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Enhanced CaSO4-induced gelation properties of soy protein isolate emulsion by pre-aggregation
Food Chemistry 242 (2018) 459–465
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Enhanced CaSO4-induced gelation properties of soy protein isolate emulsion by pre-aggregation ⁎
Xufeng Wanga, Maomao Zenga, Fang Qina, Benu Adhikarib, Zhiyong Hea, , Jie Chena,c, a b c
MARK
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State Key Laboratory of Food Science and Technology, and School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China School of Applied Sciences, RMIT University, City Campus, Melbourne, VIC 3001, Australia Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soy protein isolate Emulsion gel Pre-aggregation Rheology Microstructure
The effects of CaSO4-induced pre-aggregation on the rheological and structural properties of soy protein isolate (SPI) emulsion gels were investigated. As the Ca2+ concentration during pre-aggregation increased (from 0 mM to 7.5 mM), the elastic modulus of the gels showed substantial increase, indicating stiffer gel structures. Largedeformation rheology suggested stronger but more brittle networks formed at higher Ca2+ concentration during pre-aggregation. However, when the pre-aggregated Ca2+ concentration reached 10 mM, the corresponding gel became weaker. Water-holding capacity (WHC) of the gels were significantly improved via the pre-aggregation process. The differences in rheological properties and WHC among the gels were consistent with the variation in their microstructures. Pre-aggregation helped to form denser and more uniform structures with thicker strands, whereas over aggregation made the gel network coarser.
1. Introduction Soy cheese, also called “tofu” in Asian countries, has attracted increasing attention in recent years because of its bland taste and nutritional advantages with low levels of saturated fat and cholesterol (Ono, 2003). Traditional soy cheese is made from soy milk in a process that usually involves soaking, grinding beans in water, filtering, heating, coagulating, breaking the curd, and finally pressing and reforming the gel (Hou, Chang, & Shih, 1997). The process is complex and time consuming (Kamizake, Silva, & Prudencio, 2016). Soy protein isolate (SPI) is widely used in the food industry. During SPI extraction, most components such as lipids, soybean oligosaccharides, and isoflavones, which have been assumed to be the source of soy off-flavors, allergens, and flatulence factors, respectively, to some people, are washed out (Visser & Thomas, 1987). Hence, using SPI as a raw material for soy cheese has gained considerable attention because it makes the cheesemaking process simpler, cleaner, and more controllable compared with traditional methods (Murekatete, Hua, Chamba, Djakpo, & Zhang, 2014). Acid-induced and salt-induced are the two major methods involved in the formation of soy cheese. Previous studies have found that GDL and CaSO4 can produce more uniform soy curds with much smoother structure than those produced from other coagulates (e.g., MgCl2, MgSO4, and CaCl2) (Kao, Su, & Lee, 2003), but GDL was not suitable for
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Chinese-style tofu because of its sour flavor. In the formation of soy cheese, coagulation of soymilk is the most important step because it directly determines the quality of the product (Hou et al., 1997). It has been proposed that in a Ca2+-induced gelation process, the coagulation can be divided into two steps: (1) the soy protein forms particles (aggregates) at low calcium concentration and (2) the soluble proteins are bonded to the network by further Ca2+ addition (Guo & Ono, 2005; Ono, Katho, & Mothizuki, 1993). The aggregates formed in the first step could therefore define the final gel properties. In fact, numerous studies have been conducted to understand the relationships between the structural and textural properties of soy cheese and have suggested that the microstructure of soy cheese is affected by the protein aggregate properties (e.g., size and content) because protein aggregates are related to the thickness of gel strands and the density of the network (Doi, 1993; Lakemond et al., 2003; Lu, Lu, Yin, Cheng, & Li, 2010). Our previous study also demonstrated that larger and/or more protein aggregates helped to formed stronger gels with denser and more compact structures (Wang et al., 2017). However, most previous research has focused on investigating the factors that influence the heat-induced protein aggregation or gelation, such as preheating conditions (i.e., preheating methods, heating temperature, and holding time), pH, and ionic strength (Lu et al., 2010; Renkema, Gruppen, & Van Vliet, 2002; Zhao, Li, Qin, & Chen, 2015). Whereas for salt-induced gelation, very few studies concerning the effects of protein
Corresponding authors at: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China. E-mail addresses: [email protected] (Z. He), [email protected] (J. Chen).
http://dx.doi.org/10.1016/j.foodchem.2017.09.044 Received 20 May 2017; Received in revised form 24 August 2017; Accepted 10 September 2017 Available online 19 September 2017 0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
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of with 0.05 M phosphate buffer (pH 7.0). One milliliter of each diluted sample was put in an electrophoresis cell (DTS 1060, Malvern Instruments Ltd.) and the ζ-potential was measured using a Malvern Zetasizer Nano ZS90 (Nano ZS, Malvern Instruments, Worcestershire, UK). All measurements were replicated at least 3 times under room temperature.
aggregation induced by the coagulant itself on the gel properties are reported. The addition of Ca2+ screens electrostatic interactions between charged protein molecules and promotes the protein aggregation (Li et al., 2009). Additionally, the combination of Ca2+ and soy protein can be described as H+/Ca2+ exchanges (Canabady-Rochelle, Sanchez, Mellema, & Banon, 2009), which also neutralize electrostatic repulsion and form salt bridges, thus allowing protein molecules to form aggregates and eventually leading to the formation of three-dimensional gel network (Lu et al., 2010; Maltais, Remondetto, Gonzalez, & Subirade, 2005). In the present study, a two-step addition of the coagulate method is proposed, with a traditional one-step control, to estimate the impact of Ca2+-induced pre-aggregation of SPI emulsion on its gelation properties. In the first step, a small amount of Ca2+ was added into the SPI emulsion to make the emulsion partially aggregated (pre-aggregation). In the second step, the remaining Ca2+ was further added to form the final gel network (gelation). The effects of the Ca2+ concentration during pre-aggregation on the microstructure, rheological and physiochemical properties of the gels were investigated.
2.4.2. Oil droplet size The oil droplet size of the SPI emulsions was determined by a particle size analyzer (Microtrac S3500, Microtrac Inc., North Largo, FL, USA). Distilled water was used as the dispersant. The relative refractive index of the emulsion was taken as 1.095 (Tang, Chen, & Foegeding, n n 2011). The volume-average diameter d4,3 (∑i di4 / ∑i di3 , where ni is the number of particles with diameter di ) was recorded. 2.5. Dynamic oscillatory measurements 2.5.1. Small amplitude oscillation The viscoelastic properties of the SPI emulsion gels were characterized by a controlled-stress rheometer (HAAKE MARS III, Thermo Fisher Scientific, Karlsruhe, Germany) with a parallel plate (d = 35.002 mm, gap = 1 mm), using temperature sweep and frequency sweep mode. The emulsions were immediately loaded between the plates of the rheometer after the addition of CaSO4. Low-viscosity silicon oil was used to prevent water evaporation. The gels were oscillated at 1% strain (within the linear viscoelastic region, LVR) and a frequency of 1 Hz. The temperature was heated from 25 °C to 80 °C at 5 °C per minute, followed by incubation at this temperature for 30 min before cooling to 25 °C at 5 °C per minute. The storage modulus (G′) and loss modulus (G″) were recorded. After the gelling process was completed, frequency sweep tests were carried out at 25 °C using an angular frequency (ω) of 1–100 rad/s at a constant strain of 1%.
