Evaluation of HPCD batch treatments on enzyme inactivation kinetics and selected quality characteristics of cloudy juice from Golden delicious apples

Accepted Manuscript Evaluation of HPCD batch treatments on enzyme inactivation kinetics and selected quality characteristics of cloudy juice from Golden delicious apples A.E. Illera, M.T. Sanz, S. Beltrán, R. Melgosa, A.G. Solaesa, M.O. Ruiz PII: S0260-8774(17)30448-X DOI: 10.1016/j.jfoodeng.2017.10.017 Reference: JFOE 9050 To appear in: Journal of Food Engineering Please cite this article as: A.E. Illera, M.T. Sanz, S. Beltrán, R. Melgosa, A.G. Solaesa, M.O. Ruiz, Evaluation of HPCD batch treatments on enzyme inactivation kinetics and selected quality characteristics of cloudy juice from Golden delicious apples, Journal of Food Engineering (2017), doi: 10.1016/j.jfoodeng.2017.10.017 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. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT HPCD inactivation rate of PPO and PME increased with increasing p and T. Sensitivity to temperature change is similar for HPCD and mild thermal treatment. PME is more HPCD resistant than PPO. HPCD treatment decreased particle size of cloudy apple juice. Colloidal stability and nutritional properties were not affected by HPCD. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Evaluation of HPCD batch treatments on enzyme inactivation kinetics and selected 1 quality characteristics of cloudy juice from Golden delicious apples 2 3 A.E. Illera, M. T. Sanz ∗ , S. Beltrán, R. Melgosa. A. G. Solaesa, M. O. Ruiz 4 Department of Biotechnology and Food Science (Chemical Engineering Section), 5 University of Burgos, 09001 Burgos. Spain 6 7 Abstract 8 Cloudy apple juice has been treated by high pressure carbon dioxide (HPCD) as non-thermal 9 technology to inactive polyphenoloxidase and pectinmethylesterase in batch mode. Stirring 10 speed (from 200 to 600 rpm) induced an increase in the enzyme inactivation rate while a triple 11 cycle of pressurization/depressurization led to the same enzyme inactivation efficiency. Enzyme 12 inactivation kinetics were determined at different temperatures (from 35 to 45 ºC) and pressures 13 (from 10 to 20 MPa). Data were described by the first order kinetic model and the Weibull 14 model. For the first order kinetic model, decimal reduction time for HPCD treatment was found 15 to be smaller than for mild heating, in the same temperature range. The same tendency was 16 observed for the first decimal reduction time in the Weibull model. HPCD treatment resulted in a 17 homogenization effect reflected in the shifting of the particle size distribution towards smaller 18 diameters after treatment. HPCD treatment did not result in a change of water and oxalate 19 soluble pectin content, total phenolic compounds and hidroxymethylfurfural content. 20 21 22 ∗ Corresponding author. Tel.: +34 947 258810. Fax: ++34947258831. E-mail address [email protected] MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Keywords: Cloudy apple juice, HPCD, inactivation kinetics parameters, properties 23 1. Introduction 24 Consumption of cloudy apple juice seems to be more beneficial than consuming clear apple juice 25 due to changes in phenolic compounds and loss of pectins during clarification process 26 (Markowski et al., 2015). However, color and cloud stability are main challenges in preservation 27 of cloudy apple juice during its processing and storage. Enzymatic browning in cloudy apple 28 juice is due to the action of polyphenol oxidase (PPO) that catalyzes the oxidation of phenolic 29 compounds to quinones. On the other hand, the mechanism of cloud stability is not yet 30 understood. Among the different mechanisms proposed, electrostatic repulsion by negative 31 charges present in the partly demethylated pectin due to galacturonic residues could be 32 responsible of cloud stability avoiding aggregation (Zhao et al., 2008). In this regard, the activity 33 of pectinmethylesterase (PME) causes pectin demethylation that could precipitate with calcium 34 ions present in the juice causing clarification and the loss of turbidity. However, other 35 components with negative charge have been also reported in the literature as stabilizing agents of 36 the cloud in the serum such as phosphatide acids of cloud lipids (Krapfenbauer et al., 2006). 37 Thermal treatments have been traditionally used for the inactivation of microorganisms and 38 enzymes that affect negatively the quality of foods. However, these methods cause undesirable 39 effects in quality attributes such as flavor and loss of nutritional components. High pressure 40 carbon dioxide (HPCD) treatment has been proposed as an alternative non-thermal pasteurization 41 technique that can inactivate certain microorganisms and enzymes under mild operation 42 conditions. Typically, operating pressure does not exceed 50 MPa and temperature ranges 43 between 20 and 50 ºC, below pasteurization temperature (Manzocco et al., 2016). The 44 mechanism of pressurized CO 2 on enzyme inactivation is not yet fully understood. In the 45 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT literature different approaches can be found such as pH lowering due to dissolved CO 2 , 46 conformational changes or formation of different complex with CO 2 molecules (Hu et al., 2013). 