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Fabrication of curcumin-loaded whey protein microgels: Structural properties, antioxidant activity, and in vitro release behavior
LWT - Food Science and Technology 103 (2019) 94–100
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Fabrication of curcumin-loaded whey protein microgels: Structural properties, antioxidant activity, and in vitro release behavior
T
Mehdi Mohammadian, Maryam Salami∗, Shima Momen, Farhad Alavi, Zahra Emam-Djomeh Department of Food Science and Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Whey protein microgel Bioactive delivery Curcumin Radical scavenging activity Structural characteristics
This study evaluated the potential use of whey protein microgels (WPM) formed by heating a protein solution at pH 5.90 as carriers for curcumin. The results indicated that the loading amount of curcumin into whey protein isolate (WPI) and WPM was 1.84 ± 0.18 and 17.51 ± 0.46 μg curcumin/mg protein, respectively. Loading of microgels with curcumin significantly increased their diameter and changed their surface charge. Loading of curcumin in the whey protein microgels drastically decreased its sedimentation during storage. Fluorescence measurements revealed that the curcumin was present at the hydrophobic core of whey protein microgels. X-ray diffraction analysis also showed that curcumin was loaded inside the WPM in an amorphous form. Curcuminloaded whey protein microgels showed a high in vitro antioxidant activity which was measured by DPPH radical scavenging and reducing power assay. The in vitro release experiment also showed that the release of curcumin from WPM was significantly slower than WPI during the simulated gastrointestinal conditions. These findings suggested a possible utilization of WPI microgels in improving the applications of curcumin for the production of functional food products and drugs.
1. Introduction Curcumin is a natural polyphenol compound derived from the rhizome of turmeric (Curcuma longa) with different biological and healthpromoting properties such as antioxidant, antimicrobial, anti-inflammatory, anti-proliferative, and anticancer activity (Alavi et al., 2018; Esmaili et al., 2011; Rafiee, Nejatian, Daeihamed, & Jafari, 2018). However, the poor water dispersibility, rapid metabolism, and low stability of curcumin limit its bioavailability and also restrict its applications in food products (Alavi et al., 2018). Complexation with proteins or encapsulation within food protein-based carriers are suggested as effective methods to improve stability, solubility, and bioavailability of curcumin (Rafiee et al., 2018). A wide range of food proteins such as whey proteins (Liu, Chen, Cheng, & Selomulya, 2016; Liu et al., 2017), casein (Esmaili et al., 2011; Pan, Zhong, & Baek, 2013), bovine serum albumin (Fan, Yi, Zhang, & Yokoyama, 2018), zein (Patel, Hu, Tiwari, & Velikov, 2010), and soy proteins (Tapal & Tiku, 2012) have been employed as carriers for curcumin. Whey proteins have been widely used as functional ingredients in various food applications due to their superb technological functionalities and high nutritional value (Lin, Tian, Li, Cao, & Jiang, 2012). Heat treatment of these proteins at different pH results in the formation of aggregates with different shapes and morphologies such as ∗
nanofibrils, flexible strands, and microgels (Vahedifar, Madadlou, & Salami, 2017). Spherical whey protein microgels (WPM) with a diameter of 100–600 nm and relatively low polydispersity are formed through the heating of whey protein solution at a narrow pH range from 5.8 to 6.2 and low ionic strength (Schmitt et al., 2010; Vahedifar, Madadlou, & Salami, 2018). It was investigated that the covalent disulfide bonds and non-covalent hydrophobic and electrostatic interactions are the most important forces involved in the stability of whey protein microgels (Donato, Schmitt, Bovetto, & Rouvet, 2009). Whey protein microgels were successfully used as efficient emulsifying (Destribats, Rouvet, Gehin-Delval, Schmitt, & Binks, 2014) and foaming (Schmitt, Bovay, & Rouvet, 2014) agents. It seems that the microgelification process can be used to produce bioactive ingredients-loaded whey protein microgels which can expand their applications in the production of functional food products as well as for designing new drug delivery systems. To the best of our knowledge, there has been no research in the literature about the application of whey protein microgels as carriers for curcumin. Therefore, in this study, curcumin was loaded into the microgel particles prepared by heating of whey protein solution at pH close to its isoelectric point. The structural properties, antioxidant activity, and in vitro release behavior of the curcumin-loaded whey protein microgels were studied by different methods and techniques.
Corresponding author. E-mail address: [email protected] (M. Salami).
https://doi.org/10.1016/j.lwt.2018.12.076 Received 13 August 2018; Received in revised form 25 October 2018; Accepted 29 December 2018 Available online 31 December 2018 0023-6438/ © 2018 Published by Elsevier Ltd.
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interaction between curcumin and proteins. For fluorescence studies, samples were diluted with distilled water (pH 5.9) to a protein concentration of 0.2 mg/mL and curcumin concentration of 4 μg/mL. To assess the protein intrinsic fluorescence, the emission spectra were recorded from 300 to 450 nm with an excitation wavelength of 280 nm. The emission spectra of curcumin in different media were recorded from 450 to 700 nm with an excitation wavelength of 420 nm. Both the excitation and emission slit widths were set to 5 nm.
2. Materials and methods 2.1. Materials Whey protein isolate (WPI) with more than 90 g/100 g protein was obtained from Hilmar Ingredients (CA, USA). Curcumin, pepsin (the activity of more than 3000 units/mg), and pancreatin (200 units/mg) were obtained from Bio Basic (Bio Basic Inc., Canada). 2,2-Diphenyl-1picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (Sa. Louis, MO, USA). Other chemicals used in this study were also of analytical grade and purchased from Merck (Darmstadt, Germany) and SigmaAldrich.
2.5. Particle size and ζ-potential measurements The particle size of different samples (WPI, WPM, and C-WPM) was determined using dynamic light scattering (DLS) (Brookhaven Instruments Corp., Holtsville, NY, USA) at room temperature. The samples were diluted with double distilled water with the same pH before the measurements to avoid multiple scattering. The ζ-potential of different samples at pH 5.90 was also measured with the same DLS apparatus in ζ-potential measurement mode using the specialized zeta cell.
2.2. Preparation of curcumin-loaded whey protein microgels For the preparation of curcumin-loaded whey protein microgels (CWPM), WPI powder was dissolved in distilled water with a concentration of 50 mg/mL. Sodium azide was added as an antimicrobial agent. The protein solution was stirred at room temperature for 2 h followed by overnight storage for complete hydration and then was loaded with curcumin. For this purpose, curcumin was dissolved in anhydrous ethanol and then added to the WPI solution under stirring. The final concentration of curcumin in the mixtures was 1.0 mg/mL (curcumin to protein ratio of 1:50). This was followed by 10 h stirring at room temperature in the dark. It should be noted that the final concentration of ethanol in whey protein solution never exceeded 2.0 mL/L. At this concentration, ethanol has no significant effect on the structure of proteins (Liu et al., 2017). Subsequently, the pH of WPI-curcumin binary solution was adjusted to 5.90 ± 0.05 using NaOH and HCl (1.0 or 0.1 mol/L) and then was heated for 15 min at 80 °C under a mild condition of stirring to fabricate curcumin-loaded WPI microgels (Vahedifar et al., 2018). The microgelification process was stopped by rapid cooling of the protein solution to room temperature using cold water. The curcumin-free whey protein microgels (WPM) also were prepared according to the above-mentioned procedure without the addition of curcumin.
2.6. Optical microscopy The whey protein microgels with or without curcumin were imaged using an 18.0 MP digital USB camera attached to an optical microscope (OMAX, Gyeonggi-do, South Korea) at 10 × 100 magnification. 2.7. Fourier transform infra-red (FT-IR) spectroscopy The FT-IR spectroscopy using a Bruker FT-IR spectrometer (Billerica, Massachusetts, United States) was employed to assess the molecular attributes of different samples (pure curcumin, WPI, WPM and curcumin-loaded whey protein microgels). The lyophilized samples were powdered and compressed into the transparent potassium bromide disks and scanned from 4000 to 500/cm in the transmittance mode. 2.8. X-ray diffraction (XRD) analysis
2.3. Determination of loading amount
The XRD spectra of lyophilized samples including pure curcumin, native WPI, whey protein microgels, and curcumin-loaded whey protein microgels were collected on a Philips PW1730 X-ray diffractometer (PANalytical, Netherlands) using a Cu Kα radiation source in the 2θ range of 5°–50° with a step size of 0.05° and scan rate of 1°/s. The filament current and operating voltage were respectively set at 30 mA and 40 kV.
