Effects of chitosan coating on curcumin loaded nano-emulsion: Study on stability and in vitro digestibility

Food Hydrocolloids 60 (2016) 138e147

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Effects of chitosan coating on curcumin loaded nano-emulsion: Study on stability and in vitro digestibility Jinglei Li a, In-Cheon Hwang b, Xiguang Chen c, Hyun Jin Park a, * a

School of Life Sciences and Biotechnology, Korea University, 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea Central Research Institute, Kyung-Nong Co. Ltd., Kyungju 780-110, South Korea c College of Marine Life Science, Ocean University of China, Qingdao, 266003, Shandong, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2015 Received in revised form 6 February 2016 Accepted 6 March 2016 Available online 11 March 2016

Nano-emulsion (NE) composed of MCT oil, Tween 80 and lecithin was fabricated by ultrasonication method to encapsulate curcumin. Loading ability and efficiency of curcumin were 0.548 mg/mL and 95.10% respectively which indicated its water dispersibility was increased by 1400 fold. Chitosan with low, middle and high molecular weight (3 kDa, 30 kDa and 190e310 kDa respectively) was applied for coating the prepared NE. After chitosan coating, zeta potential value of NE was changed from negative to positive. At the same time, chitosan coating prevented NE phase separation in ionic strength test and inhibited degradation of curcumin during thermal and UV irradiation treatment. Using pH-stat method, it was found that middle and high molecular weight chitosan coating may interfere with lipolysis of NE during the in vitro digestion which also slightly decreased curcumin bio-accessibility. Therefore, NE coated with chitosan is a promising delivery system to promote the applications of curcumin in functional food and beverage system. © 2016 Published by Elsevier Ltd.

Keywords: Curcumin Nano-emulsion Chitosan Molecular weight Stability In vitro digestibility Chemical compounds studied in this article: Curcumin (PubChem CID: 969516) Chitosan (PubChem CID: 21896651) Tween 80 (PubChem CID: 5281955) Lecithin (PubChem CID: 65262)

1. Introduction Nutraceuticals are bioactive compounds contained in food at relatively low level that may be beneficial to maintain human health and prevent certain chronic diseases (McClements, 2015). Curcumin is one of the most widely investigated nutraceuticals. It is the primary active ingredient of the perennial herb Curcuma Longa (turmeric) which has been traditionally used as nutritional supplement and herbal medicine in many Asian countries for thousands of years (Aggarwal, Sundaram, Malani, & Ichikawa, 2007). Recent studies have demonstrated that curcumin has a wide spectrum of therapeutic activities, including antioxidant, antiinflammatory, anti-cancer, antimicrobial, wound healing, and potential prevention ability to neurodegenerative diseases (Aditya et al., 2013; Basnet & Skalko-Basnet, 2011; Begum et al., 2008; Takahashi, Uechi, Takara, Asikin, & Wada, 2009). Moreover, curcumin has very good safety profile as examined by thousands of years'

* Corresponding author. E-mail address: [email protected] (H.J. Park). http://dx.doi.org/10.1016/j.foodhyd.2016.03.016 0268-005X/© 2016 Published by Elsevier Ltd.

usage and several clinical trials: as high as 8 g/day dosage would not cause any adverse effects (Cheng et al., 2001). However, the utilization of curcumin in functional food and related products is restrained by several of its drawbacks. Curcumin has extremely low solubility and dissolution rate in aqueous media due to strong inter and intra-molecular hydrogen bonds (Heger, van Golen, Broekgaarden, & Michel, 2014). Meanwhile, curcumin is sensitive to several environmental factors like heating, UV irradiation, and higher pH value (Li, Lee, Shin, Chen, & Park, 2015). To effectively exert its beneficial activities, curcumin is favored to be dispersed in the aqueous medium which creates a demand for a suitable and reliable delivery system. In the past decades, several approaches such as nanodelivery system, chemical modification and physical dispersion have been exploited by many researchers to increase its solubility, stability and bioavailability (Li, Lee, Shin, Chen, & Park, 2015, Li, Shin, Chen, & Park, 2015; Paradkar, Ambike, Jadhav, & Mahadik, 2004; Shin, Chung, Kim, Joung, & Park, 2013; Wang et al., 2015). In order to better integrate curcumin into functional food and

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

beverage system, nano-delivery systems fabricated by food grade materials have attracted much attention in recent years (Li, Shin, Lee, Chen, & Park, 2016). Nano-delivery systems may confer curcumin with rapid dissolution speed, higher stability, tailored release pattern, higher permeation rate, higher bioavailability and many other advantages compared with other similar structured but micro-size or larger delivery systems (McClements, 2015; Yu & Huang, 2012). As a consequence, various kinds of nano-delivery systems have been evaluated to encapsulate curcumin in order to better incorporate it into food and beverage system (Aditya et al., 2013; Li, Lee et al., 2015; Li, Shin et al., 2015). Oil in water (O/W) nanoemulsion (NE) is an colloidal dispersion system composed of small lipid droplets in the range of 50e200 nm which are dispersed within an aqueous medium (Tadros, Izquierdo, Esquena, & Solans, 2004). NE is thermodynamically unstable and need certain emulsifiers on the oil-water interface to stabilize the colloidal system. Compared with conventional emulsion which has droplets size of several micrometers, NE is particularly attractive to encapsulate, protect and deliver lipophilic nutraceuticals for food and related applications. Wang et al. reported that NE with the size of 79.5 nm was more effective than conventional emulsion to increase the anti-inflammation activity of curcumin (Wang et al., 2008). Setthacheewakul et al. formulated curcumin loaded NE with the size about 30 nm and demonstrated that it could increase curcumin absorption by 14 fold in the animal study (Setthacheewakul, Mahattanadul, Phadoongsombut, Pichayakorn, & Wiwattanapatapee, 2010). Biopolymer is usually used for coating nano-delivery system to increase its stability, absorption rate as well as modulate the payload release pattern (Abbas, Bashari, Akhtar, Li, & Zhang, 2014; Ozturk, Argin, Ozilgen, & McClements, 2015; Sood, Jain, & Gowthamarajan, 2014). Chitosan is a natural polysaccharide widely applied for the functional food, drug delivery and tissue engineering (Kean & Thanou, 2010). Our previous study has demonstrated that nanoliposomes coated with chitosan have prolonged absorption in the gastrointestinal tract which could be contributed to its mucoadhesive properties (Shin et al., 2013). Klinkesorn and McClements studied the influence of chitosan coating on the lipase digestibility of tuna oil emulsion (Klinkesorn & McClements, 2009). They found that chitosan coated emulsion could be digested by enzyme and chitosan coating may be useful for delivering lipophilic bioactive compounds. In the present study, we formulated curcumin loaded NE via ultrasonication treatment. Tween 80 was used as surfactant to stabilize the oil droplets due to its steric effect. Lecithin was added as co-surfactant to offer strong negative charge value to NE and facilitate polymer coating. Chitosan with various molecular weight was employed to coat the prepared NE. We attempted to assess the effects of chitosan coating on the colloidal stability and storage properties of NE. Moreover, we also investigated the effects of chitosan coating on the stability of NE under environmental stresses that are common in food processing including various ionic strengths, thermal treatment and UV irradiation. At last, the in vitro digestion and in vitro bio-accessibility of curcumin loaded in NE was quantified by the pH-stat method. Our results indicate that chitosan coating is effective to increase the stability of emulsion but slightly decrease its digestibility. This study will be helpful to solve the problems associated with curcumin and better incorporate it in functional food and beverage system. 2. Materials and methods 2.1. Materials Curcumin with 98% purity was purchased from Acros Orangnics

