Whey protein stabilized nanoemulsion: A potential delivery system for ginsenoside Rg3 whey protein stabilized nanoemulsion: Potential Rg3 delivery system

Food Bioscience 31 (2019) 100427

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Whey protein stabilized nanoemulsion: A potential delivery system for ginsenoside Rg3 whey protein stabilized nanoemulsion: Potential Rg3 delivery system

T

Pingping Hou, Fengling Pu, Haoyang Zou, Mengxue Diao, Changhui Zhao, Chunyu Xi∗∗, Tiehua Zhang∗ College of Food Science and Engineering, Jilin University, Changchun, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Whey protein Nanoemulsion Stability Delivery system Ginsenoside Rg3

In the current research, we systematically investigated the physicochemical stability of the nanoemulsions that were formulated with 90% (v/v) whey protein isolate (WPI) aqueous phase and 10% (v/v) medium chain triglyceride oil phase at neutral pH using high intensity ultrasonication. We measured the physicochemical properties of nanoemulsions stabilized by WPI at different concentrations, including particle size, zeta potential, turbidity, centrifugal stability, and rheological behavior. Besides, we also evaluated the influence of processing conditions (ionic strength, freeze-thaw cycle and thermal treatment) on the nanoemulsion stability. The particle size of WPI nanoemulsions increased by approximately 5% at 25 °C and 8% at 37 °C after 7 weeks’ storage, while the particle size and zeta potential of the nanoemulsions at 4 °C were changed. The 1% WPI stabilized nanoemulsion was very sensitive to Na+ (0.1–0.5 mol/l) and freeze-thaw treatment (3 cycles) with significant increase in particle size and decrease in the absolute value of zeta potential. The turbidity of nanoemulsions decreased by over 50% after freeze-thaw cycles, especially the 1% WPI stabilized nanoemulsion. All samples were still stable under thermal conditions. Among them, the 5% WPI stabilized nanoemulsion showed the best ionic, freeze-thaw, and thermal stability. All tested nanoemulsions showed typical shear-thinning behavior, and the infinite shear viscosity of 5% WPI stabilized nanoemulsions was highest. In conclusion, the 5% WPI stabilized nanoemulsion has the best physicochemical stability under different processing conditions during storage, which has high potential in use as a delivery system for bioactive substances like ginsenoside Rg3 in food technology.

1. Introduction As a by-product of cheese production, whey protein is rich in nutrients and also plays an important role in emulsification, gelation, foaming and flavoring in food factories (Smithers, 2015; Sun et al., 2016). Because of good emulsifying property, whey protein has been widely applied in many traditional and new food processing, especially emulsion-based food preparation (Demetriades, Coupland, & Mcclements, 2010). The whey protein isolate (WPI) contains more than 90% of protein that has high commercial value (Chityala, 2016), of which β-lactoglobulin (β-lg) and α-lactalbumin (α-la) possess the main functional characteristics of WPI(Norwood et al., 2016). WPI can be used to emulsify lipophilic molecules to form oil-in-water emulsions (Berton-Carabin, Coupland, & Elias, 2013; Eric Dickinson, 2010; David Julian Mcclements, 1998). Because whey protein can be adsorbed on

∗

the surface of oil droplets due to its surfactant nature, electrostatic function and hydrophobic effect, the whey protein based emulsion is supposed to be stable by avoiding aggregation of oil droplets (Fan, Jiang, Zhang, Zhen, & Zhao, 2017). In recent years, more and more people have designed nanoemulsion systems to control the release of bioactive substances in the gastrointestinal and blood systems (Shah, Zhang, Li, & Li, 2016). After the bioactive substances are encapsulated in the nanoemulsion, its solubility in water can increase with bioavailability improved, and the bioactive substances could be released continuously and slowly. The whey protein in the nanoemulsion has antioxidant properties (Min, D Julian, & Decker, 2003), which can play a protective role in the transportation of bioactive substances from oxidative stress (Elias, Kellerby, & Decker, 2008). Zhou et al. found that WPI-stabilized nanoemulsion is an effective carrier of lipid soluble bioactive substances

Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Xi), [email protected] (T. Zhang).

∗∗

https://doi.org/10.1016/j.fbio.2019.100427 Received 26 April 2019; Received in revised form 30 June 2019; Accepted 3 July 2019 Available online 03 July 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.

