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

Accep ed Manusc ip E alua ion o HPCD ba ch ea men s on enzyme inac i a ion kine ics and selec ed quali y cha ac e is ics o cloudy juice om Golden delicious apples A.E. Ille a, M.T. Sanz, S. Bel án, R. Melgosa, A.G. Solaesa, M.O. Ruiz PII: S0260-8774(17)30448-X DOI: 10.1016/j.j oodeng.2017.10.017 Re e ence: JFOE 9050 To appea in: Jou nal o Food Enginee ing Please ci e his a icle as: A.E. Ille a, M.T. Sanz, S. Bel án, R. Melgosa, A.G. Solaesa, M.O. Ruiz, E alua ion o HPCD ba ch ea men s on enzyme inac i a ion kine ics and selec ed quali y cha ac e is ics o cloudy juice om Golden delicious apples, Jou nal o Food Enginee ing (2017), doi: 10.1016/j.j oodeng.2017.10.017 This is a PDF ile o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT HPCD inac i a ion a e o PPO and PME inc eased wi h inc easing p and T. Sensi i i y o empe a u e change is simila o HPCD and mild he mal ea men . PME is mo e HPCD esis an han PPO. HPCD ea men dec eased pa icle size o cloudy apple juice. Colloidal s abili y and nu i ional p ope ies we e no a ec ed by HPCD. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT E alua ion o HPCD ba ch ea men s on enzyme inac i a ion kine ics and selec ed 1 quali y cha ac e is ics o cloudy juice om Golden delicious apples 2 3 A.E. Ille a, M. T. Sanz ∗ , S. Bel án, R. Melgosa. A. G. Solaesa, M. O. Ruiz 4 Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), 5 Uni e si y o Bu gos, 09001 Bu gos. Spain 6 7 Abs ac 8 Cloudy apple juice has been ea ed by high p essu e ca bon dioxide (HPCD) as non- he mal 9 echnology o inac i e polyphenoloxidase and pec inme hyles e ase in ba ch mode. S i ing 10 speed ( om 200 o 600 pm) induced an inc ease in he enzyme inac i a ion a e while a iple 11 cycle o p essu iza ion/dep essu iza ion led o he same enzyme inac i a ion e iciency. Enzyme 12 inac i a ion kine ics we e de e mined a di e en empe a u es ( om 35 o 45 ºC) and p essu es 13 ( om 10 o 20 MPa). Da a we e desc ibed by he i s o de kine ic model and he Weibull 14 model. Fo he i s o de kine ic model, decimal educ ion ime o HPCD ea men was ound 15 o be smalle han o mild hea ing, in he same empe a u e ange. The same endency was 16 obse ed o he i s decimal educ ion ime in he Weibull model. HPCD ea men esul ed in a 17 homogeniza ion e ec e lec ed in he shi ing o he pa icle size dis ibu ion owa ds smalle 18 diame e s a e ea men . HPCD ea men did no esul in a change o wa e and oxala e 19 soluble pec in con en , o al phenolic compounds and hid oxyme hyl u u al con en . 20 21 22 ∗ Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess e [email protected] MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Keywo ds: Cloudy apple juice, HPCD, inac i a ion kine ics pa ame e s, p ope ies 23 1. In oduc ion 24 Consump ion o cloudy apple juice seems o be mo e bene icial han consuming clea apple juice 25 due o changes in phenolic compounds and loss o pec ins du ing cla i ica ion p ocess 26 (Ma kowski e al., 2015). Howe e , colo and cloud s abili y a e main challenges in p ese a ion 27 o cloudy apple juice du ing i s p ocessing and s o age. Enzyma ic b owning in cloudy apple 28 juice is due o he ac ion o polyphenol oxidase (PPO) ha ca alyzes he oxida ion o phenolic 29 compounds o quinones. On he o he hand, he mechanism o cloud s abili y is no ye 30 unde s ood. Among he di e en mechanisms p oposed, elec os a ic epulsion by nega i e 31 cha ges p esen in he pa ly deme hyla ed pec in due o galac u onic esidues could be 32 esponsible o cloud s abili y a oiding agg ega ion (Zhao e al., 2008). In his ega d, he ac i i y 33 o pec inme hyles e ase (PME) causes pec in deme hyla ion ha could p ecipi a e wi h calcium 34 ions p esen in he juice causing cla i ica ion and he loss o u bidi y. Howe e , o he 35 componen s wi h nega i e cha ge ha e been also epo ed in he li e a u e as s abilizing agen s o 36 he cloud in he se um such as phospha ide acids o cloud lipids (K ap enbaue e al., 2006). 37 The mal ea men s ha e been adi ionally used o he inac i a ion o mic oo ganisms and 38 enzymes ha a ec nega i ely he quali y o oods. Howe e , hese me hods cause undesi able 39 e ec s in quali y a ibu es such as la o and loss o nu i ional componen s. High p essu e 40 ca bon dioxide (HPCD) ea men has been p oposed as an al e na i e non- he mal pas eu iza ion 41 echnique ha can inac i a e ce ain mic oo ganisms and enzymes unde mild ope a ion 42 condi ions. Typically, ope a ing p essu e does no exceed 50 MPa and empe a u e anges 43 be ween 20 and 50 ºC, below pas eu iza ion empe a u e (Manzocco e al., 2016). The 44 mechanism o p essu ized CO 2 on enzyme inac i a ion is no ye ully unde s ood. In he 45 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT li e a u e di e en app oaches can be ound such as pH lowe ing due o dissol ed CO 2 , 46 con o ma ional changes o o ma ion o di e en complex wi h CO 2 molecules (Hu e al., 2013). 