139

(NJ, USA). Medium chain triglycerides (MCT) was purchased from Now Foods Co. (IL, USA). Lecithin from soybean was obtained from Junsei Chemical Co. (Tokyo, Japan). Tween 80 was provided by Samchun Chemical Co. (Pyeongtaek, Korea). Mucin from porcine stomach, bile extract from porcine, lipase from porcine pancreas and chitosan with molecular weight of 190,000e310,000 and degree of deacetylation (DD) of 85% was obtained from SigmaeAldrich (MO, USA). Chitosan with molecular weight of 30,000 and DD of 89.2% was acquired from Biotech Co. (Mokpo, Korea). Oligochitosan with molecular weight of 3000 and DD of 90% was acquired from Chitolife Co. (Pyeongtaek, Korea). All the chitosan samples were used as received with no further purification. All other reagents and solvents were of analytical grade and used as received.

2.2. Preparation of nanoemulsion (NE) Ultrasonication treatment was applied to prepare curcumin loaded NE according to previous report (Abbas et al., 2014). About 70 mg of curcumin was dispersed into 10 mL MCT oil and was fully dissolved by heating and stirring overnight. After filtration through syringe filter (0.45 mm) to remove undissolved crystals if any, MCT oil was fully mixed with lecithin. Tween 80 was dissolved into distilled water which was added to the mixture of MCT oil and lecithin. Based on our previous studies and preliminary tests, the weight ratio of MCT oil, lecithin, Tween 80 and distilled water was kept at 10:6:4:80. The mixture of oil, surfactants and water was stirred for about 10 min before subjected to a high speed blender (Ultra-Turrax T25 IKA Works Inc., Wilmington, NC, USA) at 16,000 rpm for 5 min at room temperature to prepare the coarse emulsion. After that, 100 mL of the coarse emulsion solution was treated by ultrasonication (Sonics & Materials, Inc., Newtown, CT, USA) at the power output of 150 W for 20 min in the ice bath using the continuous working model. Prepared NE sample solution was transferred to glass vials for following tests.

2.3. Preparation of chitosan coated nanoemulsion (CNE) Chitosan with various molecular weight was added into 2% acetic acid solution at the weight ratio of 1%. All the solution was stirred overnight and filtered through paper filter to remove undissolved large particles. Chitosan coating was conducted by adding 10 mL NE into 10 mL chitosan solution under stirring for overnight. Ultrasonication treatment at the power output of 75 W for 5 min was applied to decrease the size as well as break chitosan bridging that may be formed by emulsion droplets and chitosan.

2.4. Curcumin concentration and loading efficiency determination Curcumin concentration was calculated according to a calibration formula listed below: Y ¼ 0.1483*X þ 0.0487, r2 ¼ 0.995 ;

(1)

where Y was the absorption at 419 nm and X was the concentration of curcumin (mg/mL). The calibration curve was generated by the absorption readings at 419 nm of several standard curcumin ethanol solutions. NE sample was dissolved in ethanol and the absorption at 419 nm was recorded. Loading efficiency was calculated by the weight ratio of curcumin determined in the NE solution and curcumin initially added.

140

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

2.5. Hydrodynamic size and zeta potential analysis Size distribution was measured by dynamic light scattering (DLS) on a nano-ZS nanosize analyzer (Malvern, Worcestershire, U.K.). About 1 mL of sample solution was transferred to a zeta cell, and the measurement was conducted at 25  C with a detector angle of 90 and wavelength of 633 nm. Zeta potential was also recorded on the same equipment. 2.6. Morphological study by transmission electron microscopy (TEM) analysis The morphology characteristics of NE and CNE were observed by TEM analysis (Philips-FEI, Eindhoven, Netherlands). One drop of the sample solution, diluted 10 times by PBS, was deposited onto the carbon-coated grid and kept for 1 min. Excess solution was removed by filter paper. Then one drop of phosphotungstic acid solution (2% in distilled water) was applied to stain the sample for about 1 min as reported previously (Li, Joung et al., 2015, Li, Lee et al., 2015, Li, Shin et al., 2015). All the samples were dried at room temperature overnight (12 h) before capturing the TEM images. 2.7. Storage stability study NE and CNE were stored at room temperature (around 25  C) for 1 month. After that, size, PDI and zeta potential were measured to evaluate the storage stability. 2.8. Thermal stability study Considering the fact that many processed foods need thermal sterilization, it is important that formulated NE has high thermal stability for practical applications. NE and CNE was transferred into glass vials which were incubated in a water bath at 63  C for 30 min or at 100  C for 10 min to mimic the pasteurization and boiling condition. After cooling to room temperature, the size, PDI and zeta potential were measured. Thermal treated NE and CNE were stored in room temperature for 1 month and the size, PDI, zeta potential as well as the concentration of curcumin were measured. After storage in room temperature for 1 month, 0.1 mL of thermal treated emulsion solution was transferred into 5 mL of ethanol. Ethanol solution was passsed through a syringe filter (0.45 mm) and its UV absorption profile between 250 nm and 500 nm was determined and recorded on a UVevis spectrophotometer. 2.9. Ionic strength stability study During food processing, ionic strength usually increase. Hence, it is desirable for the NE system to withstand higher ionic strength with little or no size variation. NaCl solution was added into NE and CNE to adjust the final NaCl concentration at 1 mol/L, 0.5 mol/L and 0.2 mol/L. Change of size, PDI and zeta potential was monitored immediately as well as after 1 month storage in room temperature.

