400 MPa) of the Old South Brand orange juice with a lower pH (equal 3.2) was
515 ranged 145 to 264 min (Basak & Ramaswamy, 1996). Which represents the effect of HP
516 treatment on PME activity not only depends on applied pressure but also on products
517 properties. Lower PME inactivation rate was reported when the juice, containing higher
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518 soluble solid content, treated using HP method. This would be related to the protective effect
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519 of higher concentration levels on pressure inactivation of the enzyme (Basak & Ramaswamy,
520 1996; Basak, Ramaswamy, & Simpson, 2001).
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521 HP was applied to treat the orange and grapefruit juices at 600 to 900 MPa for 1 to 30 s.
522 Applying higher pressures than 600 MPa was effective in inactivation of the heat labile
523
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isoform of PME, with no effect on the heat stable isoform. HP treatment was very effective
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524 in preserving the cloudy appearance of both juices stored at 4℃ for >50 days (Goodner,
Braddock, & Parish, 1998). It was also reported that due to the existence of the stable
M
525
526 isoforms of PME, the activity of the enzyme in orange juice, Valencia variety, increases
D
527 during storage of the (Welti-Chanes, et al., 2009). These depend on the chemical nature of
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528 the enzyme, temperature, level and duration of applied pressure and the influence of the
529 environmental parameters (Basak and Ramaswamy, 1996). Pectin structure in juice was also
EP
530 changed by subjecting to the high pressure treatment due to breaking up of the covalent
531 bonds and decreasing the molecular mass of pectin making this substrate less accessible to
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532 the PME (Lacroix, et al., 2005).

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533
534 2. 2. 3. High pressure carbon dioxide
535
536 High pressure carbon dioxide is a cold pasteurization method which affects the
537 microorganisms and enzyme structures and their activities. As a non-thermal treatment, it
23
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538 has the least undesirable effects on nutritional value, appearance and organoleptic properties
539 of the food rather than the thermal processing (Truong, Boff, Min, & Shellhammer, 2002).
540 Carbon dioxide (CO2) is a non-toxic, non-flammable and cheap gas (Damar & Balaban,
541 2006). At atmospheric pressure, it is used in food packaging to control the microbial growth
PT
542 and respiration of the horticultural and agricultural products. In this case, CO2 is displaced
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543 by oxygen when it rapidly penetrates in the cellular structure (Daniels, Krishnamurthi, &
544 Rizvi, 1985; Zagory & Kader, 1988).
SC
545 When compared with HP, HPCD shows the different effect on PME activity. At high
546 pressures, CO2 dissolves in the juices and reduces pH by the formation of the carbonic acid
547
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(Eq. 9). At a constant temperature, more penetration, solubility and transport rate of CO2 in
AN
548 aqueous phase takes place at higher pressures (Bertoloni, Bertucco, De Cian, & Parton,
549 2006). According to Eqs. 10 and 11, the additional breakdown of carbonic acid brings the
M
550 formation of bicarbonate, carbonate and H+ which results in higher pH reduction in the juice
D
551 (Damar & Balaban, 2006). When pressure is removed, pH is restored to the initial one due to
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552 the release of CO2 from the juice (Bertoloni, et al., 2006).
553
EP
554 CO2 + H2O ↔ H2CO3 Eq. 9

