Inhibition of the Aggregation of Lactoferrin and (â)-Epigallocatechin

Article pubs.acs.org/JAFC

Inhibition of the Aggregation of Lactoferrin and (−)-Epigallocatechin Gallate in the Presence of Polyphenols, Oligosaccharides, and Collagen Peptide Wei Yang, Fuguo Liu, Chenqi Xu, Cuixia Sun, 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, People’s Republic of China ABSTRACT: The aggregation of lactoferrin and (−)-epigallocatechin gallate (EGCG) was inhibited by polyphenols, oligosaccharides, and collagen peptide in this study. Polyphenols, oligosaccharides, or collagen peptide can effectively prevent the formation of lactoferrin−EGCG aggregates, respectively. The addition sequence of lactoferrin, polyphenols (oligosaccharides or collagen peptide) and EGCG can affect the turbidity and particle size of the ternary complexes in the buffer solution; however, it hardly affected the ζ-potential and fluorescence characteristics. With either positive or negative charge, polyphenols and collagen peptide disrupted the formation of lactoferrin−EGCG aggregate mainly through the mechanism of its competition with EGCG molecules which surrounded the lactoferrin molecule surface with weaker binding affinities, forming polyphenols or a collagen peptide−lactoferrin−EGCG ternary complex; for neutral oligosaccharides, the ternary complex was generated mainly through steric effects, accompanied by a change in the lactoferrin secondary structure induced by gallic acid, chlorogenic acid, and xylooligosaccharide. Polyphenols, oligosaccharides, or collagen peptide restraining the formation of lactoferrin−EGCG aggregate could be applied in the design of clear products in the food, pharmaceutical, and cosmetic industries. KEYWORDS: aggregation inhibitors, lactoferrin, (−)-epigallocatechin gallate, polyphenols, oligosaccharides, collagen peptide

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INTRODUCTION Polyphenol may interact with protein reversibly by usually noncovalent forces such as hydrogen bonding, hydrophobic bonding, and van der Waals forces.1−3 Turbidity is generally due to the binding of polyphenol present in beverage and food products from plant origin with proteins to form aggregates. At the same time, the protein−polyphenol complexes are considered to be responsible for the sensation of astringency on the human palate. Astringency is often perceived as an undesirable quality factor in some beverages such as fruit juices and red wine if they are too intense.4 The interaction between polyphenol and proteins has been proven to generate negative effects, reducing the palate lubrication, causing an unpleasant sensation of roughness, dryness, and constriction and reducing absorption of nutrients.5−7 There exist a number of factors, such as pH, ionic strength, alcohol, nature of the solvent, and presence of carbohydrates, affecting the protein−polyphenol haze formation in food systems.8 Several studies have demonstrated that some polysaccharides, such as pectin, xanthan gum, and arabic gum, can be applied as food colloids, to disrupt polyphenol and protein interaction in food systems.7,9−13 There are three mechanisms responsible for the inhibition of protein− polyphenol aggregation by some polysaccharides: (1) molecular association between polysaccharides, such as arabic gum, βcyclodextrin, and polyphenols, competing with protein aggregation;7,13 (2) the formation of a protein−polyphenol− polysaccharide ternary complex, enhancing its solubility in aqueous medium, such as pectin;13 (3) steric effects, limiting the number of available binding sites of proteins to readily fit polyphenols, such as dextran.9 © 2015 American Chemical Society

It is well-known that EGCG can act as a multidentate ligand and bridge protein,2,14 forming a lactoferrin−EGCG aggregate. Yang et al. reported that EGCG was spontaneously bound to lactoferrin by a two-stage mechanism: first EGCG molecules were bound within the hydrophobic pockets of lactoferrin with strong binding affinities, and then EGCG molecules surrounded the lactoferrin molecule surface with weaker binding affinities. A further increase in EGCG concentration led to phase separation (haze formation).15 Some functional food ingredients, such as polyphenols, oligosaccharides, and collagen peptide, are widely used in beverage, food, and pharmaceutical formulations. Proteins are thought to have a number of sites bound by polyphenols, oligosaccharides, or peptides.16−18 Oligosaccharides and collagen peptide, on the other hand, have a number of ends that could bind polyphenols. Therefore, polyphenols, oligosaccharides, or peptides might affect the interaction between the protein and polyphenol when these components are intergrated together. So far, the effect of polyphenols, polysaccharides, and collagen peptide on the interaction of the polyphenol with protein has been largely overlooked. Phenolic compounds are chemically structured as a hydroxyl group bonded to an aromatic ring. Dietary polyphenols, natural compounds largely in fruits, vegetables, and cereals, are consumed as part of the human diet in significant amounts19,20 and have become an intense focus of research interest due to their health-beneficial effects, especially in the treatment and prevention of several chronic diseases. Oligosaccharides, Received: December 7, 2014 Accepted: May 4, 2015 Published: May 4, 2015 5035

