Impact of acidification and protein fortification on thermal properties of rice, potato and tapioca starches and rheological behaviour of their gels

Impac o acidi ica ion and p o ein o i ica ion on he mal p ope ies o ice, po a o and apioca s a ches and heological beha iou o hei gels Ma ina Villanue a1, Felicidad Ronda1, Thomas Moschakis2, A hina Laza idou2, Cos as G. Biliade is2* (1) Depa men o Ag icul u e and Fo es y Enginee ing, Food Technology, College o Ag icul u al and Fo es y Enginee ing, Uni e si y o Valladolid, A . Mad id, 44, 34004 Palencia, Spain. (2) Depa men o Food Science and Technology, Facul y o Ag icul u e, Fo es y and Na u al En i onmen , A is o le Uni e si y o Thessaloniki, P.O. Box 235, Thessaloniki GR-541 24, G eece * Co esponding au ho : bil[email p o ec ed] h.g ; Tel: +302310991797 Abs ac The impac o acidi ica ion and non-glu en p o ein o i ica ion (egg-albumin and soy-p o ein isola e) on he mal ansi ions o ice, po a o and apioca s a ches as well as he iscoelas ic p ope ies o hei gels p epa ed a wo cas ing empe a u es, 90ºC and 120ºC, was in es iga ed. The he mal and heological beha iou o s a ches depended on hei bo anical o igin and we e signi ican ly in luenced by he p esence and ype o p o ein added as well as by he pH o he aqueous dispe sion. Acidi ica ion o pH 4.5 inc eased he gela iniza ion empe a u e o ice s a ch in he p esence o albumin o soy p o eins, while educed i in he case o apioca s a ch, ega dless o he p esence o p o eins. Acidi ica ion o ice s a ch dispe sions dec eased signi ican ly he appa en gela iniza ion en halpy; his e ec was e en g ea e in he p esence o p o eins. The addi ion o p o eins b ough abou a s uc u ing e ec on apioca gels leading o highe iscoelas ic moduli and lowe an δ alues. In gene al, acidi ica ion led o weake gel s uc u es, wi h mo e p onounced e ec o po a o s a ch, mos likely ela ed o i s highe phospha e con en (cha ge sc eening). Much weake gels we e ob ained a 120ºC compa ed o hose p ocessed a lowe empe a u es; howe e , p o ein inco po a ion ein o ced gel s uc u e, an e ec ha was no obse ed in gels o med a 90º, as also e ealed by mic os uc u e analysis using con ocal scanning lase mic oscopy. In conclusion, p o ein addi ion and pH adjus men s o aqueous s a ch dispe sions can p o ide an e ec i e means o modula e he unc ional and ex u al p ope ies o gel-like s a ch-based glu en- ee o mula ions. Keywo ds: Acidi ica ion; s a ch; The mal p ope ies; Rheological p ope ies o s a ch gels; Non- glu en p o eins; Con ocal scanning lase mic oscopy 1. In oduc ion Glu en- ee (GF) p oduc s a e a g owing p ocessed oods sec o and he ela ed esea ch ac i i y cons i u es a p io i ized and challenging opic in he a ea o ce eal-based p oduc s. The de elopmen o new GF ood i ems is eme ging no only because he daily die a y equi emen s o essen ial nu ien s o celiac disease pa ien s a e no ully me a p esen by exis ing p oduc s, bu also because o indus y needs o p oduc di e si ica ion (Mandala & Kapsoke alou, 2011). Mo eo e , he a ge g oup o GF p oduc s is cu en ly expanding o include, in addi ion o clinically diagnosed celiac pa ien s (1-3% o he popula ion), people looking o non-alle genic ing edien s, hus leading o a new ma ke p oduc ca ego y whe e a a ie y o i ems a e being o e ed o he consume . S a ch is a mac o-cons i uen in many aw ma e ials and p ocessed oods wi h pa icula impo ance om bo h a nu i ional and a unc ional poin o iew; in many o mula ed p oduc s i s ole is pi o al as ex u e modi ie (Walkens öm and He mansson, 1998), con ibu