Assessment of interfacial viscoelastic properties of Faba bean (Vicia faba) protein-adsorbed O/W layers as a function of pH

Assessmen o in e acial iscoelas ic p ope ies o Faba bean (Vicia aba) 1 p o ein-adso bed O/W laye s as a unc ion o pH 2 Manuel Felix1, Albe o Rome o2, Cecilio Ca e a3, An onio Gue e o1,* 3 1Depa amen o de Ingenie ía Química, Escuela Poli écnica supe io , Uni e sidad de Se illa, 41011 Se illa, 4 Spain. 5 2Depa amen o de Ingenie ía Química, Facul ad de Física, Uni e sidad de Se illa, 41012 Se illa, Spain. 6 3Depa amen o de Ingenie ía Química, Facul ad de Química, Uni e sidad de Se illa, 41012 Se illa, Spain. 7 8 9 10 11 12 13 ------------------- 14 *An onio GUERRERO 15 Depa amen o de Ingenie ía Química, Escuela Poli écnica Supe io , Uni e sidad de Se illa, 16 41011 Se illa, Spain. 17 E-mail: ague e [email protected] 18 Phone: +34 954557179; ax: +34 954556447. 19 Abs ac 20 In e acial heology may be ega ded as a powe ul ool domina ing he dynamics o complex 21 luid- luid in e aces. Mo e speci ically, heological p ope ies om shea measu emen s ha e 22 been pos ula ed as he mos use ul echnique o he assessmen o he mic os uc u e o 23 complex luid- luid in e aces, and i s ela ionship o long- e m emulsion s abili y. The aim o 24 his wo k was o e alua e he in e acial p ope ies o Faba bean (FB) p o ein as a unc ion o 25 p o ein concen a ion and pH alue (3.0, 5.0 and 8.0). The compa ison be ween esul s ob ained 26 om dila a ional and in e acial shea measu emen s p o ides accu a e de ails ela ed o he 27 mic os uc u e o he p o ein a he O/W in e ace. Viscoelas ic moduli o he p o ein adso bed 28 a he O/W in e ace we e ob ained h ough dila a ional measu emen s (T acke , IT Concep ), 29 as well as in e acial SAOS measu emen s, using a double wall- ing geome y i ed o a DHR3 30 heome e (TA In umen s). Resul s om dila a ional heology indica e ha p o eins a e able o 31 o m a ilm a O/W in e ace, whose s eng h depends on he pH alue (whe e E’ anges om 32 2.0·10-2 Pa·m a pH 5.0 o 5.5·10-3 Pa·m a pH 8.0). Howe e , hese measu emen s ha e shown 33 o be less sensi i e o pH modi ica ions han in e acial shea iscoelas ic measu emen s. 34 Rega dless o he pH alue, in e acial shea measu emen s can ollow he e olu ion o he 35 iscoelas ic beha iou o he O/W in e acial laye o e he de elopmen o he p o ein gel 36 ne wo k, as p o ein adso p ion p oceeds. This e olu ion indica es ha p o ein adso p ion akes 37 place much as e a pH 3.0 and 5.0 han a pH 8.0. 38 39 Keywo ds: Faba bean p o ein; In e acial Shea Rheology; Linea iscoelas ici y; Pendan 40 D ople ; P o ein adso p ion 41 1. In oduc ion 42 In e acial heology p o ides key in o ma ion o unde s anding he dynamic beha iou o 43 complex luid- luid in e aces, which a e he basis o no only d ople o ma ion and s abili y 44 in emulsions, bu also s uc u al and mechanical p ope ies (Wijmans & Dickinson, 1998). The 45 adso p ion o model globula p o ein molecules such as β-lac oblobulin, o albumin o BSA a 46 O/W has been p e iously s udied (E Dickinson, 1999). In ac , he e a e success ul s udies in 47 which he unde s anding o complex in e aces is p o ided by hei in e p e a ion as a quasi-48 wo-dimensional ne wo k o igid sphe ical pa icles, which ep esen globula p o eins and a e 49 c oss-linked (Wijmans & Dickinson, 1998). These models a e based on he p emise ha he 50 heological p ope ies obse ed o a globula p o ein adso bed a he O/W in e ace a e simila 51 o he ones ound o bulk gels (E ic Dickinson, 1998). Howe e , in a mix u e be ween di e en 52 kinds o p o eins (i.e., di e ing in molecula weigh , composi ion, con o ma ion, e c.), 53 adso p ion is e y complex. In hese cases, he compe i i e adso p ion be ween di e en kinds 54 o p o eins mus be aken in o accoun ( an Aken, 2003). Fo compe i i e adso p ion, models 55 only wo k o p o eins o eagen g ade (mainly BSA and β-Lac oglobulin) and molecules such 56 as su ac an s (Faine man, Lucassen-Reynde s, & Mille , 2003; Mille e al., 2000). Howe e , 57 comme cial ood p oduc s con ain a la ge numbe o di e en p o eins as well as o he 58 componen s such as s a ch, lipids and sal s, which makes i impossible o de e mine he eal 59 na u e o he di e en in e ac ions a he O/W in e ace. Consequen ly, he in e p e a ion o he 60 beha iou o hese complex luid- luid in e aces in e ms o undamen al in e ac ions is a e y 61 di icul challenge. Recen ly, in e acial heology has been used o o e come his p oblem, 62 s udying p o eins ypically in ol ed in cell exchanges o ood p oduc s (Bö che , Kepple , & 63 D usch, 2017; Rome o, Ve wijlen, Gue e o, & Ve man , 2013; Sch oyen, Gunes, & Ve man , 64 2017; Vandeb il, F anck, Fulle , Moldenae s, & Ve man , 2010). 