bottom of the vial and the UV lamp was 10 cm. After irradiation for a predetermined time duration, 0.2 mL of emulsion solution was dissolved in 5 mL ethanol for determining the absorption at 419 nm on a UVevis spectrophotometer. The absorptions of sample solutions at 419 nm without UV treatment were recorded and regarded as 100% curcumin remaining. 2.11. In vitro digestion A three-step test was adopted to simulate the digestion of NE according to previous publications with certain modifications (Ahmed, Li, McClements, & Xiao, 2012; Qian, Decker, Xiao, & McClements, 2012). Step 1: Simulated saliva fluid (SSF) containing 0.5 g/L of mucin and various salts was prepared in accordance with the previous report (Sarkar, Goh, & Singh, 2009). Ten milliliter of SSF was pipetted into a glass vial and incubated in a water bath at 37  C for 10 min. The same volume of emulsion was added into pre-warmed SSF solution and then incubated at 100 rev/min in a water bath at 37  C for 10 min. Step 2: Simulated gastric fluid (SGF) was prepared by dissolving 2.0 g of NaCl and 7.0 mL of HCl (36 wt%) into 1 L of distilled water, pH value was adjusted to 1.2. After the mouth digestion phase, 20 mL of SGF was added into solution obtained after step 1. The pH value was adjusted to 2.5 and incubated under the same condition for 2 h. Step 3: After gastric digestion phase, 30 mL of digesta fluid was transferred to another glass vial and the pH value was adjusted to 7.0. Then, 4 mL of bile extract solution containing 187.5 mg of bile extract in PBS pH 7.0 was added into the above digesta sample. Then 1.0 mL of CaCl2 solution (11 wt%) was added and the pH was adjusted to 7.0. The lipolysis was initiated by adding 2.5 mL of freshly prepared lipase PBS solution. At the same time, a pH-stat (Metrohm, Switzerland) was used to record the volume of NaOH solution consumed to maintain pH value of the digesta at 7.0. The intestinal digestion was conducted for 2 h. After each step, the size, PDI and zeta potential were recorded respectively. 2.12. In vitro bio-accessibility determination In vitro bio-accessibility of curcumin was quantified as the amount of curcumin released into the digestion solution in the mixed micelles formed by bile salts and phospholipids after the in vitro intestinal digestion (Ahmed et al., 2012). The final digesta solution was centrifuged at 3000 rpm for 10 min, and then the clear upper micelle phase was mixed with ethanol which was examined on a UVevis spectrophotometer at 419 nm to determine curcumin concentration. The in vitro bio-accessibility of curcumin was calculated in the following equation: Bio-accessibility (%) ¼ curcumin in the micelle form/curcumin before digestion  100 (2)

2.10. UV irradiation stability In order to determine UV photolysis protection ability of NE and CNE for curcumin, photolysis experiment was carried out. A UV lamp (G4T5E UV-B, Sankyo Denki, Tokyo, Japan) was applied to mimic the UV rays of sunlight (290e360 nm). About 2 mL of NE and CNE solution was transferred into a small glass vial. All the emulsion solutions were irradiated under the UV illumination at room temperature in a dark box for 72 h. The distance between the

2.13. Statistical analysis All the data in this study were expressed as means and standard deviation (mean ± SD) and processed by SPSS (Version 20.0; SPSS Inc., Chicago, IL, USA). Comparisons were made using the one-way analysis of variance (ANOVA). P value of <0.05 was considered as statistically significant difference.

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

141

physicochemical characteristics of NE. For the Low-CNE, oligochitosan that has much shorter polysaccharide chain than middle and high molecular weight chitosan was used for coating. The oligo-chitosan may arrange itself more freely to interact with the emulsifier, mainly lecithin. Thus more glucosamine residues of chitosan could be attracted to the surface of NE and not displayed on the outer layer of the chitosan coating. Middle molecular weight chitosan coated NE (Mid-CNE) presented diameter of 103.57 nm which was larger than that of Low-CNE. PDI value was slightly higher but still can be regarded as unified distribution. Different from Low-CNE, the zeta potential value of Mid-CNE was much higher. PDI and zeta potential value of high molecular weight chitosan coated NE (High-CNE) was similar with that of Mid-CNE, but the size increased to 125.83 nm which may be caused by its higher molecular weight. Chitosan with high molecular weight produce thicker coating layer around the surface of NE. Our results are in line with that of previous publications. Mun et al. reported the influence of three different molecular weight chitosan (at 15, 150 and 200 kDa) on the conventional emulsion (Mun, Decker, & McClements, 2006). They found that emulsion coated with chitosan showed high positive zeta potential value and chitosan with higher molecular weight increased the mean size of emulsion. Our previous study also demonstrated that for nanoliposome, chitosan coating changed its zeta potential from negative to positive and also increased its mean size (Shin et al., 2013).

3. Results and discussion 3.1. Nanoemulsion preparation and chitosan coating In the present study, MCT oil was used as the lipid phase to solubilize curcumin to prepare NE by the ultrasonication method. Curcumin has higher solubility in MCT oil than other commonly used oil phase. Meanwhile, MCT oil fabricated NE can increase the digestibility and bio-accessibility of hydrophobic bioactive compound as demonstrated in previous study (Ahmed et al., 2012). Tween 80 was utilized as surfactant to stabilize NE due to its sterical effect. Lecithin was applied as co-surfactant that stablize and confer the prepared NE with negative charge at the same time. As listed in Table 1, NE showed mean size of 113.93 nm with low PDI value of 0.23. It had high negative zeta potential value at 36.23 mV. Prepared NE encapsulated curcumin at the concentration of 0.548 mg/ mL with loading efficiency of 95.10%. Different dispersibility values of raw curcumin in water were reported according to various preparation and measurement methods. In a recent publication, native curcumin presents water dispersibility of 0.39 ± 0.05 mg/mL (Aditya, Yang, Kim, & Ko, 2015). NE prepared in this study could enhance curcumin water dispersibility by 1400 times compared with the reported value. The physicochemical properties of NE is mainly determined by its size and interfacial characteristics. Coating with polymers significantly changes the surficial charge distribution and interfacial thickness of NE which eventually influence properties like colloidal stability, absorption and bioavailability. Herein, three different molecular weight chitosan, 3 kDa for low molecular weight chitosan, 30 kDa for middle molecular weight chitosan and 190e310 kDa for high molecular weight chitosan, were applied to coat NE to assess their effects on pertaining properties. Low molecular weight chitosan coated NE (Low-CNE) showed size of 97.65 nm which was smaller than that of NE. Sonication treatment was applied to CNE solution after chitosan coating that should be responsible for the reduced size. Additionally, Low-CNE presented smaller size than other chitosan coated NE groups. Chitosan with smaller molecular weight may form thinner coating layer as similar result have been revealed in our previous study (Li et al., 2007). Its low PDI value indicates that Low-CNE distributes in a narrow range. It was notable that zeta potential value changed from highly negative to positive which should be contributed by the coating of chitosan. Chitosan bears positive charge which could be attracted to the surface of negatively charged NE. However, zeta potential value of Low-CNE was smaller than the corresponding values of other two chitosan coated NEs which were 39.94 mV and 33.40 mV respectively. It was suggested that degree of deacetylation (DD) and concentration of chitosan may exert influence on pertaining properties of the delivery system (Chen, Zheng, Wang, Lee, & Park, 2002; Koppolu et al., 2014). In our study, chitosan samples have similar DD value which should present minor impact on

3.2. Morphological study by transmission electron microscopy (TEM) analysis Morphological properties of NE and CNE were revealed by TEM analysis as shown in Fig. 1. As depicted in Fig. 1A, NE had classic spherical shape with the diameter about 100 nm which was corroborated with the DLS test. Meanwhile, it was notable that NE had smooth surface without any rough and irregular structures. The morphology of CNE was distinctly different from NE, as presented in Fig. 1BeD. In the images of CNE samples, no prefect spheres were revealed. They showed roughly round shape with many rough and irregular structures on the surface. It has been reported that coating with polymer usually change the morphology of nanoparticles from smooth surfaced ones to slightly irregularly and rough spherical shaped ones (Li et al., 2013; Shin et al., 2013). The diameters were larger than that of NE which was also caused by chitosan coating. There was no significant size and morphology difference between CNE samples which should be resulted from the TEM sample preparation. As mentioned previously, drying and staining procedures during conventional TEM measurement may contribute to variation of size which may be inconsistent with that obtained by DLS (Goycoolea et al., 2012). Nevertheless, TEM images confirmed that chitosan coated successfully around the surface of NE.

