Impact of pH, freeze–thaw and thermal sterilization on physicochemical stability of walnut beverage emulsion

Food Chemistry 196 (2016) 475–485

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Impact of pH, freeze–thaw and thermal sterilization on physicochemical stability of walnut beverage emulsion Shuang Liu, Cuixia Sun, Yanhui Xue, Yanxiang Gao ⇑ Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China

a r t i c l e

i n f o

Article history: Received 15 May 2015 Received in revised form 15 September 2015 Accepted 18 September 2015 Available online 25 September 2015 Keywords: Walnut beverage emulsion Mixed emulsifiers Xanthan gum Freeze–thaw Thermal sterilization Stability

a b s t r a c t The effects of environment stresses on the stability of walnut emulsion were investigated. The physical stability of walnut emulsion was characterized by droplet size, zeta potential and chemical stability of walnut oil in the emulsion was evaluated by determining the peroxide concentration during the storage. The results showed that emulsion in the presence of xanthan gum and mixed emulsifiers exhibited better stability after 4 freeze–thaw cycles. At pH 3–10, the mixed emulsifiers could improve the stability through their absorption on the oil–water interface. However, xanthan gum couldn’t protect the droplets against the aggregation in high acid environment, but greatly enhanced the physical stability at pH 6–10. During the thermal sterilization process, the physical stability of walnut emulsion was decreased with a rise of sterilization temperature and the extension of sterilization time. The sterilization temperature above 121 °C and time over 25 min led to the poor physical and oxidative stability. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Walnut (Juglans regia L.) is one of the oldest tree nuts cultivated, which is native to the southeastern Europe, eastern Asia and northern America, and it is rich in vitamins B and E, which can prevent cell aging, delay senescence and enhance the memory. It is confirmed that walnut can improve the symptoms of many kinds of diseases, such as neurasthenia, hypertension, coronary heart disease, emphysema and stomach pain (Sze-Tao & Sathe, 2000). Walnut beverage emulsion is oil-in-water dispersion, which is usually composed of oil phase and aqueous phase. The oil phase is walnut oil, and the aqueous phase includes water, walnut protein and some emulsifiers. In recent years, walnut beverage emulsion market has been strengthened in many countries, however, it is a thermodynamically unstable system because of higher level of oil in walnut kernel and the poor emulsifying property of walnut protein absorbed on the oil–water interface, which tends to become unstable during the storage through a series of physicochemical mechanisms, including creaming, flocculation, coalescence, and Ostwald ripening (McClements, 2004). Due to the impact of environment stresses, such as freeze–thaw treatment and extreme pH, the beverage emulsion leads to destabilize. Therefore, the beverage emulsion, such as coconut milk, ⇑ Corresponding author at: Box 112, No. 17 Qinghua East Road, Haidian District, Beijing 100083, PR China. E-mail address: [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.foodchem.2015.09.061 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

peanut and almond emulsions, needs some emulsifiers and polysaccharides to be stabilized. Taking coconut milk for an example, to make a stable product, emulsifiers are usually applied during manufacturing. The stabilized coconut milk is subsequently preserved by chilling, freezing, pasteurization, or sterilization, which provides additional stresses on the emulsion structure. Tangsuphoom and Coupland (2009) studied the effect of thermal treatment on the stability of homogenized coconut milk and the properties of coconut milk prepared with surface-active stabilizers, the coconut milk with surface-active proteins or small-molecule surfactants of sufficient concentration exhibited better stability. On the other hand, kernel oil is chemically unstable and prone to oxidative deterioration, especially when exposed to oxygen, light, moisture and heat (Ng, Lau, Tan, Long, & Nyam, 2013). To find a solution for this problem, the emulsification, microencapsulation and other systems could be designed as delivery systems to protect walnut oil from the oxidation. Other delivery systems could be also applied to food and other industries, such as nanoemulsions, multiple emulsions, filled hydrogel particles and solid lipid particles (McClements, 2010). To design homogeneous emulsions with small particle size and high stability under the storage condition, small molecule surfactants, proteins and polysaccharides are used as emulsifiers or stabilizers. Some researchers found that proteins at the interface of the emulsion droplets were able to reduce lipid hydroperoxides by electrostatically inhibiting iron–hydroperoxide interactions and prevent oil droplet from the aggregation during the storage (Gu, Decker, & McClements, 2004). However, small

