Modulation of Formation, Physicochemical Properties, and Digestion

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Modulation of Formation, Physicochemical Properties, and Digestion of Ovotransferrin Nanofibrils with Covalent or NonCovalent Bound Gallic Acid Zihao Wei and Qingrong Huang* Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901, United States

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S Supporting Information *

ABSTRACT: The impact of covalent or non-covalent bound gallic acid (GA) on the formation, physicochemical properties, and digestion of ovotransferrin (OTF) nanofibrils was comprehensively studied. Thioflavin T fluorescence results revealed that bound GA could inhibit OTF nanofibrillation and that the fibril-inhibitory activity of bound GA was dose dependent. Covalent bound GA exerted stronger inhibition on OTF nanofibrillation than an equal amount of non-covalent bound GA. Atomic force microscopy revealed that covalent bound GA shortened OTF nanofibrils significantly, while non-covalent bound GA did not change the contour length of OTF fibrils remarkably. Bound GA altered diameter of OTF nanofibrils. Both covalent and noncovalent bound GA could alter the zeta potential, surface hydrophobicity, and rheological properties of OTF nanofibrils. Bound GA endowed OTF nanofibrils with a strong antioxidant activity. In vitro gastrointestinal digestion results showed that covalent bound GA elevated the fibril digestion rate better than non-covalent bound GA. Polyphenol binding provided a new approach to modulating the physicochemical properties of protein nanofibrils. KEYWORDS: ovotransferrin nanofibril, gallic acid, covalent interaction, non-covalent interaction, physicochemical characterization, in vitro digestion

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complexes and covalent complexes, respectively.10 Fibrillation of protein−polyphenol covalent complexes may differ from unmodified proteins and protein−polyphenol physical complexes. To the best of our knowledge, few previous studies have compared how non-covalent and covalent protein−polyphenol interactions alter protein fibrillation. In addition, interactions between proteins and different amounts of polyphenols may lead to discrepancies in the formation kinetics, physicochemical properties, and digestion of fibrils, and there are few relevant studies yet. Therefore, it is important to investigate how the fibrillation and fibril properties can be modulated by distinct protein−polyphenol interactions and varying amounts of polyphenols. Ovotransferrin (OTF), an iron-binding egg white protein containing 686 amino acids, occupies around 12% of egg white protein.12 Unlike other egg white proteins such as ovalbumin or lysozyme, relatively rare studies focusing on OTF have been carried out due to there being relatively few manufacturers, although OTF has excellent physicochemical properties such as emulsibility.13 Our previous study shows that OTF nanofibrils with no cytotoxicity may be assembled,4 so OTF nanofibrils can be chosen as the protein fibril model to be investigated in the current study. Since OTF nanofibrils are iron-bound fibrils and since the fibril theories available now may not apply to OTF nanofibrils, research about OTF nanofibrils may provide new insight into fibril engineering.

INTRODUCTION Assembly of proteins or peptides into fibrillar structures both in vitro and in vivo is an intriguing phenomenon, which has lately received increasing attention.1−4 Protein nanofibrils, which have diameters of the order of nanometers and contour lengths of the order of nanometers to micrometers, are highly ordered supramolecular assemblies with linear structures and a high aspect ratio.3,4 Protein nanofibrils synthesized in vitro have potential food applications such as Pickering emulsions, hydrogels, and mineral fortification due to unique structural characteristics and mechanical properties.2,5−7 Extensive utilization of protein nanofibrils in food systems is severely restricted owing to lack of deeper recognition to physicochemical properties and digestion of fibrils. Meanwhile, some protein fibrils accumulated in vivo at well-defined pathological states are associated with several types of human disorders such as Parkinson’s or Alzheimer’s disease.1 Hence, the fibrillation process is desirable or undesirable according to the circumstance, 8 and a thorough understanding of modulating fibril formation and modifying physicochemical properties of fibrils is essential to extend appropriate application of fibrils. Interactions between proteins and polyphenols are inevitable in food systems, which can be classified into non-covalent and covalent protein−polyphenol interactions.9 Hydrogen bondings, van der Waals forces, and hydrophobic interactions are the main non-covalent protein−polyphenol interactions, while Schiff bases and Michael addition reactions following the oxidation of polyphenols may result in covalent bonds.9−11 Non-covalent and covalent protein−polyphenol interactions may lead to the formation of protein−polyphenol physical © XXXX American Chemical Society

