- Email: [email protected]
A novel process to efficiently form transglutaminase-set soy protein isolate-stabilized emulsion gels
LWT - Food Science and Technology 53 (2013) 15e21
Contents lists available at SciVerse ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
A novel process to efficiently form transglutaminase-set soy protein isolatestabilized emulsion gels Chuan-he Tang a, b, *, Miao Yang a, Fu Liu a, Zhong Chen a a b
Department of Food Science and Technology and KLGPNPS, South China University of Technology, Guangzhou 510640, People’s Republic of China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2012 Received in revised form 19 January 2013 Accepted 5 March 2013
A novel process to efficiently form soy protein isolate (SPI)-stabilized emulsion gels, induced by microbial transglutaminase (MTGase) was reported, with the enzyme added before the emulsification (instead of after the emulsification in the conventional case). The rheological behavior and microstructure of the formed emulsion gels (at a constant protein concentration of 6 g/100 mL and oil fractions (ø) of 0.2e0.6) were characterized using dynamic oscillatory measurement and confocal laser scanning microscope. The gel stiffness of the formed gels with the novel process progressively increased with increasing enzyme concentration (1.0e5.0 U/g), or with the ø increasing from 0.2 to 0.6. Interestingly, the gel stiffness at a given ø with the novel process was considerably greater than that with the conventional process, though the enzyme concentration was less (e.g., 1.0 vs 20 U/g). Furthermore, a pre-incubation (15e60 min) of the proteins with the enzyme (before the emulsification) greatly improved the gelation. The analyses of droplet size and amount of entrapped proteins within the gel matrix indicated that during the initial gel network, the bridging flocculation of oil droplets played a prominent role, but the full development of the gel stiffness mainly depended on the inter-droplet covalent cross-linking. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Protein-stabilized emulsion gel Soy protein isolate Microbial transglutaminase (MTGase) Cold gelation
1. Introduction Protein-stabilized emulsion gels are of interest in the food industry as an important kind of food systems, including cheese and processed meats. In the recent years, there are increasing interests in developing these systems as effective controlled-release delivery systems for many bioactive nutraceuticals, especially poorly soluble compounds, e.g., carotenoids, phytosterols, polyunsatuted fatty acids (Chen, Remondetto, & Subirade, 2006; Velikov & Pelan, 2008). One of the major advantages for these systems as the carriers is that food proteins can be considered as a kind of ‘generally recognized as safe’ (GRAS) compounds, thus providing the possibilities of developing a variety of GRAS novel foods for oral administration of bioactive compounds (Chen et al., 2006). Another advantage is that the incorporating many sensitive nutraceuticals or aroma compounds into these delivery systems can greatly improve their stability and bioavailability (Lee, Choi, & Moon, 2006; Liang, Sok Line, Remondetto, & Subirade, 2010). The protein-stabilized emulsion gels can be induced by means of heat treatment, acidification with * Corresponding author. Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People's Republic of China. Tel.: þ86 20 87114262; fax: þ86 20 87114263. E-mail address: [email protected] (C.-h. Tang). 0023-6438/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2013.03.002
glucono-d-lactone (GDL), addition of divalent salts (e.g., CaCl2), and even enzymatic cross-linking with transglutaminase (TGase; Dickinson, 2012; Tang, Chen, & Foegeding, 2011). Of all these methods to induce the protein-stabilized emulsion gelation, the enzymatic method with TGase seems to be the most appropriate in the food formulations, due to considerations from processing, product safety and quality. To date, very limited information is available concerning about TGase-set protein-stabilized emulsion gels (Dickinson & Yamamoto, 1996; Kang, Kim, Woo & Moon, 2003; Lee et al., 2006; Matsumura, Kang, Sakamoto, Motoki, & Mori, 1993; Nio, Motoki, & Takinami, 1986; Tang et al., 2011; Yang, Liu, & Tang, 2011). Nio et al. (1986) for the first time reported that TGase can be applied to effectively induce the gelation of the emulsions stabilized by as1casein, soy 11S and 7S globulins. Besides, the emulsions stabilized by bovine serum albumin (BSA), sodium caseinate, soy protein isolate (SPI), and b-lactoglobulin, could also be gelled by the action of microbial TGase (MTGase) (Dickinson & Yamamoto, 1996; Kang et al., 2003; Lee et al., 2006; Tang et al., 2011; Yang et al., 2011). It is generally recognized that 1) the strength or stiffness of the formed emulsion gels increases with increasing protein concentration in continuous phase (Dickinson & Yamamoto, 1996; Matsumura et al., 1993), or with decreasing original emulsion droplet size (Matsumura et al., 1993); 2) conformational changes of
16
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
protein can improve or impair the MTGase-induced gelation of the emulsion gels, depending on the mode of pretreatments and the type of proteins (Kang et al., 2003; Tang et al., 2011); 3) increasing oil fraction (ø) progressively increases the stiffness and water holding capacity of the MTGase-set emulsion gels (Yang et al., 2011), which is similar to heat-induced protein-stabilized emulsion gels (Chen & Dickinson, 1998 a, b, 1999). In all the works mentioned above, the enzyme (MTGase) was added after the emulsion formation, and the amount of applied enzyme was relatively high ranging from 20 (Tang et al., 2011; Yang et al., 2011) to 120 (Dickinson & Yamamoto, 1996) units per gram of protein (U/g) in the continuous phase. No matter what the type of enzyme products is applied and how the cost for the application of high amount of enzyme is, there are many technical problems arising during the enzyme addition. For example, the mode and efficiency of mixing of the emulsions and the enzyme might produce a remarkable impact on the formation of the emulsion gels; the addition of the enzyme might also cause depletion flocculation of the emulsions, especially when the enzyme products contain high amount of carbohydrates, or when the emulsions with high oil fractions (associated with severe packing effect of oil droplets) are applied. Surprisingly, few articles are available addressing how to improve efficiency of MTGase-induced gelation of proteinstabilized emulsions. Recently, our research team investigated the influence of shearing during incubation on the MTGase-induced gelation of SPI-stabilized emulsions, and found that the stirring greatly improved the gelation, but the improvement was dependent on the mode of stirring (continuous or intermittent mode) and the ø (Tang, Yang, Liu, & Chen, 2013). There are also barely reports investigating the MTGase-induced gelation when the enzyme was added prior to the emulsification. Herein, another concern that should be clarified is whether the emulsification treatment decreases the catalytic activity of the enzyme. Our preliminary experiments confirmed that the activity of MTGase did not change after the emulsification at pressure levels up to 160 MPa, and in some cases, the activity on the contrary slightly increased after the treatment (data not published). Therefore, this work was to confirm the possibility to improve the gelation efficiency of MTGase-set SPI-stabilized emulsion gels, by adding the enzyme prior to the emulsification. In contrast with the conventional process (with the enzyme added after the emulsion gel formation), the process to form the emulsion gel with the enzyme added before the emulsification was denoted as the novel process. The enzyme concentration or ø dependence of the rheological behavior and microstructure of the emulsion gels obtained with the novel process was investigated, and even compared with that with the conventional process. Furthermore, the influence of a pre-incubation of the proteins with the enzyme (before the emulsification) on the gelation process, emulsion droplet size and amount of proteins entrapped with the gel network was also investigated. 2. Materials and methods 2.1. Materials SPI was prepared from defatted soybean meal, provided by Shandong Yuwang Industrial and Commercial Co. Ltd. (China), as described in our previous work (Tang et al., 2011). The protein content of this SPI was 92.9 g/100 g (wet basis) as determined by the Dumas method. L-glutamic acid g-monohydroxamate and NaCBZ-GLN-GLY were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rhodamine B (analytical grade) was purchased from Beijing DINGGUO Biological Technology Co. Ltd. (China), and Nile blue from Shanghai BAOAO Biological Technology Co. Ltd. (China).