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Fig. 1. Effect of mixed emulsifiers and xanthan gum on the droplet size (a), PdI (b), zeta-potential (c), integral transmission (d) and viscosity (e) of walnut beverage emulsion. Error bars represent standard deviations (n = 3).

molecule surfactants were preferred to stabilize the emulsion principally through steric repulsion, and hence their effects were much less sensitive to pH. Homayoonfal, Khodaiyan, and Mousavi (2014)

studied the influence of ultrasonic time, walnut oil content and the emulsifier concentration on the response variables including emulsion capacity, cloudiness, density and surface tension of walnut oil

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in water nanoemulsion by RSM in conjunction with CCRD. These studies revealed that a linear term of walnut oil concentration was the most significant parameter for the all responses. Until now, the mechanism of stabilizing real walnut beverage emulsion system was not reported by using emulsifiers and polysaccharides. To the best of our knowledge, little information is available concerning the impact of environment stresses on physicochemical stability of walnut beverage emulsion. The objective of this study was to evaluate the influence of thermal sterilization, freeze–thaw and pH value on the physical and oxidative stability of walnut beverage emulsion in the presence of mixed emulsifiers and/or xanthan gum. 2. Materials and methods 2.1. Materials Walnut kernels were purchased from Dinghui Food Co., Ltd. (Hebei, China) and stored at 19 °C until used. Xanthan gum was obtained from Xinhe Biochemical Co., Ltd. (Hebei, China). Glycerol monostearate (GMS, HLB: 3.8) was obtained from Danisco (Beijing, China) Co., Ltd. Decaglycerol monolaurate (DML, HLB: 15.5) was obtained from Evonik Food Development Co., Ltd. (Shanghai, China). Sucrose was purchased from a local retailer. Analytical grade hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Sigma Aldrich (USA).

2.2. Walnut kernel slurry The peeled walnut kernels were mixed with deionized water at the weight ratio of 1:4.5 and processed into the slurry. The crude slurry was refined through a colloid mill and filtered through three layers of gauze to remove the solid residues. 2.3. Walnut beverage emulsion Previously, we evaluated the species and amounts of emulsifiers (GMS, DML, and sucrose ester) and polysaccharides (xanthan gum, soybean polysaccharides and arabic gum) employed in the emulsion. In order to obtain stable emulsion, the formulation of walnut beverage emulsion was designed as follows: sucrose (6.5% wt), xanthan gum (0.09% wt), GMS (0.18% wt, HLB 3.8), DML (0.07% wt, HLB 15.5), refined slurry (18% wt), and deionized water (71.16% wt). The final level of walnut protein and oil in the formula was 0.85% and 1.97%, respectively. To achieve a fine emulsification with small mean particle size and narrow particle size distribution, pre-homogenization was performed by using an Ultra-Turrax at a speed of 10,000 rpm for 3 min and passing through a two-stage valve homogenizer (Niro-Soavi Panda, Parma, Italy) for three cycles at 80 MPa and 65 °C. The final emulsion was immediately cooled down to 25 °C and then transferred into screw-capped brown bottles.

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Fig. 3. Effect of freeze–thaw cycles on the droplet size (a), PdI (b) and physical stability (c) of walnut beverage emulsion in the presence of mixed emulsifiers and/or xanthan gum. Error bars represent standard deviations (n = 3).