47 Some studies have been found in the literature regarding the effect of HPCD on enzymes of 48 cloudy apple juice from Fuji apples. Niu et al. (2010) evaluated the quality of cloudy Fuji apple 49 juice processed from apple slices previously treated by HPCD. These authors found that PPO 50 could be completely inactivated at 20 MPa for 20 min and at temperature as low as 25 ºC; 51 however, residual activity of 18% was still found for PME after treatment at 65 ºC and 20 MPa 52 for 20 min. Gui at al. (2007) found higher values of residual activity than the work of Niu et al. 53 (2010), for PPO in HPCD-treated cloudy Fuji apple juice in a batch mode (40% at 30 MPa and 54 55 ºC for 60 min). When using a continuous HPCD system, holding times of 10 min were 55 needed at 22 MPa and 60 ºC to achieve complete inactivation of PPO but, at the same working 56 conditions, still PME residual activity of 42% was obtained (Xu et al., 2011). 57 From previous reported results, it can be clearly observed that there is a great variety in the 58 literature data on enzyme inactivation from cloudy apple. This is a main drawback in the 59 commercialization of this technology and reliable data on juice quality are still needed. 60 According to Wang et al. (2010) the influence of agitation on enzyme inactivation under high61 pressure environment is a key factor in the design of reactors of industrial scale volume. 62 However, the effect of stirring speed on enzyme activity has not been considered in most of the 63 studies. Wang et al. (2010) determined the effect of agitation at 40ºC on isoamylase, obtained 64 from a fermentation broth from Pseudomonas amyloderamosa after cell removal, and a 65 commercial β-amylase under supercritical carbon dioxide, at different operating times (up to 120 66 min) and different operating pressure (from 11.0 to 17.2 MPa), observing that the activity of both 67 enzymes decreased by increasing the speed of agitation. 68 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT In this work, the effect of HPCD treatment on some quality characteristics of cloudy apple juice 69 from Golden delicious has been studied. The effect of some variables, non-previously studied in 70 literature, on enzyme inactivation from cloudy apple juice, has been considered such as the effect 71 of stirring speed and the number of successive pressurization/depressurization cycles. Enzyme 72 inactivation kinetic data parameters are also necessary to scale-up HPCD process. This regard, 73 enzyme inactivation kinetics of PPO and PME have been determined at different operating 74 pressures and temperatures. Enzyme inactivation kinetic data have been described by the first 75 order kinetic model and the Weibull model as an alternative to the classical first order kinetic 76 model. Some other important quality parameters of cloudy apple juice have been also determined 77 before and right after HPCD treatment, such as pectin content, particle size distribution, color 78 and antioxidant capacity. 79 2. Materials and methods 80 2.1 Juice preparation 81 Golden delicious apples were peeled and cut in cubes and then added to a 0.3 % of L-ascorbic 82 acid solution to avoid enzymatic browning during processing. Apple cubes were wiped and 83 immediately squeezed with a screw juice extractor. The liqueur was filtered with 2 layers of 84 cheesecloth. The pH of the juice obtained was 3.89 ± 0.01 and the soluble solid content was 12.5 85 ± 0.1 °Brix. The juice was stored frozen at −18 °C until further use. 86 2.2 HPCD equipment and processing 87 The HPCD cell was made of stainless steel (SS-316) and had an internal volume of 100 mL with 88 a maximum operating pressure and temperature of 30 MPa and 80 ºC, respectively (Melgosa et 89 al., 2017). For each experiment, 40 mL of apple juice were charged into the high pressure cell 90 that was immersed in a thermostatic water bath at the operating temperature. The magnetic 91 stirring was then connected and the system was pressurized by a using a syringe pump with a 92 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT pressure controller (ISCO 260 D) up to the desired pressure. CO 2 was fed into the high pressure 93 cell through a sintered stainless steel micro-filter with a pore size of 10 µm (Briongos et al., 94 2016). This regard, Ishikawa et al. (1995) showed that the concentration of CO 2 in the sample 95 was influenced by the way CO 2 was fed into the sample, increasing by decreasing the pore size 96 of the filter at 25 MPa and 35ºC. 97 Samples were collected periodically to follow the inactivation kinetics of the enzymes. 98 Experiments were carried out in the ranges of temperature (T) from 35 to 45 ºC, pressure (p) 99 from 10 to 20 MPa and stirring speed from 200 to 600 rpm. The effect of the sampling procedure 100 and the number of pressurization/depressurization cycles on enzyme activity was also analyzed. 101 To compare the effect of the HPCD treatment on enzyme activity with mild heating treatment, 102 cloudy apple juice was heated at atmospheric pressure in the same temperature range (from 35 to 103 45 ºC). Samples were taken periodically at different treatment times up to 120 min. 104 Some other important quality attributes of cloudy apple juice, such as particle size distribution, ξ 105 potential, turbidity, pH, pectin content, total phenolic compounds, non-enzymatic browning and 106 antioxidant capacity were also determined before and right after HPCD treatment. 