Food Chemistry 161 (2014) 324–331

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Complexation of resveratrol with soy protein and its improvement on oxidative stability of corn oil/water emulsions Zhi-Li Wan a, Jin-Mei Wang a, Li-Ying Wang a, Yang Yuan a, Xiao-Quan Yang a,b,⇑ a Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China b 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

Article history: Received 12 October 2013 Received in revised form 3 March 2014 Accepted 2 April 2014 Available online 13 April 2014 Keywords: Resveratrol Soy protein isolate Protein–polyphenol interaction Oil/water emulsion Oxidative stability

a b s t r a c t This work was to evaluate the potential of soy protein isolate (SPI)–resveratrol (RES) complex as an emulsifier to improve the effectiveness of RES as a natural antioxidant in corn oil-in-water emulsions. The physical properties and oxidative stability of emulsions stabilized by the native SPI–RES and heated SPI–RES complexes were evaluated. The water solubility of RES was enhanced by complexation with SPI, which was mainly driven by hydrophobic interactions. Heat treatment favoured the formation of the SPI–RES complex and endowed it with a higher antioxidant activity. Furthermore, the emulsions stabilized by the SPI–RES complexes showed an increased oxidative stability with reduced lipid hydroperoxides and volatile hexanal. This improving effect could be attributed to the targeted accumulation of RES at the oil–water interface accompanied by the adsorption of SPI, as evidenced by the high interfacial RES concentration. These findings show that the soy protein–polyphenol complex exhibited a good potential to act as an efficient emulsifier to improve the oxidative stability of emulsions. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Oil-in-water (O/W) emulsions form the basis for many food products, such as milk, beverages, sauces and dressings. Lipid oxidation in O/W emulsions is believed to be more likely to occur due to the higher interfacial area of emulsions, where the oxidative reaction has been suggested to be initiated (Berton, Ropers, Viau, & Genot, 2011; Frankel, Huang, Kanner, & German, 1994; McClements & Decker, 2000). The oxidative degradation of lipids causes unpleasant quality changes of food emulsions, such as the development of unpalatable flavours and odours, nutrient degradation and colour changes (McClements & Decker, 2000; Shahidi & Zhong, 2010). Therefore, various antioxidants are usually incorporated into O/W emulsions to improve their oxidative stability. Recently, there has been growing interest in the use of natural polyphenols to retard lipid oxidation due to their putative health-promoting properties and remarkable antioxidant activity (Brewer, 2011).

⇑ Corresponding author at: Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China. Tel.: +86 20 87114262; fax: +86 20 87114263. E-mail address: [email protected] (X.-Q. Yang). http://dx.doi.org/10.1016/j.foodchem.2014.04.028 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Resveratrol (trans-3,5,40 -trihydroxystilbene, RES) is a natural polyphenol found in the skin of grapes, apples, peanuts and in some traditional herbs. It has been associated with many benefits for human health, including antioxidant, anti-inflammatory, anticancer, antiplatelet aggregation, cardioprotective and antiobesity effects (Vang et al., 2011). The antioxidant capacity of RES has been verified by the inhibition of lipid peroxidation induced by many in vivo and in vitro systems (Gülçin, 2010; Kimura et al., 1983; Soares, Andreazza, & Salvador, 2003). However, scarce reports of the use of RES as a food antioxidant have been found so far (Filip et al., 2003; Medina et al., 2010). In fact, the extremely low solubility of RES in both water and oil has limited its application in functional foods, especially in emulsified systems, as an antioxidant due partly to the unpredictable physical distribution of RES (Laguerre et al., 2009). Therefore, in a previous study, to strengthen the effectiveness of RES as an antioxidant in O/W emulsions, a water-soluble RES was prepared by the encapsulation of RES in stevioside self-assembled micelles, and enhanced the antioxidant efficiency of RES by purposefully accumulating RES at the oil–water interface (Wan, Wang, Wang, Yang, & Yuan, 2013). Moreover, many other approaches, such as complexation with cyclodextrin and its derivatives, or using bile acid micelles, nanoemulsions and nanoparticle delivery systems, have also been made to increase the water solubility and bioavailability of RES (Amri, Chaumeil, Sfar, & Charrueau, 2012).

