Effect of polysaccharide concentration and charge density on acid-induced soy protein isolate-polysaccharide gels using HCl

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Food Structure xxx (2016) xxx–xxx

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Effect of polysaccharide concentration and charge density on acid-induced soy protein isolate-polysaccharide gels using HCl Wee M.S.M.* , Yusoff R., Lin L., Xu Y.Y Food Innovation & Resource Centre, Singapore Polytechnic, 500 Dover Road, 139651, Singapore

A R T I C L E I N F O

Article history: Received 30 March 2016 Received in revised form 4 July 2016 Accepted 22 August 2016 Available online xxx Keywords: Soy protein isolate Carrageenan Alginate Mixed biopolymer gel Simulated gastric fluid Acid gelation

A B S T R A C T

Mixed biopolymer gels of soy protein isolate with k-carrageenan, l-carrageenan and alginate were produced via acidification with simulated gastric fluid (HCl) at pH 1.2. The gels could be formed instantaneously via dripping into the acid, or over 24 h via a dialysis membrane. The main factors studied were charge density of the polysaccharide and polysaccharide concentration (0.25
1.0% w/w). Their effects on the physical properties of the gel i.e. gel mass formed, protein concentration in supernatant, hardness, storage modulus (G0 ), water holding capacity (WHC) and microstructure were evaluated. Overall, with higher charge density of the polysaccharide (at pH 1.2) and/or increasing polysaccharide concentration, the gel formed more rapidly and had greater mechanical strength due to a denser network between the biopolymers. As a result, WHC was also increased with improved freeze-thaw stability as observed under confocal microscopy. ã 2016 Elsevier Ltd. All rights reserved.

1. Introduction Single biopolymer gels such as whey protein isolate (WPI) gels are formed by heating, whereby the proteins are denatured, unfolded and rearranged to form a gel network (Alting, 2003). With the introduction of another biopolymer i.e. a polysaccharide, interpenetrating, coupled and phase-separated networks could be formed (Turgeon & Beaulieu, 2001). These mixed biopolymer gels could enhance the mechanical and other functional properties of the gel such as taste profile and digestive breakdown (Bradbeer, Hancocks, Spyropoulos, & Norton, 2015; Turgeon & Beaulieu, 2001). Electrostatically-interacted protein-polysaccharide gels can be made by reducing the pH to below the isoelectric point of the protein such that a positively-charged protein interacts with a negatively-charged polysaccharide and traps water within the gel network. This mechanism has been explored via the concept of ‘intragastric gelation’ (Zhang & Vardhanabhuti, 2014; Zhang, Zhang, & Vardhanabhuti, 2014), since the acidic environment of gastric fluid (pH 2) would make the proteins positively charged when the solution is ingested and thereby forming gels. In this study, soy protein isolate (SPI)-polysaccharide (PS) (k-carrageenan (KC), l-carrageenan (LC) and alginate (Alg)) gels were studied. Soy protein was selected as the protein source due to

* Corresponding author. E-mail addresses: [email protected], [email protected] (M.S.M. Wee).

several reasons. Soy (plant) protein is a viable alternative to meat or dairy proteins, it is representative in the Asian context, and it is also relatively unexplored as compared to whey protein, especially in mixed biopolymer gels. Soy protein isolate is generally compatible with polysaccharides and is able to form mixed biopolymer gels with polysaccharides under certain conditions. For example, cold-set SPI-gellan gums were formed using potassium and calcium salts (Pires Vilela, Fazani Cavallieri, & da Cunha, 2011), heat-induced SPI-KC gels at high (>8% w/w) protein concentrations (Fazani Cavallieri, Garcez, Takeuchi, & da Cunha, 2010), and acid-induced gels of SPI with xanthan or guar gum using GDL (Chang, Li, Wang, Bi, & Adhikari, 2014). k-carrageenan, LC and Alg are all polyelectrolytes but differ in physical properties such as charge density, molecular weight and conformation. On its own, KC is a polysaccharide which gels in the presence of potassium (K+) ions after heating and re-cooling. Alginate gels in the presence of calcium (Ca2+) ions. l-carrageenan, however, is non-gelling. The resultant gel structure and properties are usually governed by competition between phase separation and gelation (de Jong, Klok, & van de Velde, 2009). At the high concentrations used in this study i.e. 5% w/w SPI and 0.25-1.0% w/w polysaccharide, phase separation should occur as a result of thermodynamic incompatibility. However, due to the high viscosity of polysaccharides, they also provide a stabilising effect which delays phase separation by slowing down molecular mobility (Huang, Kakuda, & Cui, 2001). Many mixed biopolymer gels found in the literature are often in the low concentration range e.g. 1–5% w/w for the protein and 0.01–

http://dx.doi.org/10.1016/j.foostr.2016.08.001 2213-3291/ã 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: M.S.M. Wee, et al., Effect of polysaccharide concentration and charge density on acid-induced soy protein isolate-polysaccharide gels using HCl, food structure (2016), http://dx.doi.org/10.1016/j.foostr.2016.08.001