65 Dila a ional heology and shea heology ha e been used o he in e p e a ion o p o ein 66 in e ac ions a luid- luid in e aces (Dano e al., 2015). The o me ela es changes in 67 in e acial ension when a d ople su e s oscilla o y dila a ions, whe eas he la e uses an 68 analogous in e p e a ion o h ee-dimensional bulk sys ems in in e acial sys ems (Maldonado-69 Valde ama & Pa ino, 2010). Howe e , bo h echniques, dila a ional and shea heology, need 70 o be ca ied ou o imp o e he in e p e a ion o he p o ein in e ac ions a he O/W in e ace. 71 Dila a ional heology has been co ela ed o sho - e m s abili y, whe eas in e acial shea 72 measu emen s ha e been ela ed o middle o long- e m s abili y o emulsions (Lucassen-73 Reynde s, Benjamins, & Faine man, 2010; Rome o e al., 2013). The e is a a ie y o de ices 74 o measu ing in e acial shea heology. Among hem, disc, bicone o double wall ing (DWR) 75 geome ies can be a ached o a highly sensi i e s ess-con olled heome e o cha ac e ise 76 complex luid- luid in e aces (Felix, Rome o, Sanchez, & Gue e o, 2019; Hoogh en e al., 77 2017; Na simhan, 2016), whe e DWR is pa icula ly use ul, since i minimizes he e ec o he 78 subphases on iscoelas ic esponse (which can be es ima ed h ough he Boussinesq numbe ) 79 (Felix, Rome o, Ve man , & Gue e o, 2016; Vandeb il e al., 2010). 80 As an al e na i e o adi ional p o ein concen a es ob ained by we pa hs, d y- ac ioning aims 81 o be mo e eco- iendly and cheape han he ypical acidic/alkaline solubilisa ion and 82 isoelec ic p ecipi a ion ollowed by sp ay-d ying; his new echnique allows a oiding bo h he 83 loss o p o ein unc ionali y, which usually akes place, and he was e p oduced in he ypical 84 isoelec ic p ecipi a ion (Clou , Walke , & Pike, 1986; Schu yse , Pelg om, an de Goo , & 85 Boom, 2015). The p o ein concen a e used in his esea ch was ob ained h ough a 86 d y- ac ioning p ocess, which is based on he di e ence o size be ween s a ch and p o ein 87 g anules. As an added bene i , his p ocedu e equi es a lowe amoun o ene gy inpu (Felix, 88 Lopez-Oso io, Rome o, & Gue e o, 2018). Mo eo e , he ood indus y is in e es ed in he use 89 o plan p o eins as an al e na i e o milk o egg p o eins o he de elopmen o O/W emulsions. 90 Plan p o eins, such as Faba bean (FB), possess a numbe o bene i s o being used in ood 91 applica ions. Thus, hey a e cos e icien , exhibi a high nu i ional alue (Ma ila e al., 2018) 92 and hey may also help o a oid some well-known alle gic p oblems associa ed o animal 93 p o eins. In addi ion, Faba bean belongs o he legume amily, which is he i h mos g own 94 c op in he wo ld, (Du, Jiang, Yu, & Jane, 2014). 95 The o e all objec i e o his wo k was o assess he in e acial p ope ies o Faba bean p o ein 96 concen a e as a unc ion o pH alue (3.0, 5.0 and 8.0) in a complex luid- luid in e aces, 97 which would con ibu e o he de elopmen o FB-s abilized emulsions. The in e acial 98 beha iou was analysed by means o dila a ional measu emen s (using a pendan d op 99 ensiome e ), and in e acial shea heology (using a DWR geome y a ached o a sensi i e 100 o a ional s ess-con olled heome e ). The compa ison o esul s ob ained by bo h echniques 101 may con ibu e o es ablish he po en ial use o FB p o ein sys em ob ained by d y ac iona ion 102 in ood p oduc s such as emulsions. 103 2. Ma e ial and me hods 104 2.1. Ma e ials 105 The Faba bean(FB) p o ein concen a e (He baP o F65) used wi hin he amewo k o his 106 esea ch was p o ided by He ba Ing edien s (San José de la Rinconada, Se ille, Spain). This 107 p o ein concen a e was ob ained om di ec ly milling d ied beans om FB, and, subsequen ly, 108 he powde ob ained was submi ed o a densi ica ion p ocess. This p ocess consis s on he 109 applica ion o a cen i ugal coun e - low. A e ha , pa icles whe e he d ag o ce is highe 110 han he cen i ugal o ce a e e used and a ligh e luen , which co esponds o he p o ein- ich 111 ac ion, is gene a ed (Felix e al., 2018). The sun lowe oil was pu chased om a local p oduce 112 (Co eysa, Se ille, Spain) and o he chemical eagen s we e pu chased om Sigma-Ald ich (S . 113 Louis, Missou i, USA). 