Cross-linked methyl cellulose films with murta fruit extract for antioxidant and antimicrobial active food packaging

1 C oss-linked me hyl cellulose ilms wi h mu a ui ex ac o an ioxidan and 1 an imic obial ac i e ood packaging 2 3 Ca ol López de Dicas illo*, Fe nanda Bus os, Abel Gua da, Ma ia José Galo o 4 Food Packaging Labo a o y (Laben-Chile), Depa men o Science and Food Technology, Facul y o 5 Technology, Cen e o he De elopmen o Nanoscience and Nano echnology (CEDENNA), Uni e sidad de 6 San iago de Chile (USACH), San iago, Chile 7 *Co esponding au ho : a[email p o ec ed]l 8 9 ABSTRACT 10 The use o biopolyme s as subs i u es o non-deg adable adi ional plas ics is an in e es ing 11 al e na i e pa icula ly o sho - e m applica ions, such as ood packaging. Addi ionally, 12 ac i e packaging has a ac ed much a en ion as an inno a i e echnology o ood 13 conse a ion. Thus, he unc ionali y o biocomposi e ilms based on me hylcellulose (MC) 14 and mu a ui (MU) (Ugni molinae Tu cz) ex ac was s udied. Mu a ui is a na i e 15 Chilean be y and good sou ce o an ioxidan and an imic obial compounds. Fi s , a MU 16 ex ac wi h he highes an ioxidan abili y and polyphenolic con en was selec ed, and ac i e 17 MC ilms we e p epa ed by cas ing using glu a aldehyde (GA) o imp o e hei wa e 18 esis ance. The e ec s o GA concen a ion and inco po a ion o MU ex ac on ma e ial 19 p ope ies and an imic obial and an ioxidan ac i i ies we e examined. The addi ion o GA 20 g ea ly dec eased swelling index, imp o ed mechanical p ope ies and an ioxidan and 21 an imic obial ac i i ies achie ed highes po en ial when GA was added a lowes 22 concen a ion. 23 24 2 Keywo ds: Na u al ex ac s, ac i e packaging, elease, me hyl cellulose, c osslinking. 25 26 Chemical compounds s udied in his a icle: 2,2´-azinobis(3-e hylbenzo hiazoline-6- 27 sulphona e) (ABTS) (PubChem CID 90658258); polye hylene glycol (PubChem CID 28 24762); glu a aldehyde (PubChem CID 3485); gallic acid (PubChem CID 370); T olox 29 (PubChem CID 40634); Folin-Ciocal eu´s phenol eagen (PubChem CID 516996); 30 anhyd ous sodium ca bona e (PubChem CID 10340). 31 32 3 1. In oduc ion 33 34 Du ing he las yea s, he esea ch on he de elopmen o ac i e ma e ials wi h 35 an imic obial and an ioxidan p ope ies has inc eased eno mously wi h he pu pose o 36 p o ec ood om oxida i e eac ions and mic obial g ow h. Fu he mo e, he applica ion o 37 na u al ing edien s is a new end in he ood indus y and ood esea ch, i.e., na u al 38 p ese a i es de i ed om na u al sou ces, ins ead o syn he ic subs ances and he educ ion 39 o addi i es in oods. Na u al subs ances such as α- ocophe ol, essen ial oils and plan 40 ex ac s ha e been inco po a ed in o polyme s o he p epa a ion o ac i e packages 41 (Galo o, Gua da, & Lopez de Dicas illo, 2015; Gómez Es aca, Lopez de Dicas illo, 42 He nandez Muñoz, Ca ala, & Ga a a, 2014). On he o he hand, he p oduc ion and he 43 use o plas ics h oughou he wo ld ha e g own eno mously, wo sening en i onmen al 44 impac and p oblems o he was e disposal. The use o biopolyme s as subs i u es o non- 45 deg adable syn he ic polyme s is becoming a sus ainable al e na i e, pa icula ly in e es ing 46 o sho - e m applica ions, such as ood packaging. Thus, he de elopmen o biodeg adable 47 ac i e packaging con aining na u al ex ac s de i ed om plan s is one in e es ing s a egy 48 la gely conside ed by he ood packaging indus y (Coope , 2013, Seydim & Sa ikus, 2006; 49 Song, Lee, Al-Mijan, & Song, 2014). One al e na i e o de elop bio-based ma e ials is by 50 ilm coa ing mul ilaye . A ilm coa ing is a hin polyme -based coa be ween 20-10 µm 51 applied o a solid suppo . Biobased coa ings o e ex a ad an ages such as low cos s due o 52 he educ ion on amoun o polyme , educing e ms o CO2 emissions, and he p ocessing 53 empe a u es a e lowe , which is e y use ul o a oid he mal deg ada ion o ac i e agen s. 54 Fo indus ial p ocesses, he echniques can be ex usion o co-ex usion o mul ilaye ilms, 55 lamina ion, and mainly oll-d ying o he sol en emo al o he polyme solu ion 56 4 (Debeau o , Quezada-Gallo, & Voilley, 1998; Va ianinen, Vaha-Nissi, M., & Ha lin, 57 2014). 