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Food proteins, such as soy protein, hold the attractive advantages of being natural, nontoxic, and widely available, are ideal binders of polyphenol by formation of protein–polyphenol complex and have been widely used to improve the water solubility, stability, and bioavailability of RES. Liang, Tajmir-Riahi, and Muriel (2008) reported that complexing with b-lactoglobulin provides a significant increase in the solubility of RES and a slight increase in its photostability. The work from Hemar, Gerbeaud, Oliver, and Augustin (2011) showed that a whey protein isolate and its b-lactoglobulin- and a-lactalbumin-rich fractions could form 1:1 complexes with RES, thus providing an excellent carrier for RES. In addition, many investigators also observed the stabilizing effects on RES of various proteins, such as collagen, sodium caseinate and bovine serum albumin (BSA) (Acharya, Sanguansri, & Augustin, 2013; Bourassa, Kanakis, Tarantilis, Pollissiou, & Tajmir-Riahi, 2010; Zhang, Mi, & Shen, 2012). Moreover, previous studies have shown that in vitro bioaccessibility and antioxidant activities of polyphenols were not influenced by the interaction with proteins (Tapal & Tiku, 2012; van der Burg-Koorevaar, Miret, & Duchateau, 2011). In searching for food proteins to form complexes with RES, we focused on soy protein isolate (SPI). SPIs are used extensively in the food industry, due to their functional properties, low cost, availability and high nutritional values. Recently, Tapal and Tiku (2012) demonstrated that SPI could be used as a carrier for water-insoluble curcumin, and the water solubility and stability of curcumin were thus enhanced. Grace et al. (2013) and Roopchand et al. (2013) found that soy proteins could stably bind and concentrate cranberry polyphenols to form a cranberry polyphenol–SPI complex, thus creating protein/polyphenol-enriched matrices. On the other hand, soy proteins are also widely used as emulsifiers in foods because of their excellent ability to form and stabilize O/W emulsions. Considering the fact that the effectiveness of antioxidants in O/ W emulsions would be promoted by their accumulation at the oil– water interface (Laguerre et al., 2009; Lucas et al., 2010), we thus postulated that the use of protein–RES complex as emulsifiers in an O/W emulsion system may result in the targeted accumulation of RES at the oil–water interface, due to the adsorption of surfaceactive proteins at oil droplet surface, and thus could improve the efficiency of RES in the interfacial resistance to lipid oxidation (Frankel et al., 1994; McClements & Decker, 2000). Therefore, the objective of this work was to improve the water solubility of RES by complexation with SPI, and then to purposefully accumulate RES at the oil–water interface by using the SPI–RES complex as an emulsifier, thus enhancing the effectiveness of RES as a natural antioxidant in O/W emulsions. The potential of SPI as a carrier for RES was first studied through interactions between SPI and RES using fluorescence spectroscopy. In general, heat treatment could induce structural changes of proteins, affecting the binding of RES, and thus the interaction between SPI and RES during heating was examined. The antioxidant activities of the native SPI–RES and heated SPI–RES complexes prepared were also evaluated. Subsequently, the physical properties and oxidative stability of corn oil/water emulsions stabilized by the native SPI–RES and heated SPI–RES complexes were investigated using lipid hydroperoxides and headspace hexanal analysis.

2. Materials and methods 2.1. Materials RES (purity >98%) was purchased from Shanxi Tianrun Phytochemical Co., Ltd., China. 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,20 -Azo-bis(2-amidinopropane) dihydrochloride (AAPH), and 6-

