Structuring diluted wheat matrices: impact of heat moisture treatment on protein aggregation and viscoelasticity of hydrated composite flours

1 Structuring diluted wheat matrices: impact of heat moisture treatment on protein aggregation and viscoelasticity of hydrated composite flours Concha Collara*, Marina Villanuevab, Felicidad Rondab aCereals and Cereal-based Products. Food Science Department. Instituto de Agroquímica y Tecnología de Alimentos (CSIC). Avda. Catedrático Agustín Escardino, 7. 46980 Paterna. Spain. bDepartment of Agriculture and Forestry Engineering, Food Technology, College of Agricultural and Forestry Engineering. University of Valladolid. Avda. Madrid, 44. 34004 Palencia, Spain *Corresponding author. Tel.: +34 963 90 00 22; Fax: +34 963 63 63 01 E-mail address: [email protected] Abstract The influence of heat-moisture treatment (HMT) and flour hydration (DY) on the restoration of dough viscoelasticity of wheat/non-wheat binary matrices was investigated by applying fundamental and empirical rheological procedures, and the protein structural reorganisation was monitored by measuring residual protein solubility in different media, and by assessing the accessibility of thiol groups inside the protein network. Single chestnut (CN), chickpea (CP), millet (MI) and teff (T) flour samples submitted to HMT (15% moisture content, 1 h and 120ºC) were blended with wheat flour at 10% (CN, CP) and 30% (MI, T) of replacement, and binary matrices hydrated at low (L), medium (M) and high (H) DY. Structuring ability of HMT was mainly observed in cereal flour blends (T, MI), where higher elastic moduli and lower loss tangent together with solid-like elastic structure over higher shear stress were observed as compared with treated non-cereal flour blends (CN. CP). Increased flour hydration significantly weakened blends structure, inducing a stepped decrease in dynamic moduli values particularly noticed in cereal blends at highest level of flour hydration, and a shift from elastic-like to viscous-like structure at lower shear stress in non-wheat cereal matrices. The formation of a protein network with reinforced compact structure associated to the presence or formation of intramolecular (CN, CP, T) and intermolecular disulphide bonds (CN, CP, MI, T), water soluble (CN, MI) and water insoluble aggregates (CP, T) is feasible to achieve with proteins of non-wheat flours submitted to HMT, particularly in high DY doughs. The lower the amount of free thiols in high molecular weight proteins encompassing high degree of crosslinking, corresponded to thermally treated samples (T, MI) blended at L and M hydration levels. For thermally treated samples, the lower the amount of free thiols in high molecular weight proteins encompassing high degree of crosslinking, corresponded to T and MI binary matrices blended at L and M hydration levels. These samples exhibited a solid-like elastic structure over higher shear stress and showed increased tolerance to stress/ strain before losing the structure. Keywords: Teff · Millet · Chestnut · Chickpea · Protein aggregation · Viscoelasticity 1. Introduction Partial wheat flour replacement by nutrient-dense and health-promoting flours such as non-wheat cereal and non-cereal flours constitutes a plausible simple strategy to create added value to baked goods. This strategy has been successfully applied recently to diluted wheat matrices blended with ancient crops, 2 minor cereals, pseudocereals, legumes, and non-traditional fruit and seed flours (Collar, 2016; Paciulli et al. 2016). Among non-wheat flours, millets, chestnut, teff and chickpea flours can provide extra nutritional, health and/or functional promoting effects when incorporated into breadmaking systems. Chestnut flour contains high-quality proteins with essential amino acids, dietary fiber, low amount of fat, and also vitamins (E, B groups) and minerals (K, P, and Mg) of nutritional interest on both wheat (Dall'Asta et al. 2013) and gluten-free (Paciulli et al. 2016) breadmaking