Article pubs.acs.org/JAFC
Fabrication Mechanism and Structural Characteristics of the Ternary Aggregates by Lactoferrin, Pectin, and (−)-Epigallocatechin Gallate Using Multispectroscopic Methods Wei Yang, Chenqi Xu, Fuguo Liu, 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, P.O. Box 112, No. 17 Qinghua East Road, Beijing 100083, People’s Republic of China ABSTRACT: The ternary aggregates were fabricated by lactoferrin (LF), pectin (high methylated pectin (HMP)/low methylated pectin (LMP)), and (−)-epigallocatechin gallate (EGCG) through three different fabrication methods at pH 5.0. The turbidity, particle size, and ζ-potential of ternary aggregates were influenced by the types of pectin, the concentration of EGCG, and fabrication methods. The fluorescence intensity of LF decreased with an increase in EGCG concentration for all ternary aggregates. Far-UV circular dichroism results indicated that EGCG could alter the secondary structure of LF with an increase in the proportion of β-sheet structure at the cost of unordered coil structure. According to near-UV circular dichroism results, EGCG could also modulate the tertiary structure of LF at the presence of pectin. In addition, EGCG could increase the viscoelasticity of the ternary aggregates with HMP, leading to better stability of the ternary aggregates. An opposite result was observed for the ternary aggregates with LMP. These findings should provide an insight into the fabrication mechanism and applications of ternary aggregates formed by protein, polysaccharide, and polyphenol in the food, pharmaceutical, and cosmetic industries. KEYWORDS: fabrication mechanism, structural characteristics, lactoferrin, pectin, (−)-epigallocatechin gallate, multispectroscopic
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INTRODUCTION Polyphenols can bind noncovalently to proteins.1−3 Some researchers have focused on the disadvantage of the use of polyphenols, since they can precipitate proteins to form an insoluble state, resulting in undesirable turbidity and colloidal haze products.4 From a nutritional point of view, the interaction between polyphenols and proteins, such as α-amylase and trypsin, has been shown to have negative effects, reducing the palate lubrication, causing an unpleasant sensation of roughness, dryness, and constriction, and reducing absorption of nutrients.5,6 Polyphenols also affect the conformational changes of proteins. It was pointed out that the conformation of proteins (such as β-lactoglobulin, casein, collagen, bovine serum albumin, and lysozyme) was modified by polyphenols (such as tea polyphenols, resveratrol, genistein, curcumin, and procyanidin).7−11 The conformational changes of proteins, signs of a partial protein unfolding upon protein−polyphenol complexes, might affect the formation and properties of the electrostatic complexes between protein and polysaccharide. Polysaccharides are widely used as stabilizers, thickening, or gelling agents in the food industry.12 The interaction between polysaccharides and polyphenols might result from cooperative hydrogen bonding between the hydroxyl groups of polysaccharide and polyphenols and from hydrophobic interactions.13−16 Sun-Waterhouse et al. reported the effect of fruit polyphenols and pectin on the properties of bread and suggested possible polyphenol complexes, via nonbinding interactions such as hydrogen bonding, with pectin.17 Hayashi et al. showed that the presence of pectin mitigated the astringency of tea infusions and attributed the observed behavior to pectin−catechin complex formation.18 Mercedes Lataza Rovaletti et al. indicated that polysaccharides could react © 2015 American Chemical Society
with tannic acid in beer, and this reaction actually caused a considerable increase in turbidity.19 Several studies have demonstrated the ability of some polysaccharides to disrupt polyphenol−protein interactions in dilute solution.6,15,16,20−22 Two hypotheses were proposed for this disrupting effect of polysaccharides toward protein and polyphenol interactions: (i) the ability of polysaccharides to form a protein−polyphenol−polysaccharide ternary complex with enhanced solubility in aqueous medium and (ii) the ability of polysaccharides to encapsulate polyphenols, competing with the binding to proteins.23 Recent studies have shown that the complexation of preheated whey protein isolate−pectin complexes inhibited the protein precipitation with polyphenols.24 As main components of foods, proteins and polysaccharides play a key role in the formation of the building blocks of structure and texture. The interactions occurring between polysaccharides and proteins are influenced by their surface charge, hydrophobicity, and the rheological characteristics. The formation of aggregates between proteins and polysaccharides might be the starting point for the development of delivery systems in the food, cosmetics, and pharmaceutical industries. These aggregates could be used to encapsulate, protect, and deliver functional or bioactive components, such as minerals, polyphenols, vitamins, enzymes, drugs, and peptides,25−31 to specific sites, controlling the rate or extent of their digestibility in different regions of the human digestive system within the Received: December 16, 2014 Accepted: May 8, 2015 Published: May 8, 2015 5046