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such as beta-carotene, as this system improves its bioavailability and stability (Zhou et al., 2018). Sari et al. studied the bioavailability of curcumin nanoemulsion stabilized by whey protein and Tween-80, which showed that curcumin was released slowly in during digestion (Sari et al., 2015). The ginsenoside Rg3 is a novel bioactive compound that has been shown to have many health beneficial activities (L. Liu et al., 2010), such as liver protection, neuroprotection, cardiovascular protection, immune promotion, fatigue relief, anti-oxidation and anti-tumor effects (Bo et al., 2012; Hoi-Hin et al., 2012; Wei et al., 2012; Wei, Fei, Su, Hu, & Hu, 2012; Y.; Xu et al., 2013). However, Rg3 is insoluble in water with low bioavailability (Yang et al., 2012), leading to limitation in use. Previous researches have reported that ginsenoside Rg3 nanoemulsion can inhibit tumor growth and metastasis (X. Dai et al., 2017; X. J. Dai et al., 2014). However, information on WPI as an emulsifier for stabilizing ginsenoside Rg3 is limited. In this work, we are committed to evaluate the physicochemical properties of nanoemulsions prepared with WPI as the emulsifier using ultrasonic technology.

Table 1 Initial particle size, PDI and zeta potential of WPI stabilized nanoemulsions. Particle size (nm) 1%WPI 3%WPI 5%WPI

PDI

a

Zeta potential (mV) a

0.459 ± 0.032 0.473 ± 0.029a 0.483 ± 0.029a

309.6 ± 7.0 324.9 ± 28.8b 321.1 ± 11.8b

−58.7 ± 2.4a −60.0 ± 1.6a −59.7 ± 1.9a

Different letters in the same column indicate significant differences at p < 0.05.

2.4. Turbidity measurement The turbidity (T) was measured with modification based on the method as previously reported (Yadav, Johnston, & Hicks, 2007). A small amount of nanoemulsion was diluted 100-fold with deionized Milli-Q water, and its absorbance value (A) was determined using a microplate reader (Synergy HT, BioTek, U.S.A.) at the wave-length of 650 nm immediately after mixing. The turbidity (T) was calculated using equation (1): T = 2.303AD/l

2. Materials and methods

(1) −1

Where T is the turbidity (cm ), A is the absorbance at 650 nm, D is the dilution factor, and l is the path length of the cuvette (cm).

2.1. Materials 2.5. Centrifugal stability constant measurement

WPI, containing 93.1% of protein (β-lactoglobulin: α-lactalbumin: = 2:7), 4.79% of moisture, 0.36% of fat, 1.60% of ash and 0.70% of lactose, was obtained from Fonterra (Auckland, New Zealand). Medium-chain triglyceride (MCT) was obtained from Yuanye Bio-Technology (Shanghai, China). Ginsenoside Rg3(S) was obtained from Chinese Institute of Jilin Ginseng (Changchun, China). Sodium azide was purchased from Sigma (St. Louis, MO, USA). All other chemical reagents were obtained from Beijing Chemical Works (Beijing, China). The deionized water used in this work was obtained by a Millipore Milli-Q™ water purification system (Millipore Corp., Milford, MA, USA).

Centrifugal stability constant (K e ) is an appropriate indicator to evaluate the stability of nanoemulsions. K e was measured with modification based on the method as reported (Zhao, Shen, & Guo, 2018). Briefly, the nanoemulsion was diluted 100-fold with deionized Milli-Q water, and its absorbance value (A1) was measured using a microplate reader (Synergy HT, BioTek, U.S.A.) at the wave-length of 490 nm immediately after mixing. Then the nanoemulsion was centrifuged at 4000 rpm for 15 min at 20 °C in a high-speed centrifuge 3K15 (SIGMA Laborzentrifugen GmBh, Germany). A small amount of subnatant was diluted 100-fold with deionized water, after mixing, measuring its absorbance value (A0) at the wave-length of 490 nm. The centrifugal stability constant (K e ) was given in equation (2):

2.2. Preparation of WPI stabilized nanoemulsions

Ke = A0/A1 × 100% WPI solutions (1%, 3%, 5%, w/v) were prepared with sodium azide solution (0.02% w/v) at normal atmospheric temperature. The WPI solution was stirred for at least 2 h and then stored at 4 °C for 12 h to hydrate completely. After restoring to room temperature, the solution was adjusted to pH 7.0 using HCl and NaOH (1M). A coarse emulsion was prepared with 10% MCT oil and 90% WPI solution using an UltraTurrax T25 high-speed blender (IKA, Staufen, Germany) at 12000 rpm for 2 min. Next, the coarse emulsion was homogenized to produce fine oil-in-water nanoemulsion through an ultrasonic processor (VCX800, Vibra-Cell, Sonics, Newtown, CT, U.S.A.) in an ice bath for 5 min (Xue Shen, Shao, & Guo, 2016).