DOI: 10.1021/acs.jafc.5b01881 J. Agric. Food Chem. 2015, 63, 5035−5045

Article

Journal of Agricultural and Food Chemistry

complexes containing lactoferrin, EGCG, and a polyphenol, oligosaccharide, or collagen peptide prepared by M2. Method III (M3). In brief, 1.0 mL of EGCG (0.1832% w/v, 4.0 mM) and 1.0 mL of a polyphenol, oligosaccharide, or collagen peptide solution in different concentrations were mixed on a vortex shaker (QL-866, Haimen QILINBEIER Instruments Co., Ltd., Haimen, People’s Republic of China) at 3000 rpm for 1 min. The mixtures were allowed to stand for 2 h. After that, 2.0 mL of lactoferrin (1.6% w/v, 0.2 mM) solution was added to these mixtures. Then these mixtures were blended for 1 min and allowed to stand for 2 h before the measurement. The abbreviation X-3 (X = gallic acid, chlorogenic acid, glucose, XOS, FOS, collagen peptide) is used to represent the ternary complexes containing lactoferrin, EGCG, and a polyphenol, oligosaccharide, or collagen peptide prepared by M3. Turbidity Measurements. Nephelometry experiments were performed with a HACH 2100N Laboratory Turbidimeter (Loveland, USA), and the aggregation behavior of lactoferrin and EGCG in the absence and presence of polyphenol, oligosaccharide, or collagen peptide solution in buffer 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. All experiments were performed in triplicate. Particle Size Measurements. Particle size and size distribution of the ternary complexes in the buffer solution were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, U.K.). The intensity of light scattered was monitored at a 90° angle. Results are described as cumulant mean diameter (size, nm) for particle size and polydispersity index (PdI) for size distribution. All experiments were performed in triplicate. ζ-Potential Measurements. The ζ potential of ternary complexes in the buffer solution was determined by a Zetasizer Nano-ZS90 particle electrophoresis instrument (Malvern Instruments, Worcestershire, U.K.). Samples were loaded in the cell, and the temperature was set at 25 °C without further dilution. The ζ-potential values are reported as the average of measurements for three freshly prepared samples, with three readings recorded for each sample. Fluorescence Spectroscopy Measurements. Fluorescence measurements were carried out using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Spectra were collected when the mixtures were kept at 25 °C for 2 h. Fluorescence emission spectra of the ternary complexes were recorded with an excitation wavelength at 292 nm. The synchronous fluorescence spectra were determined in the continuous range of 300− 600 nm with Δλ at 60 nm. Both excitation and emission band widths were set at 10 nm. Circular Dichroism Measurements. The CD spectra were recorded using a CD spectropolarimeter (Pistar π-180, Applied Photophysics Ltd. U.K.) in rhw far-UV (190−250 nm) region under constant nitrogen flush. The path length was 0.1 cm for the far-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).24,25 Each spectrum presented was obtained as the average of three consecutive scans with automatic subtraction of the buffer and/or polyphenol, oligosaccharide, or collagen peptide spectra. The concentrations of polyphenols, oligosaccharides, or collagen peptide in ternary complexes were 0.75% for gallic acid, 0.25% for chlorogenic acid, 10.25% for glucose, FOS, and XOS, and 2.25% for collagen peptide. Statistical Analysis. The experiments were conducted in duplicate, and all analyses were done in triplicate. The results are 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 significant differences of means (p < 0.05) were determined using the Duncan multiple-range test. The correlation analysis was carried out using the Pearson mode. The

especially xylo-oligosaccharide (XOS) and fructo-oligosaccharide (FOS), with a mouthfeel similar to that of sugar but being only 20−70% as sweet as table sugar, have received much attention recently as functional food ingredients.21 They are often applied to enhance the survivability and colonization of probiotic bacteria when they are enriched in foods.22 Because of its nutritional and medical benefits, collagen peptide is easily absorbed by the human body due to its low molecule weight and is often used as an ingredient and additive of functional foods and nutraceuticals.23 The objective of this work was to elucidate the effect of polyphenols, oligosaccharides, and collagen peptide on the aggregation of EGCG with lactoferrin. A clear dispersion containing lactoferrin, EGCG, and polyphenols (oligosaccharides or collagen peptide) can be applied in many fields, such as the food, pharmaceutical, and cosmetic industries.