DOI: 10.1021/acs.jafc.5b01592 J. Agric. Food Chem. 2015, 63, 5046−5054
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Journal of Agricultural and Food Chemistry
(3.2% w/v) solution was mixed with the pectin−EGCG complexes, and the mixtures were allowed to stand for 2 h before the measurement. The abbreviations TAPectin, TAHMP, and TALMP represent the ternary aggregates prepared using LF, EGCG, and pectin (both HMP and LMP), HMP, and LMP, respectively. TAPectin-x, TAHMP-x, and TALMPx (x = 1−3) were used to represent the ternary aggregates prepared by M1, M2, and M3, respectively. Turbidity Measurements. Nephelometry experiments were performed in a HACH 2100N Laboratory Turbidimeter (Loveland, TX, USA). All samples were diluted 1/10 (v/v) with acetate buffer solution (pH 5.0). The optical apparatus was equipped with a tungsten-filament lamp with three detectors: a 90° scattered-light detector, a forward-scattering light detector, and a transmitted light detector. The calibration was performed using a Gelex Secondary Turbidity Standard Kit (HACH, Loveland, TX, USA), which consists of stable suspensions of a metal oxide in a gel. All experiments were performed in triplicate. Particle Size and ζ-Potential Measurements. Particle size and ζ-potential were determined using a combined dynamic light scattering (DLS) and particle electrophoresis instrument (NanoZS90, Malvern Instruments, Worcestershire, U.K.). Basically, all samples were diluted 1/10 (v/v) with acetate buffer solution (pH 5.0) and then equilibrated for 1 min inside the instrument before dynamic light backscattering, and then data were collected over at least 10 sequential readings. The Z-average particle diameter was calculated by the instrument using the Stokes−Einstein equation. The ζ-potential of the particles was calculated using the Smoluchowski model through electrophoretic mobility measurements performed in a capillary electrophoresis device inserted into the DLS instrument. Fluorescence Spectroscopy. Fluorescence measurements were carried out using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). All samples were diluted 1/3 (v/v) with acetate buffer solution. Spectra were collected when the diluted solutions were kept at 25 °C for 2 h. Fluorescence emission spectra of LF were recorded with the excitation wavelength at 292 nm. Both excitation and emission slit widths were set at 10 nm. Circular Dichroism Measurements. The CD spectra were recorded using a CD spectropolarimeter (Pistar π-180, Applied Photophysics Ltd., Surrey, U.K.) at both far-UV (190−250 nm) and near-UV (250−320 nm) 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.) The fractions of α-helix, β-sheet, turn, and unordered coil were estimated by SELCON3. Each spectrum presented is the average of three consecutive measurements. Viscoelastic Properties. The viscoelastic properties of the samples were evaluated by optical microrheology (Rheolaser LAb from Formulaction, Union, France). In the experiment, 20 mL of samples was injected by a syringe into a flat-bottomed cylindrical glass tube (height 70 mm, external diameter 27.5 mm). The tube was immediately placed in the sample chamber of the Rheolaser LAb instrument. During the measurement, the speckle pattern of the sample, which was generated by the interfering backscattering wave of the incident laser at 650 nm caused by the Brownian motion of the particles, was collected by a multipixel detector for 3 min. The generated speckle image was treated to plot the decorrelation curve, which could quantify the particle mobility and displacement and in turn be used to characterize the viscoelastic properties of the samples. Physical Stability Measurements. The stabilities of the solutions were measured by the LUMi-Sizer (L.U.M. GmbH, Berlin, Germany), an instrument employing centrifugal sedimentation to accelerate the occurrence of instability phenomena such as sedimentation, flocculation, and creaming. The samples were subjected to centrifugal force, while near-infrared light illuminated the entire sample cell to measure the intensity of transmitted light as a function of time and position over the entire sample length simultaneously. The sample stability was shown as a space- and time-related transmission profile
