Impact of acidification and protein fortification on rheological and thermal properties of wheat, corn, potato and tapioca starch-based gluten-free bread doughs

Impac o acidi ica ion and p o ein o i ica ion on heological and he mal p ope ies o whea , co n, po a o and apioca s a ch-based glu en- ee b ead doughs Ma ina Villanue a1, Sand a Pé ez-Qui ce1, Concha Colla 2, Felicidad Ronda1 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 Ce eals and Ce eal-based P oduc s, Food Science Depa men , Ins i u o de Ag oquímica y Tecnología de Alimen os, IATA-CSIC, A da. Ca ed á ico Agus ín Esca dino 7, 46980 Pa e na, Spain. Abs ac The s udy o new glu en- ee (GF) oods is necessa y since consume s in ole an o glu en a e mo e and mo e equen ly diagnosed. The s udy e alua ed he impac o acidi ica ion -wi h ace ic+lac ic blend a 0.5 g/100 g le el- and p o ein o i ica ion -wi h caseina e (CA) o soy-p o ein isola e (SPI)- on he heological ea u es o whea , co n, po a o and apioca s a ch-based b ead doughs. Oscilla o y and c eep- eco e y es s we e ca ied ou o cha ac e ise hei iscoelas ic beha iou , and he momechanical es s we e pe o med o assess hei isco-me ic pe o mance. Dough s ickiness was also measu ed. The acid blend had a modula o e ec on dough heological p ope ies ha depended on bo h he ype o p o ein and he sou ce o he s a ch. P o eins s uc u ed and s eng hened he doughs especially hose made wi h SPI-po a o s a ch and CA-whea s a ch mix u es. Acidi ica ion dec eased G’ and G’’ moduli un il 70% wi h espec o unacidi ied doughs. The e ec was much mo e ma ked in p o ein- o i ied doughs. A signi ican inc ease in all pas ing iscosi ies was obse ed wi h p o ein addi ion, pa icula ly in he case o CA. In gene al, p o ein addi ion dec eased dough s ickiness whe eas he opposi e e ec was no ed wi h he p esence o acid. Acidi ica ion o p o ein-en iched s a ch ma ices modula e dough heological p ope ies which a e o ele ance in GF p oduc s de elopmen . Keywo ds: Ace ic acid; Glu en-F ee Doughs; Lac ic acid; P o eins; Rheology 1. In oduc ion The de elopmen o p oduc s o consume s wi h glu en- ela ed diso de s cons i u es a p io i ized and challenging opic in s a ch-based goods a ea. In addi ion o diagnosed pa ien s, also people looking o non alle genic ing edien s con ibu e o a g owing GF ma ke ca ego y; he e o e he isen a ie y o o e ed i ems seems o be an impe ious need. Unde s anding he heological cha ac e is ics o ood ma e ials is o key impo ance in designing new p oduc s. In b eadmaking applica ions, he heological p ope ies o doughs a ec bo h dough handling abili y and b eadmaking p ocess (Hoseney & Smewing, 1999), and hence inal b ead cha ac e is ics (Ronda, Pé ez-Qui ce, & Villanue a, 2017). Fundamen al and empi ical heological p ope ies o doughs also in o m abou in e ac ions among ing edien s and he c ea ion o s uc u e a mac omolecula and mac oscopic le els, espec i ely (Ronda, Villanue a, & Colla , 2014). Glu en p o ein ma ix is a key ac o in b eadmaking. Besides con ibu ing o he wa e abso p ion capaci y o he dough, glu en p o ides ex ensibili y, elas ici y and cohesi eness o b ead dough allowing he e men a ion gas o be occluded and main ained in he liquid phase du ing he dough de elopmen , leading o well-de eloped high-g ade b eads (Wiese , 2007). The elimina ion o glu en in baked p oduc s esul s in dele e ious e ec s in e ms o quali y a ibu es o p oduc s, nu i ional cha ac e is ics, and consume accep ance (Naqash, Gani, Gani, & Masoodi, 2017). The mos commonly used s a ches in GF b ead-making a e maize s a ch and po a o s a ch bu also s a ches om apioca, whea and ice among o he (Masu e, Fie ens, & Delcou , 2016). Howe e , hese s a ches ha e minimal s uc u e-building po en ial and, hus, a e equen ly used along wi h p o eins and hyd ocolloids (Cap iles & A êas, 2014). P o eins and polysaccha