C
555
H2CO3 ↔ H + + HCO3−
AC
556 Eq. 10
557
558 HCO3− ↔ H + + CO3−2 Eq. 11
559
24
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560 At low pHs, the protein bounds, existing in enzyme structure, can interact with CO2 and
561 form a bicarbonate complex which causing the enzyme inactivation. The Ca+2-protein
562 bindings are important in intracellular regulations. Another mechanism affecting the enzyme
563 activity is related to the precipitation of these Ca+2 sensitive proteins. This occurs as result of
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564 the formation of carbonate-Ca+2 complexes, depending on the binding site of the ion and
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565 chemical structure of the enzyme (Damar & Balaban, 2006). During HPCD treatment, no
566 change in the SDS-PAGE electrophoretic behavior of PME was found; while, the secondary
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567 and tertiary structures were altered, causing the enzyme inactivation (Zhou, Wu, Hu, Zhi, &
568 Liao, 2009).
569
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PME completely inactivated when Valencia orange juice was treated using supercritical
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570 CO2 treatment (29 MPa, 50℃, 4 h). This inactivation resulted in 3.5 folds rising in the juice
cloud stability. However, the PME activity increased slowly after 15 days storage at 4.4℃
M
571
572 which reflected the regeneration of the PME activity within the treated juice. No changes in
D
573 cloud stability were observed during storage representing that the cloud stability of the juice
TE
574 was not only affected by PME activity. In addition, the structure of the pectin modifies and
575 the juice becomes more turbid by subjecting to the shear forces through small openings
EP
576 during depressurization (Arreola, et al., 1991). 6.8% in PME activity was found in the
577 processed Valencia orange juice using HPCD (25 ℃ , 800 MPa, 1 min). 46.3% PME
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578 inactivation was observed when the Valencia orange juice was treated using 107 MPa for 10
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579 min without heating (0.43 ratio of CO2/juice (w/w)). Cloud stability of the juice was
580 improved after HPCD process due to the reduction in the size of cloud particles. No
581 reversible activity in the enzyme was found during storage at 1.7℃. The cloud stability of
582 the juice was enhanced during this storage conditions even though there was PME residual
25
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583 in the juice. The formed carbonic acid dissociates into bicarbonate and H+ at high
584 concentration. After releasing the pressure, bicarbonate is converted to carbonate and
585 precipitate the calcium. This prevented the calcium from acting as a bridge between pectic
586 acid chains which causing cloud loss (Kincal, et al., 2006).
PT
587 64.2% and 85.2% inactivation in PME were found in non-carbonated and carbonated
RI
588 Valencia orange juice (600 MPa, 20 ℃ , 5 min), respectively. It was also found that
589 carbonating the juice had no considerable effect on the total percentage of PME but it
SC
590 influenced the rate of its inactivation (Truong, et al., 2002).
591 It was also reported that the alteration in pH, changes in conformational of the enzyme
592
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and inhibitory effect of CO2 on the enzyme activity result in PME inactivation during HPCD
AN
593 (Damar & Balaban, 2006). The sorption of the dense CO2 by the enzyme can bring its
conformational changes, especially the secondary structure, and its inactivation (Bertoloni,
M
594
595 et al., 2006). In conclusion, the type and source of the PME beside HPCD treatment
D
596 conditions (time, temperature and applied pressure) have an effective influence on the
TE
597 inactivation of the enzyme.
598
EP
599 2. 3. Combination of thermal and non-thermal technologies
600 Considering the consumer demands for fresh-like juice, application of non-thermal
C
601 processing technologies has been increased in recent years. Non-thermal treatment in
AC
602 combination with mild thermal ones shows synergistic effects on higher inactivation of the
603 enzyme (protein structure) besides better preservation of the fresh juice quality.
604
605
606
26
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607 2. 3. 1. Combination of thermal treatment and PEF
608
609 PEF at higher frequencies increases the juice temperature, causing higher efficiency in
610 PME inactivation (Espachs-Barroso, et al., 2006). 5% rising in PME inactivation was
PT
611 observed when the orange-carrot juice (80% orange and 20% carrot) was treated in PEF
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612 using 25 kV/cm, 330 , 132 pulses, 70℃ in comparison to processing applying 25 kV/cm,
613 280 , 112 pulses, 68℃ (Rivas, Rodrigo, Martínez, Barbosa-Cánovas, & Rodrigo, 2006).
SC
614 A 90% reduction in PME activity was observed during PEF treatment of the Valencia
U
615 orange juice at 25 kV/cm for 250 ms at 50℃; only 36.3% enzyme inactivation was however
AN
616 achieved when the temperature was set at 10℃, at the same PEF treatment condition (Yeom,
617 et al., 2002).