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0.3% w/w for the polysaccharide, where phase separation occurs more readily especially with neutral polysaccharides (de Jong & van de Velde, 2007). Furthermore, acid-induced gels in other studies are usually formed using glucono-delta-lactone (GDL), whereby a longer time period is required for the pH to decrease and allowing phase separation to take place at the same time (de Jong et al., 2009). This current study differs in terms of acidification rate
the microstructure is arrested immediately upon contact with the acid, leaving minimal time for phase separation. The pH at which gelation occurs is also considerably lower in this study i.e. 1.2 as compared to 3.5 to 4.8 for GDL-induced gels. The objectives of this study were to characterise acidified soy protein-polysaccharide gels formed using simulated gastric fluid (SGF), using three different polysaccharides of different charge densities (KC, LC and Alg) at concentrations from 0.25 to 1.0% w/w. The macro- and microstructures and physical properties of the gels were evaluated. The use of SGF allowed the observation of the acid gel formed under simple simulated gastric conditions. However, gastric enzymes i.e. pepsin were not included in the SGF in order to isolate the effects of acid-induced gelation without protein breakdown in the process. Understanding the basic structure of the gel would help to provide further insight to its breakdown during digestion. In-vitro and in-vivo digestion studies of these gels will be reported in a separate study. 2. Materials and methods 2.1. Materials The protein used in this study was native soy protein isolate (SPI) powder (PRO-FAM 974 ADM, Illinois, USA) with a protein, fat, ash and moisture content of approximately 90, 4, 5 and 6% w/w respectively according to the material specifications. Polysaccharides used in this research were k-carrageenan (Grindsted Carrageenan CL 220, Danisco), l-carrageenan (Satiagum ADC 25, Cargill) and alginate (Grindsted Alginate FD 155, Danisco). Simulated gastric fluid (SGF) was prepared according to the US Pharmacopeia 33-28NF by diluting 7 ml of concentrated hydrochloric acid in 1000 ml of milli-Q water to approximately pH 1.2, with an ionic strength of 0.034 M (2 g/l) NaCl (sodium chloride). 2.2. Zeta-potential Zeta-potential of the individual protein and polysaccharide solutions (0.1% w/w) was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK) based on electrophoresis and laser Doppler velocimetry techniques. The samples were measured using universal folded capillary cells (DTS1060C; Malvern Instruments Ltd, Worcestershire, UK) at 25  0.02  C in triplicates. 2.3. Acidification by SGF 2.3.1. Dripping (Extrusion) Method Protein (10% w/w) and polysaccharide (2% w/w) stock solutions were prepared by hydrating the protein or polysaccharide in milliQ overnight under continuous stirring at room temperature (20  C). The protein-polysaccharide mixture was made by adding appropriate amounts of protein and polysaccharide stock solutions to achieve the required final concentrations. Mixtures for constructing the phase diagram had varied protein concentrations from 0 to 7.5% w/w and polysaccharide concentrations from 0 to 1% w/w. The protein concentration for all other experiments were fixed at 5% w/w. The mixture was heated at 85  C in a waterbath for 30 min with continuous stirring, and cooled to room temperature before acidification. To simulate simple intragastric gelation of the

