Effect of high pressure carbon dioxide processing on pectin methylesterase activity and other orange juice properties

2 E ec o high p essu e ca bon dioxide p ocessing on pec in 1 me hyles e ase ac i i y and o he o ange juice p ope ies 2 H. B iongos, A.E. Ille a, M. T. Sanz ∗, R. Melgosa, S. Bel án, A.G. Solaesa 3 Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), Uni e si y o 4 Bu gos, 09001 Bu gos. Spain 5 Abs ac : 6 Inac i a ion o pec inme hyles e ase (PME) and quali y pa ame e s o o ange juice ha e been 7 s udied a e high p essu e ca bon dioxide (HPCD) ea men . The HPCD ea men condi ions 8 co e ed a wide ange o empe a u e om 2 o 40 ºC, a below no mal he mal ea men , while 9 ope a ing p essu e was a ied om 10 o 30 MPa and exposu e ime om 3 o 60 min. A 10 dec ease in PME ac i i y was ound, e en a he lowes empe a u e s udied in his wo k, 2 ºC. 11 Di e en inac i a ion kine ic models we e used o co ela e he PME esidual ac i i y: he wo-12 ac ion model, he ac ional-con e sion model and he Weibull model. The wo- ac ion model 13 p esen s he lowes mean ela i e de ia ion. Some quali y pa ame e s such as colou , pH, ºB ix, 14 u bidi y, asco bic acid, o al acidi y and pa icle size dis ibu ion (PSD) we e also de e mined 15 igh a e HPCD ea men and along s o age a 4ºC up o 12 days. PSD shows ha HPCD 16 ea men esul s in a olume inc ease o small pa icles and a olume dec ease o la ge pa icles 17 ∗ Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess [email p o ec ed] 3 ega ding he non- ea ed o ange juice. Calcium con en was also de e mined be o e and a e 18 HPCD ea men o check o insoluble calcium ca bona e o ma ion bu no signi ican changes 19 we e obse ed in calcium con en a e HPCD ea men . 20 Keywo ds: O ange juice, HPCD, pec in me hyles e ase, cloud s abili y 21 1 In oduc ion 22 F ui juice and nec a s consump ion amoun ed o 9.7 million li es in 2014 in he EU, o which 23 o ange juice is one o he mos consumed (Eu opean Fui Juice Associa ion, 2015). Cloud loss is 24 a quali y de ec in o ange juice, since cloud pa icles a e in ol ed in he colou , la ou , ex u e 25 and a oma o o ange juice (Kla ons, Benne , & Vannie , 1991). Addi ionally, consume s 26 associa e he cloud loss wi h spoilage and quali y loss. Ci us cloud is a complex mix u e o 27 p o ein, pec in, lipid, hemicellulose, cellulose and o he mino componen s. Cloud pa icles o 28 ci us juices ange om 0.4 o 5 µm, being pa icles smalle han 2 µm he mos s able clouds 29 (Elle bee & Wicke , 2011). In he li e a u e, one o he mos accep ed heo ies o cloud 30 des abiliza ion is based on pec in deme hyla ion by pec inme hyles e ase (PME) (EC 3.1.1.11) in 31 a blockwise ashion. The nega i e cha ges gene a ed by PME ac i i y allow subsequen 32 o ma ion o insoluble calcium pec a e gels wi h calcium ions p esen in he juice. These gels can 33 p ecipi a e pulling he cloud wi h hem causing o ange juice cla i ica ion due o he loss o 34 u bidi y (Elle bee & Wicke , 2011). The mal ea men o o ange juice a 90 ºC o 1 minu e is 35 he me hod cu en ly used o p e en mic obial spoilage as well as he inac i a ion o he PME 36 (Oulé, Dickman, & A ul, 2013). Howe e he mal ea men causes undesi able changes in 37 se e al quali y pa ame e s such as la ou , colou and ex u e and can also des oy hea -sensi i e 38 nu i ional componen s such as i amins (Hu, Zhou, Xu, Zhang, & Liao, 2013). Non- he mal 39 4 echnologies ha e gained in e es and accep ance as ood