In vitro bioavailability, cellular antioxidant activity, and cytotoxicity of β

Jan 31, 2018 - Previously, it was shown that catechin-egg white protein (CT-EWP) conjugates were effective antioxidant emulsifiers that could form and...
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Article Cite This: J. Agric. Food Chem. 2018, 66, 1649−1657

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In Vitro Bioavailability, Cellular Antioxidant Activity, and Cytotoxicity of β‑Carotene-Loaded Emulsions Stabilized by Catechin−Egg White Protein Conjugates Luping Gu,†,‡ Che Pan,‡ Yujie Su,† Ruojie Zhang,‡ Hang Xiao,‡ David Julian McClements,*,‡ and Yanjun Yang*,† †

Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China ‡ Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States ABSTRACT: Previously, it was shown that catechin−egg white protein (CT-EWP) conjugates were effective antioxidant emulsifiers that could form and stabilize emulsions, and also inhibit the degradation of encapsulated carotenoids. The objective of the current study was to evaluate the impact of conjugation on the in vitro bioavailability, cellular antioxidant activity, and cytotoxicity of β-carotene-loaded emulsions. Lipid droplets coated with EWP or with CT-EWP conjugates exhibited quite similar behavior when they were passed through a simulated gastrointestinal tract. The β-carotene encapsulated in emulsions stabilized by CT-EWP conjugates exhibited a higher overall in vitro bioavailability, which was attributed to a greater stability of the carotenoids to chemical transformation. The emulsions stabilized by CT-EWP conjugates also exhibited greater ability in inhibiting oxidation in a cell-based assay (dichlorofluorescein diacetate). Cytotoxicity analysis suggested that β-carotene emulsions stabilized by CT-EWP conjugates only exhibited a slight cytotoxicity when used at high concentrations. These results suggest that CT-EWP conjugates can be used to formulate emulsion-based delivery systems for chemically labile hydrophobic bioactives with enhanced antioxidant activity and bioavailability. KEYWORDS: β-carotene, conjugates, bioavailability, antioxidant activity, cytotoxicity

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INTRODUCTION Some carotenoids, such as β-carotene, have been shown to be bioactive components that have potential health benefits.1,2 Numerous studies have reported that β-carotene has high provitamin A activity and acts as an effective antioxidant.3,4 Nevertheless, the utilization of this highly hydrophobic and chemically labile molecule in functional foods is currently restricted due to its poor water solubility, chemical instability, and low oral bioavailability.5 These limitations can be overcome by encapsulating β-carotene within well-designed emulsionbased delivery systems, such as emulsions or nanoemulsions.6,7 In O/W emulsions, β-carotene is present inside small oil droplets that are dispersed in a water phase. The oil droplets are coated by a layer of emulsifier molecules, which are usually selected to ensure good physical stability of the emulsions by inhibiting flocculation or coalescence, but which may also be selected to provide protection against chemical instability (such as oxidation). After ingestion, it is important that the encapsulated carotenoids have a high oral bioavailability, which depends on their bioaccessibility, transformation, and absorption characteristics.7 First, the lipid droplets should be fully digested and release the encapsulated carotenoids into the surrounding intestinal fluids in an absorbable form (bioaccessibility). In the stomach and small intestine, the triacylglycerol molecules at the surfaces of the oil droplets are hydrolyzed by lipase into free fatty acids and monoacylglycerols, which then mix with bile salts and phospholipids to form mixed micelles capable of solubilizing and transporting the β-carotene molecules released from the lipid droplets. In addition, the © 2018 American Chemical Society

efficacy of the ingested carotenoids depends on the fraction that remains in a biologically active form (transformation) and the fraction that is transported across the epithelium cells (absorption). Consequently, it is important to determine the impact of any new emulsifier used to fabricate an emulsionbased delivery system on the oral bioavailability of encapsulated nutraceuticals. Recently, it has been shown that antioxidant emulsifiers can be fabricated by covalently attaching polyphenols to proteins.8 The proteins provide good emulsifying properties,9,10 whereas the polyphenols provide strong interfacial antioxidant activity.11−13 Previous studies have shown that the protein− polyphenol conjugates synthesized using a free radical grafting method improved the physical and chemical stability of βcarotene-enriched emulsions, when compared with native proteins or conjugates prepared using other approaches.14,15 In our previous study, catechin−egg white protein (CT-EWP) conjugates were fabricated using free radical grafting and then the conjugates were utilized as a novel antioxidant emulsifier to prepare β-carotene-enriched emulsions. Compared to native proteins, CT-EWP conjugates formed emulsions with improved stability to environmental stresses (such as pH, salt, and heating), and that were better at inhibiting the degradation of encapsulated β-carotene during storage. However, to the best Received: Revised: Accepted: Published: 1649

December 16, 2017 January 31, 2018 January 31, 2018 January 31, 2018 DOI: 10.1021/acs.jafc.7b05909 J. Agric. Food Chem. 2018, 66, 1649−1657

