Whey protein isolate and flaxseed (Linum usitatissimum L.) gum electrostatic coacervates: Turbidity and rheology

Food Hydrocolloids 64 (2017) 18e27

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Whey protein isolate and flaxseed (Linum usitatissimum L.) gum electrostatic coacervates: Turbidity and rheology Jun Liu a, 1, Youn Young Shim a, b, c, *, Jianheng Shen a, Yong Wang c, Martin J.T. Reaney a, b, c, ** a

Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada Prairie Tide Chemicals Inc., 102 Melville Street, Saskatoon, SK S7J 0R1, Canada Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University, 601 Huangpu Avenue West, Guangzhou, Guangdong 510632, China

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 March 2016 Received in revised form 22 August 2016 Accepted 3 October 2016

Coacervates were prepared with flaxseed (Linum usitatissimum L.) gum (FG) and whey protein isolate (WPI) solutions in water. Structural transitions during coacervate formation were monitored by turbidimetry and zeta potentiometry. Biopolymer mixing ratio R (1:4e15:1, w/w) and pH (6.0e1.4) effects on pH-dependent phase transitions (pHc, pHf1, and pHf2) were examined. Where R ¼ 1:1 (w/w), pHc, pHf1, and pHf2 were observed at pH 5.4, pH 5.0, and pH 1.8, respectively. The highest optical density (OD600 ¼ 0.617 ± 0.009) occurred at pH 3.4, the pHmax. As R increased from 1:4 to 15:1, pHf1 and pHf2 also increased (pHf1 4.2 to 5.2 and pHf2 1.8 to 2.2). Accordingly, pHmax shifted from 3.0 to 4.8 while pHc was independent of R. The shift of pHmax was consistent with isoelectric point (IEP) of the WPI-FG mixture determined by electrophoretic mobility measurements. The maximum WPI-FG coacervate formation occurred at R ¼ 2:1 (w/w) and pHmax ¼ 3.8 with OD600 ¼ 0.783 ± 0.018. Dynamic shear viscosity and viscoelasticity of WPI-FG coacervates were determined at a range of pH and R. WPI-FG coacervates exhibited shear-thinning behavior and gel-like properties. The highest dynamic viscosity and viscoelasticity of WPI-FG coacervates were observed at pH 3.8 with R ¼ 2:1 (w/w). Electroneutrality of WPI-FG mixture favored coacervate formation with a more compact structure and improved rheological properties. Light microscopy observations revealed that uncharged WPI-FG mixtures formed coacervates that were more compact than charged coacervates and exhibited improved rheological properties. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Flaxseed gum Whey protein isolate Complex coacervate formation Turbidity Zeta potential Rheological properties

1. Introduction Proteins and polysaccharides are food biopolymers present simultaneously in most foods (Schmitt & Turgeon, 2011). They contribute to food structure, texture, and stability (Ru, Wang, Lee, Ding, & Huang, 2012). Depending on biopolymer interactions,

Abbreviations used: WPI, whey protein isolate; FG, flaxseed gum; MW, molecular weight; GA, gum Arabic; IEP, isoelectric point. * Corresponding author. Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada. ** Corresponding author. Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada. E-mail addresses: [email protected] (Y.Y. Shim), martin.reaney@usask. ca (M.J.T. Reaney). 1 Present address: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Beijing 100083, China. http://dx.doi.org/10.1016/j.foodhyd.2016.10.006 0268-005X/© 2016 Elsevier Ltd. All rights reserved.

phenomena including co-solubility, incompatibility, and complex coacervate formation might occur (de Kruif & Tuinier, 2001; Doublier, Garnier, Renard, & Sanchez, 2000; Syrbe, Bauer, & Klostermeyer, 1998; Tolstoguzov, 1991). Favorable intermolecular interactions, including electrostatic, hydrogen bonding, and hydrophobic interactions, help to form either soluble or insoluble coacervates (Espinosa-Andrews, Baez-Gonzalez, Cruz-Sosa, & Vernon-Carter, 2007; Lamprecht, Schafer, & Lehr, 2001). Many parameters, both internal and external, contribute to biopolymer mixture entropy (Ball et al., 2002; Ou & Muthukumar, 2006). Internal parameters, such as charge density, charge distribution, molecular weight (MW), flexibility, conformation, biopolymer ratio, and concentration affect complex coacervate formation (de Kruif, Weinbreck & de Vries, 2004; Schmitt, Sanchez, DesobryBanon, & Hardy, 1998). External parameters, including biopolymer solution pH, ionic strength, applied shear (mixing), pressure, and temperature also determine biopolymer coacervate