The loading amount (LA) of curcumin for WPI before (C-WPI) and after (C-WPM) microgelification process was measured according to Tapal and Tiku (2012) with some modifications. For measuring the curcumin loading amount of WPI, the WPI-curcumin solution was centrifuged at 5000 ×g for 15 min to remove any undissolved curcumin. The supernatant was diluted with ethanol to extract the curcumin and its absorbance was measured spectrophotometrically at 420 nm. The concentration of curcumin in the supernatant was calculated using a standard curve of curcumin (0.1–10 μg/mL dissolved in ethanol, R2 = 99.89%) and was reported as the loaded curcumin. In the case of whey protein microgels, the microgel solution was centrifuged at 100×g for 2 min to separate the curcumin-loaded microgels. After that, curcumin in the supernatant was extracted by ethanol and its concentration was calculated as the free curcumin. It should be noted that no curcumin precipitate was observed at the bottom of the tube after the centrifugation. Finally, the loading amount of curcumin in WPI and WPM was determined according to:
LA (μg / mg ) =
loaded amount of curcumin total amount of protein
2.9. Antioxidant activity The antioxidant activities of WPI, whey protein microgels, curcumin-loaded microgels, and the aqueous solution of curcumin were measured using DPPH radical scavenging activity test and reducing power assay according to the method of Lin et al. (2012) with minor adjustments. Different samples were diluted with distilled water of the same pH to a protein concentration of 5 mg/mL and a curcumin concentration of 0.1 mg/mL before the measurements. For DPPH radical scavenging test, 2 mL of different sample solutions was added to 2 mL of the ethanolic DPPH solution (0.2 mmol/L). 2 mL of DPPH solution was also added to 2.0 mL of distilled water as the control sample. After that, the resulting mixtures were mixed vigorously and stored for 30 min at room temperature in dark, followed by centrifuging at 1500×g for 10 min. The absorbance of the resulting supernatants was measured spectrophotometrically at 517 nm and the DPPH radical scavenging activity was calculated according to
(1)
2.4. Fluorescence measurements The fluorescence properties of different samples including free curcumin in water, WPI, WPM, C-WPM, and CeWPI (curcumin-WPI complex before microgelification) were examined using a fluorescence spectrofluorometer (Cary Eclipse, Palo Alto, CA) to study the
Radical scavenging activity (%) = 95
A C − AS × 100 AC
(2)
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where AC and AS are the absorbance of control and sample solutions, respectively. For the reducing power assay, 1 mL of diluted sample solution was mixed with 2.5 mL phosphate buffer (200 mmol/L, pH 6.6) and subsequently with 2.5 mL potassium ferricyanide (10 g/L). The resulting mixtures were incubated for 20 min in a water bath at 50 °C, followed by charging with 2.5 mL trichloroacetic acid (100 mg/mL). After that, the mixtures were centrifuged for 10 min at 1500 ×g and 2.5 mL of the supernatants was mixed with 2.5 mL distilled water and also 0.5 mL ferric chloride (1 mg/mL). The absorbance of these mixtures was read at 700 nm after 10 min incubating at room temperature using a UV–visible spectrophotometer. 2.10. In vitro release behavior The release profile of curcumin from WPI and whey protein microgels during sequential gastrointestinal digestion was studied by a dialysis bag method according to Chang et al. (2017) and Maltais, Remondetto, and Subirade (2009) with slight adjustments. Two mL of CeWPI or C-WPM solutions (pH 1.2) were filled into a dialysis bag (with 12 kDa molecular cut off) and mixed with 2.0 mL of simulated gastric fluid (SGF, consisted of NaCl, HCl, and 3.2 g/L pepsin with a final pH value of 1.2). After that, the dialysis bag was placed in a beaker containing 150 mL of the release medium consisting of 75 mL ethanol and 75 mL SGF without enzyme. This was followed by 2 h incubation at 37 °C and shaking of 100 rpm. After that, the pH of solution was adjusted to 7.5 and 4 mL of simulated intestinal fluid (SIF, consisting of monobasic potassium phosphate, NaOH, and 10 g/L of pancreatin with the final pH value of 7.5) was added to the mixture. Subsequently, the dialysis tube was placed in a beaker containing 150 mL of enzyme-free SIF-ethanol mixture and incubated for 4 h under continuous shaking (37 °C and 100 rpm). It should be noted that the ethanol was added to the release medium due to the poor solubility of curcumin in water. Two mL of the outer release medium was collected at each designated point and replaced with fresh medium. The amount of the released curcumin was determined using UV–Vis spectrophotometer at 420 nm and its concentration was calculated with the help of a curcumin standard curve which was prepared using the same release medium.
Fig. 1. Visual appearance of different sample solutions including (A) native WPI, (B) WPI-curcumin before microgelification, (C) whey protein microgels, (D) curcumin-loaded microgels, and (E) curcumin dispersed in distilled water.
Statistical analyses were performed using the one-way analysis of variance (ANOVA) by SPSS software version 16.0 (IBM software, NY, USA). The significance of difference between the means values was determined by Duncan's multiple range procedure. The level of significance was set to 0.05.
capsulated form (i.e. curcumin-loaded whey protein microgels) at the bottom of the test tube, whereas a thin layer of curcumin sediment was formed in the case of both free curcumin dispersed in distilled water and also the native whey protein isolate at the pH value of 5.9. In accordance with these observations, it was also investigated that the water dispersibility of curcumin was improved through the encapsulation within casein nanocapsules (Pan et al., 2013) and core-shell protein (zein)-polysaccharide (pectin and alginate) nanoparticles (Huang et al., 2016). Consequently, improving the water solubility of curcumin can enhance its biological activity such as antioxidant activity which was assessed in the present study. Therefore, microgelification can be considered as an efficient and facile method to encapsulate curcumin and other bioactive molecules and the resulting whey protein microgels can be considered as promising encapsulation agents for the incorporation of bioactive compounds such as curcumin into the functional food products.
3. Results and discussion
3.2. Fluorescence spectroscopy
3.1. Loading amount
The possible binding and interactions between curcumin and proteins (WPI and WPM), as well as the micro-environmental changes, were studied using fluorescence spectroscopy. The fluorescence spectra of different samples at the excitation wavelength of curcumin (420 nm) are shown in Fig. 2A. Free curcumin which was dispersed in distilled water showed a broad low-intensity emission spectrum which is in a good agreement with the results of Esamili et al. (2011). The fluorescence intensity of curcumin was slightly increased when it was bound with WPI, but the intensity was enhanced drastically when curcumin loaded in whey protein microgels. These results indicated that the curcumin was transferred from a hydrophilic environment to a more hydrophobic environment after loading in whey protein microgels. This also suggests that the curcumin was present at the core of whey protein microgels in aqueous dispersions. Pan et al. (2013) also observed a significant increase in the fluorescence intensity of curcumin after its encapsulation in casein nanoparticles. The fluorescence quenching of proteins at the excitation wavelength
2.11. Statistical analysis
In the present study, WPI and WPM were used as carriers to load curcumin and the loading amount of curcumin for WPI and WPM was 1.84 ± 0.18 and 17.51 ± 0.46 μg curcumin per mg of the protein, respectively. These results indicated that about 90% of the curcumin was encapsulated in the whey protein microgels; whereas for native WPI it was lower than 10%. Liu et al. (2016) also encapsulated curcumin in WPI using the spray drying method and reported that the encapsulation efficiency was more than 95%. The curcumin encapsulation efficiency for zein nanoparticles prepared by antisolvent precipitation also was between 71.1 and 86.8% depending on the preparation conditions (Patel et al., 2010). Moreover, as shown in Fig. 1, loading of curcumin in whey protein microgels drastically improved its water dispersibility. The aqueous solution of curcumin became completely colorless after storage due to the complete precipitation of curcumin. No sedimentation was observed for curcumin in the 96
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Table 1 Mean diameter, polydispersity index, and zeta-potential (at pH 5.9) of different samples including whey protein isolate (WPI), whey protein microgels (WPM), and curcumin-loaded microgels (C-WPM). Sample WPI WPM C-WPM
Mean diameter (nm) c
101.85 ± 2.61 461.60 ± 7.35b 513.97 ± 21.11a
Polydispersity index a
0.36 ± 0.03 0.23 ± 0.02b 0.23 ± 0.04b
Zeta-potential (mV) −19.78 ± 0.36c −24.06 ± 1.46b −30.65 ± 1.20a
Means with different superscripts in the same column differ significantly (p < 0.05).
