Controlled Release of β-Carotene in β-Lactoglobulin–Dextran

Aug 18, 2014 - Endocytosis of Corn Oil-Caseinate Emulsions In Vitro: Impacts of Droplet Sizes. Yuting Fan , Yuzhu Zhang , Wally Yokoyama , Jiang Yi...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JAFC

Controlled Release of β‑Carotene in β‑Lactoglobulin−DextranConjugated Nanoparticles’ in Vitro Digestion and Transport with Caco‑2 Monolayers Jiang Yi,†,§ Tina I. Lam,§ Wallace Yokoyama,§ Luisa W. Cheng,§ and Fang Zhong*,† †

Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China § Western Regional Research Center, ARS, U.S. Department of Agriculture, Albany, California 94710, United States ABSTRACT: Undesirable aggregation of nanoparticles stabilized by proteins may occur at the protein’s isoelectric point when the particle has zero net charge. Stability against aggregation of nanoparticles may be improved by reacting free amino groups with reducing sugars by the Maillard reaction. β-Lactoglobulin (BLG)−dextran conjugates were characterized by SDS-PAGE and CD. Nanoparticles (60−70 nm diameter) of β-carotene (BC) encapsulated by BLG or BLG−dextran were prepared by the homogenization−evaporation method. Both BLG and BLG−dextran nanoparticles appeared to be spherically shaped and uniformly dispersed by TEM. The stability and release of BC from the nanoparticles under simulated gastrointestinal conditions were evaluated. Dextran conjugation prevented the flocculation or aggregation of BLG−dextran particles at pH ∼4−5 compared to very large sized aggregates of BLG nanoparticles. The released contents of BC from BLG and BLG−dextran nanoparticles under acidic gastric conditions were 6.2 ± 0.9 and 5.4 ± 0.3%, respectively. The release of BC from BLG−dextran nanoparticles by trypsin digestion was 51.8 ± 4.3% of total encapsulated BC, and that from BLG nanoparticles was 60.9 ± 2.9%. Neither BLG−BC nanoparticles nor the Maillard-reacted BLG−dextran conjugates were cytotoxic to Caco-2 cells, even at 10 mg/mL. The apparent permeability coefficient (Papp) of Caco-2 cells to BC was improved by nanoencapsulation, compared to free BC suspension. The results indicate that BC-encapsulated β-lactoglobulin−dextran-conjugated nanoparticles are more stable to aggregation under gastric pH conditions with good release and permeability properties. KEYWORDS: β-lactoglobulin, conjugates, β-carotene, controlled release, transport, Caco-2 cells

■

INTRODUCTION β-Carotene (BC) is a highly lipophilic bioactive carotenoid. BC is an important precursor of vitamin A and has strong antioxidant activity. However, poor heat stability and extremely low solubility in vivo limit its use and bioavailability. Proteinbased nanoemulsion or nanoparticle delivery systems have been developed to encapsulate BC and protect the stability and enhance the absorption of BC.1,2 Compared to synthetic surfactants and polymers, proteins exhibit lower cell toxicity3 and stability to chemical oxidation.4,5 However, environmental factors, such as pH, ionic strength, and temperature, can cause the aggregation or sedimentation of protein-encapsulated nanoparticles that reduces its food applications. Protein-encapsulated nanoparticles may be digested at different stages of the gastrointestinal tract depending on acidity, ionic strength, or specificity of proteases (pepsin, trypsin, and chymotrypsin) to their amino acid composition. Secondary structure of proteins can also affect digestibility. For example, native β-lactoglobulin (BLG) is the main component of whey protein isolate (WPI) and has been reported to be resistant to gastric pepsin digestion due to the stability of the folded β-sheet conformation.6,7 However, denatured BLG may be degraded by enzymes in gastric conditions due to the exposure of hydrophobic amino acid. Changes in the functional properties of proteins such as solubility,8 emulsifying properties, 8 heat stability,9 and responses to other environmental stresses10 after conjugation © 2014 American Chemical Society