p ocessing me hods due o he 40 consume inc eased demand o esh-like p oduc s. Among hem, high p essu e ca bon dioxide 41 (HPCD) has been p oposed as an al e na i e non- he mal pas eu iza ion echnique o oods. 42 HPCD can also cause he inac i a ion o ce ain enzymes ha a ec he quali y o some oods 43 such as PME in he o ange juice unde mild ope a ion condi ions (Dama & Balaban, 2006). In 44 HPCD ea men s, ope a ing empe a u es can ange be ween 5 – 60 ºC and p essu es usually 45 below 50 MPa. Some o he ad an ages o using HPCD as non- he mal ea men a e ha ca bon 46 dioxide is non oxic, non lammable, inexpensi e and eadily a ailable. I can also be easily 47 emo ed a e ea men by dep essu iza ion. 48 Some p e ious s udies dealing wi h he e ec o HPCD ea men on o ange juice quali y can be 49 ound in he li e a u e. The i s wo k was ca ied ou by Balaban, A eola, Ma shall, Peplow, 50 Wei, and Co nell (1991), who ound 100 % PME inac i a ion when using a comme cial Mil on 51 Roy Supe c i ical X-10 Sys em while only 86 % PME inac i a ion was achie ed when a cus om-52 made supe c i ical sys em was used. These au ho s also ound ha , when using he cus om-made 53 sys em, cloud signi ican ly inc eased. Kincal, Hill, Balaban, Po ie , Sims, Wei, and Ma shall 54 (2006) also epo ed a high inc ease in he cloud alues (be ween 446 – 846%) in o ange juice, 55 when using a con inuous sys em bu a maximum PME inac i a ion deg ee o only 46.3%. 56 Recen ly, Zhou, Bi, Xu, Yang, and Liao (2015) e iewed he e ec s o HPCD p ocessing on 57 la ou , ex u e and colou o oods including o ange juice. Combined echnologies o high 58 powe ul asound assis ed SC-CO2 (HPU-SCCO2) ha e been also epo ed o inac i a e PME o 59 o ange juice (O uño, Balaban, & Benedi o, 2014). These au ho s ound a lowes esidual ac i i y 60 o 10.65 %. The e o e, di e en inac i a ion deg ees ha e been epo ed in he li e a u e when 61 ea ed eshly squeezed o ange juice o HPCD. This ega d, in he li e a u e i has been epo ed 62 5 an imp o emen o inac i a ion o di e en enzymes by inc easing he CO2 concen a ion in he 63 enzyme solu ions when CO2 was ed h ough a cylind ical il e nozzle (Ishikawa, Shimoda, 64 Kawano, & Osajime, 1995). Un o una ely, in mos o he p e ious s udies, no in o ma ion abou 65 he way CO2 is pu in con ac wi h he subs a e can be ound and compa ison is di icul o 66 s ablish. Addi ionally, di e ences in inac i a ion le els a e ela ed o cul i a s, o iginal pH o he 67 juice, isoenzyme o ms, o al solid con en and o he p ocessing ac o s. 68 CO2 was used unde supe c i ical condi ions in p e ious epo ed HPCD ea men s o o ange 69 juice. The main objec i e o his wo k is o assess he e ec o HPCD ea men unde 70 supe c i ical and liquid condi ions on PME ac i i y. The e ec o HPCD p ocessing on o he 71 physical and chemical pa ame e s o o ange juice will be also s udied. 72 2 Ma e ials and me hods 73 2.1 HPCD equipmen and p ocessing 74 Valencia o anges we e pu chased om a local supplie . O anges we e squeezed in an o ange 75 squeeze . The expe imen al appa a us used o he HPCD ea men has been designed in ou 76 labo a o y wi h a maximum ope a ing p essu e and empe a u e o 30 MPa and 80 ºC 77 espec i ely (Melgosa, Sanz, G. Solaesa, Bucio, & Bel án, 2015). I consis s o a CO2 ese oi , 78 a high p essu e sy inge pump wi h a p essu e con olle (ISCO 260 D) and 3 high p essu e cells 79 imme sed in a he mos a ic wa e ba h. In a ypical HPCD expe imen , o