Development of FoodâGrade Curcumin Nanoemulsion and its ...

Development of Food-Grade Curcumin Nanoemulsion and its Potential Application to Food Beverage System: Antioxidant Property and In Vitro Digestion Hee Joung Joung, Mi-Jung Choi, Jun Tae Kim, Seok Hoon Park, Hyun Jin Park, and Gye Hwa Shin

Abstract: Curcumin nanoemulsions (Cur-NEs) were developed with various surfactant concentrations by using high pressure homogenization and finally applied to the commercial milk system. Characterization of Cur-NEs was performed by measuring the droplet size and polydispersity index value at different Tween 20 concentrations. The morphology of the Cur-NEs was observed by confocal laser scanning microscopy and transmission electron microscopy. Antioxidant activity and in vitro digestion ability were tested using 2,2-diphenyl-1-picrylhydrazyl, 2,2 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt, pH-stat method, and thiobarbituric acid reactive substances assays. Cur-NEs were found to be physically stable for 1 mo at room temperature. The surfactant concentration affects particle formation and droplet size. The mean droplet size decreased from 122 to 90 nm when surfactant concentration increased 3 times. Cur-NEs had shown an effective oxygen scavenging activity. Cur-NEs-fortified milk showed significantly lower lipid oxidation than control (unfortified) milk and milk containing curcumin-free nanoemulsions. These properties make Cur-NEs suitable systems for the beverage industry.

Introduction Curcumin extracted from the rhizome of Curcuma longa is a bioactive ingredient widely used in the fields of foods, medicines, and cosmetics (Sharma and others 2012). Recent studies have demonstrated that curcumin has numerous pharmacological and biological activities such as anti-oxidant (Masuda and others 1998; Menon and Sudheer 2007; Nagarajan and others 2010), antiinflammatory (Wang and others 2008), anticancer (Bar-Sela and others 2010; Heng 2010), antimicrobial (De and others 2009), and free radical scavenger properties (Sreejayan and Rao 1996). However, curcumin has been limited in its applications due to its poor water solubility and low bioavailability (Tønnesen and others 2002; Anand and others 2007; Maiti and others 2007). In order to overcome these problems, various colloidal systems have been developed (Shin and others 2013; Li and others 2015). Many researchers have studied on the emulsion delivery system to improve bioavailability and solubility of bioactive compounds (Huang and others 2010). Nanoemulsions have been proposed to offer several advantages compared to conventional emulsions due to their higher optical clarity, better physical stability to particle aggregation and phase separation, and novel rheological properties (Mason and others 2006; McClements 2011; McClements 2015). Stability, physicochemical properties, and functional performance of nanoemulsions are significantly dependent on the MS 20151413 Submitted 8/18/2015, Accepted 12/22/2015. Authors Joung, H.J. Park, and Shin are with Department of Biotechnology, College of Life Sciences & Biotechnology, Korea Univ., Anam-dong, Sungbuk-gu, Seoul, 136-701, Korea. Author Choi is with Dept. of Bioresources & Food Science, Konkuk Univ., Seoul, 143-701, Korea. Author Kim is with Dept. of Food Science & Technology, Keimyung Univ., Daegu, 704-701, Korea. Author S.H. Park is with Dept. of Environmental Engineering, Anyang Univ., Gyeonggi-do, 430-714, Korea. Direct inquiries to authors H.J. Park and Shin (E-mails: [email protected] and [email protected]).

R C 2016 Institute of Food Technologists

doi: 10.1111/1750-3841.13224 Further reproduction without permission is prohibited

droplet concentrations, compositions, particle size distributions, and interfacial properties, which could be controlled by altering their preparation conditions (Lesmes and McClements 2009). The droplet size of nanoemulsions is crucial to determine their stability, appearance, rheological properties, and bioavailability (Acosta 2009). Typically, a high pressure homogenization process is used to decrease the mean droplet size which is dependent on the number of passes and homogenization pressure. In the case of certain emulsifiers, however, excessive processing such as higher pressures and longer emulsification times may increase the droplet size of emulsions (Jafari and others 2008). It has been known that the stability of nanoemulsions is affected by several factors such as oil type, surfactant type, compositions (ratio of oil/water/surfactant), preparation method, mechanical pressure, pH, temperature, and ionic strength (McClements 2011; Chang and McClements 2014). Recently, a number of studies have focused on the development of food-grade colloidal systems to encapsulate functional food ingredients such as flavors, food coloring agents, micronutrients, nutraceuticals, and antimicrobials (Hedren and others 2002; Fatouros and others 2007; Maldonado-Valderrama and others 2008; Li and others 2011). In this study, the compositions of oil, surfactant, and water were varied to develop the stable curcumin nanoemulsions (Cur-NEs) and their effect on the stability was examined. Antioxidant properties of Cur-NEs were tested using 2,2diphenyl-1-picrylhydrazyl (DPPH) assay and 2,2 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay. In vitro digestion of Cur-NEs was investigated using a simulated small intestine model (pH stat method). Cur-NEs were directly applied to drinking milk system. Lipid oxidation of drinking milk was investigated by measuring thiobarbituric acid reactive substances (TBARS) absorbance value to show the potential of Cur-NEs in the application to the real food systems.

