Enhancing the thermal stability of soy proteins by preheat treatment at lower protein concentration

Journal Pre-proofs Enhancing the Thermal Stability of Soy Proteins by Preheat Treatment at Lower Protein Concentration Wuchao Ma, Tao Wang, Jiamei Wang, Di Wu, Chao Wu, Ming Du PII: DOI: Reference:

S0308-8146(19)31717-0 https://doi.org/10.1016/j.foodchem.2019.125593 FOCH 125593

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Food Chemistry

Received Date: Revised Date: Accepted Date:

12 May 2019 23 September 2019 23 September 2019

Please cite this article as: Ma, W., Wang, T., Wang, J., Wu, D., Wu, C., Du, M., Enhancing the Thermal Stability of Soy Proteins by Preheat Treatment at Lower Protein Concentration, Food Chemistry (2019), doi: https://doi.org/ 10.1016/j.foodchem.2019.125593

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Enhancing the Thermal Stability of Soy Proteins by Preheat Treatment at Lower Protein Concentration Wuchao Ma a, Tao Wang b, Jiamei Wang a, Di Wu a, Chao Wu a*, Ming Du a a

National Engineering Research Center of Seafood, Collaborative Innovation Center

of Provincial and ministerial co-construction for Seafood Deep Processing, School of Food Science and Technology, Dalian Polytechnic University, Dalian, 116034, China b

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi

214122, China

*Corresponding Author Dr. Chao Wu Phone: +86-411-86332275 Fax: +86-411-86323262 1

E-mail: [email protected]

Abstract The heat-induced aggregation of edible proteins has been regarded as one of the critical challenges for their application in protein-enriched beverages. Therefore, the formulation of thermal stable proteins to improve the stability of these beverages upon heating is highly desired. In this study, soy proteins (SPs) with enhanced heat stability were obtained by low-concentration-preheating (LCPH). Results from reheating of the above samples showed that pretreatment of SPs at low concentrations (≤1.0 %, w/v) increased their resistance against aggregation. Additionally, when the suspensions of the particles were reheated at 10% (w/v) protein concentration, no gelation was found for samples prepared by LCPH, indicating collapsed protein-protein interactions, whereas gelled suspensions were obtained for native SPs and samples prepared by preheating at higher protein concentrations (≥2.0 %, w/v). Furthermore, suspensions of particles prepared at lower protein concentration showed lower viscosities and higher flow behavior index values before and after reheat treatment. These findings highlighted that LCPH would provide fundamental information on the application of SPs in high protein beverages. Keywords: Aggregation; Heat stability; Low-concentration-preheating; Soy protein;

2

1 Introduction Soy proteins (SPs) are widely applied as food ingredients and additives because of their high nutritional and functional properties. Recently, there is a growing interest in high protein foods, which are thought to be beneficial to the nutrition supply and body-weight control ( Kiilerich et al. 2015; Zuo et al. 2016). Beverages containing high content of proteins are suitable candidates for high protein foods. Although SPs can be formulated into beverages such as blended soymilk with a protein concentration of 2% to 3%, the heat-induced aggregation and gelation, leading to granular sensation and sedimentation, would have a negative impact on the sensory properties when protein concentration exceeds 6.5% (Renkema & Van Vliet, 2004). Heat treatment which is commonly used to passivate anti nutritional components and sterilize bacteria, would cause irreversible denaturation of the SPs, and the exposure of hydrophobic and sulfhydryl sites promote the aggregation behavior of the proteins, finally causing gelation when the protein concentration is sufficiently high (Phan-Xuan et al., 2013; Renkema & Van Vliet, 2004). Therefore, particulate SPs with enhanced heat stability are currently required for producing protein-enriched beverages. Controlling over size, denaturation and aggregation of protein particles can be realized by preheating, which is demonstrated to be a facile route to enhance thermal stability of proteins, as exemplified by extensive studies in whey proteins (WPs). Ryan et al (2012) reported that soluble protein aggregates with improved thermal stability as a result of increased surface charges were obtained by heating WPs or β-lactoglobulin [pH 6.8, 7% (w/w)] at 90 °C for 10 min. WP micro-aggregates formed by a combined heat treatment and microfluidization also had higher heat stability 3

compared to native WPs, and the authors suggested that the denaturation and quenching of active sites of proteins during heating, and the reformation of intra- and intermolecular bonds afterwards enhanced the heat stability (Dissanayake & Vasiljevic, 2009). Furthermore, WP particles produced by emulsification and preheat treatment also showed improved heat stability (Dissanayake & Vasiljevic, 2009; Sağlam, Venema, de Vries, Shi, & van der Linden, 2013; Sağlam, Venema, de Vries, & van der Linden, 2014; Zhang & Zhong, 2009, 2010). Zhang et al. (2010) found that WP nanoparticles obtained by W/O microemulsion and thermal pretreatment did not form a gel upon subsequent heating at 5% (w/v) protein concentration in a pH 6.8 buffer. The native WPs, however, gelled and no longer flowed following the same procedure. Similarly, Saglam et al. (2014) observed the enhanced heat stability of whey protein particles formed by a combination of two-step emulsification and heat-induced gelation, nonetheless, the whey protein particles had a size of a few micrometers, and no gelation occurred when the protein concentration was 20.1% (w/w). During heat pretreatment, protein denaturation induces aggregation, but fine particles were obtained by high pressure shearing or microemulsification. The inhibition of the second aggregation of these particles can be explained by that less reactive sites were left for further cross-linking between protein particles due to the denaturation and aggregation induced by heat pretreatment (Çakır-Fuller, 2015). However, the difficulty to remove oil induced by emulsification, as well as micrometer sized particles generated by high pressure shearing method, strictly restrict their application to produce heat stable proteins for transparent beverages. SPs contain two major fractions, i.e., β-conglycinin (7S) and glycinin (11S). The 7S fraction is a glycoprotein composed of three subunits (α’, α, and β), which are cross-linked via hydrophobic interactions. The 11S fraction is a hexamer and 4