ing o many quali y aspec s o p ocessed oods (Chen e al., 2015). Rice, po a o and apioca a e among he mos commonly used s a ches o ex u e de elopmen in many glu en- ee p oduc s. In almos all applica ions, s a chy ood ma ices a e hea ed and shea ed du ing manu ac u ing. When he s a ch g anules a e hea ed in he p esence o wa e se e al changes a a molecula and s uc u al le el occu and he o de ed (pa ially c ys alline) na i e g anules swell and o m a iscous dispe sion (Biliade is, 2009; Ca ls ed e al., 2015). The empe a u e and ene gy equi ed o hese phase ansi ions a e usually p obed by di e en ial scanning calo ime y (DSC) ha moni o s di e ences in s uc u al o ganiza ion and phase ansi ion beha io among s a ches subjec ed o he mal ea men s in aqueous media (Biliade is, 1983, 2009; Roos, 1995; Singh e al., 2003). Du ing he mal p ocessing-manu ac u ing and consump ion o s a chy oods, hyd ocolloid gels a e o med and hey a e subjec ed o la ge de o ma ions ha may cause ei he i e e sible de o ma ion o s uc u al ailu e due o ac u e (Tabilo-Munizaga and Ba bosa-Càno as, 2005). The e o e, he s udy o heological p ope ies o s a ch gels is essen ial o p ope handling and modi ica ion o senso y a ibu es o s a ch-based oods. Rice s a ch is one o he mos common ing edien s used in GF p oduc s (Ma os & Rosell, 2015), al hough i does no o m sel -suppo ing gel ne wo ks, e en a concen a ions abo e 20%, ha a e easily disin eg a ed (Abebe & Ronda 2014). S a ches om ube s and oo s, such as po a o and apioca, wi h no able highe amylose con en , a e also use ul hyd ocolloid al e na i es in gel- like glu en- ee o mula ions (Bu el, 2003). Se e al me hods o modi y ex u e o s a chy oods ha e been explo ed. Among hem, acidi ica ion o ice-based ood sys ems has been es ed by means o addi ion o ace ic o lac ic acid (Blanco e al., 2011; Kasai e al., 2001) and hei blends (Ronda e al., 2014; Villanue a e al., 2015), o hei p oduc ion om lac ic acid bac e ia e men a ion in he o m o sou dough (Moo e, Del Bello & A end , 2008), which seems o be a p omising al e na i e. Ace ic acid inc eased he anspa ency, glossiness and s ickiness and dec eased he ha dness o cooked ice (Kasai, Taniha a, Ohishi, Shimada & Ha ae, 2001). Acidi ica ion also had an e ec on complex sys ems such as ice based glu en- ee doughs (Jekle & Becke , 2012; Ohishi e al., 2007; Ronda e al., 2014), impa ing he quali y o he inal baked p oduc s (Blanco e al., 2011; Jaya am e al., 2014; Villanue a e al., 2015). Mo eo e , ace ic and lac ic acids con e accep able senso ial p ope ies o GF b eads in e ms o odou and as e, ei he when p oduced by a s a e cul u e o added as ing edien s in b ead o mula ions, leading o s aling e a da ion (Moo e, Del Bello & A end , 2008). This could be a ibu ed o an inc ease in ne p o ein cha ge, which enhances p o ein-sol en in e ac ions and p o ein un olding, bu e en ually p e en s o ma ion o a s ong ne wo k s uc u e, as no ed o glu en-based doughs (Jaya am e al., 2014). The highe solubili y and deg ada ion o ice lou p o eins in ace ic acid han in dis illed wa e migh also accele a e he abso p ion, swelling and gela iniza ion o s a ch o ice lou (Ohishi e al., 2003). Inco po a ion o exogenous p o eins is ano he al e na i e o imp o e he inal p ope ies (physical, ex u al and nu i ional) o s a chy p oduc s; i.e. besides polysaccha ides, p o eins a e known as s uc u e- and ex u e-mac omolecula modi ie s in oods (Da Sil a and Rao, 2007). Such en ichmen is e en mo e impo an o glu en- ee o mula ions ha usually ha e a poo p o ein/s a ch