Native and Thermally Modified ProteinâPolyphenol Coassemblies

Article pubs.acs.org/JAFC

Native and Thermally Modified Protein−Polyphenol Coassemblies: Lactoferrin-Based Nanoparticles and Submicrometer Particles as Protective Vehicles for (−)-Epigallocatechin-3-gallate Wei Yang, Chenqi Xu, Fuguo Liu, Fang Yuan, and Yanxiang Gao* Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, P. R. China ABSTRACT: The interactions between native, thermally modified lactoferrin (LF) and (−)-epigallocatechin-3-gallate (EGCG) at pH 3.5, 5.0, and 6.5 were investigated. Turbidity, particle size, and charge of LF−EGCG complexes were mainly dominated by pH value and secondary structure of protein. At pH 3.5 and 5.0, LF−EGCG complexes were nanoparticles which had high ζpotential, small size, and soluble state. At pH 6.5, they were submicrometer particles which exhibited low ζ-potential, large size, and insoluble state. The infrared spectra of freeze-dried LF−EGCG complexes showed that they were different from LF and EGCG alone. Far-UV CD results indicated that heat denaturation might irreversibly alter the secondary structure of LF and EGCG induced a progressive increase in the proportion of α-helix structure at the cost of β-sheet and unordered coil structure of LF at pH 3.5, 5.0, and 6.5. EGCG exhibited a strong affinity for native LF but a weak affinity for thermally modified LF at pH 5.0 and 6.5. An inverse result was observed at pH 3.5. These results could have potential for the development of food formulations based on LF as a carrier of bioactive compounds. KEYWORDS: thermally modified lactoferrin, (−)-epigallocatechin-3-gallate, nanoparticle, submicrometer particle

■

INTRODUCTION Lactoferrin (LF) is a single-chain globular glycoprotein, occurring in many mammalian secretion fluids, and possessing various biological functions.1 As an iron transporter, it may be found in both the iron-saturated (holo-LF) and the irondepleted (apo-LF) forms. It seems that when iron is bound into the LF molecule, a more closed conformation is adopted.2 LF has a relatively high pI (8−9) and therefore tends to be cationic at neutral pH whereas most other major dairy globular proteins are anionic.3 The fact that LF can form positively charged droplets at neutral pH could have a number of important practical implications. Therefore, LF has attracted strong interest as functional bioactive ingredients for applications in food, personal care, and pharmaceutical products.1 It has already been included into a range of commercial applications including infant formulas, fortified yoghurts, probiotics, skim milk, milk-type drinks, and supplemental tablets.1,4−6 (−)-Epigallocatechin-3-gallate (EGCG), the most active catechin of green tea, has become an intense focus of research interest due to its versatile biological activities including prevention and treatment of several chronic diseases, such as obesity, neurodegenerative disease, cardiovascular disease, and other adverse medical conditions.7−11 Therefore, it seems to be an important candidate for enrichment in the diet. However, the sensitivity of EGCG to oxidation limits its potential employment in functional food. Various native proteins, such as β-lactoglobulin,12 α-, β-, and κ-caseins,13 serum albumins,14,15 gelatin hydrolysates, and egg proteins16 have been studied as vehicles for EGCG. Though there were some papers to study the interactions between proteins and polyphenols, most of the selected proteins did not have outstanding features as lactoferrin, such as antibacterial, antiviral, antifungal, antiinflammatory, and immunomodulatory activities.17−20 © XXXX American Chemical Society

It is well-known that polyphenols could bind to various proteins and form soluble or insoluble complexes. EGCG contains eight hydroxyl groups, which endow it with stronger affinity to proteins, and it more easily forms precipitates than other tea polyphenols.12,13 The turbidity performance in the higher ratios of EGCG to proteins might present an obstacle for application in food systems such as clear beverages. Both LF and EGCG have various health benefits and physiological functionalities, such as antioxidant and antimicrobial properties. According to previous reports, phenolic compounds showed synergistic properties in reinforcing the antioxidant activity of lactoferrin in lipid systems,21 and combined administration of LF and tea polyphenol was more effective in inhibiting hamster buccal pouch carcinogenesis by preventing oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis.22−24 We hypothesized that the noncovalent complex between LF and EGCG might have better antioxidant and antimicrobial properties. Therefore, the interaction between LF and EGCG should be focused first in order to better understand the properties of LF−EGCG complex and its potential application in the food industry. The structure and functionality of a protein is highly dependent on environmental stresses such as temperature and pH. Recent studies have shown that protein nanoparticles and microparticles could be formed by controlled thermal denaturation of globular protein solutions.25−30 Previous studies have primarily been carried out using β-lactoglobulin to form the protein−EGCG nanoparticles, which have an Received: August 9, 2014 Revised: October 6, 2014 Accepted: October 13, 2014

A

dx.doi.org/10.1021/jf5038147 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

cell and temperature was set at 25 °C without further dilution. The ζpotential measurements were reported as the average of measurements for three fresh samples, with three readings recorded for each sample. Haze and Particle Size Measurements. LF and EGCG solutions with different concentrations were mixed to obtain final EGCG levels of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mM with a constant LF concentration of 0.1 mM. Haze formation was monitored by DLS and nephelometry when the mixture was kept at 25 °C for 2 h.39 Nephelometry experiments were performed in a HACH 2100N laboratory turbidimeter (Loveland, USA), and the aggregation behavior of LF and EGCG in aqueous solution was evaluated. The optical apparatus was equipped with a tungsten-filament lamp with three detectors: a 90° scattered-light detector, a forward-scatter light detector, and a transmitted light detector. The calibration was performed using a Gelex Secondary Turbidity Standard Kit (HACH, Loveland, USA), which consists of stable suspensions of a metal oxide in a gel.39 All experiments were performed in triplicate. Particle size and size distribution of LF−EGCG complexes were determined by dynamic light scattering (DLS) using a Zetasizer NanoZS90 (Malvern Instruments, Worcestershire, U.K.). The intensity of light scattered was monitored at a 90° angle. Results were described as cumulants mean diameter (size, nm) for particle size and polydispersity index (PDI) for size distribution. All experiments were performed in triplicate. Fourier Transform Infrared (FTIR) Spectroscopy. The infrared spectra of samples were measured with the potassium bromide (KBr) pellet method using a Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, U.K.) in the 500−4000 cm−1 range, with a resolution of 4 cm−1. Potassium bromide was used as a reference. For each measurement, 11 scans were taken. The samples analyzed were free LF, free EGCG, and freeze-dried LF−EGCG complexes. Circular Dichroism Measurements. The CD spectra were recorded using a CD spectropolarimeter (Pistar π-180, Applied Photophysics Ltd., U.K.) at both far-UV (190−250) and near-UV (250−320) regions under a constant nitrogen flush. Path lengths were 0.1 cm for the far-UV region and 1 cm for the near-UV region. Ellipticity was recorded at a speed of 100 nm/min, 0.2 nm resolution, 20 accumulations, and 2.0 nm bandwidth. The collected data were analyzed using Dichroweb: the online Circular Dichroism Web site http://dichroweb.cryst.bbk.ac.uk.40,41 Each spectrum presented was the average of three consecutive measurements. Fluorescence Spectroscopy. Fluorescence measurements were carried out using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). LF and EGCG solutions with various concentrations were mixed to prepare final EGCG levels of 10, 20, 30, 40, 50, 60, and 70 μM with a constant LF concentration of 0.1 mM. Spectra were collected when the mixture was kept at 25 °C for 2 h. The interaction between LF and EGCG was investigated using tryptophan fluorescence quenching. Fluorescence emission spectra of LF were recorded with the excitation wavelength at 292 nm. Both excitation and emission slit widths were set at 5 nm. The fluorescence quenching data were analyzed by fitting to the Stern−Volmer equation (eq 1):