J. Liu et al. / Food Hydrocolloids 64 (2017) 18e27

formation (Schmitt et al., 1998; de Kruif & Tuinier, 2001). Coacervates often introduce properties to foods that are not readily achieved with proteins and polysaccharides alone (Guzey, Kim, & McClements, 2004; Ru et al., 2012). Proteinpolysaccharide coacervate mechanical and structural properties are adaptable allowing selection or design of ideal properties for utilization in different applications (Schmitt et al., 1998; Strauss & Gibson, 2003). Since the first systematic study of fish gelatin-gum Arabic (GA) complex coacervates (Tiebackx, 1911), coacervates have been utilized for encapsulation, protein separation, protein recovery, enzyme immobilization, gelation, emulsification, and foam stabilization (Navratil & Sturdik, 2000; Roy & Gupta, 2003; Thimma & Tammishetti, 2003; Zhao et al., 2014). Proteinpolysaccharide coacervates were also used as components of fat replacers, meat analogues, coatings, edible films, and texturized foods with controlled textural and sensory properties (Azzam et al., 2002; Burova et al., 2007; Tolstoguzov, 2002; Wang, Gao, & Dubin, 1996; Yang, Chen, & Chang, 1998). Protein-polysaccharide coacervate rheological properties play important roles in determining their application as they affect food product texture and overall sensory acceptability (Espinosa-Andrews, Sandoval-Castilla, zquez-Torres, Vernon-Carter, & Lobato-Calleros, 2010; Huang, Va Xiao, Wang, & Qiu, 2015). High viscosity whey protein-GA coacervates are stabilized through attractive electrostatic interactions between biopolymer molecules (Weinbreck, Wientjes, Nieuwenhuijse, Robijn, & de Kruif, 2004). Flaxseed gum (FG) is substantially concentrated in the outermost flaxseed coat (Oomah, Kenaschuk, Cui, & Mazza, 1995). FG is easily extracted by soaking whole flaxseed in water (Ziolkovska, 2012) and it constitutes approximately 8% of dry flaxseed mass (Oomah et al., 1995). It is composed of both neutral (75%) and acidic polysaccharide fractions (25%) (Cui & Mazza, 1996; Warrand et al., 2003; Qian, Cui, Wu, & Goff, 2012) of which the latter consists mainly of pectic-like rhamnogalacturonans with both higher MW (1510 kDa) and lower MW fractions (341 kDa) (Cui & Mazza, 1996; Qian, Cui, Wu, et al., 2012). L-Rhamnose, D-galactose, and D-galacturonic acid (13.8e16.2%) were identified as major FG monomers in acidic fractions (Cui, Mazza, & Biliaderis, 1994; Cui, Mazza, Oomah, & Biliaderis, 1994). Rhamnogalacturonan-I (RG-I) that features /2)-a-L-Rhap-(1 / 4)-a-D-GalpA-(1 / diglycosyl repeats was proposed as a possible backbone structure of FG acidic fractions (Qian, Cui, Nikiforuk, & Goff, 2012). FG neutral fractions were reportedly free of uronic acid and primarily composed of arabinoxylans featuring a b-D-(1,4)-xylan backbone (Cui, Mazza, & Biliaderis, 1994). However, a small amount (1.8%) of uronic acid was observed in FG neutral fractions with a MW of 1470 kDa (Qian, Cui, Wu, et al., 2012). FG potentially has value as a dietary fiber. Similar soluble dietary fiber sources reduce risks of diabetes and coronary heart diseases, mitigate colon and rectal cancer, and decrease the incidence of obesity (Cunnane et al., 1993; Singh, Mridula, Rehal, & Barnwal, 2011; Thakur, Mitra, Pal, & Rousseau, 2009). FG incorporation into food products, such as salad dressing, meat sausage, and dairy dessert have been proposed due to its marked water-holding, swelling, rheological, and emulsification properties (Chen, Xu, & Wang, 2006; Chen, Xu, & Wang, 2007). Whey protein isolate (WPI) produced by casein precipitation (at pH 4.6 and 20  C) contains soluble milk proteins b-lactoglobulin (Isoelectric point, IEP ¼ 5.2) and a-lactalbumin (IEP ¼ 4.1) (Weinbreck, Nieuwenhuijse, Robijn, & de Kruif, 2004). WPI has excellent nutritional properties and is widely used in food formulations (Turgeon & Beaulieu, 2001; de Wit, 1998). WPI also supports emulsification, gelation, and foaming in foods (de Wit, 1998). WPI forms coacervates with polysaccharides that can be used for protein separation, encapsulation, fat replacement, and texture formation (Aberkane et al., 2010; Weinbreck, Tromp, & de Kruif, 2004;

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Capitani, Pacheco, Gumerato, Vitali, & Schmidt, 2005). In this study, WPI was employed as protein biopolymer to form FG complex coacervates. The influence of pH and biopolymer mixing ratio R (WPI:FG, w/w) on soluble and insoluble WPI-FG coacervates was investigated by turbidimetric analysis and zeta potentiometry. WPI-FG coacervates rheological properties, including dynamic viscosity and viscoelasticity, were examined as a function of pH and R. Information obtained in this study may help in designing specific future applications for WPI-FG coacervates. 2. Materials and methods 2.1. Materials Flaxseed (var. CDC Bethune), provided as a generous gift from Dr. G. Rowland, was harvested in 2011 from Floral (SK, Canada). WPI (BiPRO) used in this study was purchased from Davisco Foods International, Inc. (Le Sueur, MN, USA) with protein content of 97.7% (%N  6.38), <0.5% fat, 4.7% moisture, 1.9% ash, and <0.2% lactose. Sodium hydroxide (NaOH,  97%) was obtained from SigmaAldrich Canada Ltd. (Oakville, ON, Canada). Anhydrous ethanol (0.10% water by volume) was purchased from Commercial Alcohols Inc. (Brampton, ON, Canada) and hydrochloric acid (37% by weight) was obtained from Fisher Scientific Company (Ottawa, ON, Canada). A Milli-Q deionization reversed osmosis (RO) system (Millipore, Bedford, MA, USA) was used to prepare deionized RO water (resistivity was greater than 18.2 MU cm at 25  C). All other reagents were of analytical grade and used as received. 2.2. FG preparation Surface dust was removed by washing flaxseed with deionized RO water for 1 min at 22e23  C (RT). Thereafter, clean flaxseed was soaked in deionized RO water with water to seed weight ratio of 10:1. FG was extracted at 60  C with gentle stirring (300 rpm with a Teflon® coated magnetic stirring bar) for 24 h according to procedures previously described by Wang, Wang, Li, Xue, and Mao (2009) with small modifications. FG extracts were then collected by filtration of the solution and soaked flaxseed with cheesecloth to separate flaxseed. Centrifugation was performed at 12,700 g and 4  C for 20 min to remove insoluble particles from the FG extracts. Supernatant was mixed with anhydrous ethanol at a volume ratio of 1:1 to precipitate FG. Ethanol precipitates were collected by centrifugation (12,700 g at 4  C for 20 min), lyophilized (LABCONCO, Kansas City, MO, USA), and used as FG. The dried FG samples were kept in a desiccator at RT for subsequent analyses. 2.3. WPI and FG stock solutions Coacervates between WPI and FG were prepared from stock solutions at 0.05% (w/w) of each biopolymer. FG and WPI were dissolved in deionized RO water at RT for 2 h with constant stirring at 300 rpm, respectively. Stock solutions were maintained at 4  C for another 24 h to ensure total hydration. FG and WPI stock solutions (1.0% w/w) were prepared for rheological measurements of WPI-FG coacervates where large amounts of coacervates were required. 2.4. WPI-FG coacervate preparation Systematic effects of pH and R (WPI: FG, w/w) on WPI-FG coacervate formation were studied using turbidimetric analysis. R was varied from 1:4 to 15:1 (w/w) with a constant biopolymer concentration (CT) of 0.05% (w/w). For each biopolymer mixture pH (6.0e1.4) was adjusted via addition of glucono-d-lactone (0.005%,