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MATERIALS AND METHODS

Materials and Chemicals. Lactoferrin (purity ≥92.0%) from bovine whey was purchased from New Image International Limited (New Zealand). EGCG (purity ≥95.0%), gallic acid (purity ≥98.0%), and chlorogenic acid (purity ≥99.0%) was purchased from BSZH Science (Beijing, People’s Republic of China). Glucose (purity ≥99.0%) was purchased from Beijing Chemical Reagent (Beijing, People’s Republic of China). XOS (purity ≥95.0%) was supplied by Longlive Biotechnology Co., Ltd. (Shandong, People’s Republic of China). FOS (purity ≥95.0%) was supplied by Baolingbao Biotechnology Co., Ltd. (Shandong, People’s Republic of China). Collagen peptide (purity ≥99.0%; 2000 Da) from tilapia skin was supplied by Beijing Semnl Biotechnologies Co., Ltd. (Beijing, People’s Republic of China). Sample Preparation. Lactoferrin (1.6% w/v, 0.2 mM) and EGCG (0.1832 w/v, 4.0 mM) were dissolved in 10 mM potassium phosphate buffer solution (pH 6.0), and the solutions were each stirred constantly at 25 °C for at least 2 h. Preparation of Ternary Complexes Containing Lactoferrin, EGCG, and a Polyphenol, Oligosaccharide, or Collagen Peptide. Three addition sequences were applied to prepare the ternary complexes in the buffer solution. Different volumes of polyphenols, oligosaccharides, or collagen peptide stock solutions were mixed with designed volumes of potassium phosphate buffer solution. For the experiments, stock solutions of polyphenols (0.3% gallic acid, 1.0% chlorogenic acid), glucose and oligosaccharides (21.0%), and collagen peptide (9.0%) were prepared with potassium phosphate buffer solution. Method I (M1). Briefly, 2.0 mL of lactoferrin (1.6% w/v, 0.2 mM) and 1.0 mL of EGCG (0.1832% w/v, 4.0 mM) solutions were mixed on a vortex shaker (QL-866, Haimen QILINBEIER Instruments Co., Ltd., Haimen, People’s Republic of China) at 3000 rpm for 1 min. The mixture was allowed to stand for 2 h. After that, 1.0 mL of polyphenol, oligosaccharide, or collagen peptide solution in different concentrations was added and mixed for 1 min, and the mixture was allowed to stand for 2 h before the measurement. The abbreviation X-1 (X = gallic acid, chlorogenic acid, glucose, XOS, FOS, collagen peptide) is used to represent the ternary complexes containing lactoferrin, EGCG, and a polyphenol, oligosaccharide, or collagen peptide prepared by M1. Method II (M2). Briefly, 2.0 mL of lactoferrin (1.6% w/v, 0.2 mM) and 1.0 mL of a polyphenol, oligosaccharide, or collagen peptide solution in different concentrations were mixed on a vortex shaker (QL-866, Haimen QILINBEIER Instruments Co., Ltd., Haimen, People’s Republic of China) at 3000 rpm for 1 min. These mixtures were allowed to stand for 2 h. Then, 1.0 mL of EGCG (0.1832% w/v, 4.0 mM) solution was added to the different mixtures. After that, these mixtures were blended for 1 min and allowed to stand for 2 h before the measurement. The abbreviation X-2 (X = gallic acid, chlorogenic acid, glucose, XOS, FOS, collagen peptide) is used to represent the ternary 5036


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Figure 1. Variation (%) in turbidity of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG) with increasing concentrations of polyphenols, oligosaccharides, or collagen peptide. concordance between experimental data and calculated values was established by the root-mean-squared deviation.

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collagen peptide in the solution restrained the formation of lactoferrin−EGCG insoluble aggregates (Figure 1). The turbidity of the aggregates in the buffer solution prepared by three addition sequences progressively decreased with an increase in gallic acid, chlorogenic acid, and collagen peptide concentrations, respectively, which indicated that smaller particles were formed to scatter light for the ternary complexes. M3 was the most effective method to inhibit the formation of lactoferrin−EGCG aggregate for gallic acid and chlorogenic acid, while M2 was the most effective method for collagen peptide. These results revealed that gallic acid was the most effective (the gallic acid amount required was about 10fold lower than that of chlorogenic acid and about 100-fold lower than that of collagen peptide) in restraining the interaction between lactoferrin and EGCG. The presence of glucose, XOS, and FOS, respectively, in the solution retarded the formation of insoluble lactoferrin−EGCG aggregate with use of M2 and M3. M2 was the most effective method for glucose, while M3 was the most effective method for XOS and FOS. It should be noted that some exception was observed for chlorogenic acid, XOS, and FOS by M1 and M2. Significant (p < 0.05) increases of the insoluble aggregates were found at low concentrations, 0−0.5 g/L for chlorogenic acid and 0−10 g/L for XOS and FOS, which could be explained by their size increase, resulting from the adsorption of chlorogenic acid,