Commercial MTGase, produced by Streptomyces hygroscopicus, was purchased from TAIXIN YIMIN Fine Chemical Industry Co. Ltd. (Jiangsu province, China). The activity of the enzyme product was 200 U/g as determined by a colorimetric procedure using Na-CBZGLN-GLY as the substrate and L-glutamic acid g -monohydroxamate as the standard. The stock enzyme solution was prepared according to the same process of our previous work (Tang et al., 2011). Soy oil was purchased from the local supermarket (Guangzhou, China). All other chemicals were of analytical grade. 2.2. Preparation of SPI-stabilized emulsions The SPI stock dispersion (6 g/100 mL) was prepared in distilled water with 0.04 g/100 mL of sodium azide as an antimicrobial agent, and stirred at room temperature for 2 h. If necessary, the pH of the dispersion was adjusted to pH 7.0 with 0.2 mol/L NaOH and then the dispersion was kept overnight at 4 C. The SPI stock solution was pre-homogenized with soy oil at ø of 0.2e0.6, using a high-speed dispersing and emulsifying unit (model IKA-ULTRA-TURRAXÒ T25 basic, IKAÒ Works, Inc., Wilmington, NC) at 10,000 rpm for 2 min to obtain coarse emulsions. Then, the coarse emulsions were further homogenized at 40 MPa for one pass through a Microfluidizer (M110EH model, Microfluidics International Corporation, Newton, MA), to produce the original emulsions (for the gelation experiments with the conventional process). For the novel process, the MTGase with an enzyme concentration of 1.0e5.0 U/g was mixed with the stock SPI solution, prior to the emulsification. The protein/enzyme mixtures were immediately subject to the emulsification as the same process as described above, or incubated at 37 C for specific periods of time (15e 60 min) and then subject to the emulsification. Once the emulsions with the enzyme were obtained, and immediately subject to the next gelation experiments. 2.3. Gelation of the emulsions and dynamic viscoelasticity of the resultant emulsion gels The MTGase-induced gelation of SPI-stabilized emulsions was evaluated by low amplitude dynamic oscillatory measurements using a rheometer (RS600, HAAKE Co., Germany) with parallel plates (d ¼ 27.83 mm). The gap between the two plates was set as 1.0 mm. The equipment was driven through the RheoWin 3 Data Manager (HAAKE Co., Germany). In the conventional gelation process, the original emulsions (without addition of MTGase, prior to the emulsificaiton) were mixed with the stock enzyme solution to an enzyme concentration of 20 U/g (in the continuous phase). The mixtures were immediately loaded between the parallel plates of the rheometer. Excess sample was trimmed off and a thin layer of silicone oil applied to the exposed free edges of the sample to prevent moisture loss. During the gelation with an incubation period of 2 h, the dynamic moduli at a constant temperature (37 C) were recorded at a frequency of 1.0 Hz and a strain of 1%. In the novel process, the freshly prepared emulsions (with MTGase added before the emulsification) were directly loaded between the plates of the rheometer, and used for the gelation experiment (up to 2 h) as the above. After the above gelation experiments (with 2 h of incubation), the dynamic viscoelasticity of the resultant emulsion gels was evaluated with a frequency sweep mode. The frequency was oscillated from 0.01 to 10 rad/s and all measurements performed within the identified linear viscoelastic region and made at 1% strain. The elastic modulus (G0 ) and loss modulus (G") were recorded by RheoWin 3 Data Manager. The experiments were carried out at 37 C.
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
2.4. Confocal laser scanning microscopy (CLSM)
2.5. Droplet size determination The influence of an enzymatic pre-incubation prior to the emulsification on the droplet size of the SPI-stabilized emulsions was investigated. The pre-incubation of the SPI solutions (6 g/ 100 mL) with MTGase (at an enzyme concentration of 1.0 U/g) was conducted at 37 C for specific incubation peirods of 0, 15, 30 and 60 min, respectively. After the incubation, the activity of MTGase was stopped by addition of 1.0 M NH4Cl to a final concentration of 10 mM, according to the method of Faergemand, Jeanette, and Karsten (1998). The untreated or MTGase-preincubated SPI solutions were further mixed with soy oil at ø ¼ 0.2, and subject to the same emulsification as described above. The droplet size distribution of various emulsions, freshly prepared or after storage of 1.0 h, was determined using a Malvern MasterSizer 2000 (Malvern Instruments Ltd, Malvern, Worcestershire, UK). Distilled water was used as the dispersant. The relative refractive index of the emulsion was taken as 1.47. Droplet size measurements are reported as the volume-average diameter, d4,3 P P (¼ nid4i / nid3i , where ni is the number of particles with diameter di). All determinations were conducted at least in duplicate. 2.6. Amount of proteins adsorbed to the interface or entrapped within gel network The influence of an enzymatic pre-incubation (at 37 C for specific incubation periods of 0, 15, 30 and 60 min), on the amount of proteins adsorbed to the interface, or entrapped within the gel network, of the resultant emulsions (freshly prepared) or emulsion gels (after an incubation period of 2 h) was evaluated. The emulsions or emulsion gels (with the novel process) were prepared at a protein concentration of 6 g/100 mL and ø ¼ 0.4, and at an enzyme concentration of 1.0 U/g. The amount of proteins entrapped within the gel network was evaluated using the method of Chanyongvorakul, Matsumura, Sawa, Nio, and Mori (1997) that was applied to evaluate the adsorbed and unadsorbed proteins in the emulsions, with a few modifications. Aliquots (1.0 g) of the emulsions, freshly prepared or after incubation at 37 C for a storage period of 2 h, in microcentrifuge tubes, were centrifuged in a micro-centrifuge at 10,000 g for 30 min at room temperature. The protein concentration of separated aqueous layer was determined by the Bradford assay, using BSA as the standard. The percent of entrapped proteins was calculated as (1-protein concentration in separated aqueous layer/initial protein concentration in the aqueous phase) 100.
10000
G' (Pa)
The microstructure of the emulsion gels was evaluated with a confocal laser scanning microscope (Leica TCSSP5, Heidelberg, Germany), using Rhodamine B and Nile blue as fluorescence dyes for the protein and oil phases, with excitation wavelengths at 488 and 633 nm, respectively. Both stock dye solutions (1.0 g/100 mL) were mixed with the emulsions up to final concentrations of about 0.05 g/100 mL, prior to gel formation. The emulsion gels containing the dyes (with a total amount of 80 mL) were directly formed in concave confocal microscope slides (Sail; Sailing Medical-Lab Industries Co. Ltd., Suzhou, China), covered with glycerol-coated coverslips (that were further sealed with tin foil). The CLSM images were obtained with a 100 magnification lens. The oil phases or droplets dyed with the Nile blue are usually green, whereas the proteins dyed with the Rhodamine B are red. Actually, most of the protein-coated oil droplets would be predominantly yellow in appearance of the overlay image (green þ red / yellow).
17
1000
100
10 0.1
1
10
Frequency (Hz) Fig. 1. Frequency dependence of G0 for the SPI emulsion gels, induced by MTGase at enzyme concentrations of 0 (-), 1.0 (C), 2.5 (:) and 5.0 (;) U/g, respectively, for an incubation period of 2 h. The enzyme was added before the emulsification. The protein concentration and ø were 6.0 g/100 mL and 0.2, respectively.
2.7. Statistics An analysis of variance (ANOVA) of the data was performed, and a least significant difference (LSD) with a confidence interval of 95% was applied to compare the means. 3. Results and discussion 3.1. Rheological characteristics of emulsion gels 3.1.1. Dependence on enzyme concentration Contrasting from the conventional process to form enzyme-set emulsion gels, where the MTGase was added after the emulsion formation, the present work applied a novel process to add the enzyme before the emulsification. Once a specific amount of the enzyme was mixed with the protein solution (with a constant concentration of 6 g/100 mL), the resultant mixture was immediately mixed with various ø values of oil phase and subject to the homogenization (or emulsification). After that, the freshly formed emulsions were incubated at 37 C for an incubation period of 2 h on the rheometer, and their dynamic viscoelasticity was then evaluated. Fig. 1 shows the frequency dependence of storage modulus (G0 ) of various SPI emulsion gels, formed at various enzyme concentrations (0, 1.0, 2.5 and 5.0 U/g). We can generally observe that the G0 of all the untreated or MTGase-treated emulsions was almost independent of the frequency in the range 0.1e 10.0 Hz, which is a typical characteristic of a network consisting of permanent covalent cross-links (Dickinson & Yamamoto, 1996). In the present case, the control emulsion without the addition of MTGase also exhibited a gel-like behavior, which is consistent with the previous finding that the microfludization could produce a kind of gel-like SPI emulsions, by bridging flocculation of oil droplets, mainly through inter-droplet hydrophobic interactions between the proteins adsorbed at the interface (Tang & Liu, 2013), but seems to be different from the observation of our previous work (Yang et al., 2011; where at a comparable ø value the G0 was highly dependent on the frequency over the tested frequency range of 0.1e10 Hz). The difference might reflect that the formed original emulsions were kinetically unstable, e.g. against flocculation of oil droplets, and when the emulsions were incubated at 37 C for 2 h, severe flocculation of oil droplets might occur, thus leading to formation of gel-like network. At any test frequency, we can see that the G0 progressively and markedly increased with the enzyme concentration from 0 (control) to 5.0 U/g (Fig. 1). For example, the G0 (2300 Pa) of the emulsion gel with 1.0 U/g of MTGase was more than 20-fold than that (w100 Pa) of the control (without MTGase addition). These
18
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
3.2. Microstructure of the emulsion gels
Fig. 2. The ø dependence of the G0 (at a frequency of 1.0 Hz) of SPI-stabilized emulsion gels, formed after an incubation period of 2 h. Novel process (,): The emulsion gels were induced by the MTGase (1.0 U/g; added before the emulsification); conventional process (B): The gels were formed by 20 U/g of MTGase (added after the emulsification). Each data is the means and standard deviations of duplicate measurements. Dash lines within the figure indicate fitting of G0 data with exponential equations (ln G0 f ø).