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w/w) to lower pH to 4.4. Subsequently, HCl solutions (0.05, 0.5, 1.0, and 2.0 M) were added dropwise to achieve the desired pH. HCl solutions were used based on the following gradient: 0.05 M < pH 3.6, 0.5 M < pH 2.8, 1.0 M < pH 2.0, and 2.0 M < pH 1.4, to mitigate dilution effects (Weinbreck, de Vries, Schrooyen, & de Kruif, 2003). In order to prepare WPI-FG coacervates for rheological measurements, pH of each biopolymer mixture prepared at defined R was adjusted by 2.0 M HCl solution. WPI-FG coacervates were collected through centrifugation at 3000  g at 25  C for 20 min, followed by removing the supernatant. All measurements were performed in triplicate.

2.5. Turbidity measurement Optical density (OD) of homogenous WPI solutions, FG solutions, and their mixtures at defined R were measured at 600 nm (OD600) during turbidimetric titration. Samples were placed in Semi-Micro Acrylic (PMMA) plastic cuvettes (VWR International, LLC, Radnor, PA, USA) then OD600 was recorded with a Genesys 10S UVeVis spectrophotometer (Thermo Scientific, Madison, WI, USA). All measurements were performed in triplicate at RT and are expressed as mean ± standard deviation (SD). Turbidity (t, cm1) was defined as:

    1 I ln L I0

t¼

(1)

Where L is the light path length of 1 cm, I is the intensity of light reaching the detector when the sample is present, and I0 is the distilled water blank intensity. Turbidity curves of each biopolymer mixture were constructed as a function of solution pH. pHc was defined as the structure formation transition point where soluble “primary” WPI-FG coacervates were formed as indicated by a slight turbidity increase during acid titration. pHf1 and pHf2 were determined graphically as the intersection between two curve tangents (Weinbreck, Nieuwenhuijse, Robijn, & de Kruif, 2003), respectively. pHmax was the pH where the highest turbidity was achieved indicating the maximum interaction between WPI and FG biopolymers.

2.6. Zeta potential measurement

2ε  z  GðkaÞ 3h

Micrographs of WPI-FG coacervates were collected by OPTEC BK5000 biological microscopy (Chongqing Optec Instrument Co., Ltd., Chongqing, China) equipped with a USB 2.0 CCD digital camera (Guangzhou JPLY Electronic Technology Co., Ltd., Guangdong, China). A series of WPI-FG coacervates (CT ¼ 1.0%, w/w) were prepared at pH 3.2, 3.8, and 4.4 with a constant R of 2:1. A second series was prepared with varied biopolymer mixing ratios of R 1:1, 2:1, and 4:1 at pH 3.8. Samples (20 mL) of this WPI-FG coacervate series were transferred to glass slides then covered with a coverslip, and observed at a magnification of 40. Images were taken with a digital camera and processed with SpectrumSe image analysis software (Guangzhou JPLY Electronic Technology Co., Ltd., Guangdong, China). 2.8. Rheological measurements An AR2000ex rheometer (TA Instruments Ltd., Crawley, UK) equipped with Peltier cooling system was used for rheological analyses. WPI-FG coacervate samples were loaded onto the rheometer bottom plate and a solvent trap cover was applied to limit evaporation and mitigate interference. Before each measurement, samples were equilibrated for 2 min. A cone plate geometry (2 , 40 mm diameter) with a gap of 0.054 mm between flat surfaces of both elements was used for all rheological tests. Experimental flow curves of WPI-FG coacervates were constructed through continuous shear over a shear rate range of 0.1e100 s1 at 25  C. Two types of oscillatory measurements: strain sweep and frequency sweep, were performed to determine WPI-FG coacervate viscoelastic behavior (storage modulus G0 and loss modulus G00 ). Prior to frequency sweep tests, strain sweep measurements were conducted to determine the linear viscoelastic region (LVR), where dynamic G0 and G00 are independent of strain amplitude (Peng, Ren, Zhong, Cao, & Sun, 2011). Each strain sweep measurement was performed within a range of 0.01e100% strain under a constant frequency of 1.0 Hz (6.28 rad/s) at 25  C. Based on LVR, constant strain amplitude of 0.1% was selected for subsequent frequency sweep tests over an angular frequency range of 0.628e628 rad/s. All measurements were performed in duplicate and expressed as means. Data analysis was performed with TA Rheology Advantage Data Analysis software V 5.4.7 (TA Instruments Ltd., Crawley, UK). 3. Results and discussion