RESULTS AND DISCUSSION

The turbidity of the lactoferrin−EGCG aggregate in the buffer solution was around 160 NTU when the EGCG concentration was 1.0 mM. It should be noted that the ζ potential of the lactoferrin and lactoferrin−EGCG aggregate was around 0 mV, despite the pH of the buffer being below the reported isoelectric point of lactoferrin (pI ≈ 8). The most likely explanation for this effect is that the cationic groups on the adsorbed lactoferrin bound some anionic phosphate ions present in the buffer.26 Several assays were performed with progressively increasing concentrations of polyphenols, oligosaccharides, or collagen peptide, until practically no changes in the amount of insoluble lactoferrin−EGCG aggregate were observed. The effect of the component addition sequence on the turbidity, particle size, and structure of the ternary complexes was also investigated. Nephelometry Measurements. Nephelometry measures the formation of the structures of high-molecular-weight substances at a macromolecular level, taking into account all physicochemical driving forces involved in the formation of aggregates. It is very interesting to find that, except for a few cases, the presence of the polyphenols, oligosaccharides, or 5037


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Figure 2. Influence of polyphenol, oligosaccharide, or collagen peptide concentration on the particle size of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG).

It is interesting to find that, as the aggregation decreased, a narrow range of particle size distribution was observed for gallic acid, chlorogenic acid, and collagen peptide, respectively, by M1, M2, and M3 (a decreased PDI value, from 0.792 to 0.34 for gallic acid, to 0.314 for chlorogenic acid, and to 0.413 for collagen peptide), indicating a homogeneous population of these aggregates. As glucose, XOS, and FOS concentrations increased, the aggregation of the ternary complexes decreased and a narrow range of the particle size distribution was also observed (a decreased PDI value, from 0.792 to 0.483 for glucose, to 0.437 for XOS, and to 0.403 for FOS) by M2 and M3, also implying a heterogeneous population of these aggregates. An opposite phenomenon was observed by using M1; that is, the PDI value was increased to around 1.0 as the concentrations of glucose, XOS, and FOS, respectively, were increased, indicating the formation of more heterogeneous aggregates. Yang et al. reported that EGCG molecules might be first bound within the hydrophobic pockets of lactoferrin with strong binding affinities. As the concentration of EGCG increased, it surrounded the lactoferrin molecule surface with weaker binding affinities, acting as multidentate ligands and bridge proteins or as a protein−polyphenol complex, leading to the formation of metastable dispersions of particles; further, an increase of polyphenols led to phase separation (haze formation).15 Therefore, it is hypothesized that when lactoferrin and EGCG were mixed together in the absence of

XOS, or FOS molecules on the surface of lactoferrin−EGCG aggregate. This result was similar to that in the report of de Freitas et al., which showed that the presence of dextran in the insoluble aggregates formed by procyanidins and bovine serum albumin tended to remain constant or to slightly increase.9 In particular, for XOS and FOS by M1, but not by M2, a further increase in the concentration of XOS and FOS did not show an effective inhibition of the aggregation between lactoferrin and EGCG. These results obviously revealed that the turbidity of the ternary complexes in the buffer solution was mostly related to the addition sequence of lactoferrin, polyphenol (oligosaccharide or collagen peptide), and EGCG, especially for XOS and FOS. On the basis of the aforementioned results, it was possible to classify the capacity of polyphenols, oligosaccharides, and collagen peptide in retarding the aggregation: gallic acid > chlorogenic acid > collagen peptide > glucose > XOS > FOS. Size Measurements of Ternary Complexes. The measurement of ternary complex size in the buffer solution resulting from the interaction between lactoferrin and EGCG in the absence and presence of polyphenols, oligosaccharides, or collagen peptide was performed by DLS. Figure 2 shows the mean particle size and polydispersity of aggregates. As expected, with an increase in polyphenol, oligosaccharide, or collagen peptide concentration, the mean particle size showed changes synchronous with the turbidity. 5038


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Figure 3. Influence of polyphenol, oligosaccharide, or collagen peptide concentration on the ζ potential of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG).

polyphenols, oligosaccharides, or collagen peptide (M1), a large network structure with high PDI value was first formed, leading to phase separation. After the addition of a certain amount of polyphenols, oligosaccharides, or collagen peptide, the network was gradually disrupted by EGCG molecules which covered the lactoferrin molecule surface with weaker binding affinities, forming new smaller networks or complexes with low PDI value (for example, gallic acid and chlorogenic acid) or larger networks with a PDI value of around 1.0 (for example, XOS and FOS). When lactoferrin or EGCG and a polyphenol, oligosaccharide, or collagen peptide were first mixed together (M2 and M3, respectively), the interaction between lactoferrin and EGCG was disrupted by the polyphenol, oligosaccharide, or collagen peptide through a competition mechanism or steric effects, forming new smaller networks accompanied by a decreased PDI value. ζ-Potential Measurements. The ζ potential plays an important role in the stability of protein−polysaccharide or protein−polyphenol complexes in aqueous solution. Figure 3 shows the ζ potential of complexes presented in the solution resulting from the interaction between lactoferrin and EGCG in the absence and presence of polyphenols, oligosaccharides, or collagen peptide. In this study, the ζ-potential values of the ternary complexes increased with an increase of gallic acid and chlorogenic acid and decreased with an increase of collagen peptide, leading to particle deaggregation at higher concentration. Unlike the case