ange juice was cha ged 80 in o he high p essu e cell, which was hen placed in he he mos a ic wa e ba h a he p ese 81 empe a u e. A e wa ds, he sys em was p essu ized and main ained a cons an empe a u e and 82 p essu e o a p e-es ablished ea men ime. CO2 was ed o he high p essu e cell h ough a 83 sin e ed s ainless s eel mic o- il e wi h a po e size o 10 µm o inc ease he concen a ion o 84 6 CO2 dissol ed in he sample. The du a ion o he p essu iza ion and dep essu iza ion was less 85 han 2-3 min and i was no included in he ea men holding ime. The high p essu e cells we e 86 magne ically s i ed. Expe imen s we e ca ied ou in a empe a u e (T) ange om 2 o 40 ºC, 87 p essu e (p) om 10 o 30 MPa and exposu e ime ( ) om 3 o 60 min. Di e en p essu e cells 88 we e a anged in se ies o ca y ou expe imen s a di e en ope a ing imes. A e HPCD 89 ea men , he high p essu e cells we e dep essu ized and he ea ed o ange juice was analysed 90 (see sec ion 2.2). Du ing dep essu iza ion, a empe a u e dec ease o he o ange juice was 91 obse ed due o Joule-Thomson cooling e ec depending on applied p essu es (Zhou, Zhang, 92 Leng, Liao, & Hu, 2010). 93 PME ac i i y, pH and calcium con en we e de e mined be o e and a e HPCD ea men a 94 di e en ope a ing condi ions. To e alua e he e ec o HPCD ea men on he sel -li e o 95 o ange juice, a sample o o ange juice ea ed a 30 MPa and 40 ºC o 40 min was s o ed in he 96 e ige a o (4ºC). Aliquo s we e aken a e 5 and 12 days o s o age, and di e en quali y 97 pa ame e s o o ange juice we e de e mined and compa ed wi h o iginal eshly squeezed o ange 98 juice. 99 2.2 Physico-chemical analysis 100 2.2.1 De e mina ion o pec in me hyles e ase ac i i y. PME ac i i y was de e mined by using 101 an au oma ic i a o sys em (Me ohm Ti ando). A 1% o pec in solu ion (Al a Aesa  Pec in 102 Ci us) p epa ed in NaCl 0.3 M was used as subs a e. 50 mL o pec in solu ion mixed wi h 5 mL 103 o o ange juice we e adjus ed o pH 7.5 wi h NaOH 0.02 N. Du ing hyd olysis a oom 104 empe a u e, pH was main ained a 7.5 by adding NaOH 0.02 N. The amoun o NaOH added o 105 30 minu es was eco ded. One PME ac i i y uni (UPE) is de ined as he mic omoles o 106 7 ca boxylic g oups p oduced pe minu e and mL o juice a pH 7.5 and oom empe a u e. PME 107 ac i i y was calcula ed acco ding o he ollowing equa ion: 108 𝑈𝑈𝑈𝑈𝑈𝑈 𝑚𝑚𝑚𝑚 ⁄=(𝑚𝑚𝑚𝑚 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁)·(𝑁𝑁𝑁𝑁𝑟𝑟𝑚𝑚𝑁𝑁𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑁𝑁𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁)·(𝑜𝑜𝑁𝑁𝑓𝑓𝑚𝑚𝑁𝑁𝑟𝑟 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁)·(1000) (𝑚𝑚𝑚𝑚 𝑗𝑗𝑗𝑗𝑚𝑚𝑓𝑓𝑗𝑗)·(𝑚𝑚𝑚𝑚𝑚𝑚𝑗𝑗𝑚𝑚𝑗𝑗𝑚𝑚) [1] Resul s a e p esen ed as esidual PME ac i i y, de ined as he ela ionship be ween PME ac i i y 109 a e and be o e HPCD ea men : 110 𝑅𝑅𝑗𝑗𝑚𝑚𝑚𝑚𝑅𝑅𝑗𝑗𝑁𝑁𝑚𝑚 𝑈𝑈𝑃𝑃𝑈𝑈 𝑁𝑁𝑓𝑓𝑚𝑚𝑚𝑚𝑎𝑎𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑈𝑈𝑃𝑃𝑈𝑈 𝑁𝑁𝑓𝑓𝑚𝑚𝑚𝑚𝑎𝑎𝑚𝑚𝑚𝑚𝑚𝑚 𝑁𝑁𝑜𝑜𝑚𝑚𝑗𝑗𝑟𝑟 𝑁𝑁𝑈𝑈𝐻𝐻𝐻𝐻 𝑈𝑈𝑃𝑃𝑈𝑈 𝑁𝑁𝑓𝑓𝑚𝑚𝑚𝑚𝑎𝑎𝑚𝑚𝑚𝑚𝑚𝑚 𝑏𝑏𝑗𝑗𝑜𝑜𝑁𝑁𝑟𝑟𝑗𝑗 𝑁𝑁𝑈𝑈𝐻𝐻𝐻𝐻=𝐴𝐴 𝐴𝐴𝑜𝑜 [2] 2.2.2 De e mina ion o pH, ºB ix, o al acidi y, Vi amin C and colou . pH o o ange juice 111 was de e mined wi h a pH-me e (C ison pH & Ion-Me e GLP 22). ºB ix we e measu ed wi h 112 a Mil on Roy e ac ome e (Model 334610) a 25ºC. Tempe a u e and acidi y co ec ions we e 113 made (Kimball, 1999). 114 To al acidi y was de e mined by using an au oma ic i a o (Me ohm Ti ando). A sample o 115 2 mL o o ange juice was mixed wi h 50 mL o dis illed wa e . The mix u e was i a ed wi h 116 0.02 N NaOH. Ti able acidi y was exp essed as ci ic acid pe cen age (g ci ic acid/100g). 