Zeta potentials (z, mV) of WPI, FG, and their mixtures were determined to evaluate overall surface charge. WPI only (0.05%, w/ w) and FG only (0.05%, w/w) solutions, and their mixtures (CT ¼ 0.05%, w/w) at varied R (1:4e15:1, w/w) were titrated with a MPT-2 autotitrator (Malvern Instruments, Westborough, MA, USA) using sodium hydroxide (0.5 M), HCl (0.5 M and 0.1 M) as titrants. Electrophoretic mobility (UE) was measured at 0.4 pH unit increments using Laser Doppler Velocimetry combined with phase analysis light scattering (Malvern Zeta Nano ZS, Malvern Instrument, Worcestershire, UK). z was calculated based on the Henry equation:

UE ¼

2.7. Light microscopy observations

(2)

Where h and ε are the dispersion viscosity (Pas) and permittivity of the biopolymer solutions with varied R, respectively. k is the Debye length and a is the particle radius, where the f(ka) is equal to 1.5 based on the Smoluchowski approximation (Smoluchowski, 1903). All measurements were performed in triplicate and reported as mean ± SD.

The mechanisms involved in complex coacervate formation in protein-polysaccharide systems are not fully understood or described. Generally it is accepted that proteins form coacervates with polysaccharides when intermolecular interactions, such as electrostatic, hydrogen bonding, and hydrophobic interactions, are favorable in the biopolymer mixture (Espinosa-Andrews et al., 2007; Lamprecht et al., 2001; Liu, Shim, Wang, & Reaney, 2015). Typically, protein-polysaccharide coacervates form between the protein isoelectric point (IEP) and the polysaccharide pKa. During acid titration protein-polysaccharide coacervates undergo sequential phase changes (Fig. 1) that include: 1) formation of soluble “primary” complexes at pHc; 2) quasineutralised insoluble complex formation at pHf1 due to nucleation and growth of primary soluble complexes. With further titration, proteinpolysaccharide coacervate formation reaches a maximum (pHmax); and 3) disassociation of protein-polysaccharide coacervates after pHmax with totally disassociation occurred at pHf2 when biopolymers carry a similar net charge (de Kruif, & Tuinier, 2001; Turgeon, Beaulieu, Schmitt, & Sanchez, 2003). In this study, complex coacervate formation between WPI and FG was probed

J. Liu et al. / Food Hydrocolloids 64 (2017) 18e27

with acid titration of biopolymer mixtures from pH 6.0 to 1.4 by observing turbidity changes. The formation of WPI-FG coacervates and increased quantity and/or size of coacervates produce a solution that scatters light and has a turbid appearance. As shown in photographic images of WPI-FG dispersions (R ¼ 1:1, CT ¼ 0.05%, w/ w) (Fig. 1), the mixture is visually clear where pH is greater than 5.0 (pHf1), indicating that soluble complexes between WPI and FG are not present. WPI-FG mixtures became increasingly turbid with further pH decreases and reached a maximum from pH 3.8 to 3.0 (not visually different). Below pH 3.0, WPI-FG mixture turbidity decreased and the biopolymer mixture became almost clear at pH 1.8 (pHf2), indicating total WPI-FG coacervate disassociation. The visual appearance of WPI-FG mixtures was consistent with turbidity measured as optical density measurement at 600 nm (Fig. 1). 3.1. Effect of pH on WPI-FG coacervate formation pH plays an important role in determining biopolymer molecular charge and consequently biopolymer interactions (de Kruif, Weinbreck & de Vries, 2004). Turbidimetric titration curves (pH 6.0e1.4) were prepared for WPI solutions (0.025%, w/w), FG solutions (0.025%, w/w), and their mixtures (R ¼ 1:1, w/w) at a CT ¼ 0.05% (w/w) (Fig. 1). WPI and FG solution controls had much lower OD600 than observed for WPI-FG mixtures during acid titration. Protein aggregation and polysaccharide conformational changes caused by acid titration might cause turbidity changes observed for WPI and FG solutions (Weinbreck, de Vries, et al., 2003). This was in agreement with our previous study of a FG

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and bovine serum albumin (BSA) biopolymer system (Liu et al., 2015). Both biopolymers were negatively charged where pH was >5.4 and no intermolecular interaction was observed for WPI-FG mixtures above pH 5.4. WPI-FG solution turbidity increased slightly when pH reached 5.4 (pHc), indicating the initiation of WPIFG coacervate formation as pH decreased. A similar phenomenon was reported to occur during acid titration of whey proteins and GA biopolymer solutions (Weinbreck, de Vries, et al., 2003). Whey protein-GA mixture turbidity was low at pHc (pH 5.3) but light scattering intensity increased as pH decreased, indicating formation of soluble whey protein-GA complexes. At pHc ¼ 5.4, both WPI and FG molecules were negatively charged as the IEPWPI was 4.92 and the IEPFG was 1.73. Theoretically, WPI-FG coacervates should not occur at pHc due to repulsive forces between negatively charged WPI and FG molecules. Soluble BSA-FG coacervates formed at pH 5.4, above the BSA IEP ¼ 4.96 (Liu et al., 2015). This association is likely caused by interactions between positive patches on WPI molecules and negative carboxyl groups on FG molecules (Park, Muhoberac, Dubin, & Xia, 1992). Similar results were reported by Vinayahan, Williams, and Phillips (2010) where soluble coacervates between BSA and GA formed at pHc ¼ 5.5 substantiated by significant increases in light scattering intensity. WPI-FG mixture turbidity increased with further acid titration to pH 5.0 (pHf1), (Fig. 1). WPI molecules were positively charged and interacted with negatively charged FG molecules, driving a transition of the WPI-FG mixture from a transparent solution to a cloudy coacervate induced by electrostatic interactions (Weinbreck, de Vries, et al., 2003). The WPI-FG mixture (R ¼ 1:1, CT ¼ 0.05%, w/ w) OD600 reached a maximum of 0.617 ± 0.009 at pH 3.4 (pHmax).