2. Materials and methods 2.1. Materials SPI was extracted from defatted soybean meals (Taiwan 292, harvested in 2015) according to the method described by Guo et al. (2015). The protein content of the SPI was 92.3% (dry basis) determined by the Kjeldahl method. Soy oil and nail oil was purchased from a local supermarket; Nile red, and Rhodamin B were obtained from Sigma-Aldrich (St. Louis, Mo, USA). All other chemicals in the study were of analytical grade. 2.2. Preparation of SPI emulsions
2.5.2. Large-scale deformation Large-scale deformation tests were performed at 25 °C using the same rheometer in an oscillatory amplitude sweep mode. The strain was increased from 0.1% up to the fracture point when the stress began to decrease at a frequency of 1 Hz. The shear stress was recorded as a function of strain.
A 60 mg/mL SPI dispersion (pH 7.0, adjusted with 0.1 N NaOH or HCl) was prepared by dispersing SPI powder into deionized water, stirring mechanically at room temperature for at least 2 h and centrifuging at 10,000g for 10 min to remove insoluble materials. The dispersion was subjected to heat treatment at 95 °C for 15 min and then cooled to room temperature in an ice bath. The pretreated SPI dispersion was mixed with 5% (v/v) soy oil and pre-homogenized using a disperser homogenizer (T 18 basic ULTRA-TURRAX®, IKA Corp., Staufen, Germany) at 13,500 rpm for 2 min, followed by homogenization through a homogenizer (AH-BASIC, ATS Engineering Inc., Canada) at 40 MPa for one pass.
2.6. Water-holding capacity (WHC) The WHC of the gels was determined according to the method of Wu, Xiong, Chen, Tang, and Zhou (2009), Approximately 5 g of gel (each sample) was transferred to 50 mL centrifuge tubes and centrifuged at 10,000g for 15 min at 4 °C. WHC (%) was defined as the ratio of the water weight in the pellet to the water weight in the original gel multiplied by 100.
2.3. Gel preparation 2.3.1. Pre-aggregation of SPI emulsion The pre-aggregation treatment was conducted before the gelation process. To do this, the SPI emulsion was mixed with a stock CaSO4 dispersion to Ca2+ concentrations of 0, 2.5, 5, 7.5 and 10 mM. The emulsion/CaSO4 mixtures were mechanically stirred at 300 rpm at room temperature and allowed to aggregate for 1 h.
2.7. Confocal laser scanning microscopy (CLSM) Samples for CLSM analysis were prepared in single concave slides (Sail Brand, Jinliu Instrument Co., Ltd., Nanjing, China) covered with nail oil to prevent water evaporation. Rhodamine B and Nile red were used as fluorescence dyes for protein and oil phases (5 mL of stock emulsion + 0.05 mL of 0.1% (w/w) fluorescence dye), with excitation wavelengths at 552 and 488 nm, respectively. The gelation process was carried out as mentioned Section 2.3. The CLSM images were obtained by sequential scan (TCS SP8, Leica Microsystems Inc., Heidelberg, Germany) with a 63× magnification lens.
2.3.2. Gelation of SPI emulsion After the pre-aggregation process, additional CaSO4 was added to the samples to reach the total Ca2+ concentration of 35 mM. The mixtures were heated to 80 °C and allowed to coagulate for 30 min in a water bath. After coagulation, the gels were immediately cooled to room temperature in an ice bath and stored at 4 °C.
2.8. Scanning electron microscope (SEM) 2.4. Evaluation of emulsion characteristics after pre-aggregation The microstructure of the SPI emulsion gels were also examined by SEM (FEI Quanta 200, FEI Company, Hillsboro, OR, USA). The samples were prepared according to the method described in our previous study
2.4.1. Zeta (ζ) potential The emulsions were diluted to a protein concentration of 5 mg/mL 460
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Table 1 pH, Zeta Potential, and Oil Droplet Size of the SPI emulsion after the pre-aggregation treatment. Parameter
pH Zeta potential (mV) D4,3 (μm)
Emulsions pre-aggregated at various concentrations of Ca2+ (mM) 0
2.5
5
7.5
10
7.08 −46.5 ± 2.1a 1.82 ± 0.03a
6.90 −43.9 ± 1.1a 1.79 ± 0.10a
6.68 −39.5 ± 1.8b 1.85 ± 0.22a
6.51 −36.9 ± 0.2bc 1.95 ± 0.15a
6.40 −35.2 ± 1.3c —
Values are means and standard deviations of three determinations, the different lower case letters (a–d) in the same row indicate significant difference among the values at the p < 0.05. The character (—) means abnormal data (> 10 μm).
differences across all emulsion gels but to a varying extent, the structure was dependent upon the applied Ca2+ concentrations (2.5–10 mM). Generally, the SPI emulsion gels prepared via the “two-step” process (pre-aggregation and gelation) showed more homogeneous and compact structures. On increasing the Ca2+ concentration during pre-aggregation from 2.5 to 7.5 mM, there was a trend toward smaller pore size and the formation of denser gel structures with larger clusters (bright parts, Fig. 1). However, when the Ca2+ concentration reached 10 mM, the gel structure became coarse and porous, despite thick strands still being observed. Fig. 2 shows the SEM images of the CaSO4-induced SPI emulsion gels in which filamentous networks were observed. The variation of the gel structure was consistent with the CLSM results. Denser networks were formed as the Ca2+ pre-aggregation concentration increased. The microstructure of the SPI emulsion gel pre-aggregated at 7.5 mM Ca2+ showed the most continuous and uniform structure. However, for the gel pre-aggregated at 10 mM Ca2+, the gel structure became less uniform than those obtained at lower Ca2+ pre-aggregation concentrations; in addition, discontinuous fragments with particles sticking could be observed.
(Wang et al., 2017). The gels were cut into small pieces and pre-treated including processes of fixing, dehydration and critical point drying. Dried samples were then sputter-coated with gold using an ion sputter (SCD 005, BAL-TEC, Switzerland) and observed at 15 kV. 2.9. Statistical analysis All assays were performed in duplicate and repeated at least three times, the results are expressed as means ± standard deviation. An analysis of variance (ANOVA) of the experimental data was performed with SPSS 20.0. A least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. 3. Results and discussions 3.1. SPI emulsion characteristics The pH, zeta potential (ζ), and oil droplet size (D4,3) of the five SPI emulsions after pre-aggregation are summarized in Table 1. The pH of the emulsions with no pre-aggregation was 7.08. As the pre-aggregation concentration of Ca2+ increased, the pH progressively decreased. This can be attributed to the binding of Ca2+ onto soy proteins via the carboxyl groups of the glutamyl and asparagyl residues, resulting in H+/Ca2+ exchange (Canabady-Rochelle et al., 2009). The ζ potential of the emulsions decreased similarly to the pH values. This phenomenon was partially due to the decreased pH in the system because of preaggregation (Rasnani, Mirhosseini, Bin Baharin, & Tan, 2011), indicating that the addition of Ca2+ neutralized negative charges on the SPI surface and thereby promoted protein aggregation. The D4,3 of the emulsions was measured using a static light scattering (SLS) technique. As can be seen, the increase in the Ca2+ concentration during pre-aggregation did not change the oil droplet size significantly (p > 0.05). This could be due to the fact that in this study, the emulsion with high protein (6%) and low oil (5%) was a relatively stable system. The soy protein molecules were enough to stabilize the emulsion and prevent the oil droplets coalescence, despite the pH fluctuation. This result excludes the effects of the oil droplet size variation during pre-aggregation on the final gel properties. However, for the emulsion preaggregated at 10 mM Ca2+, no data was presented, because in that situation, partial gelation might have occurred, thus the emulsion was very viscous and the result was abnormal (> 10 μm).