Received: April 26, 2019 Revised: June 19, 2019 Accepted: August 13, 2019

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DOI: 10.1021/acs.jafc.9b02630 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

mL of OPA reagent, and absorbance was measured at 340 nm after incubation at 35 °C for 2 min. The L-leucine standard curve was used to calculate the content of free amino groups. Free Sulfhydryl Groups. The content of free sulfhydryl groups was determined using Ellman’s reagent as described previously.19 Ellman’s reagent was obtained by dissolving 200 mg of DTNB reagent in 50 mL of Tris−glycine buffer (0.086 M Tris, 0.09 M glycine, and 4 mM EDTA, pH 8.0). Samples (10−15 mg of protein equivalent) were dissolved into 5.0 mL of Tris−glycine buffer (pH 8.0) consisting of 8 M urea, followed by addition of Ellman’s reagent (50 μL). After incubation at room temperature for 1 h, the absorbance was recorded at 412 nm. The amount of free sulfhydryl groups was calculated as follows: nmol SH/mg = (73.53 × A412)/C, where A412 was the absorbance measured at 412 nm and C was the sample concentration (mg/mL). Preparation and Characterization of OTF−GA Physical Complexes. Preparation of OTF−GA Physical Complexes. To characterize the impact of distinct OTF−GA interactions on OTF nanofibrils, OTF−GA physical complexes were prepared as described previously with minor modifications.10,20 To exclude interference of a polyphenol amount, OTF−GA physical complexes with the same phenolic content as corresponding OTF−GA covalent complexes were prepared. Specifically, if 1 g of OTF−GA covalent complex consisted of M1 g of OTF and M2 g of GA, then M2 g of GA solution (at pH 5) was added into OTF solution (at pH 5) containing M1 g of OTF. The resultant mixture (at pH 5) was stirred at 25 °C in the dark without exposure to air for 24 h. The final product was lyophilized to obtain OTF−GA physical complexes. To make this paper concise, abbreviations were used to denote different OTF−GA physical complexes. OGP-L, OGP-M, and OGP-H were OTF−GA physical complexes with the same phenolic content as OGC-L, OGC-M, and OGC-H, respectively. Molecular Docking. The Surflex docking method in SYBYL 8.1 software (Certara, Princeton, USA) was employed to study interactions between GA and OTF in OTF−GA physical complexes. The crystal structure of OTF (PDB ID: 1OVT) was prepared by removing crystal water and adding hydrogen atoms. The 3D structures of GA were optimized at the B3LYP/6-31+G(d,p) level of theory using Gaussian 09. The maximum amount of output poses was set as 20, and scoring functions were utilized to evaluate the output poses. The optimal poses with the highest C-score and T-score were chosen finally.21,22 Preparation of Nanofibrils. The optimal fibrillation parameters were obtained based on procedures in our previous study,4 and optimal fibrillation parameters for OTF fibrils with and without GA were the same. The specific procedures for fibrillation of OTF and OTF−GA complexes were shown as follows. OTF and OTF−GA complexes were dispersed and dissolved in pH-adjusted Milli-Q water (pH 2, 150 mM NaCl) at a protein concentration of 40 mg/mL. Sodium azide (0.02%, w/v) was added to impede microbial growth. The samples were placed into screw-capped vials flushed with nitrogen in the dark. The vials were heated in an oil bath at 90 °C under magnetic stirring (300 rpm). After heating for 24 h, the nanofibril samples were cooled in an ice−water bath and stored at 4 °C until further analysis. ThT fluorescence, SDS-PAGE, and AFM were applied to analyze the fibrillation process. Thioflavin T (ThT) Fluorescence. The ThT working solution was prepared as described previously.4,23 The samples (40 μL) were added to 4 mL of the working solution and mixed well. ThT fluorescence was measured on a FluoroMax 3 fluorescence spectrophotometer (Horiba Scientific, Kyoto, Japan) using excitation and emission wavelengths of 440 and 482 nm, respectively. The relative fluorescence intensities were obtained by subtracting the background of the ThT working solution.23 Atomic Force Microscopy (AFM). Tapping mode AFM images of fibrils derived from OTF and OTF−GA complexes were acquired using a NanoScope IIIA Multimode AFM (Veeco Instruments Inc., Santa Barbara, USA).4 AFM data were analyzed by NanoScope Analysis Software and FiberApp,24 and the contour length distribution

Gallic acid (GA), a versatile antioxidant with a molecular weight of 170.12 from plants and fungi, has a diverse range of industrial and therapeutic applications.14 Since GA is widely present in many food products and protein−GA interactions are common, GA can be selected as a polyphenol model to explore the impact of polyphenols on protein nanofibrils. This research aimed to first modify OTF with different amounts of GA in non-covalent or covalent ways and then to investigate how the fibrillation and fibril properties can be modulated by varying amounts of GA and distinct OTF−GA interactions in terms of formation kinetics, morphology, building blocks, zeta potential, surface hydrophobicity, rheology, antioxidant property, and digestibility of fibrils. Hopefully, the current study may have practical implications in engineering structures and physicochemical properties of fibrils precisely in the near future.

