Effect of thermosonication batch treatment on enzyme inactivation kinetics and other quality parameters of cloudy apple juice

Accep ed Manusc ip E ec o he mosonica ion ba ch ea men on enzyme inac i a ion kine ics and o he quali y pa ame e s o cloudy apple juice A.E. Ille a, M.T. Sanz, O. Beni o, S. Va ona, S. Bel án, R. Melgosa, A.G. Solaesa PII: S1466-8564(17)31225-0 DOI: h ps://doi.o g/10.1016/j.i se .2018.02.001 Re e ence: INNFOO 1921 To appea in: Inno a i e Food Science and Eme ging Technologies Recei ed da e: 30 Oc obe 2017 Re ised da e: 2 Feb ua y 2018 Accep ed da e: 2 Feb ua y 2018 Please ci e his a icle as: A.E. Ille a, M.T. Sanz, O. Beni o, S. Va ona, S. Bel án, R. Melgosa, A.G. Solaesa , E ec o he mosonica ion ba ch ea men on enzyme inac i a ion kine ics and o he quali y pa ame e s o cloudy apple juice. The add ess o he co esponding au ho was cap u ed as a ilia ion o all au ho s. Please check i app op ia e. Inn oo(2017), h ps://doi.o g/10.1016/j.i se .2018.02.001 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. ACCEPTED MANUSCRIPT E ec o he mosonica ion ba ch ea men on enzyme inac i a ion kine ics and o he quali y pa ame e s o cloudy apple juice. A.E. Ille a, M. T. Sanz  , O. Beni o, S. Va ona, S. Bel án, R. Melgosa. A. G. Solaesa Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion), Uni e si y o Bu gos, 09001 Bu gos. Spain Abs ac Cloudy apple juice has been ea ed by he mosonica ion in ba ch mode as an al e na i e p ocessing o he mal ea men . The mosonica ion was ound o be e ec i e o inac i a e polyphenol oxidase; howe e , pec inme hyles e ase was ound o be mo e esis an . An inc ease o he wo king ul asound ampli ude and he amoun o ene gy supplied o he juice led o lowe enzyme esidual ac i i ies. Enzyme inac i a ion kine ics we e de e mined a di e en empe a u es ( om 44 o 67 ºC). Inac i a ion da a we e desc ibed by he i s o de kine ic model and he Weibull model, bo h models yielding good i ing. The mosonica ion ea men caused a homogeniza ion e ec e lec ed in he shi ing o he pa icle size dis ibu ion owa ds smalle diame e s. The e ec o he na u e o dissol ed gases in he juice on enzyme inac i a ion was s udied by displacing he ai dissol ed in he juice by bubbling ni ogen o ca bon dioxide, p e ious o he he mosonica ion expe imen s. Highe inac i a ion a es we e ob ained by displacing he ai wi h ni ogen.  Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess e [email protected] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Indus ial ele ance: Consume s demand o na u al and esh-like p oduc s has d i en he ood indus y o in es iga e al e na i e echnologies o eplace con en ional ood hea ea men s ha may a ec ood quali y. Among hese echnologies, he mosonica ion ea men is an a ac i e echnology ha can inac i a e mic oo ganisms and enzymes. This wo k shows ha some enzymes ha cause dele e ious e ec on cloudy apple juice can be mo e e ec i ely inac i a ed by he mosonica ion han by he mal ea men , in he same empe a u e ange, educing he damages caused by hea ing Keywo ds: Cloudy apple juice, he mosonica ion, enzyme inac i a ion, dissol ed gasses. 1. In oduc ion Cloudy apple juice is a good sou ce o bioac i e compounds such as asco bic acid, polyphenols, and pec ins. Howe e , enzyma ic b owning causes se ious p oduc de e io a ion. One o he mos impo an enzymes esponsible o enzyma ic b owning is polyphenol oxidase (PPO). PPO ca alyzes he oxida ion o phenolic compounds o quinones, which will subsequen ly eac wi h o he compounds o o m b own pigmen s ha educe juice quali y. O he impo an quali y pa ame e in cloudy apple juice is he cloud s abili y. One o he mos accep ed heo ies o cloud loss in juices, is based on he ac ion o pec inme hyles e ase (PME). This enzyme causes pec in deme hyla ion ha could expedi e pec in p ecipi a ion wi h calcium ions p esen in he juice, causing cla i ica ion and u bidi y loss (B iongos e al., 2016) The mal ea men s a e widely used in he ood indus y o inac i a e mic oo ganisms and enzymes ha cause dele e ious e ec s on oods. Howe e , consume s demand o na u al and esh-like p oduc s has d i en he ood indus y o in es iga e al e na i e echnologies o eplace con en ional ood hea ea men s, wich may a ec ood quali y in e ms o nu i ional and physicochemical pa ame e s. Among hese echnologies, ul asonica ion is an a ac i e ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT echnology ha is conside ed o be simple, eliable, en i onmen ally iendly and highly e ec i e in achie ing mic obial decon amina ion (Dias e al., 2015). Inac i a ion o mic oo ganisms and enzymes by sonica ion is a ibu ed o physical (ca i a ion, mechanical e ec s) and chemical ( o ma ion o ee adicals) e ec s (O’Donnell, Tiwa i, Bou ke, & Cullen, 2010). Inac i a ion o enzymes by ul asonica ion has been a ibu ed o he o ma ion o localized ho spo s upon collapse o bubbles, shea o ces c ea ed by mic os eaming and shock wa es, as well as gene a ion o ee adicals h ough sonolysis o wa e (Kadkhodaee & Po ey, 2008). Sonochemical ac i i y (p oduc ion o adicals) is in luenced by ope a ional pa ame e s, being he e ec o