47 Some s udies ha e been ound in he li e a u e ega ding he e ec o HPCD on enzymes o 48 cloudy apple juice om Fuji apples. Niu e al. (2010) e alua ed he quali y o cloudy Fuji apple 49 juice p ocessed om apple slices p e iously ea ed by HPCD. These au ho s ound ha PPO 50 could be comple ely inac i a ed a 20 MPa o 20 min and a empe a u e as low as 25 ºC; 51 howe e , esidual ac i i y o 18% was s ill ound o PME a e ea men a 65 ºC and 20 MPa 52 o 20 min. Gui a al. (2007) ound highe alues o esidual ac i i y han he wo k o Niu e al. 53 (2010), o PPO in HPCD- ea ed cloudy Fuji apple juice in a ba ch mode (40% a 30 MPa and 54 55 ºC o 60 min). When using a con inuous HPCD sys em, holding imes o 10 min we e 55 needed a 22 MPa and 60 ºC o achie e comple e inac i a ion o PPO bu , a he same wo king 56 condi ions, s ill PME esidual ac i i y o 42% was ob ained (Xu e al., 2011). 57 F om p e ious epo ed esul s, i can be clea ly obse ed ha he e is a g ea a ie y in he 58 li e a u e da a on enzyme inac i a ion om cloudy apple. This is a main d awback in he 59 comme cializa ion o his echnology and eliable da a on juice quali y a e s ill needed. 60 Acco ding o Wang e al. (2010) he in luence o agi a ion on enzyme inac i a ion unde high- 61 p essu e en i onmen is a key ac o in he design o eac o s o indus ial scale olume. 62 Howe e , he e ec o s i ing speed on enzyme ac i i y has no been conside ed in mos o he 63 s udies. Wang e al. (2010) de e mined he e ec o agi a ion a 40ºC on isoamylase, ob ained 64 om a e men a ion b o h om Pseudomonas amylode amosa a e cell emo al, and a 65 comme cial β-amylase unde supe c i ical ca bon dioxide, a di e en ope a ing imes (up o 120 66 min) and di e en ope a ing p essu e ( om 11.0 o 17.2 MPa), obse ing ha he ac i i y o bo h 67 enzymes dec eased by inc easing he speed o agi a ion. 68 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT In his wo k, he e ec o HPCD ea men on some quali y cha ac e is ics o cloudy apple juice 69 om Golden delicious has been s udied. The e ec o some a iables, non-p e iously s udied in 70 li e a u e, on enzyme inac i a ion om cloudy apple juice, has been conside ed such as he e ec 71 o s i ing speed and he numbe o successi e p essu iza ion/dep essu iza ion cycles. Enzyme 72 inac i a ion kine ic da a pa ame e s a e also necessa y o scale-up HPCD p ocess. This ega d, 73 enzyme inac i a ion kine ics o PPO and PME ha e been de e mined a di e en ope a ing 74 p essu es and empe a u es. Enzyme inac i a ion kine ic da a ha e been desc ibed by he i s 75 o de kine ic model and he Weibull model as an al e na i e o he classical i s o de kine ic 76 model. Some o he impo an quali y pa ame e s o cloudy apple juice ha e been also de e mined 77 be o e and igh a e HPCD ea men , such as pec in con en , pa icle size dis ibu ion, colo 78 and an ioxidan capaci y. 79 2. Ma e ials and me hods 80 2.1 Juice p epa a ion 81 Golden delicious apples we e peeled and cu in cubes and hen added o a 0.3 % o L-asco bic 82 acid solu ion o a oid enzyma ic b owning du ing p ocessing. Apple cubes we e wiped and 83 immedia ely squeezed wi h a sc ew juice ex ac o . The liqueu was il e ed wi h 2 laye s o 84 cheeseclo h. The pH o he juice ob ained was 3.89 ± 0.01 and he soluble solid con en was 12.5 85 ± 0.1 °B ix. The juice was s o ed ozen a −18 °C un il u he use. 86 2.2 HPCD equipmen and p ocessing 87 The HPCD cell was made o s ainless s eel (SS-316) and had an in e nal olume o 100 mL wi h 88 a maximum ope a ing p essu e and empe a u e o 30 MPa and 80 ºC, espec i ely (Melgosa e 89 al., 2017). Fo each expe imen , 40 mL o apple juice we e cha ged in o he high p essu e cell 90 ha was imme sed in a he mos a ic wa e ba h a he ope a ing empe a u e. The magne ic 91 s i ing was hen connec ed and he sys em was p essu ized by a using a sy inge pump wi h a 92 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT p essu e con olle (ISCO 260 D) up o he desi ed p essu e. CO 2 was ed in o he high p essu e 93 cell h ough a sin e ed s ainless s eel mic o- il e wi h a po e size o 10 µm (B iongos e al., 94 2016). This ega d, Ishikawa e al. (1995) showed ha he concen a ion o CO 2 in he sample 95 was in luenced by he way CO 2 was ed in o he sample, inc easing by dec easing he po e size 96 o he il e a 25 MPa and 35ºC. 97 Samples we e collec ed pe iodically o ollow he inac i a ion kine ics o he enzymes. 98 Expe imen s we e ca ied ou in he anges o empe a u e (T) om 35 o 45 ºC, p essu e (p) 99 om 10 o 20 MPa and s i ing speed om 200 o 600 pm. The e ec o he sampling p ocedu e 100 and he numbe o p essu iza ion/dep essu iza ion cycles on enzyme ac i i y was also analyzed. 