PR-PS mixture, the mixture (9 g) was extruded into SGF (15 g) under continuous stirring with a 10 ml syringe and syringe pump. This extrusion process allowed the PR-PS gel beads to form on contact with SGF, and therefore produce a more uniform size and surface area of the beads. 2.3.2. Membrane Dialysis In order to measure physical properties such as hardness and storage modulus, the gels should have a consistent dimension suitable for measurements. Gels for textural measurements were therefore prepared by filling the protein-polysaccharide mixtures in dialysis tubes (MWCO 12,000 25 mm, SpectraPor, USA), and dialysed against SGF for 24 h. The resultant cylindrical gel was cut into circular discs measuring approximately 10 mm in diameter and 10 mm in height for measurement. 2.4. Protein concentration in supernatant & gel weight Based on a fixed amount of PR-PS mixture (9 g total; 5% w/w SPI concentration), gel beads were formed in a fixed amount of SGF (15 g) with the dripping method and the protein content in the supernatant was quantified after centrifuging. The protein content in the supernatant would indicate the amount of protein which was involved in formation of the gel matrix. After gel formation, the mixture was centrifuged at 4000g (Sorvall Centrifuge, Thermo Scientific, Waltham, MA) for 10 min at 25  C to separate the gel beads from the SGF (supernatant) and filtered through a 0.2 mm syringe filter (Sartorius Minisart). The protein concentration of the supernatant was measured in UV cuvettes (FischerScientific, USA) at an absorbance wavelength of 280 nm (Shimadzu Spectrophotometer UV-1800, Japan) after a 20-fold dilution. Protein concentration standards were measured using bovine serum albumin (Sigma Aldrich, USA) from a concentration of 0–1600 mg/ml. Wet gel weight was obtained by weighing gels after decanting the supernatant. Dry gel weight was obtained by drying the wet gels in an oven (Memmert,USA) at 70  C for 24 h and then weighing it. 2.5. Texture analysis Textural attributes i.e. hardness was measured using a texture analyser (TA-XT2I/25 Texture Analyzer, Stable Micro System) with a cylindrical aluminium probe (f = 36 mm; 36R). The cylindrical gels (f  10 mm  10 mm) prepared using the dialysis method were compressed at a strain of 20% and test speed of 1 mm/s. The peak force at compression was recorded as the hardness of the sample. Five cylindrical gels were measured from each sample, and each sample was prepared and measured twice. 2.6. Rheological measurements Viscosity (rotational) measurements of the protein-polysaccharide solutions (all at 5% w/w SPI and 0.25–1.0% w/w polysaccharide) were made using a Paar Physica rheometer MCR 301 (Anton-Paar, Graz, Austria) in controlled shear rate (CSR) mode at 25  0.1  C (unless otherwise stated) with a cup and bob geometry. The viscosity was determined in the shear rate range of 0.01–1000 s
1 and measurement points were recorded using the no time setting option (equilibrium mode). The cylindrical gels prepared using the dialysis membrane method (section 2.3) were used to measure for storage modulus. For oscillatory rheological measurements of the gels, amplitude sweeps were carried out between 0.01 and 1000% strain at a constant frequency of 1 Hz at 25  0.1  C (unless otherwise stated) with a serrated parallel plate geometry (PP25/S, Anton-Paar, Graz, Austria) at a gap height of 10 mm. Storage modulus (G0 ) was obtained at 10% strain within the linear viscoelastic region for all samples. Measurements were

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carried out in duplicates. The gels measured were all fixed at 5% w/ w SPI concentration with polysaccharide concentrations from 0.25 to 1.0%. 2.7. Water holding capacity (WHC) The cylindrical gels prepared using the dialysis membrane method (section 2.3) were used to measure water holding capacity (WHC). The gels were placed on a filter paper (Whatman #1, 55 mm; Maidstone, UK) and inserted into a centrifuge tube. The gels were then centrifuged at 180g (Sorvall Centrifuge, Thermo Scientific, Waltham, MA) for 10 min at 25  C, and then removed from the filter paper and centrifuge tube. The water loss from the gel was calculated based on the difference in weight of the centrifuge tube with the filter paper before and after centrifuging. Water holding capacity was calculated based on the water retained per weight of the gel:  loss f rom gel Water holding capacity ¼ 1
Water  100% Gel weight 2.8. Confocal scanning laser microscopy Soy protein was labelled with rhodamine B (Sigma Aldrich, USA) by adding a few drops of 0.1% w/v rhodamine B to the gel surface. Gel samples were observed using a confocal scanning laser microscope (CSLM; FluoView 1000, Olympus, USA) at an excitation wavelength of 543 nm. The gels were placed on a microscope slide with a raised well to prevent structural damage to the gel when fixing the coverslip. The absorption and emission wavelength maxima of rhodamine B are 540 and 625 nm respectively. At least six images were taken for each gel sample at various magnifications (x10, x20, x40 and x60) to confirm that a representative image was taken. The experiment was also repeated twice and similar images were obtained to check for consistency. 3. Results and discussion 3.1. Phase diagrams A simple phase diagram was constructed for SPI-KC, SPI-LC and SPI-Alg mixtures to determine the concentration boundaries where acidic gelation occurs at room temperature (25  C) i.e. gel point, with a pre-heating step at 85  C for 30 min. Pre-heating partially unfolds the protein which exposes more functional groups for association and therefore gelation (Perrechil, Braga, & Cunha, 2013). The minimum SPI concentration for heat-induced gelation is 6% w/w (Li, 2005). However, this value would be considerably altered in the presence of another biopolymer i.e. polysaccharide (Fig. 1). Three outcomes (by visual observation) were possible with the SPI-PS mixtures after subjecting to SGF via the dripping method. The solutions either did not form gels upon contact with SGF (), formed gels upon contact with SGF (*), or gelled upon heating and cooling (). For non-gelling mixtures, there would be no distinct separation between the supernatant and mixture, hence turning the entire system turbid on gentle perturbation. In general, at low SPI (2.5% w/w) and PS (< 0.25% w/w) concentrations, gels could not be formed when added to SGF. As protein concentration increased (> 5% w/w), a lower PS concentration (< 0.25% w/w) was required to gelation. When protein and polysaccharide concentrations were both sufficiently high (e.g. 0.75 and 1.0% w/w respectively), gelation occurred after preheating and cooling. For SPI-LC and SPI-Alg mixtures, weak gels were formed after heating and cooling at SPI and LC or Alg concentrations above 5 and 0.5% w/w respectively. However, they were easily broken by shearing (stirring at 200 rpm for approximately 5 min) and