equency and gas addi ion he mos signi ican (Gielen e al., 2016). Fu he mo e, he p esence o gases (ai , oxygen o an ine gas) in he solu ion has been epo ed as necessa y o enzyme inac i a ion (G in se ich, Adze ikho, M ochek, & Me eli za, 2001). In any case, enzyme inac i a ion mechanisms a e speci ic o each enzyme and depend on hei amino acid composi ion and hei con o ma ional s uc u e (Anaya-Espa za e al., 2017). When ul asound is combined wi h hea ing, he esul ing echnique, he mosonica ion (TS), is usually mo e e ec i e as p ese a ion echnique e en TS empe a u es being lowe han hose used in he mal ea men s. TS ea men has been ound o be mo e e icien han he mal ea men o he inac i a ion o di e en ypes o enzymes esponsible o de e io a ion o ui s and ege able juices, among hem, pec in me hyles e ase, PPO, lipoxygenases and pe oxidases (Bal acioglu, Bayindi li, & Se e can, 2017). Recen ly, Anaya Espa za e al. (Anaya-Espa za e al., 2017) e iewed he ad an ages and limi a ions o e ed by he applica ion o TS on di e en ui and ege able juices. In his wo k, he e ec o TS on PPO and PME inac i a ion in Golden Delicious cloudy apple juice has been s udied. The e ec o di e en a iables, such as amoun o ene gy ansmi ed o he juice, exp essed as powe densi y, PD (W/mL), ul asoung (US) ampli ude, empe a u e and ea men ime, on enzyme ac i i y has been de e mined. In addi ion, ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT he e ec o he na u e o he dissol ed gas (ai , ni ogen and ca bon dioxide) on enzyme inac i a ion has been e alua ed. No p e ious s udies ha e been ound in he li e a u e ega ding he e ec o his a iable on ui juices p ocessed by TS. Finally, enzyme inac i a ion by TS was compa ed wi h p e ious esul s ob ained by high p essu e ca bon dioxide (HPCD) ea men , as non- he mal echnology (Ille a e al., 2018), and wi h he mal ea men alone in he same empe a u e ange. The e ec o TS on o he quali y pa ame e s o cloudy apple juice, such as pa icle size dis ibu ion (PSD), colo and o al polyphenols and hyd oxyme hyl u u al con en has been also de e mined. 2. Ma e ials and me hods 2.1 Juice p epa a ion Golden delicious apples we e peeled, cu in cubes and subme ged in a 0.3 % L-asco bic acid solu ion o a oid enzyma ic b owning du ing p ocessing. Apple cubes we e wiped and immedia ely squeezed wi h a sc ew juice ex ac o . The liqueu was il e ed wi h 2 laye s o cheeseclo h. The pH o he juice ob ained was 3.89 ± 0.01 and he soluble solids con en was 12.5 ± 0.1 °B ix. The juice was s o ed ozen a −18 °C un il u he use. Ca bon dioxide (99.9 %) was supplied by Ai Liquide S.A. (Spain). Ni ogen (98.5 %) was ob ained by a Ze i o 25 LCMS ni ogen gene a o . 2.2 The mosonica ion A 750 W Sonics Ma e ialTM wi h a 13 mm p obe was used o he mosonica ion. Samples we e p ocessed a a cons an ul asound equency o 20 kHz. Cloudy apple juice was in oduced in a he mos a ed essel (Φ = 4.8 cm, V = 199 cm3) and he p obe was subme ged in he juice a a cons an dep h o 2 cm om he bo om o he essel. Acco ding o he manu ac u e he US ampli ude is 79 m when he ampli ude con ol is se a 100 % and he 13 mm p obe can handle olumes in he ange om 50 o 150 mL. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Fi s ly, TS was pe o med in a con inuous mode and ixing he ampli ude a di e en le els ( om 25 o 100 %), keeping cons an he es o ope a ing a iables: TS ime (15 min), ea ed olume o apple juice (80 mL) and empe a u e o he jacke ed wa e (40ºC). This way, he e ec o ampli ude on PSD and PPO inac i a ion was i s analyzed. Du ing each TS expe imen , he empe a u e p o ile was egis e ed. The ene gy inpu was eco ded a e he expe imen . The ul asonic powe densi y, PD, was e alua ed as: 𝑃𝐷=𝐸 𝑡·𝑉 =𝑃 𝑉 [1] whe e E, is he ene gy inpu , J, is he ul asonica ion ime, s, P, he ul asonic powe (J/s =W) and V is he sample olume, mL. TS expe imen s we e also pe o med by a ying he ea ed sample olume om 60 o 120 mL, in con inuous and in pulse (5 s on and 5 s o ) modes, a maximum ampli ude, du ing 15 min o TS and keeping cons an he empe a u e o he jacke ed wa e a 40ºC. By a ying he sample olume a ixed ampli ude, he powe densi y was also a ied (see equa ion 1) and i s e ec on PPO inac i a ion was analyzed. Enzyme inac i a ion kine ics o PME and PPO we e de e mined a maximum ampli ude o a ea ed sample olume o 80 mL a di e en TS empe a u es, by a ying he empe a u e o he jacke ed wa e om 20 o 50ºC. P e ious o he TS ea men , cloudy apple juice was p ehea ed (90 s (Sulaiman, Soo, Yoon, Fa id, & Sil a, 2015)) o he ini ial empe a u e o TS ( empe a u e o he jacke ed wa e ) in all he expe imen s, hen empe a u e was con inuously eco ded du ing ea men . No enzyme inac i a ion was obse ed du ing he p ehea ing ime (90 s) a he ini ial TS empe a u es essayed in his wo k, om 20 o 50ºC. The e ec o he na u e o he dissol ed gas in he juice was also conside ed. P e ious o he he mosonica ion expe imen s, ai dissol ed in he juice was displaced by bubbling ni ogen o ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ca bon dioxide in he sample a a mosphe ic p essu e un il oxygen concen a ion was below 