observations indicated strengthening of gel network as a result of increased intermolecular covalent cross-links among proteins. The G0 value was even considerably higher than that previously reported for the emulsion gel (at the same protein concentration and ø) formed at a much higher enzyme concentration (20 U/g) for a much longer period (6 h) (using the conventional process; Yang et al., 2011). This suggests that the MTGase addition prior to the emulsification greatly improved the enzyme-induced gelation of SPI-stabilized emulsions. 3.1.2. Effect of oil fraction (ø) Fig. 2 shows the ø dependence of the G0 (at 1 Hz) of the MTGaseset emulsion gels, obtained with the enzyme added before (novel process) or after (conventional process) the emulsion formation, after 2 h of incubation. In the novel process, the applied enzyme concentration in the continuous phase was 1 U/g, whereas in the conventional case, it was 20 U/g. In both the cases, we can see that the G0 progressively increased, in an exponential way (ln G0 f ø, with a relationship coefficient R2 of 0.90e0.98), as the ø increased from 0.2 to 0.6 (Fig. 2). This observation is basically consistent with the fact that the protein-coated oil droplets usually act as ‘active fillers’ reinforcing the emulsion-filled gel network, wherein the reinforcing effect will be stronger with more dispersed oil droplets trapped or fully integrated into the network, at higher ø values (Chen & Dickinson, 1999; Chen, Dickinson, Langton, & Hermansson, 2000). The oil droplet packing effect can be also observed in other gel-like SPI-stabilized emulsions (Tang & Liu, 2013). The progressive increase in the stiffness of the emulsion gels upon the increase in ø was remarkably higher than that without MTGase addition (data not shown), reflecting that besides the packing effect, the enhanced reinforcing of the emulsion gel network by increasing the ø was largely dependent on the higher extent of MTGase-induced covalent cross-links. This is because in concentrated emulsions, the oil droplets are close enough to interact with one another, and as a result, inter-droplet covalent cross-links between the adsorbed proteins at the interface can be efficiently formed. By comparison, we can see that at any test ø, the G0 of the emulsion gels with the novel process was considerably higher than that with the conventional process, though in the former case, the amount of applied enzyme was much lower. The magnitude of the difference in G0 between the emulsion gels with the two processes was greater at higher ø values (Fig. 2). This comparison further indicated that the novel process with MTGase added before the emulsification exhibited excellent potential to efficiently form the protein-stabilized emulsion gels.
The microstructure of the enzyme-set SPI-stabilized gels, formed at three selected ø values of 0.2, 0.4 and 0.6, after 2 h of incubation, with the novel and conventional processes, respectively, was characterized using CLSM, as displayed in Fig. 3. As expected, the emulsion gels formed in both the cases exhibited similar microstructure, considerably varying with the applied ø. The emulsion gel at ø ¼ 0.2 was mainly composed of a homogenously filamentous network with the oil droplets entrapped and even becoming an integral part. The filamentous network, more like the protein gels in nature, was gradually transformed into the network mainly composed of packing or aggregated oil droplets, as the ø increased from 0.2 to 0.6 (Fig. 3). The microstructure of the emulsion gels formed with the conventional process is similar to that observed in our previous work (Yang et al., 2011). On the other hand, we can see that the microstructure of the emulsion gels formed with the novel process was more homogenous and compact than that with the conventional process (Fig. 3). Especially at ø ¼ 0.6, a distinct difference in microstructure could be observed for the emulsion gels between with the novel and conventional processes. With the novel process, all the oil droplets were closely associated together forming a compact and integral part of the network. In the conventional case, almost all of the oil droplets were present in the aggregated form; and there was distinct interspace observed among individual oil droplets (Fig. 3). The observations reflect that in the novel process case, the unadsorbed proteins played a major role in the network formation, which seemed to act as a kind of ‘binding agent’ reinforcing the gel network (mainly consisting of aggregated oil droplets). Thus, the differences in microstructure could well explain the difference in gel stiffness between the emulsion gels with the two processes (Fig. 2).
3.3. Effects of pre-incubation before the emulsification The above MTGase-set emulsion gels were obtained without a pre-incubation of the enzyme with the proteins prior to the emulsification. In this situation, we reasonably hypothesized that the freshly prepared emulsions in the presence of MTGase were basically identical to those in the absence of the enzyme. This means that the gelling mechanism with the novel process should be mainly through covalent cross-linking of adsorbed proteins at the interface of individual oil droplets. In this aspect, the thickness of the adsorbed protein films at the interface would produce an important role for the inter-droplet protein cross-linking. To confirm this, we investigated the influence of an enzymatic preincubation (with the protein solutions; before the emulsification) on the gelation of SPI-stabilized emulsions at a specific ø of 0.4. The pre-incubation of the protein solutions with MTGase might result in formation of high molecular weight biopolymers of soy proteins, as observed in many previous literatures (Tang, Wu, Chen, & Yang, 2006; Tang, Wu, Yu, et al., 2006). Once these biopolymers are adsorbed to the interface of oil droplets, the thickness of protein interfacial films will be much thicker than that in the uncrosslinked case. As a consequence, the MTGase-induced gelation of the emulsions should be favored. From the enzyme-induced gelation profiles, as displayed in Fig. 4, we can observe that an enzyme pre-incubation (15e60 min) of the protein solutions (before the emulsification) not only shortened the incubation period needed for the initial formation of the emulsion gel network, but also remarkably strengthened the stiffness of the correspondingly formed emulsion gels. This observation clearly indicated that the cross-linking of the proteins before the emulsification greatly favored the gelation of the emulsions.
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
19
Fig. 3. Typical CLSM images of enzyme-set SPI-stabilized emulsion gels at various ø values (0.2e0.6). Novel process: An enzyme concentration (1.0 U/g) of MTGase was added before the emulsification; conventional process: An enzyme concentration (20 U/g) of MTGase was added after emulsification. All the images were obtained with the dyes Rhodamine B (protein) and Nile blue (oil phase), excited at 488 and 633 nm, respectively. The bars indicate 10 mm in length.
Interestingly, the best improvement of the gelation was observed after a pre-incubation period of 15 min, and a further increase (30 or 60 min) in pre-incubation period on the contrary impaired the improvement (as compared to that at 15 min) (Fig. 4). To further unravel the underlying mechanism, we analyzed the microstructure of the emulsion gels, formed with three selected pre-incubation periods (0, 15 and 60 min) by CLSM, as shown in Fig. 5. Herein, we can totally see that the emulsion gel obtained with a pre-incubation period of 15 min exhibited a more homogenous network, in which all the oil droplets were evenly entrapped and dispersed in the network (Fig. 5). By comparison, in the
12000
G'(Pa)
9000 6000 3000 0
emulsion gel with 60 min of pre-incubation, the oil droplets with larger size (indicative of coalescence) were observed (Fig. 5), which might be a result of impaired emulsifying properties of proteins by an extensive cross-linking. These observations suggested that the decreased stiffness (observed after prolonged pre-incubation periods, e.g., 60 min) was largely due to both the formation of less homogenous network (relative to that after 15 min of preincubation) and impairment of emulsifying properties of proteins by the enzymatic cross-linking. Previous research had indicated that an extensive cross-linking reduced the stability of milk protein (sodium caseinate or b-lactoglobulin) emulsions prior to emulsification to coalescence or flocculation, whereas limited cross-linking improved the coalescence stability (Faergemand et al., 1998). A similar impairment of the emulsifying properties by the MTGase cross-linking has been reported for soy glycinin and/or b-conglycinin (Hu, Xu, Fan, Cheng, & Li, 2011). Thus, the decreased stiffness observed after prolonged pre-incubation periods (e.g., 30e 60 min) might be largely associated with the impaired emulsion stability of SPI by extensive cross-linking. 3.4. Changes in physicochemical parameters of the emulsions: influence of pre-incubation
0
20
40
60
80
100
120
Time (min) Fig. 4. Effects of pre-incubation [with the protein solution; 0 (,) 15 (B), 30 (6) and 60 (7) min] prior to the emulsification on the MTGase-induced gelation of SPIstabilized emulsions at ø ¼ 0.4. The enzyme concentration was 1.0 U/g. The gelation process was recorded at a frequency of 1.0 Hz.
