Development of albumen/soy biobased plastic materials processed by injection molding

1 De elopmen o albumen/soy biobased plas ic ma e ials 1 p ocessed by injec ion molding 2 M. Félix, J.E. Ma ín-Al onso, A. Rome o*, A. Gue e o 3 Depa amen o de Ingenie ía Química, Uni e sidad de Se illa, Facul ad de Química, 41012 4 Se illa, Spain 5 Abs ac 6 Biobased plas ics om enewable polyme s cons i u e a highly in e es ing ield o 7 ele an indus ial applica ions such as packaging, ag icul u e, e c., in which 8 he momechanical echniques (i.e. ex usion, comp ession molding, e c.) a e 9 inc easingly being used. In spi e o he po en ials o injec ion molding in he 10 manu ac u e o shaped p oduc s i is s ill sca cely used wi h biopolyme s. This s udy 11 e alua es injec ion molding as an al e na i e o p oduce biobased ma e ials om 12 blends p epa ed in a mixing heome e , using di e en albumen/soy a ios and glyce ol 13 as he plas icize . Viscoelas ic measu emen s and DSC o p o ein/glyce ol blends we e 14 used o selec sui able p ocessing condi ions. Physicochemical p ope ies o injec ion- 15 molded p obes we e cha ac e ized h ough dynamic mechanical he mal analysis, 16 ensile s eng h, wa e up ake and ansmi ance es s. Occu ence o shea -induced 17 e ec s o e mixing was con i med by ex ac abili y analysis o p o ein concen a es and 18 blends, pa icula ly o soy-based sys ems. Bo h p o eins and hei mix u es yield 19 injec ion-molded bioplas ics, al hough showing lowe mechanical p ope ies han LDPE 20 s anda ds. 21 Keywo ds: Albumen; Bioplas ic; Dynamic Mechanical The mal Analysis; Soy P o ein; 22 Tensile s eng h es ; T ansmi ance. 23 _______________________ 24 *A. ROMERO 25 Depa amen o de Ingenie ía Química, 26 2 Uni e sidad de Se illa, Facul ad de Química, 27 41012 Se illa (Spain) 28 E-mail: al ome [email protected] 29 Phone: +34 954557179; ax: +34 954556447. 30 1. In oduc ion 31 Plas ic ma e ials a e cu en ly conside ed e y impo an ma e ials due o hei 32 excep ional p ope ies and pe o mance o e o he ma e ials such as me al and wood 33 (Plas ic Eu ope, 2008). In ac , acco ding o a ecen epo , he demand o plas ic will 34 con inue o ise ollowing a end ha has inc eased since 1950s (Plas ics Eu ope e 35 al., 2008). Nowadays, he subs i u ion o pe oleum-based plas ics wi h bio-based 36 plas ics is seen as a p omising al e na i e because i will educe he dependency o 37 plas ics on ossil uels and he p essu e on land ills om plas ic solid was es (Al a ez- 38 Cha ez e al., 2012). In ecen yea s he e has been a g ea in e es o u ilize 39 enewable biomass in he manu ac u e o high-quali y, cos -compe i i e and 40 biodeg adable consume goods as a means o educe he consump ion and he 41 dependence on pe ochemical eeds ock and o diminish en i onmen al pollu ion 42 (Rosen a e and O ieno, 2006; Tummala e al., 2006). In pa icula , packaging ilms 43 and con aine s made o na u al biopolyme s ep esen a pa icula in e es due o hei 44 biodeg adabili y, since mos o hese p oduc s ha e a ela i e sho se ice li e ending 45 up in land ills. In his sense, p o ein-based ma e ials ha e been p o ed o be 46 comple ely deg ading in 50 days when bu ied in a mland soils (Domenek e al., 2004). 47 P o eins, lipids and polysaccha ides ha e been p oposed as biopolyme s sou ces 48 o many yea s (A e ous, 2004; De G aa , 2000; He nández-Izquie do and K och a, 49 2008; I issin-Manga a e al., 2001; Si acusa e al., 2008). Rega ding p o eins, mos 50 s udies ha e used plan p o eins such as zein, whea glu en o soybean o manu ac u e 51 bioplas ics (Cuq e al., 1998; Gomez-Ma inez e al., 2013; Je ez e al., 2005; Kim, 52 2008; Zheng e al., 2003). Mo eo e , some wo ks ha e been ocused on animal 53 3 p o eins such as milk p o eins, collagen, gela in, e c. (Cuq e al., 1998; Pomme e al., 54 2003). 55 Soy p o ein is he majo cop oduc o soybean oil and is one o he cheapes 56 p o eins in na u e (Tian e al., 2012). In ac , soy p o eins ha e commonly been used 57 o ood and animal eed o many yea s. Howe e , soy p o ein is a new polyme o 58 biodeg adable esins. Soy p o ein polyme s a e mac omolecules ha con ain a numbe 59 o amino acids and side chains ha can be used in he manu ac u e o plas ic (Sun e 60 al., 1999). The mechanical p ope ies o soy p o ein based plas ics can be con olled 61 and op imized by adjus ing he ini ial mois u e con en as well as some p ocessing 62 pa ame e s such as he molding empe a u e and/o p essu e (Wang e al., 2007; Liang 63 e al., 1999; Mo e al., 1999; Jane and Wang, 1996; Pa eau e al., 1994). Howe e , he 64 applica ion o soy p o ein plas ics is limi ed because o i s low s eng h (Tummala e al., 65 2006) and high mois u e abso p ion (Liu e al., 2005). The e o e, i can be concluded 66 ha he mos e ec i e me hod is o blend soy p o ein wi h ano he biodeg adable 67 polyme . Cu en ly, soy-based blends o plas ic applica ions