Table 1 Size, PDI, and zeta potential before and after one month storage in room temperature. Group

Original parameters

NE Low-NE Mid-NE High-NE

113.93 97.65 103.57 125.83

Size (nm) ± ± ± ±

Parameters after 1 month storage PDI

9.11b 1.16c 1.46bc 2.61a

0.23 0.20 0.29 0.30

Zeta (mV) ± ± ± ±

0.04b 0.02c 0.01a 0.04a

36.26 4.32 39.94 33.40

± ± ± ±

Size (nm) 3.45d 0.14c 0.84a 0.99b

108.70 105.53 118.17 126.53

± ± ± ±

PDI 5.53c 1.37c 3.44b 0.78a

0.23 0.18 0.19 0.19

Zeta (mV) ± ± ± ±

0.05N.S 0.03 0.01 0.02

30.47 5.58 31.07 32.07

± ± ± ±

4.09d 0.54c 3.92b 0.84a

NE, nanoemulsion; Low-NE, low molecular weight chitosan coated nanoemulsion; Mid-NE, middle molecular weight chitosan coated nanoemulsion; High-NE, high molecular weight chitosan coated nanoemulsion. Results are expressed as Mean ± SD (n ¼ 3), data with different superscripts in the same column are significantly different (p < 0.05) analyzed by ANOVA, N.S means no significant different among the data in the same column.

142

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

Fig. 1. Transmission Electron Microscopy (TEM) images of: (A) nanoemulsion, (B) low molecular weight chitosan coated nanoemulsion, (C) middle molecular weight chitosan coated nanoemulsion, (D) high molecular weight chitosan coated nanoemulsion, bar is 100 nm in each image.

3.3. Physical stability Storage stability of NE and CNE samples was investigated by measuring the variations of size, PDI and zeta potential after 1 month of storage in room temperature. As listed in Table 1, size of NE slightly decreased to 108.70 nm with similar PDI while its zeta potential value was decreased. During the storage, emulsifiers may interact with the oil droplets more compactly, thus reducing the size and zeta potential value. For both Low-CNE and Mid-CNE, sizes were slightly increased and PDI values were slightly decreased. During the storage, chitosan may re-arrange itself around the surface of NE in a more thermodynamically stable way. For High-CNE, size changed slightly to 126.53 nm which may be also caused by the re-arrangement of chitosan polymer on the NE surface. It is also notable that for both Mid-CNE and High-CNE, zeta potential decreased slightly which may be ascribed to that more glucosamine residues participate the electro-interaction with the negative charge on the surface of NE. It was obvious that both NE and CNE had high stability in the room temperature storage test that should be contributed to their steric and electrostatic stability mechanism (Goycoolea et al., 2012). Possible microbial effect on colloidal stability could be excluded as both curcumin and chitosan has strong antimicrobial ability proved in our and others' reports (Kong, Chen, Xing, & Park, 2010; Moghadamtousi et al., 2014). During food processing, NaCl or other salts are usually added for various purposes, increasing the ionic strength of the food system. Therefore, it is desirable for the NE to be stable in the increased ionic strength environment. In NaCl solution, ions have electrostatic screening effect on the emulsion which can decrease the zeta

potential value as well as the electrostatic repelling force between emulsion droplets (Klinkesorn & Namatsila, 2009). As listed in Table 2, for NE and all the CNE samples, incubation with NaCl solution increased their mean size and decreased their zeta potential values. Due to the electrostatic attraction, negatively charged NE could draw Naþ and at the same time increased its size to about 125 nm and reduced its zeta potential value to about 3 mV. For positively charged CNEs, Cl would be attracted which also greatly changed their charge distribution. Their zeta potential values tended to approach to zero, decreasing the electrostatic repelling force at the same time. However, PDI values were still at low level (<0.3), indicating all the NE samples retained the unified droplets distribution. Their respective parameters were measured after stored in room temperature for 1 month. There was no significant change of size, PDI and zeta potential comparing with that of before the storage test. However, after storage in room temperature for 3 month, obvious phase separation in NE was observed as shown in Fig. 2. In contrast, chitosan coated NEs were more stable and homogeneously dispersed in the solution. NE is mainly stabilized by electrostatic repelling force contributed by the cosurfactant. Ionic screening effect greatly decreased the zeta potential thus lowered the colloidal stability of NE. For chitosan coated NEs, steric repelling force guarantee its stability even their zeta potential values were also reduced. Chuah et al. reported that lecithin stabilized emulsion, with the mean size about 24.4 mm, was very stable under various NaCl concentrations; but chitosan coated emulsion would aggregated when NaCl concentration was higher than 0.2 M (Chuah, Kuroiwa, Kobayashi, & Nakajima, 2009). It was assumed that chitosan coated emulsion within the nano meter range has

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

143

Table 2 Size, PDI and zeta potential change of emulsion in various ionic strength solution. Parameters 0.2 M NaCl solution

0.5 M NaCl solution

1 M NaCl solution

NE Size (nm) Size, 1 month PDI PDI, 1 month Zeta (mV) Zeta, 1 month Size (nm) Size, 1 month PDI PDI, 1 month Zeta (mV) Zeta, 1 month Size (nm) Size, 1 month PDI PDI, 1 month Zeta (mV) Zeta, 1 month

Low-CNE

124.17 142.74 0.23 0.31 4.39 4.87 125.67 116.47 0.24 0.22 2.75 3.88 129.83 115.50 0.24 0.22 1.76 1.74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.23 2.94 0.05 0.03 0.43 1.27 8.81 0.71 0.05 0.07 0.48 1.03 10.74 1.26 0.01 0.03 0.80 0.77

113.21 115.63 0.23 0.23 0.94 1.70 113.93 118.10 0.22 0.23 1.61 1.91 116.80 118.23 0.23 0.19 0.71 3.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Mid-CNE 0.54 0.96 0.02 0.06 1.60 0.39 0.45 1.92 0.01 0.02 0.49 0.66 0.08 1.15 0.01 0.02 1.38 0.74

121.60 116.23 0.22 0.23 3.72 0.54 128.93 116.83 0.24 0.24 5.27 1.45 137.93 124.93 0.24 0.22 5.42 0.48

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

High-CNE 0.16 1.31 0.01 0.01 0.66 0.23 0.71 0.81 0.01 0.01 0.73 0.41 1.25 3.41 0.05 0.02 1.04 0.80

130.47 122.77 0.20 0.20 3.46 0.53 132.67 122.31 0.21 0.20 3.40 0.36 136.63 126.81 0.21 0.19 2.63 2.82

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.69 0.74 0.02 0.01 0.26 0.31 3.17 0.80 0.01 0.01 0.78 0.57 3.45 1.35 0.02 0.02 1.81 1.41

NE, nanoemulsion; Low-NE, low molecular weight chitosan coated nanoemulsion; Mid-NE, middle molecular weight chitosan coated nanoemulsion; High-NE, high molecular weight chitosan coated nanoemulsion. Results are expressed as Mean ± SD (n ¼ 3).