ides a e p esen oge he in many kinds o ood sys ems, and bo h ypes o ood mac omolecules con ibu e o he s uc u e, ex u e and s abili y o ood h ough hei hickening o gelling beha iou and su ace p ope ies (Doublie , Ga nie , Rena d, & Sanchez, 2000). The inco po a ion o p o eins in GF ma ices is ocused on he nu i ional enhancemen and on he imp o emen o b ead inal cha ac e is ics (physical and ex u al). In e - and in a-molecula in e ac ions es ablished be ween exogenous p o eins and s a ch molecules, main esponsible o dough s uc u ing, ce ainly depend on dough pH (Houben, Höchs ö e , & Becke , 2012; Ronda e al., 2014). Acidi ica ion h ough lac ic and ace ic acid addi ion con e s sui able p ope ies o inal b eads ei he when p oduced by he exogenous mic o lo a o added o b eadmaking ma ices Acidi ica ion imp o ed he odou and as e o esh b ead and inc eased he p o ease and amylase ac i i ies ha led o e a ded s aling du ing s o age (Moo e, Dal Bello, & A end , 2008). Acidi ica ion by ace ic acid and lac ic acid addi ion ha e shown o p o ide a signi ican impac in p o ein-en iched ice s a ch-based doughs p ope ies (Ronda e al., 2014) and in he quali y and shel -li e o ice s a ch-based b eads o i ied wi h CA, SPI and pea p o ein isola e (Villanue a, Mau o, Colla , & Ronda, 2015). Taken in o accoun he impo ance o o he s a ches, as po a o, apioca, co n and whea , on he de elopmen o GF p oduc s, he s udy o he e ec o acidi ica ion on p o ein-en iched doughs made wi h heses s a ches seems imely. In GF p oduc s, s a ch becomes he p ima y s uc u al elemen due o he lack o glu en, mainly du ing he baking s age, when he ba e empe a u e eaches s a ch gela iniza ion alues. Howe e , s a ches om di e en sou ces di e ma kedly on wa e binding capaci y which a ec s d ama ically dough consis ency and dough de elopmen du ing e men a ion, and he quali y o he inal p oduc s (Ronda e al., 2017). Wi h his in mind, he aim o he p esen s udy was o e alua e he impac o he addi ion o 0.5 g/100 g (s a ch+p o ein) o ace ic + lac ic acid mix u e o di e en GF b ead doughs made wi h maize, po a o, apioca o whea s a ches o i ied wi h CA o SPI (a 5 g/100 g (s a ch+p o ein) le el) on he iscoelas ici y, s ickiness and pas ing p ope ies o b ead doughs. 2. Ma e ial and me hods 2.1. Ma e ials Co n, po a o and whea s a ches we e supplied om Fe e Alimen ación S.A. (Ba celona, Spain), and apioca s a ch om Ca gill S.L. (B enn ag, Se illa, Spain). Sal , suga (Azuca e a, To o, Spain) and sun lowe oil Coosu P emium (Jaen, Spain) we e pu chased om he local ma ke . Hyd oxy-p opyl- me hyl-cellulose (HPMC, Me hocel-K4M-Food-G ade) was p o ided as a gi by Dow Chemical (Midland, USA). P o eins used in GF o mula ions we e: soybean p o ein isola e (SPI) Sup o 500-E IP gi en by P o eedo a hispano-holandesa S.A. (Ba celona, Spain) and calcium caseina e (CA) by A mo p o eines (Sain -B ice-en-Coglès, F ance). Ace ic acid and lac ic acid o analy ical g ade om Pan eac (Ba celona, Spain) we e used. Dis illed wa e was used o p epa e all he suspensions o s udy he pas ing p o iles and ap wa e was used o make GF doughs. 2.2. Me hods Dough p epa a ion A s aigh dough p ocess was pe o med in duplica e pe o mula ion, using he ollowing o mula on a 100 g s a ch (o s a ch+p o ein) basis: 6 g oil, 5 g suc ose, 1.5 g sal , 2.0 g HPMC and 75 g wa e . CA and SPI we e added a 0 o 5 g/100 g (s a ch + p o ein basis) le els and doughs we e supplemen ed wi h (0.1 + 0.4) g/100 g (s a ch + p o ein basis) o ace ic+lac ic acid when acid- ea men was applied. The expe imen al design esul ed in 24 di e en combina ions (Table 1). GF dough-making was achie ed by blending i s solid ing edien s and oil in a ki chen-aid p o essional mixe KPM5 (Michigan, USA) a speed 2. Then wa e was added and hand mixed. Finally he dough was mixed wi h dough hook a a speed 4 o 8 min. Acid blend, when