114 2.2. P o ein cha ac e isa ion 115 The p o ein con en was de e mined in quad uplica e as % N x 6.25 (Vioque, Alaiz, & Gi ón-116 Calle, 2012) using a LECO CHNS-932 ni ogen mic o analyse (Leco Co po a ion, S . Joseph, 117 MI, USA). The lipid, mois u e and ash con en s o he FB p o ein concen a e we e de e mined 118 in quad uplica e by A.O.A.C. (2000) app o ed me hods. 119 2.3. In e acial cha ac e iza ion 120 2.3.1. De e mina ion o in e acial p essu e a equilib ium 121 Measu emen s o de e mine he sa u a ion concen a ion o O/W in e ace we e pe o med 122 using a Wilhelmy pla e connec ed o a Sigma D701 ensiome e (KSV, Helsinki, Finland). The 123 measu emen s we e ca ied ou a di e en p o ein concen a e amoun s (0.1, 0.25, 0.5, 0.75, 124 1.0, 2.0, 3.0, 4.0 and 5.0 w .%) and a h ee pH alues (3.0, 5.0 and 8.0). Thus, p o ein 125 dispe sions we e p epa ed in 40 mL essels, he pH was adjus ed by adding 2M HCl o NaOH 126 and he empe a u e was kep a 20 ºC o e nigh o allow he comple e adso p ion o he p o ein 127 a he in e ace. Then, he Wilhelmy pla e was placed a he wa e /ai in e ace and he essel 128 was illed up wi h sun lowe oil (gene a ing he O/W in e ace). The in e acial p essu e was 129 measu ed a e ob aining cons an in e acial ension alues. 130 2.3.2. Pendan d ople measu emen s 131 Pendan d ople measu emen s we e ca ied ou in o de o de e mine p o ein adso p ion 132 kine ics. T ansien in e acial p essu e and in e acial dila a ional measu emen s we e ca ied 133 ou using he TRACKER pendan -d ople ensiome e (IT Concep , F ance). An axisymme ic 134 d op was o med a he ip o he needle o a sy inge whose e icali y was con olled by a 135 compu e . The d ople p o ile was digi ized and analysed h ough a CCD came a coupled o a 136 ideo image p o ile digi ize boa d connec ed o a compu e . D op p o iles we e p ocessed 137 acco ding o he Laplace equa ion as was desc ibed by Cas ellani, Al-Assa e al. (2010). 138 In addi ion, he iscoelas ic moduli o p o ein adso p ion ilms we e de e mined a 10% s ain 139 ampli ude unde non-equilib ium a 0.1 Hz, as well as a di e en equencies ( anging om 140 0.0075 o 0.1 Hz) once he pseudo-s eady s a e was achie ed a e 10,800 s. All he expe imen s 141 we e ca ied ou , a leas in iplica e. The oil was con ained in an op ical glass cu e e (8 mL), 142 whe eas he p o ein solu ion was con ained in a sy inge (200 μL). Bo h elemen s we e 143 he mos a ed a 20.0 ± 0.1 °C (Sánchez & Pa ino, 2005). 144 2.3.3. In e acial shea heological p ope ies 145 The p o ein solu ion was placed in a double wall cup and hen a double-wall- ing (DWR) was 146 ca e ully laid a he ai /wa e in e ace. Subsequen ly, sun lowe oil was ca e ully added o 147 a oid any dis u bance o he in e ace. The ing was connec ed o a highly sensi i e magne ic 148 ai -bea ing s ess-con olled heome e (DHR-3, TA Ins umen s, USA) (Vandeb il e al., 149 2010). P io o equency sweep es s, s ess swep es s we e pe o med o de e mine he linea 150 iscoelas ic egion (LVR). Then, mechanical spec a we e ob ained by means o equency 151 es s, which we e ca ied ou wi hin he linea iscoelas ic ange. The equency sweep es s 152 we e pe o med du ing he p o ein adso p ion ( equency ange om 0.314 o 6.14 ad/s) and 153 a e eaching he pseudo-equilib ium s a e ( equency ange om 0.06 o 20 ad/s). In addi ion, 154 ime sweep expe imen s we e ca ied ou o e 10,800 s a 0.1 Hz o ob ain he in e acial 155 mechanical spec a o p o ein adso p ion o elucida ing p o ein-p o ein in e ac ions. All 156 expe imen s we e ca ied ou , a leas in duplica e and he sys ems we e he mos a ed a 20.0 ± 157 0.1 °C by coupling he double wall cup di ec ly on o he Pel ie bo om pla e. Mo eo e , o 158 neglec he con ibu ion o he subphase in in e acial small ampli ude oscilla o y shea 159 measu emen s (i-SAOS), he Boussinesq numbe (Bo) was calcula ed o all he sys ems 160 s udied. This numbe can be exp essed in e ms o a p opo ion be ween he iscosi ies o he 161 in e ace and he bulk (𝜂𝜂𝑠𝑠 and 𝜂𝜂𝑏𝑏, espec i ely): 162 𝐵𝐵𝐵𝐵 = 𝜂𝜂𝑠𝑠 𝑎𝑎·𝜂𝜂𝑏𝑏 (1) 163 whe e 𝑎𝑎 is a cha ac e is ic leng h (0.07 mm o DWR geome y). 164 Bo was abo e 100 in all cases (Bo > 100). These alues indica e ha he esponse ob ained is 165 solely ela ed o he in e acial con ibu ion (E ni e al., 2003; Vandeb il e al., 2010). 166 2.4. S a is ical analysis 167 A leas h ee eplica es o each measu emen we e ca ied ou . Measu emen unce ain y was 168 de e mined by means o s anda d de ia ion. Mo eo e , signi ican di e ences (p<0.05) we e 169 analysed by means o ANOVA es s (Excel s a is ical package). 