101 To compa e he e ec o he HPCD ea men on enzyme ac i i y wi h mild hea ing ea men , 102 cloudy apple juice was hea ed a a mosphe ic p essu e in he same empe a u e ange ( om 35 o 103 45 ºC). Samples we e aken pe iodically a di e en ea men imes up o 120 min. 104 Some o he impo an quali y a ibu es o cloudy apple juice, such as pa icle size dis ibu ion, ξ 105 po en ial, u bidi y, pH, pec in con en , o al phenolic compounds, non-enzyma ic b owning and 106 an ioxidan capaci y we e also de e mined be o e and igh a e HPCD ea men . 107 2.3 Physico-chemical analysis 108 2.3.1. De e mina ion o PPO ac i i y 109 The ac i i y o PPO was de e mined spec opho ome ically by using a 0.05 M ca echol (Sigma 110 Ald ich) solu ion p epa ed in a 0.1 M phospha e bu e (pH 6.5) as subs a e. Samples we e 111 analyzed by adding 100 µL o apple juice in o 2.9 mL subs a e solu ion. Oxida ion o ca echol 112 was de e mined immedia ely by he inc ease in abso bance a 420 nm by using a Jasco V-750 113 spec opho ome e equipped wi h a Pel ie he mos a ed cell holde and a wa e pump o keep he 114 empe a u e cons an a 30 ºC. The PPO ac i i y was aken as he e y i s linea pa o he 115 eac ion cu e. 116 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2.3.2. De e mina ion o PME ac i i y 117 PME ac i i y was de e mined by using an au oma ic i a o sys em (Me ohm  Ti ando) by 118 using a 1 % o pec in solu ion (Al a Aesa  pec in ci us) p epa ed in NaCl 0.3 M as subs a e. 119 50 mL o pec in solu ion was mixed wi h 1 mL o cloudy apple juice and pH was adjus ed o 7.5 120 wi h NaOH 0.02 N. Du ing pec in hyd olysis a 30 ºC, pH was main ained a 7.5 by adding 121 NaOH 0.02 N. The amoun o NaOH added o 15 min was eco ded. One PME ac i i y uni 122 (UPE) is de ined as he mic omoles o ca boxylic g oups p oduced pe minu e and mL o juice a 123 pH 7.5 and 30 ºC (B iongos e al., 2016) . 124 Rela i e esidual ac i i ies o PPO and PME we e e alua ed as: 125 Residualac i i y,RA= !"# $%&'%·100%=,, - ·100 [1] 126 2.3.3. De e mina ion o pec ic subs ances 127 Pec ic subs ances in cloudy apple juice be o e and a e HPCD ea men ha e been de e mined 128 acco ding o Robe son (1979) by p og essi e ex ac ion o he alcohol insoluble solids by wa e 129 (high me hoxyl pec ins a e ex ac ed) and ammonium oxala e (low me hoxyl pec ins a e 130 ex ac ed). Wa e soluble pec ins play an impo an ole in he u bidi y o he cloudy apple 131 juices ac ing as colloid s abilize s, while oxala e-soluble ac ion can o m gels wi h poly alen 132 me al ions (Robe son, 1979). Pec ic subs ances a e de e mined spec opho ome ically a 133 520 nm a e o al hyd olysis o galac u onic acids by using m-hyd oxydiphenil (MHDP) as a 134 ch omogenic eagen . Solu ions we e eshly p epa ed be o e analysis: 0.15% solu ion o m- 135 hyd oxydiphenyl (Sigma Ald ich) in 0.5% NaOH and 0.0125 M sodium e abo a e (Pan eac) in 136 concen a ed sulphu ic acid. Fo pec in de e mina ion, 1 mL o wa e ex ac and 5 mL o he 137 sodium e abo a e solu ion we e mixed and placed in a wa e -ice ba h. The mix u e is hen 138 hea ed in wa e ba h a 80 ºC o 6 min and subsequen ly cooled again in a wa e -ice ba h. 139 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 0.1 mL o he m-hyd oxydiphenyl solu ion was added and a e homogeniza ion o he mix u e, 140 abso bance a 520 nm was measu ed as a unc ion o ime. Acco ding o Iba z e al. (Iba z e al., 141 2006) spec opho ome ic da a measu emen s we e aken a he highes abso bance ins ead o a a 142 ce ain eac ion ime. A calib a ion cu e was p epa ed wi h s anda d solu ions o D-galac u onic 143 acid (Sigma Ald ich) by ollowing he same colo ime ic me hod. A blank was also p epa ed 144 wi h no D-galac u onic acid. 145 2.3.4. Pa icle size dis ibu ion and ze a-po en ial 146 Pa icle size dis ibu ion (PSD) was de e mined by lase di ac ion wi h a Mas e size 2000 147 (Mal e n® Ins ., MA). The sys em uses a lase ligh a 750 nm wa eleng h o size pa icles om 148 0.4 o 2000 µm by ligh di ac ion. Pa icle size dis ibu ion was calcula ed by he F aunho e 149 model. Size dis ibu ions ( olume ac ions agains pa icle size) be o e and a e HPCD 150 ea men we e calcula ed and he weigh -a e age sizes exp essed as: 151 • The equi alen su ace a ea mean diame e : D/3,22=∑nd5 6∑n ⁄d5 8 [2] 152 • The equi alen olume mean diame e : D/4,32=∑nd5 :∑n ⁄d5 6 [3] 153 whe e d lc is he diame e o he pa icle and n c is he pe cen age o pa icles. 154 To desc ibe dis ibu ion wid h one common pa ame e is he Span: 155 Span=# =,>.@ A# =,>.B # =,>.C [4] 156 whe e D ,0.9 , D ,0.1 and D 0.5 a e he pa icle size bellow which, 90%, 50% and 10% o he 157 pa icles lies. 158 ζ-po en ial was de e mined wi h a Ze asize Nano ZS appa a us, using he Lase Dopple 159 Velocime y echniques. Samples we e dilu ed 1:50 wi h deionized wa e and il e ed h ough 160 5 µm il e . ζ-po en ial gi es an indica ion o he po en ial s abili y o a colloidal sys em and i is 161 a good index o he colloidal elec os a ic epulsi e o ces. Pa icles will epel each o he when 162 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT li e a u e abou he e ec o he numbe o cycles on enzyme inac i a ion by HPCD on esh 291 juices. 