117 Vi amin C was de e mined wi h 2,6-dichlo oindophenol i ime ic me hod (Kimball, 1999). 118 Colou was e alua ed by a Konica Minol a  CM-2600d colo ime e . The L*, a* and b* alues 119 we e ob ained ep esen ing ligh ness, ed o g een colou and yellow o blue colou , espec i ely. 120 O he condi ions a e illuminan D65 (dayligh sou ce) and a 10º s anda d obse e (pe cep ion o 121 a human obse e ) ollowing he CIE ecommenda ions. Changes in colou we e exp essed as: 122 8 ∆𝑈𝑈=��𝑚𝑚𝑏𝑏𝑏𝑏𝑏𝑏𝑜𝑜𝑏𝑏𝑏𝑏 ∗ −𝑚𝑚𝑎𝑎𝑏𝑏𝑎𝑎𝑏𝑏𝑏𝑏 ∗ �2+�𝑁𝑁𝑏𝑏𝑏𝑏𝑏𝑏𝑜𝑜𝑏𝑏𝑏𝑏 ∗ −𝑁𝑁𝑎𝑎𝑏𝑏𝑎𝑎𝑏𝑏𝑏𝑏 ∗ �2+�𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑜𝑜𝑏𝑏𝑏𝑏 ∗ −𝑏𝑏𝑎𝑎𝑏𝑏𝑎𝑎𝑏𝑏𝑏𝑏 ∗ �2 [3] Di e ences in pe cei able colou can be classi ied analy ically as no no iceable (0-0.5) sligh ly 123 no iceable (0.5-1.5), no iceable (1.5-3) well isible (3.0-6.0) and g ea (6.0-12.0) (Yuk, 124 Samped o, Fan, & Ge eke, 2014). 125 Ano he pa ame e ha can be used o e alua e al e a ions in colou o a be e age is he ch oma, 126 C, which measu es colou in ensi y: 127 𝐻𝐻=�(𝑁𝑁∗)2+(𝑏𝑏∗)2 [4] 2.2.3 De e mina ion o u bidi y and pa icle size dis ibu ion. Cloud quali y was de e mined 128 by using a spec opho ome ic me hod. The o ange juice sample was cen i uged a 9000 .p.m. 129 o 30 min. The supe na an was pou ed in o a qua z cu e e and abso bance a 660 nm was 130 measu ed in a Hi achi® spec opho om e (Model U-2000). Dis illa ed wa e was used as a blank. 131 Pe cen cloud change was calcula ed as: 132 𝑈𝑈𝑗𝑗𝑟𝑟𝑓𝑓𝑗𝑗𝑚𝑚𝑚𝑚 𝑓𝑓𝑚𝑚𝑁𝑁𝑗𝑗𝑅𝑅 𝑓𝑓ℎ𝑁𝑁𝑚𝑚𝑎𝑎𝑗𝑗=(𝐹𝐹𝑚𝑚𝑚𝑚𝑁𝑁𝑚𝑚 𝑓𝑓𝑚𝑚𝑁𝑁𝑗𝑗𝑅𝑅 𝑎𝑎𝑁𝑁𝑚𝑚𝑗𝑗𝑗𝑗−𝐼𝐼𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑁𝑁𝑚𝑚 𝑓𝑓𝑚𝑚𝑁𝑁𝑗𝑗𝑅𝑅 𝑎𝑎𝑁𝑁𝑚𝑚𝑗𝑗𝑗𝑗) 𝐼𝐼𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑁𝑁𝑚𝑚 𝑓𝑓𝑚𝑚𝑁𝑁𝑗𝑗𝑅𝑅 𝑎𝑎𝑁𝑁𝑚𝑚𝑗𝑗𝑗𝑗 ·100 [5] Pa icle size dis ibu ion (PSD) was de e mined by lase di ac ion wi h a Mas e size 2000 133 (Mal e n® Ins ., MA). The sys em uses a lase ligh a 750 nm wa eleng h o size pa icles om 134 0.4 o 2000 µm by ligh di ac ion. Pa icle size dis ibu ion was calcula ed by he F aunho e 135 model. Size dis ibu ions ( olume ac ions agains pa icle size) and he weigh a e age size 136 exp essed as he equi alen su ace a ea mean diame e , D(3,2) and he equi alen olume mean 137 diame e , D(4,3) we e calcula ed be o e and a e HPCD ea men and along s o age. 138 9 2.2.4 De e mina ion o Calcium con en . Calcium in o ange juice be o e and a e HPCD 139 ea men was de e mined by a omic abso p ion spec ome y (Pe kin Elme 3300). The o ange 140 juice was cen i uged (Eppendo Cen i ugue 5804) a 9000 pm o 30 minu es (Zhou e al., 141 2010). The p ecipi a e was discha ged and he calcium con en o he supe na an was 142 de e mined. La2O3 (Me ck) was added o samples o a inal concen a ion o 0.5% o lan hanum 143 in he medium. The addi ion o lan hanum a oids he in e e ence o phospha es in he calcium 144 de e mina ion. HCl was also added (5% in he sample) o p omo e dissolu ion o bo h calcium 145 and lan hanum in he medium. Calcium con en was ob ained by calib a ion wi h di e en 146 s anda d solu ions o calcium (Me ck Ce ipu ®, 1 g/L) by ollowing he same me hod as wi h 147 he o iginal eshly squeeze o ange juice. 