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MATERIALS AND METHODS

Materials. Ovotransferrin (OTF, purity > 88%) was purchased from Neova Technologies Inc. (Abbotsford, Canada), and OTF had an iron binding activity greater than 1000 μg Fe/g sample. Gallic acid (GA) with a purity of 98% was obtained from Acros Organics (Geel, Belgium). Pepsin was obtained from Amresco (Solon, USA), and pancreatin was purchased from Thermo Fisher Scientific, Inc. (Waltham, USA). PageRuler Plus prestained protein ladder was obtained from Thermo Fisher Scientific, Inc. (Waltham, USA). Other chemicals were obtained from Sigma-Aldrich (St. Louis, USA) unless otherwise stated. Preparation and Characterization of OTF−GA Covalent Complexes. Preparation of OTF−GA Covalent Complexes. OTF solution (4 mg/mL) was stirred overnight to ensure complete dispersion and dissolution, and sodium azide (0.02%, w/v) was added to impede microbial growth. Gallic aicd (0.1, 0.2, or 0.4 mg/mL) dissolved in 10 mL of Milli-Q water was added to 10 mL of OTF solutions under continuous stirring, respectively. Upon addition of GA, the pH of solutions was adjusted to pH 9 with 0.5 M NaOH.10 Thereafter, the mixtures were stirred continuously with free exposure to air at 25 °C for 24 h. The samples were dialyzed (molecular weight cutoff of 3500 Da) against Milli-Q water at 4 °C for 72 h to eliminate the free unbound GA, followed by freeze-drying to obtain OTF−GA covalent complexes. To make this paper clearer, abbreviations were used to denote different OTF−GA covalent complexes. OGC-L, OGC-M, and OGC-H were OTF−GA covalent complexes obtained by covalent modification of OTF (4 mg/mL) with different concentrations of GA (0.1, 0.2, or 0.4 mg/mL), respectively. The protein content of OTF−GA covalent complexes was measured using a modified Lowry method.10,15 Total Phenolic Content. The total phenolic content of OTF−GA covalent complexes was determined according to the Folin−Ciocalteu method using GA as a standard,16 and the total phenol content was expressed as milligrams of GA equivalents per gram of dry complex. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed in a vertical gel electrophoresis cell (Bio-Rad Laboratories, Hercules, USA). For SDS-PAGE of proteins without heating, SDS-PAGE was carried out using a 4% stacking gel and 10% acrylamide separating gel. For SDS-PAGE of proteins after heating at pH 2.0 and at 90 °C, SDS-PAGE was conducted employing a 4% stacking gel and 12% acrylamide separating gel. The loading volume of the diluted samples (equivalent to protein concentration of 3 mg/mL) was 10 μL, and the electrophoresis was run at a constant voltage of 100 V. SDS-PAGE bands were stained with Coomassie brilliant blue R250. Free Amino Groups. Free amino groups were determined via an OPA (o-phthaldialdehyde) method.17,18 The OPA reagent was obtained by mixing 160 mg of OPA (dissolved in 4 mL of methanol), 10 mL of 20% (w/w) sodium dodecyl sulfate, 400 μL of 2mercaptoethanol, 86 mL of water, and 100 mL of 0.1 M sodium tetraborate solution. The sample solution (200 μL) was mixed with 4 B