Fig. 2. Nano-emulsion treated with 0.2 mol/L NaCl and stored in room temperature for 3 month: (A) nanoemulsion, red arrow indicates oil layer, (B) low molecular weight chitosan coated nanoemulsion, (C) middle molecular weight chitosan coated nanoemulsion, (D) high molecular weight chitosan coated nanoemulsion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

much higher stability than its micro-sized counterpart. Variations of size, PDI and zeta potential of NE and CNE after thermal treatment were listed in Table 3. Under 63  C and 100  C conditions, the size and zeta potential values of NE, Mid-CNE and High-CNE decreased. Especially for Mid-CNE and High-CNE treated at 100  C for 10 min, the size decreased to about 75 nm. On the other hand, size of Low-CNE increased to 160 nm with little change of zeta potential. The PDI values of all samples remained at low level, suggesting that after thermal treatment all the droplets still distributed within narrow range. Relatively low zeta potential of Low-CNE may induce coalescence or Ostwald ripening which is responsible for the increased size after thermal treatment (Piorkowski & McClements, 2014). On the contrary, for NE that bear higher zeta potential value, the size decreased slightly. Our results are in conflict with previous publications. Sari et al. prepared whey protein and Tween-80 stabilized NE and they found that boiling treatment significantly increased the emulsion size and reduced its zeta potential value (Sari et al., 2015). They concluded that at higher temperature, whey protein may be denatured which induce emulsion aggregation. In one recent study, Guan et al. investigated the thermal stability of b-carotene loaded NE emulsified by lecithin and whey protein isolate (Guan, Wu, & Zhong, 2016). The authors also reported slightly decreased mean size of NE after the thermal treatment but gave no explanation for the observed phenomenon. For the decreased size of Mid-CNE and High-CNE, it was speculated

Table 3 Size, PDI and zeta potential change of emulsion under thermal treatment. Parameters 63  C, 30 min

100  C, 10 min

NE Size (nm) Size, 1 month PDI PDI, 1 month Zeta (mV) Zeta, 1 month Size (nm) Size, 1 month PDI PDI, 1 month Zeta (mV) Zeta, 1 month

98.58 109.50 0.22 0.16 18.03 17.70 100.02 108.07 0.21 0.18 20.20 16.20

Low-CNE ± ± ± ± ± ± ± ± ± ± ± ±

0.86 0.65 0.01 0.03 0.98 0.64 0.84 0.74 0.01 0.02 0.94 0.16

159.73 164.23 0.10 0.07 5.39 6.64 161.20 161.47 0.22 0.21 3.93 6.38

± ± ± ± ± ± ± ± ± ± ± ±

Mid-CNE 1.58 1.33 0.01 0.02 0.43 0.28 0.22 2.19 0.01 0.01 0.44 0.54

102.83 104.03 0.17 0.16 20.33 20.27 79.99 81.38 0.11 0.14 19.77 18.00

± ± ± ± ± ± ± ± ± ± ± ±

High-CNE 0.72 0.45 0.01 0.04 0.66 0.71 0.51 0.45 0.01 0.01 0.74 0.74

117.93 118.77 0.19 0.18 23.89 23.97 74.00 77.65 0.11 0.12 17.87 19.87

± ± ± ± ± ± ± ± ± ± ± ±

0.71 0.71 0.01 0.06 0.46 0.45 0.57 0.16 0.02 0.01 0.34 0.50

NE, nanoemulsion; Low-NE, low molecular weight chitosan coated nanoemulsion; Mid-NE, middle molecular weight chitosan coated nanoemulsion; High-NE, high molecular weight chitosan coated nanoemulsion. Results are expressed as Mean ± SD (n ¼ 3).

144

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

that higher temperature increased the water solubility of MCT oil hence ‘extracted’ it from NE. After returning to room temperature, the extracted MCT oil may form nanoparticles with either surfactants or chitosan, thus reducing the mean size. Moreover, thermal treated NE samples were stable for at least 1 month at room temperature. After 1 month, all the parameters showed no significant variations as shown in Table 3. However, it is interesting to mention that curcumin concentration of NE after the storage decreased greatly (Fig. 3). Absorption at 419 nm of NE treated at 63  C was 0.01 while for Low-CNE, Mid-CNE and High-CNE the readings were 0.064, 0.080 and 0.079 respectively. Absorption readings of 100  C treated emulsion were similar. This may be due to the barrier effect of chitosan to curcumin in two ways. First of all, curcumin may migrate into hot water since it has higher water solubility at higher temperature. And when existed outside the emulsion droplets, it would face oxidation and hydrolyzation in the aqueous solution which quickly decreased its concentration. Chitosan coating may inhibit this curcumin migration phenomenon during the thermal treatment. Additionally, chitosan can inhibit the radicals attack to curcumin inside the NE (Schreiber, Bozell, Hayes, & Zivanovic, 2013). Chitosan coating also confers NE with cationic nature that can repel reactive radicals like protons and metal ions, protecting curcumin from oxidation (Yang, Tian, Ho, & Huang, 2012). 3.4. UV irradiation stability UV irradiation is a simple and effective sterilization method during food and beverage processing (Guerrero-Beltran & BarbosaCanovas, 2004). However, curcumin is unstable to UV irradiation. In the present study, the protection ability of NE and CNEs was examined and the result was illustrated in Fig. 4. There was no significant difference among the emulsion samples until 24 h of UV treatment. After 72 h of UV irradiation, it was obvious that CNE could better protect curcumin than NE in the order of High-NCE, Mid-NCE and Low-NCE. It is possible that chitosan could protect curcumin from photochemical oxidation induced by the UV irradiation (Glaze, Lay, & Kang, 1995). After 72 h of UV treatment, size, PDI and zeta potential was measured and listed in Table 4. Size and PDI showed slightly variations, meaning that emulsion collodial system was not affected by the UV treatment. Xiao et al. observed similar UV protection ability of carboxymethyl chitosan nanoparticles to curcumin and they concluded that the barrier effect of the polymer was responsible for the enhanced UV stability (Xiao, Nian, & Huang, 2015). Zeta potential values were significantly

Fig. 4. Direct UV irradiation treatment of nanoemulsion (blue), low molecular weight chitosan coated nanoemulsion (red), middle molecular weight chitosan coated nanoemulsion (green) and high molecular weight chitosan coated nanoemulsion (purple). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 4 Size, PDI and zeta potential of emulsion after 72 h of UV irradiation treatment. Group

Size (nm)