Fig. 1. Turbidity curves of homogenous WPI (0.025%, w/w), FG (0.025%, w/w), and mixture thereof with a biopolymer mixing ratio R ¼ 1:1 (CT ¼ 0.05%, w/w) during acid titration from pH 6.0 to 1.4. pHc, pHf1, and pHf2 were critical pH transition points corresponding to structure forming events during acid titration. pHmax represented the pH where maximum OD600 was achieved. Photographic images show visible turbidity changes of WPI-FG dispersions (R ¼ 1:1, CT ¼ 0.05%, w/w) occurring between pH 6.0 to 1.4 in test tubes.

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The greatest electrostatic interaction formed between WPI and FG molecules at pHmax, resulting in accumulation of the largest amount of insoluble WPI-FG coacervate. However, insoluble WPIFG coacervates disassociated when pH was lower than pHmax where WPI-FG mixture turbidity began to decrease. FG carboxyl group's protonation with further acid addition (pH < pHmax) decreased WPI-FG mixture turbidity (de Kruif & Tuinier, 2001). WPI-FG insoluble coacervate sedimentation could also contribute to decreased biopolymer mixture turbidity when pH < pHmax. Decreased turbidity might not reflect decreased interactions between WPI and FG molecules (Wee et al., 2014). At pH 1.8 (pHf2 > FG pKa 1.73), insoluble WPI-FG coacervates were mostly disassociated, and WPI-FG mixture turbidity was near zero. This was consistent with Schmitt et al. (1998) who noted that proteinpolysaccharide complex coacervates typically form within a pH range bounded by the protein and polysaccharide IEP. 3.2. Effects of R on WPI-FG coacervate formation In mixtures of proteins and polysaccharides charge balance between biopolymers is also sensitive to biopolymer mixing ratio R. Changes in R could further affect protein-polysaccharide coacervate formation during acid titration (Ye, 2008). WPI-FG coacervate formation as a function of R from 1:4 to 15:1 (w/w) was studied by turbidimetry. CT of all WPI-FG mixtures was fixed at 0.05% (w/w) and turbidimetric analysis was performed within a pH range of 6.0e1.4. With R < 1:6 (w/w), no obvious turbidity changes of the WPI-FG mixture were observed, indicating that interactions between WPI and FG were negligible (Data not shown). Similar phenomena were reported for a pea protein isolate, GA biopolymer system when R < 1:4 (w/w) (Liu, Low, & Nickerson, 2009). Schmitt, Sanchez, Thomas, and Hardy (1999) hypothesized inter-polymeric and intra-polymeric interactions and aggregation in proteinpolysaccharide mixtures were reduced or even prevented at higher polysaccharide concentrations. Polysaccharide molecules are less effective in light scattering than protein molecules in protein-polysaccharide mixtures (Liu et al., 2009). Overlapping turbidity curves of WPI-FG mixtures were observed when R > 10:1 (w/w) (Fig. 2). This indicates that in the presence of an excess of WPI molecules in solution carboxyl FG polysaccharide chain reaction sites are limiting. Similar overlapping turbidity curves were observed for pea protein isolates mixtures with alginate polysaccharides when R was higher than 8:1 (Klemmer, Waldner, Stone, Low, & Nickerson, 2012). Turbidity curves of pea protein isolate-GA biopolymer mixtures overlapped when R was greater than 4:1 (w/ w) (Liu et al., 2009). The critical structure transition point (pHc), associated with soluble WPI-FG coacervate formation, was constant at pH 5.4 when R increased from 1:4 to 15:1 (w/w). Similarly, for a BSA-GA biopolymer system pHc was also not affected by changing R (Vinayahan et al., 2010). In solution about ten BSA molecules were complexed with each GA polysaccharide chain, producing a soluble product. This observation is consistent with complex coacervate formation observed in whey protein-carrageenan (Weinbreck, Nieuwenhuijse, et al., 2004), whey protein-exocellular polysaccharide EPS B40 (Weinbreck, Nieuwenhuijse, et al., 2003), and blactoglobulin-pectin (Girard, Sanchez, Laneuville, Turgeon, & Gauthier, 2004) biopolymer systems. However, R dependent pHc was reported by Singh, Aswal, et al. (2007) and Singh, Siddhanta, et al. (2007) when agar was used to prepare coacervates with both type-A and type-B gelatin at R < 2:1 (w/w). A possible explanation for this phenomenon could be due to interactions between polysaccharide chains with protein-protein aggregates rather than single protein molecules (Liu et al., 2009). As WPI-FG mixture R increased, more WPI protein molecules