isoelectric point of around pH 5.0 and a single thermal denaturation temperature (around 70 °C).31,32 However, to our knowledge, the interaction between thermally modified LF and EGCG was not characterized. LF has two lobes, and each one consists of two domains and a single, high affinity metal binding site, which binds, very tightly but reversibly, one ferric ion together with one carbonate as the synergistic anion.33 When LF is heated, one of the lobes unfolds at around 60 °C, and the other unfolds at around 85 °C,34−37 leading to a conformational change irreversibly in the LF molecules.30 There are some reports available on the heat-induced structural and conformational changes of LF.6,30,33 None of the papers, however, focused on the interaction between LF and EGCG. Until now, the information for the interaction and structure−affinity relationship of thermally modified LF and polyphenol was not available at different pH values. In this study, we examined whether protein−EGCG nanoparticles or submicrometer particles could be formed using native and thermally modified LF at different pH values. The effects of LF and EGCG interactions at different pH values on the structure of LF are tested. The binding constant and the number of binding sites of EGCG with native and thermally modified LF were also compared by fluorescence spectroscopy. LF has a much high isoelectric point, and LF−EGCG nanoparticles or submicrometer particles may be positively charged over a wider pH range. These LF−EGCG complexes may be useful functional ingredients in food, cosmetic, and pharmaceutical applications, e.g., to modify the optical or rheological properties of products.

■

MATERIALS AND METHODS

Materials. Lactoferrin from bovine whey was purchased from New Image International Limited (New Zealand). Its composition was protein (dry basis) 99.41%, of which LF was 92.0% of total proteins, ash 0.31%, and moisture 15.0%. The iron saturation of the LF powder was reported to be 15%. EGCG (purity ≥98%) from green tea was purchased from BSZH Science Company (Beijing, China). All other chemicals used were of analytical grade, unless otherwise stated. Solution Preparation. LF and EGCG aqueous solutions were prepared freshly by dissolving the proper amount of each powder in citrate buffer (pH 3.5, 10 mM), acetate buffer (pH 5.0, 10 mM), and phosphate buffer (pH 6.5, 10 mM) at room temperature and stirring for 2 h. If necessary, pH was adjusted with HCl (10 mM) or NaOH (10 mM). LF was dispersed in buffers to get a final concentration of 200 μM. The protein solutions were filled in plastic tubes (1 cm diameter). The thermally denatured experiments were conducted in a thermostatic water bath at 70 or 90 °C for 20 min. The two temperatures correspond approximately to the first and second unfolding transition of LF, respectively.30,34,35,38 After the thermal treatment, the tubes were immediately cooled in ice water to prevent further denaturation. All measurements were performed within 3 min after the heat treatment. The mixtures of Lf and EGCG were prepared by blending the appropriate volume of native LF or thermally modified LF and EGCG solutions in order to achieve the desired final concentrations. All spectroscopic studies were carried out on the heated−cooled protein, implying that only irreversible/permanent structural changes were detected. The abbreviations LFx (x = 3.5, 5.0, or 6.5) were used to represent the LF in the buffer solutions at pH 3.5, 5.0, and 6.5, respectively. The abbreviations LFx‑25°C were used to represent the native LF in the corresponding buffer solutions. The abbreviations LFx‑70°C and LFx‑90°C were used to represent the LF heated at 70 and 90 °C for 20 min in the corresponding buffer solutions, respectively. ζ-Potential Measurements. The ζ-potential of the complexes was determined by a particle electrophoresis instrument using a Zetasizer Nano-ZS90 (Malvern Instruments, U.K.). Samples were loaded in the

F0 = 1 + kqτ0[Q] = 1 + K sv[Q] F

(1)

where F0 and F are the fluorescence intensities in the absence and presence of quencher, kq is the biomolecular quenching constant, τ0 is the lifetime of fluorescence in the absence of a quencher, [Q] is the concentration of the quencher, and Ksv is the Stern−Volmer quenching constant.33,42 The binding constant Ka and the number of binding sites n can be calculated according to a double-logarithmic equation (eq 2):

log

F0 − F = log K a + n log[Q] F

(2)

The intercept of the double logarithmic Stern−Volmer plot provides the binding constant (Ka), and the slope yields the number of binding sites (n). B



Article

Statistical Analysis. The experiments were conducted in duplicate, and all the analyses were done in triplicate. The results were presented as the means ± standard deviation. Data were analyzed by one-way analysis of variance using the SPSS 16.0 package (SPSS Inc., Chicago, IL, USA), and the means were analyzed by Duncan′s multiple-range test. The correlations analysis was carried out using the Pearson mode. The concordance between experimental data and calculated values was established by the root mean squared deviation (RMSD).

the protein particles had a low net charge around pH 6.5 (see Figure 2), therefore, there might be a weak electrostatic

■

RESULTS AND DISCUSSION The Turbidity Measurements. The turbidity of the LF− EGCG solutions was measured to obtain some information about LF−EGCG complexes’ aggregation (Figure 1). The order of turbidity of single protein solutions was as follows: pH 6.5 > pH 3.5 > pH 5.0. At pH 6.5, native LF might undergo a pH-induced self-assembly that led to the formation of increasingly larger particles with time extension than those at pH 3.5 and 5.0.43 This result could be attributed to the fact that

Figure 2. Influence of pH and EGCG concentration on the ζ-potential of LF and LF−EGCG complexes.

repulsion between the particles. Thermally modified LF at pH 3.5 and 5.0 did not display thermal aggregation behavior whereas LF associated into large insoluble aggregates at pH 6.5. Abe et al. and Sreedhara et al. reported that LF was more thermoresistant at acidic pH whereas turbidity and sometimes gelation were observed upon heating at neutral and alkaline pH.44,45 The thermal aggregation of LF at pH 6.5 probably exposed sites normally buried inside the molecule that increased protein association by noncovalent interactions, with intermolecular thiol/disulfide reactions.46 There is a simple relationship between turbidity and ζ-potential at different thermal denaturation temperatures and pH values, that is, at pH 3.5, 5.0, and 6.5, the turbidity of free LF solution becomes higher with the increase of ζ-potential as the two lobes of LF unfold successively (see Figure 2).