58 Among he biopolyme s, anionic linea polysaccha ides de i ed om cellulose, such as 59 me hyl cellulose (MC), a e e y p omising because hey a e en i onmen ally iendly due o 60 hei biodeg adabili y. In he ood indus y, MC ilms p omise impo an po en ial o be used 61 o packaging applica ions due o hei excellen ilm-making p ope ies, la ge a ailabili y, 62 low cos , anspa ency, high-s eng h and easy abili y o be p ocessed (Aminabha i, 63 Balundgi, & Cassidy, 2008; Coope , 2013; Rimdusi , Jingjid, Dam ongsakkul, 64 Tip ipako n, & Takeichi, 2008). Me hyl cellulose a e polysaccha ides composed o linea 65 chains o β(14) glucosidic uni s wi h me hyl subs i uen s ha p o ides ilms ha a e wa e 66 soluble and oil-and g ease esis an and wi h e icien oxygen and lipid ba ie p ope ies 67 (Pa k & Ruckens ein, 2001; Ga cía, Pino i, Ma ino, & Za i zky, 2004; Nispe os- 68 Ca iedo, 1994). 69 MC ilms, like mos ilms based on biopolyme s, a e highly sensi i e o en i onmen al 70 condi ions and due o hei hyd ophilic na u e, physical p ope ies a e e y suscep ible o 71 mois u e and biodeg adabili y. As a esul , se e al esea che s ha e de eloped new 72 o mula ions h ough small chemical modi ica ions o hese polyme s, such as c osslinking 73 p ocesses which p omo e co alen linkages be ween polyme chains. C osslinking is one o he 74 mos popula me hods used o modi y wa e -soluble polyme s in o de o achie e desi ed 75 p ope ies. MC can be c oss-linked by means o di e en me hods, such as adia ion o by using 76 con enien chemical c oss-linke s consis ing on aldehydes, polyepoxy compounds, and e en 77 ea ca echins (A ala, 2002; Wach, Mi omo, Nagasawa, & Yoshii, 2003; Yu, Tsai, Lin, Lin, 78 & Mi, 2015). 79 5 Glu a aldehyde, GA, is a widely used c oss-linke which o ms a s ong co alen 80 a achmen on o a polyme su ace, p o iding he way o a igid s uc u e (He nández- 81 Muñoz, Villalobos, & Chi al , 2004; Lee e al., 2005; Mansu , Sadahi a, Souza, & 82 Mansu , 2008; Pa k, Bae, & Rhee, 2000). Some polyme cha ac e is ics could be al e ed 83 by c osslinking such as pe meabili y, mechanical p ope ies and d ug eleasing. The e ec 84 o addi ion o c oss-linke s on compounds ha a e desi ed o be eleased has been al eady 85 s udied, bu mainly wi h pha maceu ical applica ions (Aiedeh, Taha, Al-Hia i, Bus anji, & 86 Alkha ib, 2006; Ranjha, & Qu eshi, 2014; López de Dicas illo, Jo dá, Ca alá, Ga a a, 87 & He nández, 2011a; Ma inez, Ca es, Ra i, Li, & Chaiko , 2014; Teng, Campello, & 88 Wu, 2011). 89 In he de elopmen o ac i e ma e ials i is impo an o each a con olled elease o ac i e 90 componen s o he ood o p o ec i om oxida ion o mic obial spoilage, enhancing ood 91 sa e y and shel li e. In his wo k, he e ec o c osslinking on he elease o an ioxidan 92 compounds om ac i e packaging was e alua ed. Release o hese ac i e agen s will be 93 comple ely dependen on he swelling deg ee o he polyme . Due o i s hyd ophillici y, 94 me hyl cellulose was chosen in his wo k o be he polyme ma ix in he de elopmen o 95 ac i e ma e ials o ood packaging sys ems (Cala ayud e al., 2013; Lopez de Dicas illo e 96 al., 2011b). On he o he hand, GA was selec ed as c oss-linke agen due o i s p o en 97 e ec i eness in o he wo ks and o a oid deg ada ion o phenolic compounds p opi ia ed by 98 i adia ion (Beaulieu, D´Ap ano, & Lac oix, 1999, Hi ashima e al., 2013, Pa k e al., 99 2001; Rimdusi