consumer’s body: e.g., mouth, stomach, small intestine, or colon.25,32−34 Pectins are normally extracted from several raw materials (such as apple pomace, sugar beet chips, and citrus peels) and are used in the food industry as thickening, gelling, and stabilizing agents. Chemically, they are mainly composed of Dgalacturonic acid linked by α-(1,4)-glycosidic bonds, forming polygalacturonic acid. Pectins have different degrees of esterification (DE), with those higher or lower than 50% methylation corresponding to high or low methoxyl pectins, respectively.35 Lactoferrin (LF) has attracted strong interest as a functional bioactive ingredient for applications in food, personal care, and pharmaceutical products. It has a relatively high pI (8−9) and therefore tends to be cationic at a pH value below 8.0. Thus, the electrostatic interaction between LF and pectin has been harnessed to form various particulate systems and modulate emulsion functionality over a wide pH range.36,37 Recently, there has been increasing interest in the incorporation of polyphenols into protein−polysaccharide aggregates and the functionality of the ternary electrostatic aggregates between polysaccharides and proteins might be affected by the presence of polyphenols. An understanding of the fabrication mechanism and structural characteristics of the ternary aggregates is a key step for optimizing the formation of aggregates with applications in coatings, emulsions, etc. In the present work, ternary aggregates fabricated from lactoferrin (LF), pectin, and (−)-epigallocatechin gallate (EGCG) were designed to form a novel delivery systems for foods and other products. The effect of binding sequence for LF, pectin, and EGCG on the physical characteristics of the aggregates was also investigated.
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MATERIALS AND METHODS
Materials and Chemicals. Lactoferrin (purity ≥92%) from bovine whey was purchased from New Image International Limited (Auckland, New Zealand). EGCG (purity ≥95%) from green tea was purchased from BSZH Science (Beijing, People’s Republic of China). High methoxylated pectin (HMP) with a DE value of 72% (GENU pectin type YM-150-H) and low methoxylated pectin (LMP) with a DE value of 36% (GENU pectin type LM-101-AS) were obtained from CP Kelco (Copenhagen, Denmark). All other chemicals used were of analytical grade, unless otherwise stated. Solution Preparation. LF (3.2% w/v), EGCG (8.0 mM), and pectin (1.8% w/v) were dissolved in 10 mM acetate buffer (pH 5.0), and the mixture was stirred constantly at room temperature for 2 h and then stored overnight at 4 °C to achieve complete dissolution. Formation of the Ternary Aggregate Solutions (Fabricated from LF, Pectin, and EGCG). The ternary aggregate solutions were formed with three addition sequences of LF, pectin, and EGCG. All solutions were mixed on a vortex shaker (QL-866, Haimen QILINBEIER Instruments Co., Ltd., Haimen, People’s Republic of China) at 3000 rpm for 2 min. Method I (M1). First, 1.0 mL of LF (3.2% w/v) and 2.0 mL of pectin (1.8% w/v) solutions were mixed and allowed to stand for 2 h. After that, 1.0 mL of EGCG solutions with different concentrations (0−2.0 mM) were mixed with LF−pectin complexes and the mixtures allowed to stand for 2 h before the measurement. Method II (M2). First, 1.0 mL of LF (3.2% w/v) solution and 1.0 mL of EGCG solutions with different concentrations (0−2.0 mM) were mixed and allowed to stand for 2 h. After that, 2.0 mL of pectin solution (1.8% w/v) was mixed with different LF−EGCG complexes, and the mixtures were allowed to stand for 2 h before the measurement. Method III (M3). First, 2.0 mL of pectin (1.8% w/v) solution and 1.0 mL of EGCG solutions with different concentrations (0−2.0 mM) were mixed and allowed to stand for 2 h. After that, 1.0 mL of LF 5047
DOI: 10.1021/acs.jafc.5b01592 J. Agric. Food Chem. 2015, 63, 5046−5054
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Journal of Agricultural and Food Chemistry over the sample length. At the start of the measurement, the solutions were intact. As the sample was centrifuged, however, the heavier and less transparent aqueous phase moved to the bottom, which gave higher transmission; the lighter and more transparent phase moved to the top. The instrumental parameters used for the measurement were as follows: volume, 1.8 mL of dispersion; 3000 rpm; timeExp, 3600 s; time interval, 30 s; temperature, 25 °C. Statistical Analysis. The experiments were conducted in duplicate, and all of the 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 concordance between experimental data and calculated values was established by the root-mean-squared deviation (RMSD).