DOI: 10.1021/acs.jafc.9b02630 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry of fibrils was obtained after analyzing at least 100 individual fibrils for each sample. Zeta Potential. The zeta potential of OTF nanofibrils and nanofibrils derived from OTF−GA complexes was measured at pH 2−9. Measurements were carried out at 25 °C using a Zetasizer NanoZS90 instrument (Malvern Instruments, Worcestershire, U.K.). Surface Hydrophobicity (H0). The surface hydrophobicity of OTF nanofibrils and nanofibrils derived from OTF−GA complexes was determined using the 1-anilino-8-naphthalensulfonate (ANS) method, as described previously.4,25 Rheology. Rheological measurements of OTF nanofibrils and nanofibrils derived from OTF−GA complexes were carried out at 25 ± 0.1 °C using a Discovery HR-2 rheometer (TA Instruments, New Castle, USA) with a cone-and-plate geometry (diameter = 60 mm, cone angle = 4°, gap = 0.2 mm). Steady-state flow measurements were carried out, and the apparent viscosity of the samples was recorded as a function of shear rate (0.01−10 s−1). Dynamic frequency sweep test was conducted at a fixed strain amplitude of 1% (within the linear viscoelastic region), and the storage modulus (G′) as well as loss modulus (G″) of samples were recorded versus frequency (0.05−50 rad/s). ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid)) Scavenging Activity. The capacity of scavenging ABTS•+ was evaluated as described previously,26 and the antioxidant activity was expressed as nanomoles of Trolox equivalents (TE) per milligram of sample using Trolox as a standard. Fibril Digestion in Simulated Gastrointestinal Fluid. Proteolysis of fibrils was studied as described previously.27 Fibrils derived from OTF and OTF−GA complexes were diluted with simulated gastric fluid (pH 1.5, no enzyme, 34 mM NaCl) to obtain a final protein concentration of 3 mg/mL. Pepsin solution (pH 2, 10 mg/mL) was prepared freshly and added into diluted fibril samples to make a final pepsin concentration of 2 mg/mL (equivalent to an enzyme activity of 6000 FCC U/mL). The samples were incubated at 37 °C at a speed of 50 rpm in a VWR 1585 shaking incubator (VWR International, Radnor, USA). Aliquots (160 μL) were drawn after different incubation periods (0−4 h), and hydrolysis reactions were stopped by addition of 0.2 M Na2CO3. Thereafter, 4 mL of ThT working solution was added and mixed well. The in vitro digestion rate of fibrils was calculated as (I1 − I2)/I1, where I1 was the initial ThT fluorescence before proteolysis and I2 was the ThT fluorescence at any time point during proteolysis. The simulated intestinal digestion followed simulated gastric digestion. The pH of the mixture dispersions was adjusted to pH 7.0 with 0.1 M NaHCO3, and pancreatin solution (10 mg/mL) was added to initiate simulated intestinal digestion. The final pancreatin concentration was 2 mg/mL. The samples were shaken at 37 °C at a speed of 50 rpm for 2 h, and ThT fluorescence was employed to analyze the digestibility of OTF fibrils immediately. Statistical Analysis. Each experiment was performed at least in triplicate. Statistical analysis was performed by OriginPro 2017 software. One-way analysis of variance (ANOVA) followed by a Fisher LSD test was used to determine statistical differences (significant if p < 0.05).

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Table 1. Total Phenolic Content of OTF−GA Covalent Complexes sample

total phenolic contenta (mg/g sample)

OGC-L OGC-M OGC-H

24.19 ± 0.08 a 41.72 ± 0.25 b 76.16 ± 0.55 c

a

Different letters in the same column indicate significant differences (p < 0.05).

higher concentration of GA led to complexes with more bound GA. SDS-PAGE was applied to confirm the covalent binding of GA to OTF. As depicted in Figure 1, the bands of OTF−GA

Figure 1. SDS-PAGE profiles (10% separating gel) of OTF and OTF−GA covalent complexes. Lanes from left to right are protein marker, OTF, OGC-L, OGC-M, and OGC-H. The marker bands from down to up correspond to 25, 35, 55, 70, 100, 130, and 250 kDa.

covalent complexes migrated up in comparison with the bands of OTF, suggesting an increase of molecular weight upon conjugation. It was also observed that OTF−GA covalent complexes with more bound GA had a larger molecular weight. Since non-covalent interactions between proteins and polyphenols could be broken in the presence of SDS,28 the shift to higher molecular weight confirmed the covalent coupling between OTF and GA. As shown in Table 2 and Table 3, the amount of free amino groups and sulfhydryl groups in OTF−GA covalent complexes was smaller than that of OTF, which confirmed involvement of

RESULTS AND DISCUSSION

Preparation of OTF−GA Covalent Complexes and Physical Complexes. Synthesis of OTF−GA Covalent Complexes. Polyphenols may be oxidized into corresponding quinones by oxygen at alkaline pH, and these quinones can readily undergo attack by nucleophiles including lysine and cysteine in protein chains, leading to irreversible covalent linkage between proteins and polyphenols.9,10 The amount of bound GA in OTF−GA covalent complexes was quantified by the Folin−Ciocalteu method. As shown in Table 1, the total phenolic content followed the order: OGC-H > OGC-M > OGC-L, indicating that covalent modification of OTF with a

Table 2. Free Amino Groups of OTF and OTF−GA Covalent Complexes sample OTF OGC-L OGC-M OGC-H

free amino groupsa (nmol/mg protein) 636.4 530.5 458.1 338.5

± ± ± ±

11.2 16.3 19.1 15.5

d c b a

a Different letters in the same column indicate significant differences (p < 0.05).

