Low-frequency ultrasonication modulates the impact of annealing on physicochemical and functional properties of rice flour

Low-frequency ultrasonication modulates the impact of annealing on physicochemical and functional properties of rice flour Antonio J. Vela; Marina Villanueva; Felicidad Ronda* Department of Agriculture and Forestry Engineering, Food Technology, College of Agricultural and Forestry Engineering, University of Valladolid, Spain *Corresponding author. E-mail: [email protected] Abstract Ultrasonication (US) is a green technology used to physically modify flours to increase their industrial range of applicability. The aim of this work was to study the combined effect that dual US and annealing (ANN) treatments have on starch and protein structure of rice flour, at 20, 40, 50 and 60 °C. Results showed clear modifications of functional, thermal and pasting properties of flours, as well as rheological properties of gels made from them. US+ANN led to generation of small-size particles, which markedly increased the swelling power and starch damage. X-Ray Diffraction and FTIR indicated that starch crystallinity and protein secondary structure was affected by the shear forces of cavitation. The combination of US+ANN improved the crystalline structure arrangement within the starch granules, causing narrowing of the gelatinization temperature range (ΔT). Pasting viscosities were significantly decreased by ultrasonication, following an increasing trend with increasing temperature, while pasting temperature was increased, agreeing in the achievement of a thermodynamically more stable structure. The rheological properties indicated a reduction of the elastic (G') and viscous (G'') moduli after ultrasonication, as well as lower values of tan (), reflecting a higher predominance of elastic modulus versus viscous one than the non-sonicated flours. The rice flour’s properties were found to be highly sensitive to the applied treatment conditions, showing a synergetic effect when sonicating at the highest studied temperature. Keywords: Rice flour, low-frequency ultrasound treatment, annealing, thermal properties, gel rheological properties; biopolymers structure 1. Introduction Over the last decade, the market for gluten-free (GF) products has grown considerably. This demand is related to better diagnostic methods identifying an increasing number of people suffering from coeliac disease and other gluten-related disorders, and people who intake GF products as a “healthier” lifestyle (Witczak, Ziobro, Juszczak, & Korus, 2016). As a result, there are increasingly more glutenfree sources studied and transformation techniques applied in the development of novel food products with improved sensorial quality and nutritional value. Flours and starches have been modified by different techniques (genetic, chemical, enzymatic or physical) in order to improve their physical and chemical properties, and increase their industrial range of applicability. Physical modifications are better perceived by consumers for being an environment friendly technology. Physical modification of cereal products by ultrasound (US) treatments have shown many advantages in terms of higher selectivity and quality, reduced use of processing time and chemicals, linked to the concept of “green chemistry and technology”, resulting in increasing research interest in recent years (Amini, Razavi, & Mortazavi, 2015; Zhu, 2015). Physical modifications of flours by US are the result of cavitation phenomenon over the treated matter. Mechanical waves of ultrasonic frequency generate tiny bubbles as result of pressure variation, which keep growing until the oscillation of their walls equals the applied frequency and collapse (Ashokkumar, 2015; Zheng et al., 2013). Collapsing bubbles induce solvent micro-jets shooting to the suspended particles’ surface, generating very high shear forces that cause mechanical damage and surface erosion, and local rises of temperature that lead to rise of the suspension’s temperature, when the temperature of the treated medium is not controlled (Zhu, 2015). The extent of the modification depends on parameters such as duration, temperature, frequency and power of the treatment, moisture content of the system and botanical origin of the source (Zhu, 2015). Previous studies have demonstrated the great influence that temperature has on the modification achieved on starches (Amini et al., 2015; Kaur & Gill, 2019; Monroy, Rivero, & García, 2018; Sujka & Jamroz, 2013; Zheng et al., 2013). The rise of temperature in excess water represents a physical modification method called annealing (ANN). The said modification