2.4. pH stability Freshly prepared walnut beverage emulsion was of pH 7.4 and then the pH was adjusted to 3–10 using 0.1 M NaOH or 0.1 M HCl. The samples were allowed to stand for 10 h at ambient temperature (29 ± 2 °C). After that, the physical properties of the emulsions with different pH values were measured. 2.5. Freeze–thaw stability Emulsion samples (18 mL) were transferred into cryogenic test tubes (diameter 27.5 mm, height 70 mm), which were frozen in a freezer at 19 °C for 22 h and thawed in a 30 °C water bath for 2 h. The freeze–thaw cycling was repeated for 4 times. 2.6. Thermal sterilization Emulsion samples (55 mL) were transferred into brown bottles with metal caps and tightened by the automatic capper. Sealed bottles were put in the YXQ-LS-50 retort (Boxun Instrument,

Shanghai, China) under different thermal conditions. For better to understand the thermal stability of walnut beverage emulsion, following two modes were designed to treat samples: different thermal sterilization times were monitored at the same temperature (121 °C) and different thermal sterilization temperatures were adopted at the same time (25 min). After thermal sterilization, the emulsions were immediately cooled down to 25 °C and then transferred into constant temperature incubator of 37 °C and 55 °C for 4 weeks, respectively. 2.7. Analytical methods 2.7.1. Mean particle size and its distribution The mean particle size and its distribution of samples were determined with a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) at a fixed detector angle of 90°. To avoid multiple scattering effects, the samples were diluted with deionized water at a ratio of 1:800 (v/v). Droplet size and its distribution were described as cumulant mean diameter (size, nm) and polydispersity index (PdI).

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2.7.2. Zeta potential Zeta potential of the samples was determined by measuring the direction and velocity of droplet movement in the applied electric field using Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK). The samples were diluted with deionized water at the ratio of 1:800 (v/v), then the diluted emulsion was injected directly into the chamber of a Nano-ZS90 particle electrophoresis instrument prior to zeta potential analysis (Wang et al., 2014). 2.7.3. Physical stability The physical stability of the samples was measured by the LUMiSizer (L.U.M. 290 GmbH, Germany) according to the method of Lei, Liu, Yuan, and Gao (2014) with some modifications. LUMiSizer is a multi-sample analytical instrument employing centrifugal sedimentation to accelerate the occurrence of instability phenomena such as sedimentation, flocculation or creaming (Sobisch & Lerche, 2008). The integration graph shows the percentage of light absorbance as a function of time and

position over the entire sample, the ‘‘creaming rate”. The rate is correlated to the physical stability of the emulsion: the lower the creaming rate, the higher the stability. The parameters used for the measurement were set as follows: rotational speed, 4000 rpm; 7650 s; temperature, 25 °C; time interval, 30 s (Lei et al., 2014). 2.7.4. Oxidative stability Lipid hydroperoxides were measured using a modified method of Shantha and Decker (1993). The walnut beverage emulsions were stored at 37 °C and 55 °C, respectively. The lipid peroxides were measured at an interval of 1 week. Three milliliters of methanol/1-butanol (2:1, v/v) were mixed with 20 lL of an emulsion sample and 30 lL of thiocyanate/Fe2+ solution. The thiocyanate/Fe2+ solution was prepared immediately before using by mixing equal amount of thiocyanate solution (3.94 M ammonium thiocyanate) and Fe2+ solution (obtained from the supernatant of a mixture of 3 mL of 0.144 M BaCl2 in 0.4 M HCl and 3 mL of freshly prepared 0.144 M FeSO4). After 20 min, the absorbance was

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measured at the wavelength of 510 nm with a UV-1800 UV–vis spectrophotometer (Shimadzu, Japan) and lipid peroxides were quantitated from a standard curve using Fe3+. 2.7.5. Rheological behavior of walnut emulsion Rheological behavior of walnut emulsion was measured with an AR 1500 Rheometer (TA Instruments, UK), using a cone and plate geometry (cone diameter 40 mm, angle 2, gap 0.100 mm). For each measurement, 2.0 mL of emulsion was carefully deposited over the plateau of the rheometer. Steady state flow measurements were carried out at 25 ± 0.1 °C in the range of 0–100 s 1. The rheological parameters (shear stress, shear rate, apparent viscosity) were obtained from the software of TA. 2.8. Statistical analysis All experiments were performed in triplicate and the results were expressed as mean value ± standard deviation (SD) in this study. Data were collected and analyzed using the software provided with the instrument (Origin, MicroCal), and the difference was considered to be significant with p < 0.05.