balance. Soy p o ein isola e (SPI) and d ied egg albumin (EA) ha e been in es iga ed in GF applica ions due o hei nu i ional quali y and oam-s abilizing ac i i y (C ocke e al., 2011; Ma co & Rosell, 2008a, 2008b; Ronda e al., 2014; Villanue a e al., 2015). These globula p o eins ha e he abili y o hea -se in a gel s a e whe e a numbe o in a- and in e molecula disul ide bonds as well as hyd ophobic in e ac ions be ween nonpola amino acid g oups a e in ol ed (Chang e al., 2016). The s uc u e o hese gels is dependen on bo h pH and ionic s eng h (B ownsey e al., 1989). Howe e , he impac o acidi ica ion on p o ein- en iched s a ch sys ems has been li le s udied. We ha e p e iously epo ed a s uc u e weakening e ec by acidi ica ion on p o ein-en iched ice s a ch b ead doughs (Ronda e al., 2014) ha led o imp o ed ex u e o p o ein-en iched glu en ee b eads (Villanue a e al., 2015). Howe e , he impac o p o eins and he modula ing e ec o pH on he he mal p ope ies o s a ches om di e en sou ces as well as he heological beha io o hei gels ha e no been ex ensi ely s udied so a . Also, pending and impo an o he de elopmen o au ocla ed gel- like oods, is he e alua ion o he e ec o p ocessing empe a u es on o ma ion o mixed s a ch-p o ein gels and hei heological p ope ies. Conside ing all hese ac o s, he aim o he p esen wo k was o s udy he e ec o dec easing he pH o s a ch dispe sions o 4.5 on he he mal p ope ies o ice, po a o and apioca s a ches and hei mix u es wi h egg whi e and soybean p o ein isola e (a 10% by weigh le el) and on he heological p ope ies o hei gels p epa ed a 90º (common cooking empe a u es) and 120ºC (s e iliza ion empe a u es). E alua ion o he mic os uc u e o he s a ch dispe sions and gels was also ca ied ou using con ocal lase scanning mic oscopy (CLSM) o elucida ion o hei s uc u al ea u es. The knowledge gained om his wo k may help o modula e he o mula ion and imp o e ex u e o new gel-like glu en- ee ood p oduc s. 2. Ma e ial and me hods 2.1. Ma e ials Rice s a ch (9.9 % w/w mois u e, 0.5 % p o ein and <10% amylose con en e e ed o o al s a ch) and po a o s a ch (19.1 % w/w mois u e, 0.45 % w/w p o ein and 22% amylose e e ed o o al s a ch) we e supplied by Fe e Alimen ación S.A. (Ba celona, Spain), whe eas apioca s a ch (13 % mois u e, 0.1% w/w p o ein, and 17% amylose con en e e ed o o al s a ch) was supplied by Ca gill (B enn ag) S.L. Soy bean p o ein isola e was Sup o 500-E IP (pu i y ~82%) om P o eedo a hispano-holandesa S.A. (Ba celona, Spain) and egg albumin in d y powde o m (pu i y ~92%) was om Eu o o (Valladolid, Spain). A sodium ace a e (100 mM, pH 4.5) bu e was used o acidi y he s a ch-p o ein dispe sions; glacial ace ic acid o 1.0 M NaOH (analy ical g ade), supplied by Pan eac (Ba celona, Spain) we e used o adjus he pH a he desi ed le el. Double-dis illed wa e was used o p epa e all he solu ions and aqueous hyd ocolloid dispe sions. 2.2. Me hods 2.2.1. S a ch dispe sions and gel p epa a ion S a ch gels we e made by hea ing 20g o s a ch o s a ch+p o ein blend in 100 g o suspension in a he me ically sealed cylind ical s ainless s eel con aine (25 mm inne diame e and 60 mm heigh ) a 90 o 120ºC. The p o ein con en in he solid s a ch-p o ein mix u es was ei he 0 o 10 g/100 g o mix u e which means 0 o 2 g o p o eins in 100g o aqueous dispe sion. When he bu e was added, he double dis illed wa e was eplaced by he 100 mM ace a e bu e (pH=4.5). I was checked and ound ha in his case, he esul an pH in he dispe sions was always 4.5. Gels we e made by imme sion o he s ainless