107 2.3 Physico-chemical analysis 108 2.3.1. Determination of PPO activity 109 The activity of PPO was determined spectrophotometrically by using a 0.05 M catechol (Sigma 110 Aldrich) solution prepared in a 0.1 M phosphate buffer (pH 6.5) as substrate. Samples were 111 analyzed by adding 100 µL of apple juice into 2.9 mL substrate solution. Oxidation of catechol 112 was determined immediately by the increase in absorbance at 420 nm by using a Jasco V-750 113 spectrophotometer equipped with a Peltier thermostated cell holder and a water pump to keep the 114 temperature constant at 30 ºC. The PPO activity was taken as the very first linear part of the 115 reaction curve. 116 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2.3.2. Determination of PME activity 117 PME activity was determined by using an automatic titrator system (Metrohm  Titrando) by 118 using a 1 % of pectin solution (Alfa Aesar  pectin citrus) prepared in NaCl 0.3 M as substrate. 119 50 mL of pectin solution was mixed with 1 mL of cloudy apple juice and pH was adjusted to 7.5 120 with NaOH 0.02 N. During pectin hydrolysis at 30 ºC, pH was maintained at 7.5 by adding 121 NaOH 0.02 N. The amount of NaOH added for 15 min was recorded. One PME activity unit 122 (UPE) is defined as the micromoles of carboxylic groups produced per minute and mL of juice at 123 pH 7.5 and 30 ºC (Briongos et al., 2016) . 124 Relative residual activities of PPO and PME were evaluated as: 125 Residualactivity,RA= !"# $%&'%·100%=,, - ·100 [1] 126 2.3.3. Determination of pectic substances 127 Pectic substances in cloudy apple juice before and after HPCD treatment have been determined 128 according to Robertson (1979) by progressive extraction of the alcohol insoluble solids by water 129 (high methoxyl pectins are extracted) and ammonium oxalate (low methoxyl pectins are 130 extracted). Water soluble pectins play an important role in the turbidity of the cloudy apple 131 juices acting as colloid stabilizers, while oxalate-soluble fraction can form gels with polyvalent 132 metal ions (Robertson, 1979). Pectic substances are determined spectrophotometrically at 133 520 nm after total hydrolysis to galacturonic acids by using m-hydroxydiphenil (MHDP) as a 134 chromogenic reagent. Solutions were freshly prepared before analysis: 0.15% solution of m135 hydroxydiphenyl (Sigma Aldrich) in 0.5% NaOH and 0.0125 M sodium tetraborate (Panreac) in 136 concentrated sulphuric acid. For pectin determination, 1 mL of water extract and 5 mL of the 137 sodium tetraborate solution were mixed and placed in a water-ice bath. The mixture is then 138 heated in water bath at 80 ºC for 6 min and subsequently cooled again in a water-ice bath. 139 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 0.1 mL of the m-hydroxydiphenyl solution was added and after homogenization of the mixture, 140 absorbance at 520 nm was measured as a function of time. According to Ibarz et al. (Ibarz et al., 141 2006) spectrophotometric data measurements were taken at the highest absorbance instead of at a 142 certain reaction time. A calibration curve was prepared with standard solutions of D-galacturonic 143 acid (Sigma Aldrich) by following the same colorimetric method. A blank was also prepared 144 with no D-galacturonic acid. 145 2.3.4. Particle size distribution and zeta-potential 146 Particle size distribution (PSD) was determined by laser diffraction with a Mastersizer 2000 147 (Malvern® Inst., MA). The system uses a laser light at 750 nm wavelength to size particles from 148 0.4 to 2000 µm by light diffraction. Particle size distribution was calculated by the Fraunhofer 149 model. Size distributions (volume fractions against particle size) before and after HPCD 150 treatment were calculated and the weight-average sizes expressed as: 151 • The equivalent surface area mean diameter: D/3,22=∑nd5 6∑n ⁄d5 8 [2] 152 • The equivalent volume mean diameter: D/4,32=∑nd5 :∑n ⁄d5 6 [3] 153 where d lc is the diameter of the particle and n c is the percentage of particles. 154 To describe distribution width one common parameter is the Span: 155 Span=# =,>.@ A# =,>.B # =,>.C [4] 156 where D v,0.9 , D v,0.1 and D v0.5 are the particle size bellow which, 90%, 50% and 10% of the 157 particles lies. 158 ζ-potential was determined with a Zetasizer Nano ZS apparatus, using the Laser Doppler 159 Velocimetry techniques. Samples were diluted 1:50 with deionized water and filtered through 160 5 µm filter. ζ-potential gives an indication of the potential stability of a colloidal system and it is 161 a good index of the colloidal electrostatic repulsive forces. Particles will repel each other when 162 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT literature about the effect of the number of cycles on enzyme inactivation by HPCD on fresh 291 juices. 292 3.3 Effect of mild heating on PPO inactivation 293 PPO inactivation kinetics were determined at atmospheric pressure in the temperature range 294 from 35 to 45 ºC (same temperature range as for HPCD treatment, Figure 2). The maximum 295 reduction of PPO activity was nearly 40 % after 2 h of heating at 45 ºC indicating that PPO of 296 cloudy Golden delicious apple juice was rather stable under mild temperatures. Similar 297 inactivation degrees of PPO from cloudy Fuji apple juice were obtained by Gui et al. (2007). 