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hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma–Aldrich (St. Louis, MO). Defatted soy flour was provided by Shandong Yuwang Industrial and Commercial Co., Ltd., China. SPI was prepared from flour by alkaline extraction (pH 8.0) followed by precipitation at pH 4.5, as described by Wang et al. (2012). The precipitate collected was washed twice with distilled water, re-dispersed in distilled water, and then neutralised to pH 7.5 with 2 M NaOH. Subsequently, the protein solution was dialyzed against distilled water at 4 °C for 48 h before freeze-drying. The protein content of SPI was 88.8%, determined by the Dumas method (N  5.71, wet basis) in a Rapid N Cube (Elementar France, Villeurbanne, France). Corn oil was purchased from a local supermarket and used without further purification. The selection of corn oil was made due to its common use as cooking media and also relatively high reaction rate of oxidative deterioration (Naz, Sheikh, Siddiqi, & Sayeed, 2004). All other chemicals used were of analytical grade. 2.2. Preparation of SPI–RES complex The SPI dispersions (2.0%, w/v) were prepared by dissolving lyophilized SPI in distilled water and stirring at room temperature (22 °C) for 2 h. The pH of the prepared SPI dispersions was adjusted to pH 7.0 with either HCl or NaOH. RES (0.1005 g) was mixed with 50 ml of the SPI dispersions by homogenising at 8000 rpm for 5 min using an Ultra-Turrax T25 (IKA-Werke GMBH & CO., Germany). In a typical heat treatment, the resulting mixture was heated at 90 °C for 30 min in a water bath (TW12; Julabo, Seelbach, Germany), then immediately cooled in an ice bath. This heat treatment ensured extensive denaturation of SPI (Renkema & Van Vliet, 2002), which might affect the binding of RES. The free RES was removed by centrifugation (10,000g, 30 min) and the supernatant was freeze-dried in a Christ DELTA 1-24 LSC freeze-dryer (Christ, Germany) to get the dry SPI–RES complex powder. The freezedryer was set at a shelf temperature of 30 °C and a condenser temperature of 55 °C before the main drying was initiated by lowering the vacuum to 0.340 mbar. The shelf temperature was gradually increased to 30 °C, which was maintained for 24 h. The SPI–RES complex powder was suspended at 2.0% w/v in distilled water and was stirred at room temperature (22 °C) for 2 h. Then, the SPI–RES dispersions (1 ml) were added to 9 ml of acetonitrile and stirred for 5 min. After centrifugation at 15,000g for 20 min, the supernatant was used for RES quantification. Each sample was filtered through a 0.22 lm filter (Millipore, Billerica, MA, USA) prior to HPLC determination. Quantitative analysis of RES was performed according to the method described in the previous study (Wan et al., 2013) 2.3. Fluorescence spectroscopy The fluorescence spectra were recorded using an F7000 fluorescence spectrophotometer (Hitachi Co., Japan). RES was dissolved in ethanol at a concentration of 1 mg/ml as a stock solution, which was diluted with 10 mM phosphate buffer (pH 7.0) before use. The fluorescence of RES was measured by fixing its concentration at 5 lg/ml and by varying the concentration of SPI from 0 to 2.5 mg/ml. The emission spectra were recorded from 350 to 550 nm with an excitation wavelength of 320 nm. Protein intrinsic fluorescence was measured at a constant SPI concentration (0.5 mg/ml) in the presence of 0–20 lg/ml RES. Emission spectra were recorded from 300 to 500 nm at an excitation wavelength of 280 nm. Both the excitation and emission slit widths were set at 5 nm. Fluorescence quenching is described according to the Stern– Volmer equation (Eq. 1) (Lakowicz, 2006): F0/F = 1 + kqs0[Q] = 1 + KSV[Q]. In this equation F0 and F are the fluorescence

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intensities in the absence and presence of a quencher, respectively, [Q] is the quencher concentration, KSV is the Stern–Volmer quenching constant, kq is the bimolecular quenching rate constant, and s0 is the lifetime of fluorescence in the absence of a quencher. Hence, Eq. 1 was applied to determine KSV by linear regression of a plot of F0/F versus [Q]. Fluorescence quenching can be further classified as dynamic or static quenching. For the case of static quenching, the quenching data can be analysed according to a modified Stern–Volmer model (Eq. 2) (Lakowicz, 2006): F0/DF = F0/(F0  F) = 1/(faKa[Q]) + 1/fa. In this equation fa is the fraction of accessible fluorescence, and Ka is the effective quenching constant for the accessible fluorophores, which can be treated as an associative binding constant between a quencher and an acceptor. The linear regression between F0/(F0  F) and 1/[Q] enables the determination of 1/faKa (slope) and 1/fa (intercept), and therefore Ka. 2.4. Determination of antioxidant activity The DPPH radical scavenging activity, oxygen radical absorbance capacity (ORAC) and reducing power assays were carried out following the methods described by Samaranayaka and LiChan (2008) and Zheng et al. (2012). The final assay concentration of SPI/SPI–RES complex was 5 mg/ml for the DPPH radical scavenging activity and reducing power assays, while 0.1 mg/ml was used for ORAC assay. 2.5. Emulsion preparation and characterization The O/W emulsions were prepared by dispersing 10 wt% corn oil in a 10 mM phosphate buffer (pH 7.0) containing 1% (w/v) SPI, native SPI–RES, or heated SPI–RES complexes. Coarse emulsions were made using an Ultra-Turrax T25 (IKA-Werke GMBH & CO., Germany) at 6000 rpm for 2 min. Fine emulsions were prepared by passing coarse emulsions twice at 500 bar through an M110EH-30 microfludizer processor (Microfludics, U.S.A.). After the emulsification process, the pH of each prepared emulsion was measured and adjusted to pH 7.0 with either HCl or NaOH. Sodium azide (0.04 wt%) was then added to the emulsions to prevent microbial growth. Emulsions were placed in tightly sealed screwcap vials and stored in a dark oven at 40 °C for 14 days. The mean particle size and size distribution of the emulsions were measured immediately after homogenisation using a Mastersizer 2000 (Malvern Instruments Co. Ltd., Worcestershire, UK) at 25 °C. The refractive indices of corn oil and phosphate buffer were taken as 1.467 and 1.330, respectively. It was daily checked to monitor emulsion stability and reported as a volume-surface mean P P diameter d32 = nidi3/ nidi2, where ni is the number of particles with diameter di. The microstructure of the emulsions was measured using a confocal laser scanning microscope (CLSM, Leica Microsystems Inc., Heidelberg, Germany) with a 100 oil immersion objective lens. A staining solution of 40 ll containing 0.1% (w/v) Nile Red and 0.1% (w/v) Nile Blue A (fluorescent dye) was added to the aliquots (1 ml) of emulsion samples: Nile Red for the oil and Nile Blue A for the protein. The stained emulsions (50 ll) were placed on concave confocal microscope slides and examined using an argon krypton laser (ArKr, 488 nm) and a helium neon laser (HeNe, 633 nm). The oil phases stained are usually green, while the proteins are red.