procedure happens when the treated matter is kept in excess water content (>60% w/w) for an extended period of time, at a temperature above the glass transition temperature and below the onset gelatinization temperature, which allows a modest molecular reorganization (Hoover & Vasanthan, 1994; Tester & Debon, 2000; Zavareze, Renato, & Dias, 2011). ANN facilitates interaction between starch chains within the amorphous and crystalline regions, causing rearrangement to a more organized configuration, which reflects in an improvement of paste viscosity, stability and starch in vitro enzymatic digestion (Chi et al., 2019; Zavareze et al., 2011). The physical modification of starches from different botanical origin has been studied by the individual effect of both US (Amini et al., 2015; Jambrak et al., 2010; Luo et al., 2008; Zuo et al., 2009) and ANN (Chi et al., 2019; Hoover & Vasanthan, 1994; Tester, Debon, & Sommerville, 2000; Wang et al., 2017) treatments and have proven to generate changes in their physical, thermal and hydration properties. In flours, however, very little work is available (Zhu & Li, 2019, Vela et al., 2021). It is important to study both treatments in flours because they are primarily raw materials for starchy food products which are the main source of complex carbohydrates in the human diet. Flours are a more complex matrix than starches and include other components, in particular proteins which are also susceptible to be affected by treatments (Vela et al., 2021). In addition, these components interact with starch in such way that the modification effect on starch is non-comparable to flour. US treatments, as they must be performed on a water suspension of the sonicated matrix, are commonly joined with ANN treatment, resulting from temperature rise derived from cavitation; so the final characteristics of the treated matter depend on a combined effect of both treatments. However, the individual effect of each treatment is not usually evaluated, and even, not infrequently, the temperature reached in the suspension during treatment is not controlled or monitored at all. The impact of US treatments on rice flour has been previously studied at different concentration and treatment time at a constant treatment temperature of 20° C (Vela, Villanueva, Solaesa, & Ronda, 2021). However, a study of the role that temperature (ANN) plays in the range of modification achieved by ultrasonication has not been covered in the available literature so far. Therefore, the objective of this study was to evaluate the synergistic effect of temperature and ultrasonication on the physical, thermal and hydration properties of rice flour, at the temperatures of 20, 40, 50 and 60 °C. The impact of this dual treatment on flour microstructural arrangement and biopolymers (starch and proteins) structural features was also evaluated. 2. Materials and methods 2.1 Rice flour The rice flour used was supplied by Emilio Esteban SA (Valladolid, Spain). The composition was: 7.60 % protein, 5.52 % fiber, 2.06 % fat, 0.34 % ash and 13.67 % moisture (data provided by the manufacturer). The native flour was stored at 4 °C until utilization. 2.2 Ultrasound treatment The equipment used in this study consisted of Hielscher UP400St sonicator (Hielscher Ultrasonics, Germany) set with S24d22D titanium tip. Rice flour dispersions were prepared at a concentration of 10 % (g dry flour/100 g of dispersion) by suspending rice flour in distilled water (total weight of 400 g). All treatments were performed for 60 minutes at constant frequency of 24 kHz with maximum output power of 180 W (47 W/cm2), and 80 % on-off pulse (0.8 s on 0.2 s off). Treatment time was chosen to be 60 minutes to set it as basis for the annealing treatments. The control flour onset gelatinization temperature was found to be 61.1±0.2 °C, so the treatment temperatures chosen were 20, 40, 50 and 60 °C. Studied samples were sonicated at the constant target temperature, representing a combined US+ANN treatment, and were compared to their corresponding ANN treatment, which were processed following the same procedure without being sonicated (refer to Table 1). The native flour was used without any processing as control in the study. The dispersions were treated in a glass jacket containing recirculating water from a water bath to achieve the desired temperature and maintain it during treatment. During the treatments all dispersions were stirred using a magnetic stirrer to ensure a homogenous temperature and avoid sedimentation of the flour. In ANN treatments the target temperature was directly set in the water bath, while in US+ANN treatments a lower temperature was