e al., 2008). And inally, polye hylene glycol was inco po a ed as 100 plas icize . The use o plas icize is e y impo an on he ma e ials de elopmen wi h 101 biodeg adable polyme s because hey dec ease he in e molecula in e ac ions among he 102 unc ional g oups o he backbone chains, esul ing in inc eased lexibili y and ex ensibili y. 103 6 In ood indus y, polye hylene glycol (PEG) has been widely used o MC p oduc s o 104 olling, cas ing, o ex uding p ocesses because i p o ides se e al ad an ages due o 105 chemical in e ac ions be ween MC and PEG s uc u es (Pa k e al., 2001; Sa ka , & 106 Walke , 1995). 107 Ugni Molinae, also known as “mu a”, “mu illa”, “uñi” o “Chilean c anbe y”, is a na i e 108 plan om Chile, wes e n A gen ina, and ce ain egions o Boli ia. I is a wild sh ub o he 109 My aceae amily ha bea s a oma ic ed globula ui ha is said o combine he swee ness 110 o a s awbe y wi h he pungency o a gua a and he ex u e o a d ied bluebe y. P e ious 111 s udies ha e shown mu a ui is composed by high le els o la an-3-ols, such as ca echin 112 and epica echin, la onols, such as que ce in and que ce in-3-glucoside, and 113 hyd oxycinnamic acid, such as ca eic acid-3-glucoside. The o he compounds we e 114 an hocyanins, like cyanidin-3-glucoside and peonidin-3-glusocide, u in, gallic acid, 115 que ci in, lu eolin, kaemp e ol, p-couma ic and my ice in, ha could a o d in e es ing 116 an imic obial and an ioxidan ac i i ies o ood p o ec ion (Al a o e al., 2013; Junquei a- 117 Gonçal es e al., 2015; Ruiz e al., 2010; Sch eckinge , Lo on, Lila, & Gonzalez de 118 Mejia, 2010). 119 120 121 2. Ma e ials and me hods 122 123 2.1. Ma e ials and Reagen s 124 125 Me hyl cellulose (MC) was ob ained om Reu e S. L. (San iago, Chile), 2,2´-azinobis(3- 126 e hylbenzo hiazoline-6-sulphona e) (ABTS), Folin Ciocal eu phenol eagen , anhyd ous 127 7 sodium ca bona e, Gallic acid, 6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid 128 (T olox) and polye hylene glycol (PEG) (a e age molecula weigh 190-210) we e pu chased 129 om Sigma Ald ich Quimica S.A. Ugni Molinae ui , commonly known as mu a be y ui , 130 MU, was ob ained om “Na i o Li e”, an USDA and IMO o ganic g ade ce i ied company, 131 whose ui s we e al eady d ied and milled. 132 G am-posi i e bac e ia Lis e ia innocua ATCC33090 was ob ained om Labo a o y 133 Mic obiologics (Uni e si y o Chile, Chile) and s o ed in T yp one soy b o h (TSB) a -80 134 °C un il needed. Fo expe imen al use, he s ock cul u es we e main ained on T yp one soy 135 aga (TSA) slan s a 4 °C, and o expe imen a ion, a loop ul o each s ain was ans e ed 136 o 10 ml o TSB and incuba ed a 37 °C o 18 h o ob ain esh ea ly-s a iona y phase cells. 137 138 2.2. Na i e F ui S udies - An ioxidan Ac i i y and Polyphenolic Con en 139 140 Mu a ui powde was ex ac ed unde e hanol, e hanol 50% and dis illed wa e in o de 141 o s udy he pola i y o he mos an ioxidan compounds and ind he ex ac wi h highes 142 an ioxidan capaci y. The ex ac ion was ca ied ou a 40 °C du ing 3 h in an E lenmeye 143 lask in a shaking incuba o a 150 pm. Then he mix u es we e cen i uged and he uppe 144 phases we e e alua ed h ough ABTS me hod and Folin Ciocal eu es in o de o measu e 145 an ioxidan capaci y (as adical sca enging capaci y) and polyphenolic con en , espec i ely. 146 Folin-Ciocal eu me hod was done acco ding o he me hod o Single on e al., 1999, wi h 147 some modi ica ions (Single on, O ho e , & Lamuela-Ra en ós, 1999). Gallic acid was 148 used as he s anda d o he calib a ion cu e and polyphenol con en (PC) was exp essed as 149 gallic acid equi alen s (GA). 