3. Results and discussion 3.1. Effect of mixed emulsifiers and xanthan gum on physical stability of walnut beverage emulsion The effects of GMS, DML and xanthan gum on droplet size, PdI, zeta potential, physical stability and viscosity were shown in Fig. 1. In the absence of GMS, DML and xanthan gum, the zeta-potential of primary emulsion was around 15 mV, because the walnut protein in primary emulsion was negatively charged at pH 7.4 over pI (4.6–5.0). With the addition of GMS and DML, the droplet size and PdI were decreased, and the absolute value of zeta potential was increased, as shown in Fig. 1(a–c), which suggested that the emulsifiers could improve the surface activity of droplets. Emulsifiers could be absorbed on the oil–water interface through hydrogen bonding and decrease the interfacial tension against the droplets coalescence (Isaac et al., 2013). Due to the absorption of the emulsifiers around the droplets, the steric repulsion was formed among droplets. GMS and DML, as non-ionic surfactants, are lipophilic and hydrophilic emulsifiers, respectively. The blended emulsifiers with two extreme HLB values would result in the strongly lipophilic

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Fig. 6. Size (1) and oxidative stability (2) changes of the droplets in walnut beverage emulsion at different sterilization temperatures for the time of 25 min when stored at 37 °C (a) and 55 °C (b).

emulsifier mostly dissolving in the oil phase, whereas, the strongly hydrophilic emulsifier mostly dissolving in the water phase (Athas et al., 2014). That is, the emulsifiers prevent the walnut oil and water phase from separating by forming a protective barrier around the droplets (Nesterenko, Drelich, Lu, Clausse, & Pezron, 2014). The physical stability of walnut beverage emulsion was evaluated by monitoring the transmission change due to creaming and flocculation based on the space–time resolved extinction profiles (STEP). At the beginning of the measurement, the walnut beverage emulsion was intact with very little light transmitted at any point along the sample cell. Along with the centrifugation, the heavier and more transparent aqueous phase moved to the bottom, the lighter and less transparent oil phase moved to the top, thus a cream layer occurred (Zhao, Xiang, Wei, Yuan, & Gao, 2014). The more severe change of transmission with the centrifugation resulted in less stability of emulsions (Lei et al., 2014). The slope of this graph was equal to the velocity of creaming of the primary emulsion (Mert, 2012). Fig. 1(d) displayed the change of integral transmission vs centrifugation time. It could be found that the creaming rate was decreased as mixed emulsifiers were added.

Therefore, the mixed emulsifiers could enhance the physical stability of walnut beverage emulsion. As shown in Fig. 1(c), there existed a difference between the emulsions with xanthan gum and the control. In the presence of xanthan gum, the absolute value of zeta potential of the droplets in the emulsion was larger than that in the control, which implied the absorption of the anionic polysaccharide onto the surface of droplets, which implied that protein–polysaccharide complexes could be generally produced, although both walnut protein and the polysaccharide carried net negative charge (pH > pI). In this case, the attraction involved positively charged local patches on the protein interacting with the anionic polysaccharide (Dickinson, 1998). Meanwhile, the droplet size of xanthan gum coated emulsion was larger than that of the control, which interpreted that xanthan gum could form a thicker membrane around droplets. Therefore, the electrostatic and steric repulsions between droplets were stronger (Guzey & McClements, 2007), and the emulsion stabilized by xanthan gum exhibited better physical stability, which was in agreement with the result of Fig. 1(d). On the other hand, xanthan gum could increase the viscosity of the emulsion, as shown in Fig. 1(e). The higher viscosity can improve the