include polyphospha e 68 (O aigbe and Adams, 1997), polyes e s (G ai e e al., 2004; Liu e al., 2004), 69 polyu e hane (Tian e al., 2010) o na u al ibe s (Liu e al., 2005). On he o he hand, 70 no epo s on he enhancemen o soy-based plas ic ma e ials by using a combina ion 71 wi h o he p o eins ha e been ound. 72 Egg whi e p o ein (albumen), adi ionally used by he ood indus y, has ecen ly 73 p o ed he easibili y o p oduce bioplas ics (Je ez e al., 2007b). Mo eo e , i 74 compa ed o o he common p o eins like glu en, egg whi e has p o ed o be an 75 adequa e aw ma e ial in he ob aining o highly- anspa en bioplas ics wi h sui able 76 mechanical p ope ies o he manu ac u e o biodeg adable ood packaging and o he 77 plas ic p oduc s. Blends o his p o ein wi h o he p oduc s om ag icul u al sou ces, 78 biodeg adable and o lowe cos han p o ein and syn he ic addi i es ha e been 79 ecen ly p oposed (González-Gu ie ez e al., 2010). 80 4 A p o ein-based ma e ial could be de ined as a s able h ee-dimensional 81 mac omolecula ne wo k s abilized and s eng hened by hyd ogen bonds, hyd ophobic 82 in e ac ions and disul ide bonds (Pomme e al., 2003). Howe e , as p o eins 83 hemsel es do no ha e su icien plas ici y o be handled and b i le p ope ies a e 84 ypically ound in bioplas ics, a plas icize is equi ed. The ole o plas icize s in 85 educing he glass ansi ion empe a u e and p o iding mobili y o polyme ic chains 86 has been ex ensi ely desc ibed (I issin-Manga a e al., 2001; Ma ee e al., 2000; 87 Pouplin e al., 1999). Due o i s excellen p ope ies, glyce ol is among he mos 88 commonly used plas icize s o biopolyme -based biodeg adable ma e ials. 89 P o ein/plas icize blends (bioplas ics) can be p ocessed using exis ing plas ic 90 p ocessing echnologies: om he physic-chemical o cas ing me hod (Gennadios, 91 2002) o he mo-plas ic/mechanical me hod (comp ession molding o ex usion) (Je ez 92 e al., 2007a; González-Gu ié ez e al., 2011). Howe e , a ele an echnique such as 93 injec ion molding, which is among he mos common p ocessing me hods used wi h 94 syn he ic polyme s, has no been ound o be used o p o ein-based bioplas ic 95 applica ions. The po en ial use o p o ein-based injec ion molding o p oduce many 96 kinds o shaped p oduc s will en ail new a gumen s in a o o conside ing hese 97 biopolyme ma e ials as an al e na i e o syn he ic plas ics o di e en applica ions. 98 The o e all objec i e has been o s udy plas icized albumen/soy biobased plas ic 99 ma e ials p ocessed by injec ion molding. To achie e his objec i e, di e en 100 albumen/soy a ios plas icized wi h glyce ol ha e been p ocessed. In addi ion, some 101 a iables such as empe a u e and esidence ime in he p e-injec ion mixing chambe , 102 as well as he empe a u e o he mold ha e been also analyzed in o de o selec 103 sui able p ocessing pa ame e s o p o ein-based injec ion molding. 104 2. Ma e ial and me hods 105 2.1. Ma e ials 106 5 Comme cial sp ay-d ied albumen (AP) was p o ided by OVOSEC S.A and soy 107 p o ein isola e (SPI) was supplied by P o ein Technologies In e na ional (SUPRO 108 500E, Lepe , Belgium). The p o ein con en o bo h p oduc s was de e mined in 109 quad uplica e as % N x 6.25 using a LECO CHNS-932 ni ogen mic o analyze (Leco 110 Co po a ion, S . Joseph, MI, USA) (E he idge e al., 1998) being 83 w .% o AP and 91 111 w .% o SPI. Glyce ol (GL), om Pan eac Química, S.A. (Spain), was used as p o ein 112 plas icize . 113 2.2. Sample p epa a ion 114 Blends con aining 60 w .% p o ein, wi h di e en AP/SPI a ios as shown in Table 1, 115 and 40 w .% glyce ol (GL) we e mixed in a wo-blade coun e - o a ing ba ch mixe 116 (B abende Plas og aph, Ge many). Mixing p ocess was ca ied ou a 25ºC and 50 117 pm o c.a. 10 min (Je ez e al., 2005) o ob ain a dough-like ma e ial a neu al pH. 118 The inal pH alue was measu ed by a C ison pH 25 pHme e in combina ion wi h a 119 punc u e elec ode (C ison Ins umen s S.A., Ba celona, Spain). 120 The dough-like ma e ials ob ained a e he mixing p ocess we e subsequen ly 121 p ocessed by injec ion molding using a MiniJe Pis on Injec ion Molding Sys em 122 (The moHaake, Ka ls uhe, Ge many) o ob ain bioplas ic p obes. The mos sui able 123 p ocessing a iables, such as injec ion empe a u e and p essu e, as well as esidence 124 ime in he p e-injec ion mixing chambe , we e selec ed a e pe o ming empe a u e 125 amp and ime sweep es s o he dough-like ma e ials. Two ypes o molds we e used 126 o p epa e he p obes: a 60×10×1 mm ec angula shape mold o bo h DMTA 127 expe imen s and anspa ency measu emen s and a Dumpbell ype p obe de ined by 128 ISO 527-2:1993 o Tensile P ope ies o Plas ics. 129 2.3. Cha ac e iza ion 130 2.3.1. P o ein solubili y. P o ein solubili y a di e en pH alues was de e mined. 