148 Some expe imen s we e also pe o med wi h a McIl aine bu e solu ion con aining 0.05 M 149 ci ic acid and 0.1 M disodium hyd ogen phospha e, a pH close o he o ange juice (pH ≈ 4), o 150 which calcium was added o achie e a con en simila o ha in o ange juice (a ound 100 ppm -151 2.5·10-3 MCa2+) using wo ypes o calcium sal s (chlo ide, ci a e). A McIl aine bu e solu ion 152 was chosen since his solu ion had a bu e capaci y simila o ha o o ange juice (Yoshimu a, 153 Fu u e a, Shimoda, Ishikawa, Miyake, Ma sumo o, Osajima, & Hayakawa, 2002). 154 2.3 Kine ic da a analysis 155 Di e en kine ic models we e es ed o co ela e he inac i a ion kine ics o PME (Hu e al., 156 2013) . 157 Two- ac ion kine ic model. This model akes in o accoun he exis ence o se e al isoenzymes 158 o PME in o ange juice, g ouped in o wo ac ions, a labile and a s able ac ion. Bo h enzymes 159 10 we e conside ed o be inac i a ed acco ding o i s -o de kine ics, bu independen ly o each 160 o he : 161 𝐴𝐴=𝐴𝐴𝐿𝐿𝑗𝑗𝑒𝑒𝑒𝑒(−𝑘𝑘𝐿𝐿𝑚𝑚)+𝐴𝐴𝑆𝑆𝑗𝑗𝑒𝑒𝑒𝑒(−𝑘𝑘𝑆𝑆𝑚𝑚) [6] whe e AL and AS (AS = 1 - AL) a e he ac i i y o he labile and s able ac ions espec i ely and 162 kL and kS (min-1) he inac i a ion a e cons an s o bo h he labile and s able ac ions 163 espec i ely. 164 F ac ional-con e sion model. A ac ion-con e sion model is a special case o a i s o de 165 kine ic model ha akes in o accoun he non-ze o esidual ac i i y a e p olonged hea ing 166 and/o p essu e (A∞) ea men : 167 𝑚𝑚𝑚𝑚(1−𝑜𝑜)=𝑚𝑚𝑚𝑚�(𝐴𝐴−𝐴𝐴∞) (𝐴𝐴𝑜𝑜−𝐴𝐴∞)�=−𝑘𝑘𝑚𝑚 [7] 𝐴𝐴=𝐴𝐴∞+(𝐴𝐴𝑜𝑜−𝐴𝐴∞)𝑗𝑗𝑒𝑒𝑒𝑒(−𝑘𝑘𝑚𝑚) [8] Weibull model. This model can be w i en in he powe -law o m as (O uño e al., 2014): 168 𝑚𝑚𝑁𝑁𝑎𝑎 10 �𝐴𝐴 𝐴𝐴𝑜𝑜�=−𝑏𝑏𝑚𝑚𝑛𝑛 [9] whe e b is a non-linea a e pa ame e and n is he shape ac o . 169 2.4 S a is ical analysis 170 All analyses we e conduc ed using so wa e S a g aphics X64. The esul s a e p esen ed as a 171 mean ± s anda d de ia ion o a leas h ee eplica es. The signi icance o he di e ences was 172 17 sligh ly lowe alues a e HPCD ea men han in bu e solu ions, no signi ican di e ences 306 ha e been de e mined among sample means o bu e and o ange juices when applying he 307 Tukey’s HSD me hod. 308 In his ega d, he e ec o di e en expe imen al a iables on CaCO3 solubili y has been 309 ecen ly epo ed in he li e a u e (Co o, Ma os, Peña, Rod íguez, & Pas o , 2012). CaCO3 310 solubili y inc eased wi h ope a ing p essu e (p essu ized CO2) and dec eased wi h empe a u e 311 and pH o he medium. Fo ins ance, a 40ºC CaCO3 solubili y in wa e a 1 ba and 40 ba is 312 abou 4.2·10-4 MCa2+ and 2.3·10-2 MCa2+, espec i ely. Al hough CaCO3 solubili y a a mosphe ic 313 p essu e is e y small, depends s ongly on pH inc easing as pH dec eases ( o ins ance, a 25 ºC, 314 a pH = 7 MCa2+=2·10-3 and a pH = 6 MCa2+=9·10-3). The e o e, aking in o accoun ha a he 315 low pH o o ange juice, only a small amoun o dissol ed CO2 is con e ed in o bica bona e 316 dissocia ing in o ee hyd ogen ions (Zhou e al., 2015) and ha calcium ca bona e solubili y 317 inc eases by dec easing pH, calcium con en in o ange juice was in ac no expec ed o change 318 much a e HPCD p ocessing. To ou knowledge, he only measu emen o calcium con en 319 be o e and a e HPCD ea men , was epo ed by Zhou e al. (2010), who ound no signi ican 320 e ec s on he calcium con en o peach juice, which is also an acidic juice (pH ~ 3.8), a e 321 HPCD ea men . 