Vol. 81, Nr. 3, 2016 r Journal of Food Science N745

N: Nanoscale Food Science

Keywords: antioxidants, curcumin-fortified milk, curcumin, in vitro digestion, nanoemulsions

Development and application of food-grade curcumin nanoemulsion . . . Table 1–Mixing ratio and composition of MCT oil, Tween 20, and water for nanoemulsion formations. Mixture ratio

Normalization (%)

No.

Oil

Surfactant

Water

Oil

Surfactant

Water

Size(nm)

PDI

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 B1 B2 B3 B4 B5 B6 B7 B8 B9 C1 C2 C3 C4 C5 C6 C7 C8 C9 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 E5 E6 E7 E8

1

0.3

1

0.5

1

0.7

1

1

1

1.5

0.5 1.0 1.3 1.5 2.0 3.0 4.0 5.0 10.0 50.0 1.0 1.3 1.5 2.0 3.0 4.0 5.0 10.0 50.0 1.0 1.3 1.5 2.0 3.0 4.0 5.0 10.0 50.0 1.3 1.5 2.0 3.0 4.0 5.0 10.0 50.0 1.0 1.3 1.5 3.0 4.0 5.0 10.0 50.0

55.5 43.5 38.5 35.7 30.3 23.26 18.87 15.87 8.85 1.95 40.0 35.7 33.33 28.57 22.22 18.18 15.38 8.7 1.94 37.0 33.3 31.3 27.03 21.28 17.54 14.93 8.55 1.93 30.3 28.6 25.0 20.0 16.67 14.29 8.33 1.92 28.6 26.3 25.0 18.2 15.38 13.33 8.0 1.9

16.7 13.0 11.5 10.7 9.09 6.98 5.66 4.76 2.65 0.58 20.0 17.9 16.67 14.29 11.11 9.09 7.69 4.35 0.97 26.0 23.3 21.9 18.92 14.89 12.28 10.45 5.98 1.35 30.3 28.6 25.0 20.0 16.67 14.29 8.33 1.92 42.8 39.5 37.5 27.2 23.08 20.0 12.0 2.86

27.8 43.5 50.0 53.6 60.61 69.77 75.47 79.37 88.50 97.47 40.0 46.4 50.0 57.14 66.67 72.73 76.92 86.96 97.09 37.0 43.4 46.8 54.05 63.83 70.18 74.63 85.47 96.71 39.4 42.8 50.0 60.0 66.67 71.43 83.33 96.15 28.6 34.2 37.5 54.6 61.54 66.67 80.0 95.24

– – – – 94.47 ± 1.18 97.07 ± 0.30 102.30 ± 3.89 106.80 ± 5.40 116.95 ± 8.35 121.70 ± 1.59 – – 68.68 ± 2.41 65.99 ± 3.41 82.73 ± 1.50 85.09 ± 0.56 99.87 ± 2.48 116.10 ± 8.92 126.25 ± 4.90 – – – 63.28 ± 1.41 75.63 ± 3.18 82.03 ± 1.76 91.17 ± 0.35 114.15 ± 3.66 112.05 ± 1.32 – – – 69.54 ± 5.57 76.60 ± 3.72 79.98 ± 2.41 99.83 ± 3.47 118.15 ± 2.33 – – – – 70.06 ± 3.22 70.64 ± 5.95 84.85 ± 7.54 90.16 ± 0.80

– – – – 0.105 ± 0.002 0.120 ± 0.011 0.103 ± 0.020 0.126 ± 0.006 0.159 ± 0.018 0.175 ± 0.025 – – 0.121 ± 0.007 0.103 ± 0.011 0.139 ± 0.006 0.141 ± 0.006 0.143 ± 0.016 0.172 ± 0.001 0.184 ± 0.007 – – – 0.167 ± 0.001 0.150 ± 0.024 0.168 ± 0.013 0.159 ± 0.003 0.190 ± 0.012 0.214 ± 0.011 – – – 0.208 ± 0.001 0.173 ± 0.007 0.160 ± 0.008 0.182 ± 0.003 0.218 ± 0.008 – – – – 0.232 ± 0.001 0.179 ± 0.004 0.210 ± 0.004 0.240 ± 0.001


