Ionic strength and hydrogen bonding effects on whey protein isolate-flaxseed gum coacervate rheology.

Abstract Whey protein isolate (WPI) was mixed with anionic flaxseed (Linum usitatissimum L.) gum (FG), and phase transition during coacervate formation was monitored. Effects of ionic strength and hydrogen bonding on coacervation of WPI‐FG system and corresponding rheological properties were investigated. During coacervate formation, structural transitions were confirmed by both turbidimetry and confocal laser scanning microscopy. Increasing ionic strength with sodium chloride (50 mM) decreased optical density (600 nm) at pHmax. Correspondingly, pHc and pHϕ1 decreased from pH 5.4 to 4.8 and from 5.0 to 4.6, respectively, while pHϕ2 increased from pH 1.8 to 2.4. Sodium chloride suppressed biopolymer electrostatic interactions and reduced coacervate formation. Adding urea (100 mM) shifted pHϕ1, pHmax, and pHϕ2 from 4.8, 3.8, and 1.8 to 5.0, 4.0, and 2.2, respectively, while pHc was unaffected. Optical density (600 nm) at pHmax (0.536) was lower than that of control in the absence of urea (0.617). This confirmed the role of hydrogen bonding during coacervate formation in the biopolymer system composed of WPI and FG. Dynamic shear behavior and viscoelasticity of collected coacervates were measured, and both shear‐thinning behavior and gel‐like properties were observed. Addition of sodium chloride and urea reduced ionic strength and hydrogen bonding, resulting in decreased WPI‐FG coacervate dynamic viscosity and viscoelasticity. The disturbed charge balance contributed to a loosely packed structure of coacervates which were less affected by altered hydrogen bonding. Findings obtained here will help to predict flaxseed gum behavior in protein‐based foods.


2001
). Complex coacervates composed of protein and polysaccharides are widely studied as their structure and physical properties can affect performance and utility of foods, pharmaceuticals, and cosmetics (Turgeon, Schmitt, & Sanchez, 2007). Protein-polysaccharide coacervate solubility is determined by electrostatic interactions, hydrogen bonding, and/or hydrophobic interactions (Espinosa-Andrews, Baez-Gonzalez, Cruz-Sosa, & Vernon-Carter, 2007). Thus, knowledge of biopolymer interactions is helpful as a basis to engineer the structural and functional properties of food products.
Properties of protein-polysaccharide coacervates make them useful for different applications, such as nutrient encapsulation, separation and/or recovery of proteins, enzyme immobilization, emulsification, gelatinization, and/or foam stabilization (Zhao et al., 2014).
In the current study, gum was extracted from whole flaxseed and coacervates were formed with whey protein isolate (WPI). Whey protein isolate is produced by precipitation of protein, largely casein, from milk at pH 4.6 and 20°C . Beta-lactoglobulin, with an isoelectric point (IEP) of 5.2, and α-lactalbumin, with an IEP of 4.1, are primary components of WPI and have excellent nutritional properties. In food formulations, WPI contributes versatile functional properties, enabling emulsification, gelation, and foaming (Turgeon & Beaulieu, 2001). Flaxseed gum (FG) constitutes about 8% of dry flaxseed mass and is recovered mainly as an extract of flaxseed hull that is released when whole or milled seed is soaked in water (Ziolkovska, 2012). Flaxseed gum solutions are separable to yield two distinct polysaccharide fractions: a neutral arabinoxylan of 1.2 × 10 6 Da which constitutes 75% of FG mass (Qian, Cui, Wu, & Goff, 2012); and an acidic rhamnogalacturonan-I (RG-I) polysaccharide (Qian, Cui, Nikiforuk, & Goff, 2012). The acidic fraction is further separable into two fractions with molecular weights of 6.5 × 10 5 Da and 1.7 × 10 4 Da, constituting 3.75% and 21.25% of the mass of FG, respectively (Qian, Cui, Wu, et al., 2012).
The neutral fraction of FG was reported to be largely free of uronic acid. Although, uronic acid was identified to account for 1.8% of the mass of a neutral fraction of 1.47 × 10 6 Da isolated from FG (Qian, Cui, Wu, et al., 2012). Flaxseed gum solutions exhibit useful rheological properties that enable them to be considered for use in emulsification, gelation, foam formation, and foam stabilization (Singh, Mridula, Rehal, & Barnwal, 2011). Flaxseed gum solution functional properties have led to studies of FG utilization in food products of dairy dessert, sausage, salad dressing, carrot juice, etc. (Stewart & Mazza, 2000;Zhou, Meng, Li, Ma, & Dai, 2010). Moreover, FG has been reported to show health benefits when consumed as dietary fiber. Recently, Health Canada has approved a claim that consuming 40 g of flaxseed daily reduces cholesterol and this finding is consistent with the consumption of a diet high in soluble dietary fiber.
Other potential positive impacts of consuming such a diet include reduced risk of diabetes, coronary artery diseases, colon and rectal cancers, and prevention of obesity (Cunnane et al., 1993;Singh et al., 2011;Thakur, Mitra, Pal, & Rousseau, 2009 (Liu et al., 2017). In this research, we investigated the contributions of ionic strength and hydrogen bonding on WPI-FG coacervate structure and rheological properties. Phase transitions during coacervate formation were monitored, and effects of ionic strength and hydrogen bonding on WPI-FG coacervate rheology were evaluated with the addition of NaCl and urea, respectively.
Findings from these studies will provide basic information needed to predict FG behavior in protein-based food products. Flexible mechanical and structural properties of WPI-FG coacervates offer great potential for targeted food applications with desired contributions to food properties.