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physical stability of the emulsion, which could prevent the oil droplets from moving around and coalescing. In summary, the emulsion in the presence of xanthan gum exhibited better physical stability by electrostatic and steric repulsions (Harnsilawat, Pongsawatmanit, & McClements, 2006) as well as high viscosity. When both of the mixed emulsifiers and xanthan gum were added to the emulsion, the droplet size was decreased from 323.5 to 216.9 nm, which was in close proximity to the emulsion in the presence of mixed emulsifiers only. As a result, the electrostatic repulsion became much stronger. Thus, the emulsion stabilized by both mixed emulsifiers and xanthan gum was more stable than that in the presence of either mixed emulsifiers or xanthan gum. Therefore, the creaming rate of walnut beverage emulsion in the presence of mixed emulsifiers and xanthan gum was the lowest, as shown in Fig. 1(d). In summary, the mixed emulsifiers might be absorbed on the water–oil interface (steric repulsion). On the other hand, xanthan gum might form protein–polysaccharide complexes, which would increase the steric and electrostatic repulsion between droplets and reduce the van der Waals attraction (Chen, Li, Ding, & Rao, 2011). Xanthan gum could also enhance the viscosity of the continuous phase and increase the stability of the emulsion by retarding the droplets movement (Paraskevopoulou, Boskou, & Kiosseoglou, 2005). 3.2. Effect of pH on physical stability of walnut beverage emulsion Walnut beverage emulsion as a food product is consumed under different pH environments, for example, at high acid (pH < 4.6) or low acid (pH > 4.6). Moreover, the emulsion might be exposed to variable pH stresses in gastrointestinal tract (Tokle & McClements, 2011). Therefore, the stability evaluation of walnut beverage emulsion at different pH values has a practical significance. This part aimed to study the physical stability of walnut beverage emulsion at different pH values (3–10) in the presence of mixed emulsifiers and/or xanthan gum as stabilizers. Within this pH range, the droplet size, PdI, zeta potential and physical stability of walnut beverage emulsion were shown in Fig. 2. In the absence of mixed emulsifiers and xanthan gum, the zeta potential of primary emulsion droplets was changed from 17.7 to 2.9 mV as pH was decreased from 10 to 3, which was attributed to the pI (4.6–5.0) of walnut protein (Sze-Tao & Sathe, 2000). The pI of walnut protein could explain the decrease of zeta potential of the emulsion at pH 3–5. Below pI of walnut protein, the walnut protein was positively charged. However, the zeta potential of the emulsion was negative, which might be attributed to the fact that there were some negatively charged components in the emulsion, such as walnut oil bodies. In plants, seeds, and nuts, lipids are stored in oleosomes or oil bodies, which are oil droplets surrounded by a monolayer of phospholipids and embedded proteins called oleosins (Gallier, Tate, & Singh, 2013). Walnut beverage emulsion is a complicated thermodynamically unstable system. It was composed of a lot of substances, so it is difficult to understand the specific construction of walnut beverage emulsion. The mechanism responsible for the stability of walnut beverage emulsion at low pH was still not fully understood, thereby it is required for further research. When the pH was adjusted from 7.4 (original pH of the emulsion) to 10, the droplet size, PdI and zeta potential were not changed significantly. The absolute value of zeta potential of droplets in the emulsion stabilized with mixed emulsifiers and xanthan gum was greater than the control, which indicated that the anionic polysaccharide could absorb onto the surface of positively charged local patches on the protein at the pH range of 3–10. In the presence of xanthan gum, the absolute value of zeta potential of walnut beverage emulsion was decreased sharply as