Materials and Methods

cumin in oils was precipitated by centrifugation (Supra 25K, Hanil Sci., Korea) at 1750 rpm for 10 min. After removal of nondissolved Chemicals crystalline curcumin, the supernatant was diluted to an appropriate Curcumin (mixture of curcumin, demethoxycurcumin, and bis- concentration and analyzed by a UV-Vis spectrophotometer (Opdemethoxycurcumin, ࣙ98%) was purchased from Acros (New Jer- tizen 3220UV, Seoul, Korea). Curcumin concentrations in each sey, U.S.A.). Olive oil and coconut oil were purchased from Showa oils were measured at a wavelength of 415 nm. (Japan). Corn oil, DPPH, and ABTS tablet were purchased from Sigma-Aldrich (St. Louis, Mo., U.S.A.). MCT oil (45% capric acid and 55% caprylic acid) was purchased from Now Foods (Illinois, Preparation of Cur-NEs The oil phase was prepared by dissolving 0.3% (w/w) curcumin U.S.A.). Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was purchased from Samchun (Pyeongtaek, Korea). n-Hexane was powder in MCT oil. The aqueous phase was prepared by mixing the surfactant (Tween 20) and water. The compositions of purchased from Ducsan (Ansan, Korea). oil, surfactant, and water were shown in Table 1. First, oil phase and aqueous phase including surfactant were mixed and homogSolubility of curcumin enized by a high-speed homogenizer (Ultra-Turrax T18, IKA, The solubility of curcumin in each oil phase was determined us- Germany) at 13500 rpm for 15 min. Then, Cur-NEs were preing a spectrophotometric method. An excess amount of curcumin pared using a high-pressure homogenizer (M-110P Microfluidizer, was added to an oil phase and heated to 60 °C. The mixture was Microfluidics, Newton, Mass., U.S.A.) at pressures of 1000 bar for stirred for 10 min, then sonicated for 20 min. Nondissolved cur- 5 cycles. N746 Journal of Food Science r Vol. 81, Nr. 3, 2016

Development and application of food-grade curcumin nanoemulsion . . . Measurement of droplet size and polydispersity index (PDI) The mean droplet size and PDI of Cur-NEs were measured by dynamic light scattering (DLS) method using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, U.K.). Three milliliters of Cur-NEs dispersion was added to polystyrene latex cells and the mean droplet size and PDI were measured at 25 °C with a detector angle of 90° and wavelength of 633 nm. Each sample was measured at least 3 times and the average values were used.

slip, ensuring that no bubbles were trapped between the sample and the cover slip. Photo of sample was acquired using a confocal laser scanning microscope (Carl Zeiss LSM700, Jena, Germany) with an excitation wavelength at 488 nm and emission wavelength at 500 nm. The morphology of the oil droplets in the nanoemulsions was visualized by transmission electron microscopy (TEM, Tecnai G2 F30ST, Oregon, U.S.A). Nanoemulsions were diluted 10 times and a drop of diluted solution was applied to a 300 mesh copper grid. The grid was kept under ambient conditions for 30 s. As a negative staining agent, phosphotungstic acid was applied to the grid for 10 s and dried overnight and imaged.

Morphology of Cur-NEs Confocal laser scanning microscopy (CLSM) was performed to visualize Cur-NEs and bicontinuous phase. For visualization of Cur-NEs and bicontinuous phase, the fresh sample was stained Retention rate with Nile red dye at the ratio of 200:1 (w/w). One drop of the The retention rate of curcumin in Cur-NEs was determined stained sample was then placed into a slide and covered with a cover using a washing method (Padukka and others 2000). Hexane

0.35

Figure 1–Solubility of curcumin in food-grade oils.

0.30

Solubility (mg/mL)

0.25

0.20

0.15

0.10

0.05

0.00

140

MCT oil

Corn oil

Olive oil

Figure 2–The effect of surfactant ratio (w/w based on oil) and water ratio (w/w based on oil) on the droplet size of Cur-NEs.

Surfactant ratio (w/w based on oil)

Droplet size (nm)

120

0.3 0.5 0.7 1.0 1.5

100

80

60

40 1.5

2

3

4

5

10

50

Water ratio (w/w based on oil)



Coconut oil

Development and application of food-grade curcumin nanoemulsion . . . ABTS assay The ABTS assay was modified according to the method of Ak and Gülçin (2008). ABTS was produced by reacting 7.4 mM ABTS in H2 O with potassium persulfate (K2 S2 O8 ) stored in the dark for 12 h. One milliliter of ABTS solution was diluted in 30 mL methanol. Then, 1 mL of the diluted solution was added to Cur-NEs containing 130 μg of curcumin. The mixture was incubated in the dark for 30 min. The scavenging capability was calculated using the Equation 2: DPPH assay As The DPPH assay was described in the previous paper (Ak and × 100 (2) ABTS scavenging effect (%) = 1 − Ac Gülçin 2008). Briefly, 0.2 mM solution of DPPH• in 80% ethanol was prepared and 50 μL of the solution was added to 150 μL of where A is absorbance of a control (blank) lacking any radical c Cur-NEs. After incubating the samples in the dark for 30 min, scavenger and A is absorbance of the remaining ABTS in the s the absorbance was measured at 517 nm against blank samples presence of scavenger. lacking scavenger. The capability to scavenge the DPPH radical was calculated using the Equation 1: In vitro digestion test The pH stat method (in vitro digestion model) was performed As by a modification of the previously described procedure (Li and (%) × 100 (1) DPPH scaveging effect = 1− Ac McClements 2010). Cur-NEs in a flask were incubated in a water bath at 37 °C for 10 min. The system was then adjusted to pH where Ac is the absorbance of the control and As is the absorbance 7.0 using NaOH or HCl solutions. The final composition of the mixture was 1% (w/w) of oil (0.5 g), 4 mL of bile extract, 1 mL of the sample. was used as the organic solvent for extractions. One milliliter of Cur-NEs was washed out by 9 mL of hexane. The solution was mixed with a vortex at room temperature for 5 min and then separated by centrifugation at 3000 rpm for 10 min. The absorbance of the upper hexane layer containing free curcumin was measured at 415 nm wavelength using a UV-visible spectrophotometer (OPTIZEN 3220 UV, Seoul, Korea). The retention rate of curcumin was evaluated for 24 h.