for gallic acid, chlorogenic acid, and collagen peptide, it was interestingly found that the ζ potential of the ternary complexes was around 0 mV, also leading to particle deaggregation at higher concentrations of glucose, XOS, and FOS, respectively. However, the concentrations of a neutral carbohydrate, such as glucose, XOS, or FOS, were much higher in comparison to those of gallic acid, chlorogenic acid, and collagen peptide, which are thought to have an ionic character. This result interpreted the low affinity of neutral carbohydrates for polyphenols, as reported by other authors.27,28 There might be a simple relationship between the ability to inhibit the formation of lactoferrin−EGCG aggregate and ζ potential; that is, charged polyphenols and collagen peptide (whether positively or negatively charged) were more effective in the decrease of aggregation than noncharged oligosaccharides. Fluorescence Spectroscopy Measurements. There are 13 tryptophan residues in the lactoferrin structure.29 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.30 By monitoring of the emission peak change, some information could be obtained concerning the structural change and the microenvironment surrounding the fluorophore in the protein. Synchronous fluorescence spectra supplied some information about the molecular environment in the vicinity of the chromophore molecules. When Δλ = 60 nm, spectral characteristics of 5039


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Figure 4. Fluorescence emission spectra of lactoferrin, in the absence and presence of polyphenols, oligosaccharides, or collagen peptide (a1−f1), influence of increasing concentrations of polyphenols, oligosaccharides, or collagen peptide on the variation of the fluorescence of lactoferrin solution (g1), fluorescence emission spectra of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG), in the absence and presence of polyphenols, oligosaccharides, or collagen peptide (a2−f2), and the influence of increasing concentrations of polyphenols, oligosaccharides, or collagen peptide on variation of the fluorescence of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG) (g2).

The fluorescence emission spectrum obtained for lactoferrin−EGCG aggregate in the absence and presence of polyphenols, oligosaccharides, or collagen peptide is shown in Figure 4a 2 −f 2 . Similar to the effect of polyphenols, oligosaccharides, or collagen peptide on lactoferrin, a high degree of fluorescence quenching of lactoferrin−EGCG aggregate was observed for gallic acid (from 100% to 2.4− 2.6%), chlorogenic acid (from 100% to 16.8−24.3%), and XOS (from 100% to 20.8−24%) (Figure 4g1,g2). At the same time, there were obvious blue shifts of the maximum λem of fluorescence for gallic acid (from 369.6 to 343.2−351.4 nm), obvious red shifts with chlorogenic acid (from 369.6 to 440.6− 445.6 nm), and red shifts with XOS (from 369.6 to 382.4− 383.8 nm), accompanied by significant quenching of the fluorescence, which implied that binding behaviors existed between lactoferrin−EGCG aggregate and gallic acid, chlorogenic acid, or XOS, respectively. Therefore, it was inferred that the interactions of lactoferrin−EGCG aggregate with gallic acid, chlorogenic acid, or XOS, respectively, led to the compact structure of lactoferrin and increase of the hydrophobicity in the microenvironment of tryptophan residues with gallic acid, while the protein structure was unfolded and the hydrophilicity of the microenvironment of tryptophan residues for chloro-

tryptophan residues in the protein were observed.31 The shift in the position of the emission maximum (λmax) corresponded to the change in polarity around the chromophore molecule. Thus, the conformation change of lactoferrin could be estimated by this measurement.32,33 The fluorescence emission spectrum (at λex 292 nm) obtained for lactoferrin in the absence and presence of polyphenols, oligosaccharides, or collagen peptide is shown in Figure 4a1−f1. A high degree of fluorescence quenching was observed for gallic acid (from 100% to 3.9%), chlorogenic acid (from 100% to 0.09%), and XOS (from 100% to 2.7%). However, as the concentrations of glucose, FOS, and collagen peptide were respectively increased, the intensity of the fluorescence only slightly increased (for glucose and FOS) or decreased (for collagen peptide). Similar trends were also observed with synchronous fluorescence (Figure 5a1−f1). This implied that binding behavior existed between lactoferrin and gallic acid, chlorogenic acid, or XOS, respectively but that there was hardly any interaction between lactoferrin and glucose, FOS, or collagen peptide, respectively. Since M2 and M3 showed fluorescent properties closely similar to those of M1 (the fluorescence spectra data of M2 and M3 are not shown), only M1 is used for analysis in the following sections. 5040