Fig. 2. Fluorescence emission spectra of different samples including curcumin in distilled water (dash dot line), whey protein isolate (double dashed line), whey protein isolate with curcumin before microgelification (dashed line), whey protein microgels (solid line), and curcumin-loaded whey protein microgels (dotted line) at excitation wavelengths of (A) 420 nm and (B) 280 nm.
of 280 nm was determined to study the curcumin accessibility to fluorophore groups of WPI and whey protein microgels (Fig. 2B). The loading of WPI and WPM with curcumin decreased their fluorescence intensity; a greater reduction in the fluorescence intensity was observed for WPM after loading with curcumin compared to the WPI counterpart. This decrease in the intrinsic fluorescence intensity of proteins after binding to curcumin was attributed to the ability of curcumin as a ligand to bind the tryptophan and tyrosine residues of proteins which are located in the hydrophobic part of the protein primary structure (Alavi et al., 2018; Esmaili et al., 2011). The higher fluorescence quenching for WPM compared to the WPI after loading of curcumin can be due to the higher loading capacity of whey protein microgels than the native WPI which was mentioned earlier. These findings were consistent with the results of Liu et al. (2016) who reported that the fluorescence intensity of WPI was quenched after loading of curcumin through the spray drying method.
Fig. 3. Light microscopy images of (A) whey protein microgels and (B) curcumin-loaded whey protein microgels.
counterpart. Further, light microscopy (Fig. 3) showed that WPM and curcumin-loaded WPM were spherical in shape which is in a good agreement with those of Schmitt et al. (2010) and Vahedifar et al. (2018) who studied the morphology of WPM with scanning electron microscopy and atomic force microscopy and reported a spherical shape for the WPI microgels. Our results also showed that the zetapotential of WPI was significantly influenced by the microgelification. The loading of curcumin significantly (P < 0.05) changed the zetapotential value of the whey protein microgels. The curcumin-loaded microgels showed a greater absolute magnitude of zeta-potential at pH 5.9 compared to the empty microgels. Chang et al. (2017) also reported that the zeta-potential value of caseinate/zein nanoparticles was influenced by loading of curcumin.
3.3. Particle size, morphology, and zeta-potential The mean diameter, polydispersity index, and zeta-potential of different samples including WPI, whey protein microgels, and curcumin-loaded microgels are reported in Table 1. The heating of WPI at pH 5.9 and 80 °C for 15 min resulted in the formation of microgels with a diameter less than 500 nm. Vahedifar et al. (2018) also reported a hydrodynamic diameter of 300 nm for WPI microgels. The results also indicated that the size of the microgels was increased through the addition of curcumin. However, the polydispersity index of whey protein microgel solution was not significantly influenced by curcumin loading (P > 0.05). Patel et al. (2010) also reported a higher mean size for curcumin-loaded zein particles compared to the curcumin-free
3.4. FT-IR and XRD analysis FT-IR analysis (Fig. 4) showed that the microgelification process did not drastically change the spectrum of WPI and it was similar to that of the whey protein microgels. Many characteristic peaks related to different functional groups were observed in the FT-IR spectrum of pure 97
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Fig. 4. FT-IR spectra of whey protein isolate (WPI), whey protein microgels (WPM), curcumin-loaded microgels (C-WPM), and pure curcumin.
Fig. 6. DPPH radical scavenging activity (A) and reducing power (B) of different samples including curcumin in distilled water (C-DW), whey protein isolate (WPI), whey protein microgels (WPM), and curcumin-loaded microgels (C-WPM). Means followed by different letters are significantly different (p < 0.05).
Fig. 5. XRD patterns of whey protein isolate (WPI), whey protein microgels (WPM), curcumin-loaded microgels (C-WPM), and pure curcumin.
curcumin. Nevertheless, these peaks disappeared when the curcumin was loaded in the whey protein microgels. Xue et al. (2018) also observed that the characteristics peaks of curcumin disappeared after its capsulation into zein-caseinate nanoparticles probably due to the limited stretching and bending of the curcumin bonds through its incorporation into a delivery system. The peak at the wavenumber of 2959/cm in the spectrum of whey protein microgels attributing to the CeH stretching vibrations of protein functional groups, was shifted to 2930/cm after loading with curcumin. This can be due to the formation of hydrogen bonds between the proteins and curcumin which can lower the position of stretching vibrations (Alavi et al., 2018; Mohammadian & Madadlou, 2016). Alavi et al. (2018) also reported a blue shift in the position of peaks related to the stretching vibrations in the FT-IR spectrum of whey protein aggregates after binding to curcumin suggesting the generation of strong hydrogen bonding. However, the positions of the peaks related to the secondary structures of proteins in the amide I (1700–1600/cm, attributed to C]O stretching vibrations), II (1575–1480/cm, related to CeH stretching and NeH bending vibrations), and III (1400–1200/cm, attributed to CeH stretching and NeH bending vibrations) regions (Kong & Yu, 2007) in the WPM spectrum were not significantly changed through the loading with curcumin. This
Fig. 7. Cumulative release profile of curcumin from WPI (○) and whey protein microgels (□) during the sequential simulated gastric (SGF) and intestinal (SIF) digestion.
observation suggested that the secondary structures of whey protein microgels were not modified by loading of curcumin as a cargo. In accordance with our results, Liu et al. (2016) also reported that the secondary structures of β-lactoglobulin were not significantly affected by binding to curcumin. This was attributed to the high flexibility of whey proteins allowing them to bind with curcumin without demanding any drastic structural changes. The physical nature of free and encapsulated curcumin, WPI, and microgels was evaluated by X-ray diffraction analysis (Fig. 5). The native WPI showed a diffractogram without any significant peaks attributing to its amorphous nature. Moreover, the results indicated that 98
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release profile for curcumin under simulated gastrointestinal digestion from zein-caseinate composite particles. This was attributed to the encapsulation of curcumin inside of the particles and limited adsorption of curcumin on or near the surfaces of its carrier. Generally, the results of in vitro release experiment indicated that a controlled curcumin release can be achieved from microgels which can expand their uses in drug delivery applications.
the microgelification process did not affect the XRD pattern of the WPI and the whey protein microgels also were in an amorphous state. The XRD pattern of free curcumin showed different characteristic sharp and intense peaks attributing to its high crystalline structure (Mohanty & Sahoo, 2010). However, these peaks were not observed when curcumin was encapsulated in the whey protein microgels. These findings confirmed the successful loading of curcumin into the microgel particles and suggested that the amorphous curcumin was formed due to the formation of an amorphous complex with whey proteins within the microgel matrix. In accordance, Patel et al. (2010) and Pan et al. (2013) also reported that the characteristic XRD peaks of curcumin were completely disappeared after the entrapment in zein colloidal particles and casein nanocapsules, respectively. The presence of curcumin inside of whey protein microgels in an amorphous form helps its sustained release due to the easy diffusion, unlike the crystalline form which cannot diffuse effectively from the small pores and voids of the microgel particles (Mohanty & Sahoo, 2010).
4. Conclusion Whey protein microgels were successfully used to encapsulate curcumin as a bioactive cargo with a high loading capacity. The encapsulation of curcumin within the whey protein microgels significantly decreased its sedimentation and increased its antioxidant activity. The fluorescence spectroscopy showed that the curcumin was present at the hydrophobic core of whey protein microgels. Moreover, the results of FT-IR and fluorescence spectroscopy suggested that the hydrogen bonding and hydrophobic interactions were formed between curcumin and whey protein microgels. The XRD analysis also indicated that the curcumin was loaded inside the whey protein microgels in an amorphous form. The in vitro release study of the curcumin-loaded whey protein microgels also showed a controlled release profile for curcumin under simulated gastrointestinal conditions. Generally, the present study suggested that the whey protein microgels can be considered as efficient and promising carriers for the incorporation of curcumin into the functional food products with enhanced health-promoting properties.