292 3.3 E ec o mild hea ing on PPO inac i a ion 293 PPO inac i a ion kine ics we e de e mined a a mosphe ic p essu e in he empe a u e ange 294 om 35 o 45 ºC (same empe a u e ange as o HPCD ea men , Figu e 2). The maximum 295 educ ion o PPO ac i i y was nea ly 40 % a e 2 h o hea ing a 45 ºC indica ing ha PPO o 296 cloudy Golden delicious apple juice was a he s able unde mild empe a u es. Simila 297 inac i a ion deg ees o PPO om cloudy Fuji apple juice we e ob ained by Gui e al. (2007). 298 A e 1 hou o mild hea ing, hese au ho s eached a maximum educ ion o PPO ac i i y o 299 20% a 45 ºC. Howe e , Niu e al. (2010) ound ha when subjec ing apple slices o mild hea ing 300 ea men om 25 o 45 ºC o 20 min and hen p ocessing hem in o cloudy apple juice, PPO 301 ac i i y inc eased by 22-51%. Buckow e al. (2009) also ound ha a 45 ºC, PPO ac i i y om 302 cloudy apple juice inc eased 30%, a ibu ing his beha io o he elease o la en PPO. 303 Enzyme inac i a ion kine ic da a unde mild hea ing condi ions and a a mosphe ic p essu e we e 304 desc ibed by he i s -o de model. Pa ame e s a e collec ed in Table 1. Inac i a ion a e 305 cons an , k, inc eased wi h he ope a ing empe a u e, he e o e he co esponding D alues 306 dec eased wi h empe a u e. The empe a u e sensi i i y pa ame e , z T , and he ac i a ion ene gy 307 we e e alua ed as 36 ºC and 53 kJ/mol, espec i ely (see Table 1). Values o he same o de 308 we e epo ed by Gui e al. (2007) o cloudy Fuji apple juice, 27.0 ºC o z T and 72.0 kJ/mol, o 309 Ea. The mal s abili y o ex ac ed apple PPO om Golden delicious was s udied in he 310 empe a u e ange om 68 o 78 ºC by Yemenicigly e al. (1997). These au ho s epo ed a z T 311 alue o 9.9 ºC. The highe alue ob ained in his wo k o z T in he empe a u e ange om 35 o 312 45 ºC migh indica e a lowe suscep ibili y o empe a u e change a empe a u es lowe han he 313 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT one obse ed a pas eu iza ion empe a u es. In any case, he alues o he ac i a ion ene gy 314 we e simila in bo h empe a u e anges. 315 3.4. E ec o HPCD ea men on PPO inac i a ion 316 Enzyme inac i a ion kine ics o PPO o cloudy Golden deliciuos apple juice ha e been 317 de e mined a 10 MPa in he empe a u e ange om 35 o 45 ºC (Figu e 3a). By inc easing 318 empe a u e, enzyme inac i a ion a e also inc eased. In addi ion o he in insic e ec o 319 empe a u e on enzyme inac i a ion, by inc easing empe a u e, al hough CO 2 densi y dec eased, 320 he di usi i y o CO 2 can be imp o ed and accele a e he molecula collisions be ween CO 2 321 molecules and he enzyme. Figu e 3b shows he inac i a ion kine ics a 45 ºC in he p essu e 322 ange om 10 o 20 MPa. Highe ope a ing p essu es led o as e inac i a ion a es, bu he 323 ac ion o he esis an o m o he enzyme was nea ly independen o he ope a ing p essu e, 324 being a ound 6 %. This alue was lowe han he minimal esidual ac i i y o PPO om cloudy 325 Fuji apple juice epo ed by Gui e al. (2007), 37%, a 30 MPa and 55 ºC o 60 min. In any case, 326 compa ison wi h o he HPCD sys ems is di icul o es ablish since he inac i a ion eached 327 would depend no only on he ype and sou ce o he enzyme and he expe imen al condi ions 328 such as p essu e, empe a u e and ea men ime bu also on he agi a ion o he sys em and he 329 a io sample olume/cell olume since his las pa ame e de e mines he amoun o CO 2 injec ed 330 o he sys em. Un o una ely in mos o he s udies hese pa ame e s a e no de ailed. PPO om 331 cloudy Fuji apple juice was comple ely inac i a ed by a con inuous HPCD sys em a 22 MPa and 332 60 ºC o 10 min (Xu e al., 2011). This could sugges a highe e iciency o a con inuous sys em 333 due o he u bulen ene gy, compa able wi h a good agi a ion in a discon inuous sys em. 334 Howe e , 60 ºC canno be conside ed as non- he mal and esidual ac i i y as high as 47% was 335 ob ained in he con inuous sys em a 43 ºC and 25 MPa o 2 min. (Xu e al., 2011). 336 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Inac i a ion kine ic da a by HPCD we e i ed o he i s o de kine ic model and he Weibull 337 model (Table 2). Al hough he Weibull model yielded he bes i ing quali y, he i s o de 338 kine ic model desc ibed he inac i a ion cu e qui e well and he e o e, kine ic pa ame e s o 339 he linea eg ession model ha e been also lis ed in Table 2 o compa ison wi h da a epo ed in 340 he li e a u e. 341 Fo he i s o de kine ic model, inac i a ion a e cons an , k, inc eased wi h empe a u e and 342 p essu e. Consequen ly, D alues dec eased wi h inc easing p essu e and empe a u e. A each 343 ope a ing empe a u e, D alues we e smalle o HPCD ea men (Table 2) han he 344 co esponding alues o mild hea ing ea men (Table 1) p o ing ha HPCD ea men was 345 mo e e ec i e han he mal ea men alone. Fo example D was equal o 515 min a 45 ºC and 346 a mosphe ic p essu e and dec eased o 104 