0.4 mg/L (YSI P oODO op ical dissol ed oxygen me e ). A e ha , TS expe imen s we e pe o med as p e iously desc ibed. 2.3 Physical-chemical analyses 2.3.1. De e mina ion o enzyme ac i i y Polyphenol oxidases. PPO ac i i y was de e mined by adding 100 µL o apple juice in o 2.9 mL o subs a e solu ion consis ing o a 0.05 M ca echol (Sigma Ald ich) solu ion p epa ed in a 0.1 M phospha e bu e (pH 6.5). Oxida ion o ca echol was de e mined immedia ely by he abso bance inc ease a 420 nm by using a Jasco V-750 spec opho ome e equipped wi h a Pel ie he mos a ed cell holde a 30 ºC. The PPO ac i i y was de e mined by using he e y i s linea pa o he eac ion cu e (Ille a e al., 2018). Pec inme hyles e ase. Samples we e analyzed by using an au oma ic i a o sys em (Me ohm  Ti ando). A pec in solu ion (1 %) (Al a Aesa  pec in ci us) was p epa ed in NaCl (0.3 M). This solu ion was used as subs a e. 50 mL o pec in solu ion we e mixed wi h 1 mL o cloudy apple juice and pH was adjus ed o 7.5 wi h NaOH 0.005 N. Du ing pec in hyd olysis a 30 ºC, pH was main ained a 7.5 by adding NaOH 0.005 N. The amoun o NaOH added o 15 min was eco ded. One PME ac i i y uni (UPE) is de ined as he mic omoles o ca boxylic g oups p oduced pe minu e and mL o juice, a pH 7.5 and 30 ºC (B iongos e al., 2016). Rela i e esidual ac i i ies o PPO and PME we e e alua ed as: Residual ac i i y = Enzyme speci ic ac i i y a e TS ea men Enzyme speci ic ac i i y be o e TS ea men ·100% [2] As p e iously explained, du ing he p ehea ing ime (90s) no inac i a ion was obse ed, he e o e he enzyme ac i i y igh be o e TS ea men was conside ed o be he enzyme ac i i y o he un ea ed juice. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2.3.2. De e mina ion o hyd ogen pe oxide, ni a e and ni i e o ma ion One o he mechanisms p oposed on enzyme inac i a ion by US is he o ma ion o ee adicals. In he li e a u e, hyd ogen pe oxide (H2O2) o ma ion has been de e mined as an es ima ion o ca i a ion in ensi y (Ra iyan, Zhang, & Feng, 2005). Howe e , H2O2 gene a ion du ing an ul asound ea men in a ood sys em is complica ed due o he p esence o ions and o he colloidal componen s (Ra iyan e al., 2005). The e o e, in his wo k, H2O2 gene a ion du ing TS has been de e mined in a ci a e bu e solu ion a he same pH as he one o he apple juice (pH = 3.9). Fo ma ion o ni i e and ni a e in ci a e bu e solu ions was also de e mined, since i s sonochemical o ma ion in wa e has been known o a long ime (Supeno & K uus, 2000). Di e en ci a e bu e solu ions (pH = 3.9) in he p esence o dissol ed ai , ni ogen o ca bon dioxide, p epa ed as indica ed in sec ion 2.2, we e he mosonica ed (80 mL o solu ion a 60ºC) o 20 minu es. Samples we e collec ed a di e en ime in e als, o de e mine he H2O2, ni a e and ni i e o ma ion du ing he sonica ion p ocess. H2O2 was de e mined spec opho ome ically by a colo ime ic me hod based on he one desc ibed by Mead e al. (Mead, Su he land, & Ve all, 1976). 1 mL o solu ion A [1g NaOH, 33g KI, and 0.1g (NH4)6Mo7O24H2O in 500mL H2O] was mixed wi h 1 mL o solu ion B [10g C8H5O4K in 500 mL H2O] and subsequen ly mixed wi h 2 mL o he he mosonica ed bu e solu ion aken a di e en ime in e als. Abso bance a 350 nm was egis e ed wi h ime and i was ound ha 45 min was ime enough no o obse e any u he change in he eac ion medium colo . Ci a e bu e solu ion, no subjec ed o he mosonica ion, was used as a blank. The concen a ion o H2O2 was calcula ed based on a s anda d cu e a di e en H2O2 concen a ions. Ni a e and ni i e analysis we e pe o med by using D . Lange Kü e en-Tes analysis ki s and a Hach Lange DR2800 spec opho ome e . LCK 339 ki was used o measu ing ni a es. This ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT measu emen is based on he eac ion o ni a e ion wi h 2,6-dime hylphenol ha causes he o ma ion o 2,6-dime hyl-4-ni ophenol. Fo measu ing ni i es, LCK 341 ki , which is based on he eac ion o ni i e wi h a oma ic p ima y amines, p oducing he o ma ion o an azo dye, was used. 2.3.3. Pa icle size dis ibu ion Pa icle size dis ibu ion o cloudy apple juice was de e mined by lase di ac ion a 750 nm wi h a Mas e size 2000 (Mal e n® Ins ., MA). Pa icle size dis ibu ion was calcula ed by he F aunho e model. Size dis ibu ions ( olume ac ions agains pa icle size) be o e and a e he mosonica ion ea men we e calcula ed and he weigh -a e age sizes exp essed as:  The equi alen su ace a ea mean diame e : D(3,2)=∑ncdlc 3∑nc ⁄dlc 2 [3]  The equi alen olume mean diame e : D(4,3)=∑ncdlc 4∑nc ⁄dlc 3 [4] whe e dlc is he diame e o he pa icle and nc is he pe cen age o pa icles. O he use ul pa ame e s D ,0.9, D ,0.1 and D 0.5 co espond o he pa icle size bellow which, 90%, 10% and 50% o he pa icles lie espec i ely. 2.3.4. Colo , hyd oxyme hyl u u al con en and o al polyphenol con en The cloudy apple juice colo was de e mined by using a Beckman DU-650 spec opho ome e wi h diode-a ay o UV– is (Beckman Ins umen s). Following he CIE ecommenda ions, illuminan D65 (dayligh sou ce) and a 10º s anda d obse e (pe cep ion o a human obse e ) we e used. L*, a* and b* alues we e ob ained ep esen ing b igh