Japan). After centrifugation, the subnatants were carefully removed using a syringe and filtered through 0.45 lm filter (Millipore, Billerica, MA, USA). The total protein content of the subnatants was determined by the micro-Kjeldahl method (N  5.71). The interfacial protein proportion was calculated from the difference between the amount of protein used to prepare the emulsion and that measured in the subnatants after centrifugation. Determination of the distribution of RES in the emulsions was performed according to the procedure described by Panya et al. (2010). The emulsions were centrifuged as above, and the amounts of RES in subnatants were determined by HPLC system (Waters, USA). 2.7. Measurements of lipid oxidation 2.7.1. Lipid hydroperoxide Lipid hydroperoxide was measured according to the method described by Tong, Sasaki, McClements, and Decker (2000). Emulsion samples (0.2 ml) were mixed with 1.5 ml of isooctane/2-propanol (3:1, v/v) and vortexed (10 s, three times). After centrifugation at 3400g for 2 min, 200 ll of the organic solvent phase was mixed with 2.8 ml of methanol/1-butanol (2:1, v/v). Hydroperoxide detection was started by the addition of 15 ll of 3.94 M ammonium thiocyanate and 15 ll of ferrous iron solution (prepared by mixing 0.132 M BaCl2 and 0.144 M FeSO4). Twenty minutes later, the absorbance was measured at 510 nm using a UV–vis spectrophotometer (Genesys 10, Thermo Scientific, USA). Hydroperoxide concentrations were determined using a standard curve made from hydrogen peroxide. 2.7.2. Headspace analysis Fresh emulsion samples (8 ml) were transferred into special 10 ml headspace vials and sealed with silicone rubber Teflon caps with a crimper. After storage in a dark oven at 40 °C for 14 days, headspace hexanal was analysed according to the method of Panya et al. (2010) using a Trace DSQ II GC/MS (Thermo-Fisher Scientific, USA). A 75 lm carboxen/polydimethylsiloxane (Carboxen/ PDMS) stable flex solid phase microextraction (SPME) fibre (Supelco, USA) was inserted through the vial septum and exposed to the sample headspace for 10 min at 55 °C. The SPME fibre was desorbed at 250 °C for 3 min in the GC detector at a split ratio of 1:10. The chromatographic separation was performed using a TR5MS capillary column (30 m; 0.25 mm i.d.; 0.25 lm film thickness). The temperatures of the oven, injector, and flame ionisation detector were 60, 250 and 250 °C, respectively. The sample run time was 15 min. The analysis was carried out based on the results obtained from three replicates of each emulsion sample. 2.8. Statistical analysis Unless specified otherwise, three independent trials were performed, each with a new batch of sample prepared. All measurements were carried out in triplicate, and an analysis of variance (ANOVA) of the data was performed using the SPSS 19.0 statistical analysis system. The Duncan Test was used for comparison of mean values among the three treatments using a level of significance of 5%. 3. Results and discussion

2.6. Determination of protein and RES at the oil–water interface 3.1. Water solubility enhancement of RES by complexation with SPI The concentration of the protein adsorbed at the oil–water interface was determined according to the method described by Ye, Srinivasan, and Singh (2000). The fresh emulsions were centrifuged at 15000g for 2 h at 20 °C using a himac CS150NX Micro Ultracentrifuge with an S140AT rotor (Hitachi Koki Co. Ltd., Tokyo,

Fig. 1 shows the image of the RES dispersions and RES solubility for the freeze-dried powders of the native SPI–RES and heated SPI– RES complexes after their reconstitution in distilled water (2%, w/ v). At the concentration of 0.28 mg/ml, the free RES appeared very

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Fig. 1. Water solubility of RES in the native SPI–RES and heated SPI–RES powders after their reconstitution in water. The inset shows the images of free RES (a, 0.28 mg/ml) and RES in the native SPI–RES (b, 0.28 mg/ml) and heated SPI–RES (c, 0.81 mg/ml) powders redispersed in water.

