Kinetics of gluten protein-insolubilisation during pasta processing: decoupling between time- and temperature-dependent effects. Coline Martin, Marie Helene Morel, Adrien Reau, Bernard Cuq

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Coline Martin, Marie Helene Morel, Adrien Reau, Bernard Cuq. Kinetics of gluten protein- insolubilisation during pasta processing: decoupling between time- and temperature-dependent ef- fects.. Journal of Cereal Science, Elsevier, 2019, 88, pp.103-109. ￿10.1016/j.jcs.2019.05.014￿. ￿hal- 02154658￿

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Distributed under a Creative Commons Attribution| 4.0 International License Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation content, andalllegaldisclaimersthatapplytothejournalpertain. Please notethatduringtheproductionprocesserrorsmaybediscoveredwhichcouldaffect copyediting, typesetting,andreviewoftheresultingproofbeforeitispublishedinitsfinalform. our customersweareprovidingthisearlyversionofthemanuscript.Themanuscriptwillundergo This isaPDFfileofanuneditedmanuscriptthathasbeenacceptedforpublication.Asserviceto temperature-dependent effects., of glutenprotein-insolubilisationduringpastaprocessing:decouplingbetweentime-and Please citethisarticleas:ColineMartin,Marie-HélèneMorel,AdrienReau,BernardCuq,Kinetics Coline Martin,Marie-HélèneMorel,AdrienReau,BernardCuq between time-andtemperature-dependenteffects. Kinetics ofglutenprotein-insolubilisationduringpastaprocessing:decoupling Accepted Manuscript To appearin: Accepted Date: Received Date: Reference: DOI: PII: of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: Journal of Cereal Science Cereal of Journal 24 May2019 11 April2019 YJCRS 2780 10.1016/j.jcs.2019.05.014 S0733-5210(19)30274-7 Journal of Cereal Science Cereal of Journal