150 8 ABTS me hod was done acco ding o p e ious wo ks (Lopez de Dicas illo, Cas o- 151 Lopez, Lasagabas e , Lopez-Vila iño, & Gonzalez-Rod iguez, 2013). ABTS assay is 152 based on he inhibi ion o he adical ca ion 2,2´-azinobis(3-e hylbenzo hiazoline-6- 153 sulphona e), ABTS+•, by an ioxidan s, which has a cha ac e is ic wa eleng h abso p ion 154 spec um wi h maximum a 715 nm. This ca ionic adical is neu alized ei he by di ec 155 educ ion ia elec on ans e o by adical quenching ia H a om ans e (P io , Wu, & 156 Schaich, 2005). The pe cen age inhibi ion alues we e calcula ed using equa ion 1: 157 % Inhibi ion= % I = (Acon ol – Asample) /Acon ol * 100 (1) 158 Sca enging ac i i ies o he adicals we e exp essed as T olox equi alen s pe g am o 159 mu a ui sample. 160 161 2.3. Ob aining Na u al Ex ac 162 163 In o de o ob ain a ui ex ac wi h high concen a ion o be inco po a ed o ilm 164 de elopmen , mu a ui was ex ac ed wi h e hanol 50% (20 mg/mL) a 40 ºC du ing 3 h. 165 The mix u e was cen i uged, il e ed and concen a ed o a inal concen a ion o 100 mg 166 mu a/ mL. 167 168 2.4. Films P epa a ion 169 170 MC ilms we e p epa ed by solu ion-ex ension-e apo a ion (“cas ing”). This echnique 171 has he ad an age o educing he he mal deg ada ion o he na u al ex ac wi h espec o 172 con en ional ex usion and can be compa ed o ilm-coa ing echniques (Hong, Lee, & Son, 173 2005; Va ianinen e al., 2014). 1 g o MC was dissol ed in 100 mL e hanol 70% by hea ing 174 9 a 70 ºC (1% w/ ) wi h cons an s i ing un il o al dissolu ion. In o de o imp o e he wa e 175 esis ance o MC ilms, he polyme was c oss-linked wi h glu a aldehyde a di e en 176 concen a ions, 10, 15 and 20 % espec o polyme weigh . The pH o he mix u e was 177 adjus ed o 3 using a HCl solu ion (1 M), and la e he solu ion was cooled o 40 ºC o a ound 178 2 h unde agi a ion (Pa k e al., 2011; Plie a e al., 2006). Then, 233 µL o polye hylene 179 glycol (PEG) (25% w/w o MC) was added as plas icize . In he case o he ac i e ma e ials, 180 be o e he addi ion o PEG, addi ionally 2.5 mL o mu a be y ex ac (MU) (0.25:1 w . a io 181 MU:MC) ob ained on p e ious sec ion was added o he mix u e. Films we e cas on a 20 cm 182 diame e su ace pe i dish in a chambe a 55 ºC o e nigh . The inal ilms we e ca. 75-90 183 μm hick. 184 185 2.5. Physical Cha ac e iza ion o C oss-linked MC Films 186 187 2.5.1. Scanning Elec onic Mic oscopy (SEM) s udies 188 SEM mic og aphs o ac i e ilms we e ob ained om a JSM-5410 Jeol Scanning 189 Mic oscope wi h accele a ing ol age a 10 kV. Fi s ly, ilms we e ac u a ed using Tensile 190 Tes e because h ough c yo- ac u e was no possible o ob ain samples. A e , samples we e 191 coa ed wi h gold palladium using a Spu e ing Sys em Humme 6.2 and SEM mic og aphs 192 o he su ace and he c oss-sec ion o he ma e ials we e aken. 193 194 2.5.2. Op ical P ope ies 195 The ilm colo was de e mined wi h a Konica Minol a model CR-410 colo ime e . The 196 ilm specimen was placed on he su ace o a s anda d whi e pla e, and he CIELAB colo 197 space was used o de e mine he pa ame e s L*, a*, and b*. The colo was also exp essed 198 16 also a educ ion o his peak. When compa ed o MC ilm, he p esence o he plas icize 342 PEG was obse ed by he p esence o a new peak a 1350 cm-1 (C-H bending) (Shameli e 343 al., 2012). 344 Fig. 2B shows he FTIR o mu a ex ac , GA, MC and he ac i e ma e ial 345 MC_10GA_MU as an example, since all ac i e ma e ials p esen ed he same spec a. I was 346 possible o obse e mu a ex ac p esen ed a cha ac e is ic peak a a simila wa enumbe o 347 GA. Thus, he inco po a ion o mu a ui ex ac in he case o ac i e ma e ials was 348 pe cei ed only by he inc ease in abso bance o his peak a ound 1720 cm-1. 