structural properties of TAHMP-1 and TALMP-1. Our results were similar to those of Thongkaew et al., who showed that the presence of catechin, grape seed extract, and hibiscus extract to whey protein isolate−pectin complex at pH 3.5 did not show a obvious difference of mean particle diameter and the addition of catechin, grape seed extract, and tannic acid did not affect the ζ-potential of preheated whey protein isolate−pectin complex at pH 5.2.23 The increasing concentration of EGCG exhibited some effects on the turbidity and particle size of TAPectin-2 and TAPectin-3, which suggested that the ternary aggregates were formed to be large to scatter light. The larger turbidity and particle size of TAPectin-3 in comparison to those of TAPectin-2 indicated that more complex structures were formed by M3 than by M2. The effect of EGCG on the formation of large aggregates was more prominent for TALMP than for TAHMP, which was consistent with the greater change in ζ-potential of TALMP than of TAHMP (discussed below). The particle sizes of TAHMP-1, TAHMP-2, and TAHMP-3 were smaller than those of TALMP-1, TALMP-2, and TALMP-3, respectively, which suggested that the aggregates formed with HMP were more numerous than those formed with LMP. A possible explanation of this phenomenon is that LMP, containing a higher charge density than HMP, may have a greater propensity to form large complexes or sediments through neutralization of LF−EGCG complexes. On the other hand, more localized interactions between protein and polysaccharide molecules led to the formation of complexes of more limited size, because there are free segments of biopolymers that promote complex repulsion through electrostatic or steric hindrance effects.39 Thus, the particles formed with HMP were likely less compact and possessed more biopolymer segments extending into solutions in comparison to LMP particles. For TAPectin-2 and TAPectin-3, the presence of EGCG significantly (p < 0.05) affected the ζ-potential of the ternary aggregates. Interestingly, the addition of EGCG resulted in a decrease in negatively charged ζ-potential for TAHMP-2 and TAHMP-3 but an increase for TALMP-2 and TALMP-3. The charges of HMP, LMP, and EGCG at pH 5.0 were −32 ± 0.1, −40 ± 0.6, and −16 ± 1.0 mV, respectively. Therefore, it was estimated that the types of pectin and electrostatic repulsion between pectin and EGCG might have some effect on the charge distribution of those ternary aggregates. However, an EGCG concentration of more than 1.2 mM had almost no effect on the ζ-potential of the ternary aggregates, indicating that the aggregates formed may retain their charged functional groups. Fluorescence Spectroscopy Measurements. There are 13 tryptophan residues in the LF molecule.40 By monitoring of the emission peak change, some useful information could be obtained concerning the structural changes of LF. It was obvious that, for all ternary aggregates, the fluorescence intensity of LF regularly decreased with an increase in EGCG concentration, and this implied that there existed binding behavior between LF and EGCG (Figure 2a,b). The maximum λem was slightly red-shifted (from 334.4 to 335.8 nm for TAHMP and from 334.4 to 337.2−338 nm for TALMP, more red-shifted emission in comparison to TAHMP) in the presence of EGCG (Figure 2c). These results revealed that there were some changes in the immediate environment of the tryptophan residues of LF in TAHMP and TALMP and that EGCG was situated in closer proximity to the tryptophan residues in TALMP than in TAHMP for quenching to occur.