(2019),doi:10.1016/j.jcs.2019.05.014 Version postprint 13 12 extrusion. glutenprotein, insolubilisation, pasta,glutenin Keywords -Durumwheat 11 10 +33499612892 Tel: 3 9 8 7 6 5 4 UMR SupAgro,deMontpellier IATE, INRA,CIRAD,Montpellier Université Corresponding author: 2 MOREL,AdrienREAUandBernardCUQ Coline MARTIN,Marie-Hélène 1 F-34060Montpellier,France. 2 PlaceViala, ofglutenprotein-insolubilisationduringpastaKinetics processing:decouplingbetween andtemperature-dependenteffects. time- during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 1 [email protected] Version postprint 31 time. 30 cooked pasta organoleptic and 29 physical properties are not significantly impacted by a resting and Dry pasta. rested not and rested equally impacting bonds, covalent of creation by fractions 28 interactions. High temperature drying induces 27 a strong drop of solubility of all gluten protein pasta 26 extrusion leads to spontaneous insolubilisations with of the gluten network 25 structure and glutenins the impact of resting time. by Resting of freshly extruded creation of relation in discussed is weak quality Pasta cooking. after and before properties, index) viscoelasticity 24 for 23 their organoleptic (color, surface roughness) of the SDS-soluble proteins from fresh, rested 22 and and dried pasta. Final pasta were characterized physical (diameter, cooking profiles elution SE-HPLC the from assessed time, was drying and resting by induced insolubilisation 21 rested for 120 20 minutes at 20, 30 or 40°C prior drying at high temperature (90°C). Glutenins related formation of glutenin cross-linked aggregates in the pasta quality. Extruded pasta were 19 temperature and polymers glutenin large of recovery related time of contribution the investigate 18 proteins insolubilisation 17 kinetics during a resting period applied to extruded. The goal is to solubilization mechanisms 16 during pasta processing. This study aims Durum to 15 wheat investigate gluten gluten proteins are essential for pasta quality. 14 Glutenin polymers undergo Abstract during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 2 Version postprint 65 promote a firmer 64 texture and a better surface aspect of cooked pasta (Cubadda et al., 2007; to resist 63 to starch swelling mechanisms that are promoted by Wagner et al., 2011). When reticulated the during drying, the gluten protein network 62 allows pasta final cooking stage and crosslink together through new disulfide bonds (Dexter and Matsuo, 1977; Morel 61 et al., 2000; proteins initiate 60 disulfide/sulphydryl exchange reactions. Above 90°C, glutenin and gliadin SDS buffer 59 has been extensively studied. Above 55°C for glutenins and 70°C for gliadins, The impact of pasta 58 drying temperature on gluten proteins aggregation and solubility loss in 1995). al., et Ummadi 1977; Matsuo, and (Dexter fraction protein SDS-insoluble the of increase 57 dramatic a in results extrusion 96°C, Above globulin). (albumin, fractions proteins salt-soluble 56 of solubility the in decrease marked a causes it 50°C, above conducted is extrusion pasta When 55 54 &Hamer,1998). al.,2011;Weegels 2004;Wagneret Singh&MacRitchie, 2002; 53 al., et Morel 2007; al., et Lamacchia 2005; al., et Lagrain 2018; al., et (Joubert links covalent of 52 Process temperature above 55°C 51 impact gluten network structure by triggering the formation latter. the for partly and former the for fully buffer, (SDS) sulfate dodecyl sodium in soluble are 50 Both 2002). Halford, & (Shewry glutenins polymeric and gliadins monomeric of constituted are 49 weight) dry pasta of 15% to (12 proteins gluten Wheat bonds. hydrophobic and ionic hydrogen, 48 Gluten in 47 its hydrated form behaves like a viscoelastic cohesive (Guerreroetal.,2014). granules mass 46 stabilized by weak and unfolding allowing for the formation of 45 a continuous gluten network enclosing the starch the wet agglomerates 44 of semolina undergo shear deformation, that induces protein hydration processing and cooking control pasta final organoleptic qualities. During the first mixing step, 43 It is 42 commonly accepted that the wheat components and their changes ). 2015 (Sicignanoetal., consumer in 41 structure during temperature. Cooking, the 40 final food preparation step before pasta eating, is assigned to the stabilized by 39 drying, which can be either carried out at are high 38 structured (70-90°C) or into low pasta (40-55°C) by single screw extrusion of water and mixed in at order 37 to obtain crumble dough of low wet agglomerates. The agglomerates temperature. Fresh pastas are mixing, extrusion, and drying. Durum wheat semolina is first hydrated with a specific amount 36 pasta making. Pasta processing consists 35 in a succession of several unit operations: hydration, during proteins and starch between in established are that interactions chemical and physical on 34 relies structure Pasta food. cereal consumed world-wide and traditional a is pasta wheat Durum 33 32 1. Introduction during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 3 Version postprint 99 diameter distribution 98 of semolina were measured by a laser particle size analyzer dry (Coulter semolina) 97 was calculated according to TN-5.7 (AFNOR NF V03-050 method). Particle g proteins/100 g 0.26) (± (12.41 content protein crude the and method, Kjeldahl by determined 96 determined 95 according to AACC method 45-15.02 . stored Semolina 94 at total 4°C. Semolina nitrogen water content content was (13.87 (±0.12) Industrial durum wheat g 93 semolina (Panzani, France) water/100 was used as g raw material. Semolina wet was semolina) was 2.1. Rawmaterial 92 91 andmethods 2. Material 90 89 88 period. oftheresting specificimpact the process,inordertoevaluate making 87 texture) were discussed in relation 86 to the mechanisms of protein insolubilisation during pasta and aspect, surface diameter, (color, pasta cooked and time) cooking and diameter, (color, pasta 85 dried of characteristics measured The stage. drying and period resting the throughout reactions 84 protein of mechanisms the describe to allowed (SE-HPLC) chromatography liquid performance 83 extraction of SDS-soluble and SDS-insoluble proteins 82 and the analyze by size exclusion high followed by 81 a drying stage conducted under low (55°C) or high (90°C) The temperatures. resting The 80 period was conducted under different conditions of temperature (20-40°C) and after extrusion and during drying 79 stage, on protein insolubilisation kinetics in extruded pasta. The objective of the present 78 study is to investigate the impact of time during a resting period duringdryingstep. orproteinaggregation freshlyextrudedpasta in temperature 77 generated 76 either by spontaneous insolubilisation reactions kinetics. of We aim 75 to gluten investigate the potential macroscopic proteins differences in pasta at characteristics order first followed ambient temperature ambient at resting during fractions glutenins the of mechanisms 74 fractions. Gliadin 73 fractions are not impacted during the resting stage. insolubilize. The The largest 72 fractions insolubilisation of glutenin polymers are more impacted than the smallest rested 71 at ambient temperature only after stage during 70 which gluten protein extrusion, can be insolubilized. When freshly gluten extruded pasta is proteins Joubertetthatthedrying Recently, indicated stepofpastamakingprocess al. (2018) is notthe tend 69 to spontaneously dryingdiagrams. orvery hightemperature high 68 drying, temperature low to compared When 2006). Nobile, Del & (Baiano stresses mechanical 67 well with native 66 semolina gluten protein content and with gluten network ability to resist to Lamacchia et al., 2007; Matsuo & Irvine, 1970). Viscoelastic index of cooked pasta correlates during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 4 Version postprint 132 weredoneon8spaghettis. product, 8 measurements For each 131 Diameters of dried spaghetti 130 were manually determined using a caliper (Mituyo, CD-15DC). ofdriedpasta 2.3. Characterization 129 128 untilcharacterization. argon 127 nitrogen and 126 stored at -18°C. The frozen samples liquid were using frozen freeze-dried, immediately were milled Samples times. and drying and kept resting different under after and steps 125 extrusion and mixing the after immediately analysis SE-HPLC for collected were samples Pasta 124 occurred. 123 to the 122 drying phase. It was verified that at the end of extruded pasta were rested in the dryer at 20, 30 or 40°C and 88% the RH up to 120 minutes prior resting 121 time no weight loss had To investigate 120 the effect of a rest period between the extrusion and70%RH. 35°C and drying 119 stages, freshly and 86% RH, 60 min to reach 90°C and 77% RH, 60 min at 90°C and 77% RH, and 60 min at 118 starting condition at 35°C and 117 88% RH, 60 min to reach 90°C and 86% RH, 60 min at 90°C RH, and 116 150 min to reach 40°C and 72% 70% and 55°C at min 600 RH. RH, 72% and 55°C reach The to min 100 RH, 88% high-temperature and 35°C at condition drying diagram 115 was: starting was: temperature low at diagram drying The conditions. temperature high or low under 114 previously equilibrated at 35°C and 88% relative 113 humidity (RH). Pasta drying was conducted was that dryer the in placed immediately were spaghettis fresh Extruded 1989). al., et Abecassis 112 Pasta 111 drying was conducted using a laboratory vertically pasta from 110 the drying die. Extrusion machine temperature and (AFREM, pressure were France, recorded along extrusion. at 109 constant temperature (35°C) under extruded were semolina vacuum of agglomerates wet pressure. The (59 atmospheric at min, 20 MPa). for continued Fresh 108 spaghettis were extruded Tap water was equilibrated to 107 prior its spreading over the mixed semolina layer. Mixing was 40°C. Tap water was added to obtain a 106 final water content of 47.0 g water / 100 g dry matter. the mixer 105 tank. The double water-jacket of the mixing tank temperature was maintained at in introduced and weighed was kg) (7.0 Semolina 1994). al., et Abecassis France, BASSANO, 104 Semolina was 103 transformed into pasta using a laboratory pasta making 2.2. Pastamaking machine 102 (AFREM- 101 wasspan (d 100 TMLS 230, Malvern, England). The median diameter (d during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 90 -d 10 )/d 50 =1.51(±0.42). Comment citer cedocument: ACCEPTED MANUSCRIPT