with reducing sugars are well-known. Maillard conjugates have also been shown to change the digestibility of proteins encapsulating emulsions.11,12 “Dry heating” was used to prepare β-lactoglobulin−dextran conjugates. Recently, BLG has been glycated by dextrans with a wide range of molecular weights.12 Typically, a large molar excess of carbohydrates is used to increase the degree of glycation. Lesmes and McClements have also shown that large dextran can reduce the ζ-potential.12 They hypothesize that the steric barrier reduces surface charge effects with the continuous phase. In this study, we conjugated very large dextran molecules to BLG. Nanoparticles were formed using the solvent evaporation method and a mixture of mainly native BLG with a small amount of large dextran-conjugated BLG. The objective of this study was to determine if very large molecules can sterically cover large areas of the protein surface of an emulsion or particle surface and alter its physical, chemical, and in vitro biological properties. Received: Revised: Accepted: Published: 8900

June 2, 2014 July 15, 2014 August 17, 2014 August 18, 2014 dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907

Journal of Agricultural and Food Chemistry

■

Article

nanoparticles were prepared according to the method described by Tan and Nakajima15 with minor changes. Water (100 mL) was saturated with 8.3 g of ethyl acetate16 prior to formulating the protein solution to prevent precipitation of BC during homogenization. The BC in ethyl acetate solution was combined with the ethyl acetate saturated protein solution at a ratio of 1:9. The mixtures were homogenized for 10 min at 8000 rpm using an Ultra-Turrax homogenizer (T25, Ika-Werke, Staufen, Germany) to form a coarse emulsion before additional homogenization using a two-stage valve homogenizer (APV-1000 high-pressure homogenizer, Wilmington, MA, USA) for one cycle at 70 MPa. After the homogenization, ethyl acetate was removed from the emulsions on a rotary evaporator at 40 °C. The residual ethyl acetate was determined by headspace gas chromatography and found to be 350 Ω cm2; the Papp of 1 mg/mL FITC−dextran detected with a Multilabel microplate counter (Victor3, PerkinElmer) at an excitation wavelength of 487 nm and an emission of 518 nm was (4.3 ± 1.1) × 10−8 cm s−1. Both the TER, which was higher than reported values (260 ± 65 Ω cm2),17 and Papp, which was lower than reported values, indicated that the Caco-2 cell monolayers were confluent and suitable for permeation study. BC Transport with Caco-2 Monolayers. The Caco-2 monolayers were washed with DMEM three times. Both BLG and BLG−dextran conjugate-encapsulated BC nanoparticles were diluted in DMEM to a final BC concentration of 20 μg mL−1. Free BC in THF/DMSO (1:1, v/v) after dilution in DMEM (20 μg mL−1) was used as control. One and a half milliliters of the BC-encapsulated nanoparticle and control solution was added to separate inserts. The medium in the basal side was sampled at several time intervals, extracted with solvent, and analyzed for BC content by HPLC (procedure given below). The TER was also measured at several time intervals (0, 0.5, 1, 2, 3, and 4 h) to measure the integrity of Caco-2 monolayers. The apparent permeability coefficient (Papp, cm s−1) was calculated using the equation

■

RESULTS AND DISCUSSION BLG Glycation. SDS-PAGE. The characterization of BLG glycation products was determined by SDS-PAGE. The bands corresponding to native BLG, BLG glycation with dextran for 24, 48, and 72 h, and BLG heated for 72 h under reducing conditions are shown in Figure 1. One obvious band

Figure 1. SDS-PAGE analysis under reducing conditions of BLG and BLG−dextran conjugates. Lanes: 1, protein markers; 2, native BLG; 3, 4, and 5, BLG−dextran conjugates treated by glycation for 24, 48, and 72 h, respectively; 6, BLG treated for 72 h (control).