were available to bind to FG polysaccharide chains. Therefore, WPIFG mixture electroneutrality was achieved at a higher pH as R increased (Yang, Anvari, Pan, & Chung, 2012). This was in agreement with findings of this study while pHf1 and pHf2 occurred at higher pH with greater R (Fig. 2). Similar changes of pHf1 and pHf2 as a function of R were reported for whey proteins in coacervates formed with exocellular polysaccharide, EPS B40, and carrageenan (Weinbreck, Nieuwenhuijse, et al., 2003; Weinbreck, Nieuwenhuijse, et al., 2004). A critical R of 10:1 (w/w) was observed for both structural transition points of pHf1 and pHf2 as indicated by overlapping of turbidity curves (Weinbreck, Nieuwenhuijse, et al., 2004). The greatest interactions in WPI-FG mixture, pHmax, underwent a similar change as a function of R. As R increased from 1:4 to 15:1 (w/w), pHmax correspondingly increased from 3.0 to 4.8 as the electroneutrality point of WPI-FG mixture at corresponding R shifted (Fig. 3). In this study, the highest OD600 of 0.783 ± 0.018 was observed at pH 3.8 (pHmax) with R ¼ 2:1 (w/w). Higher or lower R suppressed WPI-FG coacervate formation as indicated by decreased OD600 at pHmax. Conversely, when R was greater or smaller than 2:1 (w/w), WPI-FG coacervate formation was suppressed by insufficient or excess amounts of protein molecules for binding to FG polysaccharide chains (Schmitt et al., 1998; Weinbreck, de Vries, et al., 2003; Turgeon, Schmitt, & Sanchez, 2007; Ye, 2008). 3.3. Zeta potential measurements Electrostatic interaction was identified as the major driving force involved in complex coacervate formation between proteins and anionic polysaccharides in solution (Turgeon et al., 2007).  Protein ionization due to amine (eNHþ 3 ) and carboxyl (eCOO ) groups is determined by pH. Protein charge largely determines electrostatic interactions with other biopolymers, including polysaccharides (Bowen, Hall, Pan, Sharif, & Williams, 1998). In this study, surface electrical properties of WPI and FG solutions and their mixtures with varied R were evaluated by zeta potential (z) measurement. z is the electro-kinetic potential difference between the dispersion medium and the slip plane (stationary layer of fluid attached to the dispersed particle) of moving biopolymer particles. z could provide important information regarding coacervate formation between WPI and FG and coacervate stability (Liu et al., 2015). During titration of WPI solutions (0.05%, w/w) from pH 6.19 to  1.68, surface amine (eNHþ 3 ) and carboxyl groups (eCOO ) groups of

Fig. 2. Effects of biopolymer mixing ratio R (1:415:1, w/w) on WPI-FG complex coacervate formation (CT ¼ 0.05%, w/w) during acid titration from pH 6.0 to 1.4.

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WPI molecules were protonated, and, as a result, z increased from 21.20 ± 3.97 to 17.57 ± 0.57 mV. WPI solution (0.05%, w/w) IEP was pH 4.92, where z was zero (Wee et al., 2014). WPI was negatively charged above the IEPWPI and positively charged at lower pH. FG solution (0.05%, w/w) IEP was pH 1.73. Due to decreased FG polysaccharide carboxyl residue ionization z increased from 42.3 ± 1.0 to 0.03 ± 1.15 mV during FG solution titration from pH 5.98 to 1.74 (Cui & Mazza, 1996; Warrand et al., 2003). Between the IEPFG pH and IEPWPI, FG polymers were negatively charged while WPI molecules were positively charged. At this pH attractive electrostatic interactions occur between WPI and FG polymers that lead to WPI-FG biopolymer coacervate formation (Schmitt et al., 1998). Bulk z is the sum of zs contributed by non-interacting and interacting WPI and FG and WPI-FG coacervate particles. WPI-FG mixture z increased with R from 1:4 to 15:1 (Fig. 3). This was the result of higher WPI z of than that of FG. WPI-FG mixture IEP also increased from 2.85 to 4.66 with increased R from 1:4 to 15:1 (w/w) (Fig. 3). This coincided approximately with pHmax determined by turbidimetric analysis at each given R (Fig. 2) and confirmed that electrostatic interaction was the major driving force for WPI-FG coacervate formation. At pHmax, the greatest interaction was observed due to WPI-FG neutrality (Yang et al., 2012). WPI-FG coacervates achieved the highest stability and insolubility at pHmax (Klemmer et al., 2012). 3.4. WPI-FG coacervate rheological properties 3.4.1. Dynamic shear behavior Insoluble WPI-FG coacervate enriched phases were collected by centrifugation (3000 g, 25  C, and 20 min) and subjected to dynamic shear measurement against a shear rate range of 0.1e100 s1 at 25  C. WPI-FG coacervates were either formed at pH 3.2, 3.8, and 4.4 with a fixed R of 2:1 (w/w) or formed at a constant pH of 3.8 with R varied from 1:1, 2:1, and 4:1 (w/w) to investigate pH and R effects on shear flow behavior. WPI-FG coacervates formed at different pHs all exhibited typical shear-thinning behavior within the shear rate range tested (0.1e100 s1) (Fig. 4A). With increasing shear rates from 0.1 to 100 s1, WPI-FG coacervate dynamic viscosity decreased. Shear-thinning properties may arise from WPI-FG coacervate structural breakdown or rearrangement under shear (Wee et al., 2014). WPI-FG coacervates

Fig. 3. Zeta potential (mV) of homogenous WPI (0.05%, w/w), FG (0.05%, w/w), and mixture thereof (CT ¼ 0.05%, w/w) with different biopolymer mixing ratio R (1:415:1, w/w).