Article

Journal of Agricultural and Food Chemistry

Microfluidics, Newton, MA) at 12000 psi for three cycles to form βcarotene-loaded oil-in-water emulsions. After preparation, the emulsions were immediately placed in a refrigerator before further use. Emulsions prepared using egg white protein (EWP) (rather than CT-EWP conjugates) as an emulsifier were used as controls. Cellular Antioxidant Activity of β-Carotene Emulsions. The cellular antioxidant of the samples was evaluated in Caco-2 cells using the DCFH-DA assay described previously.19 Caco-2 cells were cultured in DMEM containing 10% FBS, 1% NAA, and 1% penicillin and streptomycin at 37 °C in an incubator with 5% CO2 and 95% humidified air. When the cells reached 80% confluence, they were seeded and grown in 96-well plates (1 × 105 cells/mL). After 24 h, the cells were treated with different concentrations of β-carotene emulsions stabilized by either CT-EWP conjugates or EWP, or they were treated with aqueous solutions containing either CT-EWP conjugates or EWP. The samples were obtained by diluting initial emulsions and solutions 20, 40, 60, 80, or 100 times with DMEM and then treating the cells for 24 h. After that, the plates were treated with 100 μL of DCFH-DA (10 μM) per well and incubated at 37 °C for 30 min. The plates were then washed with PBS (1×) twice before adding 100 μL of t-BHP (500 μM) to each well (except the negative control) and then incubating for 30 min at room temperature. Finally, the fluorescence intensity of the samples was recorded using a Microplate Reader (Synergy 2 Multi-Mode, BioTek Instruments, Inc., Winooski, VT) at an emission wavelength of 528 nm and an excitation wavelength of 485 nm. The cellular antioxidant activity of the samples was then calculated using the following equation:

our knowledge, no information is currently available about the impact of these conjugates on the bioavailability of the encapsulated bioactive agents. Consequently, a major objective of the current study was to determine the impact of stabilizing emulsions with CT-EWP conjugates on the potential oral bioavailability of encapsulated β-carotene. As mentioned earlier, the overall bioavailability depends on the bioaccessibility, transformation, and absorption of the bioactive within the gastrointestinal tract (GIT). The bioaccessibility and transformation of the carotenoids was determined using a three-stage simulated gastrointestinal fate (GIT) model consisting of mouth, stomach, and small intestine phases.16 The absorption of the carotenoids was determined using a cell culture model (Caco-2 cells) that mimics the human intestinal layer.17,18 Our previous work found that CTEWP conjugates had strong antioxidant activity, but the in vitro chemical assays used in that study have low relevance for predicting the potential antioxidant effects of nutraceuticals within the human body. For this reason, the cellular antioxidant activity of the conjugates and β-carotene emulsions was also analyzed in the current study. Moreover, the potential toxicity of the CT-EWP-conjugate-stabilized emulsions was tested to evaluate their potential for applications in the food industry. The objective of this study was to evaluate the bioavailability, cellular antioxidant activity, and cytotoxicity of β-carotene emulsions stabilized by CT-EWP conjugates so as to establish the potential of this new antioxidant emulsifier for application in functional foods and beverages.

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cellular antioxdant activity (%) =

A sample − A blank Acontrol − A blank

× 100

Here, Asample is the fluorescence intensity of the cells treated with different samples, Ablank is the fluorescence intensity of the blank wells, and Acontrol is the fluorescence intensity of the cells treated with DMEM only. In Vitro Cytotoxicity of β-Carotene Emulsions. The MTT assay was utilized to determine the impact of the samples on cell viability and therefore evaluate their potential cytotoxicity.20 In brief, Caco-2 cells were incubated in 96-well plates at a density of 1 × 105 cells/mL at 37 °C in 5% CO2 incubator. After incubation for 24 h, the cells were treated with β-carotene emulsions stabilized by either CT-EWP conjugates or EWP, or with aqueous solutions containing either CTEWP conjugates or EWP. A range of concentrations was utilized for each sample tested by diluting with DMEM (20, 40, 60, 80, and 100 times) prior to analysis so as to ascertain the influence of emulsifier concentration on cytotoxicity. After a further 24 h, the cells were treated with 100 μL off MTT solution (0.5 mg/mL) and incubated for 1 h at 37 °C. Then, 100 μL of DMSO was used to dissolve the products and the absorbance was measured at a wavelength of 570 nm using a Microplate Reader (Synergy 2 Multi-Mode, BioTek Instruments, Inc., Winooski, VT). The cell viability was then calculated using the following equation:

MATERIALS AND METHODS

Materials. Fresh eggs and sunflower oil were purchased from a local supermarket. β-Carotene, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), tert-butyl hydroperoxide (t-BHP, 70%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Nile red, fluorescein isothiocyanate (FITC), mucin from porcine stomach, pepsin from porcine gastric mucosa, lipase from porcine pancreas, and porcine bile extract were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human colon carcinoma cell lines (Caco-2 cells, ATCC, Manassas, VA) were used for cellular cytotoxicity, antioxidant activity, and uptake assays after 30−45 passages. Dulbecco’s modified Eagle’s medium (DMEM) (with 4.5 g/L glucose, L-glutamine, and sodium pyruvate), fetal bovine serum (FBS), penicillin−streptomycin (10,000 U/mL), nonessential amino acid solution (NAA, 100×), HEPES (1 M), trypsin−EDTA (0.25%), phosphate buffered saline (10×), methyl tert-butyl ether (MTBE), n-hexane, chloroform, and dimethyl sulfoxide (DMSO) were obtained from Fisher Scientific (Agawam, MA, USA). All other chemicals and reagents used were analytical grade. Double distilled water was used to prepare all aqueous solutions. Preparation of β-Carotene Emulsions. Catechin−egg white protein (CT-EWP) conjugates were synthesized using a free radical grafting method, and the amount of catechin bound to the egg white protein was found to be 5.71 mg/g conjugates. Their successful fabrication was confirmed using electrophoresis and liquid chromatography−mass spectrometry, as described in our previous study.15 The CT-EWP conjugates were used as an antioxidant emulsifier to encapsulate β-carotene within oil-in-water emulsions. In brief, an aqueous phase was prepared by dissolving CT-EWP conjugates (2%, w/w) in phosphate buffer solution (5 mM, pH 3.0) and then stirring overnight at 25 °C to ensure dissolution. An oil phase was prepared by dispersing β-carotene (0.1%) in sunflower oil and then sonicating for 20 min at 50 °C in the absence of light to ensure complete dispersion and dissolution of the carotenoid crystals. Coarse emulsions were then fabricated by mixing the aqueous phase (90% w/w) and oil phase (10% w/w), using a high-shear mixer for 2 min (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland). After that, the coarse emulsions were passed through a high-pressure homogenizer (M110P,