3.3. Effects of pre-aggregation on the gelation kinetics of SPI emulsion gels The effect of pre-aggregation under different Ca2+ concentrations on the time-dependent storage modulus (G') of the SPI emulsion gels at 1 Hz is shown in Fig. 3. Herein, we can generally observe that compared to the ‘one-step’ addition of the coagulant (0 mM), the SPI emulsion gels produced by ‘two-step’ method have notably higher G′ values and lower gelling temperature (Tgel). G′ was considered the best indicator of gel structure formation and consolidation (Maltais et al., 2005), and the increase in G′ reflects the development of mechanical modulus of the SPI emulsion gel network (Tang, Luo, Liu, & Chen, 2013). This indicates that pre-aggregation of SPI emulsion before gelation could promote the formation of stronger gel networks. For example, the G' value of the gel produced from the emulsion pre-aggregated under 7.5 mM Ca2+ was approximately 80% higher than that of the gel with no pre-aggregation (2035 Pa compared with 1150 Pa). This could be attributed to the microstructural changes of the SPI emulsion gel network after different pre-aggregation treatments because during the pre-aggregation process, the addition of a small amount of Ca2+ (not enough to form a gel network) helped the emulsion to form large and compact aggregates, resulting in a stronger gel with higher G′ values. The G′ increased gradually with the increasing Ca2+ concentration during pre-aggregation (2.5–7.5 mM) but decreased at 10 mM. A reasonable explanation for this is that under certain level range of Ca2+ concentrations (during pre-aggregation), the aggregate size increased as the Ca2+ concentration increased. Generally, aggregate size was related to the thickness of the gel strands, and therefore to the gel network structure (Lu et al., 2010). Larger aggregates could help to form thicker strands during gelation. However, an excessively high concentration of Ca2+ during pre-aggregation led to faster random aggregation of the emulsion, resulting in extremely large aggregates to form an inhomogeneous structure. The resultant gel was coarser and weaker with decreased rigidity and G′ value.
3.2. Microstructures of SPI emulsion gels The microscopic structures of the SPI emulsion gels were characterized using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) techniques. The CSLM images (Fig. 1) are presented as a function of Ca2+ concentration in the pre-aggregation process. From the figure, it was confirmed that the oil droplets (green colored) showed little variation across all samples. In the “onestep” addition process, (Ca2+ concentration during pre-aggregation was 0 mM), the gel network comprised fine strands, which appeared in a loose structure with large pores. By comparison, the pre-aggregation of the SPI emulsion with Ca2+ resulted in significant microstructural 461
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Fig. 1. Typical CLSM images of the CaSO4-induced SPI emulsion gels pre-aggregated at different Ca2+ concentrations.
3.4. Large-scale deformation
The gelling temperature, Tgel, is usually defined by the loss tangent, tan δ (the ratio of G″ to G′, G″/G′) and tan δ = 1 indicates the start of gelation (Mao, Roos, & Miao, 2014). From Fig. 3B, the Tgel of the emulsion gels markedly decreased as the pre-aggregation Ca2+ concentration increased. This result suggests that protein aggregates might be precursors of gel formation (Mills, Huang, Noel, Gunning, & Morris, 2001), and that larger aggregates form a gel much more easily. Fig. 3C shows the frequency dependence of G′ for the SPI emulsion gels. In each case, G′ gradually increased with the increase of the angular frequency, ω. The G″ (ω) curves showed a similar trend but were significantly lower than G′ throughout the same frequency range (data not shown), demonstrating a gel-like behavior (Steffe, 1996). Plotting log G′ and log G″ versus log ω gave linear curves with slopes from 0.10 to 0.15 (data not shown), revealing that both modulus showed slight frequency dependency. This type of system is more similar to “weak gel” behavior (Bonacucina, Cespi, Misici-Falzi, & Palmieri, 2006; Hesarinejad, Koocheki, & Razavi, 2014). In a weak gel, the network mainly comprises non-covalent “physical” crosslinks in nature, namely hydrophobic interactions, hydrogen bonding, and van der Waals forces, which are breakable or deformable (Kohyama, Sano, & Doi, 1995).
The viscoelastic parameters determined from small-deformation experiments give useful direct information about gel strength and indirect information about gel microstructure. To obtain information about mechanical gel properties relevant to food processing or eating characteristics, large-deformation can highlight the differences in the gel structures, which are not detectable by small-strain techniques (Dickinson & Yamamoto, 1996). In general, fracture stress reflects gel hardness and strain reflects gel deformability or brittleness (Weijers, van de Velde, Stijnman, van de Pijpekamp, & Visschers, 2006). Fig. 3D shows the stress (τ) as a function of strain amplitude (γ). As can be seen, pre-aggregation at 0–7.5 mM Ca2+ resulted in an increase in the yield stress τ yield, indicating that pre-aggregation treatments can promote the formation of stronger gels. In contrast, the yield strain γ yield of the gels showed a negative relation with the concentration of Ca2+ during preaggregation, revealing that the gels became more brittle. This implies that a higher force but a smaller deformation are required to fracture the gel. The fracture properties were affected by several factors, such as the number of non-covalent protein–protein bonds per cross section of the strand, the properties of each bond, the curvature of the gel network (Renkema, 2004), and the aggregate size (Ikeda, Foegeding, & Hagiwara, 1999). For protein emulsion gels, fracture Fig. 2. Scanning electron microscopy images of the CaSO4induced SPI emulsion gels pre-aggregated at different Ca2+ concentrations.
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Fig. 3. Effect of pre-aggregation at different Ca2+ concentrations on the rheological properties of SPI emulsion gels. (A) and (B) gelation kinetics. (C) Frequency dependence of G′. (D) Large-deformation, the maximum in the curve is taken as the yield point. The different lower case letters (a–d) indicate significant difference among the values at the 95% confidence level (p < 0.05).
usually occurred outside the linear viscoelasticity region (Fig. 3D), suggesting that the limit of linearity is determined by structural changes rather than by the breaking of the weakest bonds. It has been demonstrated that fine-stranded gels with smaller flocs are more strain resistant (Ikeda et al., 1999). In this study, it is reasonable to assume that as the concentration of Ca2+ during pre-aggregation increases, the emulsion gels become less strain resistant, as larger aggregates and thicker strands were formed. A gel with larger fracture strain indicates the gel strands are more curved, and the modulus will be low (Renkema, 2004). This further verifies the results of the small amplitude rheology. However, it is notable that the gel produced from the emulsion pre-aggregated at 10 mM Ca2+ had the lowest τyield and γyield; one likely explanation for this is the coarseness of the gel. For a coarser gel, the fracture stress will be lower when the defects in the gel are larger (Renkema, 2004). As a result, the overall gel structure pre-aggregated at 10 mM Ca2+ became very coarse and discontinuous, which made the gel brittle and much easier to fracture. 3.5. Water-holding capacity (WHC)
Fig. 4. The WHC of the CaSO4-induced SPI emulsion gels pre-aggregated at different Ca2+ concentrations. The different lower case letters (a–d) indicate significant difference among the values at the 95% confidence level (p < 0.05).