M
618
619 2. 3. 2. Combination of thermal treatment and HP

D
620
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621 A collaborative effect of temperature and pressure on PME activity was reported; as,
622 applying HP in combination with mild heat treatment could be effective in PME inactivation
EP
623 (Alexandrakis, et al., 2014; Cano, Hernandez, & Ancos, 1997; Irwe & Olsson, 1994).
C
624 Several types of high pressure equipment (100 to 1000 MPa) were used lonely or in
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625 combination with water or water mixed with ethylene glycol as the pressurizing fluid, at
626 temperatures ranged from 20 to 60℃ (Torres, González-M, Klotz, & Rodrigo, 2015). 90%
627 reduction in PME activity in orange juice (Valencia late variety) was observed when treated
628 at 500 MPa at 50℃ for 15 min (Torres, et al., 2015).
27
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629 In another study, Florida orange juice was subjected to combined effect of HP treatment
630 (400, 500 and 600 MPa) and heating treatment (25, 37.5 and 50℃). PME activity reduced
631 following the first order kinetic model by rising in pressure and temperature. D-values for
632 PME inactivation during treatment of the orange juice at 400 MPa/ 25℃ rather than 400
PT
633 MPa/ 50℃, were reduced from 116.9 to 45.6 min. The Zp-values were 164, 179 and 200
RI
634 MPa when the temperatures were 25, 37.5 and 50℃, respectively (Nienaber & Shellhammer,
635 2001).
SC
636 PME activity was reduced to 50.4, 49.4 and 37.8% during high pressure treatment (250
637 MPa) in Valencia orange juice with initial temperatures of 22, 35, and 45℃, respectively
638
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(Welti-Chanes, et al., 2009). However, PME activity increased during storage (12 days at
AN
639 4℃); this shows the inefficiency of the HP alone in the inactivation of PME, especially its
M
640 resistance isoforms. The lower reversible PME activity and better cloud stability were
641 observed in the samples treated at higher pressure and temperature.

D
642 It was also observed that the dynamic high pressure homogenization (DHP) treatment at
TE
643 170 MPa decreased PME activity (20%) in Californian Valencia orange juice. Heating the
644 orange juice (50℃ for 10 min) prior to homogenization was effective in increasing the PME
EP
645 inactivation during DHP. The higher PME inactivation was reported when preheating (50℃
646 for 10 min) is combined with DHP (170 MPa); during which, the inactivation of
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647 thermostable isoform takes even place. During storage at 30℃ of this treated juice, higher
AC
648 cloud stability was observed. Higher cloud stability is maybe related to the change in pectin
649 structure due to mechanical forces and decrease of its accessibility to PME (Lacroix, et al.,
650 2005). In general, these results showed that inactivation of the PME accelerated by rising in
28
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651 pressure, the number of passes, time and temperature (Alexandrakis, et al., 2014; Welti-
652 Chanes, et al., 2009).
653
654 2. 3. 3. Combination of thermal treatment and HPCD
PT
655
RI
656 The mutual effect of pressure and CO2 on PME activity was reported; while, the rising in
657 the temperature from 25 to 50℃ showed no significant effect during treatment of Valencia
SC
658 orange juice at 800 MPa for 1 min (Corwin & Shellhammer, 2002). However, at higher
659 temperatures, a synergistic effect between the applied pressure and temperature was
660
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observed. When the temperature increased to 60℃, the D-value changed from 56.6 min to
AN
661 10.6 min under the atmospheric condition and 31 MPa, respectively. The Z-value of the
M
662 PME activity was calculated 5.2℃ with HPCD and 8.8℃ at atmospheric pressure. In this
663 study, 94% reduction in PME activity was reported in Valencia orange juice treated at 28.9
D
664 MPa at 51℃ for 173.5 min (Balaban, et al., 1991).