s eel con aine con aining he s a ch dispe sion in a p ehea ed oil ba h (a 120ºC) o in a wa e ba h (main ained a 90ºC) and kep he e o 7 min o accomplish gela ion o he s a ch/p o ein dispe sions. Du ing he hea ing pe iod he de ice was shaken manually and once he ime elapsed, he de ice was apidly cooled by imme sion in a wa e ba h a 18 ± 3 ºC o 5 min, wi hou loss o wa e o any mechanical damage (lack o shea ) o he p e o med upon hea ing gel ne wo k. 2.2.2. The mal p ope ies Di e en ial scanning calo ime y (DSC) measu emen s we e ca ied ou wi h a PL DSC-Gold calo ime e (Polyme Labs. L d, Epsom, UK). Samples o abou 4.5-6.5 mg d y ma e o s a ch o s a ch-p o ein mix u es we e dispe sed in wa e o in he acidi ied medium (20% w/w dispe sions) and sealed he me ically in o 120 µL DSC medium p essu e s ainless s eel pans (ME- 29990, Me le , Toledo, SAE). The dispe sions we e made ei he wi h dis illed wa e o wi h a 100 mM ace a e bu e (pH=4.5). Samples we e scanned om 10 o 125 °C a 5 °C/min using an emp y pan as e e ence. The onse , endse , and peak empe a u es, To, Te, Tp (ºC), o empe a u e alues a which he ansi ion s a s, ends o gi es he maximum signal, espec i ely, and he appa en en halpy, ΔH (J/g o s a ch), o s a ch gela iniza ion and amylose-lipid dissocia ion endo he ms we e e alua ed om he he mog ams. Repo ed alues a e he means o a leas duplica e measu emen s o each sys em. 2.2.3. Rheological measu emen s The gel cylinde s we e cu in o slices and placed be ween he pa allel pla es (se a ed uppe pla e wi h 25 mm diame e ) o a Physica MCR 300 heome e (Physica Mess echnic GmbH, S u ga , Ge many). Rheological es s we e ca ied ou wi h 1 mm gap. Be o e he measu emen , he gel was es ed o 10 min o allow sample elaxa ion. S ain sweeps we e ca ied ou om 0.01 o 1000% s ain a 1 Hz equency. F om s ain/s ess sweep es s o gels he maximum s ess, max, was also calcula ed as he s ess a which he elas ic modulus o he gels d opped in he LVR by 10%. F equency sweeps we e ca ied ou om 0.1 o 100 Hz in he linea iscoelas ic egion (LVR); he s ess alue chosen o he equency sweep es s o all gels was 0.5 Pa, which was in he LVR. A cons an empe a u e o 25ºC was chosen and main ained by he con olled Pel ie sys em o he heome e (TEZ 150P/MCR). Da a analysis was ca ied ou by he suppo ing so wa e US200 V2.21 o he heome e . F equency sweep da a we e i ed o he powe law model as in p e ious wo ks (Ronda e al., 2011; 2014). The eco ded iscoelas ic pa ame e s, G1’ and G1’’, and ( an )1, ep esen he elas ic and iscous moduli and he loss angen , espec i ely, a a equency o 1Hz. The a and b exponen s quan i y he dependence o he dynamic moduli on he oscilla ion equency (powe law model). Each es was ca ied ou a leas in duplica e. 2.2.4. Con ocal Lase Scanning Mic oscopy A Leica TCS SP5 con ocal lase scanning mic oscope (CLSM), moun ed on a Leica DMI 6000B in e ed mic oscope base, was ope a ed in he luo escence mode wi h a 60x oil- imme sion objec i e o nume ical ape u e 1.40. The s a ch and p o ein phases we e s ained wi h Nile Blue. Fluo escence om he sample was exci ed wi h he 633 nm o a ed HeNe lase line. The signal om he samples was collec ed and eigh scans we e a e aged o he c ea ion o each image. Fo g anula s a ches o he mixed s a ch-p o ein aqueous dispe sions, samples o ~ 5ml we e ans e ed in o a small beake . A 10µl aliquo o Nile Blue solu ion (0.01% w/ ) was added and he dispe sions we e ho oughly mixed. In he case o gels, he s a ch o s a ch/p o ein dispe sions we e ini ially s ained wi h he dye and a e wa ds he gels we e o med as desc ibed in sec ion 2.2.1. 