131 Aqueous dispe sions (c.a. 1.00 g p o ein/40 mL) we e p epa ed and pH o di e en 132 aliquo s was adjus ed o alkaline pH alues wi h 6 N NaOH, and o acid pH wi h 2 N 133 6 HCl. Samples we e homogenized and subsequen ly cen i uged o 20 min a 10,000 x 134 g a 10ºC. The supe na an we e collec ed o p o ein con en de e mina ion by means 135 o he Ma kwell me hod (Ma kwell e al., 1978). Solubili y was exp essed as a 136 pe cen age (g soluble p o ein/100 g isola e in sample). 137 2.3.2. P o ein ex ac abili y. Samples we e ex ac ed in di e en ex ac ion media (2.5 138 mL): dis illed wa e ; a dena u ing agen solu ion (Me hod A); a solu ion o dena u ing 139 and educing agen s (Me hod B). All he ma e ials we e ex ac ed o 2h a 20ºC by 140 magne ic agi a ion (app oxima ely 400 pm). Me hod A used a 0.086 mol L-1 T is-base, 141 0.045 mmol·L-1 glycine, 2 mmol·L-1 EDTA, 10 g·L-1, 5 g·L-1 Sodium Dodecyl Sul a e 142 (SDS) pH 8 bu e . In me hod B, p o eins we e dissol ed in he same bu e con aining 143 10 g·L-1 Di hio h ei ol (DTT). Dispe sions we e cen i uged a 10,000 x g o 15 min a 144 15ºC and p o ein con en was de e mined by a LECO CHNS-932 ni ogen mic o 145 analyze . Simila me hods we e p e iously used by he au ho s wi h p o ein gels 146 (Rome o e al., 2011). P o ein solubili y was de e mined om supe na an and 147 exp essed as 100x p o ein con en in he supe na an / o al p o ein con en . Th ee 148 independen ex ac ions we e ca ied ou wi h each sol en . A e age alues (±s anda d 149 de ia ion) a e epo ed. 150 2.3.3. F ee and o al sul hyd yls. F ee and o al sul hyd yl g oups o p o ein samples 151 we e de e mined using he me hod de eloped by Be e idge e al. (1974) and 152 Thannhause e al. (1984), espec i ely. Samples we e suspended (1 mg/mL) in 0.086 153 mol/L T is-HCl – 0.09 mol/L glycine – 4 mmol/L EDTA – 8 mol/L u ea – pH 8 bu e . 154 Dispe sions we e s i ed a 25 ºC du ing 10 min a 500 pm in a he momixe and hen 155 cen i uged a 15,000g (10 min, 10 ºC). Supe na an was incuba ed wi h Ellman’s 156 eagen (4mg DTNB/mL me hanol) and 1 mL NTSB was used in he case o he o al 157 sul hyd yls. Abso bance a 412 nm was measu ed in a Genesis-20 spec opho ome e 158 (The mo Scien i ic, USA). The mola ex inc ion coe icien o NTB (13,600 L·mol-1·cm-1) 159 7 was used. P o ein concen a ion o ex ac s was de e mined by he B ad o d me hod 160 (B ad o d, 1976). 161 2.3.4. Su ace hyd ophobici y o p o eins (H0). Su ace hyd ophobici y (H0) o soluble 162 p o eins om p o ein ex ac s (pH 8) was measu ed acco ding o Ka o and Nakai 163 (1980), using he luo escen p obe 1-anilino-8-naph alene-sul ona e (ANS). P o ein 164 ex ac we e dilu ed wi h pH 8-0.05 M phospha e bu e o ob ain p o ein concen a ions 165 anging om 5 o 0.005 mg/mL. Then, 40 μL o ANS (8.0 mM in he same bu e ) we e 166 added o 2 mL o sample. Fluo escence in ensi y (FI) was measu ed wi h a Pe kin- 167 Elme 2000 luo escence spec ome e (Pe kin-Elme Co p. No walk, CT, USA), a 168 wa eleng hs o 365 nm (exci a ion) and 484 nm (emission). The ini ial slope o 169 luo escence in ensi y e sus p o ein concen a ion plo was used as an index o 170 p o ein hyd ophobici y (H0). 171 2.3.5. Di e en ial Scanning Calo ime y (DSC). DSC expe imen s we e pe o med wi h 172 a Q20 (TA Ins umen s, USA), using 5 o 10 mg samples, in he me ic aluminum pans. 173 A hea ing a e o 10 ºC/min was selec ed. The sample was pu ged wi h a ni ogen low 174 o 50 mL/min. 175 2.3.6. Rheological measu emen s. Dough-like ma e ial was cha ac e ized by Small 176 Ampli ude Oscilla o y Shea (SAOS) measu emen s, using a con olled-s ain 177 heome e (ARES), in o de o selec he op imum condi ions o injec ion molding. The 178 geome y used has been a pla e and pla e geome y (dia: 25 mm) wi h a ough su ace 179 and a gap be ween pla es o 1 mm. Low iscosi y Dow Co ning 200 luid has been 180 used as sealan o a oid sample d ying. S ain sweep SAOS es s we e also pe o med 181 in o de o es ablish he linea iscoelas ici y ange. Tempe a u e amp es s we e 182 ca ied ou a 5 ºC/min om 20 o 100 ºC and ime sweep es s we e pe o med o 183 1800 s a a selec ed cons an empe a u e. In hese measu emen s, complex iscosi y 184 (  *) was moni o ed a a cons an equency o 2 ad/s. All he sys ems s udied had he 185 same he mo heological his o y be o e pe o ming any heological es . 