turbid because of its low solubility of 22.9 lg/ml (Fig. 1, inset). However, the transparent dispersions were obtained by redispersing the native SPI–RES and heated SPI–RES powders in distilled water, which suggested the solubilisation of hydrophobic RES. The solubility of RES quantitatively supported this observation (Fig. 1). The concentrations of RES solubilised in native SPI–RES and heated SPI–RES reconstituted solutions (2%, w/v) were 0.28 mg/ml and 0.81 mg/ml (Fig. 1), representing an evident increase in the water solubility of RES about 12 and 35 times, respectively, compared to that of free RES (22.9 lg/ml). It was estimated that the absolute concentrations of RES were 14.0 mg/g SPI– RES powder and 40.5 mg/g in the heated SPI–RES powder, respectively. The greater content of RES in the heated SPI–RES complex powder should be mainly attributed to the fact that the heat treatment could induce structural changes and expose previously buried hydrophobic sites of SPI, thus increasing the binding of RES in SPI. Similar results were observed by Yazdi and Corredig (2012), who reported that heat treatment increased curcumin binding in milk. 3.2. Characterization of the interaction between SPI and RES In order to better understand the underlying reason for water solubility enhancement of RES by complexation with SPI, the interactions between SPI and RES in the native and heated systems were thus studied using fluorescence spectroscopy. 3.2.1. RES fluorescence measurements The fluorescence of RES is very sensitive to the polarity of its surrounding environment. In general, the fluorescence maximum (kmax) of RES shifts to a shorter wavelength and the fluorescence intensity increases as the polarity decreases. Fig. 2 shows the fluorescence spectra of RES with different SPI concentrations in the native (Fig. 2A) and heated (Fig. 2B) systems, respectively. Free RES (5 lg/ml) showed a low broad fluorescence peak at around 396 nm in phosphate buffer (Fig. 2A), and no obvious difference was found in the fluorescence spectra of RES after heat treatment (Fig. 2B). When interacting with SPI in the native (Fig. 2A) and heated (Fig. 2B) systems, the fluorescence emission spectra of RES showed similar changes. The gradual blue shift of kmax and an increase in the RES fluorescence intensity were observed as the SPI concentration increased, suggesting the movement of RES from a hydrophilic to a more hydrophobic environment. Similar

Fig. 2. Fluorescence spectra of RES (5 lg/ml) with various amounts of SPI (0– 2.5 mg/ml) in native (A) and heated (B) systems in 10 mM phosphate buffer at pH 7.0.

results were reported for the binding of RES with b-lactoglobulin (Liang et al., 2008). The blue shift of kmax and increase in fluorescence intensity were due to the binding of RES to the non-polar regions of SPI molecules, suggesting that the interaction of RES with SPI was hydrophobic in nature. Furthermore, compared to the native SPI–RES system, the blue shift of kmax and the increase in emission intensity were more evident in the heated SPI–RES system (Fig. 2). This result indicated that the medium environment of RES in heated SPI–RES system was more hydrophobic, which was mainly due to more hydrophobic sites exposed in the heated SPI, thus assisting the binding of RES with SPI. These results are in good agreement with previous analysis (Fig. 1). 3.2.2. Fluorescence quenching measurements The fluorescence quenching method was further used to study the binding reaction between RES and SPI. The fluorescence spectra of SPI with different concentrations of RES in native (Fig. 3A) and heated (Fig. 3B) systems are shown in Fig. 3. Native SPI alone exhibited a strong fluorescence emission, with a peak at around 336 nm (Fig. 3A). After heat treatment, the fluorescence intensity of the SPI increased with a red shift of maximum emission from 336 to 338 nm (Fig. 3B), suggesting that the increased exposure of the major fluorophore tryptophan (Trp) residues to a more polar environment. Similar fluorescence phenomena were observed in

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3.3. Antioxidant activity of native SPI–RES and heated SPI–RES complexes In this study, we attempted to improve the effectiveness of RES in O/W emulsions by using the SPI–RES complex as an emulsifier. The antioxidant activity of antioxidants is usually believed to be associated with their ability to retard lipid oxidation in emulsions. Thus, prior to the study of the SPI–RES complex in emulsion oxidation, the antioxidant activities of the native SPI–RES and heated SPI–RES complexes were estimated by three common methods. The results are presented in Table 1. Under the specific concentration for the assays, the SPI and heated SPI showed similar antioxidant activities from the values of DPPH radical scavenging activity, ORAC and reducing power (Table 1). The native SPI–RES and heated SPI–RES complexes exhibited stronger antioxidant activities when compared to the SPI and heated SPI, respectively. This increase in antioxidant activity could be attributed to the presence of RES in the SPI–RES complex. Moreover, these values of antioxidant activity of heated SPI–RES complex were significantly higher than those of the native SPI–RES complex. This observation could be mainly due to the increased RES present in the heated SPI–RES complex, which has been evidenced by previous analysis (Fig. 1). 3.4. Physical characterization of emulsions