min a 45 ºC and 10 MPa. 347 z T and ac i a ion ene gy alues we e de e mined as 29 ºC and 64 kJ/mol, espec i ely, o he 348 i s o de kine ic model (see Table 2). z T and ac i a ion ene gy alues we e o he same o de as 349 hose lis ed o mild hea ing ea men (Table 1). This mean ha he sensi i i y o empe a u e 350 change was simila o HPCD as o mild hea ing ea men in he empe a u e ange co e ed in 351 his wo k. The same endency in z T and E a was ound by Liu e al. (2008) in he inac i a ion o 352 PPO o ed bee ex ac s. These au ho s ound ha unde mild hea ing ea men a a mosphe ic 353 p essu e (35-55 ºC), z T and E a we e equal o 34 ºC and 57 kJ/mol, espec i ely. A e HPCD 354 ea men a 37.5 MPa, in he same empe a u e ange, hey ob ained alues o 36 ºC and 355 54 kJ/mol o z T and E a , espec i ely. Howe e , Gui e al. (2007) ound o PPO o cloudy Fuji 356 apple juice ha z T inc eased om 27 o 108 ºC and ac i a ion ene gy dec eased om 72 o 357 18 kJ/mol unde mild hea ing ea men (35-55 ºC) and HPCD a 30 MPa in he same 358 empe a u e ange, espec i ely. This inc ease in z T and dec ease in Ea ound by Gui e al. (2007) 359 would indica e ha he enzyme inac i a ion become less empe a u e dependen unde 360 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT p essu ized CO 2 . This beha io is di icul o unde s and and i has no been obse ed in ou 361 wo k. As i has been p e iously men ioned, in addi ion o he in insic e ec o empe a u e on 362 enzyme inac i a ion, empe a u e has wo di e en e ec s on impo an p ope ies o CO 2 . An 363 inc ease in empe a u e in ol es an imp o emen o mass anspo p ope ies enhancing CO 2 364 di usi i y and he numbe o collisions be ween he CO 2 and he enzyme, bu on he o he hand, 365 CO 2 densi y and he e o e CO 2 solubili y in he aqueous medium dec eases. I seems ha hese 366 e ec s play bo h an impo an ole, since simila sensi i i y o empe a u e was ound in mild 367 hea ea men and in HPCD. 368 By using equa ions 8 and 10 z P and V a ha e been de e mined as 25 MPa and -251 cm 3 /mol, 369 espec i ely o he i s o de kine ic model. The nega i e alue o he ac i a ion olume 370 indica ed ha PPO inac i a ion was a o ed by inc easing ope a ing p essu e. O he alues o 371 ac i a ion olumes epo ed in he li e a u e o PPO inac i a ion om di e en sou ces a e o 372 he same o de , -271.7 and -307.9 cm 3 /mol o he labile and s able ac ion o PPO o wa e 373 melon a 50 ºC in he p essu e ange o 8-30 MPa (Liu e al., 2013) o -94.31 cm 3 /mol o PPO 374 om cloudy Fuji apple juice a 55ºC in he p essu e ange om 8 o 30 MPa (Gui e al., 2007). 375 The Weibull pa ame e s a e also lis ed in Table 2. Scale, α, and shape, β, pa ame e s we e used 376 o calcula e he ime equi e o inac i a e 1 log ( d=1 ) o PPO. Simila o he D- alues o he i s 377 o de kine ic model, d=1 dec eased wi h ope a ing p essu e and empe a u e. As sugges ed by an 378 Boekel (2002) is impo an o de e mine he e ec o empe a u e, and in he p esen wo k o 379 p essu e, on bo h pa ame e s o he Weibull model. Scale pa ame e was s a is ically signi ican 380 dependen on empe a u e and p essu e, when es ed a he 95% signi icance le el o a linea 381 ela ionship, dec easing wi h p essu e and empe a u e. The shape ac o , n, was less han 1 382 excep o 10 MPa and 35 ºC. The conca i y shape o he inac i a ion cu e could be unde s ood 383 as a as e inac i a ion o he labile o m o he enzyme and he p esence o a esis an o m o 384 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT he enzyme o HPCD ea men (Figu es 3a and 3b). The shape pa ame e , β, was ound o be 385 s a is ically signi ican dependen on p essu e bu al hough he e seemed o be a end in 386 empe a u e dependence o β, i was no s a is ically signi ican , when es ed a he 95% 387 signi icance le el o a linea ela ionship. In any case, i mus be highligh ed ha only h ee 388 empe a u es ha e been s udied o de e mine he e ec o empe a u e. Acco ding o an Boekel 389 (2002), he scale pa ame e , α, could be modelled in a simila way o he classical D alue o he 390 i s o de kine ic model, sugges ing a linea dependence o he log α on empe a u e and 391 conside ing ha β did no depend on empe a u e: 392 p{|n=}V−~V [16] 393 A z T ’ alue can be also de ined: 394 =V  B [17] 395 The alue o he z T ’ is lis ed in Table 2, oge he wi h he quali y o he i ing. Al hough he 396 concep o z T and z’ T is di e en since z T is ob ained om he linea pa , whe eas z’ T akes also 397 in o accoun he nonlinea pa (Van Boekel, 2002), simila alues we e ob ained o bo h 398 pa ame e s (z T = 29 ºC and z’ T = 27 ºC). 399 In his wo k, i was also ound a linea dependence o he log α on p essu e: 400 p{|n=}8−~8d [18] 401 Al hough i was ound a s a is ically signi ican linea ela ionship be ween β and p, he in e se 402 o he slope o log α e sus p was also e alua ed. Analogous o z’ T i was de ined a z’ p 403 =−V  ] [19] 404 This alue is epo ed in Table 2 and qui e simila alues can be obse ed o z p and z p ’. 