pH was adjusted from 5 to 3, which could be ascribed to the fact that the pKa of xanthan gum is around 3.1. The carboxyl group of xanthan gum would be protonized at pH 3 and 4 and walnut protein was positively charged, therefore, there was not enough electrostatic force to deposit xanthan gum on the walnut protein coated droplets. At pH values of 3 and 4, the emulsion stabilized by mixed emulsifiers carried more negative charges than that by xanthan gum, which might attribute to the fact that the nonionic surfactants (mixed emulsifiers) is the acid-resistance (Malhotra & Coupland, 2004). At pH 5–10, the absolute value of zeta potential of the emulsion in the presence of mixed emulsifiers and xanthan gum was higher than that with either mixed emulsifiers or xanthan gum. This result suggested that the anionic polysaccharide could absorb onto the surface of droplets against the aggregation and flocculation by electrostatic force. On the other hand, the increasing of electrostatic and steric repulsions due to the absorption of the anionic polysaccharide could enhance the stability of droplets, as well as decreasing the van der Waals attraction at this range of pH (Li et al., 2010), and emulsifiers absorbed on the water and oil interface to break up droplets. Fig. 2(d) showed the recorded evolution of time-dependent transmission profiles of walnut beverage emulsions at different pH levels. The results of creaming stability measurements were in agreement with the droplet size, PdI and zeta potential analysis (Fig. 2(a–c)). In summary, these results revealed that mixed emulsifiers could improve the stability through absorbing themselves on the oil and water interface at pH 3–10. However, xanthan gum could not protect the droplets from aggregation at high acid environment. On the other hand, xanthan gum could greatly enhance the stability through increasing the viscosity, electrostatic and steric repulsion at pH 6–10. 3.3. Effect of freeze–thaw process on physical stability of walnut beverage emulsion Walnut beverage emulsion may undergo freezing and thawing treatments during the storage and consumption. The purpose of following experiments was to examine the influence of freeze– thaw cycle on the stability of walnut beverage emulsion. When walnut beverage emulsion was put in the freezer, some physical properties might be changed, such as crystallization in the oil or aqueous phase (Gu, Decker, & McClements, 2007). The instability of walnut beverage emulsion could be attributed to the crystallization of the oil or aqueous phase. When walnut beverage emulsion was thawed, the droplets tended to aggregate together, leading to the droplet coalescence and oil separation. Therefore, the unstable phenomenon would occur after the freeze–thaw treatment. Many studies found that polysaccharides, proteins and emulsifiers could contribute to the freeze–thaw stability of emulsions (Thanasukarn, Pongsawatmanit, & McClements, 2004; Zhao et al., 2014). In order to better understand this point, the walnut beverage emulsions underwent the process of freeze–thaw. After the freeze–thaw treatment, the changes of droplet size, PdI and physical stability of walnut beverage emulsions were shown in Fig. 3. For the primary emulsion, the droplet size was increased from about 300 nm to 1 lm after one cycle of freeze–thaw, and the emulsion was unstable with evidently a clear cream layer, indicating that the coalescence took place after the freeze–thaw. This result suggested that the primary emulsion stabilized by walnut protein was prone to destabilization during the freeze–thaw treatment. In the presence of xanthan gum only, the droplet size was also increased with the rise of the number of freeze–thaw cycle. This phenomenon interpreted that the interfacial membrane, which was formed by the polysaccharide, was ruptured by the ice crystal. However, as shown in Fig. 3(c), the emulsion in the

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presence of xanthan gum exhibited better physical stability during freeze–thaw, this might be attributed to the fact that the polysaccharide could increase viscosity of the emulsion, and the migration rate of droplets was decreased, according to the Stokes formula (Danov, Stoyanov, Vitanov, & Ivanov, 2012). Nevertheless, in the emulsion stabilized by mixed emulsifiers only, the droplet size was not changed significantly during freeze–thaw, indicating that the nonionic surfactants could absorb on the oil and water interface and form strong and thick interfacial membrane. Consequently, the droplets would be prevented from coalescence during freezing. As Thanasukarn et al. (2004) reported, Tween 20 as a single emulsifier was added to the oil-in-water emulsion, and it could not provide better protection against partial coalescence because of the relatively thinner interfacial layers. Therefore, mixed emulsifiers might form the relative thickness interfacial layer against droplets coalescence. When both the mixed emulsifiers and xanthan gum were applied to the emulsion, the droplet size and the physical stability were scarcely increased after first freeze–thaw cycle. From the second to fourth cycle, the droplet size and physical stability of the emulsion stabilized by xanthan gum were similar, but it was more stable than that without xanthan gum, as presented in Fig. 3(c). These results revealed that both xanthan gum and mixed emulsifiers were able to better protect the emulsion droplets from coalescence and flocculation during the freeze–thaw treatment. When walnut beverage emulsion was placed in the freezer, water became crystallized, which induced the droplets to come into closer proximity as the droplets in the emulsion were regarded as non-frozen regions remaining in the aqueous phase (Ghosh, Cramp, & Coupland, 2006). Surh, Gu, Decker, and Mcclements (2005) reported that the droplets in the emulsion surrounded by multiple layers of protein–polysaccharide complexes were more resistant to environment stresses than that surrounded by the protein only (Surh et al., 2005). Similarly, we could conclude that the combination of the polysaccharide and nonionic surfactants might be more resistant to environment stresses. These findings could be ascribed to the protein–polysaccharide complexes surrounding droplets and the high viscosity of the emulsions. Therefore, walnut beverage emulsion composed of xanthan gum and mixed emulsifiers was stable to freeze–thaw cycling. 3.4. Effect of thermal sterilization parameters on physical and chemical stability of walnut beverage emulsion The purpose of following experiments was to examine the influence of thermal treatment on the physical and chemical stability of walnut beverage emulsion stabilized with xanthan gum and mixed emulsifiers. There are many factors that can influence the rate of lipid oxidation in oil-in-water emulsions, such as oxygen concentration, particle size, the thickness and rheology of interfacial membrane (Waraho, McClements, & Decker, 2011), resulting in the generation of hydroperoxides. Raghavendra and Raghavarao (2010) reported that the oil droplets would aggregate during heating, leading to the destabilization of the emulsion. Proteins, polysaccharides and emulsifiers play an important role in the stability of the emulsion and the process of walnut beverage emulsion at higher temperature caused the protein denaturation. 3.4.1. Effect of thermal sterilization time on the stability of walnut beverage emulsion As presented in Fig. 4(a), the emulsions, treated for 0, 5, 15, 25 min, respectively, at 121 °C, they exhibited higher stability since it had smaller slope, while treated for 35 and 45 min, they exhibited poorer stability. The more severe change of transmission with the centrifugation revealed less stability of the emulsion.