WHC, which is the ability to effectively immobilize water through the capillary effects of the gel matrices, is one of the most important properties in food systems (Wu et al., 2009). The WHC data of the SPI emulsion gels are shown in Fig. 4. As expected, significant differences in WHC were observed among the various gels. When compared with the “one-step” process, (0 mM), pre-aggregation with Ca2+ significantly improved the WHC of the SPI emulsion gels. The WHC of the emulsion gels increased with the increase in the Ca2+ concentration during preaggregation from 0 to 7.5 mM, whereas decreased at 10 mM. The SPI emulsion pre-aggregated at 7.5 mM Ca2+ produced gel with the highest WHC of 74.3%, which was approximately 15% higher than that of the
gel from the “one-step” process. This could be because pre-aggregation strengthened the network and helped to form dense and uniform microstructures, which strongly enabled water to be efficiently “bound” in the emulsion gel matrices (Hu et al., 2013). Gels with large pores or coarse structures tended to bind water to a lesser extent because of low capillary forces (Line, Remondetto, & Subirade, 2005).
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Fig. 5. Proposed mechanism for the effects of preaggregation on the gelation of CaSO4-induced SPI emulsions.
aggregates, thus enhancing the strength and WHC of the emulsion gels, whereas over aggregation made the final gel network coarser, which bond water to a lesser extent. The results indicate a close relation between the microstructures and properties of SPI emulsion gels. These findings will be of value for the application of SPI-stabilized emulsion gels with improved properties in the food industry, and will extend understanding of the relationships between the structural, rheological, and physiochemical properties of the gels.
3.6. Proposed mechanism for the effects of pre-aggregation on the SPI emulsion gelation The proposed mechanism for the effects of pre-aggregation on the formation of CaSO4-induced SPI emulsion gel is shown in Fig. 5. In a SPI-stabilized emulsion, the protein molecules were absorbed at the interfaces of oil droplets through hydrophobic interactions. Protein–protein interactions are generally favored by reducing the net charge on the molecules, decreasing pH values close to the isoelectric point (Boye, Alli, Ismail, Gibbs, & Konishi, 1995), screening electrostatic interactions between charged protein molecules, and forming salt bridges by divalent salts (Bryant & McClements, 2000). In a stable emulsion, the repulsion dominated the interactions between the particles, and the addition of the coagulant (CaSO4) not only formed salt bridges but also reduced the repulsion between the particles and allowed the formation of emulsion gel with filamentous gel structures (Kao et al., 2003). For the “one-step” process, the particles in the emulsion were small, thus producing gel with a loose network and fine strands. For the “two-step” process, in the first step, a small amount of CaSO4 was added to allow the emulsion to aggregate. In the second step, the remaining coagulant was added into the emulsion, the emulsion particles further aggregated to form a gel network. However, the final gel structure strongly depended on the Ca2+ concentration during the pre-aggregation process. At lower Ca2+ concentrations, the aggregating rates were relatively slow, and the emulsion had the time to arrange to form compact aggregates of various sizes; large aggregates first aggregated to constitute the primary network in the gelation process, then small aggregates and un-aggregated particles were further bonded to the network. Therefore, the appropriate (sufficient) aggregation (2.5–7.5 mM in this study) produced denser and stronger gel structures. If the Ca2+ concentration was too high (10 mM in this study), fast random aggregation occurred and the aggregates formed were too large, causing the final gel network to appear coarser and discontinuous with large holes.
Acknowledgements This research was supported by the National Natural Science Foundation of China (NSFC, 31271946), the National High-Tech Research and Development Program of China (863 program, grant No. 2013AA102200), the National Natural Science Foundation of China (NSFC, 31471583). Reference Bonacucina, G., Cespi, M., Misici-Falzi, M., & Palmieri, G. F. (2006). Rheological, adhesive and release characterisation of semisolid Carbopol/tetraglycol systems. International Journal of Pharmaceutics, 307(2), 129–140. Boye, J. I., Alli, I., Ismail, A. A., Gibbs, B. F., & Konishi, Y. (1995). Factors affecting molecular characteristics of whey protein gelation. International Dairy Journal, 5, 337–353. Bryant, C. M., & McClements, D. J. (2000). Influence of NaCl and CaCl2 on cold-set gelation of heat-denatured whey protein. Journal of Food Science, 65, 801–804. Canabady-Rochelle, L. S., Sanchez, C., Mellema, M., & Banon, S. (2009). Study of calcium−soy protein interactions by isothermal titration calorimetry and pH Cycle. Journal of Agricultural and Food Chemistry, 57(13), 5939–5947. Dickinson, E., & Yamamoto, Y. (1996). Rheology of milk protein gels and protein-stabilized emulsion gels cross-linked with transglutaminase. Journal of Agricultural and Food Chemistry, 44(6), 1371–1377. Doi, E. (1993). Gels and gelling of globular proteins. Trends in Food Science & Technology, 4(1), 1–5. Guo, S. T., & Ono, T. (2005). The role of composition and content of protein particles in soymilk on tofu curding by glucono-δ-lactone or calcium sulfate. Journal of Food Science, 70(4), C258–C262. Guo, F., Xiong, Y. L., Qin, F., Jian, H., Huang, X., & Chen, J. (2015). Surface properties of heat-induced soluble soy protein aggregates of different molecular masses. Journal of Food Science, 80(2), C279–C287. Hesarinejad, M. A., Koocheki, A., & Razavi, S. M. A. (2014). Dynamic rheological properties of Lepidium perfoliatum seed gum: Effect of concentration, temperature and heating/cooling rate. Food Hydrocolloids, 35, 583–589. Hou, H. J., Chang, K. C., & Shih, M. C. (1997). Yield and textural properties of soft tofu as affected by coagulation method. Journal of Food Science, 62(4), 824–827. Hu, H., Fan, X., Zhou, Z., Xu, X., Fan, G., Wang, L., ... Zhu, L. (2013). Acid-induced gelation behavior of soybean protein isolate with high intensity ultrasonic pretreatments. Ultrasonics Sonochemistry, 20(1), 187–195. Ikeda, S., Foegeding, E. A., & Hagiwara, T. (1999). Rheological study on the fractal nature of the protein gel structure. Langmuir, 15(25), 8584–8589. Kamizake, N. K. K., Silva, L. C. P., & Prudencio, S. H. (2016). Effect of soybean aging on
4. Conclusion In this study, a novel “two-step” addition of the coagulant method, was used to investigate the effects of pre-aggregation induced by Ca2+ before gelation on the physiochemical, rheological, and structural properties of SPI emulsion gel. Compared with the normal “one-step” process, pre-aggregation promoted the formation of stronger gels with thicker strands and denser structure. However, these strengthening effects depended to a large extent on the CaSO4 concentration during preaggregation. Sufficient aggregation produced larger and more compact 464
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X. Wang et al. the quality of soymilk, firmness of tofu and optimum coagulant concentration. Food Chemistry, 190, 90–96. Kao, F. J., Su, N. W., & Lee, M. H. (2003). Effect of calcium sulfate concentration in soymilk on the microstructure of firm tofu and the protein constitutions in tofu whey. Journal of Agricultural and Food Chemistry, 51(21), 6211–6216. Kohyama, K., Sano, Y., & Doi, E. (1995). Rheological characteristics and gelation mechanism of tofu (soybean curd). Journal of Agricultural and Food Chemistry, 43(7), 1808–1812. Lakemond, C. M., de Jongh, H. H., Paques, M., van Vliet, T., Gruppen, H., & Voragen, A. G. (2003). Gelation of soy glycinin; influence of pH and ionic strength on network structure in relation to protein conformation. Food Hydrocolloids, 17(3), 365–377. Li, X., Cheng, Y., Yi, C., Hua, Y., Yang, C., & Cui, S. (2009). Effect of ionic strength on the heat-induced soy protein aggregation and the phase separation of soy protein aggregate/dextran mixtures. Food Hydrocolloids, 23(3), 1015–1023. Line, V. L. S., Remondetto, G. E., & Subirade, M. (2005). Cold gelation of β-lactoglobulin oil-in-water emulsions. Food Hydrocolloids, 19(2), 269–278. Lu, X., Lu, Z., Yin, L., Cheng, Y., & Li, L. (2010). Effect of preheating temperature and calcium ions on the properties of cold-set soybean protein gel. Food Research International, 43(6), 1673–1683. Maltais, A., Remondetto, G. E., Gonzalez, R., & Subirade, M. (2005). Formation of soy protein isolate cold-set gels: protein and salt effects. Journal of Food Science, 70(1), C67–C73. Mao, L., Roos, Y. H., & Miao, S. (2014). Study on the rheological properties and volatile release of cold-set emulsion-filled protein gels. Journal of Agricultural and Food Chemistry, 62(47), 11420–11428. Mills, E. C., Huang, L., Noel, T. R., Gunning, A. P., & Morris, V. J. (2001). Formation of thermally induced aggregates of the soya globulin β-conglycinin. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1547(2), 339–350. Murekatete, N., Hua, Y., Chamba, M. V. M., Djakpo, O., & Zhang, C. (2014). Gelation behavior and rheological properties of salt-or acid-induced soy proteins soft tofu-type gels. Journal of Texture Studies, 45(1), 62–73. Ono, T. (2003). Soy (soya) cheeses. Encyclopedia of food science and nutrition. London: Academic5398–5402. Ono, T., Katho, S., & Mothizuki, K. (1993). Influences of calcium and pH on protein