170 3. Resul s and discussion 171 3.1. P o ein sys em cha ac e isa ion 172 The chemical composi ion o FB p o ein concen a e has been epo ed in a p e ious s udy 173 (Felix e al., 2018). The p o ein con en ob ained a e he d y- ac ioning p ocess eaches up o 174 56.4 ± 0.1 w .%. Mo eo e , he densi ica ion p ocess le o a concen a e o low a (4.6 ± 0.3 175 w .%), mois u e (4.3 ± 0.8 w .%) and ash (4.7 ± 0.1 w .%) con en s. The ca bohyd a e con en 176 (es ima ed by di e ence) was up o 30 w .%. 177 3.2 Cha ac e isa ion o he O/W in e ace 178 3.2.1. De e mina ion o in e acial p essu e a equilib ium 179 Ini ially, alues o in e acial p essu e measu ed a e equilib ium (24 h p o ein adso p ion) a e 180 de e mined by means o a Wilhelmy pla e connec ed o a ensiome e . In e acial p essu e (Π) 181 alues a e calcula ed om he in e acial ension alues (σ) as ollows: 182 𝛱𝛱=𝜎𝜎0− 𝜎𝜎 (2) 183 Whe e 𝜎𝜎0 is he O/W in e acial ension alue measu ed in he absence o su ace ac i e agen s 184 (52 mN/m) and σ he expe imen al in e acial ension alue. 185 Resul s om in e acial p essu e as a unc ion o pH (3.0, 5.0 and 8.0) and FB p o ein 186 concen a e concen a ion ( om 0.1 o 5 w .%) a e plo ed in Fig. 1. As can be obse ed, he 187 inc ease o p o ein concen a ion in ol ed an inc ease in in e acial p essu e, un il i eached a 188 cons an alue. This is he ypical beha iou ound in o he p o ein/su ac an sys ems such as 189 milk p o eins (Niño & Pa ino, 1998) o soy p o eins (O iz, Sánchez, Niño, Añon, & Pa ino, 190 2003). The in e acial p essu e inc eases when he p o ein concen a ion concen a ion is below 191 he sa u a ion concen a ion o he O/W in e ace. Howe e , once he in e ace is sa u a ed, an 192 inc ease o p o ein concen a ion in he bulk does no in ol e a highe numbe o p o ein 193 molecules a he O/W in e ace. Consequen ly, he in e acial p essu e does no ise anymo e 194 (Sánchez & Pa ino, 2005). The e o e, acco ding o he esul s ob ained, he sa u a ion 195 concen a ion o he sys ems a pH 5.0 and 8.0 is eached a 2 w .%, whe eas a pH 3.0 i is 196 eached a 1 w.%, app oxima ely. These da a a e impo an , since all u he in e acial 197 expe imen s we e ca ied ou a hese FB p o ein concen a e concen a ions, a which he 198 in e ace is sa u a ed. On he o he hand, acco ding o Felix e al. (2018), he pH alue 3.0 is 199 below he isoelec ic poin (IEP) o he p o ein (3.5). The lowe concen a ion ound o pH 3.0 200 o in e acial sa u a ion may be ela ed o p o ein un olding, which akes place a his ex eme 201 pH alue. Thus, a pH 3.0, he un olded p o ein can co e he in e ace comple ely when he 202 bulk concen a ion is lowe (O. E. Pé ez, Sánchez, Piloso , & Rod íguez Pa ino, 2008). 203 Fu he mo e, he highes alue o in e acial p essu e a e eaching he pseudo equilib ium was 204 ound o he sys em a pH 8.0, whe eas equilib ium alues o he sys ems a pH 5.0 and 3.0 205 we e e y simila . These esul s may be ela ed o he closeness o he minimum solubili y o 206 he FB p o ein concen a e, which in ol es a lowe amoun o soluble p o eins a ailable o be 207 adso bed a he in e ace and, he e o e, would lead o lowe alues o su ace p essu e. Chang 208 e al. (2015) ound simila pH-in luence o o he legume p o eins. Mo eo e , hey hypo hesised 209 ha in e acial ension is ela ed o an op imum balance be ween he cha ge and hyd ophobici y 210 o he p o ein. Thus, acco ding o p e ious esul s he e iciency o FB p o eins o s abilise he 211 in e ace a pH 8.0 may be ela ed o he inc ease in su ace cha ges, as epo ed p e iously 212 (Felix e al., 2018). Thus, he ze a po en ial alues ound o FB concen a e we e ca. 15, -20 213 and -35 mV a pH 3.0, 5.0 and 8.0, espec i ely. In addi ion, a educ ion o i s hyd ophobici y 214 whe e he i s equency sweep a e 10 min o p o ein adso p ion indica es ha he in e acial 363 shea esponse is domina ed by he iscous componen a low equency. The e olu ion o he 364 iscoelas ic moduli o e he adso p ion ime shows a c osso e poin , which shi s along p o ein 365 adso p ion om low o high equencies. Ini ially, hese esul s may no be conside ed 366 consis en wi h he ones ob ained om dila a ional heology (whe e he c osso e poin was no 367 obse ed). Resul s om in e acial shea heology seem o indica e he abili y o he adso bed 368 p o eins o exhibi cohesi e in e ac ions wi h neighbou ing molecules (Na simhan, 2016). In 369 any case, a e eaching he pseudo-equilib ium alue (i.e. a e 180 min) all in e acial O/W 370 p o ein-based ilms showed a gel-like esponse, ega dless o he pH alue. These esul s om 371 in e acial shea heology no only demons a e ha i is possible o moni o he adso p ion 372 kine ics o he O/W in e ace; hey also allow ollowing he de elopmen o he p o ein gel 373 ne wo k a he in e acial laye by measu ing he mechanical spec a du ing ime ( heo-kine ics 374 o he p ocess). 