(2)

Where A1 is the absorbance before centrifuge at 490 nm, while A0 is the absorbance after centrifuge at 490 nm. 2.6. Physicochemical stability measurements If the emulsion can maintain its performance under different conditions during storage, it is can be considered as stable. Therefore, the influence of storage time, ionic strength, freeze-thaw and thermal treatment on the physiochemical properties of nanoemulsions were evaluated by particle size, zeta potential, PDI, K e and turbidity. 2.6.1. Storage stability The nanoemulsions were stored at 4 °C, 25 °C, and 37 °C for 7 weeks. The particle size, PDI, and zeta potential were measured every week.

2.3. Particle size and zeta potential measurements The particle size, polydispersity index (PDI) and zeta potential of nanoemulsions were determined by dynamic light scattering (DLS) and electrophoretic mobility (UE) scattering by a Zetasizer Nano ZS 90 (Malvern Instruments, U.K.) as previously reported (Xue Shen et al., 2016; X. Shen, Zhao, Lu, & Guo, 2018). The samples were diluted 1:100 with deionized water, using a refractive index of 1.33 for the water and 1.52 for the oil droplets. The instrument reports the particle size values as Z-average. And the width of particle size distribution is reflected by PDI (0–1). Zeta potential is determined based on the Henry equation (Deshiikan & Papadopoulos, 1998). All measurements were conducted at 25 °C.

2.6.2. Ionic strength The same volume of NaCl solution (0.1M–0.5M) was added in the nanoemulsion. After 1 h of stirring, the mixture was stored at 25 °C for 12 h. 2.6.3. Freeze-thaw cycling The nanoemulsion was first stored in a −18 °C refrigerator for 22 h and then thawed by in a warm water bath at 40 °C for 2 h (D. Xu et al., 2010). This freeze-thaw cycle was repeated three times. 2

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Fig. 1. Changes of nanoemulsions stabilized by WPI of different concentrations during storage. (a) particle size and PDI at 4 °C, (b) zeta potential at 4 °C, (c) particle size and PDI at 25 °C, (d) zeta potential at 25 °C, (e) particle size and PDI at 37 °C, (f) zeta potential at 37 °C.

within the range from 0.1 to 500 s−1 for analysis of the flow ramp at 25 °C.

2.6.4. Thermal treatment The nanoemulsions were placed in a water bath at 60 °C, 80 °C and 100 °C for 30 min. When the emulsions were cooled down, the particle size, PDI, zeta potential, K e and turbidity were determined.

2.8. Application of Ginsenoside Rg3 to nanoemulsion 2.7. Rheological behavior

Ginsenoside Rg3 was dissolved in the MCT with ultrasonic method. The Ginsenoside Rg3 nanoemulsion was prepared with 90% WPI (5%, w/v) and 10% MCT. Then the particle size, PDI and zeta potential of nanoemulsions with Ginsenoside Rg3 (0.1, 0.2, 0.3 mM) were measured.

Rheological behavior was analyzed using a rheometer (DHR-1, TA Instrument, New Castle, DE, U.S.A.) as previously reported (X. Shen et al., 2018). The geometry of parallel steel plate with a diameter of 40 mm was used to measure 1 mm thick samples. The shear rate was 3

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Fig. 2. Impact of ionic strength on the nanoemulsions stabilized by WPI of different concentrations. (a) particle size and PDI, (b) zeta potential.

conducive to keep the physical stability of the nanoemulsion.

2.9. Statistical analysis Statistical analysis was carried out with SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed using ANOVA and Duncan multiple comparisons testing. Data were shown as mean ± standard data error (SEM). Only differences at p < 0.05 were considered as significant. All figures were acquired by Origin 9.1 (Origin Lab Corporation, Northampton, MA).