corresponding to BLG was found at about 18 kDa.18 Small amounts of BLG dimers are observed at about 40 kDa. There appears to be a slight decrease in intensity of the BLG band after heating for 72 h (control), but no absorption in the 40− 200 kDa range. After glycation with dextran for 24, 48, and 72 h, smeared zones were formed between 50 and 200 kDa. Dextran-glycated ovalbumin19 show similar smeared zones. Carbohydrate staining but not protein staining confirmed glycation by SDS-PAGE of gelatin.20 In this study the smeared zone densities appear to become more intense with increasing treatment time (from 24 to 72 h), suggesting that more conjugates were formed. The TNBS assay shows that the extent of glycation increased from 6.7 to 14.5% with increasing treatment time (from 24 to 72 h) (Table 1). The SDS-PAGE and TNBS results confirm that BLG−dextran conjugates were formed by dry-heating. CD. Changes in the secondary structure of BLG after glycation by the much larger dextran molecule were determined by far-UV CD spectroscopy (Figure 2). The negative peak at 216 nm indicates that BLG is rich in β-sheet secondary

Papp = (dQ /dt )(1/(AC0)) where dQ/dt is the permeability rate (μg s−1), A is the surface area of the filter (cm2), and C0 is the initial concentration in the donor chamber (μg mL−1). Extraction and Quantification of BC. BC was extracted from the nanoparticles with 3 mL of ethanol/n-hexane (1:2, v/v) three times. The hexane extracts were combined and brought up to 10 mL for HPLC analysis. Five microliters of 5 μg/mL trans-β-apo-8′-carotenal (internal standard) in ethanol was added to 1.0 mL samples. The transport samples (1.0 mL) were extracted three times with 3 mL of ethanol/n-hexane (1:2, v/v). Organic fractions were removed and combined with transfer pipets, and the extract was dried under a stream of nitrogen gas at 40 °C. The BC extract was dissolved in 0.1 mL of methanol/dichloromethane (1:1, v/v) containing 0.1% BHT for HPLC analysis. The recovery of trans-β-apo-8′-carotenal was >94%. HPLC quantification of BC was performed using an Agilent 1100 HPLC system with a DAD UV−vis absorption detector (Agilent, Santa Clara, CA, USA), according to the published method.2 BC 8902

dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907

Journal of Agricultural and Food Chemistry

Article

structure content (α-helix + β-sheet) was increased slightly by 4.5, 4.1, and 2.9%, corresponding to 24, 48, and 72 h of incubation time, compared to native BLG. Levels of 40−75% glycosylation of kidney bean vicilin with glucose by large molar excess of glucose resulted in marked shifts in wavelength and ellipticity in the CD spectra.24 However, no significant change of secondary structure compositions was reported by Enomoto et al. after glycation with maltopentaose and phosphorylation with pyrophosphate.21 In this study the small changes may be due to the low degree of glycosylation.24 Characterization of BC Nanoparticles. BC-loaded nanoparticles were encapsulated with native BLG and BLG−dextran conjugates (72 h) (the conjugates used below were all conjugates after 72 h of incubation) by the homogenization− evaporation method.1 The characteristics of both nanoparticles are shown in Table 3. There were no significant differences in particle diameter. The unimodal particle diameter distributions of BLG and BLG−dextran-conjugated nanoparticles are shown in Figure 3. Both polydispersity index (PDI) values were below

Table 1. Degree of Glycation of BLG and the Conjugates sample

degree of glycation (%)

BLG native BLG−dextran conjugates (24 h) BLG−dextran conjugates (48 h) BLG−dextran conjugates (72 h) BLG control

0 6.7 ± 1.5 9.4 ± 2.0 14.5 ± 1.7 0

Figure 2. Far-UV CD spectra (195−260 nm) of BLG native (A) and BLG−dextran conjugates treated by glycation for 24 h (B), 48 h (C), and 72 h (D).

structure.21 The mean residue ellipticity was decreased slightly by 72 h of Maillard-based reaction (Figure 2). The secondary structure composition of native BLG and BLG−dextran conjugates was calculated according to DichroWeb online (Table 2). The composition of native BLG in this study was αFigure 3. Particle diameter distributions of BLG and BLG−dextran conjugate (72 h)-encapsulated BC loaded nanoparticles (1.0% protein content, w/w) and (inset) transmission electron micrographs of BCloaded nanoparticles prepared with BLG (A) and BLG−dextran conjugates (B), respectively, at a final protein concentration of 1% (w/ w). Scale bar indicates 50 nm.