Figure 1. Influence of pH and EGCG concentration on the turbidity of LF and LF−EGCG complexes. C



Article

content but lower α-helix content at pH 3.5 and 6.5 (see Figure 5 (b)). It should be noted that the addition of 1.0 mM EGCG resulted in significant sedimentation of LF6.5‑25°C−EGCG aggregates. The extensive precipitation could be related to the fact that the ζ-potential of LF6.5‑25°C was very close to neutrality (0.613 ± 0.1 mV for LF6.5‑25°C) (see Figure 2) allowing the formation of large aggregates that precipitate.49 Naczk et al. studied the effect of pH on the formation of crude tannin canola extract and protein (BSA, fetuin, gelatin, and lysozyme) complexes. It was suggested that the lowest solubility of polyphenol−protein complexes occurred at 0.3−3.1 pH units below the isoelectric point of the proteins.50 Indeed, increased precipitation of protein/polyphenol complex close to the isoelectric point might be attributed to the minimum solubility of the protein at this pH. LF6.5‑70°C−EGCG complex and LF6.5‑90°C−EGCG complex, having a uniform cloudy appearance, remained relatively stable against particle aggregation until the concentrations of EGCG were more than 2.0 and 3.0 mM, respectively. The low ζ-potential (see Figure 2) explained the low stability of these LF−EGCG complexes at this pH, where particles tended to flocculate and precipitate. In general, polyphenols normally have a strong binding affinity with proteins, which often leads to precipitation,51 but some factors, such as pH, temperature, protein type, structure and concentration, and the type and structure of phenolic compounds, basically affect protein−phenolic interactions. The results from aforementioned experiments indicated that the turbidity of LF−EGCG complexes could be manipulated by controlling the pH of solutions, the structure of LF, and the concentration of EGCG. ζ-Potential Measurements. The charge of single LF and LF−EGCG complexes at different pH values is presented in Figure 2. It is interesting to note that the ζ-potential of the LF6.5‑25°C was around 0 mV, despite this pH being below the reported isoelectric point of lactoferrin. The most likely explanation for this result was that the cationic groups on the adsorbed LF bound some anionic phosphate ions present in the buffer.52 There were appreciable differences in the influence of pH and thermal treatment on the ζ-potential of LF. As shown in Figure 2, the ζ-potential of LF was increased with the rise of thermal denaturation temperature in different buffer systems. We speculated that the two lobes of the protein are so close that they mask potential interactive sites buried within the interdomains of both lobes of the protein.46 As the two lobes of LF unfold successively when the protein was heated at 70 and 90 °C, more cationic groups originally located in the hydrophobic interior of LF exposed successively to buffer solutions and/or the thermally modified LF weaken the adsorbed ability of anion present in the buffers, leading to the increased ζ-potential of the thermally modified LF in different buffer solutions. Some researchers reported that the addition of green tea polyphenols hardly affected the ζ-potential of protein.47 However, in our study, we interestingly found that the ζpotential of LF−EGCG complexes was affected by the presence of EGCG. At pH 3.5 and 5.0, the LF had a higher ζ-potential which was further increased by the addition of EGCG. The high net charge of LF−EGCG complexes played an important role in preventing precipitation, which was consistent with the turbidity result that showed a low turbidity at pH 3.5 and 5.0. It should be noted that EGCG bears negative ζ-potential at pH

In the presence of EGCG, LF−EGCG complexes were formed as indicated by the appearance of increasing turbidity at pH 3.5, 5.0, and 6.5. The precipitations of LF−EGCG complexes resulting from polyphenol−protein interactions were pH sensitive. At pH 3.5 and 5.0, no precipitation was observed for LF−EGCG complexes. This might be ascribed to two reasons: one was the high net surface charge (>13.0 mV) of protein−polyphenol complexes (see Figure 2), which prevented them from coming into close contact and forming large aggregates;47,48 the other was the small size of complexes formed (see Figure 3). The minimum level of turbidity

Figure 3. Influence of pH and EGCG concentration on the particle size of LF and LF−EGCG complexes.

occurred at pH 5.0 for LF−EGCG complexes; this might be attributed to the higher ζ-potential and smaller size of LF5.0− EGCG complex than LF3.5−EGCG complex and LF6.5−EGCG complex (see Figures 2 and 3), and/or the secondary structure profiles of LF, which presented lower unordered content but higher α-helix content at pH 5.0, while higher unordered D



Article

Figure 4. FTIR spectra of EGCG, LF, and LF−EGCG complexes (containing 1.0 mM EGCG) at pH 3.5, 5.0, and 6.5.

3.5 and 5.0, opposite to LF. This result revealed that other factors except for the net charge affected the ζ-potential of LF− EGCG complexes, for example, the molecule of EGCG is much smaller than LF, therefore, EGCG might insert into the LF molecule and surround the LF molecule surface, resulting in reorganization of protein secondary and tertiary structures, thereby changing the protein surface charge distribution. At pH 6.5, close to the pKa, the protonated phenolic groups of EGCG could be deprotonated and the generated oxygen center imparts a high negative charge density,47 which further decreased the ζ-potential value of the complexes, leading to particle aggregation at higher concentration of EGCG. The Particle Size Measurements. Figure 3 shows the particle size and the polydispersity of the LF−EGCG complexes present in buffer solutions. The particle size of thermally modified LF underwent only slight changes

compared with native LF at pH 3.5, 5.0, and 6.5. EGCG induced an increase of size of the LF−EGCG complexes at desired pH values, forming larger aggregates that enhanced the scattered light, which was in agreement with the observed increase of nephelometric measurements. Single LF showed a particle size of 19−25 nm at pH 3.5, and 17.8−20.31 nm at pH 5.0. In the presence of EGCG, this value was increased significantly (P < 0.05), but it was always smaller than 41 nm at pH 3.5, and 32 nm at pH 5.0. These nanosized particles were soluble at pH 3.5 and 5.0, in other words, the large-scale aggregation was suppressed in the mixture of LF and EGCG at these two pH values. It is interesting to find that, as the EGCG concentration was increased, the aggregation of LF−EGCG complexes at pH 3.5 and 5.0 was hardly changed, but a narrow range of the particle size distribution was observed (a decreased PDI value, from 0.6 to 0.16 at pH 3.5, and 0.78 to E



Article

slight or no obvious difference in the amide I band (at 1600− 1700 cm−1) before and after the addition of EGCG (1.0 mM), but there was some major spectral shifting for LF amide II band at 1535 cm−1 upon EGCG interaction. The peak position of amide II blue-shifted from 1535 to 1537 cm−1 for LF3.5 and LF5.0 and red-shifted from 1537.3, 1535.4, and 1533.7 cm−1 to 1535.6, 1534.9, and 1533.3 cm−1 for LF6.5‑25°C, LF6.5‑70°C, and LF6.5‑90°C, respectively. Peaks around 1448 cm−1 for LF3.5, 1449 cm−1 for LF5.0, 1446.3 cm−1 for LF6.5‑25°C, and 1453 cm−1 for LF6.5‑90°C also had obvious red shift (about 1−4 cm−1) (Tables 1−3). These results implied that EGCG interacted with the CO and C−N groups in the protein structural subunits (hydrophilic interaction).