- 5 50 ) was of 270 (±1) μm. The diameter Version postprint 164 163(Eq 1) 162 wereperformed. product,fivemeasurements usingequation1.Foreach was calculated 161 (e compression 0.5N 160 a under sec 40 after diameter (E), diameter initial measured: were (mm) values Thickness sec). 159 squeeze a 2 cm 158 cooked spaghetti sample under 0.5 N for 40 sec, before stress relaxation (20 probe(SMS aflat P/35)wasusedto MicroSystems,UK).Forviscoelasticity, XT Plus(Stable 157 Viscoelasticity and tensile strength of 156 cooked pasta were measured using a texturometer TA- visualinspection. individualsafter bythreedifferent was given 155 were disposed on a white plate 154 and compared to standardized pictures. A grade (1 to 8 scale) pasta Cooked 7304-1:2016. ISO to according evaluated was pasta cooked of roughness Surface 153 weredoneon8spaghettis. product,8measurements For each 152 CD-15DC). (Mituyo, caliper a using determined manually were spaghetti cooked of Diameters 151 Colorwasmeasuredintriplicate. (2cmheight). product 150 cooked spaghetti were arranged 149 inside the specific black box to form a homogenous layer of Color characteristics (L*, a*, and 148 b*) of cooked past was measured for each product. 40 g of gwater/100drymatter. 45-15.02.Resultsareexpressedin Method 147 Water content of cooked pasta was 146 determined in triplicate according to the approved AACC ofcookedpasta 2.5. Characterization 145 144 characterization. before conditions humidity relative saturated under temperature room at boxes 143 was drained and 142 rinsed with large amount of tap water. Pasta strands were kept under Petri Pasta were cooked at the 141 optimum cooking time + 1 minute. After cooking, spaghetti sample Optimum cooking time for dried pasta was determined according to AACC Method 66-50.01. 140 Pasta cooking was conducted in boiling water according to the French Standard NF ISO 7303. 139 2.4. Cookingbehaviorofpasta 138 137 outintriplicate. werecarried measurements 136 Colors height). cm (1.5 product of layer homogenous a form to cm) 10 x (14 box black specific 135 a inside positioned and length) cm (8 cut were spaghetti dried of g 40 product, each For (D65). 134 using a reflectance colorimeter (Konica Minolta CR - 410, France) with daily light calibration 133 Color coordinates (L* brightness, a* redness, b* yellowness) of dried pasta were measured during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation IV of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 = ( e 2 ― ( E

e ― 1 )

e 1 ( 1 ), and diameter after 20 sec of load release (e release load of sec 20 after diameter and ), E ) ― E Comment citer cedocument:

e 1 )

ACCEPTED MANUSCRIPT

- 6 2 ). Viscoelasticity index (IV) index Viscoelasticity ). Version postprint 170(Eq 3) 196 195 pastasample. extruded 194 considered, 193 after their normalisation to the corresponding area of obtained time 192 from observed the during freshly pasta resting and drying. Apparent Areas first order kinetic model (Eq. 191 F 4) was used to fit the solubility changes as a function fittingandstatisticalanalysis 2.7. Model 190 189 (F 188 were: 5.7 ±1.1 % 187 (F otherwise noted. 186 In ) 4 (Eq. percent of total protein, fractions F to F5 185(F were expressed in % of the corresponding area from freshly 184 extruded pasta, unless glutenin polymers (F 183 Chromatogram 182 of SDS-soluble proteins was divided into of the residue re-suspended in the previous buffer supplemented 5 with dithioerytritol (20 mM). 181 fractions: SDS-soluble large buffer (pH 6.9) containing 1% SDS. SDS-insoluble proteins (F 180 phosphate sodium in extracted were (Fs) protein (2000). SDS-soluble al. et to Morel according 179 (SE-HPLC) chromatography liquid performance high exclusion size by analyzed and extracted 178 SDS-soluble and 177 SDS-insoluble proteins from freeze-dried and milled pasta samples proteinsizedistribution were 2.6. Gluten 176 175 recordedforeachproduct. measurements were seven 174 strain curves were plotted 173 and elastic modulus was identified as being curves’ slope. Four to (MPa) and strain (%) were recorded from the stress-strain curves at the breaking point. Stress- 172D 171 L With 169(Eq 2) 168 grips(equations2,3). betweenthe samplevolume constant 167 strain and stress 166 values were calculated considering the thinning of the sample diameter and The tensile test was performed at constant deformation rate (3 mm.s 165 For tensile strength measurement cooked spaghettis were fixed to the tensile grips (A/SPR). during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation 0 5 3 , initial sample diameter (mm); F ). (F and ɣ-gliadins sulfur-rich -,- ); 0 푦 푆푡푟푒푠푠 푆푡푟푎푖푛 , sample length between the two jaws (mm); L (mm); jaws two the between length sample , of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 = 푦 0 + = = 푎

휋 퐿 (1 푡 퐷 4 퐿 0 2 ― × 퐹

1 0 푡 ×

); 26.7 ±2.1 % (F 퐿 1 ― 퐿 푡 퐿 ); medium range glutenin polymers (F 0 0 Comment citer cedocument: × 푒 ― 100 푏푥 ) ACCEPTED MANUSCRIPT t

, measured force at time t of extension (N). Rupture stress 2 4 ); 8.6 ±0.2 % (F ); albumin and globulins (F globulins and albumin ); - 7 t , the same after time t of extension (mm); extension of t time after same the , 3 ); 37.0 ±1.5 % (F 1 to F 2 ); ω-type sulfur-poor prolamins 1 5 to F i of the freshly extruded pasta ) were extracted by sonication 5 -1 ). Areas from fractions F1 fractions from Areas ). 5 ) until pasta rupture. The from extracts Fs were 4 ) and 13.1 ±0.4 % Version postprint 230 solubility was slower (C 229 5.0-11.9 min), and large (45.0-65.0%) insolubilisation process. Fractions 228 F high temperature phase (90°C), fractions F 227F 226 drying (C 225 phase, fractions F 224 strong decrease of 223 protein solubility, specific to each fraction (figure 1). During the heating in 45 min, 222 and a high temperature treatment phase at 90°C for 255 min. HT 88°C to drying 35 from phase led heating a to phases: two into a conducted was drying (HT) temperature High 221 fractions(F polymers 220 glutenin the impact mainly drying LT solubility. of loss (27.5-34.8%) large and min) 12.2-50.0 219 depending on gluten protein fraction (figure 218 ). 1 Fractions F by the LT drying. Low temperature (LT) drying led to specific 217 decreases of relative solubility (F fractions weight molecular low for except model, decay 216 protein fractions (F 215 gluten SDS-soluble all of decreases solubility non-linear temperature, drying the of Regardless 214 reactions. proteininsolubilisation gluten 213 were quantified to follow protein 212 insolubilisation dynamics (figure 1). Pasta drying promotes temperature. Solubility changes 211 for each gluten protein fraction as a function of drying time Impact on protein insolubilisation 210 - Extruded pasta were dried at low (55°C) or high (90°C) 3.1. Impactofdrying 209 208 Results3. 207 206 205 data. 204 was verified using single 203 factor analysis of variance (ANOVA) and Tukey test on replicated confidence level by Student tests and 202 p-value. The statistical significance of measured values of 201 fit. Statistical analysis was carried out with goodness the gauge to considered were values calculated and experimental between deviations XLSTAT 200 (Addinsoft, NY, 2017) at 95% (Microsoft, 199 Redmond, USA). Determination coefficients (R (C 198 time reaction characteristic and amplitude insolubilisation the to related respectively are b and a 197 y Where during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation 5 insolubilisation was slower (C Time ; 1/b). Coefficients were determined by least-square fitting using Excel 2016 solver 0 of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 (=100) is the normalized F normalized the is (=100) Time = 14.2-50.0 min) but of larger (52.0-50.0%) amplitude. For fractions F 1 and F 1 to F 1 andF Comment citer cedocument: Time 2 5 drops according to kinetic very similar to the one observed for LT ) were observed and were successfully fitted using an exponential = 22.2-33.3 min) but more pronounced (38.0-55.0%) than during 2 ACCEPTED MANUSCRIPT ). Time Time