ness, ed o g een colo and yellow o blue colo . Colo di e ence (E) and ch oma (C) we e e alua ed as: ∆E=√(Lbe o e ∗−La e ∗)2+(abe o e ∗−aa e ∗)2+(bbe o e ∗−ba e ∗)2 [5] 𝐶 =√(𝑎∗)2+(𝑏∗)2 [6] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT -166·PD +278, R2 = 0.987; a 67ºC: RA = -332·PD + 396 R2 = 1 (only wo poin s); o PME a 67ºC RA = -43·PD + 110, R2= 0.920). The alues o hese slopes indica e a g ea e e ec o he powe densi y in enzyme inac i a ion when wo king a highe empe a u es. This sugges s ha he syne gic e ec be ween empe a u e and ul asound is mo e e ec i e when wo king a high empe a u es. Figu e 4a also shows ha when wo king in a pulse mode, he e ec o powe densi y on PPO enzyme inac i a ion is lowe han when wo king in a con inuous mode (RA= -30·AED + 100, R2= 0.867). Al hough pulse ul asound can be conside ed as an ene gy sa ing ope a ional mode (Al-Juboo i, Yusa , & Bow ell, 2015), in his s udy, i has been ound o be less e ec i e han con inuous mode o PPO inac i a ion. O he alues ha e been ound in he li e a u e o PPO and PME inac i a ion in apple juice. Values o PPO inac i a ion in apple juice in Golden delicious by TS ea men , wi h a 22 mm diame e p obe (24 kHz) (200 mL o apple juice in a 250 mL essel) in a pulse mode (50% and 100% o cycle), we e epo ed by Basla and E ugay (Başla & E ugay, 2013). These au ho s obse ed ha he inac i a ion deg ee inc eased sha ply wi h ampli ude and empe a u e, obse ing an inc ease in he inac i a ion deg ee om 5% o 30% when ampli ude inc eased om 50 o 100 m a 50ºC, o 10 min o TS and pulse 1:1. A 60 ºC, hese au ho s epo ed much highe inac i a ion deg ees, eaching alues o 30% and 80% a 50 and 100 m o ampli ude espec i ely o 10 min o TS and pulse 1:1. Sulaimna e al. (Sulaiman e al., 2015) epo ed alues o PPO esidual ac i i y, a ound 17%, o PPO o Royal Gala apple pu ee when ea ed by TS o 15 min a 58ºC and 210 m o ampli ude in a con inuous mode, wi h a 3 mm diame e p obe, which co esponds o 1.3 W/g (25g in apple pu ee in a 34.4 mL essel). These au ho s ound ha , unde hese condi ions, nea ly comple e inac i a ion o PPO was eached a ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT empe a u es o 72-73 ºC. Abid e al. (Abid e al., 2014) s udied he TS o Fuji (Malus Domes ica) a ie y apple juice (80 mL o apple juice in a 100 mL essel), wi h a 0.5 inch p obe a 20 kHz, and a alue o he powe densi y o 0.3 W/mL in a pulse mode (5s on and 5 s o ). No alue o he ampli ude was epo ed. They epo ed esidual ac i i ies o 97, 53 and 6 % o PPO a 20, 40 and 60ºC, espec i ely a e 10 minu es o TS. Fo PME, hese au ho s epo ed simila alues o esidual ac i i ies 97, 52 and 7 % a 20, 40 and 60ºC, espec i ely a e 10 minu es o TS. In his wo k, lowe inac i a ion deg ee, i.e. highe esidual ac i i y, was ob ained o PME o Golden deliciuous, eaching he lowes esidual ac i i y o 50% a 67ºC a e 15 min o TS a 79 m o ampli ude and 1.44 W/mL. In his ega d, i mus be highligh ed ha PME om cloudy Golden deliciuous apple juice was ound o be one o he apple cul i a s p esen ing s onges he mos abili y (30% o esidual ac i i y a e hea ing 5 min a 100 ºC) (Teleszko, Nowicka, & Wojdyło, 2016), al hough mechanism o TS and he mal ea men is p obably di e en . Rega ding PPO ac i i y, highe esidual ac i i ies ha e been also ob ained compa ed o he esul s ound in he li e a u e. This could be a ibu ed o he lowe wo king ampli ude, 79 m (maximum ampli ude epo ed by he supplie o his ype o p obes). The a ie y o da a on PPO inac i a ion om apple juice indica es ha enzyme inac i a ion depends on many ac o s such as sou ce, sub- ype, en i onmen al and physicochemical condi ions (pH, empe a u e) (X. F. Cheng, Zhang, & Adhika i, 2013). Islam e al. (Islam, Zhang, & Adhika i, 2014) e iewed he inac i a ion mechanisms and ac o s a ec ing he enzyme inac i a ion by ul asound; howe e , u he in es iga ion is needed o unde s and he speci ic mechanism by which he di e en enzymes in apple juice a e inac i a ed. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT TS ea men was compa ed wi h he mal ea men by hea ing he cloudy apple juice du ing 15 min in he same empe a u e ange as o he TS p ocess, 52  1, 60  1 and 67  1ºC. Hea ing he juice a he lowes empe a u es essayed in TS expe imen s 52ºC and 60ºC did no cause any loss o enzyme ac i i y bu e en a signi ican inc ease (p  0.05), compa ed o he un ea ed juice (RA o PPO: 114 ± 12, 106 ± 16 , RA o PME: 135 ± 10, 157 ± 8 a 52ºC and 60ºC, espec i ely). A he highes empe a u e essayed, 67ºC a signi ican dec ease (p  0.05), in enzyme ac i i y by 24  4 % o PPO (RA = 76  4 %) and 9  2 % o PME (RA = 91  2 %) was obse ed. These esul s p o e ha TS signi ican ly imp o ed he inac i a ion o PPO and PME. 3.4 Inac i a ion kine ics o PPO a di e en ope a ing empe a u es. The TS inac i a ion kine ics o PPO was de e mined in he empe a u e ange om o 52 o 67ºC inal TS empe a u e. PPO o cloudy apple juice was inac i a ed as e by inc easing ope a ing empe a u e (Figu e 5). Fi s o de kine ic model has been success ully used in he li e a u e o co ela e he mosonica ion inac i a ion kine ic da a (Te e e, Buckow, & Ve s eeg, 2014). In his wo k, kine ic da a we e i ed o he i s o de kine ic model and he Weibull model. The co esponding kine ic pa ame e s a e lis ed in Table 2, oge he wi h he quali y o he i ing. Fo he i s o de kine ic model, he inac i a ion a e cons an , k, inc eased wi h empe a u e; he e o e, D- alues o PPO inac i a ion dec eased wi h empe a u e om 130 min a 52ºC o 18 min a 67ºC. Basla and E ugay (Başla & E ugay, 2013) epo ed lowe D alues o PPO o Golden delicious apple juice, 146, 66.4 and 12.9 min a 40, 50 and 60ºC espec i ely when wo king a 100 m o ampli ude by ul asound. D alues o PPO o Malus domes ica c Roya Gala apple juice anged om 49 o 4 min a 33 and 72ºC, espec i ely, a 1.3 W/g, 210 m o ampli ude (Sulaiman e al., 2015). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT The empe a u e sensi i e pa ame e s, zT and Ea, we e e alua ed h ough he slopes o plo ing equa ions 8 and 9, espec i ely, yielding 17.5 ± 0.8 ºC and 123 ± 4 kJ/mol, espec i ely. Bo h sensi i i y pa ame e s alues show ha he inac i a ion o PPO by TS is a he empe a u e sensi i e. P ocess wi h high ac i a ion ene gy alues a e e y empe a u e sensi i e (Le enspiel, 1999) as well as p ocesses wi h low zT alues. In he li e a u e, alues o he same o de we e ob ained o PPO inac i a ion o mush oom c ude ex ac in he empe a u e ange om 55 o 75ºC, wi h zT = 13.8 ºC and Ea=183 ± 32 kJ/mol (X. F. Cheng e al., 2013). Basla and E ugay (2013) epo ed a simila alue o zT o 19 ºC o PPO inac i a ion by he mosonica ion o Golden delicious apple juice, same a ie y as in his wo k. These au ho s did no epo a alue o Ea; howe e , as p e iously men ioned, hese au ho s epo ed D alues in he empe a u e ange om 40 o 60ºC and he ac i a ion ene gy could be easily e alua ed h ough A henius equa ion by using he in e se o he D- alues (D=1/k), ob aining a alue o 105 kJ/mol, simila o his wo k. On he con a y, o he alues epo ed o PPO o Malus domes ica c . Royal Gala apple pu ee inac i a ion by TS showed lowe empe a u e sensi i i y a 1.3 W/g, wi h alues o ac i a ion ene gy and zT o , 52 kJ/mol and 39ºC espec i ely, in he empe a u e ange om 33 o 72ºC (Sulaiman e al., 2015). The Weibull pa ame e s a e also lis ed in Table 2. Scale, α, and shape, , pa ame e s we e used o calcula e he ime equi ed o inac i a e 1 log ( d=1) o PPO. Simila o he D- alues o he i s o de kine ic model, d=1 dec eased wi h empe a u e. The d=1 calcula ed by he Weibull model we e lowe han he co esponding D alues ob ained by he i s o de kine ic model. The e o e, in his case, he use o he i s o de kine ic model could lead o an o e p ocessing es ima ion o PPO inac i a ion. Acco ding o an Boekel (2002), he dependence on empe a u e o bo h pa ame e s was analyzed. I was ound ha he scale pa ame e , α, was s a is ically signi ican dependen on empe a u e, when es ed a he 95% signi icance le el o a ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT linea ela ionship. The shape ac o , , was highe han 1 indica ing a downwa d conca i y o he enzyme inac i a ion cu e (Van Boekel, 2002), bu i was no s a is ically signi ican dependen on empe a u e, when es ed a he 95% signi icance le el o a linea ela ionship. Acco ding o an Boekel (2002), he scale pa ame e , α, could be modelled in a simila way o he classical D alue o he i s o de kine ic model, sugges ing a linea dependence o he log α on empe a u e and conside ing ha  did no depend on empe a u e: 𝑙𝑜𝑔𝛼=𝑎1−𝑏1𝑇 [13] A zT’ alue can be also de ined: 𝑧𝑇 ′=1 𝑏1 ⁄ [14] The alue o he zT’ is gi en in Table 2, oge he wi h he quali y o he i ing. Al hough he concep o zT and z’T is di e en since zT is ob ained om he linea pa , whe eas z’T akes also in o accoun he nonlinea pa (Van Boekel, 2002), simila alues we e ob ained o bo h pa ame e s (zT = 17.5 ± 0.8 ºC and z’T = 20 ± 1 ºC). An A henius ype equa ion was also conside ed o ela e he in e se o he scale pa ame e , 1/α, wi h empe a u e. Al hough 1/α canno be conside ed a kine ic cons an , he alue ob ained om he slope was ound o be close o he Ea ob ained by applying he i s o de kine ic model, being 107 ± 6 kJ/mol 3.4. E ec o he na u e o dissol ed gases The e ec o he na u e o dissol ed gases on PPO inac i a ion kine ics is shown in Figu e 6a a h ee di e en he mosonica ion empe a u es, 44, 52 and 60 ºC ( empe a u e o he pla eau, see Figu e 1) when ea ing 80 mL o apple juice a 100% o ampli ude. Rega ding he e ec o ni ogen, as e inac i a ion kine ics we e ob ained compa ed o he inac i a ion kine ics in he p esence o dissol ed ai in he apple juice a he same he mosonica ion empe a u e. The e ec o dissol ed ca bon dioxide was s udied only a 60ºC. In his case, ini ial inac i a ion eac ion ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT a es (ini ial slope o he cu e) in he p esence o CO2 was o he same o de as in he p esence o ai (Figu e 5), howe e , as e eac ion a es we e ob ained a longe TS imes and he e o e, lowe esidual ac i i y a e 20 min o TS was eached in p esence o CO2, 18 ± 2 %, han in he p esence o ai , 36 ± 5 % a he same ope a ing condi ions. Ne e heless, he bes esul s in e ms o inac i a ion a e and inal RA we e ob ained when he