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Furthermore, the creaming rate was estimated by plotting the integrated transmission profiles against the measuring time, and the slope of the resultant curve was inversely related to the stability (Yuan, Xu, Qi, Zhao, & Gao, 2013). These results indicated that walnut protein was prone to denaturation and coagulation (Tangsuphoom & Coupland, 2009), when the thermal sterilization temperature was over denaturation temperature (67.05 °C). Denaturation of walnut protein could lead to the destabilization of walnut beverage emulsion, which resulted in the aggregation of oil droplets. On the other hand, as mentioned above, the mixed emulsifiers could form interfacial membrane on the oil and water interface and xanthan gum could be absorbed on the droplet through forming protein–polysaccharide complexes. These properties could be benefit from protecting droplets from aggregation during thermal sterilization (Tangsuphoom & Coupland, 2009) at a short time. As the extension of thermal sterilization time, the walnut protein was destroyed more seriously by high temperature, and the interfacial layer might be damaged. Thus, the droplet size of walnut beverage emulsion was increased by thermal treatment, and the longer thermal sterilization time led to the formation of larger droplet size and could cause the plant protein denaturation seriously. During the storage, the droplet size and peroxide concentration of walnut beverage emulsion were measured. Fig. 5(1a) and (1b) showed that after 4 weeks of the storage at 37 °C and 55 °C, respectively, and the droplet size of all the thermally treated emulsions was hardly increased. The most possible explanation for this phenomenon was that the protein, polysaccharide and emulsifiers played an important role in the stability of walnut beverage emulsion. However, Fig. 5(2) showed that the peroxide concentration of walnut oil in the emulsion was significantly (p < 0.05) increased after 4 weeks of the storage both at 37 °C and 55 °C. After one week of the storage, an obvious increase of peroxide concentration was observed for all the thermally treated emulsions. At 37 °C and 55 °C, the lipid peroxide concentration in samples with different sterilization times was not changed significantly until 3 weeks of the storage. However, after 4 weeks of the storage, the lipid peroxide concentration in the emulsion with 45 min treatment at 121 °C was increased faster than others, which indicated that the rate of oil oxidation was accelerated. It revealed that longer thermal sterilization time would accelerate the lipid oxidation. However, the mechanism of lipid oxidation in oil-in-water emulsion differed from that in bulk lipids, because the emulsion had an aqueous phase containing both antioxidants and prooxidants and the oil– water interface impacted the interaction between oil and water components (Waraho et al., 2011), McClements & Decker (2000) reported that the transition metals were the major prooxidants in the oil-in-water emulsion. Meanwhile, the high temperature would result in the aggregation of oil droplets. Therefore, the most possible explanation for this phenomenon was that the heating and transition metals probably accelerated the rate of lipid oxidation in walnut beverage emulsion. On the other hand, the emulsifiers could form a barrier that decreased interactions between lipids and prooxidants in aqueous phase against lipid oxidation (Yi, Zhu, McClements, & Decker, 2014). 3.4.2. Effect of thermal sterilization temperature on the stability of walnut beverage emulsion As shown in Fig. 6(1b), with the rise of sterilization temperature, the droplet size of walnut beverage emulsion was increased significantly (p < 0.05). Subsequently the destabilization occurred extensively, which was different from the emulsions treated by extending sterilization time, as shown in Fig. 6. Thus, the destabilization induced by higher sterilization temperature was more significant (p < 0.05) than that by longer sterilization time at 121 °C. Even if walnut beverage emulsion was composed of the emulsifiers