2.2.5. S a is ical analysis S a g aphics Cen u ion .6 (Bi s eam, Camb idge, MN, USA) allowed pe o mance o ANOVA analysis o he da a; he LSD (Leas Signi ican Di e ence) es was used o e alua e signi ican di e ences (p < 0.05) be ween samples. 3. Resul s and discussion 3.1. The mal p ope ies The he mal p ope ies o di e en aqueous s a ch dispe sions (a 20 % w/w o al solids) as in luenced by pH and he p esence o p o eins a e shown in Figu e 1 and Table 1. Po a o and apioca s a ch exhibi ed one endo he mic peak on he DSC he mog ams, while ice s a ch and i s mix u es wi h p o eins showed wo peaks; i.e. he main endo he m ela ed o s a ch gela iniza ion and a much smalle peak a highe empe a u e due o he amylose-lipid complex dissocia ion (Biliade is 2009; Eliasson, 1994). The empe a u e alues o he la e (second) peak ( a he b oad ansi ion) appea ed sligh ly lowe in he bu e ed dispe sions han in dis illed wa e (83.8ºC, 95.2ºC and 106.7ºC e sus 84.8ºC, 96.1ºC and 108.1ºC o To, Tp, Te, espec i ely); he dissocia ion en halpies o he amylose-lipid complex in bo h ypes o ice s a ch dispe sions (2.1 e sus 1.9 J/g s a ch) we e no signi ican ly di e en . Fo he gela iniza ion peak, Tp, he highes empe a u e co esponded o apioca (72.6 ºC) and he lowes o po a o s a ch (64.5 ºC), and an in e media e alue o ice s a ch (68.6ºC) (Table 1). Simila indings we e epo ed by o he au ho s (da C uz F ancisco e al., 1996; Singh e al., 2003). Po a o s a ch exhibi ed he highes appa en gela iniza ion en halpy alues (13.3 J/g s a ch), ollowed by apioca s a ch (10.6 J/g s a ch), whe eas he lowes alues co esponded o ice s a ch suspensions (6.9 J/g s a ch). The di e ences obse ed may be a ibu ed o di e ences in se e al ac o s such as size, shape and dis ibu ion o s a ch g anules, amylose/amylopec in a io ( ha ollowed he same o de , po a o> apioca> ice, wi h gela iniza ion en halpy alues), mac omolecula assemblies o s a ch polyme s wi hin he g anule and he p esence o in e nal monoacyl lipids, in he case o ice s a ch, which upon hea ing o m complexes wi h amylose, an exo he mic p ocess ha educes he ‘appa en ’ gela iniza ion en halpy o he s a ch c ys alli es (Biliade is, 2009; Schi me e al., 2013). The en halpy alue e lec s he composi e he mal ene gy e ec esul ing om mel ing (endo he mic) o s a ch c ys alli es as well as he amoun o hea associa ed wi h o ma ion (exo he mic) o dis up ion (endo he mic) o sho ange o de (e.g. amylose lipid complexes) in he hea ed s a ch ma ix (Biliade is, 2009). The p esence o p o eins a he weigh a io used in he mixed sys ems (10% wi h espec o he solid s a ch-p o ein mix u e) did no seem o signi ican ly al e he Tonse , Tpeak, Tend and ΔH o s a ch gela iniza ion endo he m (Table 1), al hough he analysis o a iance did show signi ican in e ac ion e ec s (p o ein x s a ch and s a ch x p o ein x pH) (Table 1); his means ha he e ec o p o eins depended on s a ch ype and pH o he dispe sion. The appa en gela iniza ion en halpy alues o di e en s a ches signi ican ly inc eased in he p esence o soy p o ein in bo h pu e aqueous (dis illed wa e ) and acidi ied dispe sions o po a o s a ch, as well as o egg albumin in he acidi ied dispe sions o apioca s a ch, while ΔΗ dec eased when bo h p o eins we e added in he acidi ied dispe sions o ice s a ch; no change in ΔH alues was obse ed in he emaining mix u es o s a ches-p o eins. I is wo h no ing ha calo ime y o he p o ein samples alone (25 % w/w aqueous dispe sions) did no show any endo he mic peak in he DSC he mog ams o e