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Figure 5. Synchronous fluorescence spectra of lactoferrin, in the absence and presence of polyphenols, oligosaccharides, or collagen peptide (a1−f1), influence of polyphenols, oligosaccharides, or collagen peptide concentration on the variation of the synchronous fluorescence of lactoferrin (g1), synchronous fluorescence spectra of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG), in the absence and presence of polyphenols, oligosaccharides, or collagen peptide (a2−f2), and the influence of polyphenols, oligosaccharides, or collagen peptide concentration on the variation of the synchronous fluorescence of lactoferrin−EGCG aggregate (containing 1.0 mM EGCG) (g2).

genic acid and XOS increased. Additionally, this also suggested that gallic acid, chlorogenic acid, or XOS was situated in close proximity to the tryptophan residues for quenching to occur, and the microenvironment of tryptophan residues was changed. The fluorescence intensity of lactoferrin−EGCG aggregate also regularly decreased with an increase in glucose, FOS, or collagen peptide concentration. Unlike the case for gallic acid, chlorogenic acid, and XOS, a low degree of fluorescence quenching was observed for glucose (from 100% to 66.9− 89.9%), FOS (from 100% to 64.5−69.3%), and collagen peptide (from 100% to 46.8−59.6%). Meanwhile, there were only slight blue shifts for glucose (from 369.6 to 366.2−367 nm), FOS (from 369.6 to 366−367 nm), and collagen peptide (from 369.6 to 363.4 nm). These results revealed that glucose, FOS, and collagen peptide might enhance the interaction of lactoferrin and EGCG but there was hardly any binding behavior between lactoferrin and glucose, FOS, and collagen peptide, respectively, and the structure of lactoferrin interacting with 1.0 mM EGCG was not altered significantly in the presence of glucose, FOS, and collagen peptide, respectively. Additionally, this also implied that the microenvironment of

tryptophan residues did not change after the addition of glucose, FOS, or collagen peptide, respectively. The synchronous fluorescence spectra of lactoferrin−EGCG aggregate in the absence and presence of polyphenols, oligosaccharides, or collagen peptide are shown in Figure 5a2−f2, which show synchronous fluorescence trends similar to those obtained for lactoferrin in the absence and presence of polyphenols, oligosaccharides, or collagen peptide (Figure 5g1,g2). It was apparent that the emission maximum of tryptophan residues had a slight blue shift for gallic acid (from 331.6 to 328.2−328.8 nm) but a stronger red shift for chlorogenic acid (from 331.6 to 385.4−385.8 nm) and XOS (from 331.6 to 363.6 nm). These shifts revealed that the conformation of lactoferrin changed, the polarity around the tryptophan residues decreased, and the hydrophobicity increased with gallic acid, while the presence of chlorogenic acid or XOS modified the microenvironment from a nonpolar into a polar state; in other words, tryptophan residues were located in a less hydrophobic environment and were more exposed to the solution.34 These shifts in the spectra expressed some changes in conformation of lactoferrin upon binding with gallic acid, chlorogenic acid, and XOS, respectively. 5041


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Figure 6. Far-UV CD spectra of lactoferrin in the absence and presence of polyphenols, oligosaccharides, or collagen peptide with 1.0 mM EGCG (a1−a6), secondary structure of lactoferrin in the absence and presence of polyphenols, oligosaccharides, or collagen peptide with 1.0 mM EGCG (b1), changes in the secondary structure of lactoferrin in the presence of polyphenols, oligosaccharides, or collagen peptide with 1.0 mM EGCG (b2), and near-UV CD spectra of lactoferrin in the absence and presence of polyphenols, oligosaccharides, or collagen peptide with 1.0 mM EGCG (c1− c6). “Control” represents the lactoferrin−EGCG complex (containing 1.0 mM EGCG) in the absence of polyphenols, oligosaccharides or collagen peptide.

The conformation of lactoferrin in lactoferrin−EGCG aggregate was not monitored when glucose, FOS, or collagen peptide was added, respectively, since no shifts in fluorescence quenching spectra and synchronous fluorescence spectra were found. On the basis of the fluorescence results, we hypothesize that gallic acid, chlorogenic acid, and XOS not only interacted with lactoferrin and/or EGCG molecules which surrounded the lactoferrin molecule surface with weaker binding affinities via being substituted and/or adsorbed by them but also affected the internal structure of lactoferrin and/or EGCG which were bound within the hydrophobic pockets of lactoferrin with

strong binding affinities, leading to a decrease in turbidity and blue or red shifts of the maximum λem value of lactoferrin fluorescence as their concentrations were increased (except for XOS by M1, which showed increases in the turbidity with increasing concentrations of XOS). However, glucose, FOS, or collagen peptide only interacted with the surface of lactoferrin and EGCG molecules which surrounded the lactoferrin molecule surface, leading to a decrease in turbidity but no shift of the maximum λem value of lactoferrin fluorescence (except for FOS by M1, which showed an increase of the turbidity as the concentration of FOS was increased). 5042


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Figure 7. Possible mechanisms and structures involved in the inhibition of the aggregation of lactoferrin and EGCG in the presence of polyphenols, oligosaccharides, or collagen peptide.