Fig. 1. Phase diagrams of SPI with a) KC, b) LC and c) Alg; the mixtures were heated at 85  C for 30 min and then subjected to acidification with SGF via the dripping method; outcomes were determined by visual observation (non-gelling: ; gelling: *; heat-gelling: ) and the dotted lines serve as visual aid for the phase boundaries.

reversed back to solution form. Although LC and Alg (in the absence of Ca2+ ions) are non-gelling polysaccharides, they have a higher viscosity (Fig. 2) as compared to SPI-KC mixtures. This may have contributed to reduced mobility within the system, therefore facilitating gelation after heating and cooling (Ako, Durand, & Nicolai, 2011) more easily as compared to SPI-KC mixtures. 3.2. Macroscopic observations Based on visual observations (Fig. 3), the gel beads were generally off-white in colour and slightly translucent at low (0.25% w/w) polysaccharide concentrations. For SPI-KC gels, the gel beads were soft, fragile and not easily separated from SGF at the lowest KC concentration (0.25% w/w) with a swollen appearance. As concentration of KC increased to 1.0% w/w, the gel beads appeared more distinctive and discrete. The SPI-LC gels were visually similar to SPI-KC gels on increasing polysaccharide concentration. On the

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even at 1% w/w Alg. Nevertheless, Alg was able to form gels with SPI in the absence of calcium ions which further supports the presence of electrostatic interactions involved in gelation. 3.3. Zeta-potential

Fig. 2. Viscosity (at 10s
1) of SPI-PS mixtures with varying polysaccharide concentrations at 25  C (all SPI concentrations at 5% w/w).

other hand, the physical appearance of SPI-Alg gels did not differ appreciably with increasing Alg concentration. As Alg concentration increased, the gels were less swollen but were soft and fragile

The main factor attributing to the difference in physical properties between gels was likely to be the polysaccharide charge density as demonstrated in other literature (de Jong & van de Velde, 2007; Zhang, Zhang et al., 2014). Zeta-potential (ZP) of SPI and the individual polysaccharides were measured at different pH values. Interaction between SPI and the negatively charged polysaccharide is greatest when pH is below isoelectric point of the protein. Below the isoelectric point of SPI (pI  4.6; ZP = 0), the protein becomes positively charged (ZP > 0) and therefore electrostatic attraction between biopolymers can take place. No electrostatic interaction takes place between protein and neutral polysaccharides (e.g. guar gum) therefore the system does not form gels via opposite charges (Zhang, Zhang et al., 2014). The degree of electrostatic interaction between the protein and polysaccharide can be estimated from their ZP. Fig. 4a shows ZP of SPI, KC, LC and Alg individually at different pH values. Overall, LC is most negatively charged at pH < 4.6 followed by KC and then Alg. Both KC and LC carry charged sulphate (OSO3
) groups, whereby KC carries one sulphate group per repeating

Fig. 3. Visual appearance of SPI-KC (row 1), SPI-LC (row 2) and SPI-Alg (row 3) gel beads at increasing polysaccharide concentrations from 0.25 to 1.0% w/w (left to right) (all SPI concentrations at 5% w/w).

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Fig. 4. a) Zeta-potential of 0.1% w/w SPI, KC, LC and Alg solutions at various pH; shaded grey area represents the pH range at which the protein is positively charged and therefore electrostatic attraction can take place b) Absolute zeta-potential (obtained by multiplying zeta-potential of protein and the polysaccharide) at various pH.

disaccharide unit, and LC carries three sulphate groups per repeating diasaccharide unit. The charge densities for KC and LC are therefore 0.5 and 1.5 mol/mol (mol negative charge per mol monosaccharide) respectively (de Jong & van de Velde, 2007). Although the charge density of Alg is also relatively high at 1 mol/ mol, the pKa of Alg at 3.5 (Harnsilawat, Pongsawatmanit, & McClements, 2006) results in partial or complete protonation at pH < 3.5. As a result, electrostatic charges between SPI and Alg were not as strong as KC and LC at pH of the SGF. Fig. 4b shows the absolute ZP between SPI and the respective polysaccharide. The greatest interaction would be between SPI and LC at pH 2.5 since the absolute ZP is highest at this pH. The trend is similar for KC, although the absolute values were lower than LC at all pH values below 3.5. Alginate has the smallest degree of interaction with SPI at pH < 3.5.

supernatant as polysaccharide concentration increased. Overall, SPI-Alg gels had the highest protein concentration in the supernatant while SPI-LC gels had the lowest. Since SPI-Alg gels were the weakest and had the least charged interactions with the polysaccharide therefore more protein remained in the supernatant. Conversely, SPI-LC had the strongest interaction and therefore the lowest supernatant protein concentration. There were no obvious differences between SPI-KC and SPI-LC gels in terms of supernatant protein concentration, although it was slightly lower for SPI-LC gels. As seen from Fig. 5, there was a slight increase in supernatant protein concentration at 1.0% w/w LC. By visual observation, the supernatant turned slightly turbid after forming the gels. This was likely due to saturation of charges between the protein and polysaccharide, which resulted in excess protein and/ or polysaccharide in the supernatant and hence the turbid appearance.