NE Low-CNE Mid-CNE High-CNE

109.33 117.37 119.00 144.07

± ± ± ±

PDI 0.45c 0.70b 1.56b 2.45a

0.23 0.24 0.21 0.24

Zeta (mV) ± ± ± ±

0.01N.S 0.01 0.03 0.02

17.63 2.87 8.56 10.97

± ± ± ±

0.26d 0.24c 1.17b 0.48a

NE, nanoemulsion; Low-NE, low molecular weight chitosan coated nanoemulsion; Mid-NE, middle molecular weight chitosan coated nanoemulsion; High-NE, high molecular weight chitosan coated nanoemulsion. Results are expressed as Mean ± SD (n ¼ 3), data with different superscripts in the same column are significantly different (p < 0.05) analyzed by ANOVA, N.S means no significant different among the data in the same column.

reduced after the UV treatment. Both surfactants and chitosan are prone to be degraded by UV rays, altering the functional groups and charge distribution (Wasikiewicz, Yoshii, Nagasawa, Wach, & Mitomo, 2005; Zhao, Hidaka, Takamura, Pelizzetti, & Serpone, 1993). UV irradiation produce several radicals in the solution which may oxidize or react with the charged groups, thus

Fig. 3. UV absorption spectra of nanoemulsion (blue), low molecular weight chitosan coated nanoemulsion (red), middle molecular weight chitosan coated nanoemulsion (green) and high molecular weight chitosan coated nanoemulsion (purple) after thermal treatments and storage in room temperature for 1 month. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

145

‘neutralized’ the charge distributed on the surface of emulsion droplets. Nevertheless, along with results of thermal treatment, it is concluded that chitosan coating is effective to protect curcumin from oxidation and degradation. 3.5. In vitro digestion and bio-accessibility study It has been suggested that cationic polymers may inhibit lipid digestion by electrostatic interaction with lipase and bile salt during lipolysis in the small intestine (Kido et al., 2003). Considering the fact that chitosan is a widely used cationic natural polymer for fabricating nano-delivery system, it is important to determine its impact on the digestion process. In the present study, we used pHstat titration method to characterize lipid digestion process. During the lipolysis, free fatty acid is released from the oil droplets, along with decreasing of pH value. The pH-stat records volume of NaOH solution that is injected into the digesta to maintain the pH at 7.0. The volume of NaOH is used to represent the process of lipolysis indirectly (Nilsson & Belfrage, 1979). A three-step test was used to simulate the digestion process. Size changes after each step were showed in Fig. 5. After the simulated mouth and gastric digestion phase, size of NE and CNEs was not altered greatly. This was due to the fact that emulsion was not destroyed under the simulated mouth and gastric digestion conditions. In the lipolysis, fatty acid is release from the oil droplets and form micelles with bile salt that facilitates fatty acid absorption by the intestinal cell. After simulated intestinal digestion, micelle formed from NE showed size of 5348 nm. While size of Low-CNE, Mid-CNE and High-CNE formed micelle was 9509, 18,480 and 18,146 nm respectively. Zeta potential value of NE formed micelle after simulated digestion was 12.67 mV which was higher than that of CNE formed micelle. Chitosan may interact with micelle by electrostatic force as both the size and zeta potential of CNE formed micelle were notably different from that of NE formed micelle. In the simulated small intestinal digestion, NaOH consumption curve of NE was much similar with that of Low-CNE (Fig. 6). Both the turning time point and NaOH consumption level of Mid-CNE and High-CNE were lower than that of NE. Final NaOH consumption was listed in Table 5. The result implied that chitosan coating may repress lipolysis of NE, even though at a small degree. Mun et al. reported that lecithin-chitosan emulsion showed lower fatty acid release level as a result of inhibited lipase activity (Mun, Decker, Park, Weiss, & McClements, 2006). Klinkesorn et al. also mentioned that chitosan coating decreased lipid release from tuna oil-in-water emulsion (Klinkesorn & McClements, 2009). Another natural cationic polymer, ε-polylysine was investigated for its

Fig. 6. NaOH consumption of nanoemulsion (blue), low molecular weight chitosan coated nanoemulsion (red), middle molecular weight chitosan coated nanoemulsion (green) and high molecular weight chitosan coated nanoemulsion (purple) during in vitro lipolysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

effects on the digestion of corn oil-in-water emulsions by Cynthia Lopez-Pena et al. (Lopez-Pena et al., 2016). They concluded that ε-polylysine has no significant impact on the lipid digestion. However, to our best knowledge, there is no data about the influence of chitosan coating on the gastrointestinal fate of NE in the literature. As suggested in the previously studies, chitosan may inhibit lipolysis function of lipase during the in vitro digestion test in several ways (Klinkesorn & McClements, 2009). At the same time, in vitro bio-accessibility was in line with NaOH consumption level as listed in Table 5. Compared with NE, Low-CNE showed slightly higher bio-accessibility which suggest that oligo-chitosan may have little impact on the lipolysis process. However, MidCNE and High-CNE presented lower bio-accessibility than that of NE, agreeing with their NaOH consumptions. It is worthy to mention that bio-accessibility values of Mid-CNE and High-CNE is less than 5% lower than that of NE. Meanwhile, as widely investigated in previous studies, chitosan coating on the nano-delivery system is beneficial in terms of protection of encapsulated bioactive compounds, stabilization of nano-delivery system, improvement of intestinal absorption of payload and many others (Cho, Kim, & Park, 2012; Chuah et al., 2009; Pan et al., 2002; Yang et al., 2012). So, even though chitosan may interfere the digestion process of NE, its effect is very limited that chitosan coating is still advantageous for abovementioned purposes. 4. Conclusions

Fig. 5. Size of nanoemulsion and chitosan coated nanoemulsion after simulated saliva digestion (SAF), simulated gastric digestion (SGF) and simulated intestinal digestion (final).

In this study, we prepared nano-emulsion (NE) based on MCT oil, Tween 80 and lecithin to encapsulate curcumin at high loading ability and efficiency. Water dispersibility of curcumin in NE was increased by 1400 fold than that of raw curcumin. Chitosan with low, middle and high molecular weight was applied for coating the prepared NE to investigate their effects on the physical, chemical stability and bio-accessibility of curcumin. After chitosan coating, zeta potential changed from negative to positive, implying that chitosan was coated on the surface of NE. Middle and high molecular weight chitosan increased the size of NE. Both NE and chitosan coated NE were stable at room temperature. Chitosan coating could inhibit degradation of curcumin during thermal and UV irradiation treatment. However, middle and high molecular weight chitosan may interfere lipolysis of NE during the in vitro digestion which also slightly decreased its bio-accessibility. Our results

146

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147

Table 5 NaOH consumption during lipolysis and in vitro bio-accessibility of emulsion. Parameter