298 After 1 hour of mild heating, these authors reached a maximum reduction of PPO activity of 299 20% at 45 ºC. However, Niu et al. (2010) found that when subjecting apple slices to mild heating 300 treatment from 25 to 45 ºC for 20 min and then processing them into cloudy apple juice, PPO 301 activity increased by 22-51%. Buckow et al. (2009) also found that at 45 ºC, PPO activity from 302 cloudy apple juice increased 30%, attributing this behavior to the release of latent PPO. 303 Enzyme inactivation kinetic data under mild heating conditions and at atmospheric pressure were 304 described by the first-order model. Parameters are collected in Table 1. Inactivation rate 305 constant, k, increased with the operating temperature, therefore the corresponding D values 306 decreased with temperature. The temperature sensitivity parameter, z T , and the activation energy 307 were evaluated as 36 ºC and 53 kJ/mol, respectively (see Table 1). Values of the same order 308 were reported by Gui et al. (2007) for cloudy Fuji apple juice, 27.0 ºC for z T and 72.0 kJ/mol, for 309 Ea. Thermal stability of extracted apple PPO from Golden delicious was studied in the 310 temperature range from 68 to 78 ºC by Yemenicigly et al. (1997). These authors reported a z T 311 value of 9.9 ºC. The higher value obtained in this work for z T in the temperature range from 35 to 312 45 ºC might indicate a lower susceptibility to temperature change at temperatures lower than the 313 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT one observed at pasteurization temperatures. In any case, the values of the activation energy 314 were similar in both temperature ranges. 315 3.4. Effect of HPCD treatment on PPO inactivation 316 Enzyme inactivation kinetics for PPO of cloudy Golden deliciuos apple juice have been 317 determined at 10 MPa in the temperature range from 35 to 45 ºC (Figure 3a). By increasing 318 temperature, enzyme inactivation rate also increased. In addition to the intrinsic effect of 319 temperature on enzyme inactivation, by increasing temperature, although CO 2 density decreased, 320 the diffusivity of CO 2 can be improved and accelerate the molecular collisions between CO 2 321 molecules and the enzyme. Figure 3b shows the inactivation kinetics at 45 ºC in the pressure 322 range from 10 to 20 MPa. Higher operating pressures led to faster inactivation rates, but the 323 fraction of the resistant form of the enzyme was nearly independent of the operating pressure, 324 being around 6 %. This value was lower than the minimal residual activity of PPO from cloudy 325 Fuji apple juice reported by Gui et al. (2007), 37%, at 30 MPa and 55 ºC for 60 min. In any case, 326 comparison with other HPCD systems is difficult to establish since the inactivation reached 327 would depend not only on the type and source of the enzyme and the experimental conditions 328 such as pressure, temperature and treatment time but also on the agitation of the system and the 329 ratio sample volume/cell volume since this last parameter determines the amount of CO 2 injected 330 to the system. Unfortunately in most of the studies these parameters are not detailed. PPO from 331 cloudy Fuji apple juice was completely inactivated by a continuous HPCD system at 22 MPa and 332 60 ºC for 10 min (Xu et al., 2011). This could suggest a higher efficiency of a continuous system 333 due to the turbulent energy, comparable with a good agitation in a discontinuous system. 334 However, 60 ºC cannot be considered as non-thermal and residual activity as high as 47% was 335 obtained in the continuous system at 43 ºC and 25 MPa for 2 min. (Xu et al., 2011). 336 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Inactivation kinetic data by HPCD were fitted to the first order kinetic model and the Weibull 337 model (Table 2). Although the Weibull model yielded the best fitting quality, the first order 338 kinetic model described the inactivation curve quite well and therefore, kinetic parameters for 339 the linear regression model have been also listed in Table 2 for comparison with data reported in 340 the literature. 341 For the first order kinetic model, inactivation rate constant, k, increased with temperature and 342 pressure. Consequently, D values decreased with increasing pressure and temperature. At each 343 operating temperature, D values were smaller for HPCD treatment (Table 2) than the 344 corresponding values for mild heating treatment (Table 1) proving that HPCD treatment was 345 more effective than thermal treatment alone. For example D was equal to 515 min at 45 ºC and 346 atmospheric pressure and decreased to 104 min at 45 ºC and 10 MPa. 347 z T and activation energy values were determined as 29 ºC and 64 kJ/mol, respectively, for the 348 first order kinetic model (see Table 2). z T and activation energy values were of the same order as 349 those listed for mild heating treatment (Table 1). This meant that the sensitivity to temperature 350 change was similar for HPCD as for mild heating treatment in the temperature range covered in 351 this work. The same tendency in z T and E a was found by Liu et al. (2008) in the inactivation of 352 PPO of red beet extracts. These authors found that under mild heating treatment at atmospheric 353 pressure (35-55 ºC), z T and E a were equal to 34 ºC and 57 kJ/mol, respectively. After HPCD 354 treatment at 37.5 MPa, in the same temperature range, they obtained values of 36 ºC and 355 54 kJ/mol