set in the water bath (10°C below the target temperature) since previous assays had demonstrated that temperature rise derived from ultrasonication would get the dispersions to the desired temperature. Dispersions’ temperature was controlled during the whole treatment using a Testo 735-2 (Instrumentos Testo S.A., Spain) digital thermometer coupled with a flexible immersion tip. Flours were retrieved by freeze-drying, using a SP Scientific equipment, model Genesis Pilot Lyophilizer (SP Industries Inc, Warminster, U.S.A.), followed by sieving to <250µm. All treated flours were stored at 4 °C until use. Table 1. Treatment conditions and sample code given to each flour in the study. Sample Ultrasonicated Temperature (°C) Control No -- ANN-20 No 20 ANN-40 No 40 ANN-50 No 50 ANN-60 No 60 US+ANN-20 Yes 20 US+ANN-40 Yes 40 US+ANN-50 Yes 50 US+ANN-60 Yes 60 2.3 Particle size distribution The particle size distribution of all flours was determined using a Mastersizer 2000 laser diffraction particle size analyzer (Malvern Instruments Ltd, UK). The reported results are median diameter (D50) and ((D90-D10)/D50) as a dispersion measurement, as described in (Abebe, Collar, & Ronda, 2015). Measurements were made in triplicate. 2.4 Scanning electron microscopy (SEM) Images of the surface microstructure were obtained using a Quanta 200FEG scanning electron microscope (FEI, Oregon, U.S.A.) equipped with X-ray detector, without prior metallization. Visualizations were performed with accelerating voltage of 5.0 keV in low vacuum mode using a secondary electron detector at a magnification of 100x, 500x and 1500x. 2.5 Starch damage Damaged starch content was measured following the procedure indicated by Vela, Villanueva, Solaesa, & Ronda (2021) using Megazyme starch damage kit (K-SDAM, Megazyme, Ireland). Results are referred to dry matter (dm). Samples were evaluated in triplicate. 2.6 Hydration properties Water absorption capacity (WAC) was measured at 10% flour dispersion (w/v), while water absorption index (WAI), water solubility index (WSI) and swelling power (SP) were measured at 5% (w/v) following the method described by Abebe, Collar, & Ronda, (2015). All results are referred to flour dry matter. WAC was expressed in g H2O/g, WAI in g sediment/g, WSI in g soluble solids/100 g and SP in g sediment/g of insoluble solids in flour. Samples were evaluated in triplicate. 2.7 X-Ray diffraction (XRD) A Bruker-D8-Discover-A25 diffractometer (Bruker AXS, Rheinfelden, Germany) equipped with a Cu-Kα radiation (λ = 0.154 nm) at voltage of 40 kV and current of 40 mA was used to obtain the XRD patterns of the flours. Radiation intensities were measured in the range of 5° to 40° of 2θ diffraction angle, with scan step size of 0.02°, receiving slit width of 0.02nm, scatter slit width of 2.92°, divergence slit width of 1° and a rate of 1.2°/min. Samples were equilibrated at 15% humidity prior measurement using a saturated humidity ICP260 incubator at 15 °C (Memmert GmbH, Germany). Sample’s crystallinity was calculated as described in Vela, Villanueva, Solaesa, & Ronda (2020). 2.8 Fourier transform infrared spectroscopy (FTIR) FT-IR Nicolet iS50 spectrophotometer (Thermo Fisher Scientific, U.S.A.) equipped with a crystal diamond attenuated total reflectance (ATR) sampling accessory was used to record FTIR spectra of the flours, previously equilibrated at 15 % humidity (see section 2.7). Measurements were performed in wavenumber range of 400-4000 cm-1 with resolution of 4 cm-1 and accumulation of 64 scans. Amide I bands (1700-1600 cm-1) were analyzed using Origin2019b (OriginLab Corporation, U.S.A.). Individual bands were determined in deconvolved curves with second derivative analysis following iterative fitting assuming Gaussian band shapes. Peaks assignment correspond to: β-turns (1700-1660 cm-1), α-helix (1658-1650 cm-1), random coil (1650-1640 cm-1) and β-sheet (1640-1600 cm-1) (Byler & Susi, 1986). Measurements were performed in triplicate. 2.9 Differential scanning calorimetry (DSC) Flours’ thermal properties were determined with DSC3 calorimeter (Mettler Toledo, Spain). A sample of ~6 mg was weighed in 40 μL aluminum pan, and distilled water was added to reach ~30 % w/w flour concentration. The assay was carried out in excess of water, so that there was no limitation to gelatinization. Pans were sealed and kept for 30 min at room temperature prior measurement. Measurements were made from 0 to 110 °C at 5 °C/min heating rate, using an empty sealed pan as reference. Onset (TO), peak (TP) and endset (TE) temperatures (°C) and enthalpy of gelatinization (ΔH) (J/g flour dm) were quantified. A second run of the same procedure was performed after 7 days of sample storage in the pans at 4 ± 2 °C, to study retrogradation transitions. Samples were measured in duplicate. 