3.5. Antioxidant activity The in vitro antioxidant activity of different samples including WPI, WPM, curcumin-loaded WPM, and the aqueous solution of free curcumin was evaluated using the DPPH radical scavenging test and reducing power assay (Fig. 6). The native WPI alone showed antioxidant activity which is in a good accordance with previous studies (Liu et al., 2016; Mohammadian & Madadlou, 2016). The antioxidant activity of WPI was not drastically influenced by the microgelification process and the antioxidant activity of whey protein microgels was relatively similar to the native WPI. Moreover, the results indicated that the loading of curcumin in WPM significantly increased its antioxidant activity in both DPPH scavenging test and reducing power assay compared to the curcumin which was dispersed in distilled water. Among different samples, the highest antioxidant activity was related to the curcuminloaded whey protein microgels. In accordance with our results, it was shown that the DPPH radical scavenging activity of curcumin was improved through the encapsulation within the α-lactalbumin and α-lactalbumin-dextran conjugates attributing to the higher water solubility of curcumin in a capsulated form than free curcumin in water (Yi et al., 2016). In fact, the very low solubility of curcumin in water leads to the formation of large curcumin aggregates which reduces the available amount of curcumin for interacting with free radicals and decreases its ability to donate hydrogen atoms to the free radicals (Fan et al., 2018; Liu et al., 2016). Therefore, the curcumin-loaded whey protein microgels can be used as functional ingredients with high ability to prevent oxidation to produce functional foods with improved health-promoting properties.
Acknowledgements The support of University of Tehran is gratefully acknowledged. References Alavi, F., Emam-Djomeh, Z., Yarmand, M. S., Salami, M., Momen, S., & MoosaviMovahedi, A. A. (2018). Cold gelation of curcumin loaded whey protein aggregates mixed with k-carrageenan: Impact of gel microstructure on the gastrointestinal fate of curcumin. Food Hydrocolloids, 85, 267–280. Chang, C., Wang, T., Hu, Q., Zhou, M., Xue, J., & Luo, Y. (2017). Pectin coating improves physicochemical properties of caseinate/zein nanoparticles as oral delivery vehicles for curcumin. Food Hydrocolloids, 70, 143–151. Destribats, M., Rouvet, M., Gehin-Delval, C., Schmitt, C., & Binks, B. P. (2014). Emulsions stabilised by whey protein microgel particles: Towards food-grade pickering emulsions. Soft Matter, 10, 6941–6954. Donato, L., Schmitt, C., Bovetto, L., & Rouvet, M. (2009). Mechanism of formation of stable heat-induced β-lactoglobulin microgels. International Dairy Journal, 19, 295–306. Esmaili, M., Ghaffari, S. M., Moosavi-Movahedi, Z., Atri, M. S., Sharifizadeh, A., Farhadi, M., et al. (2011). Beta casein-micelle as a nano vehicle for solubility enhancement of curcumin; food industry application. LWT-Food Science and Technology, 44, 2166–2172. Fan, Y., Yi, J., Zhang, Y., & Yokoyama, W. (2018). Fabrication of curcumin-loaded bovine serum albumin (BSA)-dextran nanoparticles and the cellular antioxidant activity. Food Chemistry, 239, 1210–1218. Huang, X., Huang, X., Gong, Y., Xiao, H., McClements, D. J., & Hu, K. (2016). Enhancement of curcumin water dispersibility and antioxidant activity using core–shell protein–polysaccharide nanoparticles. Food Research International, 87, 1–9. Kong, J., & Yu, S. (2007). Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochimica et Biophysica Sinica, 39, 549–559. Lin, S., Tian, W., Li, H., Cao, J., & Jiang, W. (2012). Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin, trypsin, alcalase and flavourzyme. International Journal of Food Science and Technology, 47, 2045–2051. Liu, W., Chen, X. D., Cheng, Z., & Selomulya, C. (2016). On enhancing the solubility of curcumin by microencapsulation in whey protein isolate via spray drying. Journal of Food Engineering, 169, 189–195. Liu, J., Jiang, L., Zhang, Y., Du, Z., Qiu, X., Kong, L., et al. (2017). Binding behaviors and structural characteristics of ternary complexes of β-lactoglobulin, curcumin, and fatty acids. RSC Advances, 7, 45960–45967. Maltais, A., Remondetto, G. E., & Subirade, M. (2009). Soy protein cold-set hydrogels as controlled delivery devices for nutraceutical compounds. Food Hydrocolloids, 23, 1647–1653. Mohammadian, M., & Madadlou, A. (2016). Characterization of fibrillated antioxidant whey protein hydrolysate and comparison with fibrillated protein solution. Food Hydrocolloids, 52, 221–230.
3.6. In vitro release properties The release profiles of curcumin from WPI and whey protein microgels under consecutive simulated gastric and intestinal conditions are displayed in Fig. 7. The cumulative release of curcumin from WPI and WPM was found to be 21.05% and 3.82% after 2 h in the simulated gastric fluid, respectively. After 6 h (2 h in SGF plus 4 h in SIF), the percentage of released curcumin from WPI and WPM was about 45% and 19%, respectively. Therefore, it is easily seen that the release of curcumin from whey protein microgels was significantly slower than WPI counterpart over the entire period of release test in the simulated gastrointestinal conditions. It was investigated that the microgelification process results in the extensive cross-linking of whey proteins through the generation of new hydrophobic and disulfide bonds (Vahedifar et al., 2018) which can improve the stability of whey protein microgels against degradation by the harsh gastrointestinal condition and digestive enzymes (pepsin and pancreatin). Therefore, it can be accounted for the lower release of curcumin from whey protein microgels than native WPI. Xue et al. (2018) also investigated a sustained 99
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obtained by heat treatment. Soft Matter, 6, 4876–4884. Tapal, A., & Tiku, P. K. (2012). Complexation of curcumin with soy protein isolate and its implications on solubility and stability of curcumin. Food Chemistry, 130, 960–965. Vahedifar, A., Madadlou, A., & Salami, M. (2017). Calcium and chitosan-mediated clustering of whey protein particles for tuning their colloidal stability and flow behaviour. International Dairy Journal, 73, 136–143. Vahedifar, A., Madadlou, A., & Salami, M. (2018). Influence of seeding and stirring on the structural properties and formation yield of whey protein microgels. International Dairy Journal, 79, 43–51. Xue, J., Zhang, Y., Huang, G., Liu, J., Slavin, M., & Yu, L. L. (2018). Zein-caseinate composite nanoparticles for bioactive delivery using curcumin as a probe compound. Food Hydrocolloids, 83, 25–35. Yi, J., Fan, Y., Zhang, Y., Wen, Z., Zhao, L., & Lu, Y. (2016). Glycosylated α-lactalbuminbased nanocomplex for curcumin: Physicochemical stability and DPPH-scavenging activity. Food Hydrocolloids, 61, 369–377.