added, was dilu ed in a small pa o wa e and adjus ed o he dough be o e he mixe was powe ed on. Table 1. Randomized expe imen al design Fo mula S a ch P o ein Ace ic/Lac ic Acid* 1 Po a o SPI 0.1/0.4 2 Whea 0 0.1/0.4 3 Po a o SPI 0 4 Co n SPI 0 5 Co n SPI 0.1/0.4 6 Co n CA 0 7 Tapioca SPI 0 8 Whea 0 0 9 Tapioca SPI 0.1/0.4 10 Co n 0 0.1/0.4 11 Co n CA 0.1/0.4 12 Po a o 0 0 13 Tapioca CA 0 14 Tapioca 0 0.1/0.4 15 Po a o CA 0.1/0.4 16 Whea SPI 0.1/0.4 17 Whea CA 0 18 Tapioca CA 0.1/0.4 19 Tapioca 0 0 20 Po a o CA 0 21 Co n 0 0 22 Whea CA 0.1/0.4 23 Whea SPI 0 24 Po a o 0 0.1/0.4 P o ein: 0: wi hou p o ein, CA: Wi h 5g/100g Calcium caseina e, SPI: Wi h 5g/100g soybean p o ein isola e. *g/100g wi h espec o s a ch o s a ch+p o ein basis 2.3. Dough measu emen s pH and o al i a able acidi y o doughs To al i a able acidi y (TTA) was measu ed on en g ams o dough blended wi h 100 mL o a solu ion o ace one in wa e (5 mL/100 mL) unde cons an s i ing. The i a ion was ca ied ou agains 0.1 mol/L NaOH un il a inal pH o 8.5. The esul s we e exp essed as milliequi alen s o lac ic acid/g o dough. This measu emen was aken in iplica e on unyeas ed doughs. Fundamen al heological es s Oscilla o y and c eep– eco e y es s we e ca ied ou wi h RheoS ess-1 heome e (The mo Haake, Ka ls uhe, Ge many) wi h pa allel pla e geome y (60 mm diame e ) o se a ed su ace and wi h 3- mm gap. The excess o dough was emo ed, and aseline oil was applied o co e he exposed sample su aces. All measu emen s we e done a 25 °C. Be o e each assay he dough was allowed 10 min o elaxa ion. F equency sweeps we e ca ied ou om 10 o 1 Hz in he linea iscoelas ic egion (LVR). A cons an s ess alue o 1 Pa was chosen o he equency sweeps o all doughs. S ess sweeps we e ca ied ou om 0.1 o 100 Pa a 1 Hz. F om he cu es, he maximum s ess beyond which he dough s uc u e was b oken, τmax, was es ablished. F equency sweep da a we e i ed o he powe law model as in p e ious wo ks (Ronda e al., 2014). Wi hin he applied equency ange, he mechanical spec a i ed he powe law model wi h R2 alues abo e 0.99. C eep es s we e pe o med by imposing a s ep o shea s ess in he LVR and ou side he linea iscoelas ic egion (OLVR). Fo he c eep s udy in he LVR, a cons an shea s ess o 1 Pa was applied o 150 s, while in he eco e y phase he s ess was suddenly emo ed and he sample was allowed o 300 s o eco e he elas ic (ins an aneous and e a ded) pa o he de o ma ion. Fo he OLVR s udy, a cons an shea s ess o 50 Pa was applied o 60 s and he sample was allowed o eco e o 180 s a e emo ing he load. Each es was pe o med in iplica e. The da a om c eep es s we e modelled o he 4-pa ame e Bu ge s model (Ronda e al., 2014). Dough s ickiness S ickiness was measu ed by ollowing he p ocedu e p oposed by G ausg ube , Ha zenbichle , & Ruckenbaue (2003). A ex u ome e TA-XT2 om S able Mic osys em (Godalming, UK) p o ided wi h a SMS/Chen-Hoseney de ice whe e he sample was placed, and a me hac yla e 25 mm cylinde (P/25P) as comp ession cell, we e used. The posi i e maximum o ce (adhesi e o ce), was used o measu e s ickiness. Six eplica es we e made o each dough. Pas ing p ope ies Viscome ic p o iles o o mula ed doughs om di e en s a ch sou ces and p o eins in acidi ied/no acidi ied medium we e ob ained by using a Rapid-Visco-Analyse (RVA-4, Newpo Scien i ic, Wa iewood, Aus alia) and p o ile S anda d 1. F eeze-d ied dough samples (Colla , 2003) we e ans e ed (3.0 g o co n and whea s a ches, 2.5 g o apioca s a ch and 2.0 g o po a o s a ch o 14 g/100 g mois u e basis) in o canis e s and 25 ± 0.1 mL o dis illed wa e we e added and p ocessed ollowing s anda d me hod. The pas ing empe a u e (PT), peak ime (P- ime), peak iscosi y (PV), ough iscosi y (TV), b eakdown (BD), inal iscosi y (FV) and se back iscosi y (SB) we e calcula ed om he pas ing cu e using The mocline . 