186 8 2.3.7. Dynamic Mechanical Tempe a u e Analysis (DMTA). DMTA es s we e ca ied 187 ou wi h a RSA3 (TA Ins umen s, New Cas le, DE, USA), on ec angula p obes using 188 dual can ile e bending. All he expe imen s we e ca ied ou a cons an equency 189 (1Hz) and s ain (be ween 0.01 and 0.3%, wi hin he linea iscoelas ic egion). The 190 selec ed hea ing a e was 3ºC min−1. All he samples we e coa ed wi h Dow Co ning 191 high acuum g ease o a oid wa e loss. 192 2.3.8. Tensile s eng h measu emen s. Tensile es s we e pe o med by using he 193 Insigh 10 kN Elec omechanical Tes ing Sys em (MTS, Eden P ai ie, MN, USA), 194 acco ding o by ISO 527-2:1993 o Tensile P ope ies o Plas ics. Tensile s ess and 195 elonga ion a b eak we e e alua ed om a leas h ee duplica es o each p oduc 196 using ype IV p obes and an ex ensional a e o 100 mm·min−1 a oom empe a u e. 197 2.3.9. Wa e abso p ion capaci y. Wa e up ake o bioplas ics was de e mined ollowing 198 he ASTM D570 no m (ASTM D570-98, S anda d es Me hod o Wa e Abso p ion o 199 Plas ics) (ASTM, 2001) using a leas h ee 60×10×1 mm specimens imme sed in 200 dis illa e wa e o 2h o 24 h a oom empe a u e. 201 2.3.10. T anspa ency Measu emen s. T anspa ency measu emen s we e done by 202 means o a Genesis-20 spec opho ome e (The mo Scien i ic, USA). T ansmi ance 203 (%) o ec angula specimens, 3 mm hick, was measu ed using a wa eleng h o 600 204 nm. Ai was used as blank (100% ansmi ance). A ansmi ance index (IT) was 205 de ined in o de o compa e he anspa ency o he di e en bioplas ic. 206 𝐼𝑇=𝐵𝑖𝑜𝑝𝑙𝑎𝑠𝑡𝑖𝑐 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒 (%) 𝐴𝑙𝑏𝑢𝑚𝑖𝑛 − 𝑏𝑎𝑠𝑒𝑑 𝐵𝑖𝑜𝑝𝑙𝑎𝑠𝑡𝑖𝑐 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑎𝑛𝑐𝑒 (%) ∙100 (1) 207 2.4. S a is ical analysis 208 A leas h ee eplica es o each measu emen we e ca ied ou . S a is ical analyses 209 we e pe o med using - es and one-way analysis o a iance (ANOVA, p0.05) by 210 9 means o he s a is ical package SPSS 18. S anda d de ia ions om some selec ed 211 pa ame e s we e calcula ed. 212 3. Resul s and discussion 213 3.1. P o ein solubili y 214 Fig. 1 shows he solubili y-pH p o ile ob ained o bo h AP and SPI sys ems. AP 215 sys em displays high solubili y alues wi hin he whole expe imen al ange o pH 216 alues, showing alues highe han 80 w .%. These esul s suppo he high hyd ophilic 217 cha ac e o AP sys em which is clea ly domina ed by i s high con en in highly soluble 218 albumin p o eins. Thus, almos 70% o p o ein in egg whi e is o oalbumin and 219 conalbumin (Che el e al., 1989). This high solubili y akes place e en a he minimum 220 shown in Fig.1 obse ed a pH 5.8-6, which should co espond o he isoelec ic poin 221 (IEP). Howe e , he IEP o egg whi e has been epo ed o be sligh ly lowe since Loeb 222 (1923) epo ed a alue o 4.8, while he IEP is loca ed a 5.4 acco ding o Riddick 223 (1968). These alues a e consis en wi h he esul s ob ained om z-po en ial 224 measu emen s (da a no shown) pe o med o his AP sys em ha exhibi ed a null 225 alue a pH a ound 5. 226 SPI exhibi s much lowe solubili y alues han AP sys em, showing a maximum 227 solubili y (ca. 36%) a pH 8 and a minimum (ca. 7.2%) a pH 6. Howe e , a wide low- 228 solubili y ange (lowe han 10%) may be no iced be ween pH 3 and 6. Molina-O iz e 229 al. (2004) epo ed highe solubili y alues, pa icula ly a acidic pH, wi h a much 230 na owe minimum a pH 4.5 o SPI p epa ed om a non he mally ea ed soy lou ( o 231 a oid p o ein dena u a ion). Thus, he ex ension o he low solubili y p o ile o he 3-6 232 pH ange migh be a ibu ed o some p o ein dena u a ion du ing SPI manu ac u e. 233 I is also in e es ing o men ion ha he solubili y p o ile shows a second minimum a 234 pH 9. This minimum, al hough being less appa en , has been also de ec ed by F anzen 235 and Kinsella (1976) o a SPI showing much highe solubili y a alkaline pH. 236 3.2. Disul ide and sul hyd yl g oups 237 16 p o ein-based bioplas ic ma e ials (Je ez e al., 2007a, 2007b; Zá a e-Ramí ez e al., 401 2011), indica es ha some he mose ing po en ial s ill emains a e injec ion molding 402 a he expe imen al condi ions. These esul s con i m ha he condi ions selec ed o 403 injec ion molding, summa ized in Table 5, a e mo e sui able o SPI-based p obes. As 404 o he AP/SPI-based bioplas ics, he beha io is much close o ha one ound o he 405 SPI/GL sys em. The AP/SPI mixed sys ems show an in e media e beha io , being only 406 appa en a empe a u es highe han 60ºC. 407 The highe iscoelas ic p ope ies shown by he sys em con aining AP as he only 408 p o ein may be ela ed o some o he physicochemical p ope ies p e iously shown. 409 Thus, he lowes empe a u e o he DSC peak shown in Fig. 3 (much lowe han he 410 p ocessing empe a u e) would lead o a high deg ee o dena u a ion o his sys em. 411 This e ec would lead o a highe su ace hyd ophoboci y ha in any case is highe o 412 AP (Table 3) ha would p oduce a highe inc ease in p o ein agg ega ion. In addi ion, 413 he highe alue o ee sul hyd yls shown by AP (Table 2) would also yield a highe 414 densi y o c osslinking by o ming hyd ogen bonds. Simila esul s ha e been epo ed 415 by Bounoco e e al. (2003) ha also explained his beha io in e ms o he mal 416 induced c osslinking. 417 All he p obes s udied display simila loss angen p o iles (Fig. 4B) showing one 418 single peak, which is ela ed o a glass-like ansi ion o he plas icized p o ein-based 419 ma e ial, wi h an 𝛿 alues anging om 0.1 o 0.5, excep ing o he AP/GL sys em ha 420 shows a much lowe an 𝛿 peak a ca. 0.34. These unimodal p o iles indica e a good 421 compa ibili y be ween p o ein and glyce ol, ob ained o all he sys ems a e he 422 injec ion molding p ocess, ega dless o he p o ein used (AP, SPI o AP/SPI mix u es). 