applications. Legumes constitute wholesome imaged foods providing nutritional (high protein, mineral and fiber contents, low digestible starch), health (protective and therapeutic effects to chronic health conditions), and functional promoting effects (body, texture, and taste enhancement) to foods (Angioloni and Collar 2012a). Associated mixtures of grain (chickpea, green pea) and oilseed (soybean) legumes in highly replaced wheat flour systems provided highly nutritious breads meeting viscoelastic restrictions and sensory standards (Collar and Angioloni 2017). Teff (Eragrostis tef), a nutritious cereal wheat-type gluten-free grain, rich in carbohydrates and fibre, microelements, and phytochemicals (Abebe et al. 2007), was successfully applied in breadmaking matrices up to 40% of wheat replacement (Ronda et al. 2015). Millets contain substantial levels of various phenolic compounds (Bagdi et al., 2011), much higher than in wheat, and they exhibit cholesterol lowering properties (Cho et al., 2000). With these attributes millets exhibit a huge potential for food production, and in particular for bread baking (Angioloni and Collar, 2012b, 2013a). Extensive replacement of wheat flour by non-wheat flours to achieve nutritional and health related benefits, often encompasses an impairment of the physic-chemical attributes of resulting breads, since flours are gluten-free and unable to form a three-dimensional viscoelastic protein network. Heat moisture treatment (HMT) constitutes a clean label alternative to chemical modification for altering the aggregation/disaggregation equilibrium of proteins (Mann et al., 2013) and the gelatinization and retrogradation properties of flours (Collar, 2017) and doughs (Collar and Armero, 2018a), providing promoted viscoelasticity of hydrated gluten-free flours. HMT of flours has successfully been applied to composite non-wheat-wheat flours to improve both volume and textural profile of the resulting breads (Collar and Armero, 2018b, 2018c). In optimally kneaded or fully developed dough, protein films or sheets are the predominant structural element (Delcour et al., 2012). At dry heat treatment above 50 ºC, unfolding of gluten proteins occurs. The hydrophobic parts of the protein molecules are getting more exposed, so that the rearrangement of disulphide bonds is favoured. As a result, gluten aggregates are forming, with decreased extractability and modified molecular weight distribution (Delcour et al., 2012). Therefore, a stronger dough or more stable foam can be produced. Schober et al. (2011) suggested that hydrophobic interactions rather than disulphide bonds are responsible for the gluten-like functionality of zein and kafirin, ascribing a key importance to non-covalent bonds in the protein aggregation of nongluten forming matrices. In this work, the influence of HMT and flour hydration (dough yield) on the restoration of dough viscoelasticity of diluted binary wheat/non-wheat matrices was investigated by applying fundamental and empirical rheological procedures, and the protein structural reorganisation was monitored by measuring residual protein solubility in different media, and by assessing the accessibility of thiol groups inside the protein network. The correlation of protein structural parameters with the physic-chemical and rheological characteristics of the composite matrices were also studied, and significant relationships were established and discussed with regard to the thermal treatment and water availability in binary systems. 2. Materials and methods 3 2.1. Materials Commercial flours from wheat (WT), chestnut (CN), chickpea (CP), millets (MI) and teff (T) were purchased from Navarro (Spain). Refined WT (70% extraction rate) of 195 x 10-4 J energy of deformation W, 0.57 curve configuration ratio P/L, and 58.8% water absorption in Brabender Farinograph, was used. Moisture, protein, ash and fat contents of commercial flours were determined following the ICC Standard methods 110/1, 105/2, 104/1, and 136, respectively (ICC 1976-1996). Soluble, insoluble and total fibre contents were determined according to the AOAC method 991.43 (Table 1). Two replicates were made for each flour analysis. Digestible carbohydrates were calculated by difference. Digestible carbohydrates were calculated by indirect determination as 100 − [moisture + protein + fat + dietary fibre] (FAO 2003). 