Fig. 3. Fluorescence spectra of SPI with various amounts of RES in native (A) and heated (B) systems in 10 mM phosphate buffer at pH 7.0. (a, a0 ) 0.5 mg/ml SPI alone; (b–g, b’–g’) 0.5 mg/ml SPI with 0.5, 1, 2, 5, 10, and 20 lg/ml RES. The dotted line (B) designates native SPI. Insets: Modified Stern–Volmer plots describing fluorescence quenching of SPI in the presence of RES in native (A) and heated (B) systems.

whey protein isolates, b-lactoglobulin and a-lactalbumin (Zhang & Zhong, 2012). After the addition of RES, the fluorescence spectra of SPI in the native (Fig. 3A) and heated (Fig. 3B) systems showed similar changes. The fluorescence intensity of SPI decreased gradually with an increasing concentration of RES and a clear red shift was observed, suggesting that there was an obvious interaction between the SPI and RES in the two systems. The fluorescence quenching data were analysed using the Stern–Volmer Eq. 1. The values of kq for the two systems were calculated to be 7.03  1012 M1 s1 and 8.10  1012 M1 s1 (data not shown), respectively, which were much higher than the maximal dynamic quenching constant (2.0  1010 M1 s1). This indicated that the quenching process between the SPI and RES was mainly due to static quenching by the formation of SPI–RES complex. This result is in line with previous reports (Acharya et al., 2013; Bourassa et al., 2010; Hemar et al., 2011; Liang et al., 2008). Furthermore, the effective quenching constant Ka was calculated using the modified Stern–Volmer Eq. 2, and the corresponding Ka values were given in the insets of Fig. 3. It could be seen that the Ka value was 4.42  104 M1 for the native SPI–RES system, which was lower than that of the heated SPI–RES systems (10.52  104 M1), suggesting that heat-treated SPI was favourable for the formation of the SPI–RES complex. These results further supported previous analysis (Figs. 1 and 2).

3.4.1. Particle size distribution The particle size distributions and changes in the mean droplet diameter (d32) (data not shown) for emulsions stabilized by native SPI–RES and heated SPI–RES complexes were used to assess the emulsifying ability and physical stability, as shown in Fig. 4A. The fresh emulsion stabilized by native SPI exhibited a more narrow size distribution and smaller particle size, with d32 at 0.386 lm when compared with those of the heated SPI emulsion (d32 0.661 lm) (Fig. 4A). This suggested that heat treatment of the SPI at 90 °C resulted in a decrease in its emulsifying ability. This result might be related to the formation of protein aggregates with a larger size in the heated SPI, which hindered the fast adsorption of heated SPI to the oil droplet surface during homogenisation, thus impairing its emulsifying activity (Kiokias, Dimakou, & Oreopoulou, 2007). Similar findings have been reported by other authors (Kiokias et al., 2007; Shen & Tang, 2013). For emulsions stabilized by the native SPI–RES complex, with increasing RES concentration, no significant difference (p > 0.05) in particle size distributions or d32 (data not shown) was observed when compared with those of the SPI emulsion, suggesting no obvious changes in its emulsifying ability. Similar trends were found in emulsions stabilized by the heated SPI–RES complex (Fig. 4A). These results indicated that there was no major change in the emulsifying properties of SPI after complexation with RES. The existence of RES in the native STE–RES/heated SPI–RES complex might not influence the adsorption of the native SPI/heated SPI at the oil–water interface. These results were further supported by CLSM observations (Fig. 4A). Similar results were observed in a previous report (Tapal & Tiku, 2012). Moreover, the distribution in the size remained monomodal and there was no obvious difference in the d32 for all the emulsions during the storage studies (data not shown). 3.4.2. Interfacial protein adsorption fraction (Fads) and RES concentration Fig. 4B shows the interfacial protein adsorption fraction (Fads) and the RES concentration of the emulsions stabilized by native SPI–RES and heated SPI–RES complexes, respectively. As shown in Fig. 4B, the native SPI emulsion had a Fads of 44.9%, which was lower than that of the heated SPI emulsion (54.7%), suggesting that heat treatment significantly increased the amount of SPI adsorbed

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Z.-L. Wan et al. / Food Chemistry 161 (2014) 324–331 Table 1 Antioxidant activities of native SPI, heated SPI, native SPI–RES and heated SPI–RES complexes after reconstitution in watera.

a b c

Dispersion

DPPH radical scavenging activityb (%)

ORACc (lM trolox)

Reduing powerb (Abs at 700 nm)

Native SPI Heated SPI Native SPI–RES Heated SPI–RES

17.2 ± 0.08a 15.4 ± 0.58a 57.6 ± 0.22b 71.6 ± 0.92c

75.1 ± 2.97a 73.8 ± 3.74a 161 ± 3.88b 277 ± 9.89c

0.045 ± 0.001a 0.040 ± 0.001a 0.216 ± 0.005b 0.374 ± 0.006c

Different letters indicate significant differences between groups (p < 0.05). The concentration of samples was at 5 mg/ml for the DPPH radical scavenging activity and reducing power assays, repectively. The concentration of samples was at 0.25 mg/ml for the ORAC assays.