423 The alue o he empe a u e a he peak o he AP/GL sys em is ca. 63ºC, which is 424 a he coinciden wi h hose alues p e iously ound o albumen-based bioplas ic 425 p epa ed by comp ession molding wi h he same p o ein/glyce ol a io (Je ez e al., 426 2007a, González-Gu ie ez e al., 2010). As may be obse ed in he plo included in 427 Fig.4B, his empe a u e a he peak inc eases asymp o ically wi h he ela i e 428 17 p opo ion o SPI in he p o ein mix u e up o a alue o ca. 78.5ºC. This ea lie 429 ansi ion ound o AP/GL bioplas ics is also consis en wi h he ea lie Tg ob ained o 430 AP/GL blends om DSC measu emen s. 431 3.8. Uniaxial ensile s eng h measu emen s 432 The esul s ob ained om uniaxial s eng h measu emen s a e shown in Fig. 5. Fig. 433 5A displays he esul s o s ess-s ain cu es ob ained o p o ein-based bioplas ics 434 using AP, SPI and 50:50 AP/SPI mix u es. 435 All he cu es exhibi an ini ial linea elas ic beha io o high cons an s ess-s ain 436 slope yielding high alues o he Young’s Modulus (E), ollowed by a plas ic 437 de o ma ion s age wi h a con inuous dec ease in he s ess-s ain slope a e he elas ic 438 limi . A second cons an slope is eached a he end o his plas ic de o ma ion s age. 439 All he cu es e en ually each a maximum alue o he s ess (max) and he s ain 440 (εmax), which is immedia ely ollowed by a sudden dec ease in s ess ha co esponds 441 o he up u e o he sample. Fig. 5B shows he alues o he h ee pa ame e s (E, max 442 and εmax) om ensile es s pe o med on AP/SPI/GL bioplas ic p obes, as a unc ion o 443 he albumen con en in he AP/SPI mix u e. Pa ame e s (max) and E show a 444 p og essi e inc ease wi h inc easing albumen con en , wi h an o e all g ow h in he 445 o de o 50% o bo h pa ame e s. Pa ame e εmax also unde goes an inc ease up o 446 50% AP concen a ion. Howe e , a u he inc ease up o 100% does no lead o any 447 signi ican di e ence. The e o e, i may be gene ally s a ed ha AP-based bioplas ics 448 exhibi be e ensile p ope ies han SPI-based sys ems in acco dance o he highes 449 alues also ound o he elas ic modulus om DMA measu emen s and he lowes Td 450 ob ained wi h DSC es s. 451 In any case, all he P o ein/GL bioplas ic p obes exhibi ensile p ope ies lowe han 452 syn he ic polyme s such as LDPE. Thus, pa ame e s max, εmax and E show alues ha 453 each as much as 15%, 18% and 10%, espec i ely, o he alues o ASTM 454 no malized LDPE (ASTM D638). 455 18 3.9. Wa e up ake capaci y 456 Fig. 6 shows he esul s om wa e up ake measu emen s ob ained a e imme sion 457 o bioplas ic samples o 2 and 24 h, as well as he wa e -soluble ma e loss, as a 458 unc ion o AP con en . 459 No signi ican di e ences be ween wa e up ake pe cen ages a e 2 and 24 h a e 460 ound, ega dless o he AP/SPI a io used in he bioplas ic p epa a ion by injec ion 461 molding. This ac indica es occu ence o ela i ely as e wa e abso p ion kine ics as 462 compa ed o glu en-based bioplas ics p ocessed by comp ession molding (unpublished 463 esul s). In addi ion, an inc ease in AP con en induces a p og essi e dec ease in wa e 464 abso p ion up o hal he alue co esponding o he AP ee sample. I may be poin ed 465 ou ha his las alue is much lowe han hose p e iously epo ed (Je ez e al., 466 2007a) o simila AP/GL samples. Howe e , hese samples used highe AP/GL a io (2 467 ins ead o 1.5) and we e p epa ed by comp ession molding. Al hough he o me ac 468 may induce some changes, he abo e-men ioned di e ence in wa e up ake capaci y 469 may be mainly a ibu ed o he di e en he momechanical p ocessing condi ions used. 470 In ac , as epo ed by Je ez e al. (2007a), an inc ease in p essu e led o a ema kable 471 educ ion in wa e abso p ion. 472 On he o he hand, he ac ha AP-based bioplas ic display lowe abso p ion 473 capaci y han SPI-based samples may be ela ed o a highe s uc u e deg ee o he 474 AP p o ein ma ix. This is consis en wi h he abo e-men ioned highe p o ein 475 c osslinking ob ained o AP/GL bioplas ic p obes as deduced om DMA and ensile 476 es s. This beha io is in ag eemen wi h hose esul s ound by o he au ho s 477 (Buonoco e e al., 2003; Zheng e al., 2003), who epo ed ha he swelling a io o 478 polyme ma ices dec ease wi h inc easing p o ein c osslinking. 