2.2. Methods 2.2.1. Heat-moisture treatment (HMT) HMT conditions (15% moisture content, 1 h and 120ºC) were selected based on previous experiments (Collar and Armero, 2018a), in which maximization of viscometric profile during pasting and gelling and minimization of loss of hydration properties of flour samples were applied as criteria from conditions of total mixture reached 15%, 25%, and 35%, and heat kept for 1, 3, or 5 h at 120 ºC in a convection oven. Single CN, CP, MI and T flour samples were placed into screw-capped cylindrical glass containers (150 mm diameter, 250 mm height). Small amount of distilled water was added slowly with frequent stirring until moisture levels (w/w) of the total mixture reached 15%, and equilibrated for 24 h at room temperature. Hydrated samples that occupied 13 mm height in containers were kept for 1 h at 120 ºC in a convection oven (P-Selecta, Barcelona, Spain). Samples took 20 min to reach the preset temperature, and 30 min to reach the room temperature after heating. Untreated native flours were used as controls. Untreated (-) and HMT (+) single flours were used in binary blends in presence of WTfor dough making. 2.2.2. Visco-metric properties Pasting profiles (gelatinisation, pasting, and setback properties) of hydrated untreated and HMT treated binary blended flours prepared by WTreplacement with MI+ or T+, (30%), and with CN+ or CP+ (10%), were obtained with a Rapid Visco Analyser (RVA-4, Newport Scientific, Warriewood, Australia) using the ICC Standard method 162. Viscometric parameters were calculated from the pasting curve (Figure 1) using Thermocline v. 2.2 software (Collar, 2003). The slurry was heated to 50 C and stirred at 160 r/min for 10 s for thorough dispersion. The slurry was held at 50 ºC for up to 1 min, and then heated to 95 ºC over 3 min 42 s and held at 95 ºC for 2 min 30 s, and finally cooled to 50 ºC over 3 min 48 s, and held at 50 ºC for 2 min. The pasting temperature (ºC) (when viscosity first increases by at least 25 mPas over a 20-s period), peak time (when peak viscosity occurred), peak viscosity (maximum hot paste viscosity), holding strength or trough viscosity (minimum hot paste viscosity), breakdown (peak viscosity minus holding strength or trough viscosity), viscosity at 95 ºC (viscosity attained at the beginning of the holding period during cooking), viscosity at the end of the 95 ºC holding period, viscosity at 50 ºC (viscosity attained at the beginning of the holding period during cooling), final viscosity (end of test after cooling to 50 ºC and holding at this temperature), setback (final viscosity minus peak viscosity), and total setback (final viscosity minus holding strength) were calculated from the pasting curve using Thermocline v. 2.2 software (Collar, 2003). For each viscometric measurement, 3 replicates were made. 4 2.2.3. Dough making Binary doughs were prepared from wheat–based blended flours by WTreplacement with MI+ or T+, (30%), and with CN+ or CP+ (10%), and incorporation of water (%, flour basis) according to different hydration levels DY: low L (65% CN, CP, MI; 80% T), medium M (70% CN, CP, MI; 90% T) and high H (75% CN, CP, MI; 100% T). Binary untreated samples (-) were made at M hydration level for comparative purposes. Sampling resulted in 16 runs. A dough sample code was set referring to native (- ) and HMT (+) CN, CP, MI and T flours hydrated at L, M and H levels in sample formulation, as follows: CN+L, CN+M, CN+H, CN-M, CP+L, CP+M, CP+H, CP-M, MI+L, MI+M, MI+H, MI-M, T+L, T+M, T+H, T-M (Table 2). Blended flours (100 g), water (adapted %, flour basis), and salt (2%, flour basis were mixed in a 2 kg mixer