2.2 so wa e. All measu emen s we e pe o med in duplica e. 2.4. S a is ical analysis S a g aphics Cen u ion .6 (Bi s eam, Camb idge, MN, USA) was used o non-linea eg essions and mul i- ac o analysis o a iance. 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. pH and o al i a able acidi y o doughs The pH o unacidi ied and p o ein- ee ma ices a ied depending on he s a ch sou ce, and ollowed he o de : Tapioca (pH=5.9) < Co n (pH=6.1) < Po a o (pH=6.5) < Whea (pH=6.8) (Fig. 1a). P o ein p esence sys ema ically inc eased he dough pH alue while he ace ic-lac ic blend p o ided a dec ease ~ 2.5 uni s. The ype o p o ein and he s a ch sou ce also a ec ed he pH o he dough h ough he signi ican (p<0.05) (p o ein x s a ch x pH) 3 d o de in e ac i e e ec (Fig.1a). Dough pH inc eased wi h p o ein p esence be ween 3 % ( o whea and po a o s a ch doughs) and 18% ( o apioca s a ch dough) depending on he s a ch sou ce. Acidi ica ion o con ol ma ices educed signi ican ly (p<0.05) he pH om 6–6.7 o 3.4–3.6. Howe e , acidi ica ion o p o ein-en iched doughs only dec eased pH o 4.3–4.8. The bu e ing e ec o p o eins, esponsible o he lowe e ec o acidi ica ion on dough pH, was p e iously epo ed by Villanue a e al. (2015) o ice s a ch-based doughs. Fig.1a shows he bu e ing e ec was signi ican ly highe o CA han SPI ega dless he s a ch sou ce used o dough o mula ion; consequen ly, he pH o acidi ied CA-en iched doughs was highe han hose o SPI-en iched doughs. The TTA o con ol doughs (unacidi ied and p o ein- ee doughs) a ied signi ican ly (p<0.05) depending on he s a ch sou ces (Fig.1a): Whea (0.0028 meq/g) < Tapioca (0.0039 meq/g) < Co n (0.0077 meq/g) < Po a o (0.0100 meq/g). Acid addi ion inc eased he TTA o doughs om 0.008 meq/g o 0.034 meq/g on a e age. P o ein addi ion inc eased dough TTA bu he inc ease depended on s a ch sou ce and p o ein ype as deno ed by he signi ican (p<0.05) 3 d o de in e ac ion depic ed in Fig.1a. The inc ease was always highe o CA han SPI, in cohe ence wi h he highe bu e ing e ec o he o me , also esponsible o he lowe dec ease o pH in acidi ied doughs in CA p esence. Figu e 1. pH and TTA alues (a) and maximum s ess alues, τmax (b) eco ded o samples wi h di e en s a ch sou ce, ype o p o ein and acid addi ion. WP: doughs wi hou p o ein, CA: doughs wi h 5% calcium caseina e, SPI: doughs wi h 5% soy p o ein isola e. Void ba s (p incipal axes) and discon inue lines (seconda y axes) co espond o doughs wi hou acid addi ion, illed ba s and con inuous lines co espond o acidi ied doughs. E o ba s ep esen he mean s anda d de ia ion. Di e en le e s wi hin each g aph mean s a is ically signi ican di e ences be ween means (p<0.05). j lnkmop i pqo s b gd b he a c a c 0 0,01 0,02 0,03 0,04 0,05 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 WP CA SPI WP CA SPI WP CA SPI WP CA SPI Co n Po a o Tapioca Whea TTA (meq/g) pH aab ab ab e b a c ab ab d a ab ab ab ab ab b ab aaab ab ab 0 4 8 12 16 20 24 28 32 WPCASPI WPCASPI WPCASPI WPCASPI Co n Po a o Cassa a Whea τmax (Pa) a) b) 3.2. Dynamic oscilla o y heology The s ess sweep es s p o ided he τmax alue o maximum s ess doughs we e able o s and be o e b eaking hei s uc u e (Fig.1b). The τmax alues o all doughs we e a ound 2–4 Pa (wi hou signi ican di e ences among hem) wi h he excep ion o unacidi ied CA-en iched doughs made wi h po a o, whea o apioca s a ches (maize s a ch doughs we e no a ec ed by CA addi ion). The τmax o hese doughs we e much highe : 26, 19 and 12 Pa espec i ely. This could be due o he o ganiza ion o casein micelles ha o m la ge sup amolecula en i ies u he conside ed as sphe ical pa icles. They a e co e ed by κ-casein, which s abilizes hem in he suspension h ough s e ic and elec os a ic epulsions. Mo eo e , he hai y su ace p e en s neu al polyme s om adso bing on he micelles (Bou io , Ga nie , & Doublie , 1999) and Ca+2 ionic in e ac ions, which