composed of acid polypeptide (A) and basic polypeptide (B). Similar to WPs, SPs can also aggregate and form a gel upon heating, and the morphologies of heat-induced SP aggregates are pH-dependent. Long semiflexible fibrils with a length of few micrometers and a diameter of several nanometers can be formed at pH 2.0 and prolonged heating (Akkermans et al., 2007; Wan, Yang, & Sagis, 2016). Spherical particles with diameters ranging from hundreds of nanometers to a few micrometers are formed in a pH span around the isoelectric point. When the pH is further increased (higher than 6.5), strand-like aggregates are obtained, which could result in randomly branched clusters at higher protein concentration. Protein concentration and ionic strength greatly influence the thermal-induced aggregation behavior of SPs. For instance, higher protein concentration leads to larger size of particles, and salt can shield the surface net charges, enhancing the interaction between protein molecules during heating. However, only few studies were focused on how to enhance the heat stability of SP particles. Guo et al. (2016) reported a thermal stable SP microgel prepared by a two-step strategy, including a gel forming and a subsequent homogenizing process. As shown in the literature (Çakır-Fuller, 2015), the combined heat treatment and high pressure shearing method could produce protein particles with heat stability and controllable size, and one might be interested in whether it could be work if proteins are heated at lower protein concentration, at which heat-induced aggregation of protein molecules would be inhibited to some extent. Thus, as inspired, in this study, we present a simple yet robust pathway of preparing heat-stable SPs using a low-concentration-preheating (LCPH) technique by simply preheating the native SPs at 0.5 or 1% (w/v) and pH 6.0-6.4. The results showed that the pretreated SPs showed significantly improved heat stability when they were reheated at 10% (w/v) protein 5

concentration. Therefore, this study aimed to investigate the mechanism underlying the formation of SPs with enhanced stability, by examining physicochemical properties of modified SPs, the heat-induced aggregation behavior of native SP and preheated SPs at 1% (w/v), and the rheological properties of 10% (w/v) suspensions of native SP and preheated SPs before and after heat treatment.

2 Materials and methods 2.1 Materials Defatted soybean flakes with low denaturation ratio were gifts from Shandong Gushen Industrial & Commercial Co., Ltd (Jinan, China). The other materials and reagents were purchased from Sigma-Aldrich (Sigma-Aldrich LLC, Shanghai, China), and were used without further purifications. 2.2 Preparation of SPs SPs were extracted from soy flakes according to the method of Wu et al. (2015). Soy meals were mixed with 85% (v/v) alcohol at a ratio of 1:5 (w/v), and the suspension was stirred for 1 h at 25 °C. The slurry of meals was obtained by a filtering process, then they were mixed with 95% (v/v) alcohol at a ratio of 1:2 (w/v), and the suspensions were stirred for 1 h. The alcohol was drained off, and the soy flakes were dried at room temperature (25 °C). Dried soy flakes were mixed with deionized water at ratio of 1:10 (w/v), and the pH of the mixture was adjusted to 7.0 by 2 M NaOH, then it was stirred at room temperature (25 °C) for 2 h. After centrifugation at 15,800 g for 30 min at 4 °C, the pH of the supernatants was adjusted to 4.5 by 1 M HCl and centrifuged at 10,000 g for 20 min to obtain SP precipitates. The precipitates were dissolved by deionized water, and the pH of the suspension was adjusted to 7.0 by 2 M NaOH. The SP suspension was freeze-dried and stored at 4 °C. 6

Protein content in SPs was 93.1 ± 0.5%, which was determined by the micro-Kjeldahl method. The 7S/11S ratio in soy protein was 77.9 ± 1.2%, which was determined by a reducing SDS-PAGE (Sorgentini, Wagner, & Anon, 1995). The 7S/11S ratio (R) was calculated based on the following equation: R=

𝐼α’ + 𝐼α + 𝐼β 𝐼A3 + 𝐼A + 𝐼B + 𝐼A5

× 100%

where Iα’, Iα, Iβ, were intensities of bands α’, α, and β of 7S protein, respectively. IA3, IA, IB, and IA5, were intensities of bands A3, A, B, and A5 of 11S protein, respectively. The intensities of these bands were analyzed by Image Lab 3.0 software from the reducing SDS-PAGE of 7S and 11S proteins. 2.3 Preparation of preheated SPs SP dispersions with protein concentrations of 0.5, 1.0, 2.0, and 4.0% (w/v) were obtained by suspending the extracted SPs in deionized water. Then the pH of these dispersions was adjusted to 6.0, 6.2 and 6.4 by 4 M HCl. The dispersions were heated at 100 °C for 30 min and cooled in an ice water, then the pH of these dispersions were adjusted to 4.5. After centrifugation at 10,000 g for 20 min, the precipitates were suspended in deionized water by adjusting the pH to 7.0, then these suspensions were freeze-dried. Powders of preheated SPs in particles were obtained and stored at -20 °C before further using. 2.4 Total sulfhydryl group (SH) of preheated SPs The total sulfhydryl group content of SP particles was analyzed according to the method of Ruan et al. (2013) with some modifications. Preheated SP samples were diluted by 0.1 M PBS (pH 7.0) containing 4% (w/v) SDS to obtain suspensions at 2.5 mg/mL. 150 μL of 4, 4’-dithiodipyridine (DPS, 4 mM in 12 mM HCl) was mixed 7