2.10 Pasting properties Pasting properties were determined as described by Vela et al. (2021), using a Kinexus Pro+ rheometer (Malvern Instruments Ltd, UK) coupled with starch pasting cell geometry. Each sample (3.50 g on 14% moisture basis) was mixed with 25.0±0.1mL of distilled water in the test canister. Paddle speed was set at 160 rpm. The sample was equilibrated at 50 °C for 1 min, heated to 95 °C at 6 °C/min, maintained at 95 °C for 5 min, then cooled to 50 °C at 6 °C/min, and maintained at 50 °C for 2 min. rSpace software (Malvern Instruments Ltd, UK) was used to calculate pasting temperature (PT), peak viscosity (PV), trough viscosity (TV), breakdown viscosity (BV), final viscosity (FV) and setback viscosity (SV). Samples were measured in duplicate. 2.11 Rheological properties of gels made from studied flours Dynamic oscillatory tests were performed using Kinexus Pro+ rheometer (Malvern Instruments Ltd, UK) as described by Vela et al. (2021) with serrated parallel plate geometry (40 mm diameter) and a working gap of 1 mm. The gel samples were prepared following the protocol described in section 2.10 for testing the pasting properties and left on the bottom plate to rest for 5 min to allow relaxation. Gels were analyzed by strain sweeps from 0.1 to 1000 % strain at constant frequency of 1Hz, and frequency sweeps from 10 to 1 Hz at 1 % strain, within the linear viscoelastic region (LVR). Data obtained from frequency sweeps were adjusted to the potential equations as described by Ronda, Villanueva, & Collar (2014). Measurements were performed in duplicate. 2.12 Statistical analysis Results were statistically analyzed using Statgraphics Centurion XVIII software (Bitstream, Cambridge, MN, U.S.A.). Analysis of variance (ANOVA) by Least Significant Difference (LSD) test at p-value ≤ 0.05 was performed. 3. Results and discussion 3.1 Effect of treatment on morphology and particle size distribution Selected micrographs are presented in Fig. 1. Control flour showed a polygonal arrangement of tightly packed starch granules entwined with globular protein bodies and lipids, characteristic of rice flours (Villanueva, Harasym, Muñoz, & Ronda, 2018). Sonication led to general breakage of said compact structures (US+ANN-20 and US+ANN-60). ANN-20 (which can be seen as processing control for being handled at the lowest temperature) remains with a similar appearance to the control flour in all magnifications. ANN-60, however, presents a more unified structure, losing the visibility of individual starch granules within the arrangement, believed to be consequence of a partial melting caused by treatment temperature (60°C). The 500x and 1500x magnifications of sonicated samples (C2, C3, E2, and E3) reveal more uneven surface, with more exposed and loosen starch granules, in contrast to control and ANN samples that show a flatter and more regular surface. Figure 1. SEM images of the studied flours. (A) Control, (B) ANN-20, (C) US+ANN-20, (D) ANN- 60 and (E) US+ANN-60 at a magnification of (1) 100x, (2) 500x and (3) 1500x. A1 A2 A3 B1 B2 B3 C1 D1 E1 E2 E3 C2 C3 D2 D3 Ultrasounds have been reported to cause general disruption of rice flour particles, generating smaller size particles (Vela et al., 2021). Particle size distribution was quantified to assess granulation and uniformity of studied flours (see Supplementary Fig. 1). In US+ANN samples, the appearance of a smaller size fraction (around 1 to 10 μm) was determined, while annealed samples showed the same profile presented by the control. Results showed to be significantly dependent on both temperature and ultrasonication, and on their interaction (temperature x ultrasonication). US+ANN-20 presented the most differences compared to control flour, confirming size reduction due to action of cavitation. Median diameter (D50) values obtained were minimum at US+ANN-20 and US+ANN-40, being up to 79 % and 68 % lower than the control, while their size dispersions ((D90-D10)/ D50) were the highest (Table 2). Results also demonstrated that said small size fraction was clearly affected by higher temperatures, increasing D50 to 158 μm in US+ANN-60, which is even higher than control (146 μm). These results lead to the conclusion that destruction of small particles happens with treatments at higher temperatures, particularly temperatures closer to onset gelatinization temperature of native flour, possibly due to gelatinization of those smaller particles. Wang et al. (2017) determined that wheat starch suffered partial gelatinization and disruption of granules when annealed at 50°C, even if it was below its gelatinization temperature (55.6 °C). The 100x micrograph of US+ANN-60 (Fig 1.E1) also presented fewer proportion of small particles compared to those observed in US+ANN-20 (Fig 1.C1), despite being sonicated under the same conditions. 