Table 2. Secondary Structure Composition of Native BLG and BLG−Dextran Conjugatesa sample

incubation time (h)

α-helix (%)

β-sheet (%)

turns (%)

unordered (%)

native BLG BLG−dextran BLG−dextran BLG−dextran

24 48 72

9.3 11 10.6 10.1

39.5 40 40.2 40.1

22.5 21.7 21.4 21.1

27 26 25.8 26.2

0.200, indicating a narrow particle size distribution. The BC nanoparticles were spherically shaped, approximately 50 nm in diameter, confirming light scattering results, and uniformly dispersed (Figure 3). Although similar in diameter, the BC nanoparticles’ ζ-potential values were different. Glycation with dextran significantly decreased the apparent ζ-potential from −32.9 ± 0.8 mV for native BLG to −14.0 ± 0.7 mV for BLG− dextran (P < 0.05). Although only a fraction (∼15%) of the BLG was glycated, Lesmes and McClements have suggested that steric barriers may screen the positively charged surface from the continuous phase.12 The encapsulation efficiency of BC was 98.6 and 98.4% for native BLG and BLG−dextran conjugates, respectively, and the loading capacity was 1.07 and

a

The secondary structure composition was calculated from far-UV CD spectra data using DichroWeb online (http://dichroweb.cryst.bbk.ac. uk/html/home.shtml).

helix, 9.3%; β-sheet, 39.5%; turn, 22.5%; and unordered, 27%; whereas BLG was reported to have 15% α-helix, 50% β-sheet, and 15−20% reverse turn.22 The differences may be due to the different sources of BLG and different curve fitting analysis programs.23 SELCON3 was used in this study. After glycation, the α-helix and β-sheet structure increased, whereas the turn and unordered structure slightly decreased. The highly ordered

Table 3. Mean Particle Diameter (Z-Average), Polydispersity Index (PDI), ζ-Potential, Loading Efficiency, and Loading Capacity of BC-Encapsulated BLG and BLG−Dextran (72 h) Nanoparticlesa

a

nanoparticle

Z-average (nm)

PDI

ζ-potential

encapsulation efficiency (%)

loading capacity (%)

BLG BLG−dextran

66.6 ± 1.2 68.2 ± 2.9

0.178 ± 0.035 0.171 ± 0.034

−32.9 ± 0.8 −14.0 ± 0.7

98.6 ± 1.0 98.4 ± 1.2

1.07 ± 0.02 1.06 ± 0.03

Data are expressed as the mean ± STD. 8903

dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907

Journal of Agricultural and Food Chemistry

Article

1.06% for native BLG and BLG−dextran conjugate, respectively. pH Stability against Aggregation. Protein-based nanoparticles may flocculate or aggregate around their isoelectric point,12,25−27 leading to increased particle size28 and restricting their application in food systems, especially in mildly acidic beverages, such as juice, yogurt, and functional beverages. The glycation of lysine groups by the Maillard reaction to reduce the number of basic groups and increase steric hindrance by the use of large carbohydrates was used to overcome this problem. In this study, both native BLG and BLG−dextran conjugate-based BC nanoparticles were exposed to pH 2−7 (Figure 4). The

Figure 5. In vitro release of BC in nanoparticles encapsulated with native BLG or BLG−dextran conjugates treated with pepsin (1.0 mg/ mL) and trypsin (1.0 mg/mL) at simulated gastrointestinal juice (A, pepsin at pH 2.0; B, trypsin at pH 7.0). (#) Significant (P < 0.05) difference.