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665
666 Conclusions
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667
C
668 PME inactivation is essential in the production of acidic juice as it has higher thermal
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669 resistance than the microbial index and also affects the cloud stability of the juice. Several
670 thermal and non-thermal pasteurization methods are introduced and also applied in juice
671 processing in order to inactivate this enzyme. Conventional, microwave and ohmic heating
672 are categorized as thermal processing methods for pasteurization of different acidic juices.
673 During these treatments, protein structure of the PME altered and the enzyme inactivation
29
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674 takes place. Non-thermal treatment as pulsed electric field, high pressure processing and
675 high pressure carbon dioxide alters the physical and chemical structure of PME and
676 inactivate this enzyme. As the temperature of the juice does not considerably increase, using
677 these methods prevents or reduces the undesirable nutritional loss and changes in the
PT
678 organoleptic properties of the juices. It was approved that the combination of the non-
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679 thermal pasteurization methods with the thermal ones can enhance the PME inactivation and
680 also decreases the needed intensity of thermal treatment hence prevents juice quality
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681 deterioration.
682
683 References
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684
685 Agcam, E., Akyıldız, A., & Evrendilek, G. A. (2014). Effects of PEF and heat
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686 pasteurization on PME activity in orange juice with regard to a new inactivation kinetic
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687 model. Food Chemistry, 165, 70-76.

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688 Aghajanzadeh, S., Kashaninejad, M., & Ziaiifar, A. M. (2016). Effect of infrared
689 heating on degradation kinetics of key lime juice physicochemical properties. Innovative
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690 Food Science & Emerging Technologies, 38, 139-148.
691 Aghajanzadeh, S., Ziaiifar, A. M., Kashaninejad, M., Maghsoudlou, Y., &
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692 Esmailzadeh, E. (2016a). Thermal inactivation kinetic of pectin methylesterase and

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693 cloud stability in sour orange juice. Journal of Food Engineering.
694 Aghajanzadeh, S., Ziaiifar, A. M., Kashaninejad, M., Maghsoudlou, Y., &
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696 cloud stability in sour orange juice. Journal of Food Engineering, 185, 72-77.
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697 Ahmed, J., Ramaswamy, H. S., Kasapis, S., & Boye, J. I. (2009). Novel food
698 processing: effects on rheological and functional properties, : CRC Press.
699 Al-Qsous, S., Carpentier, E., Klein-Eude, D., Burel, C., Mareck, A., Dauchel, H.,
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701 methylesterase isoform that could be involved in flax cell wall stiffening. Planta, 219,
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702 369-378.
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704 (2014). Comparative structural changes and inactivation kinetics of pectin
705 methylesterases from different orange cultivars processed by high pressure. Food and
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707 Antunes, M. D., Dandlen, S., Cavaco, A. M., & Miguel, G. (2010). Effects of
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709 nutritional properties of fresh-cut kiwifruit. Journal of Agricultural and Food Chemistry,
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710 58, 6173-6181.

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711 Arreola, A., Balaban, M., Marshall, M., Peplow, A., Wei, C., & Cornell, J. (1991).
712 Supercritical carbon dioxide effects on some quality attributes of single strength orange
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713 juice. Journal of Food Science, 56, 1030-1033.
714 Balaban, M. O., Arreola, A. G., Marshall, M., Peplow, A., Wei, C. I., & Cornell, J.
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715 (1991). Inactivation of pectinesterase in orange juice by supercritical carbon dioxide.