for z T and E a , respectively. However, Gui et al. (2007) found for PPO of cloudy Fuji 356 apple juice that z T increased from 27 to 108 ºC and activation energy decreased from 72 to 357 18 kJ/mol under mild heating treatment (35-55 ºC) and HPCD at 30 MPa in the same 358 temperature range, respectively. This increase in z T and decrease in Ea found by Gui et al. (2007) 359 would indicate that the enzyme inactivation become less temperature dependent under 360 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT pressurized CO 2 . This behavior is difficult to understand and it has not been observed in our 361 work. As it has been previously mentioned, in addition to the intrinsic effect of temperature on 362 enzyme inactivation, temperature has two different effects on important properties of CO 2 . An 363 increase in temperature involves an improvement of mass transport properties enhancing CO 2 364 diffusivity and the number of collisions between the CO 2 and the enzyme, but on the other hand, 365 CO 2 density and therefore CO 2 solubility in the aqueous medium decreases. It seems that these 366 effects play both an important role, since similar sensitivity to temperature was found in mild 367 heat treatment and in HPCD. 368 By using equations 8 and 10 z P and V a have been determined as 25 MPa and -251 cm 3 /mol, 369 respectively for the first order kinetic model. The negative value of the activation volume 370 indicated that PPO inactivation was favored by increasing operating pressure. Other values of 371 activation volumes reported in the literature for PPO inactivation from different sources are of 372 the same order, -271.7 and -307.9 cm 3 /mol for the labile and stable fraction of PPO of water 373 melon at 50 ºC in the pressure range of 8-30 MPa (Liu et al., 2013) or -94.31 cm 3 /mol for PPO 374 from cloudy Fuji apple juice at 55ºC in the pressure range from 8 to 30 MPa (Gui et al., 2007). 375 The Weibull parameters are also listed in Table 2. Scale, α, and shape, β, parameters were used 376 to calculate the time require to inactivate 1 log (t d=1 ) of PPO. Similar to the D-values for the first 377 order kinetic model, t d=1 decreased with operating pressure and temperature. As suggested by van 378 Boekel (2002) is important to determine the effect of temperature, and in the present work of 379 pressure, on both parameters of the Weibull model. Scale parameter was statistically significant 380 dependent on temperature and pressure, when tested at the 95% significance level for a linear 381 relationship, decreasing with pressure and temperature. The shape factor, n, was less than 1 382 except for 10 MPa and 35 ºC. The concavity shape of the inactivation curve could be understood 383 as a faster inactivation of the labile form of the enzyme and the presence of a resistant form of 384 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT the enzyme to HPCD treatment (Figures 3a and 3b). The shape parameter, β, was found to be 385 statistically significant dependent on pressure but although there seemed to be a trend in 386 temperature dependence of β, it was not statistically significant, when tested at the 95% 387 significance level for a linear relationship. In any case, it must be highlighted that only three 388 temperatures have been studied to determine the effect of temperature. According to van Boekel 389 (2002), the scale parameter, α, could be modelled in a similar way to the classical D value of the 390 first order kinetic model, suggesting a linear dependence of the log α on temperature and 391 considering that β did not depend on temperature: 392 p{|n=}V−~V [16] 393 A z T ’ value can be also defined: 394 =V  B [17] 395 The value of the z T ’ is listed in Table 2, together with the quality of the fitting. Although the 396 concept of z T and z’ T is different since z T is obtained from the linear part, whereas z’ T takes also 397 into account the nonlinear part (Van Boekel, 2002), similar values were obtained for both 398 parameters (z T = 29 ºC and z’ T = 27 ºC). 399 In this work, it was also found a linear dependence of the log α on pressure: 400 p{|n=}8−~8d [18] 401 Although it was found a statistically significant linear relationship between β and p, the inverse 402 of the slope of log α versus p was also evaluated. Analogous to z’ T it was defined a z’ p 403 =−V  ] [19] 404 This value is reported in Table 2 and quite similar values can be observed for z p and z p ’. 405 3.5. Effect of HPCD on PME 406 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT To compare the HPCD resistance of PME and PPO, PME inactivation kinetics were determined 407 for the experiments performed at 45 ºC and 10 MPa and 20 MPa (Figure 4). PME inactivation 408 rate was also determined at atmospheric pressure and 45 ºC. At atmospheric pressure, it was 409 found that residual PME activity was still 90% after 120 min at 45ºC (Figure 4). Teleszko et al. 410 (2016) studied thermal treatment of different apple cultivars, founding that PME from cloudy 411 Golden deliciuous apple juice was one of the apple cultivars that presented strongest 412 thermostability (30% of residual activity after heating 5 min at 100 ºC). Niu et al. (2010) found 413 that the RA of PME from cloudy Fuji apple juice from apple slices previously preheated for 414 20 min at the corresponding working temperature, varied between 82 to 72 % at 35 and 45ºC, 415 respectively, but with error bars higher than 10% of RA at each temperature (graphical lecture). 