sites are in the interior of the protein and inaccessible for hydrolysis. BC-loaded nanoparticles were digested with trypsin (Figure 5B). Note that chymotrypsin was not used in intestinal digestion. It is possible that the combined use of trypsin− chymotrypsin could change the release rate of BC encapsulated either in BLG or BLG−dextran nanoparticles. The release of BC from native BLG nanoparticles was relatively rapid during the first 30 min, and 46.4% BC was released, whereas only 40.7% was released for BLG−dexran conjugates, suggesting BC release was prevented by glycation. After 120 min of trypsin digestion, 60.9 and 51.8% BC were released from native BLG nanoparticles and BLG−dextran nanoparticles (P < 0.05), respectively (Figure 5B). There are at least two possible explanations. First, the proteolysis of glycated Lys and Arg residues of BLG was largely reduced.9,29,30 Alternatively, glycation by the large dextran used in this study sterically hindered access of the enzyme to the BLG at the surface of the nanoparticles.12,31 Cell Toxicity of BC-Loaded Nanoparticles. The viability of Caco-2 cells treated with BC-loaded nanoparticles was evaluated after the MTT assay as previously described (Figure 6).1 The cell viability after treatment with native BLG-based nanoparticles, at protein contents of 5 and 10 mg/mL, was greater than 98 and 91 ± 2%, respectively, indicating that native BLG-based nanoparticles were nontoxic and biocompatible. There are concerns about the health risks and benefits of the intake of Maillard reaction or glycation products.32,33 The

Figure 4. Effects of pH (2.0−7.0) on mean particle diameter (A) and ζ-potential (B) of BC-loaded nanoparticles stabilized by native BLG or BLG−dextran conjugates.

diameters of native BLG nanoparticles were stable at pH 2.0− 3.0 and 6.0−7.0, but the particle diameters increased significantly to 152, 9964, and 12619 nm for pH 4.0−5.0 (Figure 4A). The large particle size about the isoelectric point suggests that the nanoparticles were strongly flocculated. Nanoparticles encapsulated by BLG−dextran conjugates did not show changes in particle diameter under the same pH conditions. The lack of aggregation and ζ-potential data support the role of steric hindrance by dextran at the interface to prevent nanoparticle flocculation or aggregation and separation of the protein surface from the continuous media.12,19,27 BC Release during in Vitro Digestion. As shown in Figure 5A, after 120 min of digestion by pepsin at pH 2.0, 6.2 and 5.4% of BC were released from BLG and BLG−dextran nanoparticles, respectively. Native BLG is resistant to proteolysis by pepsin at pH 2.06,29 because the peptic cleavage 8904

dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907

Journal of Agricultural and Food Chemistry

Article

Figure 6. In vitro cytotoxicity of BC-loaded nanoparticles encapsulated with native BLG or BLG−dextran conjugates at different protein concentrations on Caco-2 cells measured by MTT assay. Cell viability is expressed as the mean ± STD (n = 3).

results of the MTT cell viability assay of Maillard-based conjugate nanoparticles were similar to that of native BLGbased nanoparticles even at 10 mg/mL (91 ± 3%), indicating that glycation did not induce toxicity. The results above clearly showed both native BLG and BLG−dextran conjugate-based nanoparticles are safe and may be useful for BC carriers. Transport in Caco-2 Cells. The uptake of lipophilic carotenoids was studied in Caco-2 cell monolayers, a model for intestinal absorption.34−36 Caco-2 monolayers were grown to confluence, and there were no differences in TER in cells treated with free BC or BC-loaded nanoparticles (data not shown). The transport of BC into the cells was proportional to the incubation time (Figure 7A). The cumulative transport, Papp, of free BC (control, suspended in THF/DMSO) was (2.3 ± 0.5) × 10−7, whereas Papp values for native BLG and BLG− dextran conjugate nanoparticles were (3.5 ± 0.3) × 10−6 and (3.4 ± 0.3) × 10−6, respectively (P < 0.05) (Figure 7B). Compared to free BC (control), the Papp was significantly increased by 15.4- and 14.5-fold for native BLG and BLG− dextran conjugates, respectively. The results showed that the bioavailability of carotenoids can be significantly improved by nanoencapsulation with proteins. The cellular uptake of nanoparticles is believed to occur by endocytosis. There were three main endocytic pathways: clathrin-mediated endocytosis, lipid raft/caveolae-mediated endocytosis, and macropinocytosis.37,38 Conner et al. suggested that nanoparticles with mean diameters between 60 and 70 nm were mainly transported through lipid raft/caveolae-mediated endocytosis.37 After internalization, the BC nanoparticles are then intracellularly transported from endosome to lysosomes. Nanoparticles were excreted from lysosomes to basolateral compartments by exocytosis.39,40 According to a recently published paper, Golgi was involved in the nanoparticle exocytosis.38 There were no differences in the permeability rate between native BLG and BLG−dextran conjugates (P > 0.05), which indicated that polysaccharide molecules on the surface of nanoparticles had no adverse effects on bioavailability. In conclusion, we prepared BLG glycated to very large dextran molecules. Although only about 15% of the BLG was glycated, agglomeration and ζ-potential were consistent with steric hindrance. BLG−dextran conjugated nanoparticles improved the stability in simulated gastrointestinal environment around the isoelectric point (pH 4.0−5.0) to control the release of encapsulated BC. Neither native BLG nor BLG− dextran-conjugated nanoparticles affected Caco-2 cell viability.