322 To explain he cloud enhancemen a e HPCD ea men , he possible e ec o homogeniza ion 323 induced by gas expansion du ing he dep essu iza ion s ep has been s udied by de e mining he 324 pa icle size dis ibu ion, PSD. 325 3.2.3 Pa icle size dis ibu ion. PSD o o ange juice be o e and a e HPCD ea men has been 326 ep esen ed in Figu e 5. Two maximums a ound 0.8 µm and 850 µm can be obse ed. The size 327 o s able cloud pa icle has been epo ed o be in he ange o 0.4-5 μm, wi h he mos s able 328 18 cloud ha ing pa icle sizes o 2 μm and smalle (Elle bee & Wicke , 2011). The la ge pa icle 329 size in Figu e 5 is due o he p esence o some se ling pulp. In his ega d, i mus be emphasized 330 ha lase di ac ion me hods gene a es a olume dis ibu ion. The o al olume o all pa icles 331 wi h diame e s less han 5 µm ep esen s 24.5 % o he o al olume o pa icles bu ep esen s 332 100 % in numbe dis ibu ion. Tha is, al hough he numbe o bigge pa icles is e y small 333 ep esen s a high olume dis ibu ion when compa ed o cloud pa icles. Cla i ica ion o juice 334 akes place when s able cloud showed agg ega ion by shi ing he PSD dis ibu ion o la ge 335 diame e s (Co edig, Ke , & Wicke , 2001). Howe e , HPCD ea men esul s in an inc ease o 336 he olume peak o he smalle pa icles and a dec ease o la ge pa icles (Figu e 5). This 337 beha iou o o ange juice PDS helps o unde s and he cloud enhancemen a e HPCD 338 ea men . This ac has been explained in e ms o he e ec o he homogeniza ion caused by 339 HPCD ea men due o se e al easons, such as high in e nal s ess su passing he ensile 340 s eng h o he pa icles when CO2 is emo ed om he essel (Niu e al., 2010). Figu e 5 also 341 p esen s PSD o ea ed o ange juice a e 5 and 12 days s o age. I can be obse ed ha he 342 olume peak o he small pa icles inc eased du ing he s o age, while he olume peak o he 343 la ge pa icles dec eased. This beha iou could be a ibu ed o he emained ac i e PME ha 344 could decompose he high molecula weigh compound and hen educe he size o pa icles. 345 Values o D[3,2] and D[4,3] o eshly squeezed juice, a e ea men and a e 5 and 12 days 346 s o age a e p esen ed in Table 4. Acco ding o Figu e 5, he alues a e HPCD ea men we e 347 lowe han hose o eshly squeezed o ange juice and a con inuous dec ease was obse ed wi h 348 inc easing s o age ime, bu his dec ease was no signi ican di e en along s o age (Table 4). In 349 any, case, no shi o PSD o la ge diame e s can be obse ed. This endency can be also 350 19 obse ed in he alues o d(0.1), d(0.5) and d(0.9) which co espond o he size o pa icle below 351 which 10%, 50% and 90% o he sample lies, espec i ely. 352 3.2.4 pH, o al acidi y, ºB ix, asco bic acid and colou . 353 pH, ºB ix and o al acidi y did no change signi ican ly in o ange juice a e HPCD ea men and 354 emained essen ially cons an du ing s o age (Table 5). The low pH o he o iginal o ange juice 355 made di icul he dissocia ion o he ca bonic acid, o med by dissol ed CO2 in he juice, in o 356 H+ ions. Howe e , in some s udies, a dec ease in he pH o o ange juice has been epo ed du ing 357 HPCD ea men (Oulé e al., 2013; Balaban e al., 1991). The pH dec ease has been hus 358 associa ed wi h he s a e and densi y o CO2 in he juice du ing he ea men (Oulé e al., 2013). 359 Fu he in es iga ions should be pe o med o analyse he pH o he sample du ing ea men , due 360 o di e en esul s ound in he li e a u e (see sec ion 3.1). 