| Gum preparation from whole flaxseed
Surface dust on flaxseed was removed by rinsing with tap water.
Then, extraction was performed at a flaxseed to deionized RO water ratio of 10:1 (w/w) under gentle stirring (300 rpm). Extraction temperature was maintained at 60°C for 24 hr (Wang, Wang, Li, Xue, & Mao, 2009). Extracts were filtered through cheesecloth to separate soaked flaxseed. After separating the seed, FG extracts were centrifuged at 12,700 g for 20 min (4°C) to remove insoluble particles. Then, supernatants were decanted, and FG fractions were precipitated with ethanol (1:1, v/v). The precipitates were centrifuged, decanted, resuspended in water, freeze-dried, and subsequently used as FG samples.

| Stock solution preparation
Accurately weighed WPI and FG were dispersed in deionized RO water and stirred (300 rpm) at RT for 2 hr. To ensure full hydration, each biopolymer dispersion was then held at 4°C overnight to prepare stock solutions (0.05%, w/w) for coacervation. For rheological measurements, large amounts of WPI-FG coacervates were needed. Therefore, the concentration of stock solution of WPI and FG was 1.0% (w/w), respectively, and was prepared following the same procedures as described.

| WPI coacervate with FG
At a total biopolymer concentration (C T ) of 0.05% (w/w), WPIs were mixed with FG with R = 1:1 (w/w). The biopolymer mixture was titrated with HCl, and coacervate formation was observed using turbidimetric analysis. Glucono-δ-lactone solution (0.005%, w/w), an internal acidifier, was added until the pH was lowered to 4.4. Then, the pH was further decreased following the gradient of pH 4.4-3.6, pH 3.6-2.8, pH 2.8-2.0, and pH 2.0-1.4 with the dropwise addition of 0.05 M, 0.5 M, 1.0 M, and 2.0 M HCl solution, respectively (Weinbreck, Vries, Schrooyen, & Kruif, 2003). The increased molarity of HCl with decreased pH is required to mitigate dilution during titration. Urea (0, 50, and 100 mM) and NaCl (0, 25, and 50 mM) were added to disrupt hydrogen and ionic bond strength, respectively, and, thereby, determine bond effects on coacervate formation. WPI-FG coacervates prepared at C T of 1.0% (w/w) as a function of NaCl (0-200 mM) or urea (0-200 mM) were used for rheological measurements. HCl solution (2.0 M) was used to adjust the pH of biopolymer mixtures. Coacervates were collected by centrifugation at 3,000 g for 20 min (25°C).

| Turbidity measurement
During acid titration, WPI-FG mixture optical density at 600 nm (OD 600 ) was measured in the presence/absence of NaCl or urea.
Turbidity (τ, cm -1 ) of WPI-FG mixture during the acid titration was defined as follows: where L is the path length of light (1 cm), I is the radiation intensity in the presence of sample, and I 0 is the light intensity in the presence of distilled water.
WPI-FG mixture turbidity was plotted versus solution pH and used to determine transition points. Phase transition point pH c was defined as where the turbidity was slightly increased due to the soluble "primary" coacervates. The intersection of two curve tangents was regarded as pH φ1 , and pH φ2 , indicating the formation and totally dissociation of insoluble WPI-FG coacervates, respectively (Weinbreck, Nieuwenhuijse, Robijn, & Kruif, 2003). The pH where the highest OD 600 was demarcated as pH max was reached, creating the maximum interactions between WPI and FG in solution to form coacervates.
Images of stained samples were collected by confocal laser-scanning fluorescence microscope (Zeiss LSM-510) and further processed by LSM 510 image analysis software (Version 3.2). was used for analysis.