Mohanty, C., & Sahoo, S. K. (2010). The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials, 31, 6597–6611. Pan, K., Zhong, Q., & Baek, S. J. (2013). Enhanced dispersibility and bioactivity of curcumin by encapsulation in casein nanocapsules. Journal of Agricultural and Food Chemistry, 61, 6036–6043. Patel, A., Hu, Y., Tiwari, J. K., & Velikov, K. P. (2010). Synthesis and characterisation of zein–curcumin colloidal particles. Soft Matter, 6, 6192–6199. Rafiee, Z., Nejatian, M., Daeihamed, M., & Jafari, S. M. (2018). Application of different nanocarriers for encapsulation of curcumin. Critical Reviews in Food Science and Nutrition, 1–77 (just-accepted). Schmitt, C., Bovay, C., & Rouvet, M. (2014). Bulk self-aggregation drives foam stabilization properties of whey protein microgels. Food Hydrocolloids, 42, 139–148. Schmitt, C., Moitzi, C., Bovay, C., Rouvet, M., Bovetto, L., Donato, L., et al. (2010). Internal structure and colloidal behaviour of covalent whey protein microgels
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Fabrication of curcumin-loaded whey protein microgels: Structural properties, antioxidant activity, and in vitro release behavior
T
Mehdi Mohammadian, Maryam Salami∗, Shima Momen, Farhad Alavi, Zahra Emam-Djomeh Department of Food Science and Engineering, University College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Whey protein microgel Bioactive delivery Curcumin Radical scavenging activity Structural characteristics
This study evaluated the potential use of whey protein microgels (WPM) formed by heating a protein solution at pH 5.90 as carriers for curcumin. The results indicated that the loading amount of curcumin into whey protein isolate (WPI) and WPM was 1.84 ± 0.18 and 17.51 ± 0.46 μg curcumin/mg protein, respectively. Loading of microgels with curcumin significantly increased their diameter and changed their surface charge. Loading of curcumin in the whey protein microgels drastically decreased its sedimentation during storage. Fluorescence measurements revealed that the curcumin was present at the hydrophobic core of whey protein microgels. X-ray diffraction analysis also showed that curcumin was loaded inside the WPM in an amorphous form. Curcuminloaded whey protein microgels showed a high in vitro antioxidant activity which was measured by DPPH radical scavenging and reducing power assay. The in vitro release experiment also showed that the release of curcumin from WPM was significantly slower than WPI during the simulated gastrointestinal conditions. These findings suggested a possible utilization of WPI microgels in improving the applications of curcumin for the production of functional food products and drugs.
1. Introduction Curcumin is a natural polyphenol compound derived from the rhizome of turmeric (Curcuma longa) with different biological and healthpromoting properties such as antioxidant, antimicrobial, anti-inflammatory, anti-proliferative, and anticancer activity (Alavi et al., 2018; Esmaili et al., 2011; Rafiee, Nejatian, Daeihamed, & Jafari, 2018). However, the poor water dispersibility, rapid metabolism, and low stability of curcumin limit its bioavailability and also restrict its applications in food products (Alavi et al., 2018). Complexation with proteins or encapsulation within food protein-based carriers are suggested as effective methods to improve stability, solubility, and bioavailability of curcumin (Rafiee et al., 2018). A wide range of food proteins such as whey proteins (Liu, Chen, Cheng, & Selomulya, 2016; Liu et al., 2017), casein (Esmaili et al., 2011; Pan, Zhong, & Baek, 2013), bovine serum albumin (Fan, Yi, Zhang, & Yokoyama, 2018), zein (Patel, Hu, Tiwari, & Velikov, 2010), and soy proteins (Tapal & Tiku, 2012) have been employed as carriers for curcumin. Whey proteins have been widely used as functional ingredients in various food applications due to their superb technological functionalities and high nutritional value (Lin, Tian, Li, Cao, & Jiang, 2012). Heat treatment of these proteins at different pH results in the formation of aggregates with different shapes and morphologies such as ∗
nanofibrils, flexible strands, and microgels (Vahedifar, Madadlou, & Salami, 2017). Spherical whey protein microgels (WPM) with a diameter of 100–600 nm and relatively low polydispersity are formed through the heating of whey protein solution at a narrow pH range from 5.8 to 6.2 and low ionic strength (Schmitt et al., 2010; Vahedifar, Madadlou, & Salami, 2018). It was investigated that the covalent disulfide bonds and non-covalent hydrophobic and electrostatic interactions are the most important forces involved in the stability of whey protein microgels (Donato, Schmitt, Bovetto, & Rouvet, 2009). Whey protein microgels were successfully used as efficient emulsifying (Destribats, Rouvet, Gehin-Delval, Schmitt, & Binks, 2014) and foaming (Schmitt, Bovay, & Rouvet, 2014) agents. It seems that the microgelification process can be used to produce bioactive ingredients-loaded whey protein microgels which can expand their applications in the production of functional food products as well as for designing new drug delivery systems. To the best of our knowledge, there has been no research in the literature about the application of whey protein microgels as carriers for curcumin. Therefore, in this study, curcumin was loaded into the microgel particles prepared by heating of whey protein solution at pH close to its isoelectric point. The structural properties, antioxidant activity, and in vitro release behavior of the curcumin-loaded whey protein microgels were studied by different methods and techniques.
Corresponding author. E-mail address: [email protected] (M. Salami).
https://doi.org/10.1016/j.lwt.2018.12.076 Received 13 August 2018; Received in revised form 25 October 2018; Accepted 29 December 2018 Available online 31 December 2018 0023-6438/ © 2018 Published by Elsevier Ltd.
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interaction between curcumin and proteins. For fluorescence studies, samples were diluted with distilled water (pH 5.9) to a protein concentration of 0.2 mg/mL and curcumin concentration of 4 μg/mL. To assess the protein intrinsic fluorescence, the emission spectra were recorded from 300 to 450 nm with an excitation wavelength of 280 nm. The emission spectra of curcumin in different media were recorded from 450 to 700 nm with an excitation wavelength of 420 nm. Both the excitation and emission slit widths were set to 5 nm.
2. Materials and methods 2.1. Materials Whey protein isolate (WPI) with more than 90 g/100 g protein was obtained from Hilmar Ingredients (CA, USA). Curcumin, pepsin (the activity of more than 3000 units/mg), and pancreatin (200 units/mg) were obtained from Bio Basic (Bio Basic Inc., Canada). 2,2-Diphenyl-1picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (Sa. Louis, MO, USA). Other chemicals used in this study were also of analytical grade and purchased from Merck (Darmstadt, Germany) and SigmaAldrich.
2.5. Particle size and ζ-potential measurements The particle size of different samples (WPI, WPM, and C-WPM) was determined using dynamic light scattering (DLS) (Brookhaven Instruments Corp., Holtsville, NY, USA) at room temperature. The samples were diluted with double distilled water with the same pH before the measurements to avoid multiple scattering. The ζ-potential of different samples at pH 5.90 was also measured with the same DLS apparatus in ζ-potential measurement mode using the specialized zeta cell.
2.2. Preparation of curcumin-loaded whey protein microgels For the preparation of curcumin-loaded whey protein microgels (CWPM), WPI powder was dissolved in distilled water with a concentration of 50 mg/mL. Sodium azide was added as an antimicrobial agent. The protein solution was stirred at room temperature for 2 h followed by overnight storage for complete hydration and then was loaded with curcumin. For this purpose, curcumin was dissolved in anhydrous ethanol and then added to the WPI solution under stirring. The final concentration of curcumin in the mixtures was 1.0 mg/mL (curcumin to protein ratio of 1:50). This was followed by 10 h stirring at room temperature in the dark. It should be noted that the final concentration of ethanol in whey protein solution never exceeded 2.0 mL/L. At this concentration, ethanol has no significant effect on the structure of proteins (Liu et al., 2017). Subsequently, the pH of WPI-curcumin binary solution was adjusted to 5.90 ± 0.05 using NaOH and HCl (1.0 or 0.1 mol/L) and then was heated for 15 min at 80 °C under a mild condition of stirring to fabricate curcumin-loaded WPI microgels (Vahedifar et al., 2018). The microgelification process was stopped by rapid cooling of the protein solution to room temperature using cold water. The curcumin-free whey protein microgels (WPM) also were prepared according to the above-mentioned procedure without the addition of curcumin.
2.6. Optical microscopy The whey protein microgels with or without curcumin were imaged using an 18.0 MP digital USB camera attached to an optical microscope (OMAX, Gyeonggi-do, South Korea) at 10 × 100 magnification. 2.7. Fourier transform infra-red (FT-IR) spectroscopy The FT-IR spectroscopy using a Bruker FT-IR spectrometer (Billerica, Massachusetts, United States) was employed to assess the molecular attributes of different samples (pure curcumin, WPI, WPM and curcumin-loaded whey protein microgels). The lyophilized samples were powdered and compressed into the transparent potassium bromide disks and scanned from 4000 to 500/cm in the transmittance mode. 2.8. X-ray diffraction (XRD) analysis
2.3. Determination of loading amount
The XRD spectra of lyophilized samples including pure curcumin, native WPI, whey protein microgels, and curcumin-loaded whey protein microgels were collected on a Philips PW1730 X-ray diffractometer (PANalytical, Netherlands) using a Cu Kα radiation source in the 2θ range of 5°–50° with a step size of 0.05° and scan rate of 1°/s. The filament current and operating voltage were respectively set at 30 mA and 40 kV.