375 Compa ison be ween dila a ional heology and in e acial shea heology 376 Table 1 compa es he an (δ) alues om dila a ional and shea measu emen s ( an(δ)s,0.1 dand 377 an(δ)s,0.1 s, espec i ely), as well as he dila a ional and shea in e acial elas ic moduli 378 (E′(δ)s,0.1 d and G′(δ)s,0.1 d, espec i ely) ob ained a 0.1 Hz a e he adso p ion o FB p o ein a 379 he O/W in e ace. Fi s o all, he low an (δ)s,0.1 alues ob ained (ei he ( an(δ)s,0.1 d o 380 an(δ)s,0.1 𝑠𝑠) con i m he p edominan gel-like beha iou o he in e ace. Howe e , his ma ked 381 solid cha ac e is di e en , and depends on he me hod used. Thus, an(δ)s,0.1 d alues a e equal 382 o lowe han 0.10 a any pH alue. Howe e , an(δ)s,0.1 𝑠𝑠 alues oscilla e be ween 0.2 and 0.3. 383 I is wo h men ioning ha o he low alues o he dila a ional iscoelas ic pa ame e s we e 384 p e iously ob ained o a a ie y o p o ein-s abilized in e acial laye s (Rome o e al., 2012; 385 Rome o, Beaumal, Da id-B iand, Co dobes, An on, e al., 2011; Rome o, Beaumal, Da id-386 B iand, Co dobes, Gue e o, e al., 2011). In addi ion, he compa ison o G′(δ)s,0.1 d alues 387 indica es ha no only s ong in e ac ions a he O/W in e ace ake place, bu also he o ma ion 388 o a densely packed in e ace (E ni, Windhab, & Fische , 2011; F ee e al., 2004). In any case, 389 his pa ame e co obo a es he s ong dependence on pH exe ed by he FB p o ein sys em, 390 whe e he ne con ibu ion o p o ein-p o ein in e ac ions o he in e acial ilm is simila a pH 391 3.0 and 5.0 (p o ein su ace posi i ely o null ne cha ged), whe eas a pH 8.0 he p o ein 392 in e ac ions a e weake (p o ein su ace nega i ely cha ged). This e ec may be ela ed o he 393 dependence o su ace cha ges and hyd ophobici y on pH alues, wi h he in e acial ac i i y 394 o FB p o eins being educed a alkaline pH. 395 4. Concluding ema ks 396 The use o he d y- ac ioning p ocess ca ied ou a oids he was e o la ge amoun s o wa e 397 as well as he exposi ion o p o ein o ex eme pH alues and hei undesi able side e ec s, 398 p o iding a FB p o ein concen a e (56.4 w .%) wi h high in e acial ac i i y. The esul s om 399 in e acial p essu e (П) a e eaching he equilib ium a he O/W in e ace indica e ha he 400 e ec o pH on su ace cha ges and su ace hyd ophobici y (H0) has a ema kable in luence on 401 in e acial p ope ies. The highe su ace p essu e was ound in he sys em a pH 8.0, whe e he 402 su ace is high nega i ely cha ged. 403 The esul s om he dila a ional iscoelas ic measu emen s indica e ha p o eins a e able o 404 o m a ilm wi h a gel-like beha iou a he O/W in e ace, whose s eng h depends, once again, 405 on he pH alue. Howe e , hese measu emen s ha e shown o be less sensi i e o pH 406 modi ica ions han in e acial shea iscoelas ic measu emen s. Mo eo e , he e olu ion o 407 iscoelas ic p ope ies can be mo e clea ly ollowed by his la e echnique, which shows ha 408 p o ein adso p ion akes place much as e a pH 3.0 and 5.0 han a pH 8.0. Rega dless o he 409 pH alue, i-SAOS measu emen s can ollow he e olu ion o he iscoelas ic beha iou o he 410 O/W in e acial laye o e he de elopmen o he p o ein gel ne wo k. Thus, in e acial 411 heokine ics can be use ul o de e mine he sol-gel ansi ion a he in e acial laye . 412 In summa y, hese esul s con i m he ele ance o pe o ming bo h in e acial ension and 413 in e acial shea measu emen s a he O/W in e ace. The o me measu emen s a e ele an o 414 he emulsi ica ion p ocess, whe eas he la e allow ollowing he de elopmen o he p o ein 415 mic os uc u e a he in e ace (i.e. in e acial heo-kine ics) which is ega ded o be use ul o 416 emulsion s abili y. Addi ionally, hey con ibu e o con i m ha Faba bean p o ein concen a e 417 ob ained by d y ac iona ion can p o ide s ong O/W in e aces, whe e he s onges in e ace 418 was ob ained a pH 3.0. 419 5. Acknowledgmen s 420 The au ho s acknowledge o Andalusian Go e nmen o hi ing his esea ch unde he esea ch 421 p ojec TEP-6134. Au ho s also acknowledge o He ba Ing edien s o supplying he p o ein 422 powde used in his esea ch. E en ually, he au ho s also acknowledge Uni e si y o Se ille 423 o he VPPI-US g an (Re .-II.5) o Manuel Felix. 