cell viability (%) =

A sample − A blank Acontrol − A blank

× 100

Here, Asample is the absorbance of the cells treated with samples, Ablank is the absorbance of blank wells, and Acontrol is the absorbance of cells treated with DMEM only. In Vitro Digestion of β-Carotene Emulsions. A three-step simulated gastrointestinal tract (GIT) model that consisted of mouth, stomach, and small intestine phases was utilized to evaluate the potential gastrointestinal fate of the different samples and controls. The simulated saliva fluid (SSF) (sodium chloride 1.594 g/L, ammonium nitrate 0.328 g/L, potassium phosphate 0.636 g/L, potassium chloride 0.202 g/L, potassium citrate 0.308 g/L, uric acid sodium salt 0.021 g/L, urea 0.198 g/L, lactic acid sodium salt 0.146 g/ L), simulated gastric fluid (SGF) (sodium chloride 2 g/L, hydrochloric acid 83.3 mM), and simulated intestine fluid (SIF) (calcium chloride 1650

DOI: 10.1021/acs.jafc.7b05909 J. Agric. Food Chem. 2018, 66, 1649−1657

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Journal of Agricultural and Food Chemistry dihydrate 36.67 g/L, sodium chloride 219.13 g/L) were prepared as described in our previous study.21 Mouth Phase. Initially, 4 g of β-carotene emulsion was mixed with 16 g of phosphate buffer (5 mM, pH 3.0) to reach the desired oil concentration (2%, w/w). Then, 20 g of the diluted emulsions and 20 g of SSF containing mucin (30 mg/mL) were preheated to 37 °C and then mixed together. The mixture was adjusted to pH 6.8 and then swirled for 2 min at a speed of 100 rpm in an incubator shaker set at 37 °C (Innova Incubator Shaker, Model 4080, New Brunswick Scientific, Edison, NJ). Stomach Phase. 20 g of the sample resulting from the mouth phase was mixed with 20 g of SGF (37 °C) containing pepsin (3.2 mg/mL) and then adjusted to pH 2.5. After that, the mixture was placed in the incubator shaker and swirled for 2 h at 100 rpm and 37 °C. Small Intestine Phase. 30 g of the sample resulting from the stomach phase was placed in a water bath (37 °C) and adjusted to pH 7.0. Subsequently, 1.5 mL of SIF and 3.5 mL of bile extract (53.57 mg/ mL) were added, and the mixture was then adjusted back to pH 7.0. Then, 2.5 mL of lipase suspension (24 mg/mL) was added and the mixture was maintained at pH 7.0 for 2 h by adding NaOH solution (0.25 M) using a pH-stat automatic titration device (Metrohm USA Inc., Riverview, FL, USA). The volume of NaOH solution required to neutralize the FFA was recorded during the incubation. The amount of free fatty acids released was then estimated using the following equation: FFA (%) =

VNaOHmNaOHMlipid 2Wlipid

BA (%) = B*A*T * For this reason, we carried out experiments to determine the relative contribution of these three factors to the overall bioavailability of βcarotene in the emulsion-based delivery systems prepared in this study. Bioaccessibility (B*). After emulsions had passed through the threestep simulated GIT model, 20 g of small intestine sample was centrifuged (18,000 rpm, Thermo Scientific, Waltham, MA) at 4 °C for 30 min. The yellow supernatant that appeared after centrifugation of the samples was assumed to be the “micelle” fraction in which the βcarotene was solubilized, and the carotenoid concentration in this fraction was determined according to a method described previously.23 In brief, 2 mL of micelle phase was mixed with 2 mL of chloroform and 2 mL of ethanol, and then the mixture was shaken vigorously for 20 s and centrifuged for 5 min at 3500 rpm. The supernatant layer was then transferred to a glass test tube and diluted with chloroform to an appropriate concentration. Then, the absorbance of the diluted supernatant was recorded at 450 nm using a UV−visible spectrophotometer (Cary 100 UV−vis, Agilent Technologies, Santa Clara, CA, USA), with chloroform as a control. The β-carotene concentration in the total small intestine sample (without centrifugation) was also measured using the same approach. The carotenoid concentrations were determined from the absorbance measurements using a standard curve prepared using samples of known β-carotene level. The bioaccessibility of β-carotene was calculated using the following equation: m bioaccessibility (%) = micelle × 100 mdigesta