405 3.5. E ec o HPCD on PME 406 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT To compa e he HPCD esis ance o PME and PPO, PME inac i a ion kine ics we e de e mined 407 o he expe imen s pe o med a 45 ºC and 10 MPa and 20 MPa (Figu e 4). PME inac i a ion 408 a e was also de e mined a a mosphe ic p essu e and 45 ºC. A a mosphe ic p essu e, i was 409 ound ha esidual PME ac i i y was s ill 90% a e 120 min a 45ºC (Figu e 4). Teleszko e al. 410 (2016) s udied he mal ea men o di e en apple cul i a s, ounding ha PME om cloudy 411 Golden deliciuous apple juice was one o he apple cul i a s ha p esen ed s onges 412 he mos abili y (30% o esidual ac i i y a e hea ing 5 min a 100 ºC). Niu e al. (2010) ound 413 ha he RA o PME om cloudy Fuji apple juice om apple slices p e iously p ehea ed o 414 20 min a he co esponding wo king empe a u e, a ied be ween 82 o 72 % a 35 and 45ºC, 415 espec i ely, bu wi h e o ba s highe han 10% o RA a each empe a u e (g aphical lec u e). 416 A e HPCD ea men , highe inac i a ion a es we e obse ed han o mild hea ing ea men a 417 he same empe a u e. Al hough, esidual ac i i y was s ill a ound 60 % a 20 MPa and 45 ºC 418 a e 60 min. This indica es ha PME om cloudy Golden delicious apple juice is a he s able o 419 HPCD a ope a ing empe a u es conside ed as non- he mal ea men . Di e en HPCD 420 inac i a ion alues o PME om cloudy apple juice ha e been epo ed in he li e a u e. Niu e 421 al. (2010) epo ed a minimal esidual ac i i y o 18% o PME o cloudy Fuji apple juice om 422 apple slices ea ed by HPCD a 20 MPa and 65 ºC o 20 min. In a con inuous sys em, PME 423 om cloudy Fuji apple juice s ill p esen ed a esidual ac i i y o 40% a 22 MPa and 60ºC o 10 424 min (Xu e al., 2011). HPCD inac i a ion o PME ex ac s om apple juice was ound o be 425 mo e e ec i ely inac i a ed a 30 MPa o 60 min, eaching 5% o esidual ac i i y a 55 ºC (Zhi 426 e al., 2008). Howe e , in his case, esul s a e di e en o compa e since an ex ac ed enzyme 427 suspended in a bu e solu ion can gi e di e en inac i a ion esul s om hose ob ained in an 428 o iginal juice. 429 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Resul s ob ained in his wo k, indica e ha PME om cloudy apple juice was mo e esis an o 430 HPCD han PPO in he p essu e ange co e ed in his wo k. In he li e a u e, as o he mal 431 ea men , he highe s abili y has been a ibu ed o he mo e complex s uc u e o PPO wi h 432 h ee o ou subuni s, in highe plan s, han PME, wi h one subuni . This s uc u e, makes i 433 mo e suscep ible o HPCD ea men (Niu e al., 2010). In addi ion, PME in ui juices is mo e 434 di icul o be e ec i ely inac i a ed, since PME is bound o he plan cell wall, which con ains 435 na u al s abilizing ac o s (Zhou e al., 2010). 436 PME inac i a ion kine ic da a we e desc ibed by he i s o de model a a mosphe ic p essu e 437 (Table 1). The decimal educ ion ime a 45 ºC and a mosphe ic p essu e was ound o be much 438 highe han o PPO a he same condi ions. HPCD inac i a ion da a a 45ºC and 10 MPa and 439 20 MPa we e i ed o he i s o de kine ic model and he Weibull model (Table 2). The 440 Weibull model yielded he bes i ing quali y, bu s ill an accep able good i ing o he i s 441 o de kine ic model was ob ained. Fo he i s o de model, inac i a ion a e cons an inc eased 442 wi h p essu e, bu lowe alues han o PPO inac i a ion we e ob ained and he e o e highe D 443 alues. Rega ding he Weibull model, he scale and shape pa ame e s, bo h, dec eased wi h 444 ope a ing p essu e a 45ºC. These pa ame e s we e used o calcula e he ime needed o inac i a e 445 1 log ( d=1 ) o PME a he wo p essu es s udied. The d=1 calcula ed by he Weibull model is 446 much highe han he co esponding D alues by he i s o de kine ic model. The e o e, in his 447 case, he use o he i s o de kine ic model could lead o an unde p ocessing es ima ion o 448 PME inac i a ion. 449 3.6. E ec o HPCD on di e en quali y pa ame e s o cloudy apple juice 450 Some quali y pa ame e s ha e been de e mined igh a e HPCD ea men a 45 ºC, 20 MPa and 451 60 min o ea men ime. 452 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT pH. Ini ial pH o cloudy apple juice was 3.89 ± 0.01 and immedia ely a e HPCD ea men , pH 453 d opped 0.15 uni s o 3.74 ± 0.01. Signi ican di e ences ha e been de e mined among sample 454 pH a e ages be o e and a e HPCD ea men when applying he Fishe ’s leas signi ican 455 me hod a p- alue ≤ 0.05 (Table 3). Gui e al (2007) epo ed a d op o 0.3 uni s om 3.95 o 456 3.66 ± 0.03 o Fuji cloudy apple juice a e HPCD ea men a 22 MPa and 55 ºC o 60 min. 457 Acco ding o Meysammi e al. (1992), juices and be e ages can be conside ed as an aqueous 458 elec oly e sys em o mul iple ions. Unde p essu ized CO 2 a numbe o dissocia ion eac ions 459 can ake place libe a ing some o hem H + ions ha could educe he pH o he media. In any 460 case, a e 2 h a e dep essu iza ion pH e u ned o a alue close o i s ini ial alue (pH = 3.87 ± 461 0.01). 