solubility in soybean milk. Bioscience, Biotechnology, and Biochemistry, 57(1), 24–28. Rasnani, N. M., Mirhosseini, H., Bin Baharin, B. S., & Tan, C. P. (2011). Effect of pH on physicochemical properties and stability of sodium caseinate-pectin stabilized emulsion. Journal of Food, Agriculture & Environment, 9(1), 129–135. Renkema, J. M. (2004). Relations between rheological properties and network structure of soy protein gels. Food Hydrocolloids, 18(1), 39–47. Renkema, J. M., Gruppen, H., & Van Vliet, T. (2002). Influence of pH and ionic strength on heat-induced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions. Journal of Agricultural and Food Chemistry, 50(21), 6064–6071. Steffe, J. F. (1996). Rheological methods in food process engineering. Freeman Press (Chapter 5). Tang, C. H., Chen, L., & Foegeding, E. A. (2011). Mechanical and water-holding properties and microstructures of soy protein isolate emulsion gels induced by CaCl2, glucono-δlactone (GDL), and transglutaminase: Influence of thermal treatments before and/or after emulsification. Journal of Agricultural and Food Chemistry, 59(8), 4071–4077. Tang, C. H., Luo, L. J., Liu, F., & Chen, Z. (2013). Transglutaminase-set soy globulinstabilized emulsion gels: Influence of soy β-conglycinin/glycinin ratio on properties, microstructure and gelling mechanism. Food Research International, 51(2), 804–812. Visser, A., & Thomas, A. (1987). Review: Soya protein products-their processing, functionality, and application aspects. Food Reviews International, 3(1–2), 1–32. Wang, X., He, Z., Zeng, M., Qin, F., Adhikari, B., & Chen, J. (2017). Effects of the size and content of protein aggregates on the rheological and structural properties of soy protein isolate emulsion gels induced by caso 4. Food Chemistry, 221, 130–138. Weijers, M., van de Velde, F., Stijnman, A., van de Pijpekamp, A., & Visschers, R. W. (2006). Structure and rheological properties of acid-induced egg white protein gels. Food Hydrocolloids, 20(2), 146–159. Wu, M., Xiong, Y. L., Chen, J., Tang, X., & Zhou, G. (2009). Rheological and microstructural properties of porcine myofibrillar protein–lipid emulsion composite gels. Journal of Food Science, 74(4), E207–E217. Zhao, H., Li, W., Qin, F., & Chen, J. (2015). Calcium sulphate – Induced soya bean protein tofu – Type gels: Influence of denaturation and particle size. International Journal of Food Science & Technology, 51(3), 731–741.
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Enhanced CaSO4-induced gelation properties of soy protein isolate emulsion by pre-aggregation ⁎
Xufeng Wanga, Maomao Zenga, Fang Qina, Benu Adhikarib, Zhiyong Hea, , Jie Chena,c, a b c
MARK
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State Key Laboratory of Food Science and Technology, and School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China School of Applied Sciences, RMIT University, City Campus, Melbourne, VIC 3001, Australia Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soy protein isolate Emulsion gel Pre-aggregation Rheology Microstructure
The effects of CaSO4-induced pre-aggregation on the rheological and structural properties of soy protein isolate (SPI) emulsion gels were investigated. As the Ca2+ concentration during pre-aggregation increased (from 0 mM to 7.5 mM), the elastic modulus of the gels showed substantial increase, indicating stiffer gel structures. Largedeformation rheology suggested stronger but more brittle networks formed at higher Ca2+ concentration during pre-aggregation. However, when the pre-aggregated Ca2+ concentration reached 10 mM, the corresponding gel became weaker. Water-holding capacity (WHC) of the gels were significantly improved via the pre-aggregation process. The differences in rheological properties and WHC among the gels were consistent with the variation in their microstructures. Pre-aggregation helped to form denser and more uniform structures with thicker strands, whereas over aggregation made the gel network coarser.
1. Introduction Soy cheese, also called “tofu” in Asian countries, has attracted increasing attention in recent years because of its bland taste and nutritional advantages with low levels of saturated fat and cholesterol (Ono, 2003). Traditional soy cheese is made from soy milk in a process that usually involves soaking, grinding beans in water, filtering, heating, coagulating, breaking the curd, and finally pressing and reforming the gel (Hou, Chang, & Shih, 1997). The process is complex and time consuming (Kamizake, Silva, & Prudencio, 2016). Soy protein isolate (SPI) is widely used in the food industry. During SPI extraction, most components such as lipids, soybean oligosaccharides, and isoflavones, which have been assumed to be the source of soy off-flavors, allergens, and flatulence factors, respectively, to some people, are washed out (Visser & Thomas, 1987). Hence, using SPI as a raw material for soy cheese has gained considerable attention because it makes the cheesemaking process simpler, cleaner, and more controllable compared with traditional methods (Murekatete, Hua, Chamba, Djakpo, & Zhang, 2014). Acid-induced and salt-induced are the two major methods involved in the formation of soy cheese. Previous studies have found that GDL and CaSO4 can produce more uniform soy curds with much smoother structure than those produced from other coagulates (e.g., MgCl2, MgSO4, and CaCl2) (Kao, Su, & Lee, 2003), but GDL was not suitable for
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Chinese-style tofu because of its sour flavor. In the formation of soy cheese, coagulation of soymilk is the most important step because it directly determines the quality of the product (Hou et al., 1997). It has been proposed that in a Ca2+-induced gelation process, the coagulation can be divided into two steps: (1) the soy protein forms particles (aggregates) at low calcium concentration and (2) the soluble proteins are bonded to the network by further Ca2+ addition (Guo & Ono, 2005; Ono, Katho, & Mothizuki, 1993). The aggregates formed in the first step could therefore define the final gel properties. In fact, numerous studies have been conducted to understand the relationships between the structural and textural properties of soy cheese and have suggested that the microstructure of soy cheese is affected by the protein aggregate properties (e.g., size and content) because protein aggregates are related to the thickness of gel strands and the density of the network (Doi, 1993; Lakemond et al., 2003; Lu, Lu, Yin, Cheng, & Li, 2010). Our previous study also demonstrated that larger and/or more protein aggregates helped to formed stronger gels with denser and more compact structures (Wang et al., 2017). However, most previous research has focused on investigating the factors that influence the heat-induced protein aggregation or gelation, such as preheating conditions (i.e., preheating methods, heating temperature, and holding time), pH, and ionic strength (Lu et al., 2010; Renkema, Gruppen, & Van Vliet, 2002; Zhao, Li, Qin, & Chen, 2015). Whereas for salt-induced gelation, very few studies concerning the effects of protein
Corresponding authors at: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China. E-mail addresses: [email protected] (Z. He), [email protected] (J. Chen).