479 As ega ds he wa e -soluble loss ma e he AP/GL sys em shows a alue o 40% 480 and all he SPI-con aining p obes exhibi alues ha a e sligh ly highe . These esul s 481 sugges ha he loss o soluble ma e co esponds basically o he highly hyd ophilic 482 glyce ol. In ac , all he samples con ain 40% GL. The ex a-loss ma e obse ed o 483 19 SPI-based samples should co espond o mois u e con en as well as o some p o ein 484 ha is no s ongly associa ed o he ne wo k s uc u e. 485 3.10. T anspa ency measu emen s 486 Fig. 7 shows he e olu ion o he ansmi ance index (IT) wi h inc easing AP 487 pe cen age in he o al p o ein con en o he injec ion molded bioplas ic p obe. Images 488 o each p obe a e also included in Fig. 7. The e olu ion o colo and anspa ency wi h 489 inc easing AP con en is appa en , leading o a ema kable inc ease in he alue o IT. 490 The alue ob ained o AP-based p obe is 46.8  0.4, being sligh ly lowe han he 491 alue co esponding o LDPE (54.0 ± 0.3) and 52% highe han ha o he SPI-based 492 p obe. Mo eo e , as may be no ed, addi ion o 25% AP causes he highes inc ease in 493 IT alue ep esen ing abou 63% o he o al inc ease in ansmi ance. 494 4. Concluding ema ks 495 AP concen a e shows highe solubili y han SPI. AP also shows highe sul hyd yl 496 g oups and disul ide bonds, as well as highe hyd ophobici y. The e o e, AP 497 concen a e e idences highe po en ials o he momechanical p ocessing han SPI. 498 Acco ding o he ex ac abili y analysis o p o ein concen a es and p o ein/GL 499 blends, some shea -induce e ec s ook place du ing he mixing p ocess al hough being 500 less no iceable o AP han o SPI sys ems. As a esul o he mixing p ocess, glyce ol 501 educed he glass ansi ion and dena u a ion empe a u es o a highe ex en when 502 SPI p o ein was used ins ead o AP. The combina ion o empe a u e amps and ime 503 sweep es s a selec ed empe a u e has demons a ed o be sui able echniques in 504 o de o selec ope a ion condi ions o injec ion molding. These p ocessing condi ions 505 ook in o accoun he mixed he moplas ic and he mose cha ac e o he p o ein 506 sys ems s udied whe e he con ibu ion o he la e is mo e ele an . 507 Bo h p o ein sys ems used alone o in combina ion yield bioplas ics ha can be 508 he momechanically p ocessed by injec ion molding, al hough showing lowe 509 20 heological and mechanical p ope ies han LDPE s anda ds, unde he selec ed 510 p ocessing condi ions. 511 A compa ison be ween AP and SPI-based injec ion molded bioplas ic specimens 512 e ealed ha he o me show highe iscoelas ic bending p ope ies and highe 513 uniaxial ensile p ope ies. Howe e , unde he selec ed condi ions, SPI s ill showed a 514 emaining he mose ing po en ial. In any case, as a consequence o he lowe 515 heological and mechanical p ope ies o he la e , SPI showed a highe wa e up ake 516 capaci y. 517 The esul s o ansmi ance pu o wa d ha injec ion molding may yield highly 518 anspa en p o ein-based polyme ic ma e ials by adding a mode a e p opo ion o AP 519 o he aw p o ein/plas icise blend. Some o hese ma e ials showed anspa ency 520 p ope ies ha we e e en compa able o LDPE plas ic ma e ials. These esul s a e 521 consis en wi h hose epo ed by o he p e ious esea che s ha used egg whi e- 522 based comp ession molded ma e ials. 523 Acknowledgemen s 524 This wo k is pa o a esea ch p ojec sponso ed by Andalousian Go e nmen , 525 (Spain) (p ojec TEP-6134) and by “Minis e io de Economía y Compe i i idad” om 526 Spanish Go e nmen (Re . MAT2011-29275-C02-02/01). The au ho s g a e ully 527 acknowledge hei inancial suppo . 528 Re e ences 529 ASTM, 2001. S anda d es me hod o plas ics: wa e abso p ion o plas ics. Designa ion D570-98. In: 530 Annual Book o ASTM s anda ds. Ame ican Socie y o Tes ing and Ma e ials, Philadelphia, PA. 531 Al a ez-Cha ez, C. R., Edwa d, S., Mou e-E aso, R., & Geise , K. (2012). Sus ainabili y o bio-based 532 plas ics: gene al compa a i e analysis and ecommenda ions o imp o emen . Jou nal o Cleane 533 P oduc ion, 23, 47-56. 534 21 A e ous, L. (2004). Biodeg adable mul iphase sys ems based on plas icized s a ch: a e iew. Jou nal o 535 Mac omolecula Science-Polyme Re iews, 44, 231–274. 536 Baie , S., Decke , E., & McClemen s, D. J. (2004). Impac o glyce ol on he mos abili y and hea -induced 537 gela ion o bo ine se um albumin. Food Hyd ocolloids, 18, 91-100. 538 Bengoechea, C., A achid, A., Gue e o, A., Hill, S.E., & Mi chell, J. R. (2007). Rela ionship be ween he 539 glass ansi ion empe a u e and he mel low beha io o glu en, casein and soya. Jou nal o Ce eal 540 Science, 45, 275-284. 541 Be e idge, T., Toma, S. J., & Nakai, S. (1974). De e mina ion o SH- and SS-g oups in some ood p o eins 542 using Ellman’s eagen . Jou nal o Food Science, 39, 49-51. 543 Bounoco e, G. G., Del Nobile, M. A., Panizza, A., Co bo, M. R., & Nicolais, L. (2003). A gene al app oach 544 o desc ibe he an imic obial agen elease om highly swellable ilms in ended o ood packaging 545 applica ions. Jou nal o Con olled Release, 90, 97-107. 546 B ad o d, M. M. (1976). A apid and sensi i e me hod o he quan i ica ion o mic og am quan i ies o 547 p o ein u ilizing he p inciple o p o ein-dye binding. Anali ycal Biochemis y, 72, 248-254. 