416 After HPCD treatment, higher inactivation rates were observed than for mild heating treatment at 417 the same temperature. Although, residual activity was still around 60 % at 20 MPa and 45 ºC 418 after 60 min. This indicates that PME from cloudy Golden delicious apple juice is rather stable to 419 HPCD at operating temperatures considered as non-thermal treatment. Different HPCD 420 inactivation values of PME from cloudy apple juice have been reported in the literature. Niu et 421 al. (2010) reported a minimal residual activity of 18% for PME of cloudy Fuji apple juice from 422 apple slices treated by HPCD at 20 MPa and 65 ºC for 20 min. In a continuous system, PME 423 from cloudy Fuji apple juice still presented a residual activity of 40% at 22 MPa and 60ºC for 10 424 min (Xu et al., 2011). HPCD inactivation of PME extracts from apple juice was found to be 425 more effectively inactivated at 30 MPa for 60 min, reaching 5% of residual activity at 55 ºC (Zhi 426 et al., 2008). However, in this case, results are different to compare since an extracted enzyme 427 suspended in a buffer solution can give different inactivation results from those obtained in an 428 original juice. 429 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Results obtained in this work, indicate that PME from cloudy apple juice was more resistant to 430 HPCD than PPO in the pressure range covered in this work. In the literature, as for thermal 431 treatment, the higher stability has been attributed to the more complex structure of PPO with 432 three or four subunits, in higher plants, than PME, with one subunit. This structure, makes it 433 more susceptible to HPCD treatment (Niu et al., 2010). In addition, PME in fruit juices is more 434 difficult to be effectively inactivated, since PME is bound to the plant cell wall, which contains 435 natural stabilizing factors (Zhou et al., 2010). 436 PME inactivation kinetic data were described by the first order model at atmospheric pressure 437 (Table 1). The decimal reduction time at 45 ºC and atmospheric pressure was found to be much 438 higher than for PPO at the same conditions. HPCD inactivation data at 45ºC and 10 MPa and 439 20 MPa were fitted to the first order kinetic model and the Weibull model (Table 2). The 440 Weibull model yielded the best fitting quality, but still an acceptable good fitting for the first 441 order kinetic model was obtained. For the first order model, inactivation rate constant increased 442 with pressure, but lower values than for PPO inactivation were obtained and therefore higher D 443 values. Regarding the Weibull model, the scale and shape parameters, both, decreased with 444 operating pressure at 45ºC. These parameters were used to calculate the time needed to inactivate 445 1 log (t d=1 ) of PME at the two pressures studied. The t d=1 calculated by the Weibull model is 446 much higher than the corresponding D values by the first order kinetic model. Therefore, in this 447 case, the use of the first order kinetic model could lead to an underprocessing estimation for 448 PME inactivation. 449 3.6. Effect of HPCD on different quality parameters of cloudy apple juice 450 Some quality parameters have been determined right after HPCD treatment at 45 ºC, 20 MPa and 451 60 min of treatment time. 452 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT pH. Initial pH of cloudy apple juice was 3.89 ± 0.01 and immediately after HPCD treatment, pH 453 dropped 0.15 units to 3.74 ± 0.01. Significant differences have been determined among sample 454 pH averages before and after HPCD treatment when applying the Fisher’s least significant 455 method at p-value ≤ 0.05 (Table 3). Gui et al (2007) reported a drop of 0.3 units from 3.95 to 456 3.66 ± 0.03 for Fuji cloudy apple juice after HPCD treatment at 22 MPa and 55 ºC for 60 min. 457 According to Meysammi et al. (1992), juices and beverages can be considered as an aqueous 458 electrolyte system of multiple ions. Under pressurized CO 2 a number of dissociation reactions 459 can take place liberating some of them H + ions that could reduce the pH of the media. In any 460 case, after 2 h after depressurization pH returned to a value close to its initial value (pH = 3.87 ± 461 0.01). 462 Colour parameters. Table 3 lists the L*, a*, b* parameters of cloudy apple juice before and 463 after HPCD treatment. During browning, the L* values decrease, the juice’s colour becomes 464 darker, while a* and b* values increase, with more red and yellow components (Krapfenbauer et 465 al., 2006). After HPCD treatment, lightness (L*) presented slightly, but significant, lower values; 466 however, 99% of the lightness was still retained. No significant difference was observed in the 467 redness component but a significant increase of the yellowness (b*) value has been observed 468 indicating more yellow components right after HPCD treatment. The change in colour, ∆E (Eq. 469 5) is also presented in Table 3. Slightly noticeable differences in color after HPCD treatment 470 have been determined, ∆E ≈ 0.51 ± 0.05 (Yuk et al., 2014). Other authors found that L*, a* and 471 b* values remained almost constant after HPCD treatment at 55 ºC and different operating 472 pressures (8-30 MPa) for 60 min (Gui et al., 2006). 