Analysis of the Secondary Structure of Lactoferrin Using Circular Dichroism (CD). To obtain more information about the secondary structural changes of lactoferrin induced by EGCG and a polyphenol, oligosaccharide, or collagen peptide, the ternary complexes were analyzed by far-UV CD. As shown in Figure 6a1−a6, in the region of 190−260 nm, the spectrum of lactoferrin in the presence of 1.0 mM EGCG was composed of a broad negative band centered at 208−210 nm and a shoulder at 218−220 nm. In the presence of chlorogenic acid, gallic acid, and XOS, the peak showed a significant (p < 0.05) decrease for chlorogenic acid and XOS and a slight increase for gallic acid in negative ellipticities, which is characteristic of a lactoferrin secondary structure conformation change, and the peak showed little change for glucose, FOS, and collagen peptide in negative ellipticities, which indicated no change in lactoferrin secondary structure. The secondary structure contents were estimated using the DICHROWEB procedure, which is an online server for protein secondary structure analyses, from circular dichroism spectroscopic data.35 The fractions of α-helix, β-sheet, turn, and unordered coil were estimated by CDSSTR and are shown in Figure 6b1. In the presence of 1.0 mM EGCG, lactoferrin had 21% α-helix, 32% β-sheet, 22% β-turn, and 25% unordered. In the ternary complexes, lactoferrin had a relatively higher content of α-helix for chlorogenic acid, XOS, and FOS, a lower content of β-sheet for chlorogenic acid, XOS, and FOS, a lower content of β-turn for XOS, and a higher content of unordered for gallic acid, chlorogenic acid, XOS, and FOS, while the contents remained relatively unchanged for glucose and collagen peptide (Figure 6b2). The protein conformational analysis based on CD data indicated that the presence of polyphenols or oligosaccharides in the ternary complexes led to

a change in lactoferrin secondary structure; that is, polyphenols or oligosaccharides induced a progressive increase in the proportion of α-helix structure at the cost of the β-sheet structure of lactoferrin and the conformation became slightly looser due to an increase in the unordered coil fraction. These results showed that lactoferrin secondary conformational changes were consistent with the findings from fluorescence spectra. Figure 7 shows the possible mechanisms and structures involved in the inhibition of the aggregation of lactoferrin and EGCG by polyphenols, oligosaccharides, or collagen peptide. Polyphenols, such as gallic acid and chlorogenic acid, could form a protein−polyphenol complex with lactoferrin by cooperative hydrogen bonding and hydrophobic interactions. Gallic acid and chlorogenic acid molecules are too small to form insoluble aggregates with lactoferrin through effective cross-linking (data not shown). Therefore, the results obtained for gallic acid and chlorogenic acid strongly suggested that the main mechanism by which these two compounds inhibited the aggregation of lactoferrin and EGCG was to compete with EGCG molecules, which were bound on the surface of lactoferrin. The ternary complex structures EGCG−lactoferrin−gallic acid and EGCG−lactoferrin−chlorogenic acid were formed and exhibited a smaller particle size accompanied by the change of lactoferrin structure (compacting structure of lactoferrin for EGCG−lactoferrin−gallic acid aggregate and unfolding structure of lactoferrin for EGCG−lactoferrin− chlorogenic acid aggregate). Carbohydrates should have a suitable structure and composition (ionic character) and sufficient size and flexibility to be able to complex with polyphenols. The association of the neutral carbohydrates with polyphenols was thought to be 5043