The loading amount (LA) of curcumin for WPI before (C-WPI) and after (C-WPM) microgelification process was measured according to Tapal and Tiku (2012) with some modifications. For measuring the curcumin loading amount of WPI, the WPI-curcumin solution was centrifuged at 5000 ×g for 15 min to remove any undissolved curcumin. The supernatant was diluted with ethanol to extract the curcumin and its absorbance was measured spectrophotometrically at 420 nm. The concentration of curcumin in the supernatant was calculated using a standard curve of curcumin (0.1–10 μg/mL dissolved in ethanol, R2 = 99.89%) and was reported as the loaded curcumin. In the case of whey protein microgels, the microgel solution was centrifuged at 100×g for 2 min to separate the curcumin-loaded microgels. After that, curcumin in the supernatant was extracted by ethanol and its concentration was calculated as the free curcumin. It should be noted that no curcumin precipitate was observed at the bottom of the tube after the centrifugation. Finally, the loading amount of curcumin in WPI and WPM was determined according to:
LA (μg / mg ) =
loaded amount of curcumin total amount of protein
2.9. Antioxidant activity The antioxidant activities of WPI, whey protein microgels, curcumin-loaded microgels, and the aqueous solution of curcumin were measured using DPPH radical scavenging activity test and reducing power assay according to the method of Lin et al. (2012) with minor adjustments. Different samples were diluted with distilled water of the same pH to a protein concentration of 5 mg/mL and a curcumin concentration of 0.1 mg/mL before the measurements. For DPPH radical scavenging test, 2 mL of different sample solutions was added to 2 mL of the ethanolic DPPH solution (0.2 mmol/L). 2 mL of DPPH solution was also added to 2.0 mL of distilled water as the control sample. After that, the resulting mixtures were mixed vigorously and stored for 30 min at room temperature in dark, followed by centrifuging at 1500×g for 10 min. The absorbance of the resulting supernatants was measured spectrophotometrically at 517 nm and the DPPH radical scavenging activity was calculated according to
(1)
2.4. Fluorescence measurements The fluorescence properties of different samples including free curcumin in water, WPI, WPM, C-WPM, and CeWPI (curcumin-WPI complex before microgelification) were examined using a fluorescence spectrofluorometer (Cary Eclipse, Palo Alto, CA) to study the
Radical scavenging activity (%) = 95
A C − AS × 100 AC
(2)
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where AC and AS are the absorbance of control and sample solutions, respectively. For the reducing power assay, 1 mL of diluted sample solution was mixed with 2.5 mL phosphate buffer (200 mmol/L, pH 6.6) and subsequently with 2.5 mL potassium ferricyanide (10 g/L). The resulting mixtures were incubated for 20 min in a water bath at 50 °C, followed by charging with 2.5 mL trichloroacetic acid (100 mg/mL). After that, the mixtures were centrifuged for 10 min at 1500 ×g and 2.5 mL of the supernatants was mixed with 2.5 mL distilled water and also 0.5 mL ferric chloride (1 mg/mL). The absorbance of these mixtures was read at 700 nm after 10 min incubating at room temperature using a UV–visible spectrophotometer. 2.10. In vitro release behavior The release profile of curcumin from WPI and whey protein microgels during sequential gastrointestinal digestion was studied by a dialysis bag method according to Chang et al. (2017) and Maltais, Remondetto, and Subirade (2009) with slight adjustments. Two mL of CeWPI or C-WPM solutions (pH 1.2) were filled into a dialysis bag (with 12 kDa molecular cut off) and mixed with 2.0 mL of simulated gastric fluid (SGF, consisted of NaCl, HCl, and 3.2 g/L pepsin with a final pH value of 1.2). After that, the dialysis bag was placed in a beaker containing 150 mL of the release medium consisting of 75 mL ethanol and 75 mL SGF without enzyme. This was followed by 2 h incubation at 37 °C and shaking of 100 rpm. After that, the pH of solution was adjusted to 7.5 and 4 mL of simulated intestinal fluid (SIF, consisting of monobasic potassium phosphate, NaOH, and 10 g/L of pancreatin with the final pH value of 7.5) was added to the mixture. Subsequently, the dialysis tube was placed in a beaker containing 150 mL of enzyme-free SIF-ethanol mixture and incubated for 4 h under continuous shaking (37 °C and 100 rpm). It should be noted that the ethanol was added to the release medium due to the poor solubility of curcumin in water. Two mL of the outer release medium was collected at each designated point and replaced with fresh medium. The amount of the released curcumin was determined using UV–Vis spectrophotometer at 420 nm and its concentration was calculated with the help of a curcumin standard curve which was prepared using the same release medium.
Fig. 1. Visual appearance of different sample solutions including (A) native WPI, (B) WPI-curcumin before microgelification, (C) whey protein microgels, (D) curcumin-loaded microgels, and (E) curcumin dispersed in distilled water.
Statistical analyses were performed using the one-way analysis of variance (ANOVA) by SPSS software version 16.0 (IBM software, NY, USA). The significance of difference between the means values was determined by Duncan's multiple range procedure. The level of significance was set to 0.05.
capsulated form (i.e. curcumin-loaded whey protein microgels) at the bottom of the test tube, whereas a thin layer of curcumin sediment was formed in the case of both free curcumin dispersed in distilled water and also the native whey protein isolate at the pH value of 5.9. In accordance with these observations, it was also investigated that the water dispersibility of curcumin was improved through the encapsulation within casein nanocapsules (Pan et al., 2013) and core-shell protein (zein)-polysaccharide (pectin and alginate) nanoparticles (Huang et al., 2016). Consequently, improving the water solubility of curcumin can enhance its biological activity such as antioxidant activity which was assessed in the present study. Therefore, microgelification can be considered as an efficient and facile method to encapsulate curcumin and other bioactive molecules and the resulting whey protein microgels can be considered as promising encapsulation agents for the incorporation of bioactive compounds such as curcumin into the functional food products.
3. Results and discussion
3.2. Fluorescence spectroscopy
3.1. Loading amount
The possible binding and interactions between curcumin and proteins (WPI and WPM), as well as the micro-environmental changes, were studied using fluorescence spectroscopy. The fluorescence spectra of different samples at the excitation wavelength of curcumin (420 nm) are shown in Fig. 2A. Free curcumin which was dispersed in distilled water showed a broad low-intensity emission spectrum which is in a good agreement with the results of Esamili et al. (2011). The fluorescence intensity of curcumin was slightly increased when it was bound with WPI, but the intensity was enhanced drastically when curcumin loaded in whey protein microgels. These results indicated that the curcumin was transferred from a hydrophilic environment to a more hydrophobic environment after loading in whey protein microgels. This also suggests that the curcumin was present at the core of whey protein microgels in aqueous dispersions. Pan et al. (2013) also observed a significant increase in the fluorescence intensity of curcumin after its encapsulation in casein nanoparticles. The fluorescence quenching of proteins at the excitation wavelength
2.11. Statistical analysis
In the present study, WPI and WPM were used as carriers to load curcumin and the loading amount of curcumin for WPI and WPM was 1.84 ± 0.18 and 17.51 ± 0.46 μg curcumin per mg of the protein, respectively. These results indicated that about 90% of the curcumin was encapsulated in the whey protein microgels; whereas for native WPI it was lower than 10%. Liu et al. (2016) also encapsulated curcumin in WPI using the spray drying method and reported that the encapsulation efficiency was more than 95%. The curcumin encapsulation efficiency for zein nanoparticles prepared by antisolvent precipitation also was between 71.1 and 86.8% depending on the preparation conditions (Patel et al., 2010). Moreover, as shown in Fig. 1, loading of curcumin in whey protein microgels drastically improved its water dispersibility. The aqueous solution of curcumin became completely colorless after storage due to the complete precipitation of curcumin. No sedimentation was observed for curcumin in the 96
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Table 1 Mean diameter, polydispersity index, and zeta-potential (at pH 5.9) of different samples including whey protein isolate (WPI), whey protein microgels (WPM), and curcumin-loaded microgels (C-WPM). Sample WPI WPM C-WPM
Mean diameter (nm) c
101.85 ± 2.61 461.60 ± 7.35b 513.97 ± 21.11a
Polydispersity index a
0.36 ± 0.03 0.23 ± 0.02b 0.23 ± 0.04b
Zeta-potential (mV) −19.78 ± 0.36c −24.06 ± 1.46b −30.65 ± 1.20a
Means with different superscripts in the same column differ significantly (p < 0.05).