Figure 3–Ternary phase diagram and confocal laser scanning microscopy (CLMS) images of various nanoemulsion formulations containing MCT oil, Tween 20, and water contents at room temperature.

Figure 4–Transmission electron microscopy images of the nanoemulsions prepared with the ratio of MCT oil, Tween 20, and water in 1:1:50 (w/w/w; [A]: scale bar is 200 nm; [B]: scale bar is 100nm).

N748 Journal of Food Science r Vol. 81, Nr. 3, 2016

Development and application of food-grade curcumin nanoemulsion . . . of CaCl2 solution, and 2.5 mL of lipase. A pH-stat titration unit (TitraLab 906, Radiometer) was then used to maintain a constant pH of 7.0 by titrating and neutralizing the free fatty acids (FFA) generated by lipolysis with 0.1 M NaOH solution. The volume of 0.1 M NaOH consumed was recorded and calculated as the amount of FFAs hydrolyzed from the emulsion. The amount of FFAs released was calculated using the Equation 3: %FFA release =

VNaOH × m NaOH × MLipid wLipid × 2

× 100

(3)

where VNaOH is the volume of NaOH solution (mL), mNaOH is the molarity of the NaOH solution (M), MLipid is the molecular weight of MCT oil (g/mol), and wLipid is the mass of MCT oil (g).

TBARS assay Milk (Maeil milk low fat and high calcium 1%; Maeil Dairies Industry Co., Cheongyang, Korea) was purchased from a local market. Forty-five milliliters of Cur-NEs-fortified milk was prepared with 30.18 mL of milk and 12.82 mL of Cur-NEs containing 15 mg curcumin. The TBA values of the milk samples were measured according to previously described method (Hegenauer and others 1979). The TBA reagent was prepared immediately before its use to the milk sample by mixing equal volumes of freshly prepared 0.025 M TBA (neutralized with NaOH) and 2 M H3 PO4 /2 M citric acid. The reaction was initiated by mixing 5.0 mL of milk sample with 2.5 mL of the TBA reagent into a glass centrifuge tube. The mixture was heated immediately in boiling water bath for exactly 10 min and then cooled down on ice. Ten milliliters of cyclohexanone and 1 mL of 4 M ammonium sulfate were then added and

100

Figure 5–Retention rate of Cur-NEs at 4 and 25 °C for 24 h.

Retention rate (%)

90

80

70

60

50 0

5

10

15

20

25


Time (h)

Figure 6–(A) The effect of water concentration on the droplet size of Cur-NEs at the surfactant ratio of 0.3 (samples A5 to A10) and 1.5 (samples E5 to E8) after 15 d storage at 25 °C and (B) comparison of the visual appearance of (a) curcumin in MCT oil, (b) sample E7 Cur-NEs before storage, and (c) sample E7 Cur-NEs stored at 4 °C for 6 mo. The alphabet indicates significant difference (P < 0.05) and the asterisks indicate that statistically different samples after 15 d.


Development and application of food-grade curcumin nanoemulsion . . . the mixture was centrifuged (High Speed Refrigerated Centrifuge CR-21G; Hitachi, Tokyo, Japan) at room temperature under 5000 × g for 5 min. The orange-red cyclohexanone supernatant was decanted and its absorbance was measured at 532 nm using a MultiDetection Microplate Reader (Sense, HIDEX, Turku, Finland). All samples were measured 3 times and the mean values were used.

Statistical analysis All measurements were performed 3 times using freshly prepared samples and were reported as calculated means and standard deviations. Statistics on a completely randomized design were performed with the analysis of variance procedure in SAS software (Release 9.1; SAS Inst, Inc., Cary, N.C., U.S.A.). Duncan’s multiple range test (P < 0.05) was used to detect differences among the mean values of the samples.