at 60 revolutions min-1 for 10-13 min up to optimum dough development. Two trials were conducted per formulation. Preliminary tests were performed to know the range of hydration per formulation to avoid stickiness and deleterious effects on dough machinability. Preliminary tests for maximization of single non-wheat flour replacement of wheat flour showed the optimal amounts to be used without compromising dough machinability and handling ability were flourdependent. Amounts greater than 30% of cereal flours and 10% of non-cereal flours resulted in highly sticky doughs with low machinability (low cohesiveness and great firmness) and poor breadmaking performance of the blends. Analogously, previous tests conducted to know the amount of water necessary to avoid stickiness and deleterious effects on dough machinability proved that the range of 65-75% of water absorption was suitable for all the formulations except for teff formulated matrices (80-100%) to assure dough handling ability during processing. Teff flour needs higher hydration in dough-making to avoid excessive dough hardness and stickiness. 2.2.4. Oscillatory tests Dynamic oscillatory tests of the doughs were carried out with a Kinexus Pro+ rheometer (Malvern Instruments Ltd, Malvern, UK) with parallel plate geometry (40 mm diameter) of serrated surface and with 1 mm gap. The dough was placed between the plates, the surplus of sample was removed, and it was rested for 5 min to allow relaxation and to stabilize temperature at 25 ºC. A cover-hood was placed over the sample to avoid sample dryness during the test. Temperature was established by means of a Peltier device (KNX2002) and a heat exchanger (KNX2500) (Marven Instruments Ltd., Marven, UK). Frequency sweeps were carried out from 10 to 1 Hz in the linear viscoelastic region (at a constant strain of 0.03%). Frequency sweep data were fitted to the power law model as described in previous works (Ronda et al., 2013): 𝐺′(𝑤)=𝐺′1·𝑤𝑎 𝐺′′(𝑤)=𝐺′′1·𝑤𝑏 tan𝛿 (𝑤)=𝐺′′(𝑤) 𝐺′(𝑤) =(𝐺′′ 𝐺′)1· 𝑤(𝑏−𝑎) =(tan𝛿)1· 𝑤𝑐 The coefficients G’1, G’’1, and (tan 𝛿)1, represent the elastic and viscous moduli and the loss tangent at a frequency of 1 Hz. Fittings were done in the frequency range (1-10 Hz), where a linear double logarithm curve was systematically obtained. The a, b and c exponents quantify the dependence degree of dynamic moduli and the loss tangent with the oscillation frequency, ω. These tests were carried out in duplicate. Strain sweeps were carried out from 0.01 to 100 % at constant frequency of 1 Hz. From the curves, the maximum strain/stress beyond which the dough structure was broken, max/max, was calculated as the 5 strain/stress at which the elastic modulus of the gels dropped with strain 10% with respect to the value obtained in the LVR. The shear strain (cp) and stress (cp) of the crossing point, where G’ = G’’ were also recorded. These tests were carried out in duplicate. 2.2.5. Protein analysis Waterand salt-soluble proteins were extracted by using phosphate buffer containing 0.05 M NaH2PO4 and 0.1 M NaCl, pH 7.0 (buffer P). Urea-soluble proteins were extracted using buffer P with the addition of 6 M urea (buffer U). Finally, disulphide bonds were reduced by adding 0.1 M dithiothreitol (DTT) to the buffer U (buffer D). Proteins were extracted from 50 mg of freeze-dried dough dissolved in 1 ml of each buffer as previously described by Alamprese et al (2005). Samples were incubated at 25 ºC for 3.5 h under stirring. After centrifugation at 12,000 g for 1 h, the amount of extracted proteins present in the supernatants was determined according to Bradford protocol (Kruger, 2002), using bovine serum albumin as standard. 