3.2 Effect of treatment on starch damage content Damaged starch values of ANN and US+ANN samples are shown in Table 2. Damaged starch was dependent on temperature, ultrasonication and their double interaction. Values obtained for US+ANN samples increased with increasing temperature. Results indicated that sonication impacts significantly on starch structure, given that US+ANN starch damage values were significantly higher than control and their annealed counterpart, while within ANN samples only ANN-60 was significantly higher than control. Results for US+ANN-20 and US+ANN-40 indicated that flour particles disaggregation caused by cavitation does not generate important damage to the starch structure, which is more sensitive to be disrupted by high temperature. The highest increase was determined for US+ANN-60 followed by ANN-60, representing an increase of 145 % and 68 % with respect to native sample. This higher amount of damaged starch goes in agreement with results obtained in particle size distribution (section 3.1), supporting the idea that gelatinization of small particles happened in treatments carried at 60 °C. Table 4. Thermal properties of treated flours and the control flour. First scan Second scan Sample ΔHgel (J/g) To-gel (°C) TP-gel (°C) TE-gel (°C) ΔT (°C) ΔHam-lip (J/g) TO-am-lip (°C) ΔHret (J/g) To-ret (°C) TP-ret (°C) TE-ret (°C) ΔHam-lip (J/g) TO-am-lip (°C) Control 9.6a 61.1a 74.6c 81.5a 20.4a 1.37a 89.4a 4.8a 36.6ab 51.4a 63.0bc 3.3a 88.2ab ANN-20 9.2aA 61.0aA 73.9aA 81.2aB 20.2aB 1.48aA 89.2aA 4.9aB 36.8bA 51.2aA 63.4cA 3.2aA 89.4bB ANN-40 9.5aA 61.2aA 74.4bcA 81.1aB 19.9aB 1.51aA 87.9aB 4.7aA 36.9bA 51.2aA 62.3aA 3.3aA 87.3aA ANN-50 9.7aA 61.0aA 74.3bcA 80.9aB 19.6aB 1.45aA 88.7aB 4.6aB 36.9bA 51.1aA 62.7abA 3.4aA 87.6aA ANN-60 9.5aB 61.0aA 74.1abA 81.1aB 20.1aB 1.47aA 89.3aB 4.7aA 36.2aA 51.5aA 62.8abA 3.5aA 87.6aB SE 0.2 0.2 0.1 0.4 0.4 0.08 0.6 0.1 0.2 0.3 0.2 0.1 0.4 Control 9.6b 61.1a 74.6a 81.5b 20.4d 1.37a 89.4b 4.8c 36.6a 51.4a 63.0bc 3.3a 88.2b US+ANN-20 9.5bA 61.7aB 74.4aB 79.5aA 17.8cA 1.77bB 88.0abA 4.0aA 37.6bA 50.7aA 62.7abA 3.3aA 88.1bA US+ANN-40 9.7bA 61.3aA 74.3aA 79.3aA 18.0cA 1.85bB 86.1aA 4.4bA 37.6bA 50.6aA 62.5aA 3.3aA 88.1bA US+ANN-50 10.2cB 62.9bB 74.6aA 79.7aA 16.8bA 1.87bB 86.1aA 4.3bA 36.6aA 51.0aA 62.4aA 3.1aA 87.9abA US+ANN-60 8.6aA 68.7cB 75.1bB 79.4aA 10.8aA 1.89bB 87.4aA 4.8cA 37.4bB 51.4aA 63.1cB 3.2aA 86.7aA SE 0.1 0.2 0.1 0.3 0.3 0.07 0.8 0.1 0.2 0.3 0.1 0.1 0.4 Analysis of variance and significance (p-values) (F1): Temp. *** *** ns ns *** ns ns * ns ns ** ns ** (F2):Sonication ns *** * *** *** *** ** *** ** ns ns ns ns (F1) x (F2) *** *** ** ns *** ns ns *** * ns * ns * ΔHgel = Enthalpy of gelatinisation. TO-gel, TP-gel, TE-gel: Onset, peak and endset temperatures of gelatinization. ΔT = (TE-gel – TO-gel). ΔHam-lip = Enthalpy of the amylose-lipid dissociation. TO-am-lip = Onset temperature of the amylose-lipid complex dissociation. ΔHret = Enthalpy of melting of retrograded amylopectin. TO-ret, TP-ret, TE-ret: Onset, peak and endset temperatures of melting of retrograded amylopectin. ΔHgel, ΔHret, ΔHam-lip are given in J/g dry matter. SE: Pooled standard error from ANOVA. The different letters in the corresponding column within each studied factor indicate statistically significant differences between means at p < 0.05. Lowercase letters are used to compare the effect of the temperature and capital letters to compare the effect of the sonication. Analysis of variance and significance: *** p < 0.001. ** p < 0.01. * p < 0.05. ns: not significant. The second scan performed after 7 days of sample storage at 4 °C also led to two peaks; the main one associated to melting of recrystallized amylopectin, and a latter one corresponding to dissociation of amylose-lipid complex. The melting enthalpy of recrystallized amylopectin (ΔHret) of ANN samples did not show significant differences compared to control flour. However, in US-ANN samples, treatments performed at lower temperatures showed significantly lower values of ΔHret, indicating that this reduction may be generated by ultrasonication rather than annealing. Yu et al. (2013) reported decrease in ΔHret of rice starch after being ultrasonicated at 100 and 500 W. Even though US mainly attack amorphous regions, US can still destroy the branched molecule structure of amylopectin, leaving less recrystallized amylopectin in sonicated samples and finally decreasing the retrogradation enthalpy value (Yu et al., 2013). The enthalpy of the amylose-lipid complex was higher in the second scan, probably due to better conditions for complex formation after the first heating, because the leaking of amylose from granules that occurs at temperatures above gelatinization temperature range (Villanueva, Harasym, et al., 2018). 