pa ially can eplace he beha iou o disulphide b idges, could deli e simila heological cha ac e is ics o glu en sys ems (S a hopoulos & O’Kennedy, 2008). The acid blend addi ion coun e ac ed he CA s abiliza ion e ec and led o simila τmax alues han p o ein- ee ma ices. Table 2 shows he single e ec s and Fig.2a he 3 d o de in e ac i e e ec s o ac o s s udied on iscoelas ic pa ame e s ob ained om equency sweeps. Viscoelas ic beha iou o dough samples co esponded o solid-like sys ems wi h s o age modulus alues (G’1) highe han loss modulus (G’’1), sligh equency dependence (low a and b exponen s), and alues o ( an δ1) unde 1, in good acco dance wi h ea lie esul s ound o acidi ied ice s a ch doughs en iched wi h p o eins ha included SPI and CA p o eins (Ronda e al., 2014). The sligh dependence o he moduli on angula equency (a and b alues anged 0.13–0.37) and he alues o phase shi angen ( an δ) a ying in he ange 0.33–0.68 a e cha ac e is ics o he sys ems called pseudo-gels. This is in ag eemen wi h ea lie obse a ions in GF doughs (Wi czak, Ko us, Ziob o, & Juszczak, 2010). S a ch sou ce a ec ed signi ican ly (p<0.001) he iscoelas ic moduli. The highes G1’ and G1’’ moduli we e ob ained o po a o s a ch doughs (17300 Pa and 9400 Pa on a e age, espec i ely) while he lowes alues we e obse ed o whea s a ch (3000 Pa and 1700 Pa) (Table 2). Fac o s ela ed o he bo anical o igin o s a ch esponsible o s a ch swelling such as amylose/amylopec in a io, molecula weigh o amylose and amylopec in, hei dis ibu ion wi hin he g anule, g anule size, he lipid con en and o he mino componen s (such as mine als and sal s) play a c ucial ole (Wa e schoo , Gomand, Fie ens, & Delcou , 2015). The inco po a ion o p o eins also a ec ed ma kedly dough consis ency. P o eins aised bo h iscoelas ic moduli, G1’ and G1’’, leading o a e aged inc eases o 145 and 130% espec i ely wi h espec o he alues o non-p o ein added-doughs. O he au ho s also concluded ha p o eins such as soy p o eins a ec ed ice dough consis ency since hey a e he main componen s in ol ed in wa e abso p ion (Ma co & Rosell, 2008). The inc ease in ice based dough consis ency was also p e iously epo ed as esul o SPI and CA addi ion (Ronda e al., 2014; Ma os & Rosell, 2014). Table 2. Single e ec s on pH. acidi y and he heological p ope ies om oscilla o y es s o glu en- ee b ead doughs made wi h s a ches om di e en sou ces, wi hou o wi h p o ein (5 g calcium caseina e o soy p o ein isola e pe 100 g o s a ch+p o ein) wi h o wi hou acid addi ion (ace ic+lac ic acid 0.1+0.4 g/100 g s a ch+p o ein) Va iable Uni Mean Le el S a ch P o ein Acid pH o he medium pH 5.43 1 5.38 b 4.90 a 6.65 b 2 5.48 c 5.74 c 4.21 a 3 5.32 a 5.63 b 4 5.52 d SE 0.004 0.003 0.003 TTA meq/g 0.0209 1 0.0218 b 0.0190 a 0.0079 a 2 0.0233 c 0.0225 c 0.0339 b 3 0.0190 a 0.0210 b 4 0.0193 a SE 0.0002 0.0001 0.0001 Dynamic Oscilla o y Rheome y G'1 Pa 7763 1 5803 c 3942 a 9990 b 2 17309 d 9205 b 5537 a 3 4959 b 10143 c 4 2982 a SE 162 138 111 a 0.30 1 0.28 a 0.31 b 0.30 a 2 0.31 b 0.33 c 0.30 a 3 0.29 a 0.27 a 4 0.33 c SE 0.01 0.004 0.003 G''1 Pa 4126 1 2741 b 2196 a 5332 b 2 9443 c 5411 c 2920 a 3 2590 b 4771 b 4 1731 a SE 84 72 58 b 0.23 1 0.25 b 0.25 b 0.22 a 2 0.19 a 0.24 b 0.24 b 3 0.23 b 0.21 a 4 0.27 c SE 0.01 0.01 0.005 an  0.53 1 0.47 a 0.56 b 0.54 a 2 0.55 c 0.57 c 0.53 a 3 0.52 b 0.47 a 4 0.58 d SE 0.01 0.005 0.01 c -0.07 1 -0.03 c -0.06 b -0.08 a 2 -0.12 a -0.09 a -0.06 b 3 -0.07 b -0.06 b 4 -0.06 b SE 0.01 0.01 0.004 S a ch le el: 1: co n, 2: po a o, 3: apioca, 4: whea ; P o ein le el: 1: wi hou p o ein, 2: Calcium caseina e, 3: Soya p o ein isola e; Acid le el: 1: wi hou acid addi ion, 2: wi h acid addi ion. Wi hin each pa ame e , di e en le e s in he co esponding column mean s a is ically di e ences be ween means a p<0.05. TTA: o al i a able acidi y. G’1. G’’1. and ( an δ)1 ep