with 3.6 mL of the diluted sample. The mixture was kept in dark at 25 °C for 25 min. Then the absorbance was examined at 324 nm (A1). The absorbances of buffer (reagent blank) and protein solutions without addition of DPS were acquired and expressed as A2 and A3, respectively. The SH group content was calculated by the following equation (A1 ― A2 ― A3)

SH group (μmol/g) = 21000 × b × c × 106 where 21,000 is the molar extinction coefficient of DPS reagent, b the length of the light path (1.0 cm), and c the protein concentration (g/L), respectively. Measurements were performed at least in triplicates. 2.5 Surface hydrophobicity of preheated SPs The surface hydrophobicity of preheated SPs was examined based on the method of Kato and Nakai (1980). Preheated SPs were dispersed using 10 mM pH 7.0 PBS to a serial concentration of 0.02% to 0.1% (w/v). The diluted protein samples (4 mL) were mixed with 50 μL of 8 mM ANS solution. Fluorescence intensity was recorded at 390 nm excitation length and 470 nm emission length by a F2700 fluorescence spectrophotometer (Hitachi Co., Japan). The initial slope of fluorescence intensity versus protein concentration was applied as the H0 index. 2.6 Circular Dichroism (CD) Far-ultraviolet (UV) CD spectra of the protein samples were determined according to the method of Wu et al. (2019), using a J-1500 CD spectrometer (JASCO corporation, Japan) with a quartz cell of 1 cm optical path in the wavelength range of 250-300 nm. Proteins were dispersed in 10 mM pH 7.0 PBS at a concentration of 1% (w/v), which

were further diluted to 0.25 mg/ml by 10 mM pH 7.0 PBS.

8

Measurements with a step size of 1 nm, a scanning speed of 50 nm/min, and an average of 5 scans were performed to generate data. 2.7 Thermal stability of preheated SPs To assess the thermal stability of preheated SPs, the heat-induced aggregation behavior at a concentration of 1% (w/v) and gelation behavior at a concentration of 10% (w/v) were investigated separately. Preheated SPs were dispersed in deionized water (pH 7.0), stirred overnight to allow the fully hydration, and heated at 100 °C for 30 min in a sealed glass bottles (10 mL), then they were cooled in an ice water and stored at 4 °C before further usage. NaN3 (0.02%, w/w) was added as anti-bacteria agent during the treatment. 2.8 Dynamic light scattering (DLS) DLS was performed to determine the size distribution of particles in the 1% (w/v) suspension before and after reheating as shown in last section, following the method of Wu et al. (2017). The 1% (w/v) suspension was diluted to 1 mg/mL by deionized water. The apparent hydrodynamic radius (Rh) was analyzed at a fixed scattering angle (173°) by Zetasizer Nano ZS instrument (Malvern Instruments, Malvern, UK), equipped with a 633 nm laser. The CONTIN algorithm was employed to fit the intensity autocorrelation functions for obtaining the distribution of translational z-average diffusion coefficient, DT. The Stokes-Einstein equation DT = kBT/6πηRh was used to calculate the Rh. 2.9 Atomic force microscopy (AFM) The suspensions (1%, w/v) of SP aggregates were diluted to 0.01% (w/v) by deionized water. 6 μL of each diluted sample was dripped on a freshly cleaved mica sheet, then the samples were dried at room temperature (25 °C) for 1 h. Topographical images were analyzed by a Scanning Probe Microscope AFM 5500M (Hitachi 9

High-Tech Science Corporation, Japan) in an auto tuning mode equipped with a cantilever SI-DF-40P2. 2.10 Rheology measurement Storage modulus. The storage modulus of the 10% (w/v) particulate SP suspensions before and after reheat treatment (as described in section 2.6) were analyzed according to the method of Wu et al. (2018). Samples were taken out of the glass bottles by a pipette (for suspensions) or a spoon (for gels). Before testing, excess samples were removed carefully by a knife. A Discovery HR-1 rheometer (TA Instrument, New Castle, UK) equipped with a parallel plate geometry (PP25, 25 mm diameter) was used. It was found that a strain of 0.5% at 1 Hz was within the linear viscoelastic region in a preliminary experiment. Storage modulus of samples were determined at 1 mm gap and at a frequency from 0.1 to 10 Hz. The temperature was kept at 25 °C. Measurements were performed at least in triplicates. Viscosity. After storage modulus test, the shear viscosity was measured in the shear rate range of 1 to100 s-1 at a strain of 0.1% and 1 Hz, and the same parallel plate geometry was used. All measurements were performed in duplicates. Flow behavior index. The flow curves of viscosity were fitted to the Ostwald de Waele rheological model τ = K γn, then the consistency coefficient (K, Pa × S) and flow behavior index (n) were obtained (Hebishy, Buffa, Juan, Blasco-Moreno, & Trujillo, 2017). 2.11 Statistical analysis Staristical data were analyzed by analysis of standard deviations and variance using Duncan’s test (ANOVA) by SAS 9.1. The level of significance used was p < 0.05. In addition, significant differences between means of each pair of flow behavior indexes before and after reheating were determined by t-test using Excel 2017. 10