i areas at t = 0 min. The calculated coefficients of the model the of coefficients calculated The min. 0 = t at areas > 50 min) and less pronounced (8.0-30.0%). During the 1 and F - 8 2 underwent an additional very fast (C 3 to F to 1 and F 5 ) that are only slightly impacted impacted slightly only are that ) 2 ) and the sum of absolute 2 underwent a fast (C 3 , F 4 and F 3 5 , F loss of Time Time 4 and = = Version postprint 264 E the from far is value The constants. rate fitted the of plot Arrhenius an from determined 263 was kJ/moles 7.2 26.7  of energy activation An drop. solubility fasten temperature in Increase 262 minutes delay 261 16 min was noticed for samples rested at 20°C, before any significant polymers. drop. The rate of 260 protein solubility loss roughly followed a first order kinetics, but a 16 fraction F 259 polymers were impacted, and 258 large polymers from fraction F temperature rose 257 from 20 to 40°C. In accordance with Joubert et al. the as 10% to 6 about by buffer SDS in protein of extractability the decreased Resting shown). , (2018) only 256 glutenin fractions F 255 protein of decrease solubility specific to led Resting buffer – SDS in solubility protein on Impact 254 HTdrying. torestingand related for time 253 solubility and pasta 252 characteristics was investigated. Soluble gluten proteins were quantified The impact 251 of resting for 120 min at different insolubilisationduringrestingoffreshlyextrudedpasta 3.2. Protein temperatures 250 (20, 30 or 40°C) on protein 249 etal.,2007). etal.,2007;Lamacchia aspect(Cubadda surface 248 pasta characterized by a higher red 247 hue due to Maillard reaction, a firmer texture and a better strain,index.Itisthat compared toLT,HTdrying breaking and viscoelasticity admitted led to 246 stress, breaking modulus, elastic of increase an with properties, texture and 8) to 6 (from aspect 245 surface pasta cooked improved significantly drying HT drying, LT to Compared temperatures. 244 significant increase for 243 cooked pasta. Dried pasta cooking time was not impacted by drying had a significant 242 impact on pasta diameter, with a significant decrease for dried pasta and a dried pasta (L* increase) and cooked pasta (L* decrease). Compared to LT 241 drying, HT drying pasta. On the other hand, opposite impacts of HT drying on brightness (L*) were observed for 240 drying HT color. pasta on impact significant a had drying significantly HT increased drying, redness LT to (a*) 1). Compared and 239(table decreased yellowness (b*) for both dried and cooked 238 characteristics pasta impacts temperature drying expected, As - characteristics pasta on Impact 237 236 forshortduration. variationsoverlap solubility protein 235 solubility as 234(F a function of ‘time*temperature’ (figure 2). Regardless of drying temperature, 233 Petitot et 232 al., 2009). Kinetic and thermal effects on high is molecular known to increase the amount weight of SDS-insoluble 231 proteins (De Stefanis gluten and Sgrulletta, 1990; protein the heating phase. HT drying amplifies gluten protein insolubilisation mechanisms. HT drying during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation 1 +F 2 ) insolubilisation mechanisms along drying have been depicted by a curve of protein of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 2 , suggesting that solubility loss resulted from an increase in the size of glutenin 1 and F 2 (figure 3) while fractions F Comment citer cedocument: ACCEPTED MANUSCRIPT

- 9 3 , F 4 and F 5 were slightly impacted (data not 1 reacted faster than those from a value value Version postprint 298 proteins gluten specific strong and rapid to leads drying HT (46-65%). proteins cytoplasmic and 297 fractions. Glutenins (F 296 Thermal treatment during HT 295 drying induces a strong drop of solubility of all gluten protein HT drying is 294 a driving unit operation of changes of protein structure upon pasta processing. proteinsinsolubilisationupon treatment 4.1. Gluten hightemperature 293 292 Discussion4. 291 290 289 ofcookedpasta. properties timeandtexture cooking impact 288 but did 287 not significantly change cooked diameter, pasta pasta dried of decrease diameter. light a induced Resting also time Resting time (L*). brightness pasta did cooked not 286 significantly min) significantly 285 increased dried and cooked pasta yellowness (b*) and redness (a*), drying had significant and light impacts on color properties 284 of pasta (table 1). A resting time (120 Impact on pasta 283 characteristics - Compared to non-rested pasta, a resting period before HT 1c-1d). fornon-restedpasta(figure observed 282 one the to similar way a in and classes protein wheat all regarding loss, solubility protein large 281 induced 90°C at drying HT Further step. drying first the of end the at equivalent be to out turned 280 solubility loss remained 279 modest: levels of soluble proteins from rested and non-rested pasta early drying phase 278 (from 35 to 88°C for 45 min) and compared to non-rested pasta, protein loss kinetics induced by HT drying but not the total amplitude of the phenomenon. During the 277 Impact on protein 276 insolubilisation - Resting impacted the kinetic of gluten protein solubility ). 3b (figure3a, solubilityvariations ordertofollowprotein in 275 an HT drying (90°C). Soluble gluten proteins 274 were quantified along resting and drying stages Extruded pasta were sequentially submitted to 273 a resting period (at 30°C for 120 minutes) and attributes 272 3.3. Impact of pasta 271 resting on protein crosslinking during drying and on pasta quality 270 solubilityloss. forgluteninpolymers wouldaccount exchanges 269 free 268 thiol (Liang, 2011). Protein by disulfide reduction bond disulfide protein for spectroscopy clamp force single-molecule by measured bond 267 reshuffling, mediated by thiol/disulfide thiol (54-59 kJ/moles) (Bonanata et al., 2017). It approximates the E 266 albumin serum bovine of thiol unique the of oxidation the for calculated enthalpy activation the 265 from or ) 2011 (Lagrain, 100°C beyond loss solubility gliadin for reported kJ/moles 95 to 75 of during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 1 and F Comment citer cedocument: ACCEPTED MANUSCRIPT 2 ) show a stronger loss of solubility (95%) compared to gliadin