esen he elas ic and iscous moduli and he loss angen a a equency o 1 Hz. The a, b and c exponen s quan i y he dependence deg ee o dynamic moduli and he loss angen wi h he oscilla ion equency. SE: Pooled s anda d e o The esul s could be explained by he c ea ion o a obus c osslinked s uc u e in doughs by added p o eins, especially in he case o SPI by glycinin and i s high wa e e en ion abili y (C ocke , Ie, & Vodo o z, 2011). On he opposi e, dough acidi ica ion always dec eased bo h iscoelas ic moduli as was also p e iously concluded o ice s a ch (Ronda e al., 2014). The ANOVA s udy showed ha all he 2nd o de and 3 d o de e ec s signi ican ly (p<0.01) a ec ed G1’, G1’’ and an δ1. This means ha he e ec o he p o ein ype depended on bo h he s a ch sou ce and he pH o he dough. As can be seen in Fig.2a SPI p o ided he mos s eng hening e ec in po a o s a ch doughs, wi h inc eases up o 250% in G1’ wi h espec o he p o ein- ee dough. Impo an inc eases in G’ and G’’ we e also ound by Pa aşcu, Banu, Vasilean, & Ap odu (2016) when added SPI o po a o s a ch sys ems. Howe e , in he case o whea s a ch was he CA-p o ein who had he highes e ec on dough consis ency leading o inc eases in G1’ and G1’’ o 320% while SPI only led o an inc ease o 105%. The e ec o bo h p o eins was simila in he case o co n and apioca s a ch doughs (Fig.2a). The e ec o dough acidi ica ion on iscoelas ic moduli was always g ea e in he case o p o ein-en iched doughs. The acidi ica ion o p o ein- ee doughs only p o ided a signi ican (p<0.05) e ec in he case o po a o, wi h sligh dec eases in G1’ and G1’’o 14 and 18% espec i ely. Howe e , in p esence o p o ein, he dec ease in he elas ic modulus, G1’, was 41 and 74% o SPI- and CA-en iched whea doughs wi h espec o he non-acidi ied coun e pa s. Simila endency was obse ed in co n and apioca doughs (Fig.2a). In bo h cases, an δ dec eased in unacidi ied doughs as esul o p o ein addi ion, deno ing an inc ease in he p edominance o dough elas ici y. In acidi ied doughs, bo h p o eins CA and SPI, led o di e en e ec s. Acidi ica ion o CA- en iched doughs led o a ma ked inc ease in he loss angen , which indica es an inc emen in he iscous o elas ic moduli a io, while in he case o SPI-added doughs a dec ease was obse ed. The simila i ies be ween co n and apioca s a ches could be due o hei simila pa icle size and shape, comple ely di e en om po a o ( e y big size) and whea (bi-modal size dis ibu ion wi h small and big g anules) s a ches. These s uc u al di e ences and he e o e, hei unc ional p ope ies, could change he beha iou o he con inuous phase o he dough which esul s in changes o iscoelas ici y. Acco ding o Singh, Singh, Kau , Sodhi, & Gill (2003), he p esence o a high phospha e monoes e con en and he absence o lipids and phospholipids in he po a o s a ch may also be esponsible o he high G’ and G’’ o hei doughs. The p esence o phospholipids and he mo e igid g anules o co n s a ch could explain he lowe consis ency o doughs. 3.3. C eep- eco e y es s C eep- eco e y es s we e ca ied ou bo h a 1 Pa, wi hin he linea iscoelas ic egion (LVR), and a 50 Pa, ou side he linea iscoelas ic egion (OLVR). The esul s wi hin he LVR a e easie o co ela e wi h he molecula s uc u e o he sample componen s. Howe e , du ing he baking p ocess (mixing, moulding, e men a ion, baking) he doughs a e subjec ed o s ess ou side he LVR. The e o e, OLVR es s a e use ul o p edic ing he de o ma ions ha he doughs will expe ience du ing p ocessing. 