C

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Journal of Agricultural and Food Chemistry Table 3. Free Sulfydryl Groups of OTF and OTF−GA Covalent Complexes sample OTF OGC-L OGC-M OGC-H

free sulfydryl groupsa (nmol/mg protein) 6.08 0.41 0.08 0.03

± ± ± ±

0.19 0.03 0.01 0.01

d c b a

a Different letters in the same column indicate significant differences (p < 0.05).

free amino groups and sulfhydryl groups during covalent modification. When polyphenols are converted into quinones, the quinones can covalently bind to these reactive groups via Schiff bases and Michael addition reactions, leading to formation of C−N and C−S bonds, respectively.10,29 It was noteworthy that an evidently larger number of free amino groups disappeared upon covalent modification when compared with free sulfhydryl groups. It may be deduced that the major cross-linking bonds of OTF−GA covalent complexes are C−N bonds. Based on the discussion mentioned above, the proposed formation pathway of OTF− GA covalent complexes was shown in Figure S1 (Supporting Information). Preparation of OTF−GA Physical Complexes. At acidic pHs polyphenols are structurally stable, and the polyphenols interact with proteins via non-covalent forces such as hydrogen bonding and hydrophobic bonding, which result in protein− polyphenol physical complexes.10,30 In order to rule out the interference of GA content on analyzing how the fibrillation and fibril properties can be affected by distinct OTF−GA interactions during the rest of this research, the amount of GA in OTF−GA physical complexes was the same as that of corresponding OTF−GA covalent complexes. The non-covalent binding of GA to OTF was studied by Surflex-dock, which could provide pose prediction during the molecular docking study of protein−ligand complexes.21,22 The preferred OTF−GA binding mode was displayed in Figure 2. It was found that the docking sites were located in hydrophobic pockets of OTF. Since hydroxyl groups of polyphenols may interact with proteins via weak bonds such as hydrogen bonds,30 intermolecular hydrogen bonds were also observed in OTF−GA physical complexes. It may be deduced that hydrogen bondings and hydrophobic interactions are the major non-covalent interactions between OTF and GA. ThT Fluorescence. ThT fluorescence is a sensitive method to probe the presence of fibrils and to quantify the amount of fibrillar aggregates.23 As depicted in Figure 3, in the presence of bound GA, the ThT fluorescence intensity after heating at 24 h followed the order: OTF > OGP-L > OGP-M > OGP-H > OGC-L > OGC-M > OGC-H. It was apparent that interactions with GA had inhibitory effects on OTF nanofibrillation, and covalent OTF−GA interactions had stronger fibril-inhibitory activity than non-covalent OTF−GA interactions. The following explanations may account for these phenomena. First, since GA is a phenol molecule with very hydrophilic characteristic, OTF−GA complexes were less hydrophobic than unmodified OTF, as evidenced by the surface hydrophobicity result in Table S1. Considering the fact that hydrophobic interactions are the main driving forces for protein fibrillation,31 the diminished hydrophobic interactions may have antiformation and antiextension effects on OTF fibrillation. Since OTF−GA covalent complexes had a surface

Figure 2. (a) Surflex docking result of GA with OTF. (b) Partial enlarged stereoview of the docked pose of GA with OTF (dotted yellow lines indicate hydrogen bonds).

Figure 3. Thioflavin T (ThT) fluorescence of OTF and OTF−GA complexes after heating at 90 °C and pH 2.0.

hydrophobicity that was lower than that of corresponding OTF−GA physical complexes (Table S1), covalent interactions with GA repressed fibrillation more intensely than noncovalent interactions. Second, a decrease of OTF nanofibrils can be a result of disruptive effects of GA on β-sheet structures. Our previous study reveals that stacked β-sheets should be the internal structures of OTF nanofibrils,4 and a disruptive influence on the formation of β-sheet structures may lead to attenuated fibrillation. GA may have structural similarities to D

DOI: 10.1021/acs.jafc.9b02630 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry some fibril inhibitors,32 and GA may have non-covalent interactions with β-sheet structures, which prevents assembly of more building blocks into OTF fibrils. Third, the building blocks of OTF nanofibrils may be altered upon complexation with GA, which can be responsible for reduction of fibrils. Building blocks with bound GA may bring about steric hindrance during fibril assembly, which leads to fewer OTF nanofibrils. SDS-PAGE. SDS-PAGE was conducted to explore building blocks of fibrils in the absence and presence of bound GA. As shown in Figure 4, all samples were hydrolyzed into peptides