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EGCG (0.483−1.0 for glucose, 0.403−1.0 for FOS) as their concentrations were increased. A competition mechanism was postulated between collagen peptide and lactoferrin with EGCG molecules, which surrounded the lactoferrin molecule surface, leading to a decrease in turbidity. A ternary complex structure, lactoferrin−EGCG−collagen peptide, was formed and exhibited a smaller particle size without a change in lactoferrin structure. The aforementioned findings imply that polyphenols, oligosaccharides, or collagen peptide, like some anionic polysaccharides, were able to resolubilize the protein− polyphenol aggregates. Charged oligosaccharides or collagen peptide, with either positive or negative charge, disrupted the lactoferrin−EGCG aggregate mainly through the mechanism of competition of EGCG molecules which surrounded the lactoferrin molecule surface with weaker binding affinities, forming a ternary complex, accompanied by changes in lactoferrin structure (for gallic acid and chlorogenic acid) or no change (for collagen peptide), while neutral molecules disrupted the aggregate mainly through steric effects and formation of ternary complexes, also accompanied by changes in lactoferrin structure (for XOS) or no change (for glucose and FOS). Polyphenols, oligosaccharides, or collagen peptide effectively inhibit the formation of lactoferrin−EGCG aggregate by M2 or M3 instead of M1: that is, adding polyphenols, oligosaccharides, or collagen peptide to lactoferrin or EGCG buffer solution before the formation of lactoferrin−EGCG aggregate. Therefore, the pros and cons of polyphenols, oligosaccharides, or collagen peptide on the turbidity and particle size of the ternary complexes mostly depended on the addition sequence of lactoferrin, polyphenols (oligosaccharide or collagen peptide), and EGCG. Overall, the exceptional properties of polyphenols, oligosaccharides, and collagen peptide, present at high levels in food and beverage products, exhibit a possible additional contribution for reducing the astringent response or turbidity of polyphenols. The inhibition of polyphenols, oligosaccharides, and collagen peptide for the formation of lactoferrin−EGCG aggregate could be applied to the design of clear products in specific food, pharmaceutical, and cosmetic industries.

primarily a surface effect, resulting from a cooperative hydrogen-bonding interaction between the hydroxyl groups of these carbohydrates and the phenolic hydroxyl groups of polyphenols.13 According to M1, with increasing glucose concentration, there was a gradual disruption of the binding between lactoferrin and EGCG molecules which surrounded the lactoferrin molecule surface with weaker binding affinities; new smaller networks were formed. By M2 and M3, glucose limited the number of available binding sites of lactoferrin to readily fit EGCG through steric effects, leading to a decrease in the turbidity. The disruption or reconstitution of the lactoferrin−EGCG aggregate by glucose might be the main mechanism to form smaller aggregates with higher PDI values in comparison to those for gallic acid and chlorogenic acid. As mentioned above, the amount of aggregates precipitated tended to increase significantly (p < 0.05) with increasing concentration of XOS by M1, while this amount decreased by M2 and M3, which indicated that XOS, unlike gallic acid, chlorogenic acid, and glucose, did not have the ability to compete with EGCG molecules which were bound on the surface of lactoferrin. Fluorescence spectra of lactoferrin with XOS were generally similar to those of lactoferrin with chlorogenic acid, since it could form a lactoferrin−XOS complex. The results obtained strongly suggested that the main mechanism for XOS influence on the lactoferrin−EGCG aggregation was to form a complex or simple ternary complex by an interaction between lactoferrin and XOS. For example, (lactoferrin−EGCG)n-XOS, a more complex structure, exhibiting a larger particle size accompanied by a change in lactoferrin structure, was formed by M1; lactoferrin−EGCG−XOS with a ternary structure, exhibiting a smaller particle size accompanied by a change in lactoferrin structure, was formed by M2 and M3. Unlike XOS, FOS did not have the ability to influence the fluorescence characteristics of lactoferrin, only interacting with the surface of lactoferrin and EGCG molecules which surrounded the lactoferrin molecule surface. The fluorescence spectra of FOS were generally similar to those of glucose. FOS, whose molecular weight was greater than that of glucose, could adsorb onto the lactoferrin−EGCG aggregate surface (associative interaction) and the larger complex (lactoferrin− EGCG)n−FOS was formed by M1, leading to the insolubilization of the ternary complex. By M2 and M3, in addition to the formation of soluble ternary complex, another explanation could be attributed to the steric effects in solution, limiting the number of available binding sites of lactoferrin to readily fit EGCG, which was able to prevent the interaction between proteins and polyphenols, as suggested for glucose. XOS species are oligomers containing 2−10 xylose molecules linked by β 1−4-bonds;36 FOS species are composed of 2,1linked or 2,6-linked β-D-fructofuranosyl units.37 Therefore, the chemical composition of these oligosaccharides may affect their interactions with proteins or polyphenols. A more in-depth investigation should be performed to support these findings. Collagen peptide had changes of fluorescence similar to those of glucose and FOS. However, two significant differences were observed between collagen peptide and glucose or FOS: first, unlike glucose and FOS, collagen peptide could interact with EGCG, forming a new collagen peptide−EGCG complex;38 second, the PDI values of the ternary complex fabricated by collagen peptide, lactoferrin, and EGCG (0.41− 0.66), similar to those of gallic acid and chlorogenic acid (0.34− 0.372 for gallic acid, 0.314−0.452 for chlorogenic acid), were lower than those fabricated by glucose or FOS, lactoferrin, and

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AUTHOR INFORMATION

Corresponding Author

*Y.G.: 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.

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REFERENCES

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Inhibition of the Aggregation of Lactoferrin and (â)-Epigallocatechin

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