3.4.1. Volume mean droplet size (d4,3) The influence of a pre-incubation of SPI with MTGase (1.0 U/g) for various incubation periods (0e60 min) on its emulsifying properties (including emulsifying ability and emulsion stability) was evaluated by light scattering technique. After the specific periods of preincubation, the enzyme was inactivated by addition of 1.0 mol/L NH4Cl (to a final concentration of 10 mmol/L). Fig. 6 shows the volume
20
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
mean droplet size (d4,3) of the freshly prepared emulsions (ø ¼ 0.2), stabilized by SPI pre-incubated with MTGase for various periods. A low ø (0.2) was used, due to the consideration that at this ø, the formed emulsions were relatively stable against creaming, at least within a short period (e. g., 1 h). We can see that the pre-incubation with MTGase resulted in a progressive increase in d4,3 of droplets in the freshly formed emulsions, from 0.73 to 1.20 mm, as the pre-incubation period increased from 0 to 60 min (Fig. 6). However, significant increases were observed only after pre-incubation above 15 min. These observations confirmed that an extensive enzymatic cross-linking (in the present work, through a pre-incubation with MTGase above 15 min) led to impairment of emulsifying ability of SPI. On the other hand, we also evaluated the influence of a preincubation (0e120 min) with 1.0 U/g of MTGase on the d4,3 of the particles in the SPI solutions (6 g/100 mL) alone, using a Nanosizer. The results showed that the d4,3 of the particles in the solutions slightly but progressively increased from 0.052 (control) to 0.087 or 0.101 mm, as the pre-incubation period increased from 0 to 60 or 120 min (Data not shown). The increased d4,3 was clearly as a result of the polymerization or aggregation of the protiens, induced by the covalent crosslinking. Thus, the progressive increase in d4,3 of the droplets in the emulsions upon increasing the pre-incubation period could be partially attributed to the formation of high molcular biopolymers. The d4,3 of droplets in all the emulsions significantly increased after a storage period of 1.0 h (Fig. 6), indicating instability of these emulsions. Since the MTGase was inactivated before the emulsion formation, the significant increases in d4,3 could be thus largely due to bridging flocculation or coalescence of oil droplets. On the other hand, we can still observe that the instability against flocculation or coalescence was more distinct at longer periods of pre-incubation (Fig. 6), suggesting that increasing the pre-incubation period with MTGase (or the covalent cross-linking) accelerated the instability of the emulsions. This observation further confirmed that the decreased gel stiffness at prolonged pre-incubation periods was largely due to increased extent of oil droplet flocculation or coalescence (thus resulting in formation of in homogenous network). 3.4.2. Amount of the protein entrapped within gel matrix The MTGase-induced gelation of SPI emulsions is closely associated with the covalent or non-covalent interactions of proteins, adsorbed and/or unadsorbed. Once the emulsion gel network is formed, the proteins, including those adsorbed to the interface of oil droplets and even some of unabsorbed proteins, will be entrapped within the gel matrix, which can be approximately estimated using centrifugation of the emulsions or emulsion gels. Fig. 7 shows the influence of the enzymatic pre-incubation period (0e60 min) before the emulsification, on the amount of proteins entrapped within the gel matrix for the SPI emulsions (with MTGase), freshly formed or after storage of 2 h at 37 C. We can observe that the amount of entrapped proteins progressively increased from about 60 to 71 g/100 g, as the pre-incubation period increased from 0 to 60 min, indicating that the cross-linking treatment of the proteins before the emulsification led to more proteins participating in the gel network formation. After any preincubation period, the amount of entrapped proteins within the gel matrix was slightly but insignificantly increased by storage for an incubation period of 2 h (Fig. 7). These observations, together with the droplet size data (Fig. 6), suggest that at ø ¼ 0.4 (in the present work), the initiation of the
Fig. 5. Typical CLSM images of MTGase-set SPI-stabilized emulsion gels at ø ¼ 0.4, formed with various pre-incubation periods (before the emulsification) of 0 (a), 15 (b) and 60 (c) min, respectively. The arrows within figure indicate large oil droplets. The images were dyed with the same dyes as in the legend of Fig. 3.
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
21
(before the emulsification) could also greatly improve the gelation. The initiation of the emulsion gel network was largely attributed to the bridging flocculation of oil droplets, but the full development of the gel stiffness mainly depended on the inter-droplet covalent cross-linking between the protein-coated oil droplets. The findings are of great importance for the development of an important kind of emulsion gels, especially as the matrix of functional foods. Acknowledgments This work is supported by the National Natural Science Foundation of China (serial numbers: 31071504, 31171632 and 31130042), and Program for New Century Excellent Talents in University (NCET-10e0398). Fig. 6. Influence of an enzymatic pre-incubation (0e60 min) with the proteins (before the emulsification) on the mean droplet size (d4,3) of the emulsions at ø ¼ 0.2, freshly prepared ( ) or after storage of 1.0 h ( ). The pre-incubation of the protein solutions (6 g/100 mL) with 1.0 U/g of MTGase was conducted at 37 C for specific periods (0, 15, 30 and 60 min), and the enzyme then immediately inactivated. Different symbols (aec) within figure indicate significant difference at p < 0.05 level, among different pre-incubation periods. Different symbols (eef) indicate significant difference at p < 0.05 level, due to storage of 1.0 h.
emulsion gel network might be largely attributed to the bridging flocculation of oil droplets, but it should be the inter-droplet crosslinks that play a predominant role in the development of the gel stiffness, or strengthening of the gel network (though the extent of enzymatic covalent cross-linking might be low). This thus could explain why with the novel process, the application of a much less enzyme concentration (1.0 U/g) of MTGase could more efficiently form the SPI emulsion gels with much higher stiffness than the conventional process, with a much higher enzyme concentration (e.g., 20 U/g), since this low enzyme concentration seemed to be enough to induce covalent cross-links between the neighboring aggregated oil droplets. 4. Conclusions A novel process to efficiently form MTGase-set SPI-stabilized emulsion gels was proposed, wherein the enzyme was added prior to the emulsification, instead of after the emulsification in the conventional case. This novel process needed a much less enzyme amount, but produced the emulsion gels with much higher stiffness (than the conventional process). Increasing the ø progressively accelerated the MTGase-induced gelation of the emulsions. Furthermore, a pre-incubation of the proteins with the enzyme
Fig. 7. Influence of an enzymatic pre-incubation (0e60 min) before the emulsification on the amount of proteins (g/100 mL) adsorbed at the interface or entrapped within the network of SPI-stabilized emulsion gels (with 1.0 U/g of MTGase), freshly formed (,) or after 2 h of storage (B) at 37 C. Each data is the means and standard deviations of duplicate measurements. The protein concentration and ø were 6 g/100 mL and 0.4, respectively.
References Chanyongvorakul, Y., Matsumura, Y., Sawa, A., Nio, N., & Mori, T. (1997). Polymerization of b-lactoglobulin and bovine serum albumin at oilewater interfaces in emulsions by transglutaminase. Food Hydrocolloids, 11, 449e455. Chen, J., & Dickinson, E. (1998a). Viscoelastic properties of protein-stabilized emulsions: effect of protein-surfactant interactions. Journal of Agricultural and Food Chemistry, 46, 91e97. Chen, J., & Dickinson, E. (1998b). Viscoelastic properties of heat-set whey protein emulsion gels. Journal of Texture Studies, 29, 285e304. Chen, J., & Dickinson, E. (1999). Effect of surface character of filler particles on rheology of heat-set whey protein emulsion gels. Colloids and Surfaces B: Biointerfaces, 12, 373e381. Chen, J., Dickinson, E., Langton, M., & Hermansson, A.-M. (2000). Mechanical properties and microstructure of heat-set whey protein emulsion gels: effect of emulsifiers. LWT-Food Science and Technology, 33, 299e307. Chen, L., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials as nutraceutical delivery systems. Trends in Food Science & Technology, 17, 272e283. Dickinson, E. (2012). Emulsion gels: the structuring of soft solids with proteinstabilized oil droplets. Food Hydrocolloids, 28, 224e241. Dickinson, E., & Yamamoto, Y. (1996). Rheology of milk protein gels and proteinstabilized emulsion gels cross-linked with transglutaminase. Journal of Agricultural and Food Chemistry, 44, 1371e1377. Faergemand, M., Jeanette, O., & Karsten, B. Q. (1998). Emulsifying properties of milk proteins cross-linked with microbial transglutaminase. International Dairy Journal, 8, 715e723. Hu, X., Xu, X., Fan, J.-F., Cheng, Y.-Q., & Li, L. (2011). Functional properties of microbial transglutaminase modified soybean glycinin and b-conglycinin. International Journal of Food Engineering, 7. art. no. 4. Kang, Y. N., Kim, H., Shin, W. S., Woo, G., & Moon, T. W. (2003). Effect of disulfide bond reduction on bovine serum albumin-stabilized emulsion gel formed by microbial transglutaminase. Journal of Food Science, 68, 2215e2220. Lee, H. A., Choi, S. J., & Moon, T. W. (2006). Characteristics of sodium caseinate- and soy protein isolate-stabilized emulsion-gels formed by microbial transglutaminase. Journal of Food Science, 71, C352eC357. Liang, L., Sok Line, C. L., Remondetto, G. E., & Subirade, M. (2010). In vitro release of a-tocopherol from emulsion-loaded b-lactoglobulin gels. International Dairy Journal, 20, 176e181. Matsumura, Y., Kang, H.-J., Sakamoto, H., Motoki, M., & Mori, T. (1993). Filler effects of oil droplets on the viscoelastic properties of emulsion gels. Food Hydrocolloids, 7, 227e240. Nio, N., Motoki, M., & Takinami, K. (1986). Gelation of protein emulsion by transglutaminase. Agricultural and Biological Chemistry, 50, 1409e1412. 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-d-lactone (GDL), and transglutaminase: Influence of thermal treatments before and/or after emulsification. Journal of Agricultural and Food Chemistry, 59, 4071e4077. Tang, C. H., & Liu, F. (2013). Cold, gel-like soy protein emulsions by microfludization: emulsion characteristics, rheological and microstructural properties, and gelling mechanism. Food Hydrocolloids, 30, 61e72. Tang, C. H., Wu, H., Chen, Z., & Yang, X. Q. (2006). Formation and gel properties of glycinin-rich and b-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase. Food Research International, 39, 87e97. Tang, C. H., Wu, H., Yu, H. P., Li, L., Chen, Z., & Yang, X. Q. (2006). Coagulation and gelation of soy protein isolates induced by microbial transglutaminase. Journal of Food Biochemistry, 30, 35e55. Tang, C. H., Yang, M., Liu, F., & Chen, Z. (2013). Stirring greatly improves transglutaminase-induced gelation of soy protein-stabilized emulsions. LWTFood Science and Technology, 51, 120e128. Velikov, K. P., & Pelan, E. (2008). Colloidal delivery systems for micronutrients and nutraceuticals. Soft Matter, 4, 1964e1980. Yang, M., Liu, F., & Tang, C. H. (2011). Properties and microstructure of transglutaminase-set soy protein-stabilized emulsion gels. Food Research International, . http://dx.doi.org/10.1016/j.foodres.2011.11.012.