| RE SULTS AND D ISCUSS I ON
For protein-polysaccharide mixture of natural food origin, coacervates were formed when electrostatic interactions and non-Coulombic forces of hydrogen bonding, hydrophobic interactions, and steric forces were favored (Xiong et al., 2017). Such coacervates have low toxicity, are biodegradable, and potentially combine the functional properties of both biopolymers (Laneuville, Paquin, Turgeon, 2005). Accordingly, protein-polysaccharide coacervates are used foods where their rheological properties determine functionality. The magnitude and range of enthalpic factors can influence coacervate formation, microstructure, stability, and rheological properties (Chung & McClements, 2015). Viscoelasticity of coacervates formed between gum Arabic (GA) and whey proteins increased with improved electrostatic interaction as a function of acidity (Weinbreck, Wientjes, Nieuwenhuijse, Robijn, & Kruif, 2004).
Addition of NaCl might decrease enthalpic contributions by shielding charges of pectin and bovine serum albumin (BSA), thereby, conferring reduced G′ and G″ values (Ru et al., 2012). However, at pH 3.0, O-carboxymethyl chitosan (OCC) formed maximum coacervates with GA while the coacervates of the same system formed at pH 6.0 had the highest viscosity and viscoelasticity. These observations indicate that factors beyond electrostatic interactions, such as hydrogen bonding, might contribute to the rheological properties of OCC-GA coacervates (Huang et al., 2015). Similarly, agar-whey protein coacervates formed in citrate buffer produced higher modulus values than coacervates formed in water (Rocha, Souza, Magalhães, Andrade, & Gonçalves, 2014). In this study, the contributions of electrostatic interaction and hydrogen bonding to WPI-FG coacervate formation and rheological properties were studied to help resolve the above discrepancies.

| Effects of NaCl on WPI-FG coacervation
Electrostatic interactions were first identified as a primary force guiding the formation of coacervates of gelatin and acacia gum (Tiebackx, 1911). Since this pioneering work coacervate formation has been studied for many protein and polysaccharide systems, including FG and BSA (Liu, Shim, Wang, & Reaney, 2015). to give more positive charges on molecules of WPI (Girard, Turgeon, & Gauthier, 2002). However, for gelatin-agar mixtures, pH c was constant with NaCl concentrations below 200 mM while pH φ1 moved to a lower pH with salt concentrations above 50 mM (Singh et al., 2007). This could be due to the increased gelatin solubility by enhancing molecular coiling effects at a critical NaCl concentration lower than 200 mM (Schmitt, Sanchez, Thomas, & Hardy, 1999). Higher concentrations of dissolved protein increase total positive charges and thus stabilize pH c during acid titration.
During coacervate formation between β-lactoglobulin and pectin, an increased pH φ1 and independent turbidity of biopolymer system were observed with NaCl concentration lower than 100 mM and 800 mM, respectively. Aggregation of β-lactoglobulin molecules during acid titration was responsible for an increase in pH φ1 and constant solution turbidity (Wang, Lee, Wang, & Huang, 2007).
As shown in Figure 1, WPI-FG mixture pH max was independent of NaCl concentration (0-50 mM) though OD 600 at pH max decreased with addition of salt. This phenomenon could be ascribed to WPI protein composition. Whey protein isolate proteins have an isoelectric point (IEP) that compensates for decreased positive charge density on protein molecules caused by NaCl charge screening effects (Weinbreck, Vries, et al., 2003). However, pH max decreased from 4.0 to 3.6 for FG-BSA coacervates when NaCl was added at 100 mM (Liu et al., 2015). Down-shifted pH favored protein lysine ionization (−NH 2 ) and increased positive charge density on BSA molecules. This compen- With increasing NaCl concentration (0-50 mM), pH φ2 of WPI-FG increased from pH 1.8 to 2.4. Thus, the difference between pH φ1 F I G U R E 1 Effects of NaCl concentration (0-50 mM) on WPI-FG coacervation within pH 6.0-1.4 at R = 1:1 (w/w) and pH φ2 was reduced. Total dissociation of insoluble WPI-FG coacervates occurred at higher pH with the addition of salt (NaCl) than that of control. This can be ascribed to charge screening effects of dissolved Na + and Cl − ions on WPI and FG biopolymers where repulsive electrostatic interactions dominated at higher pH. Similar effects of NaCl on turbidity titration curves were reported for WPI-GA biopolymer system. With the increasing of NaCl concentration above 20.3 mM, both pH c and pH φ1 decreased while pH φ2 increased.
Biopolymer charge screening by added ions was responsible for decreased separation between pH φ1 and pH φ2 during WPI-GA coacervate formation (Weinbreck, Vries, et al., 2003). Xiong et al. (2017) also observed a narrowed region for ovalbumin-carboxymethylcellulose coacervate formation between pH φ1 and pH φ2 with increasing ionic strength (NaCl concentration from 0 to 400 mM). At higher ionic strength, the Debye length (R d = 0.3/ √ C NaCl , nm) is less than ovalbumin molecular radius (R pro ), resulting in total screened longrange repulsive interactions and weakened short-range attractive interactions (Seyrek, Dubin, Tribet, & Gamble, 2003), thus ions suppressed coacervate formation.