ai was displaced by ni ogen, eaching a RA o 5 ± 2 % a 60ºC o 20 min o TS. Figu e 6b shows he PME inac i a ion kine ics a 60ºC ( empe a u e o he pla eau) when dissol ed ai was displaced by ni ogen. A e 20 min o TS, a minimal RA o 55 % was eached while no inac i a ion o PME was obse ed a he same empe a u e when TS was pe o med in p esence o ai (Figu e 4b). Howe e , a hese condi ions, esul s ob ained wi h CO2 did no b ing any imp o emen in he inac i a ion p ocess o PME compa ed wi h he esul s ob ained in he p esence o ai and inac i a ion was nei he obse ed. PPO and PME inac i a ion kine ic da a ob ained displacing he ai by N2 o CO2 we e also i ed o he i s o de kine ic model and he Weibull model (Table 3). In gene al, a be e i ing was ob ained by he Weibull model. Fo he i s o de kine ic model, D- alues we e e alua ed. I can be clea ly obse ed ha by displacing he dissol ed ai by p e iously bubbling ni ogen in o he apple juice, D- alues we e lowe han hose epo ed in Table 2. A 60 ºC, in he p esence o ai , he D- alue o PPO was 50 min, while i dec eased down o 11 min in he p esence o ni ogen and o 36 min in he p esence o ca bon dioxide. The empe a u e sensi i e pa ame e s, zT and Ea, in he p esence o ni ogen we e e alua ed h ough he slopes o plo ing equa ions 8 and 9, espec i ely, yielding 20 ± 4 ºC and 106 ± 18 kJ/mol espec i ely. Rega ding he Weibull model, he ime equi ed o achie e one decimal educ ion, d=1, was calcula ed by using he shape and scale pa ame e s (Table 3). Displacing he ai by ni ogen led ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT o lowe alues o d=1, compa ed o he alues epo ed in he p esence o ai . I was ound ha he scale pa ame e , α, was s a is ically signi ican dependen on empe a u e, when es ed a he 95% signi icance le el o a linea ela ionship; bu he shape ac o ,  was no . Acco ding o equa ions 13 and 14, zT’ o he Weibull was e alua ed as 18 ± 4 ºC, being simila o zT o he i s o de kine ic model. An A henius ype equa ion was also conside ed o ela e he in e se o he scale pa ame e , 1/α, wi h empe a u e. F om he slope, a alue close o he ac i a ion ene gy ob ained by applying he i s o de kine ic model was ob ained, 117 ± 24 kJ/mol. These esul s indica ed ha he na u e o he dissol ed gas in he apple juice played an impo an ole in enzyme inac i a ion. The e ec o he na u e o dissol ed gases on enzyme inac i a ion has no been equen ly s udied in he li e a u e. Cheng e al. (L. H. Cheng e al., 2007) s udied he sonica ion p ocess on gua a juice quali y wi h p e ious ca bona ion o he sample. These au ho s p oposed ha dissol ed ca bon dioxide could educe su ace ension and could c ea e mo e nuclei ca i a ion. Howe e , hey ound ha a e sonica ion o a combina ion o ca bona ion + sonica ion, PPO showed highe ac i i y han he un ea ed sample. Gielen e al. (Gielen e al., 2016) discussed he di e en e ec o he dissol ed gases on sonochemis y conside ing he solubili y and he mal p ope ies o he gases as well as he di e en adical p oduc ion. In his wo k, he H2O2 a e p oduc ion was de e mined in a ci a e bu e solu ions a he same pH as apple juice (pH = 3.9) a 60 ºC, 100 % o ampli ude, 80 mL o ea ed olume du ing 20 min. H2O2 a e o ma ion was de e mined in h ee ypes o ci a e solu ions wi h dissol ed ai and by displacing he ai by bubbling ni ogen o ca bon dioxide p e ious o TS expe imen s. No H2O2 p oduc ion was obse ed du ing he i s i e minu es o TS in he p esence o ni ogen o ai , acco ding o ou analy ical me hod. A e ha , i was ound ha he H2O2 p oduc ion as a unc ion o ime ollowed a linea ela ionship, wi h a H2O2 a e p oduc ion o (3.6 ± 0.2)·10-8 mol/(Lmin) (R2 = 0.994) in ai , and (3.78 ± 0.05)·10-8 mol/(Lmin) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT (R2 = 0.999) o ni ogen. In he p esence o CO2, no H2O2 o ma ion was obse ed a e 15 min o TS; subsequen ly, H2O2 o ma ion a e was simila o ha ob ained wi h ni ogen and ai (3.81·10-8 mol/(Lmin), om 15 min o 20 min). In any case, hese esul s indica ed ha CO2 pa ially inhibi ed he o ma ion o H2O2 du ing TS. These esul s ag eed pa ially wi h he adical yield o ma ion ob ained by Gielen e al.(2016) in he p esence o di e en gases in wa e a 24  2 ºC. These au ho s also ound, ha CO2, al hough being a highly soluble gas (39.2 mmol/L e sus 0.85 and 0.71 mmol/L o ai and ni ogen espec i ely, a 293 K and 1 a m), inhibi ed adical o ma ion due o low bubble empe a u e a ibu ed o i s lowe poly opic index and a highe he mal conduc i i y compa ed o ai and ni ogen (Gielen e al., 2016). The simila H2O2 a e p oduc ion o ai and ni ogen ob ained in his wo k, could be hen a ibu ed o hei simila he mal p ope ies. Howe e , Gielen e al. (Gielen e al., 2016) ound ha ni ogen educed adical yield by he p oduc ion o adical sca enge s and a lowe bubble olume. In any case, no ela ion can be es ablished be ween H2O2 p oduc ion a e in p esence o he di e en dissol ed gases and enzyme inac i a ion, since highe inac i a ion a es we e de e mined when ni ogen displaced he dissol ed ai o he apple juice. Ni a e and ni i e a e p oduc ion was also de e mined du ing TS o he h ee bu e solu ions a