424 6. Re e ences 425 A.O.A.C. (2000). O icial Me hods o Analysis. (W. Ho wi z, Ed.) (11 h ed.). Wisconsin: 426 Associa ion o O icial Analy ical Chemis . 427 Baldu sdo i , S. G., Fulle on, M. S., Nielsen, S. H., & Jo gensen, L. (2010). Adso p ion o 428 p o eins a he oil/wa e in e ace—Obse a ion o p o ein adso p ion by in e acial shea 429 s ess measu emen s. Colloids and Su aces B: Bioin e aces, 79(1), 41–46. 430 h ps://doi.o g/h p://dx.doi.o g/10.1016/j.colsu b.2010.03.020 431 Ba nes, H. A. (2000). A Handbook o Elemen a y Rheology. Uni e si y o Wales, Ins i u e o 432 Non-New onian Fluid Mechanics. Re ie ed om 433 h p://books.google.no/books?id=zBY AQAAIAAJ 434 Bö che , S., Kepple , J. K., & D usch, S. (2017). Mix u es o Quillaja saponin and be a-435 lac oglobulin a he oil/wa e -in e ace: Adso p ion, in e acial heology and emulsion 436 p ope ies. Colloids and Su aces A: Physicochemical and Enginee ing Aspec s, 518, 46–437 56. h ps://doi.o g/h ps://doi.o g/10.1016/j.colsu a.2016.12.041 438 Cas ellani, O., Al-Assa , S., Axelos, M., Phillips, G. O., & An on, M. (2010). Hyd ocolloids 439 wi h emulsi ying capaci y. Pa 2-Adso p ion p ope ies a he n-hexadecane-Wa e 440 in e ace. Food Hyd ocolloids, 24(2–3), 121–130. 441 h ps://doi.o g/10.1016/j. oodhyd.2009.07.006 442 Chang, C., Tu, S., Ghosh, S., & Nicke son, M. T. (2015). E ec o pH on he in e -443 ela ionships be ween he physicochemical, in e acial and emulsi ying p ope ies o 444 pea, soy, len il and canola p o ein isola es. Food Resea ch In e na ional, 77, Pa 3, 445 360–367. h ps://doi.o g/h p://dx.doi.o g/10.1016/j. ood es.2015.08.012 446 Clou , P., Walke , A. F., & Pike, D. J. (1986). Ai classi ica ion o lou s o h ee legume 447 species: E ec o s a ch g anule size dis ibu ion. Jou nal o he Science o Food and 448 Ag icul u e, 37(2), 173–184. h ps://doi.o g/10.1002/js a.2740370212 449 Dano , K. D., K alche sky, P. A., Radulo a, G. M., Bashe a, E. S., S oyano , S. D., & Pelan, 450 E. G. (2015). Shea heology o mixed p o ein adso p ion laye s s hei s uc u e s udied 451 by su ace o ce measu emen s. Ad ances in Colloid and In e ace Science, 222, 148–452 161. h ps://doi.o g/h p://dx.doi.o g/10.1016/j.cis.2014.04.009 453 Dickinson, E. (1998). P o eins a in e aces and in emulsions S abili y, heology and 454 in e ac ions. J. Chem. Soc., Fa aday T ans., 94(12), 1657–1669. 455 h ps://doi.o g/10.1039/A801167B 456 Dickinson, E. (1999). Adso bed p o ein laye s a luid in e aces: in e ac ions, s uc u e and 457 su ace heology. Colloids and Su aces B-Bioin e aces, 15(2), 161–176. 458 h ps://doi.o g/10.1016/s0927-7765(99)00042-99 459 Du, S., Jiang, H., Yu, X., & Jane, J. (2014). Physicochemical and unc ional p ope ies o 460 whole legume lou . LWT - Food Science and Technology, 55(1), 308–313. 461 h ps://doi.o g/h p://dx.doi.o g/10.1016/j.lw .2013.06.001 462 E ni, P., Fische , P., Windhab, E. J., Kusnezo , V., S e in, H., & Lauge , J. (2003). S ess- 463 and s ain-con olled measu emen s o in e acial shea iscosi y and iscoelas ici y a 464 liquid/liquid and gas/liquid in e aces. REVIEW OF SCIENTIFIC INSTRUMENTS, 465 74(11), 4916–4924. h ps://doi.o g/10.1063/1.1614433 466 E ni, P., Windhab, E. J., & Fische , P. (2011). Emulsion D ops wi h Complex In e aces: 467 Globula Ve sus Flexible P o eins. Mac omolecula Ma e ials and Enginee ing, 296(3–468 4), 249–262. h ps://doi.o g/10.1002/mame.201000290 469 Faine man, V. B., Ko alchuk, V. I., Aksenenko, E. V, & Mille , R. (2016). Dila ional 470 Viscoelas ici y o Adso p ion Laye s Measu ed by D op and Bubble P o ile Analysis: 471 Reason o Di e en Resul s. Langmui , 32(22), 5500–5509. 472 h ps://doi.o g/10.1021/acs.langmui .6b01134 473 Faine man, V. B., Lucassen-Reynde s, E. H., & Mille , R. (2003). Desc ip ion o he 474 adso p ion beha iou o p o eins a wa e / luid in e aces in he amewo k o a wo-475 dimensional solu ion model. Ad ances in Colloid and In e ace Science, 106(1–3), 237–476 259. h ps://doi.o g/h p://dx.doi.o g/10.1016/S0001-8686(03)00112-X 477 Felix, M., Lopez-Oso io, A., Rome o, A., & Gue e o, A. (2018). Faba bean p o ein lou 478 ob ained by densi ica ion: A sus ainable me hod o de elop p o ein concen a es wi h 479 ood applica ions. LWT, 93, 563–569. 480 h ps://doi.o g/h ps://doi.o g/10.1016/j.lw .2018.03.078 481 Felix, M., Rome o, A., & Gue e o, A. (2017). Viscoelas ic p ope ies, mic os uc u e and 482 s abili y o high-oleic O/W emulsions s abilised by c ay ish p o ein concen a e and 483 xan han gum. Food Hyd ocolloids, 64, 9–17. 