http://dx.doi.org/10.1016/j.foodchem.2017.09.044 Received 20 May 2017; Received in revised form 24 August 2017; Accepted 10 September 2017 Available online 19 September 2017 0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
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of with 0.05 M phosphate buffer (pH 7.0). One milliliter of each diluted sample was put in an electrophoresis cell (DTS 1060, Malvern Instruments Ltd.) and the ζ-potential was measured using a Malvern Zetasizer Nano ZS90 (Nano ZS, Malvern Instruments, Worcestershire, UK). All measurements were replicated at least 3 times under room temperature.
aggregation induced by the coagulant itself on the gel properties are reported. The addition of Ca2+ screens electrostatic interactions between charged protein molecules and promotes the protein aggregation (Li et al., 2009). Additionally, the combination of Ca2+ and soy protein can be described as H+/Ca2+ exchanges (Canabady-Rochelle, Sanchez, Mellema, & Banon, 2009), which also neutralize electrostatic repulsion and form salt bridges, thus allowing protein molecules to form aggregates and eventually leading to the formation of three-dimensional gel network (Lu et al., 2010; Maltais, Remondetto, Gonzalez, & Subirade, 2005). In the present study, a two-step addition of the coagulate method is proposed, with a traditional one-step control, to estimate the impact of Ca2+-induced pre-aggregation of SPI emulsion on its gelation properties. In the first step, a small amount of Ca2+ was added into the SPI emulsion to make the emulsion partially aggregated (pre-aggregation). In the second step, the remaining Ca2+ was further added to form the final gel network (gelation). The effects of the Ca2+ concentration during pre-aggregation on the microstructure, rheological and physiochemical properties of the gels were investigated.
2.4.2. Oil droplet size The oil droplet size of the SPI emulsions was determined by a particle size analyzer (Microtrac S3500, Microtrac Inc., North Largo, FL, USA). Distilled water was used as the dispersant. The relative refractive index of the emulsion was taken as 1.095 (Tang, Chen, & Foegeding, n n 2011). The volume-average diameter d4,3 (∑i di4 / ∑i di3 , where ni is the number of particles with diameter di ) was recorded. 2.5. Dynamic oscillatory measurements 2.5.1. Small amplitude oscillation The viscoelastic properties of the SPI emulsion gels were characterized by a controlled-stress rheometer (HAAKE MARS III, Thermo Fisher Scientific, Karlsruhe, Germany) with a parallel plate (d = 35.002 mm, gap = 1 mm), using temperature sweep and frequency sweep mode. The emulsions were immediately loaded between the plates of the rheometer after the addition of CaSO4. Low-viscosity silicon oil was used to prevent water evaporation. The gels were oscillated at 1% strain (within the linear viscoelastic region, LVR) and a frequency of 1 Hz. The temperature was heated from 25 °C to 80 °C at 5 °C per minute, followed by incubation at this temperature for 30 min before cooling to 25 °C at 5 °C per minute. The storage modulus (G′) and loss modulus (G″) were recorded. After the gelling process was completed, frequency sweep tests were carried out at 25 °C using an angular frequency (ω) of 1–100 rad/s at a constant strain of 1%.
2. Materials and methods 2.1. Materials SPI was extracted from defatted soybean meals (Taiwan 292, harvested in 2015) according to the method described by Guo et al. (2015). The protein content of the SPI was 92.3% (dry basis) determined by the Kjeldahl method. Soy oil and nail oil was purchased from a local supermarket; Nile red, and Rhodamin B were obtained from Sigma-Aldrich (St. Louis, Mo, USA). All other chemicals in the study were of analytical grade. 2.2. Preparation of SPI emulsions
2.5.2. Large-scale deformation Large-scale deformation tests were performed at 25 °C using the same rheometer in an oscillatory amplitude sweep mode. The strain was increased from 0.1% up to the fracture point when the stress began to decrease at a frequency of 1 Hz. The shear stress was recorded as a function of strain.
A 60 mg/mL SPI dispersion (pH 7.0, adjusted with 0.1 N NaOH or HCl) was prepared by dispersing SPI powder into deionized water, stirring mechanically at room temperature for at least 2 h and centrifuging at 10,000g for 10 min to remove insoluble materials. The dispersion was subjected to heat treatment at 95 °C for 15 min and then cooled to room temperature in an ice bath. The pretreated SPI dispersion was mixed with 5% (v/v) soy oil and pre-homogenized using a disperser homogenizer (T 18 basic ULTRA-TURRAX®, IKA Corp., Staufen, Germany) at 13,500 rpm for 2 min, followed by homogenization through a homogenizer (AH-BASIC, ATS Engineering Inc., Canada) at 40 MPa for one pass.
2.6. Water-holding capacity (WHC) The WHC of the gels was determined according to the method of Wu, Xiong, Chen, Tang, and Zhou (2009), Approximately 5 g of gel (each sample) was transferred to 50 mL centrifuge tubes and centrifuged at 10,000g for 15 min at 4 °C. WHC (%) was defined as the ratio of the water weight in the pellet to the water weight in the original gel multiplied by 100.
2.3. Gel preparation 2.3.1. Pre-aggregation of SPI emulsion The pre-aggregation treatment was conducted before the gelation process. To do this, the SPI emulsion was mixed with a stock CaSO4 dispersion to Ca2+ concentrations of 0, 2.5, 5, 7.5 and 10 mM. The emulsion/CaSO4 mixtures were mechanically stirred at 300 rpm at room temperature and allowed to aggregate for 1 h.
2.7. Confocal laser scanning microscopy (CLSM) Samples for CLSM analysis were prepared in single concave slides (Sail Brand, Jinliu Instrument Co., Ltd., Nanjing, China) covered with nail oil to prevent water evaporation. Rhodamine B and Nile red were used as fluorescence dyes for protein and oil phases (5 mL of stock emulsion + 0.05 mL of 0.1% (w/w) fluorescence dye), with excitation wavelengths at 552 and 488 nm, respectively. The gelation process was carried out as mentioned Section 2.3. The CLSM images were obtained by sequential scan (TCS SP8, Leica Microsystems Inc., Heidelberg, Germany) with a 63× magnification lens.
2.3.2. Gelation of SPI emulsion After the pre-aggregation process, additional CaSO4 was added to the samples to reach the total Ca2+ concentration of 35 mM. The mixtures were heated to 80 °C and allowed to coagulate for 30 min in a water bath. After coagulation, the gels were immediately cooled to room temperature in an ice bath and stored at 4 °C.