548 Cha o , R. P. (1997). The mal Cha ac e iza ion o Polyme ic Ma e ials (2nd ed., pp. 484-744) Academic 549 P ess, New Yo k. 550 Che el, J. C., Cuq, J. L., & Lo ien , D. (1989). P o eínas Alimen a ias (1s ed.). Ac ibia, Za agoza 551 (España). 552 Co dobés, F., Pa al, P., & Gue e o, A. (2004). Rheology and Mic os uc u e o Hea -Induced Egg Yolk 553 Gels. Rheologica Ac a, 43, 184-195. 554 Cuq, B., Gon a d, N., & Guilbe , S. (1998). P o eins as ag icul u al polyme s o packaging p oduc ion. 555 Ce eal Chemis y, 75, 1-9. 556 De G aa , L. A. (2000). Dena u a ion o p o eins om a non- ood pe spec i e. Jou nal o Bio echnology, 557 79, 299-306. 558 Domenek, S., Feuilloley, P., G a aud, J., Mo el M-H., & Guilbe S. (2004). Biodeg adabili y o whea 559 glu en based bioplas ics. Chemosphe e, 54, 551-559. 560 22 Dono an, J. V., Mapes, C. J., Da is, J. G., & Ga ibaldi, J. A. (1975). A di e en ial scanning calo ime ic 561 s udy o he s abili y o egg whi e o hea dena u a ion. Jou nal o he Science and Food Ag icul u e, 562 26, 73-83. 563 E he idge, R. D., Pes i, G. M., & Fos e , E. H. (1998). A compa ison o ni ogen alues ob ained u ilizing 564 he Kjeldahl ni ogen and Dumas combus ion me hodologies (Leco CHS 2000) on samples ypical o 565 an animal nu i ion analy ical labo a o y. Animal Feed Science and Technology, 73, 21-28. 566 F anzen, K. L., & Kinsella, J. E. (1976). Func ional P ope ies o Succinyla ed and Ace yla ed Soy P o ein. 567 Jou nal o he Ag icul u al and Food Chemis y, 24, 788-795. 568 Gennadios, A., 2002. P o ein based ilms and coa ings (1s ed., pp. 66-115). CRC p ess, New Yo k. 569 Gomez-Ma inez, D., Pa al, P., Ma inez, I., & Gallegos, C. (2013). Glu en-based bioplas ics wi h modi ied 570 con olled- elease and hyd ophilic p ope ies. Indus ial C ops and P oduc s, 43, 704-710. 571 González-Gu ié ez, J., Pa al, P., Ga cía-Mo ales, M., & Gallegos, C. (2010). De elopmen o highly- 572 anspa en p o ein/s a ch-based bioplas ic. Bio esou ce Technology, 101, 2007-2013. 573 González-Gu ié ez, J., Pa al, P., Ga cía-Mo ales, M., & Gallegos, C. (2011). E ec o p ocessing on he 574 iscoelas ic, ensile and op ical p ope ies o albumen/s a ch-based bioplas ic. Ca bohyd a e 575 Polyme s, 84, 308-315. 576 G ai e , D., Waikul, L. H., Be ge , C., & Na ayan, R. (2004). Biodeg adable soy-p o ein polyes e blends 577 by eac i e ex usion p ocess. Jou nal o Applied Polyme Science, 92, 3231-3239. 578 Haska d, C. A., & Li-Chan, E. C. Y. (1998). Hyd ophobici y o Bo ine Se um Albumin and O albumin 579 De e mined Using Uncha ged (PRODAN) and Anionic (ANS-) Fluo escen P obes. Jou nal o he 580 Ag icul u al and Food Chemis y, 46, 2671-2677. 581 He nández-Izquie do, V. M., & K och a, J. M. (2008). The moplas ic p ocessing o p o eins o ilm 582 o ma ion – a e iew. Jou nal o Food Science, 73, 30–39. 583 I issin-Manga a, J., Gé a d, B., Be na d, B., & Gon a d N. (2001). New plas icise s o whea glu en ilms. 584 Eu opean Polyme Jou nal, 37, 1533-1541. 585 Jane, J. L., & Wang, S. (1996). Soy p o ein-based he moplas ic composi ion o p epa ing molded 586 a icles. U.S. Pa en , 5, 523,293. 587 23 Je ez, A., Pa al, P., Ma inez, I., Gallegos, C., & Gue e o, A. (2005). Rheology and p ocessing o glu en 588 based bioplas ics. Biochemical Enginee ing Jou nal, 26, 131-138. 589 Je ez, A., Pa al, P., Ma inez, I., Gallegos, C., & Gue e o, A. (2007ª). P o ein-based bioplas ics: e ec o 590 he mo-mechanical p ocessing. Rheologica Ac a, 46, 711-720. 591 Je ez, A., Pa al, P., Ma inez, I., Gallegos, C., & Gue e o, A. (2007b). Egg Whi e-based bioplas ics 592 de eloped by he momechanical p ocessing. Jou nal o Food Enginee ing, 82, 608-617. 593 Ka o, A., & Nakai, S. (1980). Hyd ophobici y de e mined by a luo escence p obe me hods and i s 594 co ela ion wi h su ace p ope ies o p o eins. Biochimica e Biophysica Ac a, 624, 13-20. 595 Kim, S. (2008). P ocessing and p ope ies o glu en/zein composi e. Bio esou . Technol. 99, 2032-2036. 596 Liang, F., Wang, Y., & Sun, X. S. (1999). Cu ing p ocess and mechanical p ope ies o p o ein-based 597 polyme s. Jou nal o Polyme Enginee ing, 19, 383-393. 598 Liu, W., Mis a, M., Askeland, P., D zal, L.T., & Mohan y, A. K. (2005). ´G een` composi es om soy based 599 plas ic and pineapple lea ibe : ab ica ion and p ope ies e alua ion. Polyme , 46, 2710-2721. 600 Liu, W., Mohan y, A. K., D zal, L. T., & Mis a, M. (2004). In luence o ibe su ace ea men on 601 mo phology and p ope ies o Indian g ass ein o ced soy p o ein based composi es. Polyme , 45, 602 7589-7596. 603 Loeb, J. (1923). On he loca ion o he o ces which de e mine he elec ical double laye be ween 604 collodion pa icles and wa e . The Jou nal o Gene al Physiology, 6, 105-129. 605 Ma kwell, M. A., Hass, S. M., Biebe , L. L., & Tolbe , N. E. (1978). A modi ica ion o he Low y p ocedu e 606 o simpli y p o ein de e mina ion in memb ane and lipop o ein samples. Analy ical Biochemis y, 87, 607 206-210. 