NE

Low-CNE

Mid-CNE

High-CNE

NaOH consumption (mL) In vitro bio-accessibility

36.80 ± 1.13N.S 79.14 ± 2.92 ab

36.74 ± 1.34 82.23 ± 2.45a

35.16 ± 0.72 76.41 ± 1.22b

34.75 ± 0.50 76.20 ± 1.52b

NE, nanoemulsion; Low-NE, low molecular weight chitosan coated nanoemulsion; Mid-NE, middle molecular weight chitosan coated nanoemulsion; High-NE, high molecular weight chitosan coated nanoemulsion. Results are expressed as Mean ± SD (n ¼ 3), data with different superscripts in the same row are significantly different (p < 0.05) analyzed by ANOVA, N.S means no significant different among the data in the same row.

suggest that NE coated with chitosan is a promising delivery system to promote the applications of curcumin in functional food and beverage system. Acknowledgments This research was supported by Kyung-Nong Company, the International Research & Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) of Korea (Grant No. 2012K1A3A1A20031356), Korea University Grant, and Institute of Biomedical Science & Food Safety, Korea University Food Safety Hall, Republic of Korea. References Abbas, S., Bashari, M., Akhtar, W., Li, W. W., & Zhang, X. M. (2014). Process optimization of ultrasound-assisted curcumin nanoemulsions stabilized by OSAmodified starch. Ultrasonics Sonochemistry, 21(4), 1265e1274. Aditya, N. P., Shim, M., Lee, I., Lee, Y., Im, M. H., & Ko, S. (2013). Curcumin and genistein coloaded nanostructured lipid carriers: in vitro digestion and antiprostate cancer activity. Journal of Agricultural and Food Chemistry, 61(8), 1878e1883. Aditya, N., Yang, H., Kim, S., & Ko, S. (2015). Fabrication of amorphous curcumin nanosuspensions using b-lactoglobulin to enhance solubility, stability, and bioavailability. Colloids and Surfaces B: Biointerfaces, 127, 114e121. Aggarwal, B. B., Sundaram, C., Malani, N., & Ichikawa, H. (2007). Curcumin: the Indian solid gold. Molecular Targets and Therapeutic Uses of Curcumin in Health and Disease, 595, 1e75. Ahmed, K., Li, Y., McClements, D. J., & Xiao, H. (2012). Nanoemulsion- and emulsionbased delivery systems for curcumin: encapsulation and release properties. Food Chemistry, 132(2), 799e807. Basnet, P., & Skalko-Basnet, N. (2011). Curcumin: an anti-inflammatory molecule from a curry spice on the path to cancer treatment. Molecules, 16(6), 4567e4598. Begum, A. N., Jones, M. R., Lim, G. P., Morihara, T., Kim, P., Heath, D. D., et al. (2008). Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease. Journal of Pharmacology and Experimental Therapeutics, 326(1), 196e208. Cheng, A. L., Hsu, C. H., Lin, J. K., Hsu, M. M., Ho, Y. F., Shen, T. S., et al. (2001). Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Research, 21(4B), 2895e2900. Chen, X. G., Zheng, L., Wang, Z., Lee, C. Y., & Park, H. J. (2002). Molecular affinity and permeability of different molecular weight chitosan membranes. Journal of Agricultural and Food Chemistry, 50(21), 5915e5918. Cho, Y., Kim, J. T., & Park, H. J. (2012). Size-controlled self-aggregated N-acyl chitosan nanoparticles as a vitamin C carrier. Carbohydrate Polymers, 88(3), 1087e1092. Chuah, A. M., Kuroiwa, T., Kobayashi, I., & Nakajima, M. (2009). Effect of chitosan on the stability and properties of modified lecithin stabilized oil-in-water monodisperse emulsion prepared by microchannel emulsification. Food Hydrocolloids, 23(3), 600e610. Glaze, W. H., Lay, Y., & Kang, J. W. (1995). Advanced oxidation processes - a kineticmodel for the oxidation of 1,2-dibromo-3-chloropropane in water by the combination of hydrogen- peroxide and Uv-radiation. Industrial & Engineering Chemistry Research, 34(7), 2314e2323. Goycoolea, F. M., Valle-Gallego, A., Stefani, R., Menchicchi, B., David, L., Rochas, C., et al. (2012). Chitosan-based nanocapsules: physical characterization, stability in biological media and capsaicin encapsulation. Colloid and Polymer Science, 290(14), 1423e1434. Guan, Y., Wu, J., & Zhong, Q. (2016). Eugenol improves physical and chemical stabilities of nanoemulsions loaded with b-carotene. Food Chemistry, 194, 787e796. Guerrero-Beltran, J. A., & Barbosa-Canovas, G. V. (2004). Review: advantages and limitations on processing foods by UV light. Food Science and Technology International, 10(3), 137e147. Heger, M., van Golen, R. F., Broekgaarden, M., & Michel, M. C. (2014). The molecular