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and polysaccharide, higher temperature (above 121 °C) could decrease the stability of the emulsion. This result was attributed to the fact that higher temperature (above 121 °C) could lead to the protein denaturation seriously, destroy the xanthan gum structure (Zhao, 1994) and the interfacial layer formed by emulsifiers (Tangsuphoom & Coupland, 2009). During the storage, the droplet size of walnut beverage emulsion was more obviously increased at 55 °C (Fig. 6(1a)) than that at 37 °C (Fig. 6(1b)). When the sterilization temperature was higher, the droplet size of walnut beverage emulsion became larger. After the storage of 4 weeks at 37 °C and 55 °C, the droplet size of walnut beverage emulsion treated for 25 min at 135 °C was increased from 451.7 to 1211.4 nm and 2108.3 nm (Fig. 6(1)), respectively, which exhibited the poorest stability with evidently clear cream layer. Thus, the destabilization of walnut beverage emulsion was enhanced with a rise of sterilization temperature. The longer sterilization time and higher sterilization temperature would induce severe denaturation of walnut protein, which could result in flocculation, sedimentation and color browning. Fig. 6(2) implied that the peroxide concentration of walnut oil in the emulsion was significantly (p < 0.05) increased with the extension of storage time at 37 °C and 55 °C. After the storage of 1 week, the obvious increase of peroxide concentration was observed for all the emulsions treated by thermal sterilization. As a result, the peroxide concentration of walnut oil was higher in the emulsion stored at 55 °C than that at 37 °C. At higher sterilization temperature, the oil droplets was easily aggregated due to the denaturation of walnut protein and destruction of xanthan gum (Zhao, 1994) and the destruction of interfacial layer formed by emulsifiers. As Zhao (1994) reported, when the temperature was above 120 °C, the property of xanthan gum was changed. Without the protection of walnut protein, mixed emulsifiers and xanthan gum, the encapsulation efficiency of walnut oil was decreased. Thus, the oil was prone to be exposed to oxygen and oxidized. However, as shown in Fig. 6(2b), after the storage of 3 weeks, the lipid peroxide concentration of walnut oil in the emulsion, which was treated at 130 °C and 135 °C, was obviously decreased. This could be attributed to the fact that the higher sterilization temperature accelerated the decomposition of hydroperoxide to form flavor compounds, such as aldehydic and ketone compounds (Alamed, Chaiyasit, McClements, & Decker, 2009). At lower sterilization temperature (100 and 110 °C), walnut beverage emulsion exhibited better physical stability and the oil in walnut beverage emulsion exhibited better oxidative stability. In summary, walnut beverage emulsion composed of walnut kernel, mixed emulsifiers and xanthan gum, could be able to withstand the thermal treatment in a certain temperature and time range. 4. Conclusion The mechanism of stabilizing walnut beverage emulsion with mixed emulsifiers and xanthan gum was proposed. The mixed emulsifiers could be absorbed on the water and oil interface, and xanthan gum could enhance the electrostatic and steric repulsion against the aggregation. They had a significant effect on the physical stability of walnut beverage emulsion under extreme conditions. During the thermal sterilization process, the walnut beverage emulsions with xanthan gum and mixed emulsifiers exhibited poorer physical and oxidative stabilities at higher temperature for longer time sterilization, which was ascribed to the denaturation of walnut protein and the destruction of xanthan gum structure and interfacial layer formed by emulsifiers.

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