after thermal unfolding and hydrolysis, indicating that major building blocks of all nanofibrils were peptides. In the case of OTF, SDS-PAGE bands with molecular weight around 10 kDa were observed, suggesting that major building blocks of OTF nanofibrils were peptides with a molecular weight around 10 kDa. It was observed that hydrolyzed OTF−GA physical complexes had a molecular weight range of around 10−15 kDa, and partial hydrolysates of OTF−GA covalent complexes had molecular weights above 15 kDa. It was apparent that peptides derived from OTF−GA complexes were generally larger than those originated from OTF, implying that major building blocks of OTF fibrils with bound GA were generally larger than those of OTF fibrils. It was worth noting that SDSPAGE bands with a molecular weight over 250 kDa were observed after heat treatment of OTF−GA covalent complexes, which could be due to molecule cross-linking. GA quinones can act as molecule cross-linkers, and some peptide fragments are coupled together to larger biomacromolecules.33 The occurrence of SDS-PAGE bands above 250 kDa implies that the GA-cross-linked peptide aggregates may be building blocks of nanofibrils derived from OTF−GA covalent complexes. Fibril Morphology. AFM was utilized to investigate whether the presence of GA induced changes in the morphology of OTF nanofibrils. Figure 5 depicts AFM images of OTF nanofibrils with covalent or non-covalent bound GA, and Figure S2 shows the contour length distribution of nanofibrils derived from OTF and GA complexes. In the case of OTF nanofibrils, apart from long and rigid fibrils, short and curly fibrils were also observed. Figure 5 and Figure S2 show that fibrils derived from OTF−GA covalent complexes were shorter than OTF nanofibrils, and the average contour length of fibrils derived from OGC-L, OGC-M, and OGC-H was 127,

Figure 4. SDS-PAGE profiles (12% separating gel) of OTF and OTF−GA complexes after heating at pH 2.0 and 90 °C for 24 h. Lanes from left to right are marker, heated OTF, OGC-L, OGC-M, OGC-H, OGP-L, OGP-M, and OGP-H.

Figure 5. AFM images of nanofibrils derived from OTF and OTF−GA complexes after heating at pH 2.0 and 90 °C for 24 h: (a) OTF fibrils, (b) OGC-L fibrils, (c) OGC-M fibrils, (d) OGC-H fibrils, (e) OGP-L fibrils, (f) OGP-M fibrils, and (g) OGP-H fibrils. The scan size is 2 μm × 2 μm, and the z scale is 20 nm. The scale bar represents 200 nm. E

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isoelectric point (pI) around pH 7.0. In comparison with the OTF fibril, the pI of fibrils derived from OTF−GA covalent complexes shifted to lower pH values, and the pI of the fibril derived from OGC-L, OGC-M, and OGC-H was about pH 5.8, 5.5, and 5.3, respectively. The following explanations may elucidate the phenomenon. Considering the fact that the hydrophilic lysines have good affinities for water,36 ε-amino groups of lysines are likely to locate at the surface of OTF fibrils. Covalent reaction of OTF with GA led to blocking of the positively charged amino groups, bringing about alterations in the net surface charge.10 Since a higher level of covalent modification results in a loss of more positively charged amino groups, the pI is shifted to a lower pH. As depicted in Figure 6, the pI of fibrils derived from OTF−GA non-covalent complexes (pH 6.2) was lower than that of OTF fibrils (pH 7.0), and it might be speculated that hydrophobic interactions and hydrogen bonds could alter the distribution of negatively charged and positively charged groups at the surface of OTF fibrils. Surface Hydrophobicity. Since the study of surface hydrophobicity can provide valuable information about how covalent or non-covalent bound GA modulate the surface structure of OTF nanofibrils, the surface hydrophobicity of OTF nanofibrils with bound GA was studied. As shown in Table 4, the surface hydrophobicity of fibrils followed the