0 10 20 30 40 50 60 70 80 90 100 0 500 1000 1500 2000 2500 3000 3500 0 200 400 600 800 Tempe a u e (ºC) Viscosi y (mPa·s) Time (s) 0 10 20 30 40 50 60 70 80 90 100 0 500 1000 1500 2000 2500 3000 3500 0 200 400 600 800 Tempe a u e (ºC) Viscosi y (mPa·s) Time (s) 0 10 20 30 40 50 60 70 80 90 100 0 500 1000 1500 2000 2500 3000 3500 0 200 400 600 800 Tempe a u e (ºC) Viscosi y (mPa·s) Time (s) 0 10 20 30 40 50 60 70 80 90 100 0 500 1000 1500 2000 2500 3000 3500 0 200 400 600 800 Tempe a u e (ºC) Viscosi y (mPa·s) Time (s) Figu e 3. E ec o acidi ica ion and p o ein o i ica ion on iscome ic p o iles o b ead doughs made om co n (a), whea (b), po a o (c) and apioca (d) s a ches. Doughs wi hou p o ein a e ep esen ed by , wi h 5% calcium caseina e by , and wi h 5% soy p o ein isola e by . Doughs wi h acid addi ion a e ep esen ed by , wi h 5% calcium caseina e acidi ied by , and wi h 5% soy p o ein isola e acidi ied by . The lines ep esen he iscome ic p o iles o aqueous s a ch dispe sions wi h a d y ma e con en iden ical o ha o he dough dispe sion. The empe a u e p o ile is ep esen ed by in he second axis. 0 20 40 60 80 100 0 2000 4000 6000 0 200 400 600 800 Tempe a u e, ºC Viscosi y, cp Time (s) a) b) c) d) 4. Conclusions Acidi ica ion and p o ein supplemen a ion modi ied he heological and pas ing p ope ies o GF b ead doughs. Those e ec s a ied acco ding o bo h he s a ch sou ce and ype o p o ein used as aw ma e ials and he p esence/absence o acid. In gene al, po a o s a ch doughs e ealed he mos signi ican esul s. The inco po a ion o p o ein s eng hened he dough, being s uc u ing especially signi ican in he case o CA addi ion o po a o, apioca and whea s a ch doughs, showing highe τmax alues. Howe e , he e ec o p o ein on iscoelas ic moduli depended on he ype o p o ein and s a ch sou ce. The acidi ica ion esul ed in a weakening o he dough ma ices s uc u e. C eep- eco e y es made in and ou side he LVR e ealed ha he addi ion o p o ein dec eased no ably he alues o maximum compliance compa ed o con ol doughs wi hou p o ein, showing highe alues wi h he addi ion o CA han SPI. In gene al, acid inco po a ion inc eased he alues o compliance o all s a ches (en iched o no wi h p o eins) in and ou side he LVR, which indica es a g ea e capaci y o de o ma ion o he doughs o a gi en s ess. P o ein p esence inc eased he pas ing p o iles, bu wi h di e ences be ween he wo p o eins s udied. The esul s o he p esen s udy can con ibu e o gene a ing new knowledge and he e o e he de elopmen and inc ease o he GF baked p oduc s quali y o b oaden he ood p oduc choices o GF p oduc s consume s. Addi ional s udies a e s ill equi ed o ex ensi e e alua ion o he e ec o acidi ica ion on hese ma ices and i s applicabili y on he b eadmaking p ocess. Acknowledgemen The au ho s hank he inancial suppo o he Minis e io de Economía y Compe i i idad and he Eu opean Regional De elopmen Fund (FEDER) (AGL2015-63849-C2-1-R 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 . The au ho s hank o Ca men M. Co as o he help wi h heological measu emen s. Re e ences A me o, E., & Colla , C. (1997). Tex u e p ope ies o o mula ed whea doughs. Rela ionships wi h dough and b ead echnological quali y. Z Lebensm Un e s Fo sch A, 204, 136-145. Bou io , S., Ga nie , C., & Doublie , J.-L. (1999). Phase sepa a ion, heology and mic os uc u e o micella casein–gua gum mix u es. Food Hyd ocolloids, 13(1), 43- 49. Cap iles, V. D., & A êas, J. A. G. (2014). No el app oaches in glu en- ee b eadmaking: In e ace be ween ood science, nu i ion, and heal h. Comp ehensi e Re iews in Food Science and Food Sa e y, 13(5), 871-890. Colla , C. (2003). Signi icance o iscosi y p o ile o pas ed and gelled o mula ed whea doughs on b ead s aling. Eu opean Food Resea ch and Technology, 216, 505-513. C ocke , R., Ie, P., & Vodo o z, Y. (2011). E ec s o soy p o ein isola e and egg whi e solids on he physicochemical p ope ies o glu en- ee b ead. Food Chemis y, 129(1), 84- 91. Doublie , J. L., Ga nie , C., Rena d, D., & Sanchez, C. (2000). P o ein-polysaccha ide in e ac ions. Cu en Opinion in Colloid and In e ace Science, 5(3-4), 202-214. G ausg ube , H., Ha zenbichle , E., & Ruckenbaue , P., 2003. Analysis o epea ed s ickiness measu es o