3. Results and discussions In this study, soy proteins were preheated, and the physicochemical properties of these particles, including total sulfhydryl group, surface hydrophobicity, and size distribution, were examined. Afterwards, the heat stability of native and preheated soy proteins, characterized by storage modulus, viscosity, and flow behavior index, were further investigated. 3.1 Total sulfhydryl group (SH) of modified SPs Sulfhydryl/disulfide transition played an important role on the heat-induced aggregation of protein molecules (Hoffmann & van Mil, 1997; Mounsey & O’Kennedy, 2007). Therefore, the total sulfhydryl content of preheated SPs was analyzed and shown in Fig. 1, which decreased significantly from 0.609-0.676 to 0.276-0.361 μmol/g with the protein concentration increased from 0.5 to 4.0%. In addition, at a given protein concentration, slightly higher content of total sulfhydryl content was found in preheated samples formed at higher pH. Upon heating, the protein molecules unfolded firstly and enabled the exposure of hydrophobic sites and sulfhydryl groups, leading to aggregation. The aggregation of protein molecules would be promoted at higher protein concentration via sulfhydryl/disulfide exchange reaction, resulting in larger particle size (Mills, Huang, Noel, Gunning, & Morris, 2001; Renkema, Gruppen, & van Vliet, 2002), because the higher protein concentration might contribute to a higher amount of intermolecular disulfide bonds due to larger density of exposed sulfhydryl groups, leading to disulfide-linked polymers with larger size. 3.2 Surface hydrophobicity (H0) of modified SPs

11

During heating, protein molecules unfolded, and the exposed hydrophobic sites would promote the aggregation of proteins (Hua, Cui, Wang, Mine, & Poysa, 2005). Fig. 1b shows that SPs preheated at lower protein concentration had lower values of H0, except for SPs modified at 4% (w/v), where lower H0 was also found, ascribed to the larger aggregation extent induced by higher protein concentration. Additionally, the hydrophobicity of SPs increased with increasing the pH in the preheat treatment processes. These results indicate that SPs preheated at lower protein concentration and pH possessed lower surface hydrophobicity, which was in accordance with the lower fluorescence intensity as shown in Fig. S1, due to more tryptophan residues were buried inside because of heat-induced aggregation of protein molecules via hydrophobic interaction (Vivian & Callis, 2001). Furthermore, red shifts were also found in the maximum fluorescence wavelength with the reduction of protein concentration (Fig. S1), suggesting larger denaturation extent of SPs preheated at lower protein concentration (Artigues, Iriarte, & Martinez-Carrion, 1994; Zhang, Takenaka, & Isobe, 2004). 3.3 Conformational changes Preheat treament at such a pH range between 6.0 and 6.4 had no impact on the primary structure of proteins as shown in Fig. S2, because similar protein compositions were found in all the modified samples and the untreated one, therefore, we further acess the effects of preheating process on the secondary structrue of SPs using the far-UV CD. As shown in Fgure 1 c-e, the far-UV CD spectra showed a typical β-sheet conformation of SP, demostrated by a negative Coton effect between 195 and 250 nm (Sharon M. Kelly, Jess, & Price, 2005). At a fixed pH, proteins preheated at lower concentration showed a larger decrease in the maximum peak wavelength, along with a blue shift from 208 nm to 206 nm. A similar result was also 12

found in SPP samples prepared by preheating at a fixed concentration (4%, for example) but at different pH as shown in Fig. 1f. These results could be explianed by that the onset denaturation temperature increased with an increasing concentration of proteins during heating (Mills et la., 2001; Zhang et al. 2004). Similarly, Mori et al. (1982) found that 11S fractions of SP at 5% (w/v) concentration did not denatured to the extent they did at 0.5% (w/v) concentraion. These findings indicated the loss of β-sheet of proteins after preheating, and the lower concentration or higher pH used during preheating, the much more loss of β-sheet. In addition, these results provide solid evidence of unfolding of protein molecules after preheating. 3.4 Heat stability of the suspensions of modified SPs 3.4.1 Particle size distribution analysis In order to investigate the heat-stability of the preheated SPs, the size distribution and aggregation behavior of the particles before and after heat treatment was firstly determined. For the samples before heat treatment (Fig. 2a-c), different size distribution profiles were observed. SP particles preheated at higher protein concentration showed larger hydrodynamic diameter (Fig. 2b and c), except for those formed at pH 6.0 which showed larger size at lower protein concentration (Fig. 2a) because of surface charge shielding. During heat pretreatment, higher protein concentration promoted the aggregation of unfolded protein molecules and resulted in larger particle size (Renkema et al., 2002). After heat treatment, the size distribution profiles of the SPs before and after preheating were shown in Fig. 2d-f. For the preheated samples obtained at pH 6.0 and 0.5% protein concentration, bimodal distributions (marked in inverted triangle in Fig. 2a and d) and no obvious changes were found before and after heat treatment, indicating that thermal stable SPs were formed. Bimodal distributions of the size of SPs formed at pH 6.0 and 1.0% (w/v) 13