3.2. Ionic stability Only do the emulsions have high ionic strength stability, can them be better applied in food system. The stability of emulsion mainly depends on electrostatic repulsion. Addition of counter-ions screens the electrostatic effect between droplets, thus reducing the droplet charge. Droplet aggregation occurs when hydrophobic and van der Waals attraction interactions exceed repulsion strength (Aoki, Decker, & McClements, 2005). As observed in Fig. 2a, the addition of salt (0.1–0.5M NaCl) increased the electrical charges (zeta potential = from about −65 to −50 mV). Regardless of these changes in zeta potential, the nanoemulsion stabilized by WPI (3% and 5%) was still resistant to aggregation up to 0.5 M NaCl (Fig. 2). As the salt concentration increased, the 1%WPI nanoemulsion had a sharp increase in particle size and PDI (p < 0.05). The maximum particle size was 744.3 ± 30.5 nm at 0.5 M NaCl (Fig. 2b). The magnitude of the charges in the 3% and 5% WPI stabilized nanoemulsions was more negative at higher salt concentration than those in the 1% WPI stabilized nanoemulsions. Therefore the electrostatic repulsion between the droplets should be greater. The 3% and 5% WPI nanoemulsions were not be influenced by NaCl (0.1–0.5 M) significantly in particle size, indicating their good ionic stability (Fig. 2a).

3. Results and discussion 3.1. Storage stability The initial particle size (below 330 nm), PDI (below 0.5) and zeta potential (about −60mV) of nanoemulsions stabilized by WPI (1%, 3%, 5%) were listed in Table 1. As shown in Fig. 1, influence of WPI concentration on the particle size and zeta potential at different temperatures was investigated over a period of 7 weeks. The PDI value can reflect the distribution of particle size. The particle size and PDI of the nanoemulsions increased slightly, while the absolute value of zeta potential decreased slowly during storage. With the prolongation of storage time, globular proteins flocculate, which led to the increase of particle size of emulsions. As the temperature increased, some non-polar groups in the hydrophobic region of the protein were gradually exposed, and the hydrophobic interaction was enhanced continuously, which promoted flocculation of the proteins. However, the emulsion droplets repelled each other due to the presence of electrostatic effect, which reduced the flocculation speed and made the particle size grow slowly (Qian, Decker, Xiao, & Mcclements, 2012). The particle size of WPI nanoemulsions increased by approximately 5% at 25 °C and 8% at 37 °C after 7 weeks’ storage, whereas the particle size and zeta potential of the nanoemulsions at 4 °C was nearly constant (p > 0.05). As the storage temperature increased, the particle size changed more and more greatly. PDI of all samples had no significant variation during 4 °C and 25 °C storage (Fig. 1). Particle size distribution is proportional to PDI value, and austenitic maturation is more likely to occur in emulsion system with wide particle size distribution (Y. Liu, Kathan, Saad, & Prudhomme, 2007). Relatively small PDI value can indicate that the nanoemulsion system has high stability (Mcclements & Rao, 2011). The physical stability of the nanoemulsion system can be evaluated by the Zeta potential on the particle surface. The greater the absolute value of zeta potential is, the higher the stability of the system is supposed to be. As shown in Fig. 1, all nanoemulsions had negative potentials. There was no significant difference in zeta potential of these samples during 4 °C storage. Above all, cryogenic storage was more

3.3. Freeze-thaw stability Food emulsions may have microbial contamination and unnecessary chemical reactions in the process of food manufacturing, storage and application (N.-N. Wu et al., 2012). The crystallization affects the physical and chemical changes of the emulsion. In the thawing process, the frozen emulsion will cause interface membrane rupture, droplet coalescence and water-oil separation. Firstly, after the emulsion is frozen, the unfrozen water in the aqueous phase decreases sharply, resulting in the oil droplets binding more closely to the partially hydrated emulsifier and ultimately promoting the droplet aggregation. Secondly, during the freezing process, ice crystals continuously permeate into the oil droplets, the interface layer is destroyed and the oil droplets combine with each other (Cornacchia & Roos, 2011). Nanoemulsions covered with WPI showed a coalesced layer of emulsion droplets and a turbid phase after freeze-thaw cycles (Fig. 3e). After one freeze-thaw cycle, all nanoemulsion samples had a decrease in particle size of turbid phase (still stable), that was why freeze-thaw led to the separation of water and oil in part of the emulsion (Fig. 3a). Similarly, centrifugal stability constant of all samples increased a lot after one cycle (Fig. 3d). Then 1% WPI nanoemulsion had a sharp increase in 4