0.298 at pH 5.0), indicating a very homogeneous population of these aggregates. At pH 6.5, the particle size of native LF was larger than 99.35 ± 6.0 nm at the original state because the ζ-potential of LF at pH 6.5 was very close to neutrality (0.613 ± 0.1 mV), and the association was promoted.53 In the presence of EGCG, this value was increased significantly (P < 0.05) and submicrometer particles (0.6−0.8 μm) could be formed. The PDI values, which were different from those at pH 3.5 and 5.0, were enhanced as the size of LF−EGCG complexes increased at pH 6.5, indicating a very heterogeneous population of the aggregates formed. Larger particles remained unstable in solution (see Figure 1) at pH 6.5, which was attributed to the low net charge, with only weak electrostatic repulsion between the particles. It can be concluded that pH and the structure of LF significantly affected the interaction between LF and EGCG. This interpreted the possibility of monitoring LF−EGCG particle size (nanometer or submicrometer) and morphology with pH and heat treatment as factors. Overall, the sensitivity of the particle size to pH and the structure of LF facilitated the control of the physical properties of LF−EGCG complexes. Fourier Transform Infrared (FTIR) Spectroscopy. EGCG is known to form complexes with proteins leading to changes in the structural, functional, and nutritional properties of both compounds. Figure 4 shows the infrared spectra of LF and LF−EGCG complexes (1.0 mM EGCG). FTIR profiles revealed some pronounced differences between native LF at different pH values, and distinguished LF−EGCG complexes from the native LF. EGCG has four typical peaks around 3550, 3476, 3365, and 3273 cm−1, probably due to the vibration of the O−H linkage of phenolic and hydroxyl groups,54−56 and two large peaks around 1691 and 1616 cm−1, probably due to the carbonyl stretching of the gallic acid,57 while those peaks were not found in the complexes at all. At the same time, the disappearance of other typical characteristic bands of EGCG, such as peaks in the regions of 1000−1500 and 700−900 cm−1, also confirmed the formation of the LF−EGCG complex by noncovalent interaction. In this study, all complexes showed a broad feature around 3300 cm−1, due to intermolecular H-bonded and O−H stretching modes. Slight change of the peak around 3300 cm−1 in all LF−EGCG complexes compared to the native LF was an indication of the interaction between LF and EGCG induced by hydrogen bonds.55 This result was in agreement with previous findings suggesting that polyphenols were bound to the protein partially via H-bonds.15,58 The absorption bands of LF located at 2936 and 2875 cm−1 were attributed to antisymmetric and symmetric CH2 stretching vibrations of proteins.59 The spectral changes of these vibrations were monitored in order to locate the presence of hydrophobic contact in the LF−EGCG complex. Upon the interaction with EGCG, no shifts of absorption peaks were observed in the LF− EGCG complex compared to native LF (Figure 4), which suggested that the hydrophobic interaction was not the main force when the LF−EGCG complex formed. Peaks around 1653 and 1535 cm−1 in the FTIR spectra of native LF were attributed to CO stretching vibrations of amide I band60,61 and N−H bending vibrations and C−N stretching vibrations of amide II band,62−65 respectively. Both the CO bonds and the C−N bonds were involved in the hydrogen bonds which took place between the different elements of secondary structure of proteins.66 LF showed

Table 1. FTIR Spectral Peak Position of LF and LF−EGCG Complexes (1.0 mM EGCG) at pH 3.5 peak wavenumber (cm−1) LF3.5‑25°C region amide amide amide amide

A B I II

amide III a

LF3.5‑70°C

LF3.5‑90°C

0 mMa

1.0 mM

0 mM

1.0 mM

0 mM

1.0 mM

3304.0 3071.0 1656.6 1535.5 1448.0 1239.0 1075.0

3298.0 3075.0 1657.8 1536.6 1446.0 1236.0 1077.0

3301.0 3072.0 1657.0 1535.0 1448.0 1238.6 1074.0

3299.9 3072.7 1656.3 1537.5 1444.5 1236.8 1077.0

3299.3 3071.7 1656.3 1535.8 1447.7 1238.5 1074.9

3299.6 3077.0 1656.1 1536.6 1445.2 1236.0 1077.3

EGCG concentration.

Table 2. FTIR Spectral Peak Position of LF and LF−EGCG Complexes (1.0 mM EGCG) at pH 5.0 peak wavenumber (cm−1) LF5.0‑25°C region amide amide amide amide

A B I II

amide III a

0 mM

a

LF5.0‑70°C

LF5.0‑90°C

1.0 mM

0 mM

1.0 mM

0 mM

1.0 mM

3299.7 3071.4 1658.0 1543.7 1447.0 1151.4 1067.6

3307.9 3069.0 1656.3 1535.1 1449.5 1160.3 1069.1

3299.2 3067.6 1657.0 1537.1 1447.0 1151.1 1067.4

3298.5 3067.5 1656.0 1536.2 1448.9 1159.9 1067.8

3299.4 3070.1 1656.0 1537.0 1448.3 1151.4 1064.7

3301.0 3066.4 1657.9 1535.5 1449.3 1160.8 1069.4

EGCG concentration.

It is interesting to find that FTIR profiles also revealed some pronounced differences in the region of 1500−1000 cm−1 at pH 3.5, 5.0, and 6.5. In this region, there existed four peaks at Table 3. FTIR Spectral Peak Position of LF and LF−EGCG Complexes (1.0 mM EGCG) at pH 6.5 peak wavenumber (cm−1) LF6.5‑25°C region amide amide amide amide

A B I II

amide III a

F

LF6.5‑70°C

LF6.5‑90°C

0 mMa

1.0 mM

0 mM

1.0 mM

0 mM

1.0 mM

3299.9 3066.6 1656.7 1537.3 1446.3 1242.2

3300.9 3069.2 1657.1 1535.6 1451.9 1240.4

3302.7 3066.7 1656.9 1535.4 1452.9 1240.7

3296.8 3070.0 1657.1 1534.9 1453.1 1238.6

3296.3 3070.2 1656.0 1533.7 1452.8 1239.0

3297.2 3071.2 1656.0 1533.3 1451.5 1238.8

EGCG concentration. dx.doi.org/10.1021/jf5038147 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX


Article

Figure 5. (a, a1−a9) Far-UV CD spectra of native and thermally modified LF in the absence and presence of 1.0 mM EGCG at pH 3.5, 5.0, and 6.5. (b) Secondary structure of native and thermally modified LF at pH 3.5, 5.0, and 6.5. (c) Changes in secondary structure of native and thermally modified LF−EGCG complexes (containing 1.0 mM EGCG) at pH 3.5, 5.0, and 6.5.

1242 cm−1 was assigned to N−H in plane bending of the amide III band;61 the band observed at 1062 cm−1 was attributed to the asymmetrical C−O vibration.67,69 The characteristic bands of LF in the region of 1500−1000 cm−1 were shifted to higher wavelength or lower wavelength, revealing the presence of

pH 3.5 and 5.0, and five peaks at pH 6.5 (Figure 4). The shape and intensity of those peaks were varied due to the pH of solutions. The two bands around 1392 and 1446 cm−1 associated with the C−O stretching bond of carboxyl groups and the C−H deformation (asymmetrical) vibration in R− CH3,67,68 respectively; the occurrence of the band at 1238 or G



Article

Figure 6. Fluorescence emission spectra of LF−EGCG complexes at different EGCG to LF molar ratios at pH 3.5, 5.0, and 6.5. Insets: Plot of F0/F versus [EGCG] as per eq 1 (top); log[(F0 − F)/F] vs log [EGCG] as per eq 2 (bottom).

specific interactions between LF and EGCG at specific pH values.