349 350 3.2.4. Swelling Resul s 351 Table 3 only p esen s swelling index (SI) esul s o c oss-linked ilms since MC ilms no 352 c oss-linked we e quickly swelled and comple ely dissol ed in wa e wi hin 1 h. 353 As i was expec ed, he inco po a ion o GA induced he chemical c osslinking o me hyl 354 cellulose which was based on he gene a ion o co alen bonds ha main ained he in eg i y 355 o he polyme ic ne wo k and educed polyme chain lexibili y and mobili y. C oss-linked 356 MC ilms wi h and wi hou mu a ui ex ac we e highly s able when imme sed in wa e , 357 he shape did no change bu ilms swelled and hei su ace a ea inc eased. Fu he mo e, SI 358 alues dec eased as GA concen a ion inco po a ion inc eased. Films wi h 20% GA showed 359 e y low alues o SI, sugges ing ha he hyd ophillici y o c oss-linked ilms was g ea ly 360 educed. 361 362 363 364 17 On he o he hand, he inco po a ion o mu a ex ac dec eased he e iciency o he 365 c osslinking p ocess and esul ed in a conside able inc ease on swelling index alues in he 366 case o ac i e ma e ials. P obably some hyd oxyl g oups om an ioxidan compounds o 367 mu a ex ac a e eac ing wi h glu a aldehyde molecules, dec easing c osslinking e iciency 368 (Yu e al., 2015). GA, as a dialdehyde, can unde go a a ie y o eac ions, and he mos 369 common a e nucleophilic addi ion eac ions. Taken in o accoun ha mu a ui ex ac is 370 composed o la onoids and o ganic acids, he main eac ions would be be ween he ca bonyl 371 g oups o GA molecule and he hyd oxyl g oups o hese molecules h ough nucleophilic 372 addi ions o he ca bon-oxygen double bonds. Reac ions o aldehydes wi h alcohols p oduce 373 ei he hemiace als (a unc ional g oup consis ing o one —OH g oup and one —OR g oup 374 bonded o he same ca bon) o ace als (a unc ional g oup consis ing o wo —OR g oups 375 bonded o he same ca bon), depending upon condi ions. 376 377 3.2.5. The mal P ope ies 378 The mal s abili y o MC and ac i e MC ilms we e e alua ed by TGA. The he mog ams 379 and hei de i a i e o he weigh loss o all MC-based ma e ials a e shown in Fig. 3. 380 The mog ams clea ly show he maximum decomposi ion o ma e ials occu ed a 381 app oxima ely 361 °C, co esponding o me hyl cellulose deg ada ion (Rimdusi e al., 382 2012; Tjong, Xu, & Meng, 1999). I can be no iced ha he inco po a ion o plas icize 383 inc eased he he mal s abili y a ound 20 °C (381 and 362 °C deg ada ion empe a u es o 384 plas icized MC_PEG and unplas icized MC, espec i ely). On he o he hand, he 385 inco po a ion o GA dec eased his e ec , and his e ec was no p opo ional o GA con en 386 as maximum deg ada ion empe a u e alues o all c oss-linked ma e ials we e e y simila , 387 18 app oxima ely a 361 °C, as Fig. 3A and B show. In his case, c osslinking a e no gene a ing 388 new bonds he mally mo e esis an o hea han ini ial bonds exis ing in MC ne wo k. 389 In gene al, he he mal decomposi ion o c oss–linked MC based ilms ollowed mul is ep 390 decomposi ion. The i s s ep was he e apo a ion o he esidual sol en om cas ing me hod 391 (e hanol-dis illed wa e ) wi h i s maximum a ound 80 ºC (~3-6 % w ). This was ollowed by 392 a decomposi ion p ocess a app oxima ely a 260 ºC, which co esponds o he new linkage 393 deg ada ion o mo e he mo-sensi i e compounds, wi h lowe molecula weigh s 394 co esponding o c osslinking p ocess. Finally he he mal MC deg ada ion p e iously 395 men ioned. 396 The inco po a ion o mu a ex ac did no o e any addi ional p o ec ion o he polyme . 