473 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Total polyphenolic compounds. Total polyphenols remained unchanged after HPCD. Gasperi 474 et al. (2009) also found no significant differences in polyphenols content after HPCD treatment 475 of a blend of Golden delicious and Granny Smith apple at 10 MPa and 36 ºC for 10 min. 476 Antioxidant capacity. Antioxidant capacity of Golden cloudy apple juice has been determined 477 by the ABTS tests (Table 3). Slightly higher values were obtained after HPCD treatment 478 although differences before and after treatment were found to be no significant (p ≤ 0.05). 479 Hydroxymethylfurfural. Table 3 presents the HMF content before and after HPCD treatment. 480 From these results, it can be concluded that non enzymatic browning reactions were not 481 significant during HPCD processing, probably due to the mild temperatures employed in HPCD 482 treatments. 483 Particle size distribution. The particle size distribution of untreated cloudy apple juice ranged 484 from 0.275 µm to 416.87 µm with three maxima at 0.63, 19.96 and 208.93 µm (see Figure 5). 485 After HPCD treatment, the particle range shifted to smaller particle sizes from 0.04 µm to 486 104.71 µm with one main maximum at 0.182 µm. Table 3 lists values of D[3,2] and D[4,3] of 487 freshly cloudy apple juice and after HPCD treatment. The values after HPCD treatment were 488 significantly lower than those of freshly cloudy apple juice. The homogenization effect of HPCD 489 can be also observed in the values of d(0.1), d(0.5) and d(0.9), which correspond to the size of 490 particle below which 10%, 50% and 90% of the sample lies, respectively. This homogenization 491 effect induced by HPCD could be attributed to the explosive action and the bubbling of CO 2 492 from the juice during decompression (Zhou et al., 2010). 493 For freshly cloudy apple juice, the total volume of all particles with diameter less than 5 µm 494 represented 56.5% of the total particles volume, while this number increased up to 96% after 495 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT HPCD treatment. This fact is also reflected in the Span values (Table 3) that shows a higher 496 degree of uniformity of the cloudy apple juice after HPCD treatment. 497 Turbidity. The turbidity of the cloudy apple juice after HPCD is considerably higher than that of 498 freshly cloudy apple juice (Table 3). It seems that there was a correlation between juice’s 499 turbidity and particle size distribution However, no correlation was found with PME residual 500 activity since, after HPCD treatment, PME activity was still more than 60%. 501 Pectin content. In this work, soluble pectin presented slightly higher values after HPCD (Table 502 4). HPCD treatment could result in a more dissolution of pectin in fruit cell into the juice, 503 increasing the value of soluble pectin. In addition, according to the PSD previously reported, 504 HPCD induced an homogenization with lower particle size diameters, which could result in a 505 higher solubility of the pectin; since, in the literature, it has been reported that pectin solubility in 506 water increases by decreasing its polymer size (Sila et al., 2009) however no significant 507 differences have been found at p ≤ 0.05. No significant differences have been also determined 508 for the oxalate-soluble pectin. 509 In the literature, Zhou et al. (2010) reported no change in water soluble pectin when treating 510 peach juice by HPCD at 30 MPa and 55 ºC at different treatment times, although these authors 511 observed an increase of the large particle after HPCD treatment. However, Yu et al (2013) 512 observed that soluble pectin of HPCD-treated banana pulp at 20 MPa, during 30 min at different 513 temperatures in the range from 45 to 60 ºC, was significantly lower than the untreated pulp. 514 These authors attributed this behavior due to interaction of pectin and protein or gelling of pectin 515 that may occur during HPCD treatment (Yu et al., 2013). 516 ξ potential. ξ potential is an indication of colloidal stability of the juice. Both, untreated and 517 treated apple juice presented negative values of -22.0 mV and -22.3 mV, respectively. These 518 values were within the range reported by Corak and Corredig (2006) of ξ potential as a function 519 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Table 1. Kinetic parameters at 0.1 MPa for the first order kinetic model for PPO inactivation by 657 mild thermal heating (35-45ºC) and PME at 45 ºC. 658 PPO First order kinetic model T, ºC k, min - 1 D, min R 2 MRD 35 40 45 0.00102 ± 0.00003 0.00166 ± 0.00005 0.00194 ± 0.00003 980 602 515 0.9814 0.9791 0.9993 1.6 1.1 1.1 z T = 36 ± 11 ºC (R 2 = 0.9188) E a = 53 ± 14 kJ/mol (R 2 = 0.9248) PME, 45ºC 0.00038 ± 0.00003 2632 0.9799 0.8 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Table 2. Kinetic parameters for the Weibull and the first order kinetic models for PPO and PME inactivation by HPCD. 659 PPO Weibull model First order kinetic model p, MPa T, ºC α , min β t d=1 , min R 2 MRD k, min - 1 D, min R 2 MRD 10 10 10 35 40 45 98 ± 2 72 ± 2 41 ± 2 1.13 ± 0.06 0.93 ± 0.04 0.89 ± 0.05 205 177 105 0.9940 0.9939 0.9960 2.3 3.4 4.9 0.0044 ± 0.0001 0.0060 ± 0.0001 0.0096 ± 0.0002 227 167 104 0.9891 0.992 0.992 3.4 3.9 6.8 z’ T = 27 ± 4 ºC; (R 2 = 0.9726) z T = 29 ± 3 ºC; (R 2 = 0.9897) E a = 64 ± 8 kJ/mol; (R 2 = 0.9834) 12.5 15 20 45 45 45 30 ± 1 23.7 ± 