It should be noted that the occurrence of a new band at 933 cm−1 in thermally modified LF and LF−EGCG complexes at H



Article

two protein domains or subdomains, or at the subunit interface in oligomeric protein systems.72 There exist 13 Trp residues in the LF structure.33 By monitoring the emission peak change, some information might be obtained concerning the structural changes and the microenvironment surrounding the fluorophore in the proteins.73 As shown in Figure 6, the thermal treatment at 70 or 90 °C induced an increase in the fluorescence intensity. The fluorescence intensities of LF solutions exhibited the increases of 6.1% and 16.3% at pH 3.5, 18.7% and 77.4% at pH 5.0, and 26% and 2.4% at pH 6.5 when heated at 70 and 90 °C for 20 min, respectively. This might be attributed to the irreversibly changed conformation of LF as the two lobes successively unfolded and the side chains of LF became more exposed to buffer solutions, leading to the Trp residues gradually exposed to water and the decrease of intramolecular quenching.33 The emission of tryptophan may be blue-shifted if the group is buried within a native protein, and its emission may be redshifted when the protein is unfolded.74 The fluorescence spectra of LF after the addition of EGCG are shown in Figure 6. It was obvious that the fluorescence intensity of LF was regularly decreased with the rise of EGCG concentration, and this implied that there existed binding behavior between LF and EGCG at pH 3.5, 5.0, and 6.5. There were no obvious shifts of the maximum λem (338 nm) of LF fluorescence at pH 3.5 and 5.0, while the maximum λem of LF6.5‑70°C and LF6.5‑90°C was obviously red-shifted (from 341 to 346 nm for LF6.5‑70°C and from 344 to 355 nm for LF6.5‑90°C) in the presence of EGCG (Figure 6h,i). These results revealed that there was a larger change in the immediate environment of the tryptophan residues of thermally modified LF at pH 6.5 than pH 3.5 and 5.0, and then EGCG was situated in close proximity to the tryptophan residues for quenching to occur. Table 4 summarizes the results correspondingly estimated according to eqs 1 and 2. Figure 6 shows the Stern−Volmer

pH 6.5 (except LF6.5‑25°C) might be associated with the denaturation of LF from thermal treatment or from the interaction with EGCG.70 Analysis of the Secondary Structures of LF and LF− EGCG Complex Using Circular Dichroism (CD). Far-UV CD was used to obtain information about the changes occurring at the secondary folding level of protein. In this section, the influence of pH on the secondary structure of LF formed by thermal treatment and EGCG-induced conformational transitions of LF (the concentration of EGCG was 1.0 mM) are monitored by far-UV CD spectroscopy (Figure 5). As shown in Figure 5a, a1−a9, in the 190−260 nm region, the spectra of LF3.5‑25°C were composed of a broad negative band centered at 208−210 nm and a shoulder at 218−220 nm. In the presence of 1.0 mM EGCG, the peak showed a significant decrease in negative ellipticities for all wavelengths of the farUV CD spectrum, which was a characteristic of LF3.5‑25°C secondary structure conformation change. Similar spectra were obtained for other native and thermally modified LF. The secondary structure content was estimated using the DICHROWEB procedure, which was an online server for protein secondary structure analyses from circular dichroism spectroscopic data.41 The fractions of α-helix, β-sheet, turn, and unordered coil were estimated by SELCON3 and are shown in Figure 5b. It should be noted that Sreedhara et al. recently studied the pH-induced change in the structure of bovine lactoferrin, which was similar to our results in this work, finding a structure change for native LF with a relatively low content of α-helix structure (18%, 21.4%, and 19.2% for LF3.5‑25°C, LF5.0‑25°C, and LF6.5‑25°C, respectively), but still rich in β-structure (32%, 33%, and 32.6% for LF3.5‑25°C, LF5.0‑25°C, and LF6.5‑25°C, respectively) (Figure 5b).45 On the other hand, some researchers reported that LF released iron at pH 3.0−4.0, resulting in a less compact structure.3,36 Therefore, the structure of LF at pH 3.5 might be less compact than those at pH 5.0 and 6.5. The results in our study were consistent with the reports for LF at pH 3.5 with a relatively higher content (29.1%) of unordered structure than those at pH 5.0 (25.4%) and 6.5 (27.2%). The protein conformational analysis based on CD data indicated that the thermal process led to changes in the secondary structure of LF. In the presence of 1.0 mM EGCG, the α-helical content was increased with a concomitant reduction of β-sheet and unordered coil contents; whereas the content of β-turn did not exhibit obvious change (Figure 5c). Therefore, it could be concluded that EGCG induced a progressive increase in the proportion of α-helix structure at the cost of β-sheet and unordered coil structure of LF (both native and thermally modified LF) at pH 3.5, 5.0, and 6.5. In other words, it indicated that EGCG contributed largely to the stability of LF helical conformation and LF became a slightly looser conformation due to the increase of unordered coil fraction. Comparison of Binding Constant and Site of EGCG with Native LF and Thermally Modified LF. In general, heating induces conformational (secondary/tertiary structure) changes of proteins, and exposes previously buried hydrophobic sites, which may influence LF affinity with EGCG. Aromatic residues (Phe, Trp, and Tyr) contribute to hydrophobic interactions that stabilize the core of protein interiors because they have relatively large apolar surface areas.71 The tryptophan (Trp) residues are often found fully or partially buried in the hydrophobic core of protein interiors, at the interface between

Table 4. Stern−Volmer Quenching Constant (Ksv), the Binding Constant (Ka), and the Number of Binding Sites (n) for the Interaction of EGCG with Native and Thermally Modified LF at pH 3.5, 5.0, and 6.5 pH 3.5

5.0

6.5

LF25°C LF70°C LF90°C LF25°C LF70°C LF90°C LF25°C LF70°C LF90°C

Ksv [104 M−1]

R2

Ka [104 M−1]

n

R2

6.30 5.77 5.60 6.50 11.40 17.60 7.61 9.43 10.60

0.988 0.994 0.959 0.932 0.961 0.965 0.980 0.965 0.927

0.46 1.44 2.55 15.23 5.31 0.41 0.24 0.18 0.03

0.96 1.09 1.14 1.32 1.17 0.85 0.86 0.82 0.61

0.970 0.998 0.914 0.954 0.951 0.965 0.954 0.948 0.987

plots for LF fluorescence quenching by EGCG. In the linear range of the Stern−Volmer regression curve, the average quenching constants (Ksv) of LF were estimated as 6.3, 5.8, and 5.6 × 104 M−1 for LF3.5‑25°C, LF3.5‑70°C, and LF3.5‑90°C, respectively; 6.5, 11.4, and 17.6 × 104 M−1 for LF5.0‑25°C, LF5.0‑25°C, and LF5.0‑25°C, respectively; 7.6, 9.4, and 10.6 × 104 M−1 for LF6.5‑25°C, LF6.5‑70°C, and LF6.5‑90°C, respectively. It is well-known that Trp τ0 depends on pH and buffer composition. In buffer solutions at pH < 7.0, Trp τ0 is in the range of 2.33 ± 0.15 ns (at pH 2.55) to 3.16 ± 0.10 ns (at pH 6.8).75 The lowest Ksv value estimated in our study was 5.6 × 104 M−1 (for I