was consistent with the analysis for the particle size distribution (Fig. 4A). Furthermore, an increase in the concentration of RES in the native SPI–RES and heated SPI–RES complexes resulted in an increase in the interfacial RES concentration (Fig. 4B). For emulsions stabilized by the native SPI–RES complex, the concentrations of RES in the aqueous phases of the native SPI–RES 0.005% and SPI– RES 0.01% emulsions were 0.0032% and 0.0064%, respectively, representing that around 64% of the initial RES was accumulated at the oil–water interface. Compared to the native SPI–RES complex emulsions, all emulsions with the heated SPI–RES complex showed a higher interfacial accumulation of RES (around 77% of initial RES), which could be mainly attributed to the higher Fads in the heated SPI–RES complex emulsions. In the present study, native SPI–RES and heated SPI–RES complexes were used as emulsifiers to form and stabilize emulsions, respectively. Therefore, it is speculated that RES may migrate to the interface, a process that is simultaneously accompanied by the adsorption of surface-active SPI at the oil–water interface, thus resulting in the high interfacial accumulation of RES.

3.5. Improved oxidative stability of emulsions

Fig. 4. (A) Particle size distributions of fresh emulsions stabilized by the native SPI– RES and heated SPI–RES complexes. The insets show the confocal micrographs of emulsions. (B) Interfacial protein adsorption fraction (Fads) and RES concentration in the aqueous phase of emulsions. Native SPI and heated SPI designate emulsions stabilized by native SPI and heated SPI, respectively. Native SPI–RES 0.005%/SPI–RES 0.01% and heated SPI–RES 0.005%/SPI–RES 0.01% designate emulsions stabilized by native SPI–RES/heated SPI–RES complex with RES concentration at 0.005% and 0.01%, respectively.

at the oil–water interface. As compared to the native SPI, the heated SPI exposed more hydrophobic parts due to protein unfolding, which facilitated protein anchoring at the oil–water interface and the formation of a thicker membrane at the interface (Kiokias et al., 2007; Wang et al., 2012), thus resulting in the increase in the Fads of emulsions. For the emulsions stabilized by the native SPI–RES complex, compared to the native SPI emulsion (44.9%), there was no significant difference (p > 0.05) on the Fads when the RES concentration was increased. This result suggested that the presence of RES had no effect on the Fads. Similar trends were also observed in emulsions with the heated SPI–RES complex. These results further indicated that the existence of RES in native STE–RES/heated SPI–RES complex could not influence the adsorption of native SPI/heated SPI at the oil droplets surface, which

Considering the fact of the high interfacial accumulation of RES, the oxidative stability of the emulsions stabilized by the native SPI–RES and heated SPI–RES complexes was further studied by lipid hydroperoxide and hexanal measurements, as shown in Fig. 5. Lipid hydroperoxides, generally accepted as the first oxidation products, were measured to observe the initial oxidation rate of the emulsions (Fig. 5A). As shown in Fig. 5A, compared with the hydroperoxides of the native SPI emulsion, it is noted that the heated SPI emulsion showed a higher level of hydroperoxides during storage (p < 0.05), suggesting that heat treatment of SPI led to a increase in the rate of lipid oxidation. For emulsions stabilized by the native SPI–RES/heated SPI–RES complex, lipid hydroperoxides were markedly lower compared to their corresponding native SPI/heated SPI emulsions (p < 0.05). Moreover, higher RES concentrations led to lower hydroperoxides, suggesting higher oxidative stability of emulsions. After 14 days of storage, the hydroperoxides for the native SPI–RES 0.01% and heated SPI–RES 0.01% emulsions were 62.0 mmol/kg oil and 68.4 mmol/kg oil, representing an evident decrease by about 45.8% and 43.0% in the formation of hydroperoxides, respectively, compared to those of their corresponding native SPI emulsion (114.3 mmol/kg oil) and heated SPI emulsion (119.9 mmol/kg oil). The results indicated that RES effectively decreased the formation of lipid hydroperoxides. In addition, the volatile compounds produced after 14 days were also analysed by headspace analysis (Fig. 5B). Hexanal is a major secondary oxidation product of corn oil. The whole chromatograms are shown with one identified peak for hexanal, and other peaks corresponding to the unidentified oxidation products. After 14 days of storage, the native SPI emulsion exhibited a peak area of hexanal (2.6  108), which was lower than that of the

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Fig. 5. Changes in lipid hydroperoxids (A) and headspace hexanal (B) of emulsions stabilized by the native SPI–RES and heated SPI–RES complexes during the storage at 40 °C for 14 days. The sample designations are exactly the same as in Fig. 4.