× 100

Here, mmicelle is the total weight of β-carotene solubilized within the mixed micelle fraction of the small intestinal fluids, and mdigesta is the total weight of β-carotene in the overall small intestine fluids. Absorption (A*). Caco-2 cells were grown in standard tissue culture dishes (Falcon) at a density of 6 × 104 cells/mL and incubated at 37 °C under atmosphere of 5% CO2−95% humidified air. The culture medium was changed every 2 days until the cell monolayers could be observed by an electron microscope. Prior to β-carotene analysis, the digesta from the small intestine was diluted 100 times with culture medium to reduce the cytotoxicity of the bile salts on the Caco-2 cells. Then, the cells were treated with diluted digesta and incubated at 37 °C for 24 h before washing by precooled PBS (1×) twice. Trypsin solution (3 mL) was used to dissociate the cell monolayers from the bottom of the plates, and then culture medium (7 mL) was added to inactivate the trypsin. The cell suspension was then centrifuged for 2 min at 2000 rpm, and the sedimented cells were collected and transferred to a new 2 mL centrifuge tube before addition of 500 μL of precooled cell lysis buffer and incubation for 30 min on ice. Then, a sonicator (model 50 Sonic Dismembrator, Fisher Scientific, Agawam, MA, USA) was used to break down the cells and allow the β-carotene to be released from the Caco-2 cells. After that, 400 μL of cell suspension was collected for the extraction of the β-carotene. 200 μL of hexane and 200 μL of alcohol were added, and the mixture was shaken vigorously and centrifuged for 5 min at 3500 rpm. The hexane layer containing β-carotene was filtered through a 0.45 μm filter (VWR International, Philadephia, PA, USA), and then the concentration of βcarotene was determined using liquid chromatography (Agilent 1100 series, Agilent Technologies, Santa Clara, CA, USA).24 A C-30 reversed phase column (250 mm × 4.6 mm id, 5 μm, YMC Carotenoid, YMC Inc., Wilmington, NC) was used as the stationary phase, and a two-solvent system at 25 °C was used as the mobile phase: A, methanol/MTBE/1 M ammonium acetate (95:3:2, v/v/v), and B, methanol/MTBE/1 M ammonium acetate (25:73:2, v/v/v). The gradient program was carried out at 1 mL/min with gradient 85%−70% A for 10 min, 70%−52% A for 2 min, 52% A for 6 min, 52%−35% A 8 min, then back to 85% A over 4 min. The β-carotene content was calculated from a standard curve and the absorption of βcarotene was then calculated using the following equation: m uptake absorption (%) = × 100 mdigesta

Here, VNaOH is the volume of NaOH solution required to neutralize the FFA, mNaOH is the molarity of the NaOH solution (0.25 M), Mlipid is the average molecular weight of the sunflower oil (876 g/mol), and Wlipid is the weight of the sunflower oil in the digestion system (0.15 g). Determination of Particle Characterization. The particle size distribution and mean particle diameter of the samples exposed to different regions of the simulated GIT were determined using a laser light scattering instrument (Mastersizer 2000, Malvern Instruments Ltd., Malvern, Worcestershire, U.K.). Prior to particle size measurements, the initial emulsions were diluted with phosphate buffer (5 mM, pH 3) to minimize multiple scattering effects. The mouth and small intestine samples were diluted with neutral phosphate buffer (5 mM, pH 7), while the stomach samples were diluted with acidic phosphate buffer (5 mM, pH 2.5). The ζ-potential values of the particles in the samples were determined using a particle electrophoresis device (Zetasizer Nano, Malvern Instruments, Worcestershire, U.K.). In this case, the initial emulsions were diluted with acidic phosphate buffer (5 mM, pH 3), the mouth samples were diluted with SSF (pH 6.8), the stomach samples were diluted with SGF (pH 2.5), and the small intestine samples were diluted with neutral phosphate buffer (5 mM, pH 7). The refractive indexes of the aqueous phase and oil phase used in the calculations were 1.33 and 1.45, respectively. For microstructure analysis, the samples exposed to different regions of the GIT were collected and then diluted with phosphate buffer as described for the particle size measurements. Then, 1 mL of diluted samples was transferred to a glass test tube, and then, 20 μL of Nile red (1 mg/mL) and 20 μL of FITC (1 mg/mL) were added to stain the lipids and proteins, respectively. After that, a drop of sample was placed on a microscope slide and covered with a thin glass coverslip. The microstructural images were captured using a Nikon confocal microscope (Nikon D-Eclipse C1 80i, Nikon, Melville, NY, USA) at a magnification of ×600 (×60 objective lens and ×10 eyepiece lens) and then analyzed using image analysis software (NISElements, Nikon, Melville, NY). Determination of β-Carotene Oral Bioavailability. The overall oral bioavailability (BA) of bioactives is determined by three main factors, namely, bioaccessibility (B*), absorption (A*), and stability to transformation (T*), as described previously by our research group:7,22 1651

DOI: 10.1021/acs.jafc.7b05909 J. Agric. Food Chem. 2018, 66, 1649−1657

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Journal of Agricultural and Food Chemistry Here, muptake is the total weight of the β-carotene taken up by the Caco-2 cells, and mdigesta is the total weight of β-carotene in the small intestine fluids. Transformation (T*). The concentration of β-carotene in the emulsions before and after passing through the three-step simulated GIT was determined using the same method described in Bioaccessibility (B*). The stability to transformation of the β-carotene was then calculated using the following equation:

transformation (%) =

mdigesta m initial

× 100

Here, mdigesta is the total weight of β-carotene in the small intestine fluids, and minitial is the total weight of β-carotene in the initial emulsions before digestion. The dilution of the samples in the different stages of the simulated GIT model was taken into account in these calculations. Statistical Analysis. In this study, a statistical analysis software (SPSS 19.0, IBM Corporation, Armonk, NY, USA) was used for data analysis. One-way variance analysis was carried out to calculate the mean and standard deviation, and Duncan’s multiple range test (p < 0.05) was used to determine significant differences among mean values.

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Figure 1. Cellular antioxidant activity of samples at different dilution times on Caco-2 cells. Values reported are mean ± standard deviation (SD), n = 6. A−D mean significant difference (p < 0.05) among one sample with different dilution times; a−d mean significant difference (p < 0.05) among different samples with the same dilution times.