Fig. 2. Fluorescence emission spectra of different samples including curcumin in distilled water (dash dot line), whey protein isolate (double dashed line), whey protein isolate with curcumin before microgelification (dashed line), whey protein microgels (solid line), and curcumin-loaded whey protein microgels (dotted line) at excitation wavelengths of (A) 420 nm and (B) 280 nm.
of 280 nm was determined to study the curcumin accessibility to fluorophore groups of WPI and whey protein microgels (Fig. 2B). The loading of WPI and WPM with curcumin decreased their fluorescence intensity; a greater reduction in the fluorescence intensity was observed for WPM after loading with curcumin compared to the WPI counterpart. This decrease in the intrinsic fluorescence intensity of proteins after binding to curcumin was attributed to the ability of curcumin as a ligand to bind the tryptophan and tyrosine residues of proteins which are located in the hydrophobic part of the protein primary structure (Alavi et al., 2018; Esmaili et al., 2011). The higher fluorescence quenching for WPM compared to the WPI after loading of curcumin can be due to the higher loading capacity of whey protein microgels than the native WPI which was mentioned earlier. These findings were consistent with the results of Liu et al. (2016) who reported that the fluorescence intensity of WPI was quenched after loading of curcumin through the spray drying method.
Fig. 3. Light microscopy images of (A) whey protein microgels and (B) curcumin-loaded whey protein microgels.
counterpart. Further, light microscopy (Fig. 3) showed that WPM and curcumin-loaded WPM were spherical in shape which is in a good agreement with those of Schmitt et al. (2010) and Vahedifar et al. (2018) who studied the morphology of WPM with scanning electron microscopy and atomic force microscopy and reported a spherical shape for the WPI microgels. Our results also showed that the zetapotential of WPI was significantly influenced by the microgelification. The loading of curcumin significantly (P < 0.05) changed the zetapotential value of the whey protein microgels. The curcumin-loaded microgels showed a greater absolute magnitude of zeta-potential at pH 5.9 compared to the empty microgels. Chang et al. (2017) also reported that the zeta-potential value of caseinate/zein nanoparticles was influenced by loading of curcumin.
3.3. Particle size, morphology, and zeta-potential The mean diameter, polydispersity index, and zeta-potential of different samples including WPI, whey protein microgels, and curcumin-loaded microgels are reported in Table 1. The heating of WPI at pH 5.9 and 80 °C for 15 min resulted in the formation of microgels with a diameter less than 500 nm. Vahedifar et al. (2018) also reported a hydrodynamic diameter of 300 nm for WPI microgels. The results also indicated that the size of the microgels was increased through the addition of curcumin. However, the polydispersity index of whey protein microgel solution was not significantly influenced by curcumin loading (P > 0.05). Patel et al. (2010) also reported a higher mean size for curcumin-loaded zein particles compared to the curcumin-free
3.4. FT-IR and XRD analysis FT-IR analysis (Fig. 4) showed that the microgelification process did not drastically change the spectrum of WPI and it was similar to that of the whey protein microgels. Many characteristic peaks related to different functional groups were observed in the FT-IR spectrum of pure 97
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Fig. 4. FT-IR spectra of whey protein isolate (WPI), whey protein microgels (WPM), curcumin-loaded microgels (C-WPM), and pure curcumin.
Fig. 6. DPPH radical scavenging activity (A) and reducing power (B) of different samples including curcumin in distilled water (C-DW), whey protein isolate (WPI), whey protein microgels (WPM), and curcumin-loaded microgels (C-WPM). Means followed by different letters are significantly different (p < 0.05).
Fig. 5. XRD patterns of whey protein isolate (WPI), whey protein microgels (WPM), curcumin-loaded microgels (C-WPM), and pure curcumin.
curcumin. Nevertheless, these peaks disappeared when the curcumin was loaded in the whey protein microgels. Xue et al. (2018) also observed that the characteristics peaks of curcumin disappeared after its capsulation into zein-caseinate nanoparticles probably due to the limited stretching and bending of the curcumin bonds through its incorporation into a delivery system. The peak at the wavenumber of 2959/cm in the spectrum of whey protein microgels attributing to the CeH stretching vibrations of protein functional groups, was shifted to 2930/cm after loading with curcumin. This can be due to the formation of hydrogen bonds between the proteins and curcumin which can lower the position of stretching vibrations (Alavi et al., 2018; Mohammadian & Madadlou, 2016). Alavi et al. (2018) also reported a blue shift in the position of peaks related to the stretching vibrations in the FT-IR spectrum of whey protein aggregates after binding to curcumin suggesting the generation of strong hydrogen bonding. However, the positions of the peaks related to the secondary structures of proteins in the amide I (1700–1600/cm, attributed to C]O stretching vibrations), II (1575–1480/cm, related to CeH stretching and NeH bending vibrations), and III (1400–1200/cm, attributed to CeH stretching and NeH bending vibrations) regions (Kong & Yu, 2007) in the WPM spectrum were not significantly changed through the loading with curcumin. This
Fig. 7. Cumulative release profile of curcumin from WPI (○) and whey protein microgels (□) during the sequential simulated gastric (SGF) and intestinal (SIF) digestion.
observation suggested that the secondary structures of whey protein microgels were not modified by loading of curcumin as a cargo. In accordance with our results, Liu et al. (2016) also reported that the secondary structures of β-lactoglobulin were not significantly affected by binding to curcumin. This was attributed to the high flexibility of whey proteins allowing them to bind with curcumin without demanding any drastic structural changes. The physical nature of free and encapsulated curcumin, WPI, and microgels was evaluated by X-ray diffraction analysis (Fig. 5). The native WPI showed a diffractogram without any significant peaks attributing to its amorphous nature. Moreover, the results indicated that 98
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release profile for curcumin under simulated gastrointestinal digestion from zein-caseinate composite particles. This was attributed to the encapsulation of curcumin inside of the particles and limited adsorption of curcumin on or near the surfaces of its carrier. Generally, the results of in vitro release experiment indicated that a controlled curcumin release can be achieved from microgels which can expand their uses in drug delivery applications.
the microgelification process did not affect the XRD pattern of the WPI and the whey protein microgels also were in an amorphous state. The XRD pattern of free curcumin showed different characteristic sharp and intense peaks attributing to its high crystalline structure (Mohanty & Sahoo, 2010). However, these peaks were not observed when curcumin was encapsulated in the whey protein microgels. These findings confirmed the successful loading of curcumin into the microgel particles and suggested that the amorphous curcumin was formed due to the formation of an amorphous complex with whey proteins within the microgel matrix. In accordance, Patel et al. (2010) and Pan et al. (2013) also reported that the characteristic XRD peaks of curcumin were completely disappeared after the entrapment in zein colloidal particles and casein nanocapsules, respectively. The presence of curcumin inside of whey protein microgels in an amorphous form helps its sustained release due to the easy diffusion, unlike the crystalline form which cannot diffuse effectively from the small pores and voids of the microgel particles (Mohanty & Sahoo, 2010).
4. Conclusion Whey protein microgels were successfully used to encapsulate curcumin as a bioactive cargo with a high loading capacity. The encapsulation of curcumin within the whey protein microgels significantly decreased its sedimentation and increased its antioxidant activity. The fluorescence spectroscopy showed that the curcumin was present at the hydrophobic core of whey protein microgels. Moreover, the results of FT-IR and fluorescence spectroscopy suggested that the hydrogen bonding and hydrophobic interactions were formed between curcumin and whey protein microgels. The XRD analysis also indicated that the curcumin was loaded inside the whey protein microgels in an amorphous form. The in vitro release study of the curcumin-loaded whey protein microgels also showed a controlled release profile for curcumin under simulated gastrointestinal conditions. Generally, the present study suggested that the whey protein microgels can be considered as efficient and promising carriers for the incorporation of curcumin into the functional food products with enhanced health-promoting properties.