he same ope a ing condi ions. The highes ni a e a e p oduc ion was ob ained when ni ogen was p e iously dissol ed in he bu e , (2.6  0.2·10-7) mol/(Lmin) (R2 = 0.982) and he same alue was ob ained o CO2 and ai , 1.8  0.2 10-7 mol/(Lmin), R2 = 0.910 and 0.960 espec i ely. Ni i e a e o ma ion was lowe han ni a e o all he gases being 3.2  0.2·10-8, 3.3  0.4·10-8 and 1.5  0.2·10-8 mol/(Lmin), o ni ogen, ai and CO2 (R2 = 0.993, 0.960, 0.967) espec i ely. These esul s could pa ially explain he highe inac i a ion a e o PPO and PME ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ound by displacing he ai wi h ni ogen. Howe e , u he s udies a e needed o unde s and he e ec o he na u e o he dissol ed gas on enzyme inac i a ion by TS. 3.5. Compa ison wi h HPCD Among he di e en non- he mal echnologies, he use o high p essu e ca bon dioxide (HPCD) ea men is ha ing also g owing a en ion o inac i a e ce ain mic oo ganisms and enzymes. Typically, ope a ing p essu e does no exceed 50 MPa and empe a u e anges be ween 20 and 50 ºC (B iongos e al., 2016), being lowe han he empe a u e employed in his wo k in TS. Figu e 7 compa es he PPO inac i a ion kine ics by TS a 44 ºC, empe a u e o he pla eau, (V=80 mL and 100 % o ampli ude) in he p esence o ai and by displacing he ai wi h ni ogen, wi h esul s ob ained in a p e ious s udy on inac i a ion kine ic o PPO om apple juice a 45ºC by HPCD a 20 MPa (Ille a e al., 2018). A 44 ºC, in he p esence o ai , sho ea men imes o TS led o an inc ease o PPO ac i i y. As indica ed in sec ion 3.2, his inc ease in enzyme ac i i y has been a ibu ed o he elease o enzymes bond o cell walls due o he acous ic shock wa es (Başla & E ugay, 2013) o o he ac i a ion o PPO la en o ms a low in ensi ies and sho ea men imes. In any case, a longe ea men imes, PPO was inac i a ed. Figu e 7 shows ha , a he same ope a ing empe a u e, HPCD ea men a 20 MPa led o lowe PPO esidual ac i i y alues. This indica es a di e en enzyme inac i a ion mechanism o hese wo non- he mal echnologies. 3.6. E ec o he mosonica ion on di e en quali y pa ame e s o cloudy apple juice Some quali y pa ame e s ha e been de e mined igh a e TS ea men a 60 ºC ( inal TS empe a u e), 100 % o ampli ude, 15 min o ea men ime and 80 mL ea ed olume o apple juice. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Colo pa ame e s. Table 4 lis s he L*, a*, b* pa ame e s o cloudy apple juice be o e and a e di e en TS ea men s. As gene al end, he e we e signi ican di e ences in all colo a ibu es a e TS. The ligh ness (L*) sligh ly inc eased, while he ed and yellow componen s dec eased compa ed o he un ea ed juice. The inc ease in ligh ness a e TS has been p e iously epo ed in he li e a u e and i has been a ibu ed o a homogeniza ion e ec o sonica ion (Tiwa i, Mu hukuma appan, O’Donnell, & Cullen, 2008). A dec ease in a* and b* pa ame e s has been also obse ed in di e en juices, such as o ange juice (Tiwa i e al., 2008) o sou sop juice (Dias e al., 2015). Colo di e ence, ΔE, (Equa ion 4) is also lis ed in Table 4. In all TS expe imen s, a colo di e ence was obse ed compa ed o he un ea ed juice. Acco ding o Yuk e al. (Yuk, Samped o, Fan, & Ge eke, 2014), he colo di e ence could be es ima ed o be no no iceable (0–0.5), sligh ly no iceable (0.5–1.5), no iceable (1.5–3), well isible (3–6) and g ea (6–12). Based on his classi ica ion, a 60ºC, he change in colo was no iceable (1.5-3) while a 67ºC was well isible (3-6). Acco ding o colo pa ame e s, he ch oma alue p esen ed he lowes alue a 67ºC and he lowes ea ed olume, 60 mL (highe powe densi y). The e o e, al hough highe TS empe a u es, led o highe inac i a ion a es, change in colo was mo e isible. Colo changes du ing TS ha e been a ibu ed o Mailla d eac ions ha may occu a long ea men ime and high empe a u e, as well as o ca i a ion, ha in ol es a ious physical, chemical and biological eac ions (Anaya-Espa za e al., 2017). To al polyphenolic compounds. To al polyphenolic compounds (TPCs) sligh ly inc eased a e TS compa ed o he un ea ed apple juice. Signi ican di e ences o he o al polyphenolic compounds we e ound a 60 and 67ºC and in he apple juice ea ed by p e iously displacing he ai wi h ni ogen (Table 4). This TPCs inc ease a e TS has been a ibu ed o he enhanced dis up ion o cell walls ha migh lead o he elease o some chemically bound phenolic ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e cap ions: Figu e 1. Tempe a u e p o ile o cloudy apple juice du ing he mosonica ion o di e en olumes o cloudy apple juice in a con inuous mode ( 60 mL-1.57 W/mL, □ 80 mL- 1.36 W/mL, ◇ 100 mL-1.21 W/mL, ○ 120 mL-1.09 W/mL) and pulse mode (x 80 mL- 1.18 W/mL). 