RESULTS AND DISCUSSION

Cellular Antioxidant Activity of β-Carotene Emulsions. Our previous study using various in vitro chemical antioxidant assays showed that CT-EWP conjugates had a stronger antioxidant activity than EWP alone, which was attributed to the covalent attachment of catechin, a polyphonic compound with high antioxidant activity, onto EWP.15 In vitro chemical assays have advantages for the initial screening of different formulations because they are relatively simple, rapid, and low cost to perform, however they do not accurately reflect the potential antioxidant activity of formulations in vivo. For this reason, the antioxidant activity of CT-EWP and EWP in βcarotene emulsions was further analyzed using a cell culture model, which more closely simulates in vivo conditions.25,26 In future studies, it will be important to determine the antioxidant activity of the samples after oral administration to animals or humans. However, these types of studies are more expensive, time-consuming, and ethically challenging than simple cell culture models, and should therefore only be carried out once the safety and potential efficacy of a new ingredient or delivery system have been established. In the present study, DCFH-DA was used as a fluorescent probe to evaluate the cellular antioxidant activity of samples applied at a range of concentrations (0.01−0.05 mg/mL) to the cells. As shown in Figure 1, the inhibition rate was 1.7−20.7% for the EWP solutions (control) and 24−93% for the CT-EWP conjugate solutions, which indicated that the conjugates exhibited a greater ability to inhibit DCFH oxidation induced by peroxyl radicals. The stronger cellular antioxidant activity of the CT-EWP conjugates was in good agreement with the previous results of our in vitro chemical antioxidant assays, and can be attributed to the presence of the antioxidant catechin moieties.15,27 Compared with the aqueous solutions containing CT-EWP conjugates, the β-carotene emulsions stabilized by the same conjugates exhibited a weaker antioxidant activity (17.9− 75.8%). This was surprising, since previous studies have reported that β-carotene has a relatively high antioxidant activity.28−30 One might therefore have expected that βcarotene-loaded lipid droplets coated with CT-EWP conjugates would have a higher antioxidant activity than CT-EWP

conjugates alone. Similarly, the antioxidant activity of the EWP-stabilized emulsions was slightly weaker than that of EWP solution. There are a number of possible reasons for the reduced antioxidant activity of the emulsifiers in the emulsions compared to the aqueous solutions. First, the molecular structure and environment of the emulsifiers are different in the emulsions and aqueous solutions, which may have altered their chemical reactivity. In the emulsions, the emulsifiers have an oil phase on one side and an aqueous phase on the other side, whereas in the solutions they are completely surrounded by aqueous phase. Second, the relatively small emulsifiers in the aqueous solutions may have been able to penetrate through the cell membranes more easily than the relatively large particles in the digested emulsions. Third, homogenization of the oil and water phases during emulsion formation may have generated heat and free radicals that reduced the antioxidant activity of the emulsifiers. Other researchers have reported that gallic acid-chitosan conjugates formed by a free radical induced grafting reaction significantly inhibited the intracellular generation of reactive oxygen species (ROS) in mouse RAW264.7 macrophages.31 In Vitro Cytotoxicity of β-Carotene Emulsions. The chemical grafting of catechin onto proteins can enhance their antioxidant activities, however, it may lead to the formation of undesirable side products during the chemical reaction, which could promote cytotoxicity. Thus, prior to utilization of the conjugates as emulsifiers in the food industry, the cytotoxicity of the conjugates and the β-carotene-loaded emulsions stabilized by the conjugates was determined using the MTT assay, which is based on evaluation of a substance’s impact on cell viability. Caco-2 cells were treated for 24 h with β-carotene emulsions stabilized by either CT-EWP conjugates or EWP of different concentrations. As shown in Figure 2, the cell viability after treatment with EWP-stabilized emulsions was greater than 93%, indicating that β-carotene emulsions stabilized by EWP were nontoxic. However, the CT-EWP-conjugate-stabilized emulsions had cell viability values ranging from 79 to 92%, indicating that they caused some cytotoxicity when used at 1652

DOI: 10.1021/acs.jafc.7b05909 J. Agric. Food Chem. 2018, 66, 1649−1657

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Journal of Agricultural and Food Chemistry

systems was determined. To this end, changes in particle size, charge, and location were measured when β-carotene-loaded emulsions stabilized by CT-EWP conjugates were passed through a simulated GIT that included mouth, stomach, and small intestine phases. The mean particle diameter (D4,3) and particle size distribution of the emulsions were determined using static light scattering (Table 1 and Figure 3), while the surface potential (ζ-potential) was determined using electrophoresis (Figure 4).

Figure 2. In vitro cytotoxicity of samples at different dilution times on Caco-2 cells measured by MTT assay. Cell viability was expressed as mean ± standard deviation (SD), n = 6. Data with different letters (A− D) are significantly different (p < 0.05) among one sample with different dilution times; data with different letters (a−d) are significantly different (p < 0.05) among different samples with the same dilution times.

relatively high concentrations. Previous studies have reported that β-carotene-loaded protein nanoparticles are nontoxic, such as those fabricated from sodium caseinate, whey protein isolate, soy protein isolate, and β-lactoglobulin.32,33 Moreover, it has also been reported that β-carotene-loaded nanoparticles fabricated from β-lactoglobulin−dextran conjugates formed by a dry-heat method were also nontoxic. These results suggest that the slight cytotoxicity observed for the CT-EWPconjugate-stabilized emulsions observed in our study may be due to the fact that the conjugates were synthesized using a free radical grafting approach, which involved the use of a hydrogen peroxide−ascorbic acid pair as a radical initiator system. Although the fabrication conditions were relatively simple and mild, some hypotoxic reaction products may have still been formed. In addition, the emulsions containing the conjugates were found to be more biocompatible (high cell viability) than the conjugate solutions. Overall, these results indicate that CT-EWP-conjugatestabilized emulsions have good antioxidant activity, and that they have relatively low toxicity. However, CT-EWP conjugates in aqueous solutions may exhibit some cytotoxicity when utilized at sufficiently high levels. Particle Characteristics during in Vitro Digestion. In this section, the impact of the antioxidant emulsifiers on the potential gastrointestinal fate of the emulsion-based delivery

Figure 3. Particle size distribution of different delivery systems after exposure to successive GIT stages: (a) β-carotene emulsions stabilized by egg white protein (EWP); (b) β-carotene emulsions stabilized by catechin−egg white protein (CT-EWP) conjugates.