2.2.6. Free thiols Free thiols (FT) were determined in protein extracts P, U and D using 25-100 μL aliquots (at least 2 nmol of protein). The procedure is based on the reaction of the thiol with Ellman’s reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to give the mixed disulphide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion (TNB2–) at 412 nm (Aitken and Learmonth, 2009). FT from low molecular weight (LMW) proteins were estimated in P extracts, FT from high molecular weight (HMW) proteins were estimated in U-P extracts, and accessible thiol content from reduced disulphide bonds were estimated in D-U extracts after subtracting the contribution of buffer D (10 μmol FT/100 μL). 2.2.7. Statistical analysis Univariate ANOVA and multivariate MANOVA analysis of data (mean comparison of samples LSD) were performed by using Statgraphics V.7.1 program (Bitstream, Cambridge, MN). 3. Results and Discussion A physicochemical approach was adopted to assess the effectiveness of HMT treatment and DY on the viscometric and viscoelastic behavior, and on the protein structure of composite doughs. Viscometric profile, dynamic rheological behavior, and protein aggregation/disaggregation were measured to quantify significant differences among samples. 3.1. Visco-metric profile of native and HMT binary doughs Heat-induced gelatinization process solubilizes amylose and amylopectin molecules (composing a continuous phase) and granule remnants (composing dispersed phases), both of which are responsible for the rheological and textural properties of the starch paste (Kim et al. 2012). Quantitative viscosity profiles of native and HMT hydrated binary blended flours of WT with MI, T, CP, and CN were lower during both cooking and cooling cycles as compared to those of hydrated WT flour counterparts (Fig. 1). The RVA profile of binary blends of WT and non-WT flours was characterized by the presence of one maximum in the 95 °C holding period representing starch gelatinization under a constant shear, smaller breakdown of viscosity at the holding period, and a variable typical increase in setback viscosity on cooling varying from discrete (T, CP, and CN), to prominent (MI) changes ascribed to the variable reassociation of constituent starch molecules, mainly amylose, into a more ordered state (Fig. 1). 6 1 Table 1. Chemical and nutritional characteristics of native and heat-moisture-treated (HMT) wheat, teff, millet, chestnut, and chickpea flours 2 Propertya %, d b. Wheat Teff Millet Chestnut Chickpea Native HMT Native HMT Native HMT Native HMT Native HMT Moisture 14.30 ± 0.12d 15.12 ± 0.11e 12.62 ± 0.13c 14.95 ± 0.21e 11.60 ± 0.09b 15.06 ± 0.23e 6.90 ± 0.05a 14.87 ± 0.33e 11.88 ± 0.09b 15.22 ± 0.09e Protein 14.12 ± 0.28c 13.94 ± 0.21c 14.08 ± 0.46c 13.96 ± 0.56c 10.29 ± 0.34c 10.51 ± 0.32b 6.44 ± 0.20a 6.56 ± 0.34a 18.82 ± 0.60d 18.03 ± 0.51d Fat 1.56 ± 0.11a 1.62 ± 0.13a 4.69 ± 0.29bc 4.79 ± 0.49c 4.03 ± 0.39b 4.19 ± 0.25b 4.08 ± 0.18b 4.21 ± 0.31bc 6.96 ± 0.12d 7.41 ± 0.31e Digestible carbohydrates 81.70 ± 2.30c 81.77 ± 2.51c 68.70 ± 4.50b 69.26 ± 6.10b 66.52 ± 5.09b 66.29 ± 6.24b 79.50 ± 3.20c 80.00 ± 4.30c 48.80 ± 1.10a 50.30 ± 1.90a Total dietary fiber 2.56 ± 0.23a 2.67 ± 0.35a 12.31 ± 1.42c 11.99 ± 1.70c 19.23 ± 2.31d 19.01 ± 1.96d 9.67 ± 1.03b 9.23 ± 1.43b 25.13 ± 2.33e 24.75 ± 3.20e Soluble dietary fiber 1.06 ± 0.11a 0.96 ± 0.21a 4.84 ± 0.55b 4.96 ± 0.66b 4.07 ± 0.63b 3.98 ± 0.59b – – 6.55 ± 0.42c 6.39 ± 0.65c Insoluble dietary fiber 1.49 ± 0.28a 1.71 ± 0.33a 7.46 ± 1.10b 7.03 ± 1.23b 15.16 ± 1.96c 15.03 ± 1.79c – – 18.62 ± 1.40c 18.36 ± 2.10c Mean values ± standard deviation 3 a Within rows, mean values with different following letter do differ significantly from each other (p < 0.05) 4 5 6 7 8 7 On the opposite, qualitative RVA profile of WT flour clearly defined a maximum viscosity during the holding period (3777 mPa s), and a subsequent breakdown on cooking (1231 mPa s), followed by a discreet setback on cooling (2141 mPa s), as reported earlier (Collar 2003, 2017). Fig. 1. Rapid Visco Analyzer profiles of native (-) and heat-moisture-treated (+) doughs from chestnut (CN), chickpea (CP), millets (MI), and teff (T) binary blended flours with native wheat flour (WT). RVA profiles for untreated and HMT hydrated blended flours exhibited some quantitative differences (Fig. 1), depending on both the non-wheat