608 Ma ee , Y., G inbe g, V., & Tols oguzo , V. (2000). The plas icising e ec o wa e on p o eins, 609 polysaccha ides and hei mix u es. Glassy s a e o biopolyme s, oods and seeds. Food 610 Hyd ocolloids, 14, 425-437. 611 Mine, Y. (1996). E ec o pH du ing he d y hea ing on he gelling p ope ies o egg whi e p o eins. Food 612 Resea ch In e na ional, 29, 155-161. 613 Mine, Y., Nou omi, T., & Haga, N. (1991). Emulsi ying and s uc u al-p ope ies o o albumin. Jou nal o 614 he Ag icul u al and Food Chemis y, 39, 443-446. 615 24 Mo, X., & Sun, X. S. (2002). Plas iciza ion o soy p o ein polyme by polyol-based plas icize s. Jou nal o 616 Ame ican Oil Chemis s’ Socie y, 79, 197-202. 617 Mo, X., Sun, X. S., & Wang, Y. (1999). E ec s o molding empe a u e and p essu e on p ope ies o soy 618 p o ein polyme s. Jou nal o Applied Polyme Science, 73, 2595-2602. 619 Mohamed, A. A. (2002). E ec o co n oil and amylase on he he mal p ope ies o na i e soy p o ein and 620 comme cial soy p o ein isola e. Food Chemis y, 78, 291-303. 621 Molina-O iz, S. E., Puppo, M. C., & Wagne , J. R. (2004). Rela ionship be ween s uc u al changes and 622 unc ional p ope ies o soy p o ein isola es-ca ageenan sys ems. Food Hyd ocolloids, 18, 1045- 623 1053. 624 Oa es, C. G., Ledwa d, D. A., Mi chell, J. R., & Hogson, I. (1987). Physical and chemical changes esul ing 625 om hea ea men o soya and soya algina e mix u es. Ca bohyd a e Polyme s, 7, 17-33. 626 O aigbe, J. U., & Adams, D. O. (1997). Bioabso bable soy p o ein plas ic composi es: E ec o 627 polyphospha e ille s on wa e abso p ion and mechanical p ope ies. Jou nal o En i onmen al 628 Polyme Deg ada ion, 5, 199-208. 629 Pa eau, I., Chen, C. Z., & Jane, J. (1994). Biodeg adable plas ic made om soybean p oduc s. 1. E ec o 630 p epa a ion and p ocessing on mechanical p ope ies and wa e abso p ion. Indus ial and 631 Enginee ing Chemis y Resea ch, 33, 1821-1827. 632 Plas ics Eu ope. (2008). Annual Repo 2007. Sa egua ding he Plane by Reaching Ou . 633 www.plas icseu ope.o g/cus /documen eques .apx?DocID=493 634 Plas ics Eu ope, EUPC ( he Eu opean Plas ics Con e e s), EPRO (Eu opean Associa ion o Plas ics 635 Recycling and Reco e y O ganisa ions), & EuP ( he Eu opean Plas ics Recycl es). (2008). The 636 Compelling Fac s abou Plas ics. An Analysis o Plas ics P oduc ion, Demand and Reco e y o 2006 637 in Eu ope. h p://ep o-plas ics ecycling.com/_ e wal ung/membe s/downloads/B ochu e_Fac s.pd 638 Pomme , M., Redl, A., Mo el, M.H., & Guilbe , S. (2003). S udy o whea glu en plas icisa ion wi h a y 639 acids. Polyme , 44, 115-122. 640 Pouplin, M., Redl, A., & Gon a d, N. (1999). Glass ansi ion o whea glu en plas icized wi h wa e , 641 glyce ol, o so bi ol. Jou nal o he Ag icul u al and Food Chemis y, 47, 538-543. 642 25 Rao, Q., & Labuza, T. P. (2012). E ec o mois u e con en on selec ed physicochemical p ope ies o wo 643 comme cial hen egg whi e powde s. Food Chemis y, 132, 373-384. 644 Riddick. T. M. (1968). Con ol o colloid s abili y h ough ze a po en ial Vol. 11. (1s ed.) Ze a-me e . Inc., 645 New Yo k. 646 Rome o, A., Co dobes, F., Puppo, M. C., Villanue a, A., Ped oche, J., & Gue e o, A. (2009a). Linea 647 iscoelas ici y and mic os uc u e o hea -induced c ay ish p o ein isola e gels. Food Hyd ocolloids, 648 23, 964-972. 649 Rome o, A., Bengoechea, C., Co dobes, F., & Gue e o, A. (2009b). Applica ion o he mal ea men s o 650 enhance gel s engh and s abili y o highly concen a ed c ay ish based emulsions. Food 651 Hyd ocolloids, 23, 2346-2353. 652 Rome o, A., Co dobes, F., Gue e o, A., & Puppo, M. C. (2011). C ay ish p o ein isola ed gels. A s udy o 653 pH in luence. Food Hyd ocolloids, 25, 1490-1498. 654 Rosen a e , K. U., & O ieno, A. W. (2006). Conside a ions o manu ac u ing bio-based plas ic p oduc s. 655 Jou nal o Polyme s and he En i onmen , 14, 335-346. 656 Shimada, K., & Che el, J. C. (1988). De e mina ion o Sul hyd yl G oups and Disul ide Bonds in Hea - 657 Induced Gels o Soy P o ein Isola e. Jou nal o he Ag icul u al and Food Chemis y, 36, 147-153. 658 Si acusa, V., Rocculi, P., Romani, S., & Dalla Rosa, M. (2008). Biodeg adable polyme s o ood 659 packaging: a e iew. T ends in Food Science & Technology, 19, 634–643. 660 S uik, L. C. E., 1978. Physical Aging in Amo phous Polyme s and O he Ma e ials (1s ed., pp. 35-49). 661 Else ie , Ams e dam. 662 Sun, X. S., Kim, H. R., & Mo, X. (1999). Plas ic pe o mance o soybean p o ein componen s. Jou nal o 663 Ame ican Oil Chemis s’ Socie y, 76, 119-123. 664 Thannhause , T. W., Konishi, Y., & Sche aga, H. A. (1984). Sensi i e quan i a i e analysis o disul e bonds 665 in polypep ides and p o eins. Analy ical Biochemis y, 138, 181-188. 666 Tian, H., Wang, Y., Zhang, L., Quan, C, & Zhang, X. (2010). Imp o ed lexibili y and wa e esis ance o 667 soy p o ein he moplas ics con aining wa e bo ne polyu e hane. Indus ial C ops and P oduc s, 32, 668 13-20. 669