3.4. Protein concentration in supernatant 3.5. Gel weight Fig. 5 shows the protein concentration remaining in the supernatant with increasing polysaccharide concentration for SPI-KC, SPI-LC and SPI-Alg gels. All three polysaccharides showed the same trend of decreasing protein concentration in the

Fig. 5. Protein concentration in supernatant (mg/ml) after formation of SPI-KC, SPILC and SPI-Alg gels (all SPI concentrations at 5% w/w).

Apart from protein concentration in supernatant, the gel weight was also used as an indicator for the interaction between protein and polysaccharide. Fig. 6a shows the wet gel weight of the beads with increasing polysaccharide concentration. The wet gel weight overall decreased with increasing polysaccharide concentration as a result of less water being adsorbed by the gel beads (Fig. 3). The dry gel weight (Fig. 6b) however, increased with polysaccharide concentration. This was in agreement with the supernatant protein concentration (Fig. 5), as more protein (and also polysaccharide) would be entrapped in the gel matrix, resulting in a higher gel mass. For SPI-LC gels, there was a slight decrease in dry gel weight at 1.0% w/w LC, along with an increase in supernatant protein concentration (Fig. 5). Further increasing the polysaccharide concentration to 1.0% w/w could lead to repulsion of negative charges amongst polysaccharides in the solution and thus a looser network between protein and polysaccharide when forming the gel. For LC which has a higher charge density, it is possible that saturation of the positive charges on the protein was reached at a lower polysaccharide concentration. As mentioned, the supernatant turned turbid at this LC concentration. The higher dry gel weight observed at 0.25% w/w is not well understood at this moment. It could be due to the lower mixture viscosity (Fig. 2) which allowed better interaction between protein and polysaccharide as the biopolymer molecules have greater mobility. Although with increasing polysaccharide concentration there would be more interaction with the protein, this may come with other opposing effects to gel formation, such as increasing

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Fig. 6. a) Wet weight and b) dry weight of SPI-KC, SPI-LC and SPI-Alg gels with increasing polysaccharide concentration (all SPI concentrations at 5% w/w).

electrostatic repulsion between polysaccharides or higher viscosity of the continuous phase. 3.6. Hardness and storage modulus (G0 ) In order to measure physical properties such as hardness and storage modulus, the gels were prepared via dialysis membrane to obtain a cylindrical gel shape. The resultant appearance of the gels are as shown on Fig. 7. Only SPI-KC and SPI-LC gels were analysed as the SPI-Alg gels were non-self-supporting and could not be taken out of the dialysis tubing intact. It was expected that the gels produced using the dialysis method may have certain microstructural differences from those by gel bead extrusion (Fig. 3), since gelation did not occur instantaneously and other effects such as phase separation may have been predominant. This will be examined using confocal microscopy in a later section. For the visual appearance of SPI-KC gels, increasing polysaccharide concentration led to a firmer gel although the crosssectional surface became coarser and uneven. Less syneresis was also observed. The same was seen for SPI-LC gels, although a hollow centre was found at 0.75 and 1.0% LC concentrations. This hollow centre was likely a result of rapid gelation on the exterior,

thus preventing the diffusion of acid into the interior of the gel (within 24 h). It could also be due to the higher viscosity (Fig. 2) of SPI-LC gels which slowed down the rate of acid diffusion into the gel centre. The mechanical strength of the gels were tested at both large (nonlinear) and small (linear) deformations measuring hardness and storage modulus respectively. The hardness of the SPI-KC gels increased with polysaccharide concentration. However, the hardness of SPI-LC gels did not increase with polysaccharide concentration but rather decreased dramatically above 0.75% w/w LC. For both SPI-KC and SPI-LC gels, G0 increased exponentially with increasing polysaccharide concentration, and SPI-LC gels had an overall higher G0 than SPI-KC gels except at 1% w/w polysaccharide. An increasing hardness or G0 with polysaccharide concentration indicated a denser gel network with more interactions (Yamamoto & Cunha, 2007) since there would be more negatively charged groups on the polysaccharide available to bind to the positively charged groups on the protein. This was also observed in many protein-polysaccharide gel systems, including heat-induced SPIKC gels which had increasing rupture stress with increasing polysaccharide concentration (Fazani Cavallieri et al., 2010), and acid-induced WPI-xanthan gum and WPI-carrageenan gels which

Fig. 7. Visual appearance of SPI-KC (top row) and SPI-LC (bottom row) gels at 0.25, 0.5, 0.75 and 1.0% w/w polysaccharide concentrations formed via dialysis against SGF for 24 h (f  10 mm x 10 mm) (all SPI concentrations at 5% w/w).