Initial Samples. The initial emulsions stabilized by CT-EWP conjugates had monomodal particle size distributions with mean particle diameters around 0.30 μm, which was fairly similar to that for the emulsions produced using EWP alone (0.29 μm). The slightly larger particle size for the emulsions produced using CT-EWP conjugates may have been because they had a higher molecular weight than EWP, and therefore adsorbed to the droplet surfaces more slowly during homogenization. The confocal microscopy images showed

Table 1. Mean Particle Diameter of β-Carotene Emulsions Stabilized by Egg White Proteins (EWP) and Catechin−Egg White Protein (CT-EWP) Conjugates after Exposure to Different GIT Regionsa mean particle diameter (μm)

a

samples

initial

mouth

stomach

intestine

EWP CT-EWP conjugates

0.288 ± 0.004 a 0.304 ± 0.006 b

9.70 ± 0.99 a 8.88 ± 0.58 a

24.89 ± 0.45 a 22.76 ± 0.18 b

22.20 ± 1.91 a 16.77 ± 1.03 b

Data with different letters (a, b) in a column are significantly different (p < 0.05). 1653

DOI: 10.1021/acs.jafc.7b05909 J. Agric. Food Chem. 2018, 66, 1649−1657

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Journal of Agricultural and Food Chemistry

because of a reduction in the electrostatic repulsion between the lipid droplets arising from a change in pH and ionic strength of the aqueous solution surrounding them.34 Alternatively, it may have occurred due to bridging or depletion flocculation induced by the presence of mucin molecules within the oral phase.34 The surface potential of the particles in the emulsions also changed appreciably after exposure to the mouth stage. Indeed, the ζ-potential changed from highly positive (+51.9 mV) to moderately negative (−15.8 mV), which can mainly be attributed to the change in the pH from the initial samples (pH 3) to the simulated oral conditions (pH 6.8). As a result, the pH of the aqueous solution surrounding the particles in the emulsions went from below to above the isoelectric point of the adsorbed egg white proteins, thereby leading to charge reversal. In addition, the surface potential of the lipid droplets may have also changed due to the presence of mineral ions and mucin in the simulated saliva. Stomach Phase. After incubation in the stomach phase, the particle size distribution of the emulsions stabilized by CTEWP conjugates remained monomodal (Figure 3), but the mean particle diameter increased considerably to around 23 μm (Table 1). The confocal microscopy images suggested that the increase in particle size observed in the stomach phase was mainly due to droplet flocculation, i.e., clustering of the individual droplets (Figure 5). Previous studies have suggested that the instability of protein-coated lipid droplets under simulated gastric conditions can at least partly be attributed to the hydrolysis of the adsorbed proteins by pepsin.35 In addition, the high ionic strength of the gastric fluids may also promote flocculation of the lipid droplets by reducing the electrostatic repulsion between them. Moreover, the presence of anionic mucin molecules arising from the mouth phase may also lead to some bridging flocculation. This hypothesis is supported by the fact that the measured ζ-potential of the droplets in the stomach phase was close to zero (−2.4 mV), whereas it would be expected to be highly positively charged for protein-coated droplets under highly acidic conditions. This phenomenon is most likely due to the adsorption of anionic mucin molecules onto the surfaces of cationic protein-coated lipid droplets.36 Small Intestine Phase. After passage through the small intestine phase, a further increase in mean particle diameter was detected and the particle size distribution became multimodal, indicating the presence of a variety of particles with different dimensions. The confocal microscopy images showed that most of the original fat droplets had been digested, but that there were some large lipid-rich particles present, which may have been undigested lipid droplets or mixed micelles. The surface potential of the particles in the samples became highly negative after exposure to the small intestine phase, which can be attributed to the fact that the colloidal particles present are mainly constructed from anionic species under these pH conditions, such as bile salts, phospholipids, peptides, and free fatty acids.37 Overall, the emulsions stabilized by EWP and CT-EWP conjugates exhibited quite similar behavior when they were passed through the simulated GIT model, which could be explained by the fact that the EWP dominated the overall emulsifying properties of both systems. Consequently, the utilization of protein−polyphenol conjugates to stabilize the emulsions did not appear to have a major impact on their gastrointestinal fate when compared to using proteins alone. Lipid Digestion. The rate and extent of lipid digestion of βcarotene-loaded emulsions stabilized by either CT-EWP

Figure 4. Electrical characteristics (ζ-potential) of β-carotene emulsions stabilized by egg white protein (EWP) and catechin−egg white protein (CT-EWP) conjugates after exposure to successive GIT stages. Data with different letters (a, b) are significantly different (p < 0.05).

that the lipid droplets in the emulsions were uniformly dispersed throughout the system, without any appreciable droplet aggregation being observed (Figure 5). This phenom-

Figure 5. Laser confocal microscopy of β-carotene emulsions stabilized by egg white protein (EWP) and catechin−egg white protein (CT-EWP) conjugates after exposure to different regions of the simulated GIT (scale bar is 1 μm). Green: proteins stained with FITC. Red: sunflower oil stained with Nile red.

enon may be attributed to the relatively high positive surface potential on the emulsifier-coated lipid droplets (+56.4 mV for EWP and +51.9 mV for CT-EWP conjugates), as shown in Figure 4. As a result, there would be a strong electrostatic repulsion between the droplets that prevented them from coming into close contact. The high magnitude of the ζpotential on the droplets occurs because the pH of the aqueous phase (∼3.0) was well below the isoelectric point of the egg white proteins (∼5.0). Mouth Stage. After exposure to the mouth stage, the particle size distribution of the CT-EWP-conjugate-stabilized emulsions remained monomodal, but the mean particle diameter increased appreciably (8.88 μm), which indicated that extensive droplet aggregation occurred under simulated oral conditions. Confocal laser scanning microscopy also indicated that extensive droplet aggregation occurred under simulated mouth conditions. This instability may have occurred 1654

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Journal of Agricultural and Food Chemistry conjugates or EWP alone was determined using a pH-stat method (Figure 6). The two types of emulsions exhibited fairly

Figure 7. Bioaccessibility (B*), absorption (A*), and transformation (T*) of β-carotene in emulsions stabilized by egg white protein (EWP) and catechin−egg white protein (CT-EWP) conjugates. Values reported are mean ± standard deviation (SD), n = 3. Data with different letter (a, b) are significantly different (p < 0.05).