Article

LF3.5‑25°C), yielding kq above 2.4 × 1013 M−1 s−1, at least 3 orders of magnitude higher than 2.0 × 1010 M−1 s−1, the maximum collisional (dynamic) quenching constant for various quenchers interacting with biopolymers.76,77 These results thus implied that the quenching process was static quenching involving the formation of LF−EGCG complexes. For the static mechanism of quenching, the double logarithmic Stern−Volmer equation (eq 2) could be used to analyze the quenching data and calculation of the apparent binding constants (Ka) of the LF−EGCG complexes formed. As illustrated in Table 4, the binding constants (Ka) and the number of binding sites per LF molecule (n) for EGCG with LF were increased with the rise of denaturation temperature at pH 3.5, while they were decreased at pH 5.0 and 6.5. Most of the Ka values were in the range of 103−105 M−1, except LF6.5‑90°C, which displayed great thermal aggregation behavior at pH 6.5. The association constants obtained were in agreement with previously published data for other proteins (such as bovine serum albumin, whey protein isolate, sodium caseinate) and EGCG by applying the same methodology (104−105 M−1).12,13,31 The Ka values were estimated as LF5.0‑25°C > LF5.0‑70°C > LF3.5‑90°C > LF3.5‑70°C > LF3.5‑25°C > LF5.0‑90°C > LF6.5‑25°C > LF6.5‑70°C > LF6.5‑90°C. Based on the aforementioned result, we concluded that if we do not take LF5.0‑90°C into account, the affinity of EGCG toward LF was pH dependent and had the following order: pH 5.0 > pH 3.5 > pH 6.5. Lower pH led to stronger binding because the dissociation of protein had more binding sites at lower pH.78 Therefore, we hypothesized that when pH changes away from the pI of LF, the electrostatic repulsion between LF molecules increases, which inhibits the self-association of the protein and facilitates the binding of EGCG to LF. The interactions between chlorogenic acid and several proteins such as BSA, lysozyme, and α-lactalbumin at pH ≤ 7 resulted from noncovalent bonds and the amount of chlorogenic acid bound by BSA (per molecule) was somewhat higher at lower pH.79 Some researchers reported that the EGCG-binding capacity of thermally modified β-lactoglobulin could be enhanced compared to the native protein at pH 7.0,31 while, with the rise of thermal denaturation temperature, the binding constant decreased at pH 5.0 and 6.5, which implied that EGCG had strong affinity for native LF but weak affinity for thermally modified LF. It seemed that the probably more unfolded structure of LF5.0‑70°C and LF5.0‑90°C decreased the exposure of noncovalent sites normally located on the surface of both lobes of the protein, reducing the binding constant when LF−EGCG complexes formed. Moreover, the thermal aggregation of LF at pH 6.5 probably exposed nonpolar groups normally buried in the LF nonpolar interior, which increased the self-association of the protein through hydrophobic attraction and decreased the binding of EGCG to LF. In contrast, EGCG had weak affinity for native LF but strong affinity for thermally modified LF at pH 3.5. This might be ascribed to the difference of pH in the solution and the secondary structure of the protein at pH 3.5. In this study, the native and thermally modified LF had potential to form coassembled vehicles for the delivery of EGCG. The changes of pH in the solution and the structure of protein were the crucial factors influencing the noncovalent interaction between LF and EGCG. LF−EGCG nanoparticles have good transparency at pH 3.5 and 5.0, which could be applied in clear beverages and liquid foods. At the same time, LF−EGCG submicrometer particles remained relatively stable to particle aggregation and were formed at pH 6.5, which could

be applied in cloudy beverages and liquid foods. Overall, LF− EGCG nano- and submicrometer particles as protective vehicles for EGCG could be obtained by choosing the appropriate reaction parameters, which was beneficial for the development of controlled release of other bioactive materials.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-62737034. Fax: +86-10-62737986. E-mail: [email protected]. Funding

The research work was funded by the National Natural Science Foundation of China under Grant No. 31371835. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Wakabayashi, H.; Yamauchi, K.; Takase, M. Lactoferrin research, technology and applications. Int. Dairy J. 2006, 16, 1241−1251. (2) Levay, P. F.; Viljoen, M. Lactoferrin: a general review. Acta Haematol. 1995, 80, 252−267. (3) Steijns, J. M.; Van Hooijdonk, A. C. M. Occurrence, structure, biochemical properties and technological characteristics of lactoferrin. Br. J. Nutr. 2000, 84, 11−17. (4) Satué-Gracia, M. T.; Frankel, E. N.; Rangavajhyala, N.; German, J. B. Lactoferrin in infant formulas: effect on oxidation. J. Agric. Food Chem. 2000, 48, 4984−4990. (5) Soejima, T.; Yamauchi, K.; Yamamoto, T.; Ohara, Y.; Nagao, E.; Kanbara, K.; Fujisawa, M.; Okuda, Y.; Namba, S. Determination of bovine lactoferrin in lactoferrin-supplemented dairy products and raw milk by an automated latex assay. J. Dairy Res. 2007, 74, 100−105. (6) Sui, Q.; Roginski, H.; Williams, R. P. W.; Versteeg, C.; Wan, J. Effect of pulsed electric field and thermal treatment on the physicochemical properties of lactoferrin with different iron saturation levels. Int. Dairy J. 2010, 20, 707−714. (7) Kao, Y. H.; Chang, H. H.; Lee, M. J.; Chen, C. L. Tea, obesity, and diabetes. Mol. Nutr. Food Res. 2006, 50, 188−210. (8) Levites, Y.; Weinreb, O.; Maor, G.; Youdim, M. B. H.; Mandel, S. Green tea polyphenol (−)-epigallocatechin-3-gallate prevents Nmethyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. J. Neurochem. 2001, 78, 1073−1082. (9) Chyu, K. Y.; Babbidge, S. M.; Zhao, X. N.; Dandillaya, R.; Rietveld, A. G.; Yano, J.; Dimayuga, P.; Cercek, B.; Shah, P. K. Differential effects of green tea-derived catechin on developing versus established atherosclerosis in apolipoprotein E-null mice. Circulation 2004, 109, 2448−2453. (10) Cabrera, C.; Artacho, R.; Gimenez, R. Beneficial effects of green tea-a review. J. Am. Coll. Nutr. 2006, 25, 79−99. (11) Nagle, D. G.; Ferreira, D.; Zhou, Y. D. Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives. Phytochemistry 2006, 67, 1849−1855. (12) Kanakis, C. D.; Hasni, I.; Bourassa, P.; Tarantilis, P. A.; Polissioiu, M. G.; Tajmir-Riahi, H.- A. Milk β-lactoglobulin complexes with tea polyphenols. Food Chem. 2011, 127, 1046−1055. (13) Hasni, I.; Bourassa, P.; Hamdani, S.; Samson, G.; Carpentier, R.; Tajmir-Riahi, H.-A. Interaction of milk α-and β-caseins with tea polyphenols. Food Chem. 2011, 126, 630−639. (14) Ishii, T.; Ichikawa, T.; Minoda, K.; Kusaka, K.; Ito, S.; Suzuki, Y.; Nakayama, T. Human serum albumin as an antioxidant in the oxidation of (−)-epigallocatechin gallate: participation of reversible covalent binding for interaction and stabilization. Biosci., Biotechnol., Biochem. 2011, 75, 100−106. (15) Wang, X.; Ho, C.-T.; Huang, Q. Investigation of Adsorption Behavior of (−)-Epigallocatechin Gallate on Bovine Serum Albumin Surface Using Quartz Crystal Microbalance with Dissipation Monitoring. J. Agric. Food Chem. 2007, 55, 4987−4992. J