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had higher G0 as polysaccharide to protein ratio increases (Zhang, Zhang et al., 2014). The type of interactions formed during complexation e.g. electrostatic, covalent (sulphide) or hydrophobic also determines the strength of the gel (Alting, Hamer, de Kruif, & Visschers, 2003). At 0.75–1.0% w/w KC however, there was an unexpected increase in gel hardness as well as G0 . The gel strength was expected to plateau with increasing polysaccharide concentration since the electrostatic interaction between charges would saturate and no further interactions could be formed. Therefore it is possible that there is a synergistic effect between KC and SPI and not between LC and SPI. It has been found in other studies that KC may exhibit synergy with proteins such as soy and pea protein (Ipsen, 1995), and dairy proteins (Ould Eleya & Turgeon, 2000). Furthermore, KC is a gelling polysaccharide while LC is non-gelling. Since KC gels upon heating and cooling, the preheating step prior to acidification may have resulted in partial gelation of a KC network, which contributed to additional elasticity of the gel at sufficiently high KC concentrations (Ako et al., 2011). The discrepancy between small and large deformation behaviour for SPI-LC gels may be due to the difference in length scales measured under small and large deformation (Vliet, 1995), where stress is applied in the form of compression (uniaxial to strain) for hardness vs. shear (parallel to strain) for storage modulus. Gel hardness is determined by both the number of effective strands in the gel and the modulus of the protein strands (van Vliet, 1999), while the elastic modulus (G0 ) is governed by the number of elastic effective junctions between strands (Alting et al., 2004). Under compression (large deformation), it is likely that the hollow centres of the SPI-LC gels at 0.75 and 1.0% w/w contributed significantly to their structural weakness as observed in their visual appearances (Fig. 7). For G0 , since the deformation is within the linear region at mm length scales, the gel maintains its structure and is not affected at length scales of the hollow centre for the SPI-LC gels (i.e. >1 mm). Charge density, and therefore consequently the gelation rate were likely to have played an important factor in determining the mechanical strength of the gels. l-carrageenan, which has a higher charge density than KC, has an overall higher G0 for SPI-LC gels than SPI-KC gels due to more interactions between the protein and polysaccharide. At the same time, however, gelation then occurred more rapidly for SPI-LC as compared to SPI-KC gels as manifested by the hollow centre in the cylindrical gels (Fig. 7). This in turn led to a weaker gel strength under compression. Although Alg has a high charge density at neutral pH, the total absolute charges between SPI and Alg were low at acidic pH (Fig. 4b), due to the high pKa of Alg (pKa  3.5) (Draget, Moe, Skjak-Braek, & Smidsrod,

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2006). As a result, the SPI-Alg gels formed were weak and non-selfsupporting. On the other hand, the OSO3
groups in KC and LC have a lower pKa (<2) than alginate (Gu, Decker, & McClements, 2004). Furthermore, the OSO3
groups have a strong attraction to the local protein amino groups (NH3+) which contributed to the compatibility of the biopolymers and therefore the strength of the gels (Grinberg & Tolstoguzov, 1997). This is somewhat in agreement with Zhang, Hsieh et al. (2014); Zhang, Zhang et al. (2014); de Jong and van de Velde’s (2007) work on the effects of charge density on gel strength, whereby the polysaccharide with a higher charge density formed stronger gels. In de Jong et. al.’s (2007) work, it was found that for polysaccharides with a high charge density (>0.7 mol/mol) i.e. LC, increasing polysaccharide concentration in the mixed gel also increased the gel fracture stress. On the other hand, polysaccharides with intermediate charge density (0.3–0.7 mol/mol) i.e. KC, have decreasing fracture stress with increasing polysaccharide concentration. Their results do not reflect the findings in this paper, possibly due to the different acidification mechanism, as well as the concentration range investigated. In their paper, the authors used GDL to form the gels which would have led to phase separation in the duration in which the pH was decreased. Additionally, the polysaccharide concentrations used was up to 0.3% w/w, where viscosity effects were not as pronounced. The high viscosity (Fig. 2) would have helped to slow down phase separation and formed a different microstructure. Therefore this method of rapid acidification using HCl may be a viable and effective method for making gels at high polysaccharide concentrations. 3.7. Water holding capacity (WHC) The water holding capacity (WHC) of SPI-KC and SPI-LC gels (prepared via dialysis method) are as shown on Fig. 9. SPI-Alg gels were not determined for WHC as the gels were not self-supporting and prone to disintegration. The WHC increased with polysaccharide concentration for both KC and LC, although the SPI-KC gels still had an overall lower WHC than SPI-LC gels. The gels at 0.25% w/w polysaccharide were also observed to exude more serum under its own weight when left to stand (Fig. 7). If one were to take the weight difference between the wet (Fig. 6a) and dry (Fig. 6b) gels, there would be a decreasing amount of water loss to the contrary of an increasing WHC with increasing polysaccharide concentration. Therefore the WHC would refer to the water tightly bound to the protein-polysaccharide gel which is not removed by centrifugation whereas parameters such as swelling ratio or water-binding capacity would take into account