Figure 7. Accumulation transport (A) and apparent permeability coefficient (Papp) (B) of BC by Caco-2 cell monolayers incubated with BC THF/DMSO suspension, native BLG-stabilized BC-loaded nanoparticles, and BLG−dextran conjugate-stabilized BC-loaded nanoparticles at a BC concentration of 20 μg/mL. Values are expressed as the mean ± STD (n = 3). (#) Significant (P < 0.05) difference.

Transport of BC by BLG and BLG−dextran-conjugated nanoparticles increased about 15 times compared to that of free BC. The results found in this study can be used in better designing carotenoid delivery systems.

■

AUTHOR INFORMATION

Corresponding Author

*(F.Z.) Phone: +86-510-85197876. Fax: +86-510-85197876. Email: [email protected]. Funding

This work was financially supported by National 125 Program 2011BAD23B02, 2013AA1022207; NSFC 31171686; NSFJiangsu-BK2012556; 111 Project B07029; PCSIRT0627 and JUSRP11422. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED BC, β-carotene; BLG, β-lactoglobulin; WPI, whey protein isolate; PDI, polydispersity index; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco’s modified Eagle medium; THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; SDS-PAGE, sodium dodecyl sulfate− polyacrylamide gel electrophoresis; CD, circular dichroism 8905

dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907

Journal of Agricultural and Food Chemistry

■

Article

(20) Zhou, H.; Sun, X.; Zhang, L.; Zhang, P.; Li, J.; Liu, Y.-N. Fabrication of biopolymeric complex coacervation core micelles for efficient tea polyphenol delivery via a green process. Langmuir 2012, 28, 14553−14561. (21) Enomoto, H.; Li, C.-P.; Morizane, K.; Ibrahim, H. R.; Sugimoto, Y.; Ohki, S.; Ohtomo, H.; Aoki, T. Glycation and phosphorylation of β-lactoglobulin by dry-heating: effect on protein structure and some properties. J. Agric. Food Chem. 2007, 55, 2392−2398. (22) Creamer, L. K.; Parry, D. A. D.; Malcolm, G. N. Secondary structure of bovine β-lactoglobulin B. Arch. Biochem. Biophys. 1983, 227, 98−105. (23) Greenfield, N. J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2007, 1, 2876−2890. (24) Tang, C.-H.; Sun, X.; Foegeding, E. A. Modulation of physicochemical and conformational properties of kidney bean vicilin (phaseolin) by glycation with glucose: implications for structure− function relationships of legume vicilins. J. Agric. Food Chem. 2011, 59, 10114−10123. (25) Diftis, N. G.; Biliaderis, C. G.; Kiosseoglou, V. D. Rheological properties and stability of model salad dressing emulsions prepared with a dry-heated soybean protein isolate−dextran mixture. Food Hydrocolloids 2005, 19, 1025−1031. (26) Evans, M.; Ratcliffe, I.; Williams, P. A. Emulsion stabilisation using polysaccharide−protein complexes. Curr. Opin. Colloid Interface Sci. 2013, 18, 272−282. (27) Wong, B. T.; Day, L.; Augustin, M. A. Deamidated wheat protein−dextran Maillard conjugates: effect of size and location of polysaccharide conjugated on steric stabilization of emulsions at acidic pH. Food Hydrocolloids 2011, 25, 1424−1432. (28) Charoen, R.; Jangchud, A.; Jangchud, K.; Harnsilawat, T.; Naivikul, O.; McClements, D. J. Influence of biopolymer emulsifier type on formation and stability of rice bran oil-in-water emulsions: whey protein, gum arabic, and modified starch. J. Food Sci. 2011, 76, E165−E172. (29) Luz Sanz, M.; Corzo-Martínez, M.; Rastall, R. A.; Olano, A.; Moreno, F. J. Characterization and in vitro digestibility of bovine βlactoglobulin glycated with galactooligosaccharides. J. Agric. Food Chem. 2007, 55, 7916−7925. (30) Wang, W.; Zhong, Q. Properties of whey protein−maltodextrin conjugates as impacted by powder acidity during the Maillard reaction. Food Hydrocolloids 2014, 38, 85−94. (31) Mun, S.; Decker, E.; Park, Y.; Weiss, J.; McClements, D. J. Influence of interfacial composition on in vitro digestibility of emulsified lipids: potential mechanism for chitosan’s ability to inhibit fat digestion. Food Biophys. 2006, 1, 21−29. (32) Ames, J. M. Evidence against dietary advanced glycation endproducts being a risk to human health. Mol. Nutr. Food Res. 2007, 51, 1085−1090. (33) Somoza, V. Five years of research on health risks and benefits of Maillard reaction products: an update. Mol. Nutr. Food Res. 2005, 49, 663−672. (34) Claudie, D.-m.; Alexandrine, D.; Bertrand, C.; Franck, T.; MarieJosephe, A. Citrus flavanones enhance carotenoid uptake by intestinal Caco-2 cells. Food Funct. 2013, 4, 1625−1631. (35) During, A.; Harrison, E. H. An in vitro model to study the intestinal absorption of carotenoids. Food Res. Int. 2005, 38, 1001− 1008. (36) Sy, C.; Gleize, B.; Dangles, O.; Landrier, J.-F.; Veyrat, C. C.; Borel, P. Effects of physicochemical properties of carotenoids on their bioaccessibility, intestinal cell uptake, and blood and tissue concentrations. Mol. Nutr. Food Res. 2012, 56, 1385−1397. (37) Conner, S. D.; Schmid, S. L. Regulated portals of entry into the cell. Nature 2003, 422, 37−44. (38) He, B.; Lin, P.; Jia, Z.; Du, W.; Qu, W.; Yuan, L.; Dai, W.; Zhang, H.; Wang, X.; Wang, J.; Zhang, X.; Zhang, Q. The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells. Biomaterials 2013, 34, 6082−6098. (39) Chiu, Y.-L.; Ho, Y.-C.; Chen, Y.-M.; Peng, S.-F.; Ke, C.-J.; Chen, K.-J.; Mi, F.-L.; Sung, H.-W. The characteristics, cellular uptake and