Article

(16) Bohin, M. C.; Vincken, J. P.; van der Hijden, H. T.; Gruppen, H. Efficacy of food proteins as carriers for flavonoids. J. Agric. Food Chem. 2012, 60, 4136−4143. (17) Rodríguez-Franco, D. A.; Vázquez-Moreno, L.; Ramos-Clamont, M. G. Actividad antimicrobiana de la lactoferrina: Mecanismos y ́ aplicaciones clinicas potenciales. Rev. Latinoam. Microbiol. 2005, 47, 101−11. (18) Takakura, N.; Wakabayashi, H.; Ishibashi, H.; Teraguchi, S.; Tamura, Y.; Yamaguchi, H.; Abe, S. Oral lactoferrin treatment of experimental oral candidiasis in mice. Antimicrob. Agents Chemother. 2003, 47, 2619−2623. (19) Ward, P. P.; Uribe-Luna, S.; Conneely, O. M. Lactoferrin and host defense. Biochem. Cell Biol. 2002, 80, 95−102. (20) Yamauchi, K.; Tomita, M.; Giehl, T. J.; Ellison, R. 3. Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. Infect. Immun. 1993, 61, 719−728. (21) Medina, I.; Tombo, I.; Satué-Gracia, M. T.; German, J. B.; Frankel, E. N. Effects of natural phenolic compounds on the antioxidant activity of lactoferrin in liposomes and oil-in-water emulsions. J. Agric. Food Chem. 2002, 50, 2392−2399. (22) Mohan, K. V. P.; Gunasekaran, P.; Varalakshmi, E.; Hara, Y.; Nagini, S. In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells. Cell. Biol. Int. 2007, 31, 599−608. (23) Chandra Mohan, K. V. P.; Letchoumy, P. V.; Hara, Y.; Nagini, S. Combination chemoprevention of hamster buccal pouch carcinogenesis by bovine milk lactoferrin and black tea polyphenols. Cancer. Invest. 2008, 26, 193−201. (24) Letchoumy, P. V.; Mohan, K. C.; Stegeman, J. J.; Gelboin, H. V.; Hara, Y. In vitro antioxidative potential of lactoferrin and black tea polyphenols and protective effects in vivo on carcinogen activation, DNA damage, proliferation, invasion and angiogenesis during experimental oral carcinogenesis. Oncol. Res. 2008, 17, 193. (25) Bromley, E. H. C.; Krebs, M. R. H.; Donald, A. M. Mechanisms of structure formation in particulate gels of β-lactoglobulin formed near the isoelectric point. Eur. Phys. J. E: Soft Matter Biol. Phys. 2006, 21, 145−152. (26) Donato, L.; Schmitt, C.; Bovetto, L.; Rouvet, M. Mechanism of formation of stable heat-induced β-lactoglobulin microgels. Int. Dairy J. 2009, 19, 295−306. (27) Hoffmann, M. A. M.; Roefs, S. P. F. M.; Verheul, M.; Mil, P. J. J. M. v.; Kruif, K. G. d. Aggregation of β-lactoglobulin studied by in situ light scattering. J. Dairy Res. 1996, 63, 423−440. (28) Jones, O. G.; Decker, E. A.; McClements, D. J. Comparison of protein−polysaccharide nanoparticle fabrication methods: Impact of biopolymer complexation before or after particle formation. J. Colloid Interface Sci. 2010, 344, 21−29. (29) Santipanichwong, R.; Suphantharika, M.; Weiss, J.; McClements, D. J. Core-Shell Biopolymer Nanoparticles Produced by Electrostatic Deposition of Beet Pectin onto Heat-Denatured β-Lactoglobulin Aggregates. J. Food Sci. 2008, 73, N23−N30. (30) Bengoechea, C.; Peinado, I.; McClements, D. J. Formation of protein nanoparticles by controlled heat treatment of lactoferrin: Factors affecting particle characteristics. Food Hydrocolloids 2011, 25, 1354−1360. (31) Shpigelman, A.; Israeli, G.; Livney, Y. D. Thermally-induced protein−polyphenol co-assemblies: beta lactoglobulin-based nanocomplexes as protective nanovehicles for EGCG. Food Hydrocolloids 2010, 24, 735−743. (32) Chanasattru, W.; Jones, O. G.; Decker, E. A.; McClements, D. J. Impact of cosolvents on formation and properties of biopolymer nanoparticles formed by heat treatment of β-lactoglobulin-Pectin complexes. Food Hydrocolloids 2009, 23, 2450−2457. (33) Stănciuc, N.; Aprodu, I.; Râpeanu, G.; van der Plancken, I.; Bahrim, G.; Hendrickx, M. Analysis of the thermally induced structural changes of bovine lactoferrin. J. Agric. Food Chem. 2013, 61, 2234− 2243. (34) Kussendrager, K. D. Effects of heat treatment on structure and iron-binding capacity of bovine lactoferrin. IDF Bull. 1994,

No. Indigenous antimicrobial agents of milk: recent developments, 133−146. (35) Paulsson, M. A.; Svensson, U.; Kishore, A. R.; Satyanarayan Naidu, A. Thermal behavior of bovine lactoferrin in water and its relation to bacterial interaction and antibacterial activity. J. Dairy Sci. 1993, 76, 3711−3720. (36) Baker, E. N.; Baker, H. M. Molecular structure, binding properties and dynamics of lactoferrin. Cell. Mol. Life Sci. 2005, 62, 2531−2539. (37) Baker, E. N.; Baker, H. M. A structural framework for understanding the multifunctional character of lactoferrin. Biochimie 2009, 91, 3−10. (38) Bokkhim, H.; Bansal, N.; Grøndahl, L.; Bhandari, B. Physicochemical properties of different forms of bovine lactoferrin. Food Chem. 2013, 141, 3007−3013. (39) de Freitas, V.; Carvalho, E.; Mateus, N. Study of carbohydrate influence on protein-tannin aggregation by nephelometry. Food Chem. 2003, 81, 503−509. (40) Lobley, A.; Whitmore, L.; Wallace, B. A. DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 2002, 18, 211−212. (41) Whitmore, L.; Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 2004, 32 (Suppl. 2), W668− W673. (42) Zhang, Y.; Zhong, Q. Probing the binding between norbixin and dairy proteins by spectroscopy methods. Food Chem. 2013, 139, 611− 616. (43) Persson, B. A.; Lund, M.; Forsman, J.; Chatterton, D. E. W.; Åkesson, T. Molecular evidence of stereo-specific lactoferrin dimers in solution. Biophys. Chem. 2010, 151, 187−189. (44) Abe, H.; Saito, H.; Miyakawa, H.; Tamura, Y.; Shimamura, S.; Nagao, E.; Tomita, M. Heat stability of bovine lactoferrin at acidic pH. J. Dairy Sci. 1991, 74, 65−71. (45) Sreedhara, A.; Flengsrud, R.; Prakash, V.; Krowarsch, D.; Langsrud, T.; Kaul, P.; Devold, T. G.; Vegarud, G. E. A comparison of effects of pH on the thermal stability and conformation of caprine and bovine lactoferrin. Int. Dairy J. 2010, 20, 487−494. (46) Brisson, G.; Britten, M.; Pouliot, Y. Heat-induced aggregation of bovine lactoferrin at neutral pH: Effect of iron saturation. Int. Dairy J. 2007, 17, 617−624. (47) Von Staszewski, M.; Jara, F. L.; Ruiz, A. L. T. G.; Jagus, R. J.; Carvalho, J. E.; Pilosof, A. M. R. Nanocomplex formation between βlactoglobulin or caseinomacropeptide and green tea polyphenols: Impact on protein gelation and polyphenols antiproliferative activity. J. Funct. Foods 2012, 4, 800−809. (48) Wang, S. H.; Liu, F. F.; Dong, X. Y.; Sun, Y. Thermodynamic analysis of the molecular interactions between amyloid β-peptide 42 and (−)-epigallocatechin-3-gallate. J. Phys. Chem. B 2010, 114, 11576− 11583. (49) Charlton, A. J.; Baxter, N. J.; Khan, M. L.; Moir, A. J.; Haslam, E.; Davies, A. P.; Williamson, M. P. Polyphenol/peptide binding and precipitation. J. Agric. Food Chem. 2002, 50, 1593−601. (50) Naczk, M.; Oickle, D.; Pink, D.; Shahidi, F. Protein precipitating capacity of crude canola tannins: effect of pH, tannin, and protein concentrations. J. Agric. Food Chem. 1996, 44, 2144−2148. (51) Xia, S. Q.; Li, Y.; Xia, Q. Y.; Zhang, X. M.; Huang, Q. R. Glycosylation of bovine serum albumin via maillard reaction prevents epigallocatechin-3-gallate-induced protein aggregation. Food Hydrocolloids 2014, DOI: 10.1016/j.foodhyd.2014.05.022. (52) Tokle, T.; Lesmes, U.; McClements, D. J. Impact of electrostatic deposition of anionic polysaccharides on the stability of oil droplets coated by lactoferrin. J. Agric. Food Chem. 2010, 58, 9825−9832. (53) Von Staszewski, M.; Jagus, R. J.; Pilosof, A. M. R. Influence of green tea polyphenols on the colloidal stability and gelation of WPC. Food Hydrocolloids 2011, 25, 1077−1084. (54) Robb, C. S.; Geldart, S. E.; Seelenbinder, J. A.; Brown, P. R. Analysis of green tea constituents by HPLC-FTIR. J. Liq. Chromatogr. Relat. Technol. 2002, 25 (5), 787−801. K