flour considered and the thermal treatment applied. In general, MI batters develop higher viscometric profiles during pasting and particularly during gelling (p < 0.05) than the other flour batters provide (Fig. 1). During the cooking cycle, mean values for viscosity of MI vs CP/CN/T were as follows: peak viscosity (2534 mPa s vs 2315-2426 mPa s), holding strength (1412 vs 1351-1414 mPa s), and breakdown (1122 mPa s vs 964-1102 mPa s). During cooling cycle, gelling characteristics (mean values) of MI vs CP/CN/T varied from 2459 to 1290-1535 mPa s (total setback), from 2583 to 2064-2225 mPa s (viscosity at 50 °C), and from 3871 to 2647-2949 mPa s (final viscosity), respectively. Despite HMT significantly (p < 0.05) increased the magnitude of some cooking and cooling parameters, changes were discreet (5-9%) except for MI batters (Fig. 1), specifically peak viscosity (+ 16%), holding strength (+ 24%), final viscosity (+ 15%), and viscosity at 50 °C (+ 22%). Since no significant change was observed in the chemical and nutritional composition analysis (Table 1), the differences of pasting/gelling characteristics may be attributed to the changes in the structure, as noted earlier (Lim et al. 2003). Changes of HMT on pasting and flow properties were collectively attributed to changes in granular volume (decreases in wheat, potato and lentil starches, but increases in oat starch), which modifies the resistance of the granule to deformation and disintegration, the interactions between starch chains and the amylose leaching (Hoover 2010). In millet flours submitted to dry heating, similar findings have been reported (Sun et al. 2014), suggesting that an ester bond could be formed when the starch and non-starch components were dry heated, similar to the interaction of starch and gum crosslinking during dry heating treatments (Lim et al. 2003). After dry heat, flour granules formed into big lumps under microscopy observation, and the gel structure of the 8 flour had more compact holes and it had formed into a stronger and closer structure The denser structure of the dry heat sample gels illustrated that the dry heating process made the interactions stronger in the flour/starch compositions, providing functionality that is equivalent to chemical crosslinking (Sun et al. 2014). In hydrothermal treatments such as HMT, crosslinking effects are not so favored due to water mobility, despite the high amount of dietary fiber (19%) present in millet flour (Table 1) can induce polymeric association with the polysaccharide functional groups in the starch after HMT at low moisture levels of 15%. Table 2. Sample code of native and heat-moisture-treated (HMT) binary matrices Sample code Wheat flour replacement, % Hydration level, % Native (-) HMT (+) Medium (M) Low (L) Medium (M) High (H) Chestnut (CN) 10 CN + L 65 CN + M 70 CN + H 75 CN - M 70 Chickpea (CP) 10 CP + L 65 CP + M 70 CP + H 75 CP - M 70 Millet (MI) 30 MI + L 65 MI + M 70 MI + H 75 MI - M 70 Teff (T) 30 T + L 80 T + M 90 T+ H 100 T - M 90 3.2. Fundamental rheological properties of native and HMT binary doughs Small deformation oscillatory measurements provide complementary information to large deformations using empirical methods in terms of structure of the material. 3.2.1. Frequency sweeps The profiles of mechanical spectra for native and HMT blended doughs showed variable structured systems. The spectra exhibited a dependence of the viscoelastic moduli on oscillatory frequency in terms of increase of both moduli G’ and G” with the increase in frequency (1–10 Hz), associated to a high overall chain mobility within the network (Lopes-da-Silva et al. 2007). The a, b, and c exponents quantify the dependence degree of dynamic moduli and the loss tangent respectively with the oscillation frequency. Values ranged from 0.1251 to 0.2262 (a), 0.2199 to 0.2808 (b), and 0.0488 to 0.1049 (c). In good accordance, the frequency sweep data showed that all dough matrices displayed higher G’, G”, and tan δ at higher frequencies compared