0.4 17.2 ± 0.9 0.74 ± 0.02 0.70 ± 0.02 0.59 ± 0.02 93 78 71 0.9983 0.9995 0.9981 2.9 1.2 5.9 0.015 ± 0.001 0.019 ± 0.001 0.026 ± 0.004 67 53 38 0.966 0.953 0.965 23.6 15.6 44.3 z’ P = 27 ± 3 MPa; (R 2 = 0.9729) z P = 25 ± 5 MPa; (R 2 = 0.9226) Va=- 251 ± 48 cm 3 /mol; (R 2 = 0.9332 ) PME α, min β t d=1 , min R 2 MRD k, min -1 D, min R 2 MRD p, MPa T, ºC 10 45 785 ± 294 0.55 ± 0.09 3576 0.9587 2.3 0.0015 ± 0.0001 667 0.9424 5.2 20 45 373 ± 74 0.45 ± 0.04 2380 0.9940 1.1 0.0037 ± 0.0006 270 0.9181 8.6 660 661 662 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Table 3. Some quality parameters before and right after HPCD treatment of cloudy apple juice at 663 20 MPa and 45 ºC after 60 min. 664 Parameter Before HPCD After HPCD pH 3.89 ± 0.006 a 3.74 ± 0.006 b Color ∆E= 0.51 ± 0.05 L*= 37.30 ± 0.03 a a*= 1.6 ± 0.1 a b*= 7.90 ± 0.03 a L*= 36.98 ± 0.01 b a*= 1.59 ± 0.08 a b*= 8.26 ± 0.07 b Total polyphenols, mg galic/L 456.1 ± 13.1 a 455.4 ± 4.3 a ABTS, % of inhibition 20 min, 49 ± 2 a 60 min, 65 ± 2 a 120 min, 81 ± 3 a 20 min, 52 ± 3 a 60 min, 68 ± 4 a 120 min, 85 ± 4 a Non-enzimatic browning, (mg HMF/L) 2 ± 0.3 a 1.70 ± 0.09 a PSD D(3,2) = 1.7 ± 0.07 µm a D(4,3) = 109 ± 3 µm a d(0,1) = 0.59 ± 0.01 µm a d(0,5) = 13 ± 1 µm a d(0,9) = 337 ± 7 µm a Span = 26 ± 7 µm a D(3,2) = 0.21 ± 0.01 µm b D(4,3) = 2.4 ± 0.2 µm b d(0,1) = 0.099 ± 0.001 µm b d(0,5) = 0.265 ± 0.006 µm b d(0,9) = 1.63 ± 0.07 µm b Span = 5.8 ± 0.1 µm b Turbidity, NTU 105 ± 2 a 168 ± 3 b Water soluble pectin Oxalate soluble pectin (mg galacturonic/L) 22 ± 2 a 23 ± 4 a 28 ± 3 a 23 ± 3 a ξ-potential -22.0 ± 0.3 a -22.3 ± 0.4 a Values with different letters in each row (a, b) are significantly different when applying the Fisher’s least significant 665 method at p-value ≤ 0.05. 666 667 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT List of Figure captions 668 Figure 1. (a) Effect of stirring speed on PPO inactivation from cloudy apple juice by HPCD at 669 20 MPa and 45 ºC ( ◇ 200 rpm, ∆ 400 rpm, ○ 600 rpm). (b) Inverse of residual activity of PPO 670 versus the product of number of revolutions per time and treatment time (N·t) at 20 MPa and 45 671 ºC (stirring speed range: 200 to 600 rpm). The continuous line corresponds to the linear fitting 672 (Eq. 15). 673 Figure 2. Inactivation of PPO in cloudy apple juice at atmospheric pressure at different mild 674 temperatures (35 ºC ◇ , 40 ºC ○, 45 ºC □). The continuous lines represent the first order model 675 (Table 1). 676 Figure 3. Inactivation of PPO in cloudy apple juice by HPCD (a) 10 MPa and different 677 temperatures (35 ºC ◇ , 40 ºC ○, 45 ºC □) (b) 45ºC and different pressures (10 MPa □, 12.5 MPa 678 ∆, 15 MPa ◇ , 20 MPa ○). The continuous lines represent the Weibull model (Table 2). 679 Figure 4 . Inactivation of PME in cloudy apple juice under mild heating treatment at 45ºC (∆ 680 atmospheric pressure) and by HPCD at 45 ºC (10 MPa □, 20 MPa ○). The continuous lines at 681 atmospheric pressure represent the first order kinetic model (Table 1), while in HPCD treatment 682 represent the Weibull model (Table 2). 683 Figure 5. Particle Size Distribution (PSD) of cloudy apple juice before treatment ( ─ ) and after 684 HPCD treatment at 45 ºC, 20 MPa for 60 min (-·-·). 685 686 687 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Figure 1. (a) Effect of stirring speed on PPO inactivation from cloudy apple juice by HPCD at 20 MPa and 45 ºC (◇ 200 rpm, ∆ 400 rpm, ○ 600 rpm). (b) Inverse of residual activity of PPO versus the product of number of revolutions per time and treatment time (N·t) at 20 MPa and 45 ºC (stirring speed range: 200 to 600 rpm). The continuous line corresponds to the linear fitting (Eq. 15). 0 20 40 60 80 100 0 20 40 60 80 Residual PPO activity, A/Ao·100 time, min 1 3 5 7 9 11 0 10000 20000 30000 40000 Ao/A N(rpm)·t (b) (a) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 688 689 Figure 2. Inactivation of PPO in cloudy apple juice at atmospheric pressure at different mild temperatures (35 ºC ◇, 690 40 ºC ○, 45 ºC □). The continuous lines represent the first order model (Table 1). 691 692 693 0 20 40 60 80 100 0 20 40 60 80 100 120 PPO RA, A/Ao·100 time, min MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 694 695 Figure 3. Inactivation of PPO in cloudy apple juice by HPCD (a) 10 MPa and different temperatures (35 ºC ◇, 696 40 ºC ○, 45 ºC □) (b) 45ºC and different pressures (10 MPa □, 12.5 MPa ∆, 15 MPa ◇, 20 MPa ○). The continuous 697 lines represent the Weibull model (Table 2). 698 699 0 20 40 60 80 100 0 20 40 60 80 100 120 PPO RA, A/Ao·100 time, min (a) 0 20 40 60 80 100 0 20 40 60 80 100 120 PPO RA, A/Ao·100 time, min (b) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 700 Figure 4. Inactivation of PME in cloudy apple juice under mild heating treatment at 45ºC (∆ atmospheric pressure) 701 and by HPCD at 45 ºC (10 MPa □, 20 MPa ○). The continuous lines at atmospheric pressure represent the first order 702 kinetic model (Table 1), while in HPCD treatment represent the Weibull model (Table 2). 703 704 705 706 707 708 0 20 40 60 80 100 0 20 40 60 80 100 120 PME RA, A/Ao·100 time, min MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 709 710 Figure 5. Particle Size Distribution (PSD) of cloudy apple juice before treatment ( ─ ) and after HPCD treatment at 711 45 ºC, 20 MPa for 60 min (-·-·). 712 713 0 1 2 3 4 5 6 7 0.01 0.1 1 10 100 1000 Volume (%) Particle size (µm)