Article

solution as a function of pH and buffer composition. Can. J. Chem. 1981, 59, 1037−1044. (76) Lange, D. C.; Kothari, R.; Patel, R. C.; Patel, S. C. Retinol and retinoic acid bind to a surface cleft in bovine β-lactoglobulin: a method of binding site determination using fluorescence resonance energy transfer. Biophys. Chem. 1998, 74, 45−51. (77) Gonçalves, R.; Mateus, N.; De Freitas, V. Study of the interaction of pancreatic lipase with procyanidins by optical and enzymatic methods. J. Agric. Food Chem. 2010, 58, 11901−11906. (78) Sastry, M. C. S.; Rao, M S N. Binding of chlorogenic acid by the isolated polyphenol-free 11 S protein of sunflower (Helianthus annuus) seed. J. Agric. Food Chem. 1990, 38, 2103−2110. (79) Prigent, S. V. E.; Gruppen, H.; Visser, A. J. W. G.; Van Koningsveld, G. A.; De Jong, G. A.; Voragen, A. G. Effects of noncovalent interactions with 5-O-caffeoylquinic acid (chlorogenic acid) on the heat denaturation and solubility of globular proteins. J. Agric. Food Chem. 2003, 51, 5088−5095.

(55) Chen, Y. C.; Yu, S. H.; Tsai, G. J.; Tang, D. W.; Mi, F. L.; Peng, Y. P. Novel technology for the preparation of self-assembled catechin/ gelatin nanoparticles and their characterization. J. Agric. Food Chem. 2010, 58, 6728−6734. (56) Zhu, B.; Li, J.; He, Y.; et al. Hydrogen-Bonding Interaction and Crystalline Morphology in the Binary Blends of Poly (ε-caprolactone) and Polyphenol Catechin. Macromol. Biosci. 2003, 3, 684−693. (57) Peña, C.; Larranaga, M.; Gabilondo, N.; Tejado, A.; Echeverria, J. M.; Mondragon, I. Synthesis and characterization of phenolic novolacs modified by chestnut and mimosa tannin extracts. J. Appl. Polym. Sci. 2006, 100, 4412−4419. (58) Frazier, R. A.; Deaville, E. R.; Green, R. J.; Stringano, E.; Willoughby, I.; Plant, J.; Mueller-Harvey, I. Interactions of tea tannins and condensed tannins with proteins. J. Pharmaceut. Biomed. 2010, 51, 490−495. (59) Ye, J.; Fan, F.; Xu, X.; Liang, Y. Interactions of black and green tea polyphenols with whole milk. Food Res. Int. 2013, 53, 449−455. (60) Renugopalakrishnan, V.; Chandrakasan, G.; Moore, S.; Hutson, T. B.; Berney, C. V.; Bhatnagar, R. S. Bound water in collagen: evidence from Fourier transform infrared and Fourier transform infrared photoacoustic spectroscopic study. Macromolecules 1989, 22, 4121−4124. (61) Sessa, D. J.; Mohamed, A.; Byars, J. A. Chemistry and physical properties of melt-processed and solution-cross-linked corn zein. J. Agric. Food Chem. 2008, 56, 7067−7075. (62) Beauchemin, R.; N’Soukpoe-Kossi, C. N.; Thomas, T. J.; Thomas, T.; Carpentier, R.; Tajmir-Riahi, H. A. Polyamine analogues bind human serum albumin. Biomacromolecules 2007, 8, 3177−3183. (63) Bandekar, J. Amide modes and protein conformation. Biochim. Biophys. Acta 1992, 1120, 123−143. (64) Krimm, S.; Bandekar, J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv. Protein Chem. 1986, 38, 181−364. (65) Eissa, A. S.; Puhl, C.; Kadla, J. F.; Khan, S. A. Enzymatic crosslinking of β-lactoglobulin: conformational properties using FTIR spectroscopy. Biomacromolecules 2006, 7, 1707−1713. (66) Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. 2007, 39, 549−559. (67) Constantinescu, C.; Palla-Papavlu, A.; Rotaru, A.; Florian, P.; Chelu, F.; Icriverzi, M.; Nedelcea, A.; Dinca, V.; Roseanu, A.; Dinescu, M. Multifunctional thin films of lactoferrin for biochemical use deposited by MAPLE technique. Appl. Surf. Sci. 2009, 255, 5491− 5495. (68) Sadeghi, S.; Madadlou, A.; Yarmand, M. Microemulsification− cold gelation of whey proteins for nanoencapsulation of date palm pit extract. Food Hydrocolloids 2014, 35, 590−596. (69) Bagheri, L.; Madadlou, A.; Yarmand, M.; Mousavi, M. E. Nanoencapsulation of date palm pit extract in whey protein particles generated via desolvation method. Food Res. Int. 2013, 51, 866−871. (70) Yang, W.; Liu, F.; Xu, C.; Fang, Y.; Gao, Y. Molecular interaction between (−)-epigallocatechin-3-gallate and bovine lactoferrin using multi-spectroscopic method and isothermal titration calorimetry. Food Res. Int. 2014, 64, 141−149. (71) Kuznetsova, I. M.; Turoverov, K. K.; Uversky, V. N. Use of the phase diagram method to analyze the protein unfolding-refolding reactions: fishing out the “invisible” intermediates. J. Proteome Res. 2004, 3, 485−494. (72) Royer, C. A. Probing protein folding and conformational transitions with fluorescence. Chem. Rev. 2006, 106, 1769−1784. (73) Xiao, J.; Shi, J.; Cao, H.; Wu, S.; Ren, F.; Xu, M. Analysis of binding interaction between puerarin and bovine serum albumin by multi-spectroscopic method. J. Pharm. Biomed. Anal. 2007, 45, 609− 615. (74) Lakowicz, J. R.; Masters, B. R. Principles of fluorescence spectroscopy. J. Biomed. Opt. 2008, 13, 9901. (75) Gudgin, E.; Lopez-Delgado, R.; Ware, W. R. The tryptophan fluorescence lifetime puzzle: A study of decay times in aqueous L


Native and Thermally Modified ProteinâPolyphenol Coassemblies

Recommend Documents

Native and Thermally Modified ProteinâPolyphenol Coassemblies