- 10 a value of 30-40 kJ/moles Version postprint 332 structures. polymeric formationofsupramolecular the 331 predict to possible always not is it complexity, gluten to Due hierarchically. occurs assembling 330 structures derived from gluten protein aggregation could emerge during pasta drying and their 329 proportion of the 328 various classes of proteins. The hypothesis was made that supramolecular architecture and 327 size of protein aggregates created during drying depend on the nature and insolubilisation and creating large protein La aggregates. Gatta et (2017) al. suggested that the 326 significant. HT drying cycle homogenizes the level of SDS-extractible proteins by 325 forcing the insolubilisation and the 324 influence of weak interactions established during resting is then not rested pasta. 323 The creation of disulfide bonds during thermal treatment drives gluten protein proteins insolubilisation rates for rested pasta 322 are not significantly different from those of not the creation 321 of weak interactions (figure 5). After a thermal We hypothesized that glutenin insolubilisation upon treatment resting of 320 extruded pasta could be during due to HT drying, bonds. tocreatedisulfide arelessable and 319 during mixing and 318 extruding stages. Thiol groups of cysteine residues might not be exposed and all involved in intrachain disulfide bonds. 317 Their quaternary structure might not be altered positions conserved highly at located for γ-type) cysteines 8 and for α-type cysteines (6 residues 316 inducing high levels of insolubilisation. Sulfur-rich α-,- and ɣ-gliadins (F 315 treatment (90°C) 314 allows the creation of numerous disulfide bonds axis (figure ). 2 LT between 313 drying reinforces to proteins, a certain extent pasta thus protein network. A HT thermal bonds, as indicated 312 by the superposed curves when plotted according to "time*temperature" treatment (55°C) of 311 glutenins leads to the creation of intra-chains thermal and LT A inter-chains bonds. disulfide disulfide new of creation the and reactions exchange disulphide/sulfhydryl 310 established 309 that heating Lagrain et 308 glutenins al., 2005; Petitot, above 2009; Wagner 2007; al., et Lamacchia 1999; et Feillet, & (Icard-Vernière studied 55°C extensively been al., has HT under 2011; 307 Zweifel and et al., 2003). It gliadins is well additional bond involving above glutenins reduces 306 their solubility. The behaviour of gluten proteins 70°C allows components. The 305 protein chains get closer and could generate wheat of additional compaction induce interactions. could stress mechanical Each generated The diameter. pasta of reduction 304 significant a to leads drying pasta during elimination Water 5). (figure drying and resting pasta 303 We proposed a phenomenological 302 model to describe the changes in protein solubility during fractions. 14minfortheglutenin timeslowerthan withcharacteristic mechanisms, 301 glutenins are 300 highly sensitive to HT treatments. HT drying allows very fast insolubilisation gliadins and 40% of the cytoplasmic proteins. Compared to 299 gliadins, higher molecular weight insolubilisation. HT drying induces the insolubilisation of 95% of the glutenins, 60% of the during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 11 4 ) contain cysteine Version postprint 366 interactions weak build to able become they resting, During resting. upon solubility of loss min) 365 of rest. Low molecular weight glutenins (F 364 (C fastest the and (33%) strongest 363 7.65%) and cytoplasmic proteins 362F F 361 Glutenins fractions. protein gluten of solubility of loss specific a to leads pasta extruded Resting 360 proteinsinsolubilisationkinetic duringrestingat30°CandHTdrying 4.3. Gluten 359 358 2011). bonds(Lagrain, disulfide 357 ofnew creation promote the that reactions exchange the disulphide/sulfhydryl allowsfor 70°C 356 observed. Indeed, it is 355 well established that heating glutenins above 55°C and gliadins above 60°C, 354 no further protein solubility loss induced by observed at the beginning of 353 additional LT drying. Because during this disulfide step temperature remains below crosslinking was temperature monitored drying chamber. It 352 accounts for the modest drop in glutenin polymers reshuffling. 351 The reaction also occurs when After extruded extrusion, 350 stress pasta relaxation of are stretched glutenin directly polymers would placed drive this into reshuffling by disulfide thiol/disulfide exchange, which 349(Lagrain, ranges 2011). between It the is 30-40 compatible kJ/moles with the (Liang, activation 2011). energy needed to promote disulfide bond 348 far from 347 the E remains it Nevertheless, it. consolidate to needed be would experiments additional and accuracy 346 and 7.02 min (40°C). The corresponding 345 activation energy (E (30°C) min 11.3 to (20°C) min 30.3 from reaction the hastens 40°C) to 20 (between temperature 344 F polymers glutenin smaller the for min 26.9 in 16.4% of drop a against 343(F 342 weight molecular largest the of Polymers loss. solubility polymers glutenin significant but slight 341 In this work it was found that resting at 20 340 to 40°C of freshly extruded pasta coincided with a willbedetected. peptide-ordisulfide-bonds bonds like 339 during extraction in 338 the SDS-buffer used here. Only irreversible changes involving covalent of weak interactions, even if 337 maintained after sample freeze-drying are going to be disrupted glutenin polymers facilitating their alignment and hydrophobic and H-bond pairing. Such type 336 unfold would forces shear and compression extrusion, pasta During chains. protein of mobility 335 The water content of fresh extruded 334 pasta is high enough (30% wet basis) to allow molecular reactionSS-SHduringrestingafterextrusionatvarioustemperatures 4.2. Exchange 333 during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation 1 1 and F and ) reacted more readily than glutenin from F from glutenin than readily more reacted ) 2 of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 show a stronger loss of solubility (16.4-33.0%) compared to gliadins F gliadins to compared (16.4-33.0%) solubility of loss stronger a show a value reported for the gliadin solubility loss observed during pasta drying Comment citer cedocument: ACCEPTED MANUSCRIPT Times 5