protein concentration were also found before and after heat treatment, but the sizes increased from 100 and 500 nm to 200 and 800 nm, respectively, suggesting aggregation occurred after heat treatment. Samples pretreated at pH 6.0, protein concentrations of 2.0 and 4.0 % (w/v) showed apparent changes of sizes from unimodal distributions to bimodal ones, and larger particles size with one or few microns diameter were found. Similar size increments were observed in preheated SPs obtained at the same concentrations but at a pH of 6.2. Interestingly, no obvious changes of the size were found for the preheated samples prepared at pH 6.2 at both 0.5 and 1.0% (w/v) protein concentration, indicating that these particles were heat stable. The size of the particles prepared at pH 6.4 and 4.0% (w/v) protein concentration increased from 150 to 200 nm after heat treatment, but no apparent changes in size were found for its counterparts pretreated at lower concentrations. It could be concluded that particles formed at higher protein concentration were of less thermal stability because of greater increases in their sizes, and the equal of which was observed for the particles formed at lower pH. The above results indicated that thermal stable SPs was obtained using LCPH. During the heat pretreatment, higher protein concentration promoted the formation of larger particles via exterior hydrophobic interaction and disulfide bonds, whereas the protein interior might be not denatured completely (Zhang & Zhong, 2010). Therefore, during the subsequent reheating, intensified denaturation of these proteins led to externalization of hydrophobic sites, resulting in larger particles due to aggregation (Sağlam et al., 2014). In literatures, it was reported that the forming of disulfide bonds would induce an enhanced heat-induce aggregation behavior of proteins (Hoffmann & van Mil, 1997; Mounsey & O’Kennedy, 2007), however, this was not the case in this study, because larger aggregates were found in suspensions of SPs with a less content of 14

total sulfhydryl group (Fig. 1a) preheated at higher protein concentrations. This result could explained by that, for one thing, sulfhydryl sites buried inside the proteins or aggregates did not participate the aggregation during the secondary heating, whereas these sulfhydryl sites was detected as free ones in the present of denature agent (SDS, as shown in method 2.4). For another reason, some researchers found that the hydrophobic interaction drive the forming of larger sized aggregates or gelation of soy proteins during heating (Hua, Cui, Wang, Mine, & Poysa, 2005; Ruan, Chen, Kong, & Hua, 2014). 3.4.2 AFM analysis In order to further verify the effect of reheat treatment on the size and morphology of preheated SPs formed at different pH and protein concentration, the suspensions (1%, w/v) of samples before and after reheating were diluted and analyzed by AFM. A clear difference was found for the size of the control sample (SPs without preheating) before and after heat treatment at 100 °C for 30 min, which increased markedly after heating. This finding was in agreement with the DLS result in Fig. 2, indicating that SP aggregates were formed during heating. At pH 6.0, particles with larger size were found at lower protein concentrations (0.5 and 1.0% (w/v)) than at higher ones (2.0 and 4.0% (w/v)) before reheating, and a slight increase was found in the size of particles formed at 0.5% (w/v) after heat treatment, whereas apparent increases in size and larger particles were observed for the soy protein particles formed at 1.0, 2.0, or 4.0% (w/v) after reheating. Upon increasing the pH to 6.2 and 6.4, the size of preheated SPs increased with increasing protein concentration before or after reheating. While at a same protein concentration, larger particles could be observed at pH 6.2 than at pH 6.4, attributing to the decrease of electrostatic repulsion (Sağlam, Venema, de Vries, van Aelst, & van der Linden, 2012). Moreover, 15

smaller changes of the size of SP particles before and after reheat treatment were observed at lower protein concentrations, suggesting that LCPH effectively promoted the thermal stability of SPs. During heat pretreatment, slower aggregation of protein molecules via hydrophobic and disulfide bonds was expected at lower protein concentration. Thus, the hydrophobic and disulfide interplays were confined within limited molecules and reacted to form compact internal structures. However, larger scale intermolecular aggregation occurred when the same proteins were heated at a sufficiently higher concentration resulting from high-density active binding sites. 3.5 Heat stability of the suspensions at 10% (w/v) protein concentration In protein-enriched beverages, the thermal stability and flow behavior of the system are of crucial importance for the acceptability of customers, and heating process is commonly applied to sterilize bacteria and inactivate enzymes. During heating process, protein molecules in the beverage would be denatured and unfolded, and the exposed hydrophobicity sites and sulfhydryl groups would promote the interaction between protein molecules, thus forming aggregates or gels when the protein concentration is above a critical value. In the section 3.3, it had been demonstrated that heat pretreatment of SPs could improve the stability of the protein particles below 1% (w/v) protein concentration during reheating. Therefore, in this section, the heat stability of preheated SPs at higher protein concentration (10%, w/v) was further investigated by rheological characteristics and flow behavior index before and after reheat treatment (100 °C for 30 min). 3.5.1 The appearance of the suspensions after heating The appearances of 10% (w/v) suspensions of native soy protein (the control) and preheated samples after heat treatment (100 °C, 30 min) were shown in Fig. S3. The 16