397 No mally he inco po a ion o an ioxidan s inc eases he mal polyme s abili y bu he 398 opposi e e ec obse ed in his wo k was al eady seen in o he wo ks (A ie a, Lopez, 399 Fe ándiz, & Pel ze , 2013, Lopez de Dicas illo, Alonso, Ca alá, Ga a a, & He nandez- 400 Muñoz, 2010). A TGA s udy o me hyl cellulose wi h mu a ex ac wi hou c osslinking 401 was ca ied ou o obse e he deg ada ion p ocesses. As Fig.3B shows, simila deg ada ion 402 p ocesses we e displayed when compa ed o ac i e c oss-linked ma e ials. MU ex ac was 403 also analyzed, showing a b oad deg ada ion band ha s a s a 120 ºC app oxima ely, wi h a 404 maximum a app oxima ely 200 ºC. This deg ada ion can be associa ed o he 405 “ca ameliza ion” p ocess o he suga s om he la onoids o MU ex ac , mainly la onols, 406 such as my ice in and que ce in, which a e pa ially glycosyla ed (Shene e al., 2008). So, 407 his peak a 250 °C can be associa ed o a c osslinking e ec o ca bonyl g oups om 408 la onoids om his ui ex ac . This ac has been shown ecen ly in some wo ks (Yu e 409 al., 2015; Benbe aieb, Ka bowiak, B achais, & Debeau o , 2015). 410 411 19 3.2.6. Mechanical P ope ies 412 The ensile p ope ies including elonga ion a b eak (EB), Young´s modulus (YM) and 413 ensile s eng h (TS) o all he ilms de eloped we e e alua ed, and he esul s a e shown in 414 Fig. 4. As i was expec ed, he alue o YM o MC_PEG dec eased conside ably due o i s 415 plas icizing e ec . Ne e heless, as he inc ease o YM alues o all c oss-linked ilms 416 shows, he inco po a ion o GA led o a ema kable inc ease in igidi y due o i s c oss-linke 417 unc ion. 418 Resul s e ealed c oss-linking p ocesses also had a signi ican e ec on inc easing he 419 mechanical s eng h o he ilms. The MC chains mobili y dec eased due o he s ong 420 in e ac ion be ween polyme ic ma ix and c oss-linke GA, enhancing he polyme s eng h 421 bu educing he lexibili y o he co esponding ilms. This e ec esul ed in an enhancemen 422 o TS alues and a ema kable educ ion in EB alues. Simila esul s we e obse ed when 423 MC was c oss-linked wi h ea-ca echins (Yu e al., 2015). Yu e al. showed he igid a oma ic 424 and he e ocyclic ings om la onoids can physically c oss-link MC. Such inc ease in TS 425 was also obse ed o gela in-soy p o ein ilms in he p esence o e ulic acid and que ce in 426 (Benbe aieb e al., 2015). In his wo k, la onols, such as que ce in and my ice in, om 427 mu a ex ac could be he eason o he highes alues o TS ob ained in he case o ac i e 428 ilms. The inco po a ion o MU ex ac had also a singula e ec inducing ilms o a 429 conside able inc ease on EB alues. This e ec could be due o he inc ease in polyme 430 chains mobili y caused by he p esence o some componen s om mu a ex ac be ween 431 polyme ic chains ha inc eased he lexibili y. This plas icizing e ec can be no iced also 432 om YM esul s. I is in e es ing ha when GA was a highes concen a ion his e ec was 433 diminished, as i some MU ex ac componen s we e eac ing wi h GA molecules. 434 435 20 3.3. An ioxidan ac i i y esul s 436 437 Fig. 5 p esen s he adical sca enging ac i i ies o MC ma e ials exposed o ABTS+· 438 solu ion exp essed as T olox equi alen s. As i was expec ed, ilms wi hou he inco po a ion 439 o MU ex ac did no show an ioxidan ac i i y, as Fig. 5 shows in he case o ilm wi h 20% 440 GA (MC_20GA), as an example. 441 The elease o an ioxidan componen s o MU ex ac om ma e ials ad anced owa ds 442 an equilib ium alue ha co esponded o 50.6, 40.4 and 32.4 % o he nominal alue o 443 ma e ials wi h 10, 15 and 20% GA, espec i ely. The ex en o elease a equilib ium depends 444 on he compa ibili y o he mig an wi h bo h he ood simulan and he polyme ic ilm. The 445 kine ics o all ac i e c oss-linked ma e ials p esen ed simila p o iles, ollowing an 446 “exponen ial g ow h o a maximum” ype p o ile. The inco po a ion o PEG, as plas icize , 447 su ely acili a ed he elease o ac i e agen s because molecula and polyme chain 448 mo emen s we e enhanced. Se e al wo ks ha e used plas icize s o modi y polyme 449 p ope ies ela ed o he mass anspo o ac i e agen s (Lopez de Dicas illo, A es-Pe nas, 450 Cas o-López, Vila iño, & Gonzalez-Rod íguez, 2013; Siepmann, Sielpmann, Wal he , 451 MacRae, & Bodmeie , 2008). The e ec o he GA concen a ion