(7.73%). High molecular weight glutenins (F = 11.3 min) loss of solubility during the first 60 minutes 60 first the during solubility of loss min) 11.3 = 2 ) have a smaller (16.4%) and slower (C - 12 2 . In less than 15 min, F min, 15 than less In . a =26.4  7.2 kJ/moles) lacks of 1 2 area decreased by 33% by decreased area . An increase in resting in increase An . 3 and F and 1 Times ) show the 4 = 26.9 (3.24- Version postprint 400 a significant impact on HT dried pasta quality. Weak interactions contribution to pasta quality 399 have not do time resting during created interactions weak additional and changes protein Gluten 398 and time (table ). 1 HT drying confers identical texture qualities 397 to rested and not rested pasta. temperature resting of regardless resting, by impacted significantly not is quality pasta Cooked 396 timeonpasta4.4. Impactofaresting quality 395 394 1999). etal., otherglutenins(Lee with 393 low molecular weight glutenins show 392 a greater number of cysteines available for interactions able to insolubilize during the resting period. It has been shown that pasta 391 quality increases as ratio might be related to a 390 strong capacity of spontaneous insolubilisation, as glutenins F glutenin/gliadin high A 2018 ). al., et (Joubert qualities protein gluten and variety wheat durum 389 The spontaneous 388 insolubilisation potential of gluten proteins upon resting may be linked to ofsemolina. components 387 of solubility of 386 cytoplasmic protein (F additional intra-gliadins weak interactions 385 does not impact their solubility. The apparent loss create inter-protein weak interactions and to insolubilize during resting 384 (figure 4). Creation of Compared to glutenins, their open surface 383 could be smaller, giving them few opportunities to molecular 382ɣ-gliadins weight (F proteins are less likely to unfold 381 during mixing and extruding stages. (F gliadins sulfur-poor ω-type The 380 They experience very small 379 losses of solubility and low insolubilisation rates during resting. Gliadins (F 378 2011). 377 glutenins and 376 higher than 70°C for gliadins (Icard-Vernière & Feillet, 1999; Wagner et al., during resting is less probable, as their creation is 375 driven by temperature higher than 55°C for and increase probability of efficient interactions. The contribution of 374 covalent disulfide bonds An increase of rest temperature might favor molecular mobility of protein chain in fresh 373 pasta F and 372 aggregates. A slight increase of resting temperature (from 20 to 30°C) speeds up glutenins (F 371 be due 370 to the creation of hydrophobic interactions between adjacent chains, to form allow molecular mobility of the protein chains. Glutenins insolubilisation during resting could larger 369 thermal stress. The 368 water content of fresh extruded pasta is high (30% wet basis) enough to insolubilisation of 367 glutenins during resting occurs spontaneously, without between mechanical nor adjacent chains (figure 4), thus leading to a loss of solubility. This kinetic during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation 2 ) and gliadins (F ) andgliadins of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 3 and F 4 ) and cytoplasmic proteins (F 4 ) and cytoplasmic proteins (F 3 Comment citer cedocument: andF ACCEPTED MANUSCRIPT 4 ). 1 (table ) spontaneousinsolubilisation 3 - and sulfur-rich α-,- while solubility of losses small show )