361 The con en o asco bic acid dec eased a e HPCD ea men (a ound 14%) and con inued 362 dec easing du ing s o age. Howe e he dec ease o he asco bic con en a e HPCD ea men is 363 lowe han a e pas eu iza ion ea men s. Oule e al. (2013) epo ed a dec ease o he i amin C 364 con en o 13% a e HPCD ea men (25 MPa, 40ºC) while a e pas eu iza ion i amin C 365 dec eased 43% (90ºC and 60 s). This di e ence is a ibu ed no only o he lowe empe a u es 366 employed in HPCD ea men s bu also o he O2- ee en i onmen and he low solubili y o 367 i amin C in SC-CO2. In his wo k, highe loss o i amin C has been obse ed du ing s o age 368 (10% and 24% a e 5 and 12 days espec i ely) han o he alues ound in he li e a u e du ing 369 s o age. Fo ins ance, Oulé e al. (2013) ound a ound 5% i amin C loss a e 56 days s o age a 370 4 ºC. This high pe cen age loss could be due o he p esence o O2 du ing s o age, bu also o he 371 pH o he o ange juice used in his wo k (4.11-4.12), since i is known ha mo e acidic 372 condi ions s abilized asco bic acid (Bull e al., 2004). 373 20 Table 6 lis s he L*, a*, b* pa ame e s o eshly squeezed o ange juice, a e HPCD ea men 374 and du ing s o age. Ligh ness (L*) and yellowness (b*) signi ican ly dec eased indica ing he 375 da kening o he o ange juice and less yellow and mo e blue colou a e HPCD p ocessing. On 376 he con a y, edness (a*) was no signi ican di e en in he un ea ed and HPCD p ocessed 377 o ange juice. In he li e a u e, di e ences ha e been epo ed o he ligh ness, edness and 378 yellowness in HPCD- ea ed o ange juice (Zhou e al., 2015). Du ing s o age, ligh ness (L*), 379 edness (a*) and yellow (b*) did no change signi ican ly a e 5 days s o age; howe e edness 380 and yellowness dec eased signi ican ly a e 12 days s o age. Acco ding o Zhou e al. (2015) he 381 colou o oods can be in luenced by biochemical o chemical eac ion as well as physical e ec s 382 induced by HPCD. Among o he mechanisms, oxida ion o asco bic acid could also lead o he 383 colou change (Zhou e al., 2015). This would ag ee wi h he esul s epo ed in Table 5 o 384 asco bic acid con en . The change in colou , ∆E (Eq 3) is also p esen ed in Table 6 and isible 385 di e ences in colou a e HPCD ea men ha e been de e mined (∆E ≈ 5). Kincal e al. (2006) 386 also epo ed ∆E alues as high as 13.83, a 72 MPa and a a io o 0.64 CO2/juice (w/w). Ch oma 387 alues lis ed in Table 6 show ha HPCD ea men esul s in a signi ican lowe colou in ensi y 388 juice a e p ocessing bu no signi icance di e ences ha e been obse ed along s o age. 389 4. Conclusions 390 F eshly squeezed o ange juice has been ea ed by HPCD unde di e en ope a ing condi ions. 391 PME in o ange juice was e ec i ely inac i a ed by HPCD showing a as ini ial dec ease ha 392 emained nea ly cons an a e p olonged HPCD ea men . The inac i a ion deg ee inc eased 393 wi h p essu e and empe a u e. Di e en inac i a ion kine ic models we e used o co ela e he 394 esidual PME ac i i y, being he wo- ac ion model he bes wi h he lowes mean ela i e 395 de ia ion. Inac i a ion o PME in o ange juice a e HPCD ea men seems o be e e sible 396 21 since i s ac i i y is sligh ly eco e ed along s o age a 4 ºC. PSD shows an inc ease o he 397 olume peak o he smalle pa icles (0.3-5 μm) and a dec ease o la ge pa icles a e HPCD 398 ea men , suppo ing he cloud enhancemen obse ed. Calcium con en does no change 399 signi ican ly a e HPCD ea men , p o ing ha insoluble calcium con en was no o med. 400 Fu he in es iga ion should be done o analyse he e ec o cloud enhancemen a e HPCD 401 ea men . 402 ACKNOWLEDGMENTS 403 To Hype ba ic. To he Spanish Go e nmen h ough MINECO (CTQ2015-64396-R). R. 404 Melgosa acknowledges MINECO o a g an (BES-2013-063937). A. G. Solaesa acknowledges 405 he Bu gos Uni e si y o a p e-doc o al ellowship. 