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RESULTS AND DISCUSSION A fixed concentration of pectin (0.9% w/v) was used in the experiments to saturate the LF (0.8% w/v) molecules, as no further change in ζ-potential was observed above this concentration (data not shown). Effect of Addition Sequence of LF, Pectin, and EGCG on Turbidity, Particle Size, and ζ-Potential of the Ternary Aggregates. It is well-known that EGCG can bind to LF with strong affinity and form nanosized particles at pH 5.0.38 At the same time, pectin and EGCG can form a pectin− EGCG complex by cooperative hydrogen bonding between the hydroxyl groups of polysaccharide and groups of polyphenols. Therefore, the addition sequence of LF, pectin, and EGCG may directly affect the interactions of these compounds, thereby affecting the structural characteristics of the ternary aggregates. The turbidity, particle size, and ζ-potential of the ternary aggregates are shown in Figure 1. The magnitude of the
Figure 1. Turbidity, particle size, and ζ-potential of TAHMP (a) and TALMP (b) as a function of EGCG concentration (0−2.0 mM) at pH 5.0.
negative charge on the LF−pectin complexes was greater for LMP than for HMP, which could be attributed to the fact that LMP has a higher linear charge density: i.e., more carboxylic acid groups per unit chain length. The turbidity and particle size exhibited a synchronous trend with the rise of EGCG concentration. For TAPectin-1, the turbidity, particle size, and ζ-potential were not apparently changed with different concentrations of EGCG; in other words, the aggregates large enough to scatter light strongly were not formed, and EGCG hardly affected the 5048
DOI: 10.1021/acs.jafc.5b01592 J. Agric. Food Chem. 2015, 63, 5046−5054
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Figure 2. Fluorescence spectra of LF in TAHMP (a) and TALMP (b) at different EGCG concentrations (0−2.0 mM) at pH 5.0. Influence of increasing concentration of EGCG on the fluorescence variation of LF in TAHMP and TALMP (c).
In the solution prepared by M1, LF−pectin aggregates were formed first; then, EGCG was added to the aggregates, and it might be embedded in the gaps of LF−pectin aggregates and interact with LF, leading to almost complete loss of fluorescence intensity in TAPectin-1, as in TAPectin-2 and TAPectin-3. Meanwhile, with increasing concentrations of EGCG, the outer layer of pectin prevented the formation of a larger “−(LF−EGCG)n−” network structure, leading to little change in turbidity, particle size, and ζ-potential of TAPectin-1 (see Figure 1). Therefore, the structure of TAPectin-1 was LF in the inner layer, pectin in the outer layer, and EGCG in the middle layer (Figure 3a). In the solution prepared by M2, “−(LF−EGCG)n−” complex showed a small or large particle size formed at relatively low or high concentration of EGCG first; after pectin was added to the solution, the pectin had a sufficient capacity to
Figure 3. Schematic representation of possible mechanisms (a−c) involved in the formation of the ternary aggregates with LF, pectin, and EGCG. Legend: LF, green square; pectin, blue ribbon; EGCG, red streak. The red arrow indicates increasing concentrations of EGCG.
coat the “−(LF−EGCG)n−” complex. Therefore, the structure of TAPectin-2 was “−(LF−EGCG)n−” complex in the inner layer 5049
DOI: 10.1021/acs.jafc.5b01592 J. Agric. Food Chem. 2015, 63, 5046−5054
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Journal of Agricultural and Food Chemistry
Figure 4. Far-UV CD spectra of LF in LF−pectin aggregates and TAPectin containing 1.0 mM EGCG (a, b). Secondary structure of LF in LF−pectin aggregates and TAPectin containing 1.0 mM EGCG (c). Changes in secondary structure of LF in TAPectin containing 1.0 mM EGCG (d). Near-UV CD spectra of LF in LF−pectin aggregates and TAPectin containing 1.0 mM EGCG (e, f).