5 ) might be induced by a linkage of proteins to other - 13 5 ), experience no loss of solubility. These low 5 ) are seldomly impacted by a resting period. 1 are 1 Version postprint 432 JournalofFoodEngineering 76,341–347. quality. cooking 430 429 71(3),247-253. Chemestry extrusion speed, temperature 427 and pressure in the extruder on pasta quality. Cereal J.Sci.FoodAgric47,475-485. byheattreatment. products 424 St Paul,MN:AACC50.01. International. 431 Baiano, A., Del Nobile, M.A., 2006. Influence of the drying temperature on the spaghetti 428 Abecassis, J., Abbou, R., Chaurand, M., Morel, M.-H., Vernoux, P., 1994. 426 Influence of Abecassis, J., Faure, J., 425 Feillet, P., 1989. Improvement of cooking quality of maize AFNOR, 1970.NF V03-050. pasta 423 AACC International. Approved Methods of Analysis. 11th ed. Method 422 44–15.02. Method 66- 421 References 420 419 “DefiBleDur”. throughtheresearchprogram work wassupportedbyBPIFrance 418 This semolina. wheat durum providing for France) (Marseille, Panzani analyses, biochemichal 417 help 416 in pasta making and characterization, their for Reau A. and Rodriguez, F. Maraval, G. Lhomond, L. acknowledge J. kindly authors The Bonicel, 415 C. Fabre and T-M. Lasserre Acknowledgments for 414 413 restingtime. establishedduring weakinteractions comparedto pastaquality cooked 412 to contribution higher a have drying during created bonds Covalent pasta. rested not and rested 411 of solubility of 410 all gluten protein fractions by creation of covalent bonds, drop impacting strong a equally induces drying HT proteins. cytoplasmic and gliadins to compared glutenins more 409 weak interactions. This spontaneous insolubilisation follows a first order 408 kinetics and impacts Pasta resting 407 after extrusion leads to gluten protein insolubilisation reactions by creation of 406 Conclusion5. 405 404 403 colorchanges. infreshpasta,thusdoesnotallow activity 402 not impacted by 401 a resting time before drying. Resting does not significantly impact enzyme is texture and color pasta Dry drying. during up set bonds covalent to compared lower much is during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 14 Version postprint 460 459 46,58–63. Science JournalofCereal behaviour. cooking to relationship their and cycles drying by induced proteins pasta in Changes 2007. 457 Food59(5),2034–2039. ofAgriculturaland Chemistry ofgliadin.Journal polymerization 455 454 42,327–333. Science Cereal cooling on the physico-chemical properties of wheat gluten-water suspensions. Journal of 452 451 73,76-83. Science flours.JournalofCereal monococcum) (Triticum supramolecular arrangements in pasta from durum wheat (Triticum 449 durum) and einkorn drypasta.FoodChemistry240,189–195. semolinato from 447 Chemistry76,558–565. Changes.Cereal andBiochemical Development 441 54,882–894. Chemistry Cereal 439 438 55. and drying temperature on the cooking of durum wheat pasta. Cereal 436 Chemistry 84, 48– 435 952–962108, 434 mechanistic insights on its oxidation to sulfenic acid. Free Radical Biology and Medicine Coitiño, L., 2017. The thiol of human serum albumin: Acidity, microenvironment and 458 Lamacchia, C., Di Luccia, A., Baiano, A., Gambacorta, G., la Gatta, B., Pati, S., La Notte, E., 456 Lagrain, B., Rombouts, I., Brijs, K., Delcour, J. A., 2011. Kinetics 453 of heat-induced Lagrain, B., Brijs, K., Veraverbeke, W.S., Delcour, J.A., 2005. The impact of heating and 450 different for Evidence 2017. A., Luccia, Di G., Petrella, G., Rusco, M., Rutigliano, B., Gatta, La 448 polymers glutenin SDS-insoluble of Fate 2018. M.-H., Morel, V., Lullien-Pellerin, M., Joubert, 446 Icard-Vernière, C., 445 Feillet, P., 1999. Effects 444 of Mixing protein–polysaccharide of characterization Conditions FTIR 2014. K., Caba, la de J.P., Kerry, P., on Guerrero, Polymers111,598–605. inextrudedblends.Carbohydrate interactions Pasta 443 Dough 442 De 12,97–104. ofCerealScience properties ofpasta.Journal Stefanis, E., Sgrulletta, D., 1990. Effects of high-temperature drying on technological 440 Dexter, J.E., Matsuo, R.R., 1977. Changes in semolina proteins during spaghetti processing. 437 Cubadda, R.E., Carcea, M., Marconi, E., Trivisonno, M.C., 2007. Influence of gluten proteins 433 Bonanata, J., Turell, L., Antmann, L., Ferrer-Sueta, G., Botasini, S., Méndez, E., Alvarez, B., during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 15 Version postprint 488 487 43,179-208. Nutrition glutenin in relation to breadmaking functionality. 485 Critical Review in Food Science and 72,564-567. Chemistry duetoextrusionprocessing.Cereal proteins semolina 483 Science39,297–301. JournalofCereal temperature. high 481 ofFoodandAgriculture95,2579–2587. JournaloftheScience stepbystep. quality 479 Botany53,947–958. JournalofExperimental grainutilization. in 477 476 116,401–412. Chemistry 475 Food hydrolysates. protein of allergenicity potential the and fractions starch and protein of digestibility vitro in the on Effects temperatures. drying high by induced structure pasta of 473 3,488–497. uponmixing.Biomacromolecules wheatglutenprotein of 471 470 Chemistry77,685–691. Cereal Chromatography. 469 Liquid High-Performance Size-Exclusion by Determined as Proteins Flour Wheat Total of Extractability and Distribution Size on Settings Power and Time, Sonication Temperature, 467 47,173–180. Chemistry 465 133,3528-3534. Society AmericanChemical Journalofthe Cleavage. 463 462 98,149–155. andAppliedGenetics genes.Theoretical individual from products of properties Functional IV. wheats. primitive of proteins subunit glutenin weight 486 Veraverbeke, W. S., Delcour, J.A., 2002. Wheat protein composition and properties of wheat 484 Ummadi, P., Chenoweth, W.L., Ng, P.K.W., 1995. Changes in solubility and distribution of 482 Singh, H., MacRitchie, F., 2004. Changes in proteins induced by heating gluten dispersions at 480 Sicignano, A., Di Monaco, R., Masi, P., Cavella, S., 2015. From raw material to dish: Pasta 478 role and properties structures, proteins: storage seed Cereal 2002. N.G., Halford, P.R., Shewry, 474 Petitot, M., Brossard, C., Barron, C., Larré, C., Morel, M.H., Micard, V., 2009. Modification 472 Morel, M.H., Redl, A., Guilbert, S., 2002. Mechanism of heat and shear mediated aggregation 468 Morel, M.H., Dehlon, P., Autran, J.C., Leygue, J.P., Bar-L’Helgouac’h, C., 2000. Effects of 466 Matsuo, R.R., Irvine, G.N., 1970. Effect of gluten on the cooking quality of spaghetti. Cereal 464 Liang, J., Fernandez, J. M., 2011. Kinetic Measurements on Single-Molecule Disulfide Bond 461 Lee, Y.K., Bekes, F., Gras, P., Ciaffi, M., Mrell, M.K., Appels, R., 1999. The low-molecular- during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 16 Version postprint 494 493 95-123. Paul. Interaction: The Keys to Cereal Quality; Hamer, R. J., Hoseney, R. C., 491 Eds.; AACC: St. 490 59,3146-3154. Food Chemistry aggregation of wheat gluten protein upon pasta processing. Journal of Agricultural and 497 167. 496 drying on structural and textural 495 properties of durum wheat pasta. Cereal Chemistry 80, 159- Zweifel, C., Handschin, S., Escher, F., Conde-Petit, B., 2003. Influence of high-temperature 492 Weegels, P.L., Hamer, R.J., 1998. Temperature-induced changes of wheat products. In 489 Wagner, M., Morel, M.H., Bonicel, J., Cuq, B., 2011. Mechanisms of heat-mediated during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 17 Version postprint Pasta rested for 120 min at 40°C before dryingathightemperature at40°Cbefore Pasta restedfor120min dryingathightemperature at30°Cbefore Pasta restedfor120min dryingathightemperature at20°Cbefore Pasta restedfor120min hightemperature dried at Pasta directly lowtemperature dried at Pasta directly Cooked pasta Dried pasta Cooked pasta Dried pasta Cooked pasta Dried pasta Cooked pasta Dried pasta Cooked pasta Dried pasta during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Products Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation N/A: Not N/A: Not applicable. aspect. surface and diameter color, for made were measurements Eight triplicates. in out carried were times cooking and measurement Rheological 20,30or40°C)andHTdrying. temperature 1: Table Characteristics of dried pasta and cooked pasta produced by HT drying, LT drying, or coupling resting phase (for 120 min at different of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Brightness 81.7 ±1.4 74.9 ±5.1 54.9 ±0.8 76.3 ±4.6 79.8 ±0.9 54.1 ±0.1 55.5 ±1.7 71.5 ±0.6 56.0 ±0.3 55.2 ±0.1 (N/A) L* b d b a c a a b c a Comment citer cedocument: -0.533 ±0.049 0.988 ±0.459 0.707 ±0.076 0.803 ±0.290 0.203 ±0.047 4.29 ±0.57 3.88 ±0.02 5.36 ±0.53 2.39 ±0.03 3.43 ±0.14 Redness (N/A) a* b b a a a