406 407 22 Re e ences: Agcam, E., Akyıldız, A., & E endilek, G. A. (2014). E ec s o PEF and hea pas eu iza ion on PME ac i i y in o ange juice wi h ega d o a new inac i a ion kine ic model. FOOD CHEMISTRY, 165, 70–76. h p://doi.o g/10.1016/j. oodchem.2014.05.097 A eola, A. G., BalaBan, M. O., Ma shall, M. R., Peplow, A. J., Wei, C., & Co nell, J. A. (1991). Supe c i ical Ca bon Dioxide E ec s on Some Quali y A ibu es o Single S eng h O ange Juice. Jou nal o Food Science, 56(4), 1030–1033. Balaban, M. O., A eola, A. 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Food Chemis y, 115, 449–455. h p://doi.o g/10.1016/j. oodchem.2008.12.028 Zhou, L., Zhang, Y., Leng, X., Liao, X., & Hu, X. (2010). Accele a ion o p ecipi a ion o ma ion in peach juice induced by high-p essu e ca bon dioxide. Jou nal o Ag icul u al and Food Chemis y, 58, 9605–9610. h p://doi.o g/10.1021/j 101430j 25 Table 1. PME inac i a ion alues in o ange juice a e HPCD and o he non- he mal ea men s Sys em pH p, MPa T, ºC , min Inac i a ion Re e ence Mil on Roy Sys em 3.8 29 50 240 100% (Balaban e al., 1991) Cus om made sys em 3.8 29 45 120 84% (Balaban e al., 1991) S a ic 3.9 40 55 10 36% (Niu e al., 2010) S a ic 3.9 40 55 60 95% (Niu e al., 2010) Con inuous 3.7 72 24 10* 56% (Kincal e al., 2006) S a ic 4.12 30 40 20-60 90-92 % This wo k HPU-SCCO 2 3.8 23 41 10 89 % (O uño e al., 2014) Pas eu iza ion 3.5 0.1 90 10 - 20 s 93 – 96 % (Agcam e al., 2014) (*) esidence ime; HPU-SCO2 = high powe ul asound assis ed supe c i ical CO2. 26 Table 2. Es ima ed kine ic pa ame e s o o ange juice PME inac i a ion a di e en ope a ing condi ions, o di e en kine ic models Kine ic model T, ºC p, MPa Model pa ame e s D alue, min MRD 2 (p<0.05) Two- ac ion 40 30 k L = 0.74 ± 0.07 A L = 0.85 ± 0.02 D L = 3.3 ± 0.3 5.8 0.999 k S = 0.010 ± 0.003 A S = 0.15 ± 0.02 D S = 230 ± 69 21 20 k L = 0.24 ± 0.01 A L = 0.652 ± 0.008 D L = 9.6 ± 0.4 1.4 0.999 k S = 0.0048 ± 0.0005 A S = 0.348 ± 0.007 D S = 480 ± 69 21 10 k L = 0.123 ± 0.003 A L = 0.628 ± 0.009 D L = 18.7 ± 0.5 1.2 0.999 k S = 0.0020 ± 0.0005 A S = 0.372 ± 0.009 D S = 1152 ± 288 F ac ional- con e sion 40 30 k = 0.63 ± 0.06 A ∞ = 0.11 ± 0.01 D = 3.7 ± 0.3 11.4 0.996 21 20 k = 0.18 ± 0.02 A ∞ = 0.28 ± 0.01 D = 13 ± 1 4.8 0.997 21 10 k = 0.112 ± 0.003 A ∞ = 0.336 ± 0.004 D = 20.6 ± 0.6 1.4 0.999 Weibull 40 30 b = 0.58 ± 0.03 n = 0.15 ± 0.02 D = 1.7 ± 0.1 5.8 0.990 21 20 b = 0.27 ± 0.02 n = 0.19 ± 0.02 D = 3.7 ± 0.3 2.8 0.998 21 10 b = 0.14 ± 0.03 n = 0.31 ± 0.06 D = 7 ± 2 5.3 0.985 MRD: mean ela i e de ia ion (Eq. 10) 33 Figu e 3. E ec o ope a ing ime on PME inac i a ion a di e en ope a ing condi ions (△ 40ºC and 30 MPa,  21ºC, 20 MPa; ○, 21ºC, 10 MPa). Da a: mean ± SD (n=3). Con inuous lines co espond o he wo- ac ion model. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 010 20 30 40 50 60 70 Residual ac i i y, A/Ao ime, min 34 Figu e 4. E olu ion o PME esidual ac i i y and cloud pe cen age (Eq. 5) in HPDC ea ed (30 MPa, 40 ºC and 40 min) o ange juice du ing s o age a 4ºC. (◇ PME esidual ac i i y,  cloud pe cen age). Da a: mean ± SD (n=3). 0 5 10 15 20 25 30 35 40 0 5 10 Residual ac i i y, %; Cloud change % S o age, days 35 Figu e 5. Pa icle Size Dis ibu ion (PSD) o o ange juice eshly squeezed ( ─ ), immedia ely a e ea men by HPCD a 30 MPa, 40ºC o 40 min (····); a e 5 days s o age a 4ºC (-·-·) ; a e 12 days s o age a 4ºC (− − −). 0 1 2 3 4 5 6 7 0.01 0.1 1 10 100 1000 10000 Volume (%) Pa icle size, (µm)