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716 Journal of Food Science, 56, 743-746.
717 Basak, S., & Ramaswamy, H. (1996). Ultra high pressure treatment of orange juice: a
718 kinetic study on inactivation of pectin methyl esterase. Food Research International, 29,
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720 Basak, S., Ramaswamy, H., & Simpson, B. (2001). High pressure inactivation of
721 pectin methyl esterase in orange juice using combination treatments. Journal of Food
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724 strength for electroplasmolysis of vegetable tissues. Biosystems Engineering, 86, 339-
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725 345.
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727 Comparison of microwaves and conventional thermal treatment on enzymes activity and
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879 on bacterial spores, enzymes, bioactive components and quality factors in food. The
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923 pulp. Journal of Food Science, 53, 162-164.
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924 Yeom, H., Streaker, C., Zhang, Q., & Min, D. (2000). Effects of pulsed electric fields
925 on the activities of microorganisms and pectin methyl esterase in orange juice. Journal
926 of Food Science, 65, 1359-1363.
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936 Zhao, X., Huang, K., & Yan, L. (2011). Review of numerical simulation of
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938 Zhou, L., Wu, J., Hu, X., Zhi, X., & Liao, X. (2009). Alterations in the activity and
939 structure of pectin methylesterase treated by high pressure carbon dioxide. Journal of
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940 Agricultural and Food Chemistry, 57, 1890-1895.
941
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942
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947
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948 Figure Captions
949
950 Fig.1. Pectin molecule (a) characteristic of galacturonic acid and esterified methoxy
951 groups, (b) calcium pectate structure (Kimball, 1999).
PT
952
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953 Fig.2. (a) Residual PME activity in sour orange juice as a function of heating time at
954 various temperatures and (b) expanded view of thermal inactivation of heat resistance
SC
955 isoform (derived from (Aghajanzadeh, Ziaiifar, Kashaninejad, Maghsoudlou, &
956 Esmailzadeh, 2016b)
957
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958 Fig.3. Dipole rotation and ionic polarization mechanisms during MW heating as applying
M
959 an alternating electric field (derived from (Ahmed, et al., 2009)
960
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Fig.4. Proposed model of high pressure processing effect on structure of enzyme (derived
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961
962 from Chakraborty et al., 2014)

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963
964
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965
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967
968
969
970
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971 Table 1. Study the PME inactivation during different thermal treatment methods
972
Mechanisms of
Treatment Treatment NO of
enzyme Juice Other obtained results References
method conditions isoforms
inactivation
D –value: 2.85 to 1240 s at 60 to 90℃
Higher PME inactivation at higher soluble solids
(Tajchakavit &
60 to 90℃ Two content and lower pH
Ramaswamy, 1997b)
PT
Orange Estimation of the CUT effectiveness based the Z-
value of PME
(Wicker & Temelli,
60 to 90℃ Two D-value: 0.23 s to higher than 174 s at 60 to 90℃
1988)
Water bath Thermal
(Aghajanzadeh,
RI
denaturation of Key D-value: 7.21 - 126.54 min at 60 - 90℃
60 to 90℃ Two Kashaninejad, et al.,
PME as a result lime Cloud stability affected by the PME activity
2016)
of rising in the
D-value: 22.1 - 152.5 min at 60 - 90℃
temperature
Sour Two & Calculation of CUT effectiveness using the Z-value (Aghajanzadeh, Ziaiifar,
SC
60 to 90℃
orange three Dependency of the cloud stability and the PME et al., 2016b)
activity
Plate heat Mixed 82.5 to PME inactivation influencing by the pH of the
Two (Tribess & Tadini, 2006)
exchanger orange 87.5℃ juice
Estimate the juice thermal pasteurization using D-
U
Oil bath Orange 75 to 95℃ Two (Sio, et al., 2001)
values
55 to 70℃
Higher rate of enzyme inactivation in MW than (Tajchakavit &
AN
(700W- One
conventional heating treatment Ramaswamy, 1997b)
2450MHz)
89.7℃ and
Observation of the same effects between MW and
96.4℃
- conventional heating methods on PME activity and (Villamiel, et al., 1998)
(525 W,
some nutritional compounds
M
Involving of 2450 MHz)

dipole rotation 60 to 85℃ PME inactivation and desirable stabilization of the
Orange
and ionic (3000W, Two juice by controlling the temperature during MW (Cinquanta, et al., 2010)
Microwave polarization 2.45 GHz) processing
D
mechanisms in 900 W and

increasing 2450 MHz:
temperature 40 ml/min, Higher PME inactivation during continuous MW (Demirdöven & Baysal,
-
TE
83℃ and 50 treatment in comparison to the conventional heating 2016)