REFERENCES

(1) Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F. Cellular uptake of β-carotene from protein stabilized solid lipid nanoparticles prepared by homogenization−evaporation method. J. Agric. Food Chem. 2014, 62, 1096−1104. (2) Yi, J.; Li, Y.; Zhong, F.; Yokoyama, W. The physicochemical stability and in vitro bioaccessibility of β-carotene in oil-in-water sodium caseinate emulsions. Food Hydrocolloids 2014, 35, 19−27. (3) He, W.; Tan, Y. N.; Tian, Z. Q.; Chen, L. Y.; Hu, F. Q.; Wu, W. Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats. Int. J. Nanomed. 2011, 6, 521−533. (4) Mao, L. K.; Xu, D. X.; Yang, J.; Yuan, F.; Gao, Y. X.; Zhao, J. Effects of small and large molecule emulsifiers on the characteristics of β-carotene nanoemulsions prepared by high pressure homogenization. Food Technol. Biotechnol. 2009, 47, 336−342. (5) Qian, C.; Decker, E. A.; Xiao, H.; McClements, D. J. Physical and chemical stability of β-carotene-enriched nanoemulsions: influence of pH, ionic strength, temperature, and emulsifier type. Food Chem. 2012, 132, 1221−1229. (6) Reddy, I. M.; Kella, N. K. D.; Kinsella, J. E. Structural and conformational basis of the resistance of β-lactoglobulin to peptic and chymotryptic digestion. J. Agric. Food Chem. 1988, 36, 737−741. (7) Teng, Z.; Li, Y.; Luo, Y.; Zhang, B.; Wang, Q. Cationic βlactoglobulin nanoparticles as a bioavailability enhancer: protein characterization and particle formation. Biomacromolecules 2013, 14, 2848−2856. (8) Li, Y.; Zhong, F.; Ji, W.; Yokoyama, W.; Shoemaker, C. F.; Zhu, S.; Xia, W. Functional properties of Maillard reaction products of rice protein hydrolysates with mono-, oligo- and polysaccharides. Food Hydrocolloids 2013, 30, 53−60. (9) Liu, G.; Zhong, Q. Thermal aggregation properties of whey protein glycated with various saccharides. Food Hydrocolloids 2013, 32, 87−96. (10) Wooster, T. J.; Augustin, M. A. β-Lactoglobulin−dextran Maillard conjugates: their effect on interfacial thickness and emulsion stability. J. Colloid Interface Sci. 2006, 303, 564−572. (11) Corzo-Martínez, M.; Soria, A. C.; Belloque, J.; Villamiel, M.; Moreno, F. J. Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin. Int. Dairy J. 2010, 20, 742−752. (12) Lesmes, U.; McClements, D. J. Controlling lipid digestibility: response of lipid droplets coated by β-lactoglobulin-dextran Maillard conjugates to simulated gastrointestinal conditions. Food Hydrocolloids 2012, 26, 221−230. (13) Liu, G.; Zhong, Q. Glycation of whey protein to provide steric hindrance against thermal aggregation. J. Agric. Food Chem. 2012, 60, 9754−9762. (14) Dyballa, N.; Metzger, S. Fast and sensitive colloidal Coomassie G-250 staining for proteins in polyacrylamide gels. J. Vis. Exp. 2009, DOI: 10.3791/1431. (15) Tan, C. P.; Nakajima, M. β-Carotene nanodispersions: preparation, characterization and stability evaluation. Food Chem. 2005, 92, 661−671. (16) Altshuller, A. P.; Everson, H. E. The solubility of ethyl acetate in water. J. Am. Chem. Soc. 1953, 75, 1727−1727. (17) Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2007, 2, 2111−2119. (18) Chen, Y.-j.; Liang, L.; Liu, X.-m.; Labuza, T. P.; Zhou, P. Effect of fructose and glucose on glycation of β-lactoglobulin in an intermediate-moisture food model system: analysis by liquid chromatography−mass spectrometry (LC−MS) and data-independent acquisition LC−MS (LC−MSE). J. Agric. Food Chem. 2012, 60, 10674−10682. (19) Li, Z.; Gu, L. Fabrication of self-assembled (−)-epigallocatechin gallate (EGCG) ovalbumin−dextran conjugate nanoparticles and their transport across monolayers of human intestinal epithelial Caco-2 cells. J. Agric. Food Chem. 2014, 62, 1301−1309. 8906

dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907

Journal of Agricultural and Food Chemistry

Article

intracellular trafficking of nanoparticles made of hydrophobicallymodified chitosan. J. Controlled Release 2010, 146, 152−159. (40) Roger, E.; Lagarce, F.; Garcion, E.; Benoit, J.-P. Biopharmaceutical parameters to consider in order to alter the fate of nanocarriers after oral delivery. Nanomedicine 2010, 5, 287−306.

8907

dx.doi.org/10.1021/jf502639k | J. Agric. Food Chem. 2014, 62, 8900−8907