3.7 Effect of treatment on pasting properties Pasting properties of studied flours are shown in Table 5. All pasting properties were significantly influenced by temperature, sonication and their double interaction. Pasting temperature (PT) denoted a delay in the beginning of gelatinisation of treated samples. Said delay supports the fact that annealing induces strengthening of intragranular bonded forces, resulting in starch requiring more heat before structural disintegration and paste formation occurs (Zavareze et al., 2011). US+ANN samples showed that US had a synergetic effect with ANN potentiating PT delay, following an increasing trend with increasing treatment temperature, getting to be up to 3.9 °C higher (US+ANN- 50) than control. Ultrasonication led to general reduction of pasting profiles, being more prone with increasing temperature (Fig. 3). Pasting development in flours depends on their water-binding capacity, which results from combined starch, protein, and fiber water-binding capacities, susceptible to be modified by ultrasonication (Harasym et al., 2020). Similar behavior has been reported in quinoa (Zhu & Li, 2019), buckwheat (Harasym et al., 2020) and rice (Vela et al., 2021) flours, as well as maize (Luo et al., 2008), sweet potato (Zheng et al., 2013) and rice (Zuo et al., 2009) starches after sonication. The effect of ANN on pasting properties of flours and starches, on the contrary, is not as clear, leading to the conclusion that it mainly depends on treatment conditions and structural characteristic of the treated matter (Zavareze et al., 2011). Results seem to indicate that these profile reductions after sonication are potentiated with ANN, following a decreasing trend with increasing temperature. Table 5. Pasting and rheological parameters of the studied flours. Sample PT (°C) PV (Pa · s) TV (Pa · s) FV (Pa · s) BV (Pa · s) SV (Pa · s) G1' (Pa) a G1'' (Pa) b tan()1 c τmax (Pa) Cross over (Pa) Control 80.3a 4.06c 1.77c 4.15d 2.37b 2.37c 187c 0.092a 31c 0.342a 0.165a 0.25a 121c 174b ANN-20 81.4bA 3.82bB 1.70bcA 3.93cB 2.12aB 2.23bB 157bA 0.107abB 28bcB 0.344aA 0.172aB 0.24aA 85abA 141aA ANN-40 81.5bA 3.61aB 1.53aA 3.65bA 2.08aB 2.13bB 152abA 0.105abB 27abcB 0.337aA 0.176abB 0.23aA 96bA 147aA ANN-50 81.4bA 3.58aB 1.56aA 3.38aA 2.02aB 1.82aB 118aA 0.121bB 23aB 0.344aA 0.192bB 0.23aA 73aA 125aA ANN-60 82.5cA 3.67aB 1.65abB 3.54bB 2.02aB 1.89aB 140abA 0.106abB 24abB 0.345aA 0.170aB 0.24aA 90bA 138aA SE 0.3 0.04 0.05 0.05 0.07 0.06 12 0.008 2 0.005 0.007 0.01 6 9 Control 80.3a 4.06d 1.77b 4.15d 2.37d 2.37e 187c 0.092b 31c 0.342a 0.165b 0.25a 121b 174c US+ANN-20 82.9bB 3.53cA 1.81bB 3.87cA 1.74cA 2.06dA 174bcA 0.078aA 20abA 0.367cB 0.123aA 0.29bB 147cB 174cB US+ANN-40 83.1bB 3.53cA 1.81bB 3.66bA 1.75cA 1.85cA 159abA 0.087abA 22bA 0.358bcB 0.133aA 0.28bB 138cB 155bA US+ANN-50 84.2cB 3.33bA 1.91cB 3.60bB 1.42bA 1.69bA 165bB 0.080abA 20abA 0.363bcB 0.126aA 0.28bB 107aB 151bB US+ANN-60 83.9cB 2.38aA 1.49aA 2.86aA 0.88aA 1.36aA 141aA 0.083abA 19aA 0.355bB 0.137aA 0.27bB 101aA 123aA SE 0.2 0.02 0.04 0.04 0.05 0.02 6 0.006 1 0.004 0.006 0.01 6 6 Analysis of variance and significance (p-values) (F1): Temp. *** *** *** *** *** *** * ns * * * ns *** ** (F2):Sonication *** *** *** *** *** *** ** *** *** *** *** *** *** * (F1) x (F2) ** *** *** *** *** *** ns * ns ns ** ns *** ** PT = Pasting Temperature. PV = Peak Viscosity. TV = Trough Viscosity. FV = Final Viscosity. BV = Breakdown Viscosity. SV = Setback Viscosity. The power law model was fitted to frequency sweeps experimental data (G’ = G’1·a; G’’ = G’’1·b; tan  = (tan  )1· ·c) where G1’, G1’’ and tan()1 are the coefficients obtained from the fitting and represent the elastic and viscous moduli and loss tangent respectively, at a frequency of 1Hz. The a, b and c exponents quantify the dependence degree of dynamic moduli and the loss tangent with the oscillation frequency. τmax represents the maximum stress tolerated by the sample in the LVR. SE: Pooled standard error from ANOVA. The different letters in the corresponding column within each studied factor indicate statistically significant differences between means at p < 0.05. Lowercase letters are used to compare the effect of the temperature and capital