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collected at pH 3.8 (181.6 Pas at 1.0 s1) had higher apparent viscosity as a result of more tightly packed structures where the greatest electrostatic attractive interactions between WPI and FG molecules occurred (Stone, Teymurova, & Nickerson, 2014). Light micrographs (Fig. 4A) of WPI-FG coacervates revealed the presence of small droplets suspended in the biopolymer mixture when pH was 4.4. These small coacervates agglomerated to produce larger particles and more packed structures when pH was 3.8 (pHmax). Further pH decreases lead to dissociation of these WPI-FG coacervate agglomerates due to similar charges carried by biopolymers in solution. This was in agreement with our turbidity analysis and WPI-FG mixture z measurements at R ¼ 2:1 (w/w) and pH 3.8 (pHmax) where the maximum electrostatic attractive interactions between WPI and FG molecules produced the highest OD600 (0.783 ± 0.018). Either increasing or decreasing pH of WPI-FG mixture reduced coacervate formation generating a more loosely packed coacervate structure. Espinosa-Andrews et al. (2008; 2010; 2013) reported similar results where GA-chitosan coacervate viscosity was correlated with electrostatic interaction strength, which in turn was highly pH-dependent. Interestingly, O-carboxymethyl chitosan (O-CMC)-GA coacervates formed at pH 3.0 demonstrated typical shear-thinning property at shear rates below 60 s1. While a structural rearrangement of O-CMC-GA coacervates was observed as indicated by a marked increase in viscosity when shear rate increased from 60 to 100 s1. A more compact structure could be formed with shear rate beyond a critical value (Huang et al., 2015). R affects charge balance (Ye, 2008). Here effects of R (1:1, 2:1, and 4:1, w/w) on WPI-FG coacervate shear flow properties were investigated at pH 3.8. WPI-FG coacervates formed at varied R demonstrated typical shear-thinning flow behavior within the tested shear rates (0.1e100 s1). Both increasing and decreasing protein in WPI-FG biopolymer mixtures from R ¼ 2:1 (w/w) decreased apparent viscosity at a shear rate of 1.0 s1 (hR ¼ 1:1 ¼ 30.4 Pas; hR ¼ 4:1 ¼ 109 Pas). For a WPI-FG biopolymer system with R ¼ 2:1 (w/w) and pH ¼ 3.8 electro neutrality was achieved which produced the greatest electrostatic interaction (Yang et al., 1998). As a consequence, the strongest WPI-FG coacervates formed and the highest shear resistance was measured (Espinosa-Andrews et al., 2013). For increasing or decreasing R, positive charged amine groups on WPI molecules were not sufficient to neutralize negatively charged carboxyl groups on FG polysaccharides. Consequently, a WPI-FG mixture at pH 3.8 with R other than 2:1 (w/w) was charged. The net charges of WPI-FG mixture suppressed the extent and strength of electrostatic attractive interactions between WPI and FG molecules and reduced WPI-FG coacervate formation (Schmitt et al., 1999). This contributed to lower apparent viscosity observed for WPI-FG coacervate solutions at pH 3.8 and either R ¼ 1:1 and 4:1 (w/w) than for solutions prepared at R ¼ 2:1 (w/w). This was confirmed by light images (Fig. 4B) taken of WPI-FG coacervates formed at varied R (1:1, 2:1, and 4:1) under a constant pH of 3.8. WPI-FG coacervates demonstrated a more compact network structure at an R of 2:1 (w/ w) and pH of 3.8. Higher or lower R than 2:1 produced a nonneutralized WPI-FG system charge, resulting in more loosely packed structures and correspondingly lowers apparent viscosity. Similar results were observed for sodium caseinate-gum tragacanth coacervates that produced the greatest viscosity at pH 4.04, the pH of charge balance (Gorji, Gorji, Mohammadifar, & Zargaraan, 2014). 3.4.2. Viscoelastic properties WPI-FG coacervates samples were also subjected to strain sweep tests over a strain amplitude range of 0.01e100% under a constant frequency of 1.0 Hz (6.28 rad/s) and a temperature of 25  C (Fig. 5A and B). The LVR was determined based strain sweep data

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J. Liu et al. / Food Hydrocolloids 64 (2017) 18e27

Fig. 4. Dynamic flow behavior of WPI-FG coacervates prepared at CT ¼ 1.0% (w/w) as a function of pH (A) and biopolymer mixing ratio R (B) over a shear rate range of 0.1100 s1. Micrographs on the right are optical microscopy images of WPI-FG coacervates obtained at varied pH and R (40 magnification; scale bar: 200 mm).

where dynamic G0 and G00 were independent of strain amplitude (Peng et al., 2011). A constant strain amplitude of 0.1% strain was used for subsequent frequency sweep measurements over an angular frequency range of 0.628e628 rad/s for all WPI-FG coacervate samples. All measurements were performed in duplicate and expressed as means. For all WPI-FG coacervate mixtures prepared at varied pH but fixed R (2:1, w/w), storage modulus G0 was higher than loss modulus G00 over the frequency range tested, indicating a highly interconnected gel-like network structure. Prior to acid titration, WPI molecules were positively charged but they began interactions with negatively charged FG molecules to form a strongly entangled network as pH decreased. Similar gel-like properties were observed for coacervates formed between bovine serum albumin (BSA) and pectin (Ru et al., 2012) and b-lactoglobulin and pectin (Wang, Lee, Wang, & Huang, 2007). However, predominantly viscous behavior was also observed for coacervates formed in agar-gelatin (Singh, Aswal, & Bohidar, 2007) and whey protein-GA biopolymer systems (Weinbreck et al., 2004). Dynamic G0 and G00 were greater at pH 3.8 than at pH 3.2. Both rheological parameters indicate the