ml/min,
75℃
Introducing the MW heating as an effective and
Grapefr
900W, 30 s - novel method in the PME inactivation and (Igual, et al., 2010)
EP
uit
preservation of the nutritional value
20cm
electrode
gap, 50Hz, Introducing the OH as an effective method in juice (Leizerson & Shimoni,
-
8 kV processing 2005)
C
maximum
Electrically
Ohmic voltage
heating and Orange
AC
heating 42 V/cm,
electroporation
50 Hz, D-value: 0.71 min (42 V/cm) and 0.74 min (44
69 °C and V/cm) (Demirdöven & Baysal,
-
44 V/cm, Higher PME inactivation by rising in temperature 2014)
50 Hz, 70 even in moderate electric field intensity
°C
973
974
975
976
977
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978 Table 2. Study the PME inactivation during different non- thermal treatment methods.
979
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D
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44
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Mechanisms of
Treatment %PME
method
enzyme Juice Processing conditions
inactivation
Other obtained results References
inactivation
Batch A:25 kV/cm, 280 μs
(process time), 2.5 μs, 112
pulses, 1 ml/s (flow rate), Reduction in PME activity in addition to
Batch A:
Orange- 68℃ (outlet temperature) the preservation of the sensorial aspects (Rivas, et al.,
75.6
carrot Batch B:25 kV/cm, 330 and the physicochemical quality similar to 2006)
Batch B: 81
(process time), 132 pulses, the fresh juice
1 ml/s(flow rate),
70℃ (outlet temperature)
PT
25 kV/cm, 59 (process
time), 600 Hz, 1.4 s
(pulse duration), Irreversible inactivation of PME (Yeom, et al.,
90%
Electroporation monopolar square-shape 2000)
and changes of pulse,27.2 ml/s (flow rate),
RI
conformational 60.1℃ (outlet temperature)
35kV/cm, 184 ms (
structure of
PEF process time), 2.2 ms
PME as result (pulse duration), 700 Hz, Reduction in PME activity by rising in PEF (Yeom, et al.,
of rising in the 83.2
0.42 ml/s (flow rate), intensity and temperature 2002)
SC
electrical bipolar square-shape pulse,
conductivity Orange 61.9℃ (outlet temperature)
35 kV/cm, 1500 s ( PME activity affecting by applied electric
process time), 200 Hz, 4 field intensity, pulse width, frequency and
s pulses (pulse width), treatment time (Elez-Martinez,
80
U
bipolar square-shape pulse, The first-order fractional conversion, et al., 2007)
1 ml/s (flow rate), 37.5℃ Fermi’s and Hulsheger’s models were used
(outlet temperature) to describe the PME inactivation
AN
2.35 kV/cm, 1206.2 μs
Higher PEF intensity resulted in higher
(process time), 500 Hz,
PME inactivation (Agcam, et al.,
3 μs, 402.1 pulses, 0.633 93.8%
Irreversible inactivation of PME as it 2014)
ml/s (flow rate), 58.2℃
maintained constant during storage
(outlet temperature)
M
Decrease in the inactivation rate at of PME (Basak &

270 to 400 MPa, 0 to 120
26 to 90 by increasing in soluble solid content and Ramaswamy,
min
pH 1996)
Introducing the combination of the low
(Cano, et al.,
200 MPa, 15 min, 30℃ 25 pressure and mild heat treatment as an
1997)
D
effective method in PME inactivation

Effectiveness of the applied pressure and
time of treatment on PME inactivation (Basak, et al.,
400 MPa, 60 min 92
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Decreasing in PME inactivation by rising 2001)

Orange in soluble solid content
(Nienaber &
400 MPa, 12 and 15 min, Accelerating in the PME inactivation by
10.1 to 49.9 Shellhammer,
25, 37.5 & 50℃ combination of HP and mild heat treatment
Folding or 2001)
HP unfolding of the Existing a synergistic effect between HP
EP
100 to 800 MPa, 30 – 60 (Polydera, et al.,

PME structure 5 to 20 and heating treatments on PME
℃ 2004)
inactivation
Representing a synergistic effect between
(Torres, et al.,
500 MPa, 15 min, 50℃ 90.05 moderate thermal treatment and HP on
2015)
PME inactivation
C
orange & Extending the juice shelf life in addition to (Goodner, et al.,
500 MPa, 15 min, 50℃ 10 to 93
grapefruit preservation of its taste and appearance 1998)
Protection effect on PME activity at higher
AC
soluble solids content of the juice