462 Colou pa ame e s. Table 3 lis s he L*, a*, b* pa ame e s o cloudy apple juice be o e and 463 a e HPCD ea men . Du ing b owning, he L* alues dec ease, he juice’s colou becomes 464 da ke , while a* and b* alues inc ease, wi h mo e ed and yellow componen s (K ap enbaue e 465 al., 2006). A e HPCD ea men , ligh ness (L*) p esen ed sligh ly, bu signi ican , lowe alues; 466 howe e , 99% o he ligh ness was s ill e ained. No signi ican di e ence was obse ed in he 467 edness componen bu a signi ican inc ease o he yellowness (b*) alue has been obse ed 468 indica ing mo e yellow componen s igh a e HPCD ea men . The change in colou , ∆E (Eq. 469 5) is also p esen ed in Table 3. Sligh ly no iceable di e ences in colo a e HPCD ea men 470 ha e been de e mined, ∆E ≈ 0.51 ± 0.05 (Yuk e al., 2014). O he au ho s ound ha L*, a* and 471 b* alues emained almos cons an a e HPCD ea men a 55 ºC and di e en ope a ing 472 p essu es (8-30 MPa) o 60 min (Gui e al., 2006). 473 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT To al polyphenolic compounds. To al polyphenols emained unchanged a e HPCD. Gaspe i 474 e al. (2009) also ound no signi ican di e ences in polyphenols con en a e HPCD ea men 475 o a blend o Golden delicious and G anny Smi h apple a 10 MPa and 36 ºC o 10 min. 476 An ioxidan capaci y. An ioxidan capaci y o Golden cloudy apple juice has been de e mined 477 by he ABTS es s (Table 3). Sligh ly highe alues we e ob ained a e HPCD ea men 478 al hough di e ences be o e and a e ea men we e ound o be no signi ican (p ≤ 0.05). 479 Hyd oxyme hyl u u al. Table 3 p esen s he HMF con en be o e and a e HPCD ea men . 480 F om hese esul s, i can be concluded ha non enzyma ic b owning eac ions we e no 481 signi ican du ing HPCD p ocessing, p obably due o he mild empe a u es employed in HPCD 482 ea men s. 483 Pa icle size dis ibu ion. The pa icle size dis ibu ion o un ea ed cloudy apple juice anged 484 om 0.275 µm o 416.87 µm wi h h ee maxima a 0.63, 19.96 and 208.93 µm (see Figu e 5). 485 A e HPCD ea men , he pa icle ange shi ed o smalle pa icle sizes om 0.04 µm o 486 104.71 µm wi h one main maximum a 0.182 µm. Table 3 lis s alues o D[3,2] and D[4,3] o 487 eshly cloudy apple juice and a e HPCD ea men . The alues a e HPCD ea men we e 488 signi ican ly lowe han hose o eshly cloudy apple juice. The homogeniza ion e ec o HPCD 489 can be also obse ed in he alues o d(0.1), d(0.5) and d(0.9), which co espond o he size o 490 pa icle below which 10%, 50% and 90% o he sample lies, espec i ely. This homogeniza ion 491 e ec induced by HPCD could be a ibu ed o he explosi e ac ion and he bubbling o CO 2 492 om he juice du ing decomp ession (Zhou e al., 2010). 493 Fo eshly cloudy apple juice, he o al olume o all pa icles wi h diame e less han 5 µm 494 ep esen ed 56.5% o he o al pa icles olume, while his numbe inc eased up o 96% a e 495 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT HPCD ea men . This ac is also e lec ed in he Span alues (Table 3) ha shows a highe 496 deg ee o uni o mi y o he cloudy apple juice a e HPCD ea men . 497 Tu bidi y. The u bidi y o he cloudy apple juice a e HPCD is conside ably highe han ha o 498 eshly cloudy apple juice (Table 3). I seems ha he e was a co ela ion be ween juice’s 499 u bidi y and pa icle size dis ibu ion Howe e , no co ela ion was ound wi h PME esidual 500 ac i i y since, a e HPCD ea men , PME ac i i y was s ill mo e han 60%. 501 Pec in con en . In his wo k, soluble pec in p esen ed sligh ly highe alues a e HPCD (Table 502 4). HPCD ea men could esul in a mo e dissolu ion o pec in in ui cell in o he juice, 503 inc easing he alue o soluble pec in. In addi ion, acco ding o he PSD p e iously epo ed, 504 HPCD induced an homogeniza ion wi h lowe pa icle size diame e s, which could esul in a 505 highe solubili y o he pec in; since, in he li e a u e, i has been epo ed ha pec in solubili y in 506 wa e inc eases by dec easing i s polyme size (Sila e al., 2009) howe e no signi ican 507 di e ences ha e been ound a p ≤ 0.05. No signi ican di e ences ha e been also de e mined 508 o he oxala e-soluble pec in. 509 In he li e a u e, Zhou e al. (2010) epo ed no change in wa e soluble pec in when ea ing 510 peach juice by HPCD a 30 MPa and 55 ºC a di e en ea men imes, al hough hese au ho s 511 obse ed an inc ease o he la ge pa icle a e HPCD ea men . Howe e , Yu e al (2013) 512 obse ed ha soluble pec in o HPCD- ea ed banana pulp a 20 MPa, du ing 30 min a di e en 513 empe a u es in he ange om 45 o 60 ºC, was signi ican ly lowe han he un ea ed pulp. 514 These au ho s a ibu ed his beha io due o in e ac ion o pec in and p o ein o gelling o pec in 515 ha may occu du ing HPCD ea men (Yu e al., 2013). 