100, and 92 nm, respectively. No significant changes in the contour length of OTF fibrils were induced upon non-covalent complexations with GA. It was noteworthy that although complexations with GA could disrupt conversion of OTF into amyloid fibrils, very few amorphous aggregates were viewed in these AFM pictures, suggesting that the transformation of amyloid fibrils into amorphous aggregates is not a major antifibrillation mechanism of the bound GA. To sum up, covalent complexations of OTF with GA could shorten OTF nanofibrils and higher levels of covalent modification led to shorter OTF nanofibrils, while non-covalent OTF−GA interactions did not alter the contour length of OTF nanofibrils remarkably. Protein nanofibrils are multistranded structures with many filaments,4,34,35 and it was intriguing to investigate how the diameter of OTF nanofibrils was affected by bound GA. As shown in Figure S3 and Table S2, OTF nanofibrils had a height of 3.0 ± 0.7 or 6.0 ± 0.7 nm. Because each filament of protein fibrils may have a height of 1.5−2.0 nm and because the cross-section height of fibrils is related to the number of filaments,34 it may be postulated that OTF fibrils with heights around 3.0 nm are multistranded structures consisting of two filaments.4 Since two 2-filament fibrillar structures can interact to yield one 4-filament fibril,35 OTF fibrils with heights around 6.0 nm should be 4-filament fibrils.4 It was observed that fibrils derived from OGC-L, OGC-M, and OGC-H had heights around 4.5 ± 0.3 nm, and the populations with heights around 3.0 or 6.0 nm disappeared. Because each filament of OTF nanofibrils had a diameter around 1.5 nm, fibrils prepared from OTF−GA covalent complexes could be obtained via the cooperative association of protofibrils into 3-filament mature fibrils. When compared with fibrils derived from unmodified OTF, thicker fibrils with heights around 9.0 ± 0.9 nm were detected in the presence of non-covalent bound GA, implying the emergence of 6-filament mature fibrils. Overall, both covalent and non-covalent bound GA could alter the diameter of OTF nanofibrils. Zeta Potential. Zeta potential is a prime factor when it comes to the formulation stability of protein fibrils and application of fibrils in food systems, which deserves an indepth study. Since bound GA might alter the number and distribution of charged groups on OTF fibril surfaces, the impact of bound GA on the zeta potential of OTF fibrils was investigated. As shown in Figure 6, the OTF fibril had an

Table 4. Surface Hydrophobicity of Fibrils Derived from OTF and OTF−GA Complexes sample OTF fibril OGC-L fibril OGC-M fibril OGC-H fibril OGP-L fibril OGP-M fibril OGP-H fibril

surface hydrophobicitya 1021.4 373.4 243.9 158.9 831.0 717.4 628.9

± ± ± ± ± ± ±

19.2 g 24.9 c 17.5 b 10.3 a 9.7 f 28.4 e 19.6 d

a Different letters in the same column indicate significant differences (p < 0.05).

order: OTF fibril > OGP-L fibril > OGP-M fibril > OGP-H fibril > OGC-L fibril > OGC-M fibril > OGC-H fibril. The surface hydrophobicity results demonstrated that bound GA made surfaces of OTF fibrils more hydrophilic, and OTF fibrils with covalent bound GA had a lower surface hydrophobicity than fibrils with an equal amount of non-covalent bound GA. The phenomenon may be explained by that covalent complexations of OTF with GA can introduce more hydrophilic groups to fibril surfaces than corresponding noncovalent bound GA.10 Rheological Properties. The influence of bound GA on rheological properties of OTF fibril dispersions was studied. As depicted in Figure 7, all fibril samples exhibited shear-thinning behavior, which could partly arise from a decreased relative contribution of entropic forces.37 Another explanation for the shear-thinning behavior is that OTF fibril networks may orient themselves in the flow direction and partly disentangle at an increasing shear rate, which leads to a lower resistance to gradual deformation.38 It was noted that storage modulus G′ was larger than G″ in all cases, indicating gel-like properties of all fibrils. It was noteworthy that all fibril dispersions derived from OTF−GA complexes had a lower viscosity and storage modulus than the OTF fibrils. The following speculations may

Figure 6. Zeta potential of fibrils derived from OTF and OTF−GA complexes as a function of pH. F

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Table 5. ABTS Scavenging Activity of Fibrils Derived from OTF and OTF−GA Complexes sample OTF fibril OGC-L fibril OGC-M fibril OGC-H fibril OGP-L fibril OGP-M fibril OGP-H fibril

antioxidant activitya (nmol TE/mg sample) 110.8 864.1 1505.2 2821.1 909.8 1589.7 2939.3

± ± ± ± ± ± ±

9.1 a 37.5 b 53.3 c 94.6 d 30.7 b 71.0 c 65.9 d

a

Different letters in the same column indicate significant differences (p < 0.05).

significantly, which could be due to the introduction of a large amount of phenolic hydroxyl groups.41 It was noted that fibrils with covalent bound GA exhibited a slightly weaker antioxidant capacity than fibrils with a corresponding amount of non-covalent bound GA. Considering the fact that part of hydroxyl groups may act as cross-linkers in OTF−GA covalent complexes, the loss of hydroxyl groups may account for the differences in antioxidant activity of fibrils with covalent or non-covalent bound GA. In Vitro Digestion of Nanofibrils Derived from OTF− GA Complexes. Table 6 shows the digestion rates of Table 6. Digestion Rate of Fibrils Derived from OTF and OTF−GA Complexes after Simulated Gastric Digestion sample OTF fibril OGC-L fibril OGC-M fibril OGC-H fibril OGP-L fibril OGP-M fibril OGP-H fibril