Satsuma 100 to 400 MPa, 10 min, Irreversible PME activity during storage of (Ogawa, et al.,
100
mandarin 25℃ the processed juice 1990)
Preserving the natural flavor and taste of
the juice
Higher PME inactivation as result of
applying DHP in combination of heating
Thermal Higher cloud stability of the juice during
Pre-heating: 50℃ for 10
denaturation and storage
Pre-heating min About 90% (Lacroix, et al.,
folding or Orange Reduction in size of suspended solid
and DHP DHP: 170 MPa for five 2005)
unfolding of the particles in the juice
passes
PME structure
45
ACCEPTED MANUSCRIPT
107 MPa, 10 min, 0.43 Irreversible Effect on PME inactivation

(Kincal, et al.,
ratio of CO2/juice (w/w), 46.3 Enhancement of the cloud stability during
2006)
24℃ storage
The inefficiency of this condition in
(Corwin &
complete PME inactivation
25℃, 800 MPa, 1 min 93.2 Shellhammer,
Existence of an interaction effect between
2002)
Interaction pressure and CO2
between protein Reduction in Process time (Truong, et al.,
600 MPa, 20℃, 5 min 85.2
and CO2 and Increasing the rate of PME inactivation 2002)
HPCD Orange
formation of Enhancement in cloud stability by PME
PT
bicarbonate inactivation.
(Arreola, et al.,
complex 29 MPa, 50℃, 4 h 100 Reversible PME activity during storage
1991)
Effect of shearing forces on pectin
structure and improving the cloud stability
Reduction in d-value by rising in the
RI
28.9 MPa at 51℃ for 173.5 applied temperature and pressure (Balaban, et al.,
94
min Reversible PME activity after 15 days 1991)
storage at 4.4℃
980
SC
981
982
983
U
AN
984
985
M
986
D
987
TE
988
989
EP
990
991
C
992
AC
46
ACCEPTED MANUSCRIPT
PT
RI
993
SC
994 (a)
995
U
AN
M
D
TE
996
997 (b)
EP
998 Fig. 1. Pectin molecule (a) characteristic of galacturonic acid and esterified methoxy groups, (b)
999 calcium pectate structure (Kimball, 1999).

C
1000
AC
1001
1002
1003
1004
1005
1006
1007
47
ACCEPTED MANUSCRIPT
2.5
Log [(PEU/PEU0)×100]
2
1.5
PT
1
60℃
70℃
0.5 80℃
RI
90℃
0
0 2 4 6 8 10
Time (min)
SC
1008
1009 (a)
U
1010 AN
1.6
Log[(PEU/PEU0)×100]
1.4
M
1.2
60℃
1
70℃
D
0.8 80℃
90℃
TE
0.6
0 2 4 6 8 10
1011 Time (min)
EP
1012 (b)
1013 Fig. 2. (a) Residual PME activity in sour orange juice as a function of heating time at various
C
1014 temperatures and (b) expanded view of thermal inactivation of heat resistance isoform (derived from
1015 (Aghajanzadeh, Ziaiifar, et al., 2016b)

AC
1016
1017
1018
1019
1020
1021
48
ACCEPTED MANUSCRIPT
PT
1022
RI
1023 Fig. 3. Dipole rotation and ionic polarization mechanisms during MW heating as applying an
1024 alternating electric field (derived from (Ahmed, et al., 2009)
SC
1025
1026
U
1027
AN
1028
1029
1030
M
1031
1032
D
1033
TE
1034
1035
1036
EP
1037
1038
C
1039
1040
AC
1041
49
ACCEPTED MANUSCRIPT
PT
RI
1042
SC
1043 Fig. 4. Proposed model of high pressure processing effect on structure of enzyme (derived from
1044 Chakraborty et al., 2014)
U
AN
M
D
TE
C EP
AC
50
ACCEPTED MANUSCRIPT
Highlights
- PME influences the physicochemical properties of the juice by de-esterification
of the pectin.
PT
- PME is introduced as a pasteurization index in high acidic juice.
RI
- Thermal pasteurization inactivates the PME by denaturation of its protein
structure.
SC
- Non-thermal treatments inactivate the PME by
different physicochemical mechanisms.
U
- Combination of the thermal and non-thermal pasteurization methods enhances the
AN
PME inactivation.
M
D
TE
C EP
AC