Figure 6. Amount of free fatty acids released from β-carotene emulsions stabilized by egg white protein (EWP) and catechin−egg white protein (CT-EWP) conjugate measured using a pH-stat in vitro digestion model. Values reported are mean ± standard deviation (SD), n = 3. Data with different letters (a, b) are significantly different (p < 0.05).

micelles. For example, the conjugates may have bound to the mixed micelles through hydrophobic interactions, and reduced their water solubility. Nevertheless, further research is required to identify the molecular and physicochemical origin of this effect, which we intend to perform in future studies. Absorption. After being solubilized within mixed micelles, carotenoids must be transported through the lumen and mucus layer, and then taken up by the epithelium cells.40 In this section, a Caco-2 cell culture model was used to simulate the uptake of the solubilized β-carotene by epithelium cells. Initially, the Caco-2 cells were investigated by electron microscopy and no significant difference was observed in the appearance before and after being treated with the samples (data not shown), which was an indication of that the samples tested did not appreciably impact cell growth. The level of βcarotene absorption from the emulsions stabilized by CT-EWP conjugates (56.3%) was significantly lower than in the emulsions stabilized by EWP alone (71.2%) (Figure 7), which suggested that the conjugates interfered with carotenoid absorption. This may have occurred because the conjugates interfered with the solubilization capacity, transport, or uptake of the mixed micelles. Again, further research is required to identify the molecular origin of this phenomenon. For instance, studies could be carried out to compare the interactions of the EWP and CT-EWP with the mixed micelles using turbidity, isothermal titration calorimetry, or electrophoresis techniques. Transformation. The impact of the conjugates on the stability of the β-carotene to chemical degradation under simulated GIT conditions was also measured. The fraction of βcarotene remaining in its original form at the end of the small intestine phase was much higher in the emulsions stabilized by the CT-EWP conjugates (72.9%) than in the emulsions stabilized by EWP alone (49.9%). These results suggest that encapsulation of β-carotene in lipid droplets coated by CTEWP conjugates gave better protection against chemical degradation, which can be attributed to the increase in the antioxidant activity of the egg proteins after covalent attachment of a catechin moiety. Overall Bioavailability. Finally, the overall bioavailability of the β-carotene in the two different emulsions systems was calculated from the measured bioaccessibility, absorption, and transformation values (Figure 8). Overall, encapsulation of βcarotene within emulsions stabilized by CT-EWP conjugates

similar trends in their free fatty acid (FFA) release profiles. During the first 20 min, there was a progressive increase in the amount of FFAs released from the emulsions, followed by a more gradual increase at longer times, until a relatively constant final value was attained. The relatively rapid initial rate of lipid digestion observed in this study can be attributed to the relatively small size and high surface area of the lipid droplets, which enabled the lipase molecules to access the oil−water interfaces more readily.38 Although these two emulsions had fairly similar initial rates of lipid digestion, the final extent of lipid digestion was somewhat different. The final amount of FFAs released was about 79% for the EWP stabilized system, but around 86% for the CT-EWP-conjugate-stabilized system, which suggests that the conjugates facilitated more complete lipid digestion. The origin of this effect is currently unknown, but it may be due to alterations in the nature or amount of lipid droplet surfaces exposed to the lipase molecules caused by the presence of polyphenols attached to the protein. Bioavailability of β-Carotene. As discussed earlier, the overall oral bioavailability of β-carotene may be limited by its bioaccessibility (B*), absorption (A*), or transformation (T*) characteristics.7 In this section, experiments were therefore carried out to determine the main factors limiting the bioavailability of β-carotene in emulsions stabilized either by CT-EWP conjugates or by EWP alone. Bioaccessibility. Initially, the bioaccessibility of β-carotene in the emulsion-based delivery systems was determined (Figure 7). A slightly lower bioaccessibility of β-carotene was observed in the CT-EWP-stabilized emulsions (89.9%) than in the EWPstabilized emulsions (95.4%). One might have expected that the CT-EWP-stabilized emulsions would have exhibited greater βcarotene bioaccessibility than the EWP-stabilized emulsions because the lipid phase was digested more completely. Indeed, previous studies have reported that the level of carotenoids released from lipid droplets and solubilized within mixed micelles increases as the extent of lipid droplet digestion increases.23,39 Hence, the lower bioaccessibility of β-carotene in the CT-EWP-conjugate-stabilized emulsions must be attributed to other factors. It is possible that the conjugates interfered with the transfer of carotenoids from the lipid droplets to the mixed micelles, or with the solubilization capacity of the mixed 1655