Fig. 8. a) Hardness at g = 20% based on compression test (large deformation) and b) G0 at g = 1% based on oscillatory rheological test of SPI-KC and SPI-LC gels (f  10 mm x 10 mm) (all SPI concentrations at 5% w/w).

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bound liquid within the gel network that cannot be removed under the current centrifugation conditions. The hydrophilic nature of polysaccharides would also contribute to increased water-binding. However, an increase in polysaccharide concentration does not always necessarily translate to a higher WHC in mixed proteinpolysaccharide gels. The gelation mechanism governs the microstructure of the gel which in turn affects the WHC. In whey proteinpectin gels, increasing pectin concentration led to a decrease in WHC due to thermodynamic incompatibility resulting in a coarse micro-phase separated network. Therefore the WHC of these gels will be further discussed in the next section in relation to its microstructure. 3.8. Microstructure

Fig. 9. Water holding capacity (%) of SPI-KC and SPI-LC gels (all SPI concentrations at 5% w/w).

the water adsorbed by the gel. It has been reported that with smaller crosslink densities there would be increased swelling due to solubilisation of the whey protein (Peters, Luyten, Alting, Boom, & van der Goot, 2015). At lower polysaccharide concentrations, there would also be fewer protein-polysaccharide interactions and therefore more sites on the protein and/or polysaccharide are available to bind free water (Zhang, Hsieh, & Vardhanabhuti, 2014), giving the swollen gel appearance with a high wet gel weight. The hardness and G0 of the gels correlated with its WHC i.e. increasing hardness or G0 and WHC with polysaccharide concentration. Furthermore, LC with a higher charge density (and firmer gel) (Fig. 8) formed gels with higher WHC as compared to the SPIKC gels. Higher polysaccharide concentrations and charge density resulted in a denser (firmer) gel network which is able to trap more

The microstructure of SPI-KC, SPI-LC and SPI-Alg gel beads are as shown on Fig. 10. The red (bright) areas represent the gel (protein-polysaccharide network) stained by Rhodamine B while the dark areas represent the pores. The SPI-KC and SPI-LC gels were fairly similar in their microstructure with increasing polysaccharide concentrations. There were distinct pores in the gel network at polysaccharide concentrations from 0.25 to about 0.625% w/w, which initially increased in size (from 5 to 20 mm) but decreased in pore numbers within this concentration range. These pores were no longer visible at 0.75 or 1.0% w/w within the red areas. Instead, there were large regions of dark areas which were likely to be due to the coarseness and uneven surface of the gel viewed on a single focal plane. It may appear at first glance that phase inversion has occurred at 1.0% w/w KC and LC. However, based on visual observations, the gel beads took shape more rapidly at 1.0% w/w (as compared to 0.25% w/w) KC and LC, which contradictorily reduces the time for phase separation before gelation. This rapid gelation combined with the high solution viscosity may have prevented slow rearrangement of the gel structure after gelation, hence the coarse and uneven texture appearing as dark areas. The SPI-Alg gels have a distinctively different microstructure from SPI-KC and SPI-LC gels. The pores were much larger (20 mm compared to 5 mm for SPI-KC and SPI-LC gels) especially at 0.375-

Fig. 10. Microstructure of SPI-KC (top row), SPI-LC (middle row) and SPI-Alg (bottom row) gel beads; scale bar represents 100 mm (image size: 210  210 mm).

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Fig. 11. Microstructure of 5% SPI-0.375% Alg gel; scale bar represents 100 mm (image size: 317  317 mm).

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0.5% w/w concentration, where the pores were enclosed by a weblike thin-stranded network distributed among gel clusters. The overall microstructure was better viewed at x10 magnification (Fig. 11). Numerous pores (10 mm) formed a web-like structure around clusters of the gel. It is unclear whether the clusters were solely protein-rich clusters or the strands were made of interacted protein and polysaccharide complexes. The web-like strands running through the gel network may be the most important factor contributing to its weak mechanical strength and hence not self-supporting gel when made via the dialysis method. At 0.75 and 1.0% w/w Alg, the network became particulate-like (bright red dots, possibly concentrated dye spots) which could be due to protein aggregation as a result of phase separation in excess polysaccharide concentration (van den Berg, Rosenberg, van Boekel, Rosenberg, & van de Velde, 2009). The microstructures of SPI-KC and SPI-LC were not distinctively different, and their other properties also had similar trends and values. Increasing polysaccharide concentration reduced the porosity of the gel, which correlated with an improvement in WHC, hardness and G0 . Coarsening of the gel texture and reduced pore sizes suggests an increase in localised protein concentration of the gel complex, which could improve the mechanical strength of the gel (de Jong & van de Velde, 2007) since the hardness of the gel is related to the number of effective protein strands within the matrix (van Vliet, 1999). The polysaccharide may be bound and buried within large clusters of protein aggregates upon interaction, which results in larger pores when interaction is stronger and

Fig. 12. Microstructure of SPI-KC (top row), SPI-LC (middle row) and SPI-Alg (bottom row) gels (image size: 210  210 mm).