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A review of pectin methylesterase inactivation in citrus juice during pasteurization

Sara Aghajanzadeh, Aman Mohammad Ziaiifar

To appear in: Trends in Food Science & Technology

Received Date: 26 June 2017

7 pectin. Iinactivation of PME is introduced as a pasteurization index in citrus juices,

9 Scope and approach

14 inactivation in different citrus juices.

15 Key finding and conclusion

16 Using non-thermal methods in combination with moderate thermal methods can

19 Keywords: Pectin methylesterase; Citrus juice; Pasteurization; Thermal methods;

1 A review of pectin methylesterase inactivation in citrus

2 juice during pasteurization

4 Sara Aghajanzadeh a*, Aman Mohammad Ziaiifar a

33 Scope and approach

38 different citrus juices.

39 Key finding and conclusion

40 Using non-thermal methods in combination with moderate thermal methods can be

43 Keywords: Pectin methylesterase; Citrus juice; Pasteurization; Thermal methods; Non-

52 phenolic compounds which are effective in prevention of cardiovascular disease, various

70 characteristic in citrus juice (Kimball, 1999).

75 reactions including polymerization and gelatinization (Kimball, 1999). De-esterification of

84 Inactivation of PME is assumed to follow a semi- logarithmic first order kinetic as a

92 activity of PME, respectively. The negative slope of regression of Ln ( ) vs. treatment

93 time denotes the reaction rate constant.

114 the rate of PME inactivation.

117 Which, T2 and T1 are temperatures corresponding to D2 and D1, respectively. In HP

148 constant, respectively.

150 1. 2. Isoforms of PME

179 isoforms (Aghajanzadeh, Ziaiifar, Kashaninejad, Maghsoudlou, & Esmailzadeh, 2016a;

190 2. Juice pasteurization

202 several thermal and non-thermal methods.

206 2. 1. Thermal pasteurization of juice

224 2. 1. 1. Conventional thermal pasteurization

268 eliminate these unwanted effects.

270 2. 1. 2. Microwave heating

275 Commission in food industrial applications. In pasteurization process, the choice of MW

278 characteristics (dielectric properties, homogeneity and quantity) (Ahmed, Ramaswamy,

288 between these molecules or ions during this rearrangement.

289 In comparison to the conventional treatment, come up time (CUT) is shorter in MW

292 by an electrical field of MW (Tajchakavit & Ramaswamy, 1997a). MW energy may be

294 hydrophobic, electrostatic and hydrogen bonds may be disrupted.

313 70℃ with Z-value of 22.1℃ (Cinquanta et al., 2010).

321 in a uniform heating (Cinquanta, et al., 2010). Additionally, applying a continuous MW

327 temperature using 40 ml/min-83 ℃ and 50 ml/min-75 ℃ heating conditions, respectively.

336 González, et al., 2014).

342 2. 1. 3. Ohmic heating

351 operation (N. Kaur & Singh, 2015).

356 thermal effect, electro-plasmolysis (electropermeabilization) as a non-thermal phenomenon

387 key factors affecting the PME inactivation.

396 2. 2. Non- thermal pasteurization of juice

408 2. 2. 1. Pulsed electric field

(Kulshrestha & Sastry, 2006).

combination of thermal and non-thermal treatments on PME inactivation during juice

467 2. 2. 2. High pressure processing

487 (N. Kaur & Singh, 2015).

506 tertiary one (Alexandrakis, et al., 2014).

511 months (Ogawa, Fukuhisa, Kubo, & Fukumoto, 1990).

520 1996; Basak, Ramaswamy, & Simpson, 2001).