was clea ly no iceable, 452 diminishing an ioxidan ac i i y o ma e ials as GA concen a ion inc eased. This was 453 p obably due o he eac ions be ween an ioxidan compounds om na u al ex ac and GA 454 ha dec eased hei a ailabili y, and his ac was al eady e idenced by o he esul s, such as 455 mechanical p ope ies. An ioxidan ac i i y o ilms was p opo ional o an ioxidan s eleased 456 om ac i e biocomposi es o adical solu ion. This is he eason he kine ic p o ile o adical 457 sca enging ac i i ies o ma e ials p esen ed he same p o ile as common ac i e compound 458 elease om ac i e ma e ials (Lopez de Dicas illo e al., 2011a,b; Masche oni, Guilla d, 459 21 Nalin, Mo a, & Pie gio anni, 2010). Release o componen s om polyme ma ix is also 460 based on swelling deg ee o polyme s. As Table 2 indica es, highes swelling deg ee was 461 ob ained om ma e ials wi h 10% GA, as well as he highes an ioxidan ac i i y. 462 The ex en o he an ioxidan ac i i y can be cha ac e ized by he pa i ion coe icien , K, 463 which is de ined as he a io o he amoun o an ioxidan s e ained in he polyme ic phase 464 ela i e o he concen a ion o eleased an ioxidan s. As Fig. 5 shows, K coe icien s alues 465 ob ained we e e y simila and app oxima ely equal o alue 1, which co esponds when 466 equilib ium is eached ending up in a simila concen a ion o an ioxidan s in he polyme 467 han in he ood. Ne e heless, esul s showed highes an ioxidan ex en elease occu ed 468 when GA was lowes because as GA concen a ion inc eased, mo e an ioxidan compounds 469 we e e ained in he polyme s due o eac ions be ween an ioxidan molecules and GA. 470 471 3.4. An imic obial ac i i y esul s 472 473 Table 4 shows he an imic obial esul s ob ained o ac i e MC ilms de eloped wi h MU 474 ex ac agains Lis e ia innocua. The dilu ion me hod clea ly displayed ha ilms con aining 475 MU ex ac we e able o signi ican ly educe he g ow h o bac e ia om he i s day o 476 con ac . As obse ed in Table 4, he dilu ion me hod was use ul o quan i y he bac e ial 477 educ ion caused by ilms. Resul s showed a dec ease in mic obial g ow h o all samples 478 assayed including he blank ilm wi h highes GA con en . This ilm may con ain esidual 479 GA which can elease om he ilms o educe he g ow h o bac e ia because GA p esen s 480 some an imic obial ac i i y. 481 As i was expec ed, an imic obial esul s ollowed he same endency as an ioxidan 482 ac i i y. Films wi h lowes GA concen a ion p esen ed highes an imic obial powe , 483 22 educing almos 4 logs, and esul ed in he mos e icien an imic obial ilm. In spi e o i s 484 in e es ing p ope ies, speci ic mig a ion es s should be ca ied ou be o e any applica ion o 485 ensu e ood sa e y. 486 487 4. Conclusions 488 489 In his wo k ac i e ilms based on me hyl cellulose (MC) and mu a ui ex ac (MU) 490 we e ob ained h ough cas ing p ocess. MC polyme was c oss-linked wi h glu a aldehyde 491 (GA) a di e en concen a ions owing o s udy he e ec o c osslinking deg ee on physical 492 p ope ies o he polyme and he elease o ac i e compounds. The mal p ope ies we e 493 sligh ly a ec ed by he inco po a ion o GA and MU, bu he he mal s abili y o he 494 de eloped ma e ials is s ill app op ia e o he in ended use. C osslinking p ocesses inc eased 495 mechanical s eng h and dec eased swelling index o de eloped ma e ials and he amoun o 496 ac i e agen s a ailable om MU ex ac in he polyme ma ixes. As GA concen a ion 497 inco po a ed inc eased, swelling index, an ioxidan and an imic obial ac i i ies o ac i e 498 ma e ials dec eased. 499 500 Acknowlegmen s 501 The au ho s acknowledge he Financial suppo o CONICYT h ough he P ojec 502 FONDEF “P og ama IDeA Ciencia Aplicada” (P ojec CA13I10334) and “P og ama de 503 Financiamien o Basal pa a Cen os Cien íficos y Tecnológicos de Excelencia” (P ojec 504 FB0807). 