he same empe a u e ange (da a no shown). This means he p o eins used in his s udy we e comple ely dena u ed as a consequence o hei manu ac u ing p ocess; o he wise some endo he mic ansi ions would ins ead occu as ound o na i e p o ein sys ems (e.g. a double endo he mic ansi ion in he case o SPI, co esponding o 7S and 11S globulins o oilseeds and legume seeds, espec i ely; Biliade is, 1983). Acidi ica ion o he s a ch dispe sions did no ha e a signi ican single e ec on he gela iniza ion he mal ansi ion pa ame e s (Table 1). An in luence was only no ed, depending on he s a ch sou ce and he ype o s a ch-p o ein mix u e used, h ough signi ican in e ac ions e ec s (s a ch x pH and s a ch x p o ein x pH). A pH 4.5, he gela iniza ion en halpy o he ice s a ch suspension was signi ican ly dec eased (~26 %) and his e ec was mo e p onounced in he p esence o p o eins (-29% and -35% o EA and SPI, espec i ely). Simila e ec s o acidi ica ion we e ecen ly epo ed o ice s a ch by Colussi e al. (2015). Howe e , no signi ican e ec s o he acidi ied medium on po a o and apioca s a ch gela iniza ion en halpy we e no ed (Table 1). These wo ube s a ches ha e simila i ies be ween hem and di e om ice s a ch (e.g. signi ican ly highe amylose con en and absence o lipids) ha could explain hei simila beha iou upon acidi ica ion e sus he ice s a ch. On he o he hand, di e ences be ween s a ches om po a o and apioca, such as he highe phospho us con en and he longe a e age chain leng h o amylopec in in he o me (da C uz F ancisco e al., 1996), may explain di e ences be ween hei gela iniza ion empe a u e and en halpy alues (Table 1); o apioca s a ch highe To, Tp, and Te alues and lowe ΔH we e ound compa ed o hose om po a o. The change o he dispe sion medium om dis illed wa e o ace a e bu e (pH 4.5) inc eased he gela iniza ion empe a u e, Tp, o ice s a ch by app oxima ely 2ºC in he p esence o EA o SPI p o eins, while i did no exe any e ec on pu e ice s a ch dispe sions. Ohishi e al. (2007) ound ha addi ion o 0.2 M ace ic acid esul ed in a signi ican ly lowe gela iniza ion empe a u e (~ 1ºC) o ice s a ch ob ained om a b own ice japonica a ie y. The au ho s a ibu ed his shi o accele a ion o wa e abso p ion by he s a ch g anules (i.e. p omo ion o i s hyd a ion by adding ace ic acid). Hibi (2002) also epo ed ha he endo he mic peak empe a u e o ice s a ch was lowe in he p esence o 0.33M ace ic acid. In bo h p e ious cases he esul an pH o he hea ed s a ch dispe sions was no epo ed by he au ho s, bu i should be lowe han ha o he p esen s udy, whe e he ice s a ch dispe sion was adjus ed o pH 4.5; his migh explain why we did no obse e any e ec on gela iniza ion empe a u e o he pu e ice s a ch dispe sions. In con as , acidi ica ion o apioca s a ch dispe sions acili a ed he gela iniza ion, as demons a ed by he lowe onse and peak empe a u es o he acidi ied s a ch samples, compa ed o hei dis illed wa e coun e pa s, ega dless he p esence o absence o p o eins (Table 1). Acidi ica ion o apioca s a ch also led o a wide gela iniza ion ange, i.e. (Te-To) di e ence, implying a mo e complex phase ansi ion beha iou (Table 1 and Figu e 1). Table 1. S a ch gela iniza ion pa ame e s o ice, po a o and apioca s a ches and hei mix u es wi h egg albumin and soy p o ein isola e in aqueous (dis illed wa e ) and pH 4.5 (ace a e bu e 100mM) dispe sions (20 % w/w). S a ch P o ein Ace a e Bu e To (ºC) Tp (ºC) Te (ºC) ΔH (J/g s a ch) Rice 0 0 60.3 abcd 68.6 c 77.2 bcd 6.9 b 1 59.9 abc 68.8 c 77.0 bcd 5.2 a EA 0 59.9 abcd 68.9 c 77.8 cd 7.0 b 1 63.5 