Results and Discussion Lipid screening by curcumin solubility The solubility of curcumin in various oil types was measured to identify the appropriate oil phase. For the production of Cur-NEs, common food-grade oils such as coconut oil, olive oil, corn oil, and medium chain triglyceride (MCT) oil were screened. Coconut oil contains a mixture of MCT and long chain triglyceride (LCT), whereas olive oil and corn oil are mainly composed of LCT. As shown in Figure 1, the solubility of curcumin was much

120

A

100

80

c

60

b b

40

Physicochemical properties of Cur-NEs The physicochemical properties of nanoemulsions were significantly dependent on the types of surfactants and oils and the ratio of oil, surfactant, and water in the mixture. In generally, hydrophilic lipophilic balance (HLB) of surfactant should be over 10 to develop O/W emulsions. In this study, Tween 20 (HLB value = 16.7) was selected as a surfactant for Cur-NEs. As a nonionic surfactant, Tween 20 is less toxic and less affected by ionic strength and pH changes as compared to ionic surfactants. Various ratios of oil, surfactant, and water were tested to optimize the O/W emulsion formations. Cur-NEs were stabilized and optimized with 5 cycles of high pressure homogenization. The droplet size and PDI of Cur-NEs were summarized in Table 1. The droplet size of Cur-NEs was significantly dependent on the ratios of oil, surfactant, and water. In order to form stable nanoemulsions, the amount of aqueous phase should be at least 2 to 3 times higher than the total amount of oil phase and surfactant. The mean droplet size

a,b

a

a

20

Radical Scavenging Capacity (%)


120

higher in MCT oil compared to other oils such as coconut oil, corn oil, and olive oil. The solubility of curcumin at 60 °C was found to be 0.25 mg/mL in MCT oil, 0.1 mg/mL in coconut oil, 0.08 mg/mL in olive oil, and 0.07 mg/mL in corn oil, respectively. However, the bioaccessibility of curcumin was reported to be higher in MCT oil than in LCT and short chain triglyceride oil (Ahmed and others 2012). Therefore, MCT oil was chosen as an oil phase for the preparation of Cur-NEs.

0 B6

B7

B8

60

b 40

a

a

B5

B6

a

a

a

20

B9

B4

B7

B8

B9

120

C 100

d 80

c c

60

b 40

a

0



B5

120


80

0 B4

20

B 100

D

c

100

c b

80

60

a

a

A9

B8

40

20

0 A9

B8

C8

D7

E7

C8

D7

E7

Figure 7–The effect of water and surfactant concentrations on the radical scavenging capacity of Cur-NEs using DPPH assay (A) and (C) and ABTS assay (B) and (D).


Development and application of food-grade curcumin nanoemulsion . . .

Morphology of Cur-NEs Figure 4 shows the TEM images of Cur-NEs (sample D8, oil : surfactant : water = 1 : 1 : 50). The droplet size of Cur-NEs (sample D8) in the TEM images was approximately 120 nm coinciding with that measured using a zeta size analyzer in the previous section. The droplet was oval and spherical shape in TEM images while it was spherical shape in CLMS images. The change of shape might occur during the drying process for the sample preparation.

Retention rate and stability of Cur-NEs Retention rate of Cur-NEs was tested at different temperatures (4 and 25 °C) for 24 h. As shown in Figure 5, the retention rates of Cur-NEs at 4 and 25 °C were 98.0% and 91.3%, respectively. Cur-NEs stored at 4 °C showed higher retention rate than that stored at 25 °C. This is because Cur-NEs in higher temperature are susceptible to break down by Ostwald ripening (Lesmes and McClements 2009). The stability of Cur-NEs was evaluated by measuring the change in their mean droplet size during the storage at 25 °C for 15 d as shown in Figure 6A. The droplet sizes at low (samples A5 to A10) and high (samples E5 to E8) concentrations of the surfactant were slightly increased after 15 d but most of them were not significantly different (P > 0.05) except samples A6 and E8. High concentrations of surfactant can produce smaller droplets because surfactant molecules can stabilize a larger oil-water interface (Chang and McClements 2014). However, these systems are thermodynamically unstable and therefore tend to be inhomogeneous and undergo an increase in the droplet size (McClements 2011). Although the droplet size was slightly increased after 15 d, the PDI value was still lower than 0.2 and phase separation was not observed. Thus, Cur-NEs were sufficiently stable and can be applied for beverage systems such as milk or yogurt. Figure 6B shows the pictures of (a) solubilized curcumin in MCT oil, (b) Cur-NEs at 0 d, and (c) Cur-NEs after storage at 4 °C for 6 mo. Based on the observation with naked eye, there was no phase separation even in the Cur-NEs stored at 4 °C for 6 mo. After 6 mo storage at 4 °C, the droplet size of the sample (Figure 6B) increased from

Figure 8–The effect of surfactant concentration on the percentage of free fatty acid (FFA) release of Cur-NEs.


of Cur-NEs was ranged from 63 to 126 nm. The PDI values of Cur-NEs were less than 0.2 in all conditions, which indicates the high stability of Cur-NEs. The results show that the droplet size of Cur-NEs decreases with the increase of surfactant concentration and increases with water concentrations as shown in Figure 2. The ternary phase diagram shows the regions of O/W emulsion formation as shown in Figure 3. Nanoemulsions were only observed in the conditions of over 50% of aqueous phase, less than 33% of oil phase, and less than 32% of surfactant content. Phase separation was observed in the out of these conditions and a bicontinuous phase was observed nearby the nanoemulsion formation area. In order to detect clear droplet formation and bicontinuous phase, CLMS images were taken by incorporation of Nile Red into the nanoemulsion as shown in Figure 3. The droplets of CurNEs were clearly round-shaped and homogeneously distributed. However, CLMS image shows massive clots in the bicontinuous phase.