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Fig. 3. Impact of freeze-thaw cycling on nanoemulsions stabilized by WPI of different concentrations. (a) particle size, (b) zeta potential, (c) turbidity, (d) K e , (e) visual observation of nanoemulsions after freeze-thaw cycling.

shown in Fig. 3c, all samples had significant decrease in turbidity after one cycle (p < 0.05). The 5% WPI nanoemulsion samples decreased by 52.8% in turbidity, the 3% WPI nanoemulsion samples decreased by 67.3%, and 1% WPI nanoemulsion samples decreased by 86.7%. Above all, the 5% WPI nanoemulsion showed the best freeze-thaw stability.

particle size by 56 nm after 3 cycles (p < 0.05), while 3% and 5% WPI nanoemulsion samples had no significant change (Fig. 3a). When the concentration of WPI was 1%, its emulsification was not strong enough, causing aggregation. The absolute value of zeta potential decreased significantly for 1% WPI enanoemulsion with freeze-thaw cycle (Fig. 3b). The 3% and 5% WPI stabilized nanoemulsions have relatively thick interfacial layers, which can generate large repulsive forces on droplet appearance, and it is difficult for ice crystals to break through the relatively thick protein interfacial film, thus reducing coalescence. Part of protein detached from the droplet membrane during freezing and thawing process, resulting in reduced the absolute potential value. Higher concentration of protein can form a more compact and stable oil-water interface layer, which has better freeze-thaw stability. As

3.4. Thermal stability The nanoemulsions were obtained by ultrasonic homogenization. The application of ultrasound treatment can improve the physicochemical properties and emulsification properties of protein, thus improving the stability of emulsion (O'Sullivan, Arellano, Pichot, & Norton, 2014; D. Wu et al., 2019). When the emulsion is applied to 5

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Fig. 4. Impact of thermal treatment on the nanoemulsions stabilized by WPI of different concentrations. (a) particle size and PDI, (b)zeta potential, (c) turbidity, (d) Ke. Table 2 Influence of the WPI concentration of nanoemulsions on the fitting parameters of the Sisko equation for flow curves at 25 °C. WPI

η∞ (mPa.s)

ks (Pasn)

n

R2

1% 3% 5%

2.16 ± 0.02a 2.47 ± 0.02b 2.74 ± 0.01c

1.87 ± 0.09a 1.84 ± 0.12a 1.98 ± 0.08b

0.895 ± 0.025a 0.882 ± 0.022a 0.88 ± 0.034a

0.999 0.999 0.999

Different letters in the same column indicate significant differences at p < 0.05. Table 3 Particle size, PDI and zeta potential of ginsenoside Rg3 nanoemulsions.

Fig. 5. Apparent viscosity and shear rate relationship of the nanoemulsions stabilized by WPI of different concentrations.

food, it may undergo thermal treatment such as sterilization. After the unfolded protein in the emulsion reaches the deformation temperature, the core reaction group is exposed and protein polymerization occurs, leading to flocculation and condensation of the emulsion (Demetriades et al., 2010; Kim, Decker, & Mcclements, 2002). The influence of thermal treatment (60, 80, 100 °C for 30 min) on the particle size, zeta potential, turbidity and K e (Fig. 4) of nanoemulsions covered with WPI were measured. According to Fig. 4, no significant influence of thermal treatment on the particle size, PDI, zeta potential, and turbidity of the

Ginsenoside Rg3 (mM)

Particle size (nm)

Initial 0.1 0.2 0.3 0.4 0.5

321.1 325.5 331.0 338.4 362.8 393.3

± ± ± ± ± ±

11.8a 28.6a 21.5a 10.6a 21.2b 13.3c

PDI

0.483 0.487 0.491 0.489 0.491 0.490

Zeta potential (mV) ± ± ± ± ± ±

0.029a 0.034a 0.013a 0.033a 0.014a 0.026a

−59.7 −58.8 −58.6 −58.9 −58.4 −57.7

± ± ± ± ± ±

1.9a 1.7b 1.2b 1.2b 1.7b 0.6b

Different letters in the same column indicate significant differences at p < 0.05.