| Effects of urea on WPI-FG coacervation
It is well accepted that electrostatic interactions contribute to pro- influence of urea addition on OD 600 at pH max was smaller than that of NaCl at the same concentration. This was consistent with previous research on coacervate formation which showed that electrostatic interactions were the primary interaction for BSA-FG coacervate stabilization while hydrogen bonding had less effect (Liu et al., 2015).
Turbidity changes during titration were consistent with confocal laser scanning microscopy observations (Figure 4). The quantity of WPI-FG coacervates observed when urea concentration increased from 0 to 50 mM was slightly reduced (Figure 4a This indicated that hydrogen bonding played a minor role during formation of complex coacervates of β-lactoglobulin and highly methylated pectin (Girard et al., 2002).
Consistently, with urea concentration below 150 mM, pH c value was not affected for BSA-FG mixtures. However, pH φ1 , pH max, and pH φ2 were shifted from 5.0, 4.0, and 2.2 to 4.8, 3.8, and 1.8, respectively, and OD 600 at pH max was decreased (0.818 to 0.664) (Liu et al., 2015). Data support the hypothesis that hydrogen bonding stabilizes WPI-FG coacervates formed between with electrostatic attractive interactions as the primary driving force.

| Dynamic shear behavior
Coacervates formed between WPI and FG at pH 3.4 in the presence of NaCl (0-200 mM) were used as representatives (C T = 1.0%, w/w). As shown in Figure 5a, the dynamic viscosity of coacervates decreased with increasing shear rate, indicating shear-thinning behavior. Flaxseed gum polysaccharide chains in WPI-FG coacervates possibly contributed much of the observed unique dynamic shear behavior (Liu et al., 2016). Consistently, NaCl was reported to decrease intramolecular charge repulsion between FG polysaccharide chains, thus reducing number of junction zones and consequently FG gel strength (Chen, Xu, & Wang, 2006).
Similar effects of NaCl (0-100 mM) on dynamic shear behavior were observed on coacervates formed between sodium caseinate and pectin (Weinbreck, Wientjes, et al., 2004). Structural breakdown or rearrangement of the WPI-FG coacervates under applied shearing force might contribute to shear-thinning behavior (Wee et al., 2014). Flaxseed gum polysaccharide chains formed the highest strength of electrostatic attractive forces with WPI molecules at pH 3.4 with no charge screening effects caused by dissolved salt ions. Low salt environment resulted in a tightly packed WPI-FG coacervate structure and higher apparent viscosity (Stone, Teymurova, & Nickerson, 2014 (Huang et al., 2015). However, Chung and McClements (2015) observed increased apparent viscosity of sodium caseinate-pectin coacervates when NaCl concentration was above 100 mM. Hydrogel particles comprised of sodium caseinate-pectin coacervates and water were formed with casein-rich particles coated with pectin molecules. The increased apparent viscosity could be ascribed to increased porosity and nonsphericity, thus improved effective volume fraction of caseinate-pectin coacervates formed at NaCl concentrations higher than 100 mM.
Coacervates formed from WPI-FG biopolymer mixtures were also responsive to urea as it decreases hydrogen bonding (Ye, 2008).
Here, effects of urea concentration (0-200 mM) on shear flow properties of WPI-FG coacervates (pH 3.4, C T = 1.0%, and R = 1:1, w/w) were investigated (Figure 5b). Typical shear-thinning flow behavior was observed for WPI-FG coacervates formed in the presence of urea. Electroneutrality was achieved at pH 3.4 for the biopolymer mixture which contributed to the highest electrostatic interaction strength (Yang, Chen, & Chang, 1998 (Schmitt et al., 1999).