516 ξ po en ial. ξ po en ial is an indica ion o colloidal s abili y o he juice. Bo h, un ea ed and 517 ea ed apple juice p esen ed nega i e alues o -22.0 mV and -22.3 mV, espec i ely. These 518 alues we e wi hin he ange epo ed by Co ak and Co edig (2006) o ξ po en ial as a unc ion 519 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Table 1. Kine ic pa ame e s a 0.1 MPa o he i s o de kine ic model o PPO inac i a ion by 657 mild he mal hea ing (35-45ºC) and PME a 45 ºC. 658 PPO Fi s o de kine ic 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. Kine ic pa ame e s o he Weibull and he i s o de kine ic models o PPO and PME inac i a ion by HPCD. 659 PPO Weibull model Fi s o de kine ic model p, MPa T, ºC α , min β 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 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 quali y pa ame e s be o e and igh a e HPCD ea men o cloudy apple juice a 663 20 MPa and 45 ºC a e 60 min. 664 Pa ame e Be o e HPCD A e HPCD pH 3.89 ± 0.006 a 3.74 ± 0.006 b Colo ∆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 To al polyphenols, mg galic/L 456.1 ± 13.1 a 455.4 ± 4.3 a ABTS, % o inhibi ion 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-enzima ic b owning, (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 Tu bidi y, NTU 105 ± 2 a 168 ± 3 b Wa e soluble pec in Oxala e soluble pec in (mg galac u onic/L) 22 ± 2 a 23 ± 4 a 28 ± 3 a 23 ± 3 a ξ-po en ial -22.0 ± 0.3 a -22.3 ± 0.4 a Values wi h di e en le e s in each ow (a, b) a e signi ican ly di e en when applying he Fishe ’s leas signi ican 665 me hod a p- alue ≤ 0.05. 666 667 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Lis o Figu e cap ions 668 Figu e 1. (a) E ec o s i ing speed on PPO inac i a ion om cloudy apple juice by HPCD a 669 20 MPa and 45 ºC ( ◇ 200 pm, ∆ 400 pm, ○ 600 pm). (b) In e se o esidual ac i i y o PPO 670 e sus he p oduc o numbe o e olu ions pe ime and ea men ime (N· ) a 20 MPa and 45 671 ºC (s i ing speed ange: 200 o 600 pm). The con inuous line co esponds o he linea i ing 672 (Eq. 15). 673 Figu e 2. Inac i a ion o PPO in cloudy apple juice a a mosphe ic p essu e a di e en mild 674 empe a u es (35 ºC ◇ , 40 ºC ○, 45 ºC □). The con inuous lines ep esen he i s o de model 675 (Table 1). 676 Figu e 3. Inac i a ion o PPO in cloudy apple juice by HPCD (a) 10 MPa and di e en 677 empe a u es (35 ºC ◇ , 40 ºC ○, 45 ºC □) (b) 45ºC and di e en p essu es (10 MPa □, 12.5 MPa 678 ∆, 15 MPa ◇ , 20 MPa ○). The con inuous lines ep esen he Weibull model (Table 2). 679 Figu e 4 . Inac i a ion o PME in cloudy apple juice unde mild hea ing ea men a 45ºC (∆ 680 a mosphe ic p essu e) and by HPCD a 45 ºC (10 MPa □, 20 MPa ○). The con inuous lines a 681 a mosphe ic p essu e ep esen he i s o de kine ic model (Table 1), while in HPCD ea men 682 ep esen he Weibull model (Table 2). 683 Figu e 5. Pa icle Size Dis ibu ion (PSD) o cloudy apple juice be o e ea men ( ─ ) and a e 684 HPCD ea men a 45 ºC, 20 MPa o 60 min (-·-·). 685 686 687 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Figu e 1. (a) E ec o s i ing speed on PPO inac i a ion om cloudy apple juice by HPCD a 20 MPa and 45 ºC (◇ 200 pm, ∆ 400 pm, ○ 600 pm). (b) In e se o esidual ac i i y o PPO e sus he p oduc o numbe o e olu ions pe ime and ea men ime (N· ) a 20 MPa and 45 ºC (s i ing speed ange: 200 o 600 pm). The con inuous line co esponds o he linea i ing (Eq. 15). 0 20 40 60 80 100 0 20 40 60 80 Residual PPO ac i i y, A/Ao·100 ime, min 1 3 5 7 9 11 0 10000 20000 30000 40000 Ao/A N( pm)· (b) (a) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 688 689 Figu e 2. Inac i a ion o PPO in cloudy apple juice a a mosphe ic p essu e a di e en mild empe a u es (35 ºC ◇, 690 40 ºC ○, 45 ºC □). The con inuous lines ep esen he i s o de model (Table 1). 691 692 693 0 20 40 60 80 100 0 20 40 60 80 100 120 PPO RA, A/Ao·100 ime, min MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 694 695 Figu e 3. Inac i a ion o PPO in cloudy apple juice by HPCD (a) 10 MPa and di e en empe a u es (35 ºC ◇, 696 40 ºC ○, 45 ºC □) (b) 45ºC and di e en p essu es (10 MPa □, 12.5 MPa ∆, 15 MPa ◇, 20 MPa ○). The con inuous 697 lines ep esen he Weibull model (Table 2). 698 699 0 20 40 60 80 100 0 20 40 60 80 100 120 PPO RA, A/Ao·100 ime, min (a) 0 20 40 60 80 100 0 20 40 60 80 100 120 PPO RA, A/Ao·100 ime, min (b) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 700 Figu e 4. Inac i a ion o PME in cloudy apple juice unde mild hea ing ea men a 45ºC (∆ a mosphe ic p essu e) 701 and by HPCD a 45 ºC (10 MPa □, 20 MPa ○). The con inuous lines a a mosphe ic p essu e ep esen he i s o de 702 kine ic model (Table 1), while in HPCD ea men ep esen he 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 ime, min MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 709 710 Figu e 5. Pa icle Size Dis ibu ion (PSD) o cloudy apple juice be o e ea men ( ─ ) and a e HPCD ea men a 711 45 ºC, 20 MPa o 60 min (-·-·). 712 713 0 1 2 3 4 5 6 7 0.01 0.1 1 10 100 1000 Volume (%) Pa icle size (µm)