Development and application of food-grade curcumin nanoemulsion . . . the dissolution of curcumin in the oil phase, leading to a higher antioxidant activity. In another work, Tween 20 was found to increase the dispersion of oil into the water phase, thereby increasing the antioxidant activity of the O/W emulsion (Kiralan and others 2014). It was also reported that the surfactant can affect the physical location of antioxidants in the O/W emulsion by solubilizing lipid soluble antioxidants into the aqueous phase (Richards and others 2002). As a result, concentrations of oil and surfactant Antioxidant properties of Cur-NEs affect the radical scavenging activity of Cur-NEs by influencing In vitro antioxidant activity of Cur-NEs was tested by DPPH the amount of solubilized curcumin serving as antioxidant. assay and ABTS assay. To determine the antioxidant capacity of functional foods or various bioactive compounds, both assays are In vitro digestion of Cur-NEs the most popular and simple analyzing methods by using specIn order to gain further insight on the influence of the surfactant trophotometer (Hedren and others 2002). Figure 7A shows the concentration on Cur-NEs, in vitro lipid digestibility of the Cureffect of water concentration on the radical scavenging activity NEs was examined using a simulated small intestine model (pH (RSA) of Cur-NEs samples B4 to B9 by DPPH assay, although stat method). The more lipid in the Cur-NEs was digested, the the concentrations of surfactant and oil were fixed. Sample B5 more amount of FFA was released. Except the highest surfactant showed the lowest antioxidant activity (20.3%), whereas the high- concentration (sample E7), the rate of lipid digestion in Curest antioxidant activity was found in sample B9 (69%). Although NEs was same until 1st 20 min whereas the lipid digestion was sample B9 showed the significant difference (P < 0.05) in the significantly (P < 0.05) decreased with surfactant concentration RSA with other samples, the RSA of Cur-NEs was not demon- over the time as shown in Figure 8. Higher FFA release of about strated a tendency by water concentration. In case of ABTS assay 130% was observed in samples A9 and B8 which contain the (Figure 7B), the lowest RSA value was 25% in sample B9 but the lower surfactant concentration. However, the lowest FFA release highest RSA value was 40% in sample B8. Based on both DPPH was about 110% which was observed in the highest surfactant and ABTS assays, the change of water content in Cur-NEs did concentration (sample E7). not show any significant factors to RSA values of Cur-NEs. To This result suggests that the surfactant concentration in Curobserve the effect of surfactant concentration on the antioxidant NEs affected %FFA release property and low concentrations of properties of Cur-NEs, 5 types of Cur-NEs were selected by dif- surfactant allow relatively rapid FFA release of Cur-NEs. Previous ferent surfactant ratio with fixed oil to water ratio (1:10). The studies also showed similar results that the rate of lipid digestion was ratio of surfactant to oil was varied from 0.3 to 1.5. Figure 7C relatively slow at high surfactant concentration (Hegenauer and and D show the effect of surfactant concentration on the antioxi- others 1979; Mun and others 2007; Li and McClements 2011). dant activity of Cur-NEs by DPPH and ABTS assays with samples Surfactants could work to be absorbed on to the droplets surfaces A9, B8, C8, D7, and E7. All samples have only different amount and to control the adsorption of lipase molecules. It has been proof surfactant, although oil and water contents were constant as posed that lipase cannot bind to the surface of the oil droplets shown in Table 1. The RSA of Cur-NEs was significantly (P < because of the greater steric hindrance and the higher surface 0.05) increased with surfactant (Tween 20) concentration. This re- activity of Tween 20, which can competitive bind to the oil sult indicated that higher surfactant concentration could facilitate droplets with lipase. 84.9 to 113.9 nm, whereas the PDI value decreased from 0.210 to 0.008 (data were not shown). Although the droplet size of CurNEs increased during 6 month storage, more homogeneous size distribution was observed because of the Ostwald ripening of the Cur-NEs during storage (Mason and others 2006). The droplet size of Cur-NEs could increase over time because larger particles are more thermodynamically favored than smaller particles.

0.6

Figure 9–Lipid oxidation of control milk, nanoemulsion-fortified milk, and Cur-NEs-fortified milk for 10 d storage.

0.4

A532


Milk Milk + Nanoemulsions Milk + Cur-NEs

0.5

0.3

0.2

0.1

0.0

0

2

4

6

Time (days)


8

10

12

In all samples, %FFA release of Cur-NEs was more than 100% after 33 min of releasing time. MCT oil in the Cur-NEs contains 3 FFA in one glycerol and it could be possible to produce more than 2 FFAs per 1 triacylglycerol during digestion (Hegenauer and others 1979; Ahmed and others 2012). Other studies showed that the rate of %FFA release increased with decreasing droplet sizes because of the high surface area of the lipid phase exposed to the aqueous phase (Lundin and Golding 2009; Li and McClements 2010). The different results may be due to the different experimental conditions such as lipid concentrations, emulsifier type, and simulated small intestinal fluid compositions.