Contents lists available at SciVerse ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
A novel process to efficiently form transglutaminase-set soy protein isolatestabilized emulsion gels Chuan-he Tang a, b, *, Miao Yang a, Fu Liu a, Zhong Chen a a b
Department of Food Science and Technology and KLGPNPS, South China University of Technology, Guangzhou 510640, People’s Republic of China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 November 2012 Received in revised form 19 January 2013 Accepted 5 March 2013
A novel process to efficiently form soy protein isolate (SPI)-stabilized emulsion gels, induced by microbial transglutaminase (MTGase) was reported, with the enzyme added before the emulsification (instead of after the emulsification in the conventional case). The rheological behavior and microstructure of the formed emulsion gels (at a constant protein concentration of 6 g/100 mL and oil fractions (ø) of 0.2e0.6) were characterized using dynamic oscillatory measurement and confocal laser scanning microscope. The gel stiffness of the formed gels with the novel process progressively increased with increasing enzyme concentration (1.0e5.0 U/g), or with the ø increasing from 0.2 to 0.6. Interestingly, the gel stiffness at a given ø with the novel process was considerably greater than that with the conventional process, though the enzyme concentration was less (e.g., 1.0 vs 20 U/g). Furthermore, a pre-incubation (15e60 min) of the proteins with the enzyme (before the emulsification) greatly improved the gelation. The analyses of droplet size and amount of entrapped proteins within the gel matrix indicated that during the initial gel network, the bridging flocculation of oil droplets played a prominent role, but the full development of the gel stiffness mainly depended on the inter-droplet covalent cross-linking. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Protein-stabilized emulsion gel Soy protein isolate Microbial transglutaminase (MTGase) Cold gelation
1. Introduction Protein-stabilized emulsion gels are of interest in the food industry as an important kind of food systems, including cheese and processed meats. In the recent years, there are increasing interests in developing these systems as effective controlled-release delivery systems for many bioactive nutraceuticals, especially poorly soluble compounds, e.g., carotenoids, phytosterols, polyunsatuted fatty acids (Chen, Remondetto, & Subirade, 2006; Velikov & Pelan, 2008). One of the major advantages for these systems as the carriers is that food proteins can be considered as a kind of ‘generally recognized as safe’ (GRAS) compounds, thus providing the possibilities of developing a variety of GRAS novel foods for oral administration of bioactive compounds (Chen et al., 2006). Another advantage is that the incorporating many sensitive nutraceuticals or aroma compounds into these delivery systems can greatly improve their stability and bioavailability (Lee, Choi, & Moon, 2006; Liang, Sok Line, Remondetto, & Subirade, 2010). The protein-stabilized emulsion gels can be induced by means of heat treatment, acidification with * Corresponding author. Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People's Republic of China. Tel.: þ86 20 87114262; fax: þ86 20 87114263. E-mail address: [email protected] (C.-h. Tang). 0023-6438/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2013.03.002
glucono-d-lactone (GDL), addition of divalent salts (e.g., CaCl2), and even enzymatic cross-linking with transglutaminase (TGase; Dickinson, 2012; Tang, Chen, & Foegeding, 2011). Of all these methods to induce the protein-stabilized emulsion gelation, the enzymatic method with TGase seems to be the most appropriate in the food formulations, due to considerations from processing, product safety and quality. To date, very limited information is available concerning about TGase-set protein-stabilized emulsion gels (Dickinson & Yamamoto, 1996; Kang, Kim, Woo & Moon, 2003; Lee et al., 2006; Matsumura, Kang, Sakamoto, Motoki, & Mori, 1993; Nio, Motoki, & Takinami, 1986; Tang et al., 2011; Yang, Liu, & Tang, 2011). Nio et al. (1986) for the first time reported that TGase can be applied to effectively induce the gelation of the emulsions stabilized by as1casein, soy 11S and 7S globulins. Besides, the emulsions stabilized by bovine serum albumin (BSA), sodium caseinate, soy protein isolate (SPI), and b-lactoglobulin, could also be gelled by the action of microbial TGase (MTGase) (Dickinson & Yamamoto, 1996; Kang et al., 2003; Lee et al., 2006; Tang et al., 2011; Yang et al., 2011). It is generally recognized that 1) the strength or stiffness of the formed emulsion gels increases with increasing protein concentration in continuous phase (Dickinson & Yamamoto, 1996; Matsumura et al., 1993), or with decreasing original emulsion droplet size (Matsumura et al., 1993); 2) conformational changes of
16
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
protein can improve or impair the MTGase-induced gelation of the emulsion gels, depending on the mode of pretreatments and the type of proteins (Kang et al., 2003; Tang et al., 2011); 3) increasing oil fraction (ø) progressively increases the stiffness and water holding capacity of the MTGase-set emulsion gels (Yang et al., 2011), which is similar to heat-induced protein-stabilized emulsion gels (Chen & Dickinson, 1998 a, b, 1999). In all the works mentioned above, the enzyme (MTGase) was added after the emulsion formation, and the amount of applied enzyme was relatively high ranging from 20 (Tang et al., 2011; Yang et al., 2011) to 120 (Dickinson & Yamamoto, 1996) units per gram of protein (U/g) in the continuous phase. No matter what the type of enzyme products is applied and how the cost for the application of high amount of enzyme is, there are many technical problems arising during the enzyme addition. For example, the mode and efficiency of mixing of the emulsions and the enzyme might produce a remarkable impact on the formation of the emulsion gels; the addition of the enzyme might also cause depletion flocculation of the emulsions, especially when the enzyme products contain high amount of carbohydrates, or when the emulsions with high oil fractions (associated with severe packing effect of oil droplets) are applied. Surprisingly, few articles are available addressing how to improve efficiency of MTGase-induced gelation of proteinstabilized emulsions. Recently, our research team investigated the influence of shearing during incubation on the MTGase-induced gelation of SPI-stabilized emulsions, and found that the stirring greatly improved the gelation, but the improvement was dependent on the mode of stirring (continuous or intermittent mode) and the ø (Tang, Yang, Liu, & Chen, 2013). There are also barely reports investigating the MTGase-induced gelation when the enzyme was added prior to the emulsification. Herein, another concern that should be clarified is whether the emulsification treatment decreases the catalytic activity of the enzyme. Our preliminary experiments confirmed that the activity of MTGase did not change after the emulsification at pressure levels up to 160 MPa, and in some cases, the activity on the contrary slightly increased after the treatment (data not published). Therefore, this work was to confirm the possibility to improve the gelation efficiency of MTGase-set SPI-stabilized emulsion gels, by adding the enzyme prior to the emulsification. In contrast with the conventional process (with the enzyme added after the emulsion gel formation), the process to form the emulsion gel with the enzyme added before the emulsification was denoted as the novel process. The enzyme concentration or ø dependence of the rheological behavior and microstructure of the emulsion gels obtained with the novel process was investigated, and even compared with that with the conventional process. Furthermore, the influence of a pre-incubation of the proteins with the enzyme (before the emulsification) on the gelation process, emulsion droplet size and amount of proteins entrapped with the gel network was also investigated. 2. Materials and methods 2.1. Materials SPI was prepared from defatted soybean meal, provided by Shandong Yuwang Industrial and Commercial Co. Ltd. (China), as described in our previous work (Tang et al., 2011). The protein content of this SPI was 92.9 g/100 g (wet basis) as determined by the Dumas method. L-glutamic acid g-monohydroxamate and NaCBZ-GLN-GLY were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rhodamine B (analytical grade) was purchased from Beijing DINGGUO Biological Technology Co. Ltd. (China), and Nile blue from Shanghai BAOAO Biological Technology Co. Ltd. (China).