reference system (10%, w/v) containing native SPs gelled after heat treatment. Suspensions of SPs preheated at 0.5 and 1.0% protein concentrations and pH 6.0 had opaque, milk-like appearances, and no gelation occurred after reheat treatment, indicating thermal stability of both samples. Besides, no gelation was found for 10% (w/v) suspensions of samples preheated at pH 6.2 with a concentration of 0.5% (w/v), and pH 6.4 with concentrations of 0.5 and 1.0% (w/v) after reheating. By contrast, sols were observed for the suspensions obtained at pH 6.2 with a concentration of 1.0% (w/v), and pH 6.4 with a concentration 2.0% (w/v). Furthermore, noticeable gels were found at each pH beyond a concentration of 2% (w/v). The results again confirmed that LCPH was a robust approach for gaining SPs of high heat stability. 3.5.2 Storage modulus analysis Storage

modulus

can

gather

information

on

the

cross-links

in

the

three-dimensional network of heat-induced protein gels by measuring the energy elastically stored in the sample and the deformation energy introduced by the motor movement. Interactions between protein molecules through covalent and non-covalent bonds have significant influence on the sustaining of gel network. Fig. 4a-f shows the storage modulus of 10% (w/v) suspensions of native (the control) and preheated SPs before and after reheat treatment. As expected, before reheat treatment, larger values of G’ were found for samples formed at higher protein concentrations due to larger particles after preheating as shown in Fig. 2b-c. Furthermore, particles formed at higher protein concentrations had larger content of disulfide bonds (Fig. 1a) and higher hydrophobicity (Fig. 1b), enhancing the covalent and non-covalent interaction between proteins. After reheating, larger increases in G’ values were found for the 17

suspensions of samples preheated at concentrations ≥2% (w/v) in all the test except at pH 6.4, and slight increases were found for native SPs and those formed at pH 6.4 with a concentration 2.0% (w/v). However, negligible changes of G’ values were observed for the SPs preheated at 0.5 and 1.0% (w/v) after reheat treatment. These results were in agreement with the findings of Fig. S3, further demonstrating the thermal stability of SPs prepared by LCPH. 3.5.3 Viscosity and flow behavior index Viscosities of dispersions containing preheated or native SPs before (g-i) and after (j-l) reheating as a function of shear rates were shown in Fig. 4. Shear-thinning behaviors were found for all the samples, shown by the decreased viscosities with increasing shear rate. For the suspensions before reheating in Fig. 4g-i, clearly higher viscosities were found for samples formed by heat pretreatment at higher protein concentration, which was consistent with the result in Fig. 4a-c. It had been shown that upon heating SPs unfolded and aggregated via hydrophobic interaction and disulfide bonds, and larger aggregates could be formed at higher protein concentration (Chen, Zhao, Chassenieux, & Nicolai, 2017; Mills et al., 2001; Renkema et al., 2002), which would increase the viscosity of suspensions (Kelsey N. Ryan & Foegeding, 2015). However, this was not the case for the protein particles with larger size (Fig. 2a) formed at pH 6.0 and lower protein concentrations (0.5 and 1.0%, w/v), where lower viscosities were also found probably as a result of higher internal structural density (Sağlam et al., 2012), and less swelling ratio during reheat treatment (Sağlam et al., 2014). It was reported that particle swelling during heating might be responsible for the increase in viscosity after thermal treatment (Sağlam et al., 2013). After reheating, the apparent viscosity of dispersions of the native and preheated SPs obtained by heat pretreatment at 2.0 and 4.0 % (w/v) protein concentration greatly increased, which 18

could be explained by the increased interaction between proteins and the subsequent formation of aggregates (Çakır-Fuller, 2015; McSwiney, Singh, & Campanella, 1994; Ryan et al., 2012; Vardhanabhuti & Foegeding, 1999). When an external force was applied to the suspension, larger aggregates and more cross-links presented greater resistance to flow than smaller ones and less cross-links occurred. It has been known that SPs are sensitive to heating that is capable of denaturing and aggregating proteins. Interestingly, negligible changes (pH 6.2, 1.0% (w/v)) in the viscosities of the samples for SPs preheated at lower protein concentrations (0.5 and 1.0%, w/v) were observed. The flow behavior index values of suspensions before and after heat treatment were shown in Table 2. The fluid is Newtonian if the flow behavior index ≈1, and suspensions with higher flow behavior index value (approaching 1) had better fluidity (Wang, Wang, Li, Adhikari, & Shi, 2011; Xu, Wang, Jiang, Yuan, & Gao, 2012). In general, heat treatment elicited decreases in flow behavior index of suspensions. For the suspensions without reheating, the n values were higher for the SPs preheated at lower protein concentrations (0.5 and 1.0%, w/v), which ranged from 0.6430 to 0.9053, then they slightly decreased to the values ranging from 0.5583 to 0.8916 after reheating. However, the n values ranging from 0.2713 to 0.6998 for the suspensions of SPs preheated at higher protein concentrations (2.0 and 4.0%, w/v) remarkably decreased to the values between 0.1625 and 0.3000 after reheating. Similarly, the n value of native SPs decreased significantly and considerably from 0.7339 to 0.2803. These results further demonstrated that preheated SPs prepared at lower protein concentration had higher thermal stability, as indicated by less decreases in the values of flow behavior index after reheat treatment at 100 °C for 30 min. The findings in the present study illustrated that SPs with distinct heat stability could be obtained by LCPH. Fig. 5 shows the possible mechanism underlying the 19