gh 70.7 de 77.6 cd 5.0 a SPI 0 61.0 bcde 68.4 c 78.1 d 7.8 bc 1 62.1 cde g 70.1 d 76.9 bcd 5.1 a Po a o 0 0 59.3 ab 64.5 a 75.9 bc 13.3 hij 1 59.6 abc 64.9 ab 78.7 de 13.7 ij EA 0 59.3 ab 64.7 ab 75.2 b 14.3 jk 1 57.9 a 64.2 a 72.4 a 14.6 jk SPI 0 60.1 abcd 64.5 ab 78.5 d 15.4 kl 1 60.5 abcde 65.5 b 80.8 e 16.6 l Tapioca 0 0 63.1 e gh 72.6 81.2 10.6 de 1 60.3 abcd 70.6 de 82.2 9.3 cd EA 0 64.0 gh 72.8 82.9 11.8 gh 1 62.6 de g 71.3 e 85.5 g 12.4 ghi SPI 0 65.3 h 73.0 83.1 11.2 e g 1 61.0 bcde 70.9 de 82.3 9.7 de SE 0.90 0.36 0.77 0.56 Analysis o a iance and significance (p- alues) Fac o 1 (s a ch) *** *** *** *** Fac o 2 (p o ein ype) ns ns ns ns Fac o 3 (pH) ns ns ns ns Fac o 1x2 ns ns *** * Fac o 1x3 ** *** ns ** Fac o 2x3 ns ns ns ns Fac o 1x2x3 *** *** *** *** P o ein: 0: wi hou p o ein; EA: egg albumen; SPI: soy p o ein isola e. Ace a e bu e (pH=4.5): 0: wi hou ace a e bu e (aqueous medium); 1: Ace a e bu e medium. Di e en le e s in he co esponding column indica e s a is ically signi ican di e ences be ween means a p<0.05. SE: Pooled s anda d e o ob ained om ANOVA analysis. Analysis o a iance and signi icance: *** p<0.001. ** p<0.01. * p<0.05. ns: no signi ican . Figu e 1. DSC he mog ams ob ained om aqueous (dis illed wa e ) o acidi ied (ace a e bu e 0,1M pH 4.5) dispe sions o di e en s a ches ( ice, po a o and apioca) and hei mix u es wi h p o eins (egg albumen o soy p o ein isola e). R: ice s a ch; P: po a o s a ch; and T: apioca s a ch. EA: egg albumin; SPI: soy p o ein isola e. -A: Acidi ied dispe sion. 3.2. Rheological p ope ies 3.2.1. S ain sweeps The esponses o iscoelas ic moduli o s a ch gels, p epa ed a 90ºC and 120ºC as a unc ion o s ain a e shown in Figu es 2 and 3, espec i ely. In ice s a ch gels i was possible o disc imina e wo di e en egions, he linea iscoelas ic egion in which G’ and an δ (da a no shown) alues we e p ac ically cons an , and he non-linea domain in which G’ s a ed o dec ease, and a he same ime he an δ began o inc ease wi h inc easing s ain un il he cu es o G’ and G’’ in e sec ed ( an δ=1). Howe e , o apioca (a 90ºC and 120ºC) and po a o s a ch (a 120ºC) gels, he loss angen was main ained well below 1 o e he en i e s ain ange, e ealing no c oss-o e o he G’ and G’’ cu es. The maximum s ess, max, ha gels can esis be o e s uc u e dis up ion (end o LVR) is depic ed in Table 2. Figu es 2 and 3 show ha he elas ic modulus (G’) o pu e ice gels ( o med a bo h 90ºC and 120ºC) yielded a lowe s ain alues han apioca and po a o s a ch gels, indica ing a mo e suscep ible s uc u e which b eaks easie unde he applied s ess han hose o ube s a ch gels. In ac , i was no possible o de e mine he maximum s esses, max, o he la e gels o e he en i e s ain ange eco ded. Rice s a ch gels o med a 90oC aised hei max alues wi h EA p o ein addi ion (Table 2). On he o he hand, o i ica ion wi h SPI and acidi ica ion (pH = 4.5) o s a ch gels p epa ed a 90ºC and 120ºC did no signi ican ly a ec his pa ame e . Wi h inc easing gel se ing empe a u e he e was dec eased gel esis ance o b eakage; i.e. much lowe max (be ween 35% and 100% dec eased alues) o gels o med a 120ºC han hei espec i e samples p epa ed a 90ºC. P e ious wo ks showed simila e ec s and epo ed a signi ican dec ease o he igidi y and s eng h o gels when p epa ed a au ocla ing empe a u es (Ch is ianson e al., 1986; Doublie e al., 1987). Due o he high solids con en o he hea ed aqueous s a ch dispe sions (20 % w/w) and he lack o shea o ces du ing hea -se ing o he gel ne wo k s uc u es i is unlikely ha ex ensi e phase sepa