whea dough using a ex u e analyze . Jou nal o Tex u e S udies 34 (1), 69-82. Hi ashima, M., Takahashi, R., & Nishina i, K. (2012). The gela iniza ion and e og ada ion o co ns a ch gels in he p esence o ci ic acid. Food Hyd ocolloids, 27(2), 390-393. Hoseney, R.C. & Smewing, J. (1999). Ins umen al measu emen o s ickiness o doughs and o he oods. Jou nal o Tex u e S udies, 30, 123-136. Houben, A., Höchs ö e , A., & Becke , T. (2012). Possibili ies o inc ease he quali y in glu en- ee b ead p oduc ion: An o e iew. Eu opean Food Resea ch and Technology, 235(2), 195-208. Majzoobi, M., Ka eh, Z., & Fa ahnaky, A. (2016). E ec o ace ic acid on physical p ope ies o p egela inized whea and co n s a ch gels. Food Chemis y, 196, 720-725. Ma co, C., & Rosell, C. M. (2008). B eadmaking pe o mance o p o ein en iched, glu en- ee b eads. Eu opean Food Resea ch and Technology, 227(4), 1205-1213. Masu e, H. G., Fie ens, E., & Delcou , J. A. (2016). Cu en and o wa d looking expe imen al app oaches in glu en- ee b ead making esea ch. Jou nal o Ce eal Science, 67, 92-111. Ma os, M. E., & Rosell, C. M. (2014). Unde s anding glu en- ee dough o eaching b eads wi h physical quali y and nu i ional balance. Jou nal o he Science o Food and Ag icul u e, 95(4), 653-661. Moo e, M. M., Dal Bello, F., & A end , E. K. (2008). Sou dough e men ed by Lac obacillus plan a um FST 1.7 imp o es he quali y and shel li e o glu en- ee b ead. Eu opean Food Resea ch and Technology, 226(6), 1309-1316. Naguleswa an, S., Vasan han, T., Hoo e , R., & Liu, Q. (2010). S uc u e and physicochemical p ope ies o palmy ah (Bo assus labelli e L.) seed-shoo s a ch g own in S i Lanka. Food Chemis y, 118(3), 634-640. Naqash, F., Gani, A., Gani, A., & Masoodi, F. A. (2017). Glu en- ee baking: Comba ing he challenges - A e iew. T ends in Food Science and Technology, 66, 98-107. Pa aşcu, L., Banu, I., Vasilean, I., & Ap odu, I. (2016). Rheological and The mo-mechanical Cha ac e iza ion o S a ch – P o ein Mix u es. Ag icul u e and Ag icul u al Science P ocedia, 10, 280-288. Ribo a, P. D., & Rosell, C. M. (2010). E ec s o enzyma ic modi ica ion o soybean p o ein on he pas ing and heological p o ile o s a ch-p o ein sys ems. S a ch, 62(7), 373-383. Ronda, F., Villanue a, M., & Colla , C. (2014). In luence o acidi ica ion on dough iscoelas ici y o glu en- ee ice s a ch-based dough ma ices en iched wi h exogenous p o ein. LWT - Food Science and Technology, 59(1), 12-20. Ronda, F., Pé ez-Qui ce, S., & Villanue a, M. (2017). Rheological P ope ies o Glu en-F ee B ead Doughs. Rela ionship wi h B ead Quali y. In Ahmed, J., P aszek, P., Basu, S (Eds.), Ad ances in ood heology and applica ions. (pp. 297-334). ELSEVIER, Camb idge, UK. Singh, N., Singh, J., Kau , L., Sodhi, N. S., & Gill, B. S. (2003). Mo phological, he mal and heological p ope ies o s a ches om di e en bo anical sou ces. Food Chemis y, 81(2), 219-231. S a hopoulos, C. E., & O’Kennedy, B. T. (2008). A heological e alua ion o concen a ed casein sys ems as eplacemen o glu en: Calcium e ec s. In e na ional Jou nal o Dai y Technology, 61(4), 397-402. Villanue a, M., Mau o, R. R., Colla , C., & Ronda, F. (2015). Acidi ica ion o p o ein-en iched ice s a ch doughs: e ec s on b eadmaking. Eu opean Food Resea ch and Technology, 240(4), 783-794. Villanue a, M., Ronda, R., Moschakis, T., Laza idou, A., & Biliade is, C.G. (2018) 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. Food Hyd ocolloids 79: 20-29. Wa e schoo , J., Gomand, S. V., Fie ens, E., & Delcou , J. A. (2015). S a ch blends and hei physicochemical p ope ies. S a ch/S ae ke, 67, 1-13. Wiese , H. (2007). Chemis y o glu en p o eins. Food Mic obiology, 24(2), 115–119. Wi czak, M., Ko us, J., Ziob o, R., & Juszczak, L. (2010). The e ec s o mal odex ins on glu en- ee dough and quali y o b ead. Jou nal o Food Enginee ing, 96, 258-265.