| Viscoelastic properties
As shown in Figure 6a (Xiong et al., 2017). However, a few exceptions have been reported. Wang, Wang, and Heuzey (2016) found that viscoelasticity of gelatin type B-chitosan coacervates was enhanced in the presence of NaCl due to reduced water content in the coacervate gel structure. For coacervates formed between β-lactoglobulin and pectin, G′ increased within NaCl concentration range of 0.01-0.21 mol/L. However, further increases in NaCl concentration caused a large decrease in G′ (Wang et al., 2007), and a great increase in G″ was observed for N,O-carboxymethyl chitosan (NOCC)-GA coacervates when NaCl reached 250 mmol/L. These findings can be interpreted as an effect of lowered water content while more compact coacervate structures occurred with higher NaCl concentration.
Thus, coacervates produced in higher NaCl were more resistant to shear and displayed increased modulus values at high frequencies (Huang, Du, Xiao, & Wang, 2017).
The contribution of hydrogen bonding to viscoelasticity of WPI-FG coacervates (pH 3.4 and R = 1:1, w/w) with varied urea concentration was recorded (Figure 7b). Both G′ and G″ decreased F I G U R E 6 Effects of NaCl concentration (a) and urea concentration (b) on G′ and G″ of WPI-FG coacervates as determined by strain sweep at 6.28 rad/s F I G U R E 7 Effects of NaCl concentration (a) and urea concentration (b) on G′ and G″ of WPI-FG coacervates as determined by frequency sweep tests at 0.1% strain amplitude with increased urea concentration. Hydrogen bonds were decreased by urea, leading to diminished coacervate viscoelasticity due to the loosely packed structural properties. Urea effects on WPI-FG coacervate viscoelasticity were not as prominent as caused by NaCl at the same concentration. This confirmed our previous conclusion based on observations of BSA-FG coacervates where we determined that electrostatic interaction primarily contributed to coacervate stability while hydrogen bonding played a secondary role (Girard et al., 2002). Fish gelatin-GA coacervates formed at lower temperatures had higher G′ within the frequency range tested. FT-IR analysis confirmed that fish gelatin formed helical structure at 10°C due to favored hydrogen bonding caused by decreased temperature. Thus, lower temperature facilitated the formation of a sponge-like porous microstructure of the fish gelatin-GA coacervates with the water vacuoles entrapped became smaller and more homogenous in size.
This results in a more compact coacervate microstructure due to favored non-Coulombic interactions, such as hydrogen bonding, which can be related to greater elasticity of the coacervate phase (Anvari, Pan, Yoon, & Chung, 2015).

| CON CLUS IONS
Effects of ionic strength and hydrogen bonding on WPI-FG coacervate formation and coacervate rheological properties were investigated in this study. Increased ionic strength with added NaCl decreased WPI-FG pH c and pH φ1 but increased pH φ2 . However, pH max was not affected by increasing NaCl concentration to 100 mM. Suppressed biopolymer electrostatic interactions induced by added NaCl were responsible for the narrower pH range of WPI-FG coacervate stability with reduced WPI-FG coacervate particle size and number. Urea, a standard denaturant that decreases hydrogen bonding, shifted pH φ1 , pH max , and pH φ2 to lower pHs while pH c was constant with urea concentrations up to 100 mM.
Structures of WPI-FG coacervates were loosely packed due to increased ionic strength and decreased hydrogen bonding caused by NaCl and urea addition, respectively, resulting in reduced dynamic viscosity and viscoelasticity.

ACK N OWLED G M ENTS
Financial support for the authors' work was obtained from Agricultural Development Fund (Grant number: 20140276) of the Saskatchewan Ministry of Agriculture.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest regarding the publication of this paper.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.