a ion in ol ing amylose leaching ou o he swollen g anules akes place. Howe e , chain o de ing (sho scale c ys alliza ion) o he linea s a ch ac ion in he high empe a u e cas ed gels, o ming he con inuous phase, could be mo e p onounced and i migh con ibu e o he obse ed weakening o he ne wo k s uc u e o hese samples (Biliade is, 2009). In he mixed gels, he included p o eins mos likely pa i ion a he in e g anula egions ollowing swelling o he g anules upon gel se ing. 3.2.2. F equency sweeps The iscoelas ic moduli a 1 Hz, G1’, G1’’, and ( an δ)1 and he ob ained exponen “a” and “b” alues by i ing he powe law model o he equency sweep da a ( om 0.01 o 10 Hz) a e summa ized in Table 2. F equency sweeps showed ha G’ exceeded G’’ o e he en i e equency ange o all he gels es ed, wi h he an δ alues being low enough (Table 2) o classi y such heological esponses as being o “ ue” gels (K ys yjan e al., 2015; Ribo a and Rosell, 2010). The na u al loga i hms o G1’ and G1’’ we e used o he ANOVA analysis, allowing mo e conclusi e esul s on he impac o he s udied ac o s (Ronda e al., 2011). A) 90ºC Wi hou p o ein Wi h EA Wi h SPI Rice Po a o Tapioca B) 120ºC Wi hou p o ein Wi h EA Wi h SPI Rice Po a o Tapioca Figu e 4. Con ocal lase scanning mic og aphs o s a ch gels p epa ed a 90ºC (A) and 120ºC (B). EA: Egg albumin; SPI: Soy p o ein isola e. 4. Conclusions The addi ion o egg albumin and soy p o ein isola e a 10% (by weigh in he solid s a ch-p o ein mix u es) combined wi h a educ ion o pH o 4.5 p o ed o be an e icien way o modi y he he mal and heological p ope ies o ice, po a o and apioca s a ches and s a ch gels. Gels p epa ed a 120 °C we e much weake (lowe consis ency and mo e p one o b eak-down upon de o ma ion) han hose o med a 90 °C; he ice s a ch being he mos sensi i e o inc easing geli ica ion empe a u e and po a o he mos esis an . In gene al, p o eins wo ked as s uc u e enhancing agen s a bo h empe a u es; in gels p epa ed a 120ºC he s eng hening e ec was mo e p onounced. In con as , acidi ica ion weakened he s uc u e o hese gels. I can be concluded ha bo h pH and addi ion o exogenous p o eins a e use ul app oaches o modi y he unc ional p ope ies o gel-like s a chy ood p oduc s. The heological obse a ions made in his s udy could be help ul in a emp ing o modi y glu en- ee o mula ions, which a e mos ly based on non-whea ce eal o ube s a ches o op imize he end-p oduc quali y a ibu es ( ex u e, mic os uc u e, he mal s abili y o au ocla ed ood p oduc s o ex end hei shel li e). Fu he s udies a e needed o cla i y he ole o acidi ica ion in o hea ed s a ch-p o ein mix u es by employing a ange o s uc u e p obing analy ical echniques (calo ime y, a ious heological es s, ch oma og aphy, di e en spec oscopies, e c.) o un a el he molecula in e ac ions among he hyd ocolloids in ol ed and he eby ine une end-p oduc quali y. Acknowledgemen The au ho s g a e ully acknowledge he inancial suppo o he Spanish Ins i u ion Minis e io de Economia y Compe i i idad and he Eu opean Regional De elopmen Fund (FEDER) (P ojec s AGL2012-35088 and AGL2015-63849-C2-2-R) and Conseje ia de Educa ion (Jun a de Cas illa y Leon) / FEDER (P ojec VA072P17). Ma ina Villanue a hanks he Jun a de Cas illa y León o he doc o a e g an . Re e ences Abebe, W. and Ronda, F. (2014). Rheological and ex u al p ope ies o e [E ag os is e (Zucc.) T o e ] g ain lou gels. Jou nal o Ce eal Science, 60, 122-130. Biliade is, C. G. (1983). Di e en ial Scanning Calo ime y in Food esea ch—A Re iew. 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