letters to compare the effect of the sonication. Analysis of variance and significance: *** p < 0.001. ** p < 0.01. * p < 0.05. ns: not significant. Figure 3. Pasting profile of control and treated flours. ANN treatments are represented by a discontinuous line and US+ANN treatments by a continuous line. The grey line corresponds to temperature (°C). Previous studies agree on this behavior when combining sonication with high temperatures (Amini et al., 2015; Zuo et al., 2009). Peak viscosity (PV) reduction varied from 13 % (US+ANN-20) to 41 % (US+ANN-60). Amini et al. (2015) reported similar trend when sonicating corn starch at different temperatures, obtaining the most marked PV drop in sample sonicated at 65 °C. PV was achieved at a longer time in sonicated samples, graphically reflected as a profile displacement to the right (see Fig. 3). PT and PV results indicate reorganization within the starch molecule to thermodynamically more stable arrangement after US+ANN, requiring higher temperatures to hydrate amylose chains and longer exposure to 95 °C for water to completely hydrate the amorphous regions and fully gelatinize treated samples. The lowest PV value obtained for US+ANN-60 agrees with partial gelatinization observed in previous sections (sections 3.1, 3.2, 3.3 and 3.6), since the absence of the previously gelatinized particles limits the flour’s pasting development capacity, in comparison to those samples sonicated at lower temperatures. Trough viscosity (TV) was significantly reduced by ANN, while, in general, a significant increase was determined for sonicated counterparts. US+ANN- 60 was the only sonicated sample with TV significantly lower than the control, following the same trend showed by ANN. Final (FV), breakdown (BV) and setback (SV) viscosities also followed decreasing trend with increasing treatment temperature. Lower SV values indicate lower amylose 30 40 50 60 70 80 90 100 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 0200 400 600 800 1000 1200 1400 Temperature (°C) Viscosity (Pa · s) Time (s) Control ANN-20 US+ANN-20 ANN-40 US+ANN-40 ANN-50 US+ANN-50 ANN-60 US+ANN-60 retrogradation. Few differences were shown by FV values between ANN and US+ANN at 20, 40 and 50 °C. However, at 60 °C a marked decrease was obtained after sonication. It indicates greater damaged caused to amylose chains resulting from synergetic effect of US and ANN at this temperature. BV was markedly reduced after sonication, with values being up to 63 % (US+ANN- 60) lower than the control. This parameter gives information about stability of the samples, indicating their ability to withstand stress and heating. Significantly lower results can be related to internal granular structure rearrangement, as well as the structure of flour’s proteins which by limiting starch swelling promote lower water absorption and lower viscosities. 3.8 Effect of treatment on the rheological properties of gels formed with the treated flours Rheological properties of gels formed with the studied flours are shown in Table 5. All properties were found to be more significantly influenced by sonication than by temperature. Strain sweeps assays indicate two different regions in gels behavior, the linear viscoelastic region (LVR), where the elastic (G') and viscous (G'') moduli as well as the loss tangent (tan() = G''/G') were constant, and the non-linear region, where gels quickly lose their structure’s integrity, reaching the cross over point (G' = G''). The end of LVR is denoted by τmax, which indicates the maximum value that gels are able to resist before disruption of their structure. Results showed significant increase of τmax for US+ANN samples at 20 and 40 °C, and significant reduction at 50 and 60 °C, and progressive cross over decrease with increasing temperature. Cross over of all sonicated samples was found closer to τmax than in control, indicating a quick collapse of gel structures after leaving the LVR. It is believed that the US effect in the combined treatment was the increase of max given that at lower temperatures this value was increased in comparison to control, while the ANN effect seems to be the opposite, given that increasing treatment temperature led to a decrease of said parameter. A reduction of both τmax and cross over was determined in all ANN samples, agreeing with this observed behavior. An increase of τmax has been reported after sonication exposures as low as 10 min (Vela et al., 2021). Reduced values obtained with higher temperature are indicative of weaker gel structures with reduced resistance to breakage. Frequency sweeps showed that G1' exceeded G1'' over the entire studied range, resulting