3.5. Antioxidant activity The in vitro antioxidant activity of different samples including WPI, WPM, curcumin-loaded WPM, and the aqueous solution of free curcumin was evaluated using the DPPH radical scavenging test and reducing power assay (Fig. 6). The native WPI alone showed antioxidant activity which is in a good accordance with previous studies (Liu et al., 2016; Mohammadian & Madadlou, 2016). The antioxidant activity of WPI was not drastically influenced by the microgelification process and the antioxidant activity of whey protein microgels was relatively similar to the native WPI. Moreover, the results indicated that the loading of curcumin in WPM significantly increased its antioxidant activity in both DPPH scavenging test and reducing power assay compared to the curcumin which was dispersed in distilled water. Among different samples, the highest antioxidant activity was related to the curcuminloaded whey protein microgels. In accordance with our results, it was shown that the DPPH radical scavenging activity of curcumin was improved through the encapsulation within the α-lactalbumin and α-lactalbumin-dextran conjugates attributing to the higher water solubility of curcumin in a capsulated form than free curcumin in water (Yi et al., 2016). In fact, the very low solubility of curcumin in water leads to the formation of large curcumin aggregates which reduces the available amount of curcumin for interacting with free radicals and decreases its ability to donate hydrogen atoms to the free radicals (Fan et al., 2018; Liu et al., 2016). Therefore, the curcumin-loaded whey protein microgels can be used as functional ingredients with high ability to prevent oxidation to produce functional foods with improved health-promoting properties.
Acknowledgements The support of University of Tehran is gratefully acknowledged. References Alavi, F., Emam-Djomeh, Z., Yarmand, M. S., Salami, M., Momen, S., & MoosaviMovahedi, A. A. (2018). Cold gelation of curcumin loaded whey protein aggregates mixed with k-carrageenan: Impact of gel microstructure on the gastrointestinal fate of curcumin. Food Hydrocolloids, 85, 267–280. Chang, C., Wang, T., Hu, Q., Zhou, M., Xue, J., & Luo, Y. (2017). Pectin coating improves physicochemical properties of caseinate/zein nanoparticles as oral delivery vehicles for curcumin. Food Hydrocolloids, 70, 143–151. Destribats, M., Rouvet, M., Gehin-Delval, C., Schmitt, C., & Binks, B. P. (2014). Emulsions stabilised by whey protein microgel particles: Towards food-grade pickering emulsions. Soft Matter, 10, 6941–6954. Donato, L., Schmitt, C., Bovetto, L., & Rouvet, M. (2009). Mechanism of formation of stable heat-induced β-lactoglobulin microgels. International Dairy Journal, 19, 295–306. Esmaili, M., Ghaffari, S. M., Moosavi-Movahedi, Z., Atri, M. S., Sharifizadeh, A., Farhadi, M., et al. (2011). Beta casein-micelle as a nano vehicle for solubility enhancement of curcumin; food industry application. LWT-Food Science and Technology, 44, 2166–2172. Fan, Y., Yi, J., Zhang, Y., & Yokoyama, W. (2018). Fabrication of curcumin-loaded bovine serum albumin (BSA)-dextran nanoparticles and the cellular antioxidant activity. Food Chemistry, 239, 1210–1218. Huang, X., Huang, X., Gong, Y., Xiao, H., McClements, D. J., & Hu, K. (2016). Enhancement of curcumin water dispersibility and antioxidant activity using core–shell protein–polysaccharide nanoparticles. Food Research International, 87, 1–9. Kong, J., & Yu, S. (2007). Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochimica et Biophysica Sinica, 39, 549–559. Lin, S., Tian, W., Li, H., Cao, J., & Jiang, W. (2012). Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin, trypsin, alcalase and flavourzyme. International Journal of Food Science and Technology, 47, 2045–2051. Liu, W., Chen, X. D., Cheng, Z., & Selomulya, C. (2016). On enhancing the solubility of curcumin by microencapsulation in whey protein isolate via spray drying. Journal of Food Engineering, 169, 189–195. Liu, J., Jiang, L., Zhang, Y., Du, Z., Qiu, X., Kong, L., et al. (2017). Binding behaviors and structural characteristics of ternary complexes of β-lactoglobulin, curcumin, and fatty acids. RSC Advances, 7, 45960–45967. Maltais, A., Remondetto, G. E., & Subirade, M. (2009). Soy protein cold-set hydrogels as controlled delivery devices for nutraceutical compounds. Food Hydrocolloids, 23, 1647–1653. Mohammadian, M., & Madadlou, A. (2016). Characterization of fibrillated antioxidant whey protein hydrolysate and comparison with fibrillated protein solution. Food Hydrocolloids, 52, 221–230.
3.6. In vitro release properties The release profiles of curcumin from WPI and whey protein microgels under consecutive simulated gastric and intestinal conditions are displayed in Fig. 7. The cumulative release of curcumin from WPI and WPM was found to be 21.05% and 3.82% after 2 h in the simulated gastric fluid, respectively. After 6 h (2 h in SGF plus 4 h in SIF), the percentage of released curcumin from WPI and WPM was about 45% and 19%, respectively. Therefore, it is easily seen that the release of curcumin from whey protein microgels was significantly slower than WPI counterpart over the entire period of release test in the simulated gastrointestinal conditions. It was investigated that the microgelification process results in the extensive cross-linking of whey proteins through the generation of new hydrophobic and disulfide bonds (Vahedifar et al., 2018) which can improve the stability of whey protein microgels against degradation by the harsh gastrointestinal condition and digestive enzymes (pepsin and pancreatin). Therefore, it can be accounted for the lower release of curcumin from whey protein microgels than native WPI. Xue et al. (2018) also investigated a sustained 99
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obtained by heat treatment. Soft Matter, 6, 4876–4884. Tapal, A., & Tiku, P. K. (2012). Complexation of curcumin with soy protein isolate and its implications on solubility and stability of curcumin. Food Chemistry, 130, 960–965. Vahedifar, A., Madadlou, A., & Salami, M. (2017). Calcium and chitosan-mediated clustering of whey protein particles for tuning their colloidal stability and flow behaviour. International Dairy Journal, 73, 136–143. Vahedifar, A., Madadlou, A., & Salami, M. (2018). Influence of seeding and stirring on the structural properties and formation yield of whey protein microgels. International Dairy Journal, 79, 43–51. Xue, J., Zhang, Y., Huang, G., Liu, J., Slavin, M., & Yu, L. L. (2018). Zein-caseinate composite nanoparticles for bioactive delivery using curcumin as a probe compound. Food Hydrocolloids, 83, 25–35. Yi, J., Fan, Y., Zhang, Y., Wen, Z., Zhao, L., & Lu, Y. (2016). Glycosylated α-lactalbuminbased nanocomplex for curcumin: Physicochemical stability and DPPH-scavenging activity. Food Hydrocolloids, 61, 369–377.
Mohanty, C., & Sahoo, S. K. (2010). The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials, 31, 6597–6611. Pan, K., Zhong, Q., & Baek, S. J. (2013). Enhanced dispersibility and bioactivity of curcumin by encapsulation in casein nanocapsules. Journal of Agricultural and Food Chemistry, 61, 6036–6043. Patel, A., Hu, Y., Tiwari, J. K., & Velikov, K. P. (2010). Synthesis and characterisation of zein–curcumin colloidal particles. Soft Matter, 6, 6192–6199. Rafiee, Z., Nejatian, M., Daeihamed, M., & Jafari, S. M. (2018). Application of different nanocarriers for encapsulation of curcumin. Critical Reviews in Food Science and Nutrition, 1–77 (just-accepted). Schmitt, C., Bovay, C., & Rouvet, M. (2014). Bulk self-aggregation drives foam stabilization properties of whey protein microgels. Food Hydrocolloids, 42, 139–148. Schmitt, C., Moitzi, C., Bovay, C., Rouvet, M., Bovetto, L., Donato, L., et al. (2010). Internal structure and colloidal behaviour of covalent whey protein microgels
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