improved heat stability of SPs. SPs prepared at lower protein concentration were of thermal stability, the gelling properties of which were deteriorated. This observation could be explained by that particles with smaller size, denser structure, and larger denaturation extent were formed at lower protein concentration, so that at the reheating step, the aggregation of particles was inhibited. However, when the SPs were modified at higher protein concentration, faster aggregation might limit the unfolding of proteins molecules, and particles with less density and smaller unfolding ratio were formed. During the second heating, further unfolding of protein molecules and higher swelling ratio of the particles would promote the aggregation (Purwanti, Moerkens, van der Goot, & Boom, 2012; Sağlam et al., 2014). When SPs were modified at different pH values, particles with different heat stability were also observed. As the pH (6.0) closer to pI, the surface charge of proteins was shielded, and lower electrostatic repulsion between proteins favored the aggregation, resulting in particles with larger size, and denser, finally, less swelling capacity during second heating led to higher thermal stability of these particles. When the pH (6.4) was shifted away from pI, the increase in net negative charge of proteins would contribute to suspensions with better dispersity. During heat pretreatment, unfolding of protein molecules was promoted as a result of higher long range charge (Donato, Schmitt, Bovetto, & Rouvet, 2009; Xiong, Dawson, & Wan, 1993), and particles with smaller size and higher unfolding extent might be responsible for their higher heat stability.

4. Conclusions This study demonstrated that SPs with desirable thermal stability can be prepared by a simple, robust, and effective LCPH technique, and the obtained SPs prepared at higher pH had smaller size and transparent appearances, having promising prospects 20

meeting the current demands of high protein beverages. When suspensions were reheated at 1% (w/v) protein concentration, no appreciable increase of the size was found for LCPH-treated SPs, whereas those treated in larger concentrations were subjected to a higher size increment. Most importantly, after reheat treatment no gelation was found for 10% (w/v) suspensions of LCPH-treated SPs, further indicating enhanced heat stability. This study provided fundamental insights into preparation of heat-stable SPs via a facile LCPH technique, which would possibly contribute to meet the desperate needs of high protein food in drink and beverage industry.

Acknowledgements This research was supported by the General Program of Liaoning Science & Technology Department (Grant No.201800462).

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Figure Captions Figure 1. The total sulfhydryl group (a) and surface hydrophobicity (b) of soy protein particles modified at different protein concentration and pH. The symbols in Figure 1b share the same meaning as shown in Figure 1a. Far-UV CD spectra of SPP samples prepared by preheating at different pH (c, 6.0, d, 6.2, and e, 6.4) and different concentration. f, representative Far-UV CD spectra of SPP samples preheated at 4% concentration but different pH. Figure 2. Size distribution of modified soy protein particles before (a, b, and c) and after (d, e, and f) reheat treatment. The symbols in Figure a-f share the same meaning as shown in Figure 2a. Soy protein particles were obtained at different protein concentration (0.5, 1.0, 2.0, and 4.0%) and pH (6.0-a and d, 6.2-b and e, and 6.4-c and f). Figure 3. Atomic force microscopy topographical images of soy protein particles (1%, w/v) before and after reheat treatment. The size of each picture is 2×2 μm. The heat pretreatment conditions soy protein particles formed were shown on the top and right sides. Figure 4. Storage modulus (G’) (a-f) and viscosities (g-l) of SPs suspensions (10%, w/v) before (a-c and g-i) and after (d-f and j=l) reheat treatment (100 °C, 30 min): a, d, g, and j, soy protein modified at pH 6.0, b, e, h, and k, soy protein modified at pH 6.2, c, f, i, and l, soy protein modified at pH 6.4. Soy proteins preheated at different protein concentrations were marked in different symbols, the meanings of which were shown in a and g. 24

Figure 5. Proposed mechanism of enhanced heat stability of soy protein particles prepared at lower protein concentration. C represents protein concentration.

Figure 1

2.8

400

pH 6.0 pH 6.2 pH 6.4

h

Surface hydrophobicity

Sulfhydryl group (μmol/g)

2.9

2.7 2.6

f

0.6

fg g de

e e bc

0.4

cd de a

b bc

0.2 0.0

Control

1.0

0.5

2.0

Protein concentration (%,w/v)

300

e

e

a

100

Control

0.5

1.0

4.0

0.5% 1% 2% 4%

10 5

CD[mdeg]

5

CD[mdeg]

15

Control 0.5% 1% 2% 4%

10

0 -5

-10

0 -5 -10 -15

-15 200

210

220

230

240

-20 190

250

210

220

230

Wavelength/nm

c

d 15

0.5% 1% 2% 4%

10

-5

0 -5

-10

-10

-15

-15 220

230

240

250

5

0

210

240

pH 6.0 pH 6.2 pH 6.4

10

CD[mdeg]

5

200

200

Wavelength/nm

15

CD[mdeg]

2.0

Protein concentration (%, w/v)

b

15

-20 190

d d

c

b

b

a

-20 190

f

f

200

0

4.0

i

h

g

250

-20 190

200

210

220

230

Wavelength/nm

Wavelength/nm

e

f 25

240

250

Figure 1. The total sulfhydryl group (a) and surface hydrophobicity (b) of soy protein particles modified at different protein concentration and pH. The symbols in Figure 1b share the same meaning as shown in Figure 1a. Far-UV CD spectra of SPP samples prepared by preheating at different pH (c, 6.0, d, 6.2, and e, 6.4) and different concentration. f, representative Far-UV CD spectra of SPP samples preheated at 4% concentration but different pH.