Lipid oxidation of Cur-NEs in milk system Three types of milk samples, control (unfortified) milk, milk with empty nanoemulsion (without curcumin), and milk with Cur-NEs containing 15 mg of curcumin, were tested to investigate the lipid oxidation during storage (Figure 9). The TBA absorbance of the original milk sample increased from 0.024 to 0.095 after 6 d, and went up to 0.427 after 10 d. However, the TBA absorbance in the milk containing Cur-NEs slightly increased from 0.039 to 0.142 after 10 d. Control milk (not containing Cur-NEs) also showed lower TBA absorbance than original milk. A previous study proposed the beneficial effect of MCT addition in soybean oil or in frying oil on the oxidative stability during storage in the dark (Toyosaki and others 2008). This result suggested that CurNEs are significantly effective to reduce lipid oxidation of milk during storage.

Conclusions The solubility of curcumin in MCT oil was 0.25 mg/mL, which was more than 3 times higher than those of other screened oils such as coconut oil, olive oil, and corn oil. The stable nanoemulsions were only prepared when the aqueous phase was over 50%. Physicochemical properties of Cur-NEs were significantly affected by surfactant concentration. The droplet size of Cur-NEs was greatly decreased with surfactant concentration because surfactant molecules make a larger oil–water interface. The radical scavenging activity of Cur-NEs was not significantly changed with water content but significantly increased with surfactant concentration. The high surfactant concentration might facilitate the dissolution of curcumin in the oil phase then increase the antioxidant activity. In vitro lipid digestibility test supported that higher surfactant concentration could retard the lipid degradation because more surfactant molecules disturb the adsorption of lipase molecules to the droplet surface. Cur-NEs-fortified milk could significantly reduce the lipid oxidation than those of control (unfortified) milk and milk containing curcumin free nanoemulsions. It showed the potential application of Cur-NEs to the beverage industry.

Acknowledgments This work was supported by Korea Univ. Grant, Korea Food & Drug Administration (15162MFDS050) from Ministry of Food and Drug Safety in 2015 and by the Intl. Research & Development Program of the Natl. Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) of Korea (Grant No. 2012K1A3A1A20031356).

References Acosta E. 2009. Bioavailability of nanoparticles in nutrient and nutraceutical delivery. Curr Opin Colloid Interface Sci 14:3–15. Ahmed K, Li Y, McClements DJ, Xiao H. 2012. Nanoemulsion- and emulsion-based delivery systems for curcumin: encapsulation and release properties. Food Chem 132:799–807.