2.8. Scanning electron microscope (SEM) 2.4. Evaluation of emulsion characteristics after pre-aggregation The microstructure of the SPI emulsion gels were also examined by SEM (FEI Quanta 200, FEI Company, Hillsboro, OR, USA). The samples were prepared according to the method described in our previous study
2.4.1. Zeta (ζ) potential The emulsions were diluted to a protein concentration of 5 mg/mL 460
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Table 1 pH, Zeta Potential, and Oil Droplet Size of the SPI emulsion after the pre-aggregation treatment. Parameter
pH Zeta potential (mV) D4,3 (μm)
Emulsions pre-aggregated at various concentrations of Ca2+ (mM) 0
2.5
5
7.5
10
7.08 −46.5 ± 2.1a 1.82 ± 0.03a
6.90 −43.9 ± 1.1a 1.79 ± 0.10a
6.68 −39.5 ± 1.8b 1.85 ± 0.22a
6.51 −36.9 ± 0.2bc 1.95 ± 0.15a
6.40 −35.2 ± 1.3c —
Values are means and standard deviations of three determinations, the different lower case letters (a–d) in the same row indicate significant difference among the values at the p < 0.05. The character (—) means abnormal data (> 10 μm).
differences across all emulsion gels but to a varying extent, the structure was dependent upon the applied Ca2+ concentrations (2.5–10 mM). Generally, the SPI emulsion gels prepared via the “two-step” process (pre-aggregation and gelation) showed more homogeneous and compact structures. On increasing the Ca2+ concentration during pre-aggregation from 2.5 to 7.5 mM, there was a trend toward smaller pore size and the formation of denser gel structures with larger clusters (bright parts, Fig. 1). However, when the Ca2+ concentration reached 10 mM, the gel structure became coarse and porous, despite thick strands still being observed. Fig. 2 shows the SEM images of the CaSO4-induced SPI emulsion gels in which filamentous networks were observed. The variation of the gel structure was consistent with the CLSM results. Denser networks were formed as the Ca2+ pre-aggregation concentration increased. The microstructure of the SPI emulsion gel pre-aggregated at 7.5 mM Ca2+ showed the most continuous and uniform structure. However, for the gel pre-aggregated at 10 mM Ca2+, the gel structure became less uniform than those obtained at lower Ca2+ pre-aggregation concentrations; in addition, discontinuous fragments with particles sticking could be observed.
(Wang et al., 2017). The gels were cut into small pieces and pre-treated including processes of fixing, dehydration and critical point drying. Dried samples were then sputter-coated with gold using an ion sputter (SCD 005, BAL-TEC, Switzerland) and observed at 15 kV. 2.9. Statistical analysis All assays were performed in duplicate and repeated at least three times, the results are expressed as means ± standard deviation. An analysis of variance (ANOVA) of the experimental data was performed with SPSS 20.0. A least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. 3. Results and discussions 3.1. SPI emulsion characteristics The pH, zeta potential (ζ), and oil droplet size (D4,3) of the five SPI emulsions after pre-aggregation are summarized in Table 1. The pH of the emulsions with no pre-aggregation was 7.08. As the pre-aggregation concentration of Ca2+ increased, the pH progressively decreased. This can be attributed to the binding of Ca2+ onto soy proteins via the carboxyl groups of the glutamyl and asparagyl residues, resulting in H+/Ca2+ exchange (Canabady-Rochelle et al., 2009). The ζ potential of the emulsions decreased similarly to the pH values. This phenomenon was partially due to the decreased pH in the system because of preaggregation (Rasnani, Mirhosseini, Bin Baharin, & Tan, 2011), indicating that the addition of Ca2+ neutralized negative charges on the SPI surface and thereby promoted protein aggregation. The D4,3 of the emulsions was measured using a static light scattering (SLS) technique. As can be seen, the increase in the Ca2+ concentration during pre-aggregation did not change the oil droplet size significantly (p > 0.05). This could be due to the fact that in this study, the emulsion with high protein (6%) and low oil (5%) was a relatively stable system. The soy protein molecules were enough to stabilize the emulsion and prevent the oil droplets coalescence, despite the pH fluctuation. This result excludes the effects of the oil droplet size variation during pre-aggregation on the final gel properties. However, for the emulsion preaggregated at 10 mM Ca2+, no data was presented, because in that situation, partial gelation might have occurred, thus the emulsion was very viscous and the result was abnormal (> 10 μm).
3.3. Effects of pre-aggregation on the gelation kinetics of SPI emulsion gels The effect of pre-aggregation under different Ca2+ concentrations on the time-dependent storage modulus (G') of the SPI emulsion gels at 1 Hz is shown in Fig. 3. Herein, we can generally observe that compared to the ‘one-step’ addition of the coagulant (0 mM), the SPI emulsion gels produced by ‘two-step’ method have notably higher G′ values and lower gelling temperature (Tgel). G′ was considered the best indicator of gel structure formation and consolidation (Maltais et al., 2005), and the increase in G′ reflects the development of mechanical modulus of the SPI emulsion gel network (Tang, Luo, Liu, & Chen, 2013). This indicates that pre-aggregation of SPI emulsion before gelation could promote the formation of stronger gel networks. For example, the G' value of the gel produced from the emulsion pre-aggregated under 7.5 mM Ca2+ was approximately 80% higher than that of the gel with no pre-aggregation (2035 Pa compared with 1150 Pa). This could be attributed to the microstructural changes of the SPI emulsion gel network after different pre-aggregation treatments because during the pre-aggregation process, the addition of a small amount of Ca2+ (not enough to form a gel network) helped the emulsion to form large and compact aggregates, resulting in a stronger gel with higher G′ values. The G′ increased gradually with the increasing Ca2+ concentration during pre-aggregation (2.5–7.5 mM) but decreased at 10 mM. A reasonable explanation for this is that under certain level range of Ca2+ concentrations (during pre-aggregation), the aggregate size increased as the Ca2+ concentration increased. Generally, aggregate size was related to the thickness of the gel strands, and therefore to the gel network structure (Lu et al., 2010). Larger aggregates could help to form thicker strands during gelation. However, an excessively high concentration of Ca2+ during pre-aggregation led to faster random aggregation of the emulsion, resulting in extremely large aggregates to form an inhomogeneous structure. The resultant gel was coarser and weaker with decreased rigidity and G′ value.
3.2. Microstructures of SPI emulsion gels The microscopic structures of the SPI emulsion gels were characterized using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) techniques. The CSLM images (Fig. 1) are presented as a function of Ca2+ concentration in the pre-aggregation process. From the figure, it was confirmed that the oil droplets (green colored) showed little variation across all samples. In the “onestep” addition process, (Ca2+ concentration during pre-aggregation was 0 mM), the gel network comprised fine strands, which appeared in a loose structure with large pores. By comparison, the pre-aggregation of the SPI emulsion with Ca2+ resulted in significant microstructural 461
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Fig. 1. Typical CLSM images of the CaSO4-induced SPI emulsion gels pre-aggregated at different Ca2+ concentrations.