26

Figure 2

control 0.5% 1.0% 2.0% 4.0%

4

8

8

Intensity

8

12

12

Intensity

Intensity

12

4

0

0

0 10

100 Size/nm

10

1000

100 Size/nm

a

8 4 0

10

100 1000 Size/nm

10000

1000

12

Intensity

Intensity

Intensity

0

100 Size/nm

c

12

4

10

1000

b

12 8

4

8 4 0

10

100 Size/nm

d

e

1000

10

100 Size/nm

1000

f

Figure 2. Size distribution of modified soy protein particles before (a, b, and c) and after (d, e, and f) reheat treatment. The symbols in Figure a-f share the same meaning as shown in Figure 2a. Soy protein particles were obtained at different protein concentration (0.5, 1.0, 2.0, and 4.0%) and pH (6.0-a and d, 6.2-b and e, and 6.4-c and f).

27

Figure 3

Control-pH 7.0

0.5%

1.0%

2.0%

4.0%

pH 6.0

Before Heating heating

6.2 pH 6.0

After heating

Before

pH 6.2

heating

After

pH 6.2

heating

pH 6.4

Before heating

After

pH 6.4

heating

28

Figure 3. Atomic force microscopy topographical images of soy protein particles (1%, w/v) before and after reheat treatment. The size of each picture is 2×2 μm. The heat pretreatment conditions soy protein particles formed were shown on the top and right sides.

29

Figure 4

100

Storage modulus/Pa

Storage modulus/Pa

150

200

200

control 0.5% 1.0% 2.0% 4.0%

50

Storage modulus/Pa

200

150 100 50

50 0

0.1

10

1 Frequency/Hz

0.1

1 Frequency/Hz

a

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10

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800 Storage modulus/Pa

Storage modulus/Pa

600

600 400 200

0

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0 1

0

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50 40 30 20 10 0

70

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60

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40 30 20 10

40 30 20 10

0 10

-1

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0 1

Shear rate/s

10 -1 Shear rate/s

g

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1

800

800

200

Viscosity/Pa·s

800 Viscosity/Pa·s

1000

Viscosity/Pa·s

1000

400

600

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400

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0

0 10 -1 Shear rate/s

100

100

i

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10 -1 Shear rate/s

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f

Viscosity/Pa·s

60

Viscosity/Pa·s

control 0.5% 1% 2% 4%

1

1 Frequency/Hz

e

70

10

c

800

0.1

1 Frequency/Hz

b

800 Storage modulus/Pa

100

0

0

Viscosity/Pa·s

150

0 1

10 -1 Shear rate/s

j

k

100

1

10 -1 Shear rate/s

l

Figure 4. Storage modulus (G’) (a-f) and viscosities (g-l) of SPs suspensions (10%, w/v) before (a-c and g-i) and after (d-f and j=l) reheat treatment (100 °C, 30 min): a, d, 30

100

g, and j, soy protein modified at pH 6.0, b, e, h, and k, soy protein modified at pH 6.2, c, f, i, and l, soy protein modified at pH 6.4. Soy proteins preheated at different protein concentrations were marked in different symbols, the meanings of which were shown in a and g.

31

Figure 5

Figure 5. Proposed mechanism of enhanced heat stability of soy protein particles prepared at lower protein concentration. C represents protein concentration.

Table 1. Flow behavior index of the soy protein particles suspensions (10%, w/v) before and after reheat treatment (100 °C, 30 min). Means with different letters are significantly different at 5% level. Samples pH

6.0

6.2

6.4

Flow behavior index

Concentration (%, w/v)

Before reheat treatment

After reheat treatment

0.5

0.87 ± 0.02f

0.89 ± 0.04h

1.0

0.64 ± 0.01c**

0.56 ± 0.02e**

2.0

0.43 ± 0.02b*

0.30 ± 0.01c*

4.0

0.27 ± 0.01a

0.23 ± 0.03ab

0.5

0.91 ± 0.03f**

0.69 ± 0.03g**

1.0

0.78 ± 0.03e**

0.59 ± 0.02a**

2.0

0.42 ± 0.01b**

0.25 ± 0.00bc**

4.0

0.31 ± 0.01a**

0.16 ± 0.02a**

0.5

0.78 ± 0.00e**

0.71 ± 0.01d**

1.0

0.78 ± 0.02e**

0.61 ± 0.01f**

32

2.0

0.70 ± 0.07d**

0.19 ± 0.00a**

4.0

0.31 ± 0.02a*

0.19 ± 0.02a*

0.73 ± 0.01de**

0.28 ± 0.02bc**

Control Note: **: p < 0.01. *: 0.01 < p < 0.05.

Highlights：

► Preheating at lower concentration produce heat stable soy proteins. ► Larger particles were induced via disulfide bonds and hydrophobic interaction. ► Smaller particles were obtained by heating at lower concentration and higher pH. ► Heat stable soy proteins possess larger unfolding extent.

33