Commercial MTGase, produced by Streptomyces hygroscopicus, was purchased from TAIXIN YIMIN Fine Chemical Industry Co. Ltd. (Jiangsu province, China). The activity of the enzyme product was 200 U/g as determined by a colorimetric procedure using Na-CBZGLN-GLY as the substrate and L-glutamic acid g -monohydroxamate as the standard. The stock enzyme solution was prepared according to the same process of our previous work (Tang et al., 2011). Soy oil was purchased from the local supermarket (Guangzhou, China). All other chemicals were of analytical grade. 2.2. Preparation of SPI-stabilized emulsions The SPI stock dispersion (6 g/100 mL) was prepared in distilled water with 0.04 g/100 mL of sodium azide as an antimicrobial agent, and stirred at room temperature for 2 h. If necessary, the pH of the dispersion was adjusted to pH 7.0 with 0.2 mol/L NaOH and then the dispersion was kept overnight at 4 C. The SPI stock solution was pre-homogenized with soy oil at ø of 0.2e0.6, using a high-speed dispersing and emulsifying unit (model IKA-ULTRA-TURRAXÒ T25 basic, IKAÒ Works, Inc., Wilmington, NC) at 10,000 rpm for 2 min to obtain coarse emulsions. Then, the coarse emulsions were further homogenized at 40 MPa for one pass through a Microfluidizer (M110EH model, Microfluidics International Corporation, Newton, MA), to produce the original emulsions (for the gelation experiments with the conventional process). For the novel process, the MTGase with an enzyme concentration of 1.0e5.0 U/g was mixed with the stock SPI solution, prior to the emulsification. The protein/enzyme mixtures were immediately subject to the emulsification as the same process as described above, or incubated at 37 C for specific periods of time (15e 60 min) and then subject to the emulsification. Once the emulsions with the enzyme were obtained, and immediately subject to the next gelation experiments. 2.3. Gelation of the emulsions and dynamic viscoelasticity of the resultant emulsion gels The MTGase-induced gelation of SPI-stabilized emulsions was evaluated by low amplitude dynamic oscillatory measurements using a rheometer (RS600, HAAKE Co., Germany) with parallel plates (d ¼ 27.83 mm). The gap between the two plates was set as 1.0 mm. The equipment was driven through the RheoWin 3 Data Manager (HAAKE Co., Germany). In the conventional gelation process, the original emulsions (without addition of MTGase, prior to the emulsificaiton) were mixed with the stock enzyme solution to an enzyme concentration of 20 U/g (in the continuous phase). The mixtures were immediately loaded between the parallel plates of the rheometer. Excess sample was trimmed off and a thin layer of silicone oil applied to the exposed free edges of the sample to prevent moisture loss. During the gelation with an incubation period of 2 h, the dynamic moduli at a constant temperature (37 C) were recorded at a frequency of 1.0 Hz and a strain of 1%. In the novel process, the freshly prepared emulsions (with MTGase added before the emulsification) were directly loaded between the plates of the rheometer, and used for the gelation experiment (up to 2 h) as the above. After the above gelation experiments (with 2 h of incubation), the dynamic viscoelasticity of the resultant emulsion gels was evaluated with a frequency sweep mode. The frequency was oscillated from 0.01 to 10 rad/s and all measurements performed within the identified linear viscoelastic region and made at 1% strain. The elastic modulus (G0 ) and loss modulus (G") were recorded by RheoWin 3 Data Manager. The experiments were carried out at 37 C.
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
2.4. Confocal laser scanning microscopy (CLSM)
2.5. Droplet size determination The influence of an enzymatic pre-incubation prior to the emulsification on the droplet size of the SPI-stabilized emulsions was investigated. The pre-incubation of the SPI solutions (6 g/ 100 mL) with MTGase (at an enzyme concentration of 1.0 U/g) was conducted at 37 C for specific incubation peirods of 0, 15, 30 and 60 min, respectively. After the incubation, the activity of MTGase was stopped by addition of 1.0 M NH4Cl to a final concentration of 10 mM, according to the method of Faergemand, Jeanette, and Karsten (1998). The untreated or MTGase-preincubated SPI solutions were further mixed with soy oil at ø ¼ 0.2, and subject to the same emulsification as described above. The droplet size distribution of various emulsions, freshly prepared or after storage of 1.0 h, was determined using a Malvern MasterSizer 2000 (Malvern Instruments Ltd, Malvern, Worcestershire, UK). Distilled water was used as the dispersant. The relative refractive index of the emulsion was taken as 1.47. Droplet size measurements are reported as the volume-average diameter, d4,3 P P (¼ nid4i / nid3i , where ni is the number of particles with diameter di). All determinations were conducted at least in duplicate. 2.6. Amount of proteins adsorbed to the interface or entrapped within gel network The influence of an enzymatic pre-incubation (at 37 C for specific incubation periods of 0, 15, 30 and 60 min), on the amount of proteins adsorbed to the interface, or entrapped within the gel network, of the resultant emulsions (freshly prepared) or emulsion gels (after an incubation period of 2 h) was evaluated. The emulsions or emulsion gels (with the novel process) were prepared at a protein concentration of 6 g/100 mL and ø ¼ 0.4, and at an enzyme concentration of 1.0 U/g. The amount of proteins entrapped within the gel network was evaluated using the method of Chanyongvorakul, Matsumura, Sawa, Nio, and Mori (1997) that was applied to evaluate the adsorbed and unadsorbed proteins in the emulsions, with a few modifications. Aliquots (1.0 g) of the emulsions, freshly prepared or after incubation at 37 C for a storage period of 2 h, in microcentrifuge tubes, were centrifuged in a micro-centrifuge at 10,000 g for 30 min at room temperature. The protein concentration of separated aqueous layer was determined by the Bradford assay, using BSA as the standard. The percent of entrapped proteins was calculated as (1-protein concentration in separated aqueous layer/initial protein concentration in the aqueous phase) 100.
10000
G' (Pa)
The microstructure of the emulsion gels was evaluated with a confocal laser scanning microscope (Leica TCSSP5, Heidelberg, Germany), using Rhodamine B and Nile blue as fluorescence dyes for the protein and oil phases, with excitation wavelengths at 488 and 633 nm, respectively. Both stock dye solutions (1.0 g/100 mL) were mixed with the emulsions up to final concentrations of about 0.05 g/100 mL, prior to gel formation. The emulsion gels containing the dyes (with a total amount of 80 mL) were directly formed in concave confocal microscope slides (Sail; Sailing Medical-Lab Industries Co. Ltd., Suzhou, China), covered with glycerol-coated coverslips (that were further sealed with tin foil). The CLSM images were obtained with a 100 magnification lens. The oil phases or droplets dyed with the Nile blue are usually green, whereas the proteins dyed with the Rhodamine B are red. Actually, most of the protein-coated oil droplets would be predominantly yellow in appearance of the overlay image (green þ red / yellow).
17
1000
100
10 0.1
1
10
Frequency (Hz) Fig. 1. Frequency dependence of G0 for the SPI emulsion gels, induced by MTGase at enzyme concentrations of 0 (-), 1.0 (C), 2.5 (:) and 5.0 (;) U/g, respectively, for an incubation period of 2 h. The enzyme was added before the emulsification. The protein concentration and ø were 6.0 g/100 mL and 0.2, respectively.
2.7. Statistics An analysis of variance (ANOVA) of the data was performed, and a least significant difference (LSD) with a confidence interval of 95% was applied to compare the means. 3. Results and discussion 3.1. Rheological characteristics of emulsion gels 3.1.1. Dependence on enzyme concentration Contrasting from the conventional process to form enzyme-set emulsion gels, where the MTGase was added after the emulsion formation, the present work applied a novel process to add the enzyme before the emulsification. Once a specific amount of the enzyme was mixed with the protein solution (with a constant concentration of 6 g/100 mL), the resultant mixture was immediately mixed with various ø values of oil phase and subject to the homogenization (or emulsification). After that, the freshly formed emulsions were incubated at 37 C for an incubation period of 2 h on the rheometer, and their dynamic viscoelasticity was then evaluated. Fig. 1 shows the frequency dependence of storage modulus (G0 ) of various SPI emulsion gels, formed at various enzyme concentrations (0, 1.0, 2.5 and 5.0 U/g). We can generally observe that the G0 of all the untreated or MTGase-treated emulsions was almost independent of the frequency in the range 0.1e 10.0 Hz, which is a typical characteristic of a network consisting of permanent covalent cross-links (Dickinson & Yamamoto, 1996). In the present case, the control emulsion without the addition of MTGase also exhibited a gel-like behavior, which is consistent with the previous finding that the microfludization could produce a kind of gel-like SPI emulsions, by bridging flocculation of oil droplets, mainly through inter-droplet hydrophobic interactions between the proteins adsorbed at the interface (Tang & Liu, 2013), but seems to be different from the observation of our previous work (Yang et al., 2011; where at a comparable ø value the G0 was highly dependent on the frequency over the tested frequency range of 0.1e10 Hz). The difference might reflect that the formed original emulsions were kinetically unstable, e.g. against flocculation of oil droplets, and when the emulsions were incubated at 37 C for 2 h, severe flocculation of oil droplets might occur, thus leading to formation of gel-like network. At any test frequency, we can see that the G0 progressively and markedly increased with the enzyme concentration from 0 (control) to 5.0 U/g (Fig. 1). For example, the G0 (2300 Pa) of the emulsion gel with 1.0 U/g of MTGase was more than 20-fold than that (w100 Pa) of the control (without MTGase addition). These
18
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
3.2. Microstructure of the emulsion gels
Fig. 2. The ø dependence of the G0 (at a frequency of 1.0 Hz) of SPI-stabilized emulsion gels, formed after an incubation period of 2 h. Novel process (,): The emulsion gels were induced by the MTGase (1.0 U/g; added before the emulsification); conventional process (B): The gels were formed by 20 U/g of MTGase (added after the emulsification). Each data is the means and standard deviations of duplicate measurements. Dash lines within the figure indicate fitting of G0 data with exponential equations (ln G0 f ø).