Ak T, Gülçin I. 2008. Antioxidant and radical scavenging properties of curcumin. ChemBiol Interact 174:27–37. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. 2007. Bioavailability of curcumin: problems and promises. Mol Pharm 4:807–18. Bar-Sela G, Epelbaum R, Schaffer M. 2010. Curcumin as an anticancer agent: review of the gap between basic and clinical applications. Curr Med Chem 17:190–7. Chang YH, McClements DJ. 2014. Optimization of orange oil nanoemulsion formation by isothermal low-energy methods: influence of the oil phase, surfactant, and temperature. J Agric Food Chem 62:2306–12. De R, Kundu P, Swarnakar S, Ramamurthy T, Chowdhury A, Nair GB, Mukhopadhyay AK. 2009. Antimicrobial activity of curcumin against helicobacter pylori isolates from India and during infections in mice. Antimicrob Agents and Chemother 53:1592–7. Fatouros DG, Bergenstahl B, Mullertz A. 2007. Morphological observations on a lipid-based drug delivery system during in vitro digestion. Eur J Pharm Sci 31:85–94. Hedren E, Diaz V, Svanberg U. 2002. Estimation of carotenoid accessibility from carrots determined by an in vitro digestion method. Eur J Clin Nutr 56:425–30. Hegenauer J, Saltman P, Ludwig D, Ripley L, Bajo P. 1979. Effects of supplemental iron and copper on lipid oxidation in milk. 1. Comparison of metal complexes in emulsified and homogenized milk. J Agric Food Chem 27:860–7. Heng MCY. 2010. Curcumin targeted signaling pathways: basis for antiphotoaging and anticarcinogenic therapy. Int J Dermatol 49:608–22. Huang Q, Yu H, Ru Q. 2010. Bioavailability and delivery of nutraceuticals using nanotechnology. J Food Sci 75:R50–R7. Jafari SM, Assadpoor E, He Y, Bhandari B. 2008. Re-coalescence of emulsion droplets during high-energy emulsification. Food Hydrocolloids 22:1191–202. Kiralan SS, Dogu-Baykut E, Kittipongpittaya K, McClements DJ, Decker EA. 2014. Increased antioxidant efficacy of tocopherols by surfactant solubilization in oil-in-water emulsions. J Agric Food Chem 62:10561–6. Lesmes U, McClements DJ. 2009. Structure-function relationships to guide rational design and fabrication of particulate food delivery systems. Trends Food Sci Tech 20:448–57. Li J, Shin GH, Chen X, Park HJ. 2015. Modified curcumin with hyaluronic acid: combination of pro-drug and nano-micelle strategy to address the curcumin challenge. Food Res Int 69:202–8. Li Y, Hu M, McClements DJ. 2011. Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: proposal for a standardized pH-stat method. Food Chem 126:498–505. Li Y, McClements DJ. 2010. New mathematical model for interpreting pH-stat digestion profiles: impact of lipid droplet characteristics on in vitro digestibility. J Agric Food Chem 58:8085–92. Li Y, McClements DJ. 2011. Inhibition of lipase-catalyzed hydrolysis of emulsified triglyceride oils by low-molecular weight surfactants under simulated gastrointestinal conditions. Eur J Pharm Biopharm 79:423–31. Lundin L, Golding M. 2009. Structure design for healthy food. Aust J Dairy Technol 64: 68–74. Maiti K, Mukherjee K, Gantait A, Saha BP, Mukherjee PK. 2007. Curcumin–phospholipid complex: preparation, therapeutic evaluation, and pharmacokinetic study in rats. Int J Pharm 330(1–2):155–63. Maldonado-Valderrama J, Woodward NC, Gunning AP, Ridout MJ, Husband FA, Mackie AR, Morris VJ, Wilde PJ. 2008. Interfacial characterization of beta-lactoglobulin networks: displacement by bile salts. Langmuir 24:6759–67. Mason TG, Wilking JN, Meleson K, Chang CB, Graves SM. 2006. Nanoemulsions: formation, structure, and physical properties. J Phys-Condens Mat 18:R635–R66. Masuda T, Hidaka K, Shinohara A, Maekawa T, Takeda Y, Yamaguchi H. 1998. Chemical studies on antioxidant mechanism of curcuminoid: analysis of radical reaction products from curcumin. J Agric Food Chem 47:71–7. McClements DJ. 2011. Edible nanoemulsions: fabrication, properties, and functional performance. Soft Matter 7:2297–316. McClements DJ. 2015. Nanoscale nutrient delivery systems for food applications: improving bioactive dispersibility, stability, and bioavailability. J Food Science 80:1602N–11N. Menon VP, Sudheer AR. 2007. Antioxidant and anti-inflammatory properties of curcumin. Adv Exp Med Biol 595:105–25. Mun S, Decker EA, McClements DJ. 2007. Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase. Food Res Int 40:770–81. Nagarajan S, Kubra IR, Rao LJM. 2010. Separation of curcuminoids enriched fraction from spent turmeric oleoresin and its antioxidant potential. J Food Sci 75:158H–62H. Padukka I, Bhandari B, D’Arcy B. 2000. Evaluation of various extraction methods of encapsulated oil from β-cyclodextrin-lemon oil complex powder. J Food Compos Anal 13:59–70. Richards MP, Chaiyasit W, McClements DJ, Decker EA. 2002. Ability of surfactant micelles to alter the partitioning of phenolic antioxidants in oil-in-water emulsions. J Agric Food Chem 50:1254–9. Sharma J, Ponnusamy Pazhaniandi P, Tanwar VK, Das SK, Goswami M. 2012. Antioxidant effect of turmeric powder, nitrite, and ascorbic acid on stored chicken mince. Int JFood SciTech 47:61–6. Shin GH, Chung SK, Kim JT, Joung HJ, Park HJ. 2013. Preparation of chitosan-coated nanoliposomes for improving the mucoadhesive property of curcumin using the ethanol injection method. J Agric Food Chem 61:11119–26. Sreejayan N, Rao MN. 1996. Free radical scavenging activity of curcuminoids. Arzneimittelforschung 46:169–71. Tønnesen HH, Másson M, Loftsson T. 2002. Studies of curcumin and curcuminoids. XXVII. Cyclodextrin complexation: solubility, chemical, and photochemical stability. Int J Pharm 244:127–35. Toyosaki T, Sakane Y, Kasai M. 2008. Oxidative stability, trans, trans-2,4-decadienals, and tocopherol contents during storage of dough fried in soybean oil with added medium-chain triacylglycerols (MCT). Food Res Int 41:318–24. Wang X, Jiang Y, Wang YW, Huang MT, Ho CT, Huang Q. 2008. Enhancing antiinflammation activity of curcumin through O/W nanoemulsions. Food Chem 108: 419–24.



Development and application of food-grade curcumin nanoemulsion . . .

Development of FoodâGrade Curcumin Nanoemulsion and its ...

Development of FoodâGrade Curcumin Nanoemulsion and its ...

Development of FoodâGrade Curcumin Nanoemulsion and its ...

Development of FoodâGrade Curcumin Nanoemulsion and its ...