to those at low frequencies, indicating the predominance of viscous characteristics in the dough and decrease in the elastic characteristics at higher frequencies. G’ 9 was greater than G” at all the frequencies studied (1–10 Hz), indicating viscoelastic soft solid nature of matrices (data not shown) and predominance of elastic character regardless the non-wheat flour used and the dough hydration applied in dough making. In good accordance, the frequency sweep data showed that G’ values were greater than G” at all the frequencies studied (1– 10 Hz), indicating the predominance of elastic character regardless the non-wheat flour used and the dough hydration applied in dough making. A viscoelastic soft solid nature of matrices can be concluded from the tan δ values obtained that ranged 0.20–0.36. In addition, all dough matrices displayed higher G’, G”, and tan δ at higher frequencies compared to those at low frequencies. However, the viscous moduli showed always higher frequency dependence than elastic moduli, with “b” exponent values (0.22–0.28) always above the “a” exponent values (0.13– 0.23). These results indicate a decrease in the solid-like characteristic predominance in doughs and the relative increase in the viscous behavior at higher frequencies. The coefficients G’1, G”1, and (tan δ)1 represent the elastic and viscous moduli and the loss tangent at a frequency of 1 Hz (Table 3), respectively. For HMT blended samples, storage moduli values (G’1) varied from 4050 to 17,823 Pa, loss moduli (G”1) ranged from 809 to 4555 Pa, and loss tangent (tan δ)1 as the ratio of viscous and elastic response of the material being tested ranked from 0.1997 to 0.3614 (Table 3). The lower (tan δ)1 values are indicative of a more elastic structure, hence, blends with cereal flours T+ (0.2097–0.1997) and MI+ (0.2555–0.2581) exhibited the highest elasticity range at small deformations compared to non-cereal flours CP+ (0.3614–0.3508) and CN+ (0.3217–0.3014). Native blended samples made at intermediate (M) hydration level presented compared to their HMT blended counterparts (Table 3), lower viscoelastic moduli values in non-wheat cereal flour blends (− 14– 17% T-M, − 27–28% MI-M) but higher dynamic moduli values in non-cereal flour blends (9–11% CP-M, 37–41% CN-M). The mentioned behavior is consistent with a more structured nature of cereal blends after HMT, particularly for MI+ samples. HMT allows control of molecular mobility at high temperatures by limiting the amount of water. HMT-induced changes in starch structure and properties have been found to vary with starch source and amylose content. For instance, tuber starches are more sensitive to HMT than legume or cereal starches (Gunaratne and Hoover 2002). The different effect of HMT on cereal than non-cereal flour blends may be related to the different granular and crystalline structure of cereal starches (A-type X-ray pattern) versus non-cereal (mainly Ctype X-ray pattern) (Hoover 2010). Cereal starches did not exhibit an altered X-ray diffraction pattern after HMT (Jacobs and Delcour 1998). Also, the marked different nature and content of proteins, mainly insoluble storage proteins in cereals and predominantly soluble globulins and albumins in non-cereals flours, affect the impact of HMT on both flour proteins and starch-protein interactions. Single and interactive effects of the type of non-wheat flour and the level of hydration in HMT blends were significant (p < 0.05) on main fundamental rheological parameters (Table 4). Effects of flour type are particularly significant for G’1 and (tan δ)1. Higher G’1 and lower (tan δ)1 values were observed for non-wheat cereal flour blends (10358– 11,074 Pa, 0.2033–0.2545) as compared with non-cereal flour blends (6880–7947 Pa, 0.3556–0.3190). Flour hydration systematically induced a stepped decrease of 40–43% in dynamic moduli values from L to M and from M to H blended doughs. The larger drops in dynamic moduli were noticed in cereal blends at highest level of flour hydration (H), reaching values of 4050 (G’1) and 810 Pa (G”1) for T and 5122 (G’1) and 1323 Pa (G”1) for MI (Table 4). On the contrary, cereal blends made at lower level of hydration (L) showed the greatest values for dynamic moduli, particularly for G’1 (> 17,700 Pa), in accordance with a more solid and structured nature. 