nanoemulsions were observed (p > 0.05). The results showed that the nanoemulsions were stable upon heating, especially 5% WPI nanoemulsion had higher K e (32.9 ± 2.7%, 33.3 ± 3.3%) after 80 °C and100 °C thermal treatment (p < 0.05). Possibly, because of changes in the structure of the protein during the thermal treatment (Christophe 6

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Conflicts of interest statement

et al., 2009), the rearranged protein binds more tightly to the fat droplets. Protein at denaturation temperature above, the formation of a smaller biopolymer, its freeze-thaw, salt ion and thermal stability are better than the polymer formed at ambient temperature. Above all, the WPI nanoemulsions had good thermal stability.

The authors declare that there is no conflict of interest. Acknowledgement This work was supported by the “13.5” National Keypoint Research and Invention Program (grant number 2017YFD0400603); Research and Industrialization with Key Technologies for High Value Processing and Safety Control of Agricultural Products (grant number SF2017-6-4); The National Natural Science Foundation of China (grant number 31871717).

3.5. Rheological properties As shown in Fig. 5, The viscosity of all nanoemulsion samples decreased with the increase of shear rate, which showed typical shearthinning characteristics. The typical characteristic of shear-thinning emulsions is pseudoplasticity (E Dickinson & Golding, 1997; Taherian, Fustier, Britten, & Ramaswamy, 2008). The viscosity of emulsion is related to the interaction between droplets, which is weakened by high shear rate (Mantovani, Fattori, Michelon, & Cunha, 2016). Flow curves (Fig. 5) were described by the Sisko model (Hermoso, Martinez-Boza, & Gallegos, 2014), and the corresponding parameters were listed in Table 2. The Sisko model had a high correlation coefficient R2 (> 0.999), which indicated that the flow curve can be well fitted, and it was given in equation (3):

η = η∞ + ks

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where η stands for the apparent viscosity, η∞ is the infinite shear viscosity, ks is the consistency index, and n is the flow index. As we can see in Fig. 5, 5% WPI stabilized nanoemulsions always had higher viscosity, whose η∞ was 3.17 mPa s (Table 2). Due to the weak interaction of shear disruption, η∞ is an appropriate indicator to characterize the properties of samples. The rheological properties of emulsion samples changed during the preparation process due to stirring, and η∞ can avoid the errors (Wan, Qixin, & Zhixiong, 2013). The consistency index ks reflects the viscosity of the emulsion. And the higher the ks is, the higher the viscosity will be (Hou et al., 2012). And the ks of 5% was higher than others (p < 0.05), which was consistent with the result of η∞. All samples showed the pseudoplastic (n < 1) behavior (Taherian, Fustier, & Ramaswamy, 2006). The flow behavior index (n) had no significant difference by all treatments (p > 0.05). 3.6. Ginsenoside Rg3 nanoemulsion According to the results above, 5% WPI stabilized nanoemulsion had the best physicochemical stability, which has high potential in use as a nanoemulsion-based encapsulating or delivery system in food technology. We applied ginsenoside Rg3 to the nanoemulsion. And the particle size, PDI, and zeta potential of nanoemulsions containing different concentration of ginsenoside Rg3 (0.1–0.5 mM) were listed in Table 3. As the results showed, particle size rise became insignificant (p > 0.05) as Rg3 concentration increased from 0.1 to 0.3 mM, while particle size) increased by 24.4 and 29.1 nm significantly with Rg3 concentration from 0.4 to 0.5 mM(p < 0.05. The absolute value of zeta potential decreased after the addition of Rg3 (p < 0.05). The PDI of all samples were relatively stable (p > 0.05). We can conclude that the addition of Rg3 (0.1–0.3 mM) had little influence on the stability of nanoemulsions, and can be applied to food production. 4. Conclusion This study has extensively investigated the physicochemical properties of WPI-based nanoemulsions. In conclusion, 5% WPI stabilized nanoemulsion showed the best physicochemical stability, which was still stable under common processing conditions (ionic strength, freezethaw and thermal treatment) after 7 weeks’ storage. Then the successful application of ginsenoside Rg3 makes it possible to design WPI-stabilized nanoemulsion as a delivery system for lipophilic bioactive substances. 7

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