484 h ps://doi.o g/h p://dx.doi.o g/10.1016/j. oodhyd.2016.10.028 485 Felix, M., Rome o, A., Sanchez, C. C., & Gue e o, A. (2019). Modelling he non-linea 486 in e acial shea heology beha iou o chickpea p o ein-adso bed complex oil/wa e 487 laye s. Applied Su ace Science, 469, 792–803. 488 h ps://doi.o g/h ps://doi.o g/10.1016/j.apsusc.2018.11.074 489 Felix, M., Rome o, A., Ve man , J., & Gue e o, A. (2016). In e acial p ope ies o highly 490 soluble c ay ish p o ein de i a i es. Colloids and Su aces A: Physicochemical and 491 Enginee ing Aspec s, 499, 10–17. 492 h ps://doi.o g/h p://dx.doi.o g/10.1016/j.colsu a.2016.03.037 493 F ee , E. M., Yim, K. S., Fulle , G. G., & Radke, C. J. (2004). In e acial heology o globula 494 and lexible p o eins a he hexadecane/wa e in e ace: Compa ison o shea and 495 dila a ion de o ma ion. Jou nal o Physical Chemis y B, 108(12), 3835–3844. 496 h ps://doi.o g/10.1021/jp037236k 497 Hoogh en, R. Van, Blai , V. E., Vanan oye, A., Scho ield, A. B., Ve man , J., & Thijssen, J. 498 H. J. (2017). In e acial Rheology o S e ically S abilized Colloids a Liquid In e aces 499 and I s E ec on he S abili y o Picke ing Emulsions. Langmui , 33(17), 4107–4118. 500 h ps://doi.o g/10.1021/acs.langmui .6b04365 501 Lucassen-Reynde s, E. H., Benjamins, J., & Faine man, V. B. (2010). Dila ional heology o 502 p o ein ilms adso bed a luid in e aces. Cu en Opinion in Colloid & In e ace 503 Science, 15(4), 264–270. h ps://doi.o g/h p://dx.doi.o g/10.1016/j.cocis.2010.05.002 504 Maldonado-Valde ama, J., & Pa ino, J. M. R. (2010). In e acial heology o p o ein–505 su ac an mix u es. Cu en Opinion in Colloid & In e ace Science, 15(4), 271–282. 506 h ps://doi.o g/h ps://doi.o g/10.1016/j.cocis.2009.12.004 507 Ma ila, P., Mäkinen, S., Eu ola, M., Jala a, T., Pihla a, J.-M., Hells öm, J., & Pihlan o, A. 508 (2018). Nu i ional Value o Comme cial P o ein-Rich Plan P oduc s. Plan Foods o 509 Human Nu i ion, 73(2), 108–115. h ps://doi.o g/10.1007/s11130-018-0660-7 510 Mille , R., Faine man, V. B., Makie ski, A. V, K ägel, J., G igo ie , D. O., Kazako , V. N., 511 & Sinyachenko, O. V. (2000). Dynamics o p o ein and mixed p o ein/su ac an 512 adso p ion laye s a he wa e / luid in e ace. Ad ances in Colloid and In e ace Science, 513 86(1–2), 39–82. h ps://doi.o g/h p://dx.doi.o g/10.1016/S0001-8686(00)00032-4 514 Na simhan, G. (2016). Cha ac e iza ion o In e acial Rheology o P o ein-S abilized Ai --515 Liquid In e aces. Food Enginee ing Re iews, 8(3), 367–392. 516 h ps://doi.o g/10.1007/s12393-015-9133-z 517 Niño, M. R. R., & Pa ino, J. M. R. (1998). Su ace ension o p o ein and insoluble lipids a 518 he ai -aqueous phase in e ace. Jou nal o he Ame ican Oil Chemis s’ Socie y, 75(10), 519 1233. h ps://doi.o g/10.1007/s11746-998-0168-7 520 Nino, M. R. R., Sanchez, C. C., Ruiz-Henes osa, V. P., & Pa ino, J. M. R. (2005). Milk and 521 soy p o ein ilms a he ai -wa e in e ace. Food Hyd ocolloids, 19(3), 417–428. 522 h ps://doi.o g/10.1016/j. oodhyd.2004.10.008 523 Nosko , B. A., Mikhailo skaya, A. A., Lin, S. Y., Loglio, G., & Mille , R. (2010). Bo ine 524 Se um Albumin Un olding a he Ai /Wa e In e ace as S udied by Dila ional Su ace 525 Rheology. Langmui , 26(22), 17225–17231. h ps://doi.o g/10.1021/la103360h 526 O iz, S. E. M., Sánchez, C. C., Niño, M. R. R., Añon, M. C., & Pa ino, J. M. R. (2003). 527 S uc u al cha ac e iza ion and su ace ac i i y o sp ead and adso bed soy globulin ilms 528 a equilib ium. Colloids and Su aces B: Bioin e aces, 32(1), 57–67. 529 h ps://doi.o g/h ps://doi.o g/10.1016/S0927-7765(03)00146-2 530 Pe ei a, L. G. C., Theodoly, O., Blanch, H. W., & Radke, C. J. (2003). Dila a ional heology 531 o BSA con o me s a he ai /wa e in e ace. Langmui , 19(6), 2349–2356. 532 h ps://doi.o g/10.1021/la020720e 533 Pe ez, A. A., Ca e a, C. R., Sanchez, C. C., San iago, L. G., & Pa ino, J. M. R. (2009). 534 In e acial dynamic p ope ies o whey p o ein concen a e/polysaccha ide mix u es a 535 neu al pH. Food Hyd ocolloids, 23(5), 1253–1262. 536 h ps://doi.o g/10.1016/j. oodhyd.2008.08.013 537 Pe ez, A. A., Sánchez, C. C., Pa ino, J. M. R., Rubiolo, A. C., & San iago, L. G. (2010). Milk 538 whey p o eins and xan han gum in e ac ions in solu ion and a he ai –wa e in e ace: A 539 heokine ic s udy. Colloids and Su aces B: Bioin e aces, 81(1), 50–57. 