observations indicated strengthening of gel network as a result of increased intermolecular covalent cross-links among proteins. The G0 value was even considerably higher than that previously reported for the emulsion gel (at the same protein concentration and ø) formed at a much higher enzyme concentration (20 U/g) for a much longer period (6 h) (using the conventional process; Yang et al., 2011). This suggests that the MTGase addition prior to the emulsification greatly improved the enzyme-induced gelation of SPI-stabilized emulsions. 3.1.2. Effect of oil fraction (ø) Fig. 2 shows the ø dependence of the G0 (at 1 Hz) of the MTGaseset emulsion gels, obtained with the enzyme added before (novel process) or after (conventional process) the emulsion formation, after 2 h of incubation. In the novel process, the applied enzyme concentration in the continuous phase was 1 U/g, whereas in the conventional case, it was 20 U/g. In both the cases, we can see that the G0 progressively increased, in an exponential way (ln G0 f ø, with a relationship coefficient R2 of 0.90e0.98), as the ø increased from 0.2 to 0.6 (Fig. 2). This observation is basically consistent with the fact that the protein-coated oil droplets usually act as ‘active fillers’ reinforcing the emulsion-filled gel network, wherein the reinforcing effect will be stronger with more dispersed oil droplets trapped or fully integrated into the network, at higher ø values (Chen & Dickinson, 1999; Chen, Dickinson, Langton, & Hermansson, 2000). The oil droplet packing effect can be also observed in other gel-like SPI-stabilized emulsions (Tang & Liu, 2013). The progressive increase in the stiffness of the emulsion gels upon the increase in ø was remarkably higher than that without MTGase addition (data not shown), reflecting that besides the packing effect, the enhanced reinforcing of the emulsion gel network by increasing the ø was largely dependent on the higher extent of MTGase-induced covalent cross-links. This is because in concentrated emulsions, the oil droplets are close enough to interact with one another, and as a result, inter-droplet covalent cross-links between the adsorbed proteins at the interface can be efficiently formed. By comparison, we can see that at any test ø, the G0 of the emulsion gels with the novel process was considerably higher than that with the conventional process, though in the former case, the amount of applied enzyme was much lower. The magnitude of the difference in G0 between the emulsion gels with the two processes was greater at higher ø values (Fig. 2). This comparison further indicated that the novel process with MTGase added before the emulsification exhibited excellent potential to efficiently form the protein-stabilized emulsion gels.
The microstructure of the enzyme-set SPI-stabilized gels, formed at three selected ø values of 0.2, 0.4 and 0.6, after 2 h of incubation, with the novel and conventional processes, respectively, was characterized using CLSM, as displayed in Fig. 3. As expected, the emulsion gels formed in both the cases exhibited similar microstructure, considerably varying with the applied ø. The emulsion gel at ø ¼ 0.2 was mainly composed of a homogenously filamentous network with the oil droplets entrapped and even becoming an integral part. The filamentous network, more like the protein gels in nature, was gradually transformed into the network mainly composed of packing or aggregated oil droplets, as the ø increased from 0.2 to 0.6 (Fig. 3). The microstructure of the emulsion gels formed with the conventional process is similar to that observed in our previous work (Yang et al., 2011). On the other hand, we can see that the microstructure of the emulsion gels formed with the novel process was more homogenous and compact than that with the conventional process (Fig. 3). Especially at ø ¼ 0.6, a distinct difference in microstructure could be observed for the emulsion gels between with the novel and conventional processes. With the novel process, all the oil droplets were closely associated together forming a compact and integral part of the network. In the conventional case, almost all of the oil droplets were present in the aggregated form; and there was distinct interspace observed among individual oil droplets (Fig. 3). The observations reflect that in the novel process case, the unadsorbed proteins played a major role in the network formation, which seemed to act as a kind of ‘binding agent’ reinforcing the gel network (mainly consisting of aggregated oil droplets). Thus, the differences in microstructure could well explain the difference in gel stiffness between the emulsion gels with the two processes (Fig. 2).
3.3. Effects of pre-incubation before the emulsification The above MTGase-set emulsion gels were obtained without a pre-incubation of the enzyme with the proteins prior to the emulsification. In this situation, we reasonably hypothesized that the freshly prepared emulsions in the presence of MTGase were basically identical to those in the absence of the enzyme. This means that the gelling mechanism with the novel process should be mainly through covalent cross-linking of adsorbed proteins at the interface of individual oil droplets. In this aspect, the thickness of the adsorbed protein films at the interface would produce an important role for the inter-droplet protein cross-linking. To confirm this, we investigated the influence of an enzymatic preincubation (with the protein solutions; before the emulsification) on the gelation of SPI-stabilized emulsions at a specific ø of 0.4. The pre-incubation of the protein solutions with MTGase might result in formation of high molecular weight biopolymers of soy proteins, as observed in many previous literatures (Tang, Wu, Chen, & Yang, 2006; Tang, Wu, Yu, et al., 2006). Once these biopolymers are adsorbed to the interface of oil droplets, the thickness of protein interfacial films will be much thicker than that in the uncrosslinked case. As a consequence, the MTGase-induced gelation of the emulsions should be favored. From the enzyme-induced gelation profiles, as displayed in Fig. 4, we can observe that an enzyme pre-incubation (15e60 min) of the protein solutions (before the emulsification) not only shortened the incubation period needed for the initial formation of the emulsion gel network, but also remarkably strengthened the stiffness of the correspondingly formed emulsion gels. This observation clearly indicated that the cross-linking of the proteins before the emulsification greatly favored the gelation of the emulsions.
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
19
Fig. 3. Typical CLSM images of enzyme-set SPI-stabilized emulsion gels at various ø values (0.2e0.6). Novel process: An enzyme concentration (1.0 U/g) of MTGase was added before the emulsification; conventional process: An enzyme concentration (20 U/g) of MTGase was added after emulsification. All the images were obtained with the dyes Rhodamine B (protein) and Nile blue (oil phase), excited at 488 and 633 nm, respectively. The bars indicate 10 mm in length.
Interestingly, the best improvement of the gelation was observed after a pre-incubation period of 15 min, and a further increase (30 or 60 min) in pre-incubation period on the contrary impaired the improvement (as compared to that at 15 min) (Fig. 4). To further unravel the underlying mechanism, we analyzed the microstructure of the emulsion gels, formed with three selected pre-incubation periods (0, 15 and 60 min) by CLSM, as shown in Fig. 5. Herein, we can totally see that the emulsion gel obtained with a pre-incubation period of 15 min exhibited a more homogenous network, in which all the oil droplets were evenly entrapped and dispersed in the network (Fig. 5). By comparison, in the
12000
G'(Pa)
9000 6000 3000 0
emulsion gel with 60 min of pre-incubation, the oil droplets with larger size (indicative of coalescence) were observed (Fig. 5), which might be a result of impaired emulsifying properties of proteins by an extensive cross-linking. These observations suggested that the decreased stiffness (observed after prolonged pre-incubation periods, e.g., 60 min) was largely due to both the formation of less homogenous network (relative to that after 15 min of preincubation) and impairment of emulsifying properties of proteins by the enzymatic cross-linking. Previous research had indicated that an extensive cross-linking reduced the stability of milk protein (sodium caseinate or b-lactoglobulin) emulsions prior to emulsification to coalescence or flocculation, whereas limited cross-linking improved the coalescence stability (Faergemand et al., 1998). A similar impairment of the emulsifying properties by the MTGase cross-linking has been reported for soy glycinin and/or b-conglycinin (Hu, Xu, Fan, Cheng, & Li, 2011). Thus, the decreased stiffness observed after prolonged pre-incubation periods (e.g., 30e 60 min) might be largely associated with the impaired emulsion stability of SPI by extensive cross-linking. 3.4. Changes in physicochemical parameters of the emulsions: influence of pre-incubation
0
20
40
60
80
100
120
Time (min) Fig. 4. Effects of pre-incubation [with the protein solution; 0 (,) 15 (B), 30 (6) and 60 (7) min] prior to the emulsification on the MTGase-induced gelation of SPIstabilized emulsions at ø ¼ 0.4. The enzyme concentration was 1.0 U/g. The gelation process was recorded at a frequency of 1.0 Hz.