pro-oxidant agents present in the aqueous phase (Kiokias et al., 2007; McClements & Decker, 2000). However, partially aggregated SPI that resulted from heat treatment might be softer and have an increased porosity compared to the native SPI, and thus probably did not cover the oil–water interface as well as the native SPI. This would lead to a more heterogenous interfacial coverage and a higher oxygen permeability, thus offsetting the advantage of increased interfacial protein coverage. Similarly, recent studies have also reported that heat-treated whey proteins or b-lactoglobulin also slightly increased the rate of oxidation or did not improve the oxidative stability of the emulsions (Berton-Carabin, Genot, Gaillard, Guibert, & Ropers, 2013; Kellerby, McClements, & Decker, 2006; Phoon, Narsimhan, & Martin-Gonzalez, 2013), which were consistent with the present work. It is generally considered that the most likely mechanism for the acceleration of lipid oxidation in emulsions is the decomposition of lipid hydroperoxides into highly reactive free radicals by pro-oxidants, such as transition metals (McClements & Decker, 2000). Hydroperoxides in emulsion droplets are often surface active and, thus, accumulate at the oil surface. For this reason, lipid oxidation in O/W emulsions is thought to occur at the oil–water interface. Based on these understandings, how to inhibit interfacial oxidation may mainly determine the oxidative stability of emulsions. In general, the phenolic antioxidant RES could accept the free radicals by donation of phenolic hydrogen atoms, thus delaying or terminating the lipid oxidation reaction. This might be one of the dominant antioxidation mechanisms of RES in emulsions. In the present study, we employed SPI–RES complexes as emulsifiers for preparing emulsions. Thus, RES might accumulate at the oil– water interface accompanied by the adsorption of surface-active SPI at the droplet surface during the emulsification process, resulting in a high interfacial RES concentration (Fig. 4B). Such a high accumulation of RES at the interface would enhance the effectiveness of RES to scavenge free radicals produced from the decomposition of lipid hydroperoxides (Frankel, Huang, Kanner, & German, 1994; McClements & Decker, 2000), and thus the lipid oxidation in the emulsions stabilized by the native SPI–RES and heated SPI–RES complexes was effectively inhibited (Fig. 5). 4. Conclusion

heated SPI emulsion (3.3  108) (p < 0.05), which impled higher oxidative stability of the native SPI emulsion. For emulsions stabilized by the native SPI–RES and heated SPI–RES complexes, with increasing RES concentrations, a gradual decrease was observed for the peak area of hexanal in emulsions. The peak areas of hexanal for native SPI–RES 0.01% and heated SPI–RES 0.01% emulsions were 1.4  108 and 1.9  108, indicating an marked decrease by about 46.2% and 42.4% in the peak areas of hexanal, respectively, compared to those of their corresponding native SPI emulsion (2.6  108) and heated SPI emulsion (3.3  108). These results showed good correlation with those obtained from the lipid hydroperoxides analysis (Fig. 5A) and provided further evidence that RES effectively improved the oxidative stability of emulsions. These findings are in line with those observed in our previous study (Wan et al., 2013) and the work form Medina et al. (2010), where around 0.005–0.01% RES exhibited an evident improvement on the oxidative stability of emulsions. Based on the results of lipid hydroperoxides and headspace analysis, it is evident that native SPI emulsion showed a higher oxidative stability as compared to the heated SPI emulsion. Considering the higher Fads of the heated SPI emulsion (Fig. 4B), it was proposed that there was an increased oxidative stability for the heated SPI emulsion due to the formation of thicker protein films at the oil droplet surface, which may offer a stronger barrier to

In the present study, it was shown that the corn oil/water emulsions stabilized by a SPI–RES complex exhibited an enhanced oxidative stability. The complexation with SPI, mainly driven by hydrophobic interactions, markedly increased the water solubility of RES. Heat treatment favoured the formation of a SPI–RES complex and endowed it with an enhanced antioxidant activity. Furthermore, the corn oil/water emulsions stabilized by the native SPI–RES and heated SPI–RES complexes showed an increased oxidative stability with reduced lipid hydroperoxides and volatile hexanal. This improved antioxidant effectiveness of RES in emulsions should be mainly attributed to the high interfacial accumulation of RES. These findings show that SPI could be used as a carrier for RES in functional foods, and opens up the possibility of the use of a protein–polyphenol complex as an efficient emulsifiers to improve the oxidative stability of O/W emulsions. Acknowledgements This research was supported by grants from the Fundamental Research Funds for the Central Universities (SCUT, 2013ZM0052), the Chinese National Natural Science Foundation (Nos. 31371744 and 31130042), and the Open Project Program of Process of Starch and Vegetable Protein Engineering Research Center of Ministry of Education (2012-ERC-04).

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