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(2) Tan, C.; Feng, B.; Zhang, X.; Xia, W.; Xia, S. Biopolymer-coated liposomes by electrostatic adsorption of chitosan (chitosomes) as novel delivery systems for carotenoids. Food Hydrocolloids 2016, 52, 774−784. (3) Sies, H.; Stahl, W. Vitamins E and C, beta-carotene, and other carotenoids as antioxidants. Am. J. Clin. Nutr. 1995, 62, 1315S−1321S. (4) Coronel-Aguilera, C. P.; San Martín-Gonzál ez, M. F. Encapsulation of spray dried β-carotene emulsion by fluidized bed coating technology. LWT - Food Sci. Technol. 2015, 62, 187−193. (5) McClements, D. J.; Xiao, H. Potential biological fate of ingested nanoemulsions: influence of particle characteristics. Food Funct. 2012, 3, 202−220. (6) Chen, J.; Li, F.; Li, Z.; McClements, D. J.; Xiao, H. Encapsulation of carotenoids in emulsion-based delivery systems: Enhancement of βcarotene water-dispersibility and chemical stability. Food Hydrocolloids 2017, 69, 49−55. (7) McClements, D. J. Nanoscale nutrient delivery systems for food applications: improving bioactive dispersibility, stability, and bioavailability. J. Food Sci. 2015, 80, N1602−N1611. (8) Liu, F.; Ma, C.; Gao, Y.; McClements, D. J. Food-grade covalent complexes and their application as nutraceutical delivery systems: A review. Compr. Rev. Food Sci. Food Saf. 2017, 16, 76−95. (9) Ye, A.; Singh, H. Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: influence of polysaccharides. Food Hydrocolloids 2006, 20, 269−276. (10) Ye, A.; Zhu, X.; Singh, H. Oil-in-Water emulsion system stabilized by protein-coated nanoemulsion droplets. Langmuir 2013, 29, 14403−14410. (11) Gu, L.; Peng, N.; Chang, C.; McClements, D. J.; Su, Y.; Yang, Y. Fabrication of surface-active antioxidant food biopolymers: conjugation of catechin polymers to egg white proteins. Food Biophysics 2017, 12, 198−210. (12) Tan, C.; Selig, M. J.; Abbaspourrad, A. Anthocyanin stabilization by chitosan-chondroitin sulfate polyelectrolyte complexation integrating catechin co-pigmentation. Carbohydr. Polym. 2018, 181, 124−131. (13) Liu, F.; Sun, C.; Yang, W.; Yuan, F.; Gao, Y. Structural characterization and functional evaluation of lactoferrin-polyphenol conjugates formed by free-radical graft copolymerization. RSC Adv. 2015, 5, 15641−15651. (14) You, J.; Luo, Y.; Wu, J. Conjugation of ovotransferrin with catechin shows improved antioxidant activity. J. Agric. Food Chem. 2014, 62, 2581−2587. (15) Gu, L.; Su, Y.; Zhang, M.; Chang, C.; Li, J.; McClements, D. J.; Yang, Y. Protection of β-carotene from chemical degradation in emulsion-based delivery systems using antioxidant interfacial complexes: Catechin-egg white protein conjugates. Food Res. Int. 2017, 96, 84−93. (16) Zhang, Z.; Chen, F.; Zhang, R.; Deng, Z.; McClements, D. J. Encapsulation of pancreatic lipase in hydrogel beads with selfregulating internal pH microenvironments: retention of lipase activity after exposure to gastric conditions. J. Agric. Food Chem. 2016, 64, 9616−9623. (17) Katsikari, A.; Patronidou, C.; Kiparissides, C.; Arsenakis, M. Uptake and cytotoxicity of poly(d,l-lactide-co-glycolide) nanoparticles in human colon adenocarcinoma cells. Mater. Sci. Eng., B 2009, 165, 160−164. (18) Raveendran, R.; Bhuvaneshwar, G.; Sharma, C. P. In vitro cytotoxicity and cellular uptake of curcumin-loaded Pluronic/ Polycaprolactone micelles in colorectal adenocarcinoma cells. J. Biomater. Appl. 2013, 27, 811−827. (19) Bakuradze, T.; Lang, R.; Hofmann, T.; Stiebitz, H.; Bytof, G.; Lantz, I.; Baum, M.; Eisenbrand, G.; Janzowski, C. Antioxidant effectiveness of coffee extracts and selected constituents in cell-free systems and human colon cell lines. Mol. Nutr. Food Res. 2010, 54, 1734−1743. (20) Hu, B.; Ting, Y.; Zeng, X.; Huang, Q. Cellular uptake and cytotoxicity of chitosan−caseinophosphopeptides nanocomplexes loaded with epigallocatechin gallate. Carbohydr. Polym. 2012, 89, 362−370.

Figure 8. Oral bioavailability (BA) of β-carotene in emulsions stabilized by egg white protein (EWP) and catechin−egg white protein (CT-EWP) conjugate.

led to a slightly higher bioavailability (36.9%) than encapsulation within emulsions stabilized by EWP alone (33.9%). This effect was mainly attributed to the stronger antioxidant activity of the conjugates, leading to a higher fraction of β-carotene remaining in its original form after exposure to the simulated GIT model. In summary, β-carotene-loaded emulsions stabilized by CTEWP conjugates were fabricated and their in vitro cellular antioxidant activity, cytotoxicity, and oral bioavailability were examined. The emulsions stabilized by the conjugates displayed stronger cellular antioxidant activity than those stabilized by proteins alone, however, they also exhibited a greater cytotoxicity when used at high levels. Oral bioavailability analysis indicated that the encapsulation of β-carotene within CT-EWP conjugate emulsions slightly improved their bioavailability, which was mainly attributed to the strong antioxidant activity of the conjugates. Our results suggested that CT-EWP conjugates can be utilized to develop antioxidant emulsifiers that can be used for the encapsulation and protection of bioactive components. However, further studies are needed to test their efficacy and toxicity in animal and human studies.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Luping Gu: 0000-0002-6760-0727 David Julian McClements: 0000-0002-9016-1291 Funding

The work was supported by the National Natural Science Foundation of China [Grants 31501428 and 31671809] and Jiangsu province “Collaborative Innovation Center for Food safety and quality control” industry development program. This material was also partly based upon work supported by the National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491) and USDA, AFRI Grants (2014-67021). Notes

The authors declare no competing financial interest.

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