c a d c c Yellowness 43.8 ±0.7 35.9 ±0.3 41.6 ±1.4 38.0 ±0.5 39.9 ±0.1 38.3 ±0.3 42.4 ±0.1 33.8 ±0.3 41.9 ±0.2 (N/A) b* d b c a b c a c a ACCEPTED MANUSCRIPT 2.36 ±0.09 2.15 ±0.07 1.47 ±0.03 2.38 ±0.04 2.50 ±0.04 1.47 ±0.02 2.68 ±0.01 1.50 ±0.03 1.53 ±0.02 1.48 ±0.04 Diameter (mm) d b d a a a d b a c - 18 Cooking (min) time N/A N/A N/A N/A N/A 11 10 11 10 10 Surface aspect (N/A) N/A N/A N/A N/A N/A 8 8 8 8 6 121.6 ±8.9 159.4 ±9.8 113.2 ±7.0 121.6 ±6.4 86.8 ±8.0 modulus Elastic (MPa) N/A N/A N/A N/A N/A a b b b c 263 ±63 342 ±67 153 ±10 295 ±44 271 ±38 Rupture stress (Pa) N/A N/A N/A N/A N/A b a a a a 2.09 ±0.38 1.68 ±0.21 2.62 ±0.42 2.44 ±0.32 2.05 ±0.36 Rupture strain (N/A) N/A N/A N/A N/A N/A b a a a a Viscoelasticity 8.63 ±0.53 12.4 ±0.5 14.4 ±1.1 13.0 ±1.7 14.4 ±1.0 index (N/A) N/A N/A N/A N/A N/A a a b a b Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation high or low temperature drying in pasta. dryingin high orlowtemperature upon and resting upon proteins gluten wheat of insolubilisation of model Mechanistic 5: Figure standard(120 to420min).Errortherelative deviation. barsfeature as a function of time during successive resting phase at 30°C (0 to 120 min) and HT drying Figure 4: Evolution of relative contents of soluble proteins for durum wheat glutenin in pasta overlap. notdisplayed) 30, or40°C).Errorbars(relativestandarddeviation; F fractions protein gluten wheat different the for proteins soluble of contents relative of Evolution 3: Figure overlap. notdisplayed) (HT). Errorbar(relativestandarddeviation; drying temperature high or (LT) drying temperature low during temperature time*drying drying Figure 2: Evolution of relative contents of soluble glutenin fractions in pasta as a function of overlap. displayed(a;b),errorbarsstandarddeviation) (c-d). Whennot (relative fractions (F protein gluten wheat different the for proteins soluble of contents relative of Evolution 1: Figure of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 1 and F and 1 to F 5 2 ) in pasta as a function of drying time during LT drying (a-b) or HT drying in pasta as a function of time during resting at different temperatures (at 20, (at temperatures different at resting during time of function a as pasta in Comment citer cedocument: ACCEPTED MANUSCRIPT

- 19 Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation When not displayed (a;b), error bars (relative standard deviation) overlap. displayed(a;b),errorbarsstandarddeviation) (c-d). Whennot (relative fractions (F protein gluten wheat different the for proteins soluble of contents relative of Evolution 1: Figure 1 Protein fraction (%) Protein fraction (%) 1 2 4 6 8 2 4 6 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 1 to F 2 2 0 0 0 0 5 ) in pasta as a function of drying time during LT drying (a-b) or HT drying D D r r y y 4 4 i i 0 0 n n 0 0 g g Comment citer cedocument:

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Protein fraction (%) 1 Protein fraction (%) 1 6 8 2 4 0 2 4 6 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5 0 0 1 1 0 0 0 0 D D r r y y i i n n g g 1 1

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Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation Error bar (relative standard deviation; not displayed) overlap. notdisplayed) (HT). Errorbar(relativestandarddeviation; drying temperature high or (LT) drying temperature low during temperature time*drying drying Figure 2: Evolution of relative contents of soluble glutenin fractions in pasta as a function of of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Protein fraction (%) Protein fraction (%) 1 1 1 1 2 4 6 8 2 4 6 8 0 2 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Comment citer cedocument: 1 0 0 1 0 0 ACCEPTED MANUSCRIPT D D 0 r r 0 y y 0 i i 2 n n 0 g g

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( 0 ( ° ° 0 C C B H B H 4 . . T T T 0 T m m 0 - - - - i i 0 n n 6 % % % 0 % ) ) 0 ( ( ( ( 0 F F F F 0 1 1 1 1 + + + + F F F F 2 2 2 2 5 ) ) ) 7 ) 0 0 0 0 0 0 0 Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation Error bars (relative standard deviation; not displayed) overlap. notdisplayed) 30, or40°C).Errorbars(relativestandarddeviation; F fractions protein gluten wheat different the for proteins soluble of contents relative of Evolution 3: Figure of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 1 and F and Protein fraction (%) 1 1 9 5 6 7 8 0 1 0 0 0 0 0 0 0 0 2 in pasta as a function of time during resting at different temperatures (at 20, (at temperatures different at resting during time of function a as pasta in Comment citer cedocument: ACCEPTED MANUSCRIPT

6 T - 22 0 i m e

( m i n ) 4 3 2 0 0 0 ° ° ° C C C - - - % % % 1 ( ( ( 2 F F F 0 1 1 1 + + + F F F 2 2 2 ) ) ) Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation Error bars feature the relative standard(120 to420min).Errortherelative deviation. barsfeature as a function of time during successive resting phase at 30°C (0 to 120 min) and HT drying Figure 4: Evolution of relative contents of soluble proteins for durum wheat glutenin in pasta of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 23 Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation high or low temperature drying in pasta. dryingin high orlowtemperature upon and resting upon proteins gluten wheat of insolubilisation of model Mechanistic 5: Figure of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT

- 24 Version postprint during pasta processing: decouplingbetween time- andtemperature-dependent effects. Journal Martin, C., Morel,M.H., Reau,A.,Cuq, B.(2019).Kinetics ofgluten protein-insolubilisation - Proteins insolubilisation has no significant impact on cooked pasta properties. oncookedpasta impact hasnosignificant - Proteinsinsolubilisation resting. comparedto onpastaqualities impact dryinghasahigher - Hightemperature bondsthroughSS-SHexchangereactions. covalent creation - Restingenables ofproteininsolubilisation. tohigherlevels lead - Highrestingtemperatures orderkinetics. uponrestingfollowsafirst insolubilisation - Glutenprotein Highlights of Cereal Science, 88,103-109. , DOI:10.1016/j.jcs.2019.05.014 Comment citer cedocument: ACCEPTED MANUSCRIPT