16 between proteins, which include covalent interactions (such as disulfide bonds) and non-covalent interactions (such as ionic bonds, hydrogen bonds, and hydrophobic interactions). Authors suggested that wheat gluten unfolded during heat treatment, and the hydrophobic regions and free SH groups were exposed. When the gelation temperature was greater than 60 °C, crosslinking reactions among the wheat gluten molecules occurred, and subsequently, the unfolded protein chains aggregated via the formation of disulfide bonds and hydrophobic interactions, and the aggregates agglomerated into a threedimensional network. In non-wheat flours, an analogous two-step process (unfolding-aggregation) may take place, being the rate and the extent of the formation of intermolecular disulfide bridges dependent on the protein chain length, number, and exposure of free thiols after unfolding, sterical hindrance, and co-protein effects as stated before (Wang et al. 2016). The level of accessible free SH groups and the surface hydrophobicity of unfolded proteins were the main protein characteristics determining coprotein effects in mixtures with gluten. In fact, the amount of free thiols from high molecular weight proteins negatively correlated (p < 0.05, coefficient of correlation r) with τcp (− 0.5018), γmax (− 0.5484), and τmax (− 0.6085), evidencing that the lower the amount of free thiols/high crosslinking corresponded to samples that exhibited a solid like elastic structure over higher shear stress and showed increased tolerance to stress/strain before loosing the structure. 4. Conclusions HMT treatment appears as an efficient and clean strategy to enhance in variable extent the viscoelasticity of T, MI, CN, and CP binary blends with wheat flour, depending on both the nature of the non-wheat flour and the hydration level of the matrices. Structuring ability of HMT was mainly observed in cereal flour blends. Higher elastic moduli and lower loss tangent together with solid-like elastic structure over higher shear stress were observed for treated non-wheat cereal flour blends (T, MI) as compared with treated non-cereal flour blends (CN, CP). Increased flour hydration significantly weakened blends structure, inducing a stepped decrease in dynamic moduli values particularly noticed in cereal blends at highest level of flour hydration, and a shift from elastic-like to viscouslike structure at lower shear stress in non-wheat cereal matrices. Protein denaturation of individual polypeptides and dissociation of non-covalent aggregates significantly led to an increase in the number of accessible thiols in CN, CP and MI blends on HMT. The formation of SH-containing water soluble aggregates (CN, MI) and the formation of water insoluble aggregates of higher molecular weight (CP, T) take place on HMT. Results are consistent with changes in the protein molecular conformation that resulted in the exposure of hydrophobic region and SH groups that were initially buried inside the native molecule. With HMT, exposed free SH groups were oxidized to interor intramolecular disulfide bonds. The formation of a protein network with reinforced compact structure associated to the presence or formation of intra- (CN, CP, T) and intermolecular S-S bonds (CN, CP, MI, T), water-soluble (CN, MI) and water-insoluble aggregates (CP, T) is feasible to achieve with proteins of non-wheat flour submitted to HMT, particularly in high DY doughs. The lower the amount of free thiols in high molecular weight proteins encompassing high degree of crosslinking corresponded to thermally treated samples (T+, MI+) blended at low (L) and medium (M) hydration levels. 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