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smaller pores evenly distributed in gel network when interaction is not as strong (Zhang, Zhang et al., 2014). However, it should be noted pore sizes do not necessarily always correlate to mechanical strength
gels with similar cluster and pore sizes may have contrasting rheological behaviour depending on network connectivity, therefore pore sizes may not be a good indicator on gel strength (Eugenia Hidalgo et al., 2015; Olsson, Langton, & Hermansson, 2002). Microstructures of the gel beads were also taken before freezing and after thawing (Fig. 12). On freeze-thaw, the microstructure of the gels at 0.25% w/w polysaccharide appeared to have undergone extensive rearrangement with the appearance of large dark regions. Pores were also no longer visible within the red (bright) areas and became smoother as well. These dark areas were likely to be porous regions created by the freezing and melting of ice crystals, which is commonly observed in freeze-thawed matrices (Charoenrein, Tatirat, Rengsutthi, & Thongngam, 2011). Since ice

has a larger volume than water, this was likely to result in an excluded volume effect on the gel, thus concentrating the gel regions and therefore tightening the pores. At low polysaccharide concentrations, the gels had a weaker network i.e. poorer hardness, G0 and WHC, which exacerbated the freeze-thaw effect as the gel structure could not maintain its integrity on formation of ice crystals. In contrast, the microstructure of the gels at 1.0% w/w polysaccharide appear unchanged by freeze-thaw. Therefore the polysaccharide concentration plays a critical role in the gel network structure and integrity, whereby a denser network would result in better mechanical strength and water-binding. The microstructures of the gels made by two different methods i.e. dripping (fast acidification) and membrane dialysis (slow acidification) were also compared (Fig. 13). The difference between slow and fast acidification were most prominent at low polysaccharide concentrations i.e. 0.25% w/w. For SPI-KC gels at 0.25% w/w KC, the microstructure with slow acidification had more dark regions with less connectivity seen in the microstructure without the pores which could be due to phase separation during the process of gelation. Similar observations were made for the SPI-LC gels at 0.25% LC, whereby the microstructure was coarser and more phase separated with slow acidification. At 1% w/w polysaccharide concentration, however, the microstructure of the gel did not differ much in terms of the non-protein rich areas for both SPI-KC and SPI-LC gels. It should also be noted that the cylindrical gel samples made from slow acidification were taken from within the crosssectional area whereas the gel beads made from fast acidification were viewed from its surface. The difference in sampling methods may have contributed to differences in microstructure. 4. Conclusion Polysaccharide concentration and charge density were the main factors determining the mechanical strength, physical properties and microstructure of the gels. Polysaccharide concentration correlated well with the extent of interaction and therefore mechanical strength of the gels. With increasing polysaccharide concentrations, there were less protein in the supernatant and greater gel mass formed. The gels were also harder and more elastic, which improved its WHC and freeze-thaw stability. The presence of numerous pores distributed among the gel network may be the contributor of poor gel strength. Nevertheless, factors such as rate of acidification and viscosity of the proteinpolysaccharide mixture should also be taken into account since they affect the overall thermodynamic stability of the system and hence final microstructure. The polysaccharide with the highest negative charge density at pH 1.2 i.e. LC also formed the strongest gels under small deformation, followed by KC, and Alg which has a low charge density at pH 1.2 did not form self-supporting gels using the dialysis method. Overall, this method of forming acidinduced gels using HCl would be suitable for systems with high polysaccharide concentrations (therefore viscosity). It can also be used for simple simulation of intragastric gelation. Future work include in-vivo and in-vitro digestion studies of these gels, as well as using a different plant protein i.e. pea protein to form these acid gels. Industrial relevance

Fig. 13. Microstructures of SPI-KC (rows 1 & 2) and SPI-LC (rows 3 & 4) gels at 0.25 and 1.0% w/w polysaccharide concentrations made via fast (left) and slow (right) acidification.

The formation of soy protein isolate-polysaccharide gels in the presence of simulated gastric fluid shows a possibility of conferring satiety in consumers. It is also an alternative method of forming acid-induced gels as opposed to using glucono-delta-lactone (GDL). Soy-based products are very popular in Asia and therefore this research would be even more relevant in the Asian context.

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