3.4. Large-scale deformation
The gelling temperature, Tgel, is usually defined by the loss tangent, tan δ (the ratio of G″ to G′, G″/G′) and tan δ = 1 indicates the start of gelation (Mao, Roos, & Miao, 2014). From Fig. 3B, the Tgel of the emulsion gels markedly decreased as the pre-aggregation Ca2+ concentration increased. This result suggests that protein aggregates might be precursors of gel formation (Mills, Huang, Noel, Gunning, & Morris, 2001), and that larger aggregates form a gel much more easily. Fig. 3C shows the frequency dependence of G′ for the SPI emulsion gels. In each case, G′ gradually increased with the increase of the angular frequency, ω. The G″ (ω) curves showed a similar trend but were significantly lower than G′ throughout the same frequency range (data not shown), demonstrating a gel-like behavior (Steffe, 1996). Plotting log G′ and log G″ versus log ω gave linear curves with slopes from 0.10 to 0.15 (data not shown), revealing that both modulus showed slight frequency dependency. This type of system is more similar to “weak gel” behavior (Bonacucina, Cespi, Misici-Falzi, & Palmieri, 2006; Hesarinejad, Koocheki, & Razavi, 2014). In a weak gel, the network mainly comprises non-covalent “physical” crosslinks in nature, namely hydrophobic interactions, hydrogen bonding, and van der Waals forces, which are breakable or deformable (Kohyama, Sano, & Doi, 1995).
The viscoelastic parameters determined from small-deformation experiments give useful direct information about gel strength and indirect information about gel microstructure. To obtain information about mechanical gel properties relevant to food processing or eating characteristics, large-deformation can highlight the differences in the gel structures, which are not detectable by small-strain techniques (Dickinson & Yamamoto, 1996). In general, fracture stress reflects gel hardness and strain reflects gel deformability or brittleness (Weijers, van de Velde, Stijnman, van de Pijpekamp, & Visschers, 2006). Fig. 3D shows the stress (τ) as a function of strain amplitude (γ). As can be seen, pre-aggregation at 0–7.5 mM Ca2+ resulted in an increase in the yield stress τ yield, indicating that pre-aggregation treatments can promote the formation of stronger gels. In contrast, the yield strain γ yield of the gels showed a negative relation with the concentration of Ca2+ during preaggregation, revealing that the gels became more brittle. This implies that a higher force but a smaller deformation are required to fracture the gel. The fracture properties were affected by several factors, such as the number of non-covalent protein–protein bonds per cross section of the strand, the properties of each bond, the curvature of the gel network (Renkema, 2004), and the aggregate size (Ikeda, Foegeding, & Hagiwara, 1999). For protein emulsion gels, fracture Fig. 2. Scanning electron microscopy images of the CaSO4induced SPI emulsion gels pre-aggregated at different Ca2+ concentrations.
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Fig. 3. Effect of pre-aggregation at different Ca2+ concentrations on the rheological properties of SPI emulsion gels. (A) and (B) gelation kinetics. (C) Frequency dependence of G′. (D) Large-deformation, the maximum in the curve is taken as the yield point. The different lower case letters (a–d) indicate significant difference among the values at the 95% confidence level (p < 0.05).
usually occurred outside the linear viscoelasticity region (Fig. 3D), suggesting that the limit of linearity is determined by structural changes rather than by the breaking of the weakest bonds. It has been demonstrated that fine-stranded gels with smaller flocs are more strain resistant (Ikeda et al., 1999). In this study, it is reasonable to assume that as the concentration of Ca2+ during pre-aggregation increases, the emulsion gels become less strain resistant, as larger aggregates and thicker strands were formed. A gel with larger fracture strain indicates the gel strands are more curved, and the modulus will be low (Renkema, 2004). This further verifies the results of the small amplitude rheology. However, it is notable that the gel produced from the emulsion pre-aggregated at 10 mM Ca2+ had the lowest τyield and γyield; one likely explanation for this is the coarseness of the gel. For a coarser gel, the fracture stress will be lower when the defects in the gel are larger (Renkema, 2004). As a result, the overall gel structure pre-aggregated at 10 mM Ca2+ became very coarse and discontinuous, which made the gel brittle and much easier to fracture. 3.5. Water-holding capacity (WHC)
Fig. 4. The WHC of the CaSO4-induced SPI emulsion gels pre-aggregated at different Ca2+ concentrations. The different lower case letters (a–d) indicate significant difference among the values at the 95% confidence level (p < 0.05).
WHC, which is the ability to effectively immobilize water through the capillary effects of the gel matrices, is one of the most important properties in food systems (Wu et al., 2009). The WHC data of the SPI emulsion gels are shown in Fig. 4. As expected, significant differences in WHC were observed among the various gels. When compared with the “one-step” process, (0 mM), pre-aggregation with Ca2+ significantly improved the WHC of the SPI emulsion gels. The WHC of the emulsion gels increased with the increase in the Ca2+ concentration during preaggregation from 0 to 7.5 mM, whereas decreased at 10 mM. The SPI emulsion pre-aggregated at 7.5 mM Ca2+ produced gel with the highest WHC of 74.3%, which was approximately 15% higher than that of the
gel from the “one-step” process. This could be because pre-aggregation strengthened the network and helped to form dense and uniform microstructures, which strongly enabled water to be efficiently “bound” in the emulsion gel matrices (Hu et al., 2013). Gels with large pores or coarse structures tended to bind water to a lesser extent because of low capillary forces (Line, Remondetto, & Subirade, 2005).
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Fig. 5. Proposed mechanism for the effects of preaggregation on the gelation of CaSO4-induced SPI emulsions.
aggregates, thus enhancing the strength and WHC of the emulsion gels, whereas over aggregation made the final gel network coarser, which bond water to a lesser extent. The results indicate a close relation between the microstructures and properties of SPI emulsion gels. These findings will be of value for the application of SPI-stabilized emulsion gels with improved properties in the food industry, and will extend understanding of the relationships between the structural, rheological, and physiochemical properties of the gels.
3.6. Proposed mechanism for the effects of pre-aggregation on the SPI emulsion gelation The proposed mechanism for the effects of pre-aggregation on the formation of CaSO4-induced SPI emulsion gel is shown in Fig. 5. In a SPI-stabilized emulsion, the protein molecules were absorbed at the interfaces of oil droplets through hydrophobic interactions. Protein–protein interactions are generally favored by reducing the net charge on the molecules, decreasing pH values close to the isoelectric point (Boye, Alli, Ismail, Gibbs, & Konishi, 1995), screening electrostatic interactions between charged protein molecules, and forming salt bridges by divalent salts (Bryant & McClements, 2000). In a stable emulsion, the repulsion dominated the interactions between the particles, and the addition of the coagulant (CaSO4) not only formed salt bridges but also reduced the repulsion between the particles and allowed the formation of emulsion gel with filamentous gel structures (Kao et al., 2003). For the “one-step” process, the particles in the emulsion were small, thus producing gel with a loose network and fine strands. For the “two-step” process, in the first step, a small amount of CaSO4 was added to allow the emulsion to aggregate. In the second step, the remaining coagulant was added into the emulsion, the emulsion particles further aggregated to form a gel network. However, the final gel structure strongly depended on the Ca2+ concentration during the pre-aggregation process. At lower Ca2+ concentrations, the aggregating rates were relatively slow, and the emulsion had the time to arrange to form compact aggregates of various sizes; large aggregates first aggregated to constitute the primary network in the gelation process, then small aggregates and un-aggregated particles were further bonded to the network. Therefore, the appropriate (sufficient) aggregation (2.5–7.5 mM in this study) produced denser and stronger gel structures. If the Ca2+ concentration was too high (10 mM in this study), fast random aggregation occurred and the aggregates formed were too large, causing the final gel network to appear coarser and discontinuous with large holes.
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