in tan ()1 values that classify their rheological behavior as “true” gels (Villanueva, Ronda, Moschakis, Lazaridou, & Biliaderis, 2018). Both ANN and US+ANN treatments led to similar reduction of G1' with respect to control with slight differences among them. Only the 50 °C samples (ANN-50 and ANN+US-50) led to significantly different G1' values (118 Pa versus 169 Pa). G1'' showed significant differences between all studied temperature pairs, being lower in sonicated samples. Kaur & Gill (2019) said that the decrease of G' and G'' after sonication is caused by severe damaged over starch granules under shear forces induced by US, leading to straightening out of amylose molecules, reducing the shear action within the fluid layers and resulting in decreased viscosity. The linear amylose chains released during gelatinization were unable to form a consolidated compact network (Carmona-García et al., 2016). US+ANN led to lower tan()1 values than their ANN counterpart and control, indicative of higher solid-like behavior. This behavior could result from structural disorganization caused by US and the consequent association of polymeric chains to form the viscoelastic network (Monroy et al., 2018). No significant differences were found in tan()1 values among sonicated samples, indicating that change was not as dependent on treatment temperature, as it was on sonication time, since previous data has shown progressive reduction with increasing sonication time (Vela et al., 2021). It is believed that ultrasonication does not randomly break polymeric chains, since it is limited by a minimum chain length, once this limit is reached no further chain scission happens (Jambrak et al., 2010), which could explain these statistically equal results. 4. Conclusion Treatment temperature was a determinative variable in the extent of modifications caused by US treatments on rice flour, modifying morphology, as well as functional, thermal, pasting and rheological properties. The closer the treatment temperature was to TO-gel of the native flour, the more marked the modification achieved. Cavitation effect led to general particle disruption and generation of smaller particles, causing a dramatic increase of WAI and SP, reaching values up to 134 % and 132 % (US+ANN-20) higher than control flour. The highest damaged starch and the lowest ΔHgel obtained for US+ANN-60, as well as other evidence detected in particle size, pasting and rheological properties, reflect partial gelatinization at this treatment, even though 60 ºC was below control flour’s onset gelatinization temperature. This behavior was not observed in ANN-60, which denotes synergistic effect of ANN and US treatments on flour physical modification. XRD and FTIR assays indicated changes caused to starch’s long range and short range crystallinity, believed to be mainly due to increased proportion of short-chained amylose chains, and enhanced interaction among amylose chains and between amylose and outer branches of amylopectin due to mobility facilitated by annealing, leading to a thermodynamically more stable arrangement. Thermal properties agree on structural rearrangement after treatments, obtaining a decrease of TO-gel and TP-gel, decrease of TE-gel, and narrowing of ΔT, associated to closer packing of starch granule’s crystallites. FTIR also showed significant modification of proteins secondary structure by both US and ANN, affecting all structures, indicating that proteins are prone to be modified by cavitation, leading to modifications of the flour’s pasting and gel’s rheological behavior, as they result from interaction among flour’s components. US decreased viscosity during pasting event, and caused a delay in the beginning of gelatinization and profile shift to the right, indicative of thermodynamically more stable arrangement. Ultrasonication led to formation of gels with higher strength, while higher treatment temperature led to weaker structures. Results prove that the effect of ultrasonication on the achieved flour modification can deeply vary depending on treatment temperature, so it is recommended to control the temperature during the whole treatment to avoid unwanted rises and sample degradation that could come from samples’ gelatinization. Acknowledgement Authors thank the financial support of Ministerio de Economía y Competitividad and the European Regional Development Fund (FEDER) (AGL2015-63849-C2-2-R), the Ministerio de Ciencia e Innovación (PID2019-110809RB-I00) and the Junta de Castilla y León/FEDER VA195P20. A. Vela thanks the Junta de Castilla y León for the doctorate grant and M. 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