3.4.1. Volume mean droplet size (d4,3) The influence of a pre-incubation of SPI with MTGase (1.0 U/g) for various incubation periods (0e60 min) on its emulsifying properties (including emulsifying ability and emulsion stability) was evaluated by light scattering technique. After the specific periods of preincubation, the enzyme was inactivated by addition of 1.0 mol/L NH4Cl (to a final concentration of 10 mmol/L). Fig. 6 shows the volume
20
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
mean droplet size (d4,3) of the freshly prepared emulsions (ø ¼ 0.2), stabilized by SPI pre-incubated with MTGase for various periods. A low ø (0.2) was used, due to the consideration that at this ø, the formed emulsions were relatively stable against creaming, at least within a short period (e. g., 1 h). We can see that the pre-incubation with MTGase resulted in a progressive increase in d4,3 of droplets in the freshly formed emulsions, from 0.73 to 1.20 mm, as the pre-incubation period increased from 0 to 60 min (Fig. 6). However, significant increases were observed only after pre-incubation above 15 min. These observations confirmed that an extensive enzymatic cross-linking (in the present work, through a pre-incubation with MTGase above 15 min) led to impairment of emulsifying ability of SPI. On the other hand, we also evaluated the influence of a preincubation (0e120 min) with 1.0 U/g of MTGase on the d4,3 of the particles in the SPI solutions (6 g/100 mL) alone, using a Nanosizer. The results showed that the d4,3 of the particles in the solutions slightly but progressively increased from 0.052 (control) to 0.087 or 0.101 mm, as the pre-incubation period increased from 0 to 60 or 120 min (Data not shown). The increased d4,3 was clearly as a result of the polymerization or aggregation of the protiens, induced by the covalent crosslinking. Thus, the progressive increase in d4,3 of the droplets in the emulsions upon increasing the pre-incubation period could be partially attributed to the formation of high molcular biopolymers. The d4,3 of droplets in all the emulsions significantly increased after a storage period of 1.0 h (Fig. 6), indicating instability of these emulsions. Since the MTGase was inactivated before the emulsion formation, the significant increases in d4,3 could be thus largely due to bridging flocculation or coalescence of oil droplets. On the other hand, we can still observe that the instability against flocculation or coalescence was more distinct at longer periods of pre-incubation (Fig. 6), suggesting that increasing the pre-incubation period with MTGase (or the covalent cross-linking) accelerated the instability of the emulsions. This observation further confirmed that the decreased gel stiffness at prolonged pre-incubation periods was largely due to increased extent of oil droplet flocculation or coalescence (thus resulting in formation of in homogenous network). 3.4.2. Amount of the protein entrapped within gel matrix The MTGase-induced gelation of SPI emulsions is closely associated with the covalent or non-covalent interactions of proteins, adsorbed and/or unadsorbed. Once the emulsion gel network is formed, the proteins, including those adsorbed to the interface of oil droplets and even some of unabsorbed proteins, will be entrapped within the gel matrix, which can be approximately estimated using centrifugation of the emulsions or emulsion gels. Fig. 7 shows the influence of the enzymatic pre-incubation period (0e60 min) before the emulsification, on the amount of proteins entrapped within the gel matrix for the SPI emulsions (with MTGase), freshly formed or after storage of 2 h at 37 C. We can observe that the amount of entrapped proteins progressively increased from about 60 to 71 g/100 g, as the pre-incubation period increased from 0 to 60 min, indicating that the cross-linking treatment of the proteins before the emulsification led to more proteins participating in the gel network formation. After any preincubation period, the amount of entrapped proteins within the gel matrix was slightly but insignificantly increased by storage for an incubation period of 2 h (Fig. 7). These observations, together with the droplet size data (Fig. 6), suggest that at ø ¼ 0.4 (in the present work), the initiation of the
Fig. 5. Typical CLSM images of MTGase-set SPI-stabilized emulsion gels at ø ¼ 0.4, formed with various pre-incubation periods (before the emulsification) of 0 (a), 15 (b) and 60 (c) min, respectively. The arrows within figure indicate large oil droplets. The images were dyed with the same dyes as in the legend of Fig. 3.
C.-h. Tang et al. / LWT - Food Science and Technology 53 (2013) 15e21
21
(before the emulsification) could also greatly improve the gelation. The initiation of the emulsion gel network was largely attributed to the bridging flocculation of oil droplets, but the full development of the gel stiffness mainly depended on the inter-droplet covalent cross-linking between the protein-coated oil droplets. The findings are of great importance for the development of an important kind of emulsion gels, especially as the matrix of functional foods. Acknowledgments This work is supported by the National Natural Science Foundation of China (serial numbers: 31071504, 31171632 and 31130042), and Program for New Century Excellent Talents in University (NCET-10e0398). Fig. 6. Influence of an enzymatic pre-incubation (0e60 min) with the proteins (before the emulsification) on the mean droplet size (d4,3) of the emulsions at ø ¼ 0.2, freshly prepared ( ) or after storage of 1.0 h ( ). The pre-incubation of the protein solutions (6 g/100 mL) with 1.0 U/g of MTGase was conducted at 37 C for specific periods (0, 15, 30 and 60 min), and the enzyme then immediately inactivated. Different symbols (aec) within figure indicate significant difference at p < 0.05 level, among different pre-incubation periods. Different symbols (eef) indicate significant difference at p < 0.05 level, due to storage of 1.0 h.
emulsion gel network might be largely attributed to the bridging flocculation of oil droplets, but it should be the inter-droplet crosslinks that play a predominant role in the development of the gel stiffness, or strengthening of the gel network (though the extent of enzymatic covalent cross-linking might be low). This thus could explain why with the novel process, the application of a much less enzyme concentration (1.0 U/g) of MTGase could more efficiently form the SPI emulsion gels with much higher stiffness than the conventional process, with a much higher enzyme concentration (e.g., 20 U/g), since this low enzyme concentration seemed to be enough to induce covalent cross-links between the neighboring aggregated oil droplets. 4. Conclusions A novel process to efficiently form MTGase-set SPI-stabilized emulsion gels was proposed, wherein the enzyme was added prior to the emulsification, instead of after the emulsification in the conventional case. This novel process needed a much less enzyme amount, but produced the emulsion gels with much higher stiffness (than the conventional process). Increasing the ø progressively accelerated the MTGase-induced gelation of the emulsions. Furthermore, a pre-incubation of the proteins with the enzyme
Fig. 7. Influence of an enzymatic pre-incubation (0e60 min) before the emulsification on the amount of proteins (g/100 mL) adsorbed at the interface or entrapped within the network of SPI-stabilized emulsion gels (with 1.0 U/g of MTGase), freshly formed (,) or after 2 h of storage (B) at 37 C. Each data is the means and standard deviations of duplicate measurements. The protein concentration and ø were 6 g/100 mL and 0.4, respectively.
References Chanyongvorakul, Y., Matsumura, Y., Sawa, A., Nio, N., & Mori, T. (1997). Polymerization of b-lactoglobulin and bovine serum albumin at oilewater interfaces in emulsions by transglutaminase. Food Hydrocolloids, 11, 449e455. Chen, J., & Dickinson, E. (1998a). Viscoelastic properties of protein-stabilized emulsions: effect of protein-surfactant interactions. Journal of Agricultural and Food Chemistry, 46, 91e97. Chen, J., & Dickinson, E. (1998b). Viscoelastic properties of heat-set whey protein emulsion gels. Journal of Texture Studies, 29, 285e304. Chen, J., & Dickinson, E. (1999). Effect of surface character of filler particles on rheology of heat-set whey protein emulsion gels. Colloids and Surfaces B: Biointerfaces, 12, 373e381. Chen, J., Dickinson, E., Langton, M., & Hermansson, A.-M. (2000). Mechanical properties and microstructure of heat-set whey protein emulsion gels: effect of emulsifiers. LWT-Food Science and Technology, 33, 299e307. Chen, L., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials as nutraceutical delivery systems. Trends in Food Science & Technology, 17, 272e283. Dickinson, E. (2012). Emulsion gels: the structuring of soft solids with proteinstabilized oil droplets. Food Hydrocolloids, 28, 224e241. Dickinson, E., & Yamamoto, Y. (1996). Rheology of milk protein gels and proteinstabilized emulsion gels cross-linked with transglutaminase. Journal of Agricultural and Food Chemistry, 44, 1371e1377. Faergemand, M., Jeanette, O., & Karsten, B. Q. (1998). Emulsifying properties of milk proteins cross-linked with microbial transglutaminase. International Dairy Journal, 8, 715e723. Hu, X., Xu, X., Fan, J.-F., Cheng, Y.-Q., & Li, L. (2011). Functional properties of microbial transglutaminase modified soybean glycinin and b-conglycinin. International Journal of Food Engineering, 7. art. no. 4. Kang, Y. N., Kim, H., Shin, W. S., Woo, G., & Moon, T. W. (2003). Effect of disulfide bond reduction on bovine serum albumin-stabilized emulsion gel formed by microbial transglutaminase. Journal of Food Science, 68, 2215e2220. Lee, H. A., Choi, S. J., & Moon, T. W. (2006). Characteristics of sodium caseinate- and soy protein isolate-stabilized emulsion-gels formed by microbial transglutaminase. Journal of Food Science, 71, C352eC357. Liang, L., Sok Line, C. L., Remondetto, G. E., & Subirade, M. (2010). In vitro release of a-tocopherol from emulsion-loaded b-lactoglobulin gels. International Dairy Journal, 20, 176e181. Matsumura, Y., Kang, H.-J., Sakamoto, H., Motoki, M., & Mori, T. (1993). Filler effects of oil droplets on the viscoelastic properties of emulsion gels. Food Hydrocolloids, 7, 227e240. Nio, N., Motoki, M., & Takinami, K. (1986). Gelation of protein emulsion by transglutaminase. Agricultural and Biological Chemistry, 50, 1409e1412. 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-d-lactone (GDL), and transglutaminase: Influence of thermal treatments before and/or after emulsification. Journal of Agricultural and Food Chemistry, 59, 4071e4077. Tang, C. H., & Liu, F. (2013). Cold, gel-like soy protein emulsions by microfludization: emulsion characteristics, rheological and microstructural properties, and gelling mechanism. Food Hydrocolloids, 30, 61e72. Tang, C. H., Wu, H., Chen, Z., & Yang, X. Q. (2006). Formation and gel properties of glycinin-rich and b-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase. Food Research International, 39, 87e97. Tang, C. H., Wu, H., Yu, H. P., Li, L., Chen, Z., & Yang, X. Q. (2006). Coagulation and gelation of soy protein isolates induced by microbial transglutaminase. Journal of Food Biochemistry, 30, 35e55. Tang, C. H., Yang, M., Liu, F., & Chen, Z. (2013). Stirring greatly improves transglutaminase-induced gelation of soy protein-stabilized emulsions. LWTFood Science and Technology, 51, 120e128. Velikov, K. P., & Pelan, E. (2008). Colloidal delivery systems for micronutrients and nutraceuticals. Soft Matter, 4, 1964e1980. Yang, M., Liu, F., & Tang, C. H. (2011). Properties and microstructure of transglutaminase-set soy protein-stabilized emulsion gels. Food Research International, . http://dx.doi.org/10.1016/j.foodres.2011.11.012.