It was found that LF in the LF−HMP aggregate contained 20% α-helix, 30% β-sheet, 21% β-turn, and 29% unordered, and 21% α-helix, 29% β-sheet, 22% β-turn, and 29% unordered fractions occurred in LF−LMP aggregate. The slight difference in the secondary structure of LF in LF−HMP and LF−LMP aggregates might be attributed to the different physiochemical characteristics of HMP and LMP. It is well-known that the noncovalent interactions of EGCG and proteins affect the structure of proteins.7,8,41,42 Hence, in the presence of EGCG, the secondary structural composition of LF in TAHMP and TALMP possibly changed when LF−EGCG complex was formed. As is shown in Figure 4a,b, in the presence of 1.0 mM EGCG, the broad negative band centered at 208−210 nm showed a slight decrease in negative ellipticities for TAHMP and TALMP, while the shoulder at 218−220 nm showed a decrease or an increase in negative ellipticities, which is a characteristic of LF secondary structure conformation change. The protein conformational analysis based on CD data indicated that the addition of EGCG led to changes in the secondary structure of LF in TAHMP and TALMP. In the presence of 1.0 mM EGCG, the β-sheet and β-turn contents
and pectin in the outer layer (Figure 3b). Pectin, which forms the outer layer of TAPectin-2, also could prevent further aggregation of these compounds; thus, TAPectin-2 exhibited a particle size relatively larger than that of TAPectin-1 but a smaller particle size than TAPectin-3 (see Figure 1). In the solution prepared by M3, “−pectin−EGCG−” complex was formed first; after LF was added to the solution, “−[LF−EGCG−pectin−EGCG−LF]n−” aggregates, more complicated than those of TAPectin-2, were formed and showed the greatest turbidity and largest particle size at corresponding concentrations of EGCG (see Figure 1). The structure of TAPectin-3 was a complex network structure fabricated by LF, EGCG, and pectin (Figure 3c). Circular Dichroism (CD) Measurements. Far-UV CD is used to obtain the information about the changes occurring at a secondary folding level of proteins. In this section, the influence of pectin type on the secondary structure of LF- and EGCGinduced conformational transitions of LF in TAHMP and TALMP (containing 1.0 mM EGCG) was monitored by far-UV CD spectroscopy (Figure 4a,b). The fractions of α-helix, β-sheet, turn, and unordered coil are shown in Figure 4c,d. 5050
DOI: 10.1021/acs.jafc.5b01592 J. Agric. Food Chem. 2015, 63, 5046−5054
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Figure 5. MVI and EI of TAHMP (a, c) and TALMP (b, d) as a function of EGCG concentration (0−2.0 mM) at pH 5.0.
increased with a concomitant reduction of α-helical and unordered coil fractions in TAHMP-1, TAHMP-2, TAHMP-3, TALMP-1, and TALMP-2. Therefore, it could be concluded that, for these ternary aggregates, EGCG contributed largely to the stability of LF β-sheet conformation and LF assumed a slightly compact conformation due to the decrease of unordered coil fraction. It is interesting to find that an inverse result, an increase of αhelix (about 2%) and decrease of β-turn (about −1%), was observed in TALMP-3, which might be one reason for the lower viscoelasticity and less stability of TALMP-3 in comparison to TAHMP-3. Near-UV CD is a powerful tool for probing the tertiary structure of proteins. CD spectra in the region of 250−320 nm were attributed to aromatic amino acids, and the real shape and magnitude of the near-UV CD spectrum of a protein were determined by the number of each type of aromatic amino acids present, their mobility, the nature of the environment (hydrogen bonds, polar groups, and polarizability), and spatial disposition in the protein.43 From Figure 4e,f, the near-UV CD spectra of LF in TAHMP and TALMP showed a broad distribution range from 250 to 300 nm, comprising four maxima at 254, 277, 285, and 292 nm, which were attributed to phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), respectively.43 The presence of EGCG led to a remarkable decrease in the ellipticities, which suggested that the LF tertiary conformation had changed. It should be noted that LF in TALMP showed a greater decrease in the magnitudes of the near-UV CD bands in comparison to that in TAHMP, which indicated that the LMP− EGCG complex had a greater impact than HMP−EGCG complex on the structure of LF. As EGCG caused different changes of LF secondary and tertiary structures in TAHMP and TALMP, respectively, it may alter the stoichiometry of the LF−pectin complexes, which might increase the tendency for charge neutralization and lead to stronger bridging forces between LMP and LF and therefore the nature of ternary aggregates with larger particle size formed by LMP in comparison to HMP (see Figure 1).
Thus, EGCG could interact with LF in TAHMP and TALMP, leading to changes of LF secondary and tertiary structures, and the effects of EGCG on LF secondary structure mainly depended on the types of pectin and fabrication methods. Viscoelastic Properties. Passive particle tracking is a direct method consisting of monitoring the Brownian motion of tracer particles within a system and interpreting such movements in terms of the local viscoelasticity, or microrheology.44 Particle tracking microrheology is a powerful technique that enables researchers to measure the viscoelastic properties of complex fluids with relatively low viscoelastic moduli (typically