540 h ps://doi.o g/h ps://doi.o g/10.1016/j.colsu b.2010.06.021 541 Pé ez, O. E., Sánchez, C. C., Piloso , A. M. R., & Rod íguez Pa ino, J. M. (2008). Dynamics 542 o adso p ion o hyd oxyp opyl me hylcellulose a he ai –wa e in e ace. Food 543 Hyd ocolloids, 22(3), 387–402. h ps://doi.o g/10.1016/j. oodhyd.2006.12.005 544 Pé ez, O., Sánchez, C., Piloso , A., & Rod íguez, J. M. (2009). chang 2015. Jou nal o 545 Colloid and In e ace Science, 336(2), 485–496. 546 h ps://doi.o g/10.1016/j.jcis.2009.04.011 547 Rod íguez Pa ino, J. M., Ca e a Sánchez, C., Molina O iz, S. E., Rod íguez Niño, M. R., & 548 Añón, M. C. (2004). Adso p ion o Soy Globulin Films a he Ai −Wa e In e ace. 549 Indus ial & Enginee ing Chemis y Resea ch, 43(7), 1681–1689. 550 h ps://doi.o g/10.1021/ie0302443 551 Rod iguez Pa ino, J. M., Rod iguez Nino, M. R., & Sanchez, C. C. (1999). Adso p ion o 552 whey p o ein isola e a he oil-wa e in e ace as a unc ion o p ocessing condi ions: a 553 heokine ic s udy. Jou nal o Ag icul u al and Food Chemis y, 47(6), 2241–2248. 554 h ps://doi.o g/10.1021/j 981119i 555 Rome o, A., Beaumal, V., Da id-B iand, E., Co dobes, F., An on, M., & Gue e o, A. (2011). 556 In e acial and emulsi ying beha iou o c ay ish p o ein isola e. Lw -Food Science and 557 Technology, 44(7), 1603–1610. h ps://doi.o g/10.1016/j.lw .2011.03.005 558 Rome o, A., Beaumal, V., Da id-B iand, E., Co dobes, F., Gue e o, A., & An on, M. (2011). 559 In e acial and Oil/Wa e Emulsions Cha ac e iza ion o Po a o P o ein Isola es. Jou nal 560 o Ag icul u al and Food Chemis y, 59(17), 9466–9474. 561 h ps://doi.o g/10.1021/j 2019853 562 Rome o, A., Beaumal, V., Da id-B iand, E., Co dobes, F., Gue e o, A., & An on, M. (2012). 563 In e acial and emulsi ying beha iou o ice p o ein concen a e. Food Hyd ocolloids, 564 29(1), 1–8. h ps://doi.o g/10.1016/j. oodhyd.2012.01.013 565 Rome o, A., Ve wijlen, T., Gue e o, A., & Ve man , J. (2013). In e acial beha iou o 566 c ay ish p o ein isola e. Food Hyd ocolloids, 30(1), 470–476. 567 h ps://doi.o g/10.1016/j. oodhyd.2012.07.009 568 Sánchez, C. C., & Pa ino, J. M. R. (2005). In e acial, oaming and emulsi ying cha ac e is ics 569 o sodium caseina e as in luenced by p o ein concen a ion in solu ion. Food 570 Hyd ocolloids, 19(3), 407–416. 571 h ps://doi.o g/h p://dx.doi.o g/10.1016/j. oodhyd.2004.10.007 572 Sch oyen, B., Gunes, D. Z., & Ve man , J. (2017). A e sa ile subphase exchange cell o 573 in e acial shea heology. Rheologica Ac a, 56(1), 1–10. 574 Schu yse , M. A. I., Pelg om, P. J. M., an de Goo , A. J., & Boom, R. M. (2015). D y 575 ac iona ion o sus ainable p oduc ion o unc ional legume p o ein concen a es. 576 T ends in Food Science & Technology, 45(2), 327–335. 577 h ps://doi.o g/h ps://doi.o g/10.1016/j. i s.2015.04.013 578 Tamm, F., He bs , S., B odko b, A., & D usch, S. (2016). Func ional p ope ies o pea p o ein 579 hyd olysa es in emulsions and sp ay-d ied mic ocapsules. Food Hyd ocolloids, 58, 204–580 214. h ps://doi.o g/h p://dx.doi.o g/10.1016/j. oodhyd.2016.02.032 581 Tang, C.-H., & Shen, L. (2015). Dynamic adso p ion and dila a ional p ope ies o {BSA} a 582 oil/wa e in e ace: Role o con o ma ional lexibili y. Food Hyd ocolloids, 43, 388–399. 583 h ps://doi.o g/h p://dx.doi.o g/10.1016/j. oodhyd.2014.06.014 584 an Aken, G. A. (2003). Compe i i e adso p ion o p o ein and su ac an s in highly 585 concen a ed emulsions: e ec on coalescence mechanisms. Colloids and Su aces A-586 Physicochemical and Enginee ing Aspec s, 213(2–3), 209–219. 587 h ps://doi.o g/10.1016/s0927-7757(02)00514-9 588 Vandeb il, S., F anck, A., Fulle , G. G., Moldenae s, P., & Ve man , J. (2010). A double wall-589 ing geome y o in e acial shea heome y. Rheologica Ac a, 49(2), 131–144. 590 h ps://doi.o g/10.1007/s00397-009-0407-3 591 Vioque, J., Alaiz, M., & Gi ón-Calle, J. (2012). Nu i ional and unc ional p ope ies o Vicia 592 aba p o ein isola es and ela ed ac ions. Food Chemis y, 132(1), 67–72. 593 h ps://doi.o g/h p://dx.doi.o g/10.1016/j. oodchem.2011.10.033 594 Wa d, A. F. H., & To dai, L. (1946). Time-dependence o bounda y ensions o solu ions. 1. 595 The ole o di usion in ime-e ec s. Jou nal o Chemical Physics, 14(7), 453–461. 596 h ps://doi.o g/10.1063/1.1724167 597 Figu e 4 634 635 636 Figu e 5 637 638 639 Figu e 6 640 641 0.1 110 F equency ( ad/s) G' s G'' s G' s G'' s 10min 60min 20min 90min 40min 180min C 0.1 110 F equency ( ad/s) G' s G'' s G' s G'' s 10min 60min 20min 90min 40min 180min B 0,1 110 10 -3 10 -2 F equency ( ad/s) G' s , G'' s / (Pa·m) G' s G'' s G' s G'' s 10min 60min 20min 90min 40min 180min A