formation of stronger gel-like network structures at the higher pH, which was close to IEP of WPI-FG system at R ¼ 2:1 (w/w), indicating that maximum interactions occurred. Both dynamic G0 and G00 were lower at pH 4.4 than at pH 3.8. The WPI-FG biopolymer system formed at higher pH had a lower net positive charge and consequently a more loosely packed WPI-FG coacervate structure with greater amounts of bound water. Similar findings were reported for sodium caseinate-Astragalus rahensis biopolymer system. The highest G0 and G00 were observed at pH 4.04 and higher or lower pH produced mixtures with lower G0 and G00 (Gorji et al., 2014). R (1:1, 2:1, and 4:1, w/w) effects on WPI-FG coacervate viscoelastic properties were investigated at pH 3.8 (Fig. 6). For all samples tested storage modulus G0 was always higher than loss modulus G00 over the frequency range tested (0.628e628 rad/s) indicating gel-like properties. Where R ¼ 2:1 (w/w) maximum G0 and G00 were observed indicating the strongest electrostatic attractive interactions involved in coacervate formation between WPI and FG. This was in agreement with turbidity and z measurements where WPI-FG mixture electroneutrality was achieved

J. Liu et al. / Food Hydrocolloids 64 (2017) 18e27

25

mouthfeel. In addition, FG-WPI coacervates could also be used as food emulsifiers that provide improved surface activity when hydrophilic polysaccharide and hydrophobic protein are mixed together. Furthermore, encapsulation of food sensitive nutrients for controlled release and targeted delivery might be possible using FG-WPI coacervates as a barrier material. Such an approach could improve bioavailability and bioactivity of encapsulated food nutrients. 4. Conclusion

Fig. 5. Strain dependent of storage modulus (G0 ) and loss modulus (G00 ) at a fixed frequency of 6.28 rad/s for WPI-FG coacervates prepared at CT ¼ 1.0% (w/w) as a function of pH (A) and biopolymer mixing ratio R (B).

at pH 3.8 with R ¼ 2:1 (w/w). Similar phenomena were also reported for a whey protein-l-carrageenan coacervate system where charge neutralization between both macromolecules occurred (Weinbreck, Nieuwenhuijse, et al., 2004). R that was either higher or lower than 2:1 decreased the WPI-FG mixture charge balance at pH 3.8. This could suppress WPI-FG coacervate formation and limit the strength of gel-like coacervate structures. Findings of this study supported the assumption that both dynamic G0 and G00 of WPI-FG coacervates formed with R of 1:1 and 4:1 (w/w) were lower than that of R ¼ 2:1 (w/w). At pH 3.8, WPI-FG mixtures were either positively or negatively charged with R of 1:1 or 4:1 (w/w), respectively. A loosely packed coacervate structure was formed as biopolymer molecules repelled each other, resulting in both decreased dynamic G0 and G00 . For b-lactoglobulin/pectin coacervates, stronger gel-like structures were formed as R was increased and coacervate rheological properties were highly dependent on composition (Wang et al., 2007). Similar patterns were reported with BSA-pectin coacervates. However, with R > 20:1 (w/w), reduction of rheological properties was observed as an excess amounts of BSA molecules (Ru et al., 2012). Studies of FG and WPI coacervate formation will provide useful information regarding textural or sensory properties contributed to WPI based food products when FG is included as a food additive. For example, FG could be introduced into yogurt as food thicker and/or dietary fiber with potential health benefits. Substantial coacervate formation between FG and WPI in yogurt could lead to precipitation, thus reducing the product storage stability and consumer acceptance. Conversely, FG-WPI coacervate properties can be controlled by manipulating solution pH and biopolymer mixing ratio R to achieve a yogurt with superior textural properties and

Electrostatic coacervates were formed between WPI and FG during acid titration (pH 6.0e1.4) and investigated by turbidimetric analysis. The maximum coacervate formation of WPI-FG mixtures was observed at pH 3.8 and a CT of 0.05% (w/w) with R ¼ 2:1 (w/w) as substantiated by the highest OD600 ¼ 0.783 ± 0.018. Influence of R (1:4e15:1, w/w) was also evaluated and critical pH dependent phase transition points of pHf1, pHf2, and pHmax increased to higher pH with increasing R, while pHc was independent of R. WPIFG mixture pHmax at each R was coincident with the corresponding IEP measured by zeta potentiometry, indicating the major role of electrostatic interaction in WPI-FG coacervate formation. Typical shear-thinning behavior and gel-like properties were observed through dynamic shear, stain sweep, and frequency sweep measurements on WPI-FG coacervates. The highest apparent viscosity and viscoelasticity were observed on WPI-FG coacervates prepared at pH 3.8 and R ¼ 2:1 (w/w). pH higher or lower than 3.8 and R higher or lower than 2:1 reduced both dynamic viscosity and viscoelasticity of WPI-FG coacervates. This was induced by unbalanced charges in WPI-FG mixture that led to a loosely packed coacervate structure. Following successful coacervate formation of

Fig. 6. Frequency dependent of storage modulus (G0 ) and loss modulus (G00 ) at a fixed strain amplitude of 0.1% for WPI-FG coacervates prepared at CT ¼ 1.0% (w/w) as a function of pH (A) and biopolymer mixing ratio R (B).

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