100 % o ampli ude (76 m), 40ºC o he jacke ed wa e . Figu e 2. E ec o he ampli ude on PPO esidual ac i i y (□), inal empe a u e o he juice () and powe densi y, W/mL (○) o a ea ed olume o 80 mL and 40ºC o he jacke ed wa e . Lines ep esen he linea eg ession. Figu e 3. Pa icle size dis ibu ion o esh cloudy apple juice (a ) and ea ed by TS a di e en ampli udes 19 m (25% o maximum ampli ude, b − − − −), 38 m (50% o maximum ampli ude, c ∙−∙−∙−∙), 57 m (75% o maximum ampli ude, d ▬ ▬ ▬ ), 76 m (100% o maximum ampli ude, e ∙∙∙∙∙∙∙). T ea ed olume, 80 mL and 40ºC o he jacke ed wa e . Figu e 4. Enzyme esidual ac i i y a e 15 min o he mosonica ion a 100 % o ampli ude (79 m) and di e en empe a u es ( 52ºC, □ 60ºC, Δ 67ºC, T o he pla eau) in a con inuous mode and in pulse mode a 54ºC (T o he pla eau) () as a unc ion o AED, W/mL (a) PPO (b) PME. Lines ep esen he linea ela ionship. Figu e 5. PPO inac i a ion kine ics du ing TS ea men a 100 % o ampli ude (79 m) a di e en ope a ing empe a u es ( 52ºC, □ 60ºC, ◇ 64ºC, ○ 67ºC, T o he pla eau). T ea ed olume = 80 mL. Con inuous lines ep esen he Weibull model. Figu e 6. PPO (6a) and PME (6b) inac i a ion kine ics du ing TS ea men a 100 % o ampli ude (79 m) a di e en ope a ing empe a u es (◇ 44ºC;  52ºC, □, ■ 60ºC, T o he pla eau) in p esence o ni ogen (hollow symbols) and ca bon dioxide ( ull symbols). T ea ed olume = 80 mL. Lines ep esen he Weibull model (con inuous: N2, dashed line: CO2). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 7. Compa ison o PPO inac i a ion kine ics a 44 - 45 ºC o di e en non- he mal ea men s: Δ HPCD a 20 MPa, ◇ TS in he p esence o N2 (V=80 mL, 100 % o ampli ude), □ TS in he p esence o ai (V=80 mL, 100 % o maximum ampli ude). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 1. Tempe a u e p o ile o cloudy apple juice du ing he mosonica ion o di e en olumes o cloudy apple juice in a con inuous mode ( 60 mL-1.57 W/mL, □ 80 mL- 1.36 W/mL, ◇ 100 mL-1.21 W/mL, ○ 120 mL-1.09 W/mL) and pulse mode (x 80 mL-1.18 W/mL). 100 % US ampli ude (76 m), 40ºC o he jacke ed wa e . 35 40 45 50 55 60 65 0 2 4 6 8 10 12 14 16 Tempe a u e (ºC) Time, min ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 2. E ec o he ampli ude on PPO esidual ac i i y (□), inal empe a u e o he juice () and powe densi y, W/mL (○), o a ea ed olume o 80 mL and 40ºC o he jacke ed wa e . Lines ep esen he linea eg ession. -0.2 0.2 0.6 1.0 1.4 1.8 0 20 40 60 80 100 120 140 0 20 40 60 80 Powe densi y, W/mL PPO Residual ac i i y (%), T (ºC) Ampli ude, m ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 3. Pa icle size dis ibu ion o esh cloudy apple juice (a ) and ea ed by TS a di e en ampli udes 19 m (25% o maximum ampli ude, b − − − −), 38 m (50% o maximum ampli ude, c ∙−∙−∙−∙), 57 m (75% o maximum ampli ude, d ▬ ▬ ▬ ), 76 m (100% o maximum ampli ude, e ∙∙∙∙∙∙∙). T ea ed olume, 80 mL and 40ºC o he jacke ed wa e . 0 1 2 3 4 5 6 7 8 9 10 0.01 0.1 1 10 100 1000 10000 Volume (%) Pa icle size, m a b c d e ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 4. Enzyme esidual ac i i y a e 15 min o he mosonica ion a 100 % o ampli ude (79 m) and di e en empe a u es ( 52ºC, □ 60ºC, Δ 67ºC, T o he pla eau) in a con inuous mode and in pulse mode a 54ºC (T o he pla eau) () as a unc ion o powe densi y, W/mL (a) PPO (b) PME. Lines ep esen he linea ela ionship (con inuous mode , pulse mode - - - ). 0 20 40 60 80 100 0.8 1.0 1.2 1.4 1.6 1.8 PPO Residual ac i i y (%) Powe densi y, W/mL 0 20 40 60 80 100 120 0.8 1.0 1.2 1.4 1.6 1.8 PME Residual ac i i y (%) Powe densi y, W/mL (b) (a) ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 5. PPO inac i a ion kine ics du ing TS ea men a 100 % o ampli ude (79 m) a di e en ope a ing empe a u es ( 52ºC, □ 60ºC, ◇ 64ºC, ○ 67ºC, T o he pla eau). T ea ed olume = 80 mL. Con inuous lines ep esen he Weibull model. 0 20 40 60 80 100 0 5 10 15 20 25 PPO Residual ac i i y (%) Time, min ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 6. PPO (6a) and PME (6b) inac i a ion kine ics du ing TS ea men a 100 % o ampli ude (79 m) a di e en ope a ing empe a u es (◇ 44ºC;  52ºC, □, ■ 60ºC (T o he pla eau) in p esence o ni ogen (◇, , □) and ca bon dioxide (■). T ea ed olume = 80 mL. Lines ep esen he Weibull model (con inuous: N2, dashed line: CO2). 0 20 40 60 80 100 0 5 10 15 20 25 PPO Residual ac i i y (%) Time, min 0 20 40 60 80 100 0 5 10 15 20 25 PME Residual ac i i y (%) Time, min ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Figu e 7. Compa ison o PPO inac i a ion kine ics a 44 - 45 ºC o di e en non- he mal ea men s: Δ HPCD a 20 MPa, ◇ TS in he p esence o N2 (V=80 mL, 100 % o ampli ude), □ TS in he p esence o ai (V=80 mL, 100 % o ampli ude) (Lines a e o guide he eye). 0 20 40 60 80 100 120 010 20 30 40 PPO esidual aci i y (%) Time, min ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Table 1. E ec o ampli ude in he he mosonica ion ea men on he pa icle size dis ibu ion (m) o cloudy apple juice Time D[3,2] D[4,3] d(0.1) d(0.5) d(0.9) Cloudy apple juice 2.24 ± 0.04d 70 ± 2d 0.690 ± 0.003d 20.9 ± 0.8c 249 ± 4c 19 m (25 %) 0.82 ± 0.02c 44 ± 2c 0.36 ± 0.01c 0.88 ± 0.01b 158 ± 4b 38 m (50 %) 0.168 ± 0.003b 4.66 ± 0.06b 0.086 ± 0.003b 0.202 ± 0.003a 0.70 ± 0.06a 57 m (75 %) 0.140 ± 0.001a 0.238 ± 0.001a 0.077 ± 0.000a 0.164 ± 0.001a 0.513 ± 0.001a 76 m (100 %) 0.138 ± 0.001a 0.217 ± 0.001a 0.078 ± 0.001a 0.160 ± 0.001a 0.434 ± 0.001a Da a: mean ± SD (n=3). Di e en le e s in a column indica e signi ican di e ences by he Tukey’s hones ly signi ican di e ence (HSD) me hod a p- alue ≤ 0.05. ACCEPTED MANUSCRIPT