505 506 Re e ences 507 23 508 Aiedeh, K. M., Taha, M. O., Al-Hia i, Y., Bus anji, Y., & Alkha ib, H. S. (2006). E ec o ionic 509 c osslinking on he d ug elease p ope ies o chi osan diace a e ma ices. Jou nal o 510 Pha maceu ical Sciences, 96, 38-43. 511 512 Al a o, S., Mu is, A., Palma, R., Qui oz, A., Seguel, I., & Scheue mann, E. (2013). In luence 513 o geno ype and ha es yea on polyphenol con en and an ioxidan ac i i y in mu illa 514 (Ugni molinae Tu cz) ui . Jou nal o Soil Science and Plan Nu i ion, 13, 67-78. 515 516 Aminabha i, T. M., Balundgi, R. H., & Cassidy, P. E. 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Assesmen o an ioxidan and an ibac e ial ac i i y. 579 Molecules, 20, 5698-5713. 580 581 Lee, M., Lee, S., Ma, Y., Pa k, S., Bae, D., Ha, S., & Song, B. (2005). E ec o plas icize and 582 c osslinking agen on he physical p ope ies o p o ein ilms. Jou nal o Food Science and 583 Nu i ion, 10, 88-91. 584 585 Lopez de Dicas illo, C., Alonso, J. M., Ca alá, R., Ga a a, R., & He nandez-Muñoz, P. (2010). 586 Imp o ing he an ioxidan p o ec ion o packaged ood by inco po a ing na u al la onoids 587 in o e hylene- inyl alcohol copolyme (EVOH) ilms. Jou nal o Ag icul u al and Food 588 Chemis y, 58, 10958-10964. 589 590 López de Dicas illo, C., Jo dá, M., Ca alá, R., Ga a a, R., & He nández, P. (2011a). 591 De elopmen o ac i e poly inyl alcohol/β-cyclodex in composi es o sca enge undesi able 592 ood componen s. Jou nal o Ag icul u al and Food Chemis y, 59, 11026-11033. 593 594 Lopez de Dicas illo, C., Ne in, C., Al a o, P., Ca ala, R., Ga a a, R., & He nandez-Muñoz, P. 595 (2011b). De elopmen o new an ioxidan ac i e packaging ilms based on e hylene inyl 596 alcohol copolyme (EVOH) and g een ea ex ac , Jou nal o Ag icul u al and Food 597 Chemis y, 59, 7832-7840. 598 599 32 Table 3 723 Swelling esul s. 724 Sample Swelling Index (SI) MC_10GA 156 ± 4.8 e MC_15GA 88.9 ± 7.6 b MC_20GA 78.7 ± 0.9 a MC_10GA_MU 237.7 ± 1.6 MC_15GA_MU 131.6 ± 2.3 d MC_20GA_MU 105.6 ± 1.6 c Lowe case le e s a- indica e signi ican di e ences in 725 swelling among he ma e ials. 726 727 728 33 Table 4 729 An imic obial ac i i y o de eloped ilms. 730 Reduc ion Film UFC/mL % Reduc ion Log Cycles Con ol 7 x 107 - - MC_20GA 1 x 107 85,7 0,85 MC_10GA_MU 1 x 104 99,9 3,85 MC_15GA_MU 6,8 x 104 99,9 3,01 MC_20GA_MU 7 x 104 99.9 3 731 732 34 Figu e cap ions 733 734 Fig. 1. C oss-sec ion SEM images o he MC ilms de eloped: A: MC; B: MC_PEG; C: 735 MC_10GA; D: MC_10GA_MU; E: MC_20GA; F: MC_20GA_MU. 736 737 Fig. 2. FTIR spec a o : (A) i) MC; ii) MC_10GA; iii) MC_15GA; i ) MC_20GA; (B) i) 738 MC; ii) MC_15GA_MU; iii) mu a ex ac ; and i ) GA. 739 740 Fig. 3. A) TGA and de i a i e cu es (inse ) o he weigh loss o MC-based ma e ials 741 wi hou MU ex ac and GA; B) TGA de i a i e cu es o ac i e MC-based ma e ials and 742 MU ex ac . 743 744 Fig. 4. Young´s modulus (YM), Tensile s eng h (TS) and Elonga ion a b eak (EB) o MC 745 de eloped ma e ials (le e s a- indica e signi ican di e ences in each p ope y alues among he samples 746 in a mechanical p ope y). 747 748 Fig. 5. ABTS+· adical sca enge ac i i ies o mu a ui MC ilms. 749 750 35 751 752 A E C D B F 36 753 37 754 755 38 756 757 758 MC MC_PEG 10GA 15GA 20GA 10GA_MU15GA_MU20GA_MU YM (N/mm2) 0 200 400 600 800 1000 Young´s Modulus MC MC_PEG 10GA 15GA 20GA 10GA_MU15GA_MU20GA_MU TS ( N/mm2) 0 2 4 6 8 10 12 14 16 18 Tensile S eng h MC MC_PEG 10GA 15GA 20GA 10GA_MU15GA_MU20GA_MU EB (%) 0 10 20 30 40 50 60 70 Elonga ion a b eak a e cd de bb c aa c b b e c cc a e d c bc ab 39 759 760 ime (hou s) 0,0 2,0 4,0 6,0 23,0 23,5 24,0 24,5 AA (mg T olox/g ilm) 0,0 0,3 0,6 0,9 3,0 4,0 5,0 MC_20GA MC_10GA_MU MC_15GA_MU MC_20GA_MU K = 3.1 K = 4 K = 4.8