basis for the pharmacokinetics and pharmacodynamics of curcumin and its metabolites in relation to cancer. Pharmacological Reviews, 66(1), 222e307. Kean, T., & Thanou, M. (2010). Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug Delivery Reviews, 62(1), 3e11. Kido, Y., Hiramoto, S., Murao, M., Horio, Y., Miyazaki, T., Kodama, T., et al. (2003). Ɛpolylysine inhibits pancreatic lipase activity and suppresses postprandial hypertriacylglyceridemia in rats. Journal of Nutrition, 133(6), 1887e1891. Klinkesorn, U., & McClements, D. J. (2009). Influence of chitosan on stability and lipase digestibility of lecithin-stabilized tuna oil-in-water emulsions. Food Chemistry, 114(4), 1308e1315. Klinkesorn, U., & Namatsila, Y. (2009). Influence of chitosan and NaCl on physicochemical properties of low-acid tuna oil-in-water emulsions stabilized by nonionic surfactant. Food Hydrocolloids, 23(5), 1374e1380. Kong, M., Chen, X. G., Xing, K., & Park, H. J. (2010). Antimicrobial properties of chitosan and mode of action: a state of the art review. International Journal of Food Microbiology, 144(1), 51e63. Li, Y. Y., Chen, X. G., Liu, C. S., Cha, D. S., Park, H. J., & Lee, C. M. (2007). Effect of the molecular mass and degree of substitution of oleoylchitosan on the structure, rheological properties, and formation of nanoparticles. Journal of Agricultural and Food Chemistry, 55(12), 4842e4847. Li, J., Joung, H. J., Lee, I. W., Chen, X., & Park, H. J. (2015). The influence of different water types and brewing durations on the colloidal properties of green tea infusion. International Journal of Food Science & Technology, 50(11), 2483e2489. Li, J., Lee, I. W., Shin, G. H., Chen, X., & Park, H. J. (2015). Curcumin-Eudragit® E PO solid dispersion: a simple and potent method to solve the problems of curcumin. European Journal of Pharmaceutics and Biopharmaceutics, 94, 322e332. Li, J., Shin, G. H., Chen, X. G., & Park, H. J. (2015c). Modified curcumin with hyaluronic acid: combination of pro-drug and nano-micelle strategy to address the curcumin challenge. Food Research International, 69, 202e208. Li, X. Y., Qi, J. P., Xie, Y. C., Zhang, X., Hu, S. W., Xu, Y., et al. (2013). Nanoemulsions coated with alginate/chitosan as oral insulin delivery systems: preparation, characterization, and hypoglycemic effect in rats. International Journal of Nanomedicine, 8, 23e32. Li, J., Shin, G. H., Lee, I. W., Chen, X., & Park, H. J. (2016). Soluble starch formulated nanocomposite increases water solubility and stability of curcumin. Food Hydrocolloids, 56, 41e49. Lopez-Pena, C. L., Zheng, B., Sela, D. A., Decker, E. A., Xiao, H., & McClements, D. J. (2016). Impact of ε-polylysine and pectin on the potential gastrointestinal fate of emulsified lipids: In vitro mouth, stomach and small intestine model. Food Chemistry, 192, 857e864. McClements, D. J. (2015). Nanoscale nutrient delivery systems for food applications: improving bioactive dispersibility, stability, and bioavailability. Journal of Food Science, 80(7), N1602eN1611. Mun, S., Decker, E. A., & McClements, D. J. (2006). Effect of molecular weight and degree of deacetylation of chitosan on the formation of oil-in-water emulsions stabilized by surfactant-chitosan membranes. Journal of Colloid and Interface Science, 296(2), 581e590. Mun, S., Decker, E. A., Park, Y., Weiss, J., & McClements, D. J. (2006). Influence of interfacial composition on in vitro digestibility of emulsified lipids: potential mechanism for chitosan's ability to inhibit fat digestion. Food Biophysics, 1(1), 21e29. Nilsson, N. O., & Belfrage, P. (1979). Continuous Monitoring of free fatty-acid release from adipocytes by pH-stat titration. Journal of Lipid Research, 20(4), 557e560. Ozturk, B., Argin, S., Ozilgen, M., & McClements, D. J. (2015). Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural biopolymers: whey protein isolate and gum arabic. Food Chemistry, 188, 256e263. Pan, Y., Li, Y. J., Zhao, H. Y., Zheng, J. M., Xu, H., Wei, G., et al. (2002). Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo. International Journal of Pharmaceutics, 249(1e2), 139e147. Paradkar, A., Ambike, A. A., Jadhav, B. K., & Mahadik, K. R. (2004). Characterization of curcumin-PVP solid dispersion obtained by spray drying. International Journal of Pharmaceutics, 271(1e2), 281e286. Piorkowski, D. T., & McClements, D. J. (2014). Beverage emulsions: recent developments in formulation, production, and applications. Food Hydrocolloids, 42, 5e41. Koppolu, B. P., Smith, S. G., Ravindranathan, S., Jayanthi, S., Kumar, T. K. S., & Zaharoff, D. A. (2014). Controlling chitosan-based encapsulation for protein and vaccine delivery. Biomaterials, 35(14), 4382e4389. Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2012). Nanoemulsion delivery systems: influence of carrier oil on beta-carotene bioaccessibility. Food

J. Li et al. / Food Hydrocolloids 60 (2016) 138e147 Chemistry, 135(3), 1440e1447. Sari, T. P., Mann, B., Kumar, R., Singh, R. R. B., Sharma, R., Bhardwaj, M., et al. (2015). Preparation and characterization of nanoemulsion encapsulating curcumin. Food Hydrocolloids, 43, 540e546. Sarkar, A., Goh, K. K., & Singh, H. (2009). Colloidal stability and interactions of milkprotein-stabilized emulsions in an artificial saliva. Food Hydrocolloids, 23(5), 1270e1278. Schreiber, S. B., Bozell, J. J., Hayes, D. G., & Zivanovic, S. (2013). Introduction of primary antioxidant activity to chitosan for application as a multifunctional food packaging material. Food Hydrocolloids, 33(2), 207e214. Setthacheewakul, S., Mahattanadul, S., Phadoongsombut, N., Pichayakorn, W., & Wiwattanapatapee, R. (2010). Development and evaluation of selfmicroemulsifying liquid and pellet formulations of curcumin, and absorption studies in rats. European Journal of Pharmaceutics and Biopharmaceutics, 76(3), 475e485. Shin, G. H., Chung, S. K., Kim, J. T., Joung, H. J., & Park, H. J. (2013). Preparation of chitosan-coated nanoliposomes for improving the mucoadhesive property of curcumin using the ethanol injection method. Journal of Agricultural and Food Chemistry, 61(46), 11119e11126. Sood, S., Jain, K., & Gowthamarajan, K. (2014). Optimization of curcumin nanoemulsion for intranasal delivery using design of experiment and its toxicity assessment. Colloids and Surfaces B: Biointerfaces, 113, 330e337. Tadros, T., Izquierdo, R., Esquena, J., & Solans, C. (2004). Formation and stability of nano-emulsions. Advances in Colloid and Interface Science, 108, 303e318. Takahashi, M., Uechi, S., Takara, K., Asikin, Y., & Wada, K. (2009). Evaluation of an oral carrier system in rats: bioavailability and antioxidant properties of liposome-encapsulated curcumin. Journal of Agricultural and Food Chemistry,

147

57(19), 9141e9146. Wang, L. J., Hu, Y. Q., Yin, S. W., Yang, X. Q., Lai, F. R., & Wang, S. Q. (2015). Fabrication and characterization of antioxidant pickering emulsions stabilized by zein/ chitosan complex particles (ZCPs). Journal of Agricultural and Food Chemistry, 63(9), 2514e2524. Wang, X. Y., Jiang, Y., Wang, Y. W., Huang, M. T., Ho, C. T., & Huang, Q. R. (2008). Enhancing anti-inflammation activity of curcumin through O/W nanoemulsions. Food Chemistry, 108(2), 419e424. Wasikiewicz, J. M., Yoshii, F., Nagasawa, N., Wach, R. A., & Mitomo, H. (2005). Degradation of chitosan and sodium alginate by gamma radiation, sonochemical and ultraviolet methods. Radiation Physics and Chemistry, 73(5), 287e295. Xiao, J., Nian, S., & Huang, Q. (2015). Assembly of kafirin/carboxymethyl chitosan nanoparticles to enhance the cellular uptake of curcumin. Food Hydrocolloids, 51, 166e175. Yang, X. Q., Tian, H. X., Ho, C. T., & Huang, Q. R. (2012). Stability of citral in emulsions coated with cationic biopolymer layers. Journal of Agricultural and Food Chemistry, 60(1), 402e409. Yu, H. L., & Huang, Q. R. (2012). Improving the oral bioavailability of curcumin using novel organogel-based nanoemulsions. Journal of Agricultural and Food Chemistry, 60(21), 5373e5379. Zhao, J., Hidaka, H., Takamura, A., Pelizzetti, E., & Serpone, N. (1993). Photodegradation of surfactants 11 zeta-Potential measurements in the photocatalytic oxidation of surfactants in aqueous titania dispersions. Langmuir, 9(7), 1646e1650. Moghadamtousi, S. Z., Kadir, H. A., Hassandarvish, P., Tajik, H., Abubakar, S., & Zandi, K. (2014). A review on antibacterial, antiviral, and antifungal activity of curcumin. BioMed Research International, 2014, 1e12.