Figure 7. (a) Apparent viscosity of fibrils derived from OTF and OTF−GA complexes as a function of shear rate. (b) Storage modulus (G′) and loss modulus (G″) of fibrils derived from OTF and OTF− GA complexes as a function of oscillatory frequency.

digestion ratea (%) 50.3 63.9 70.1 72.8 54.7 57.9 58.1

± ± ± ± ± ± ±

2.8 1.1 0.8 1.2 1.3 1.0 0.8

a d e f b c c

a

Different letters in the table indicate significant differences (p < 0.05).

explain these phenomena. First, as mentioned earlier, fewer fibrils were generated in the presence of bound GA. The decrease of fibrillar structures may attenuate entanglement networks, contributing to a lower viscosity and storage modulus. Second, bound GA may weaken hydrophobic interactions in fibril systems due to reduced surface hydrophobicity. Interconnections contributing to entanglement networks include hydrophobic interactions,39 and the decrease of hydrophobic interactions may imply looser entanglement networks. Third, the persistence length is related to the micromechanical fibril property,40 and rigid fibrils may enhance interactions between the microstructural elements better than flexible fibrils due to longer persistence length. As stated above, the curly fibrils in the presence of covalent bound GA had a shorter persistence length, which could be responsible for the lower viscosity and storage modulus. Last but not least, in comparison with short fibrils, long fibrils may facilitate more frequent contacts among the microstructural elements and formation of a tighter entanglement network.38 As OTF nanofibrils in the presence of covalent bound GA were shorter than OTF nanofibrils, nanofibrils prepared from OTF− GA covalent complexes possessed a lower viscosity and storage modulus than OTF nanofibrils. Antioxidant Activity. As shown in Table 5, bound GA enhanced the antioxidant activity of OTF nanofibrils

nanofibrils prepared from OTF and OTF−GA complexes after simulated gastric digestion. It was noted that 50.3% of fibrillar structures remained after proteolysis of OTF nanofibrils, suggesting incomplete digestion of OTF nanofibrils by pepsin in simulated gastric fluid. Two speculations may explain the phenomenon. First, since proteases possess cleavage site selectivity,42 pepsin may have limited cleavage sites within specific amino acid sequences. Pepsin may not act on some peptide chains, contributing to the preservation of part of the fibrils. Second, since the internal structures of fibrils are typically stabilized by strong hydrophobic interactions and electrostatic interactions,43 it may be reasonably hypothesized that some specific regions in the fibrils are so tightly packed that the regions are not accessible to proteolysis by pepsin. Interestingly, although previous research reveals that polyphenols may inhibit activity of proteases,44 it was found that the digestion rate of fibrils prepared from OTF−GA complexes was higher than that of OTF fibrils. Meanwhile, the digestion rate of OTF fibrils with covalent bound GA was greater than that of OTF fibrils with non-covalent bound GA. The following explanation may account for these phenomena. As discussed earlier, peptide building blocks of OTF fibrils with bound GA were different from those of OTF fibrils without G

DOI: 10.1021/acs.jafc.9b02630 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry GA. It may be assumed that peptide building blocks of OTF fibrils with bound GA have more potential pepsin cleavage sites than those of OTF fibrils without GA. Since building blocks of OTF fibrils with bound GA are more prone to proteolysis of pepsin, it is not hard to infer that OTF fibrils with bound GA have a higher digestion rate. Table 7 shows the digestion rate of fibrils derived from OTF and OTF−GA complexes after simulated intestinal digestion.

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OTF fibril OGC-L fibril OGC-M fibril OGC-H fibril OGP-L fibril OGP-M fibril OGP-H fibril

*E-mail: [email protected]. Tel.: +1 (848) 932-5514. Fax: +1 (732) 932-6776. ORCID

Zihao Wei: 0000-0002-1942-7907 Qingrong Huang: 0000-0001-8637-0229 Notes

digestion ratea (%) 83.1 92.7 94.1 94.4 88.2 88.6 89.2

± ± ± ± ± ± ±

1.0 0.5 0.7 0.8 0.3 0.4 0.6

The authors declare no competing financial interest.

■

a d e e b bc c

ACKNOWLEDGMENTS This work was supported by the United States Department of Agriculture, the National Institute of Food and Agriculture (2019-67017-29176). We acknowledge financial support from the China Scholarship Council for the first author. We thank Guizhao Liang from the School of Bioengineering in Chongqing University for his assistance in molecular docking.

a

Different letters in the table indicate significant differences (p < 0.05).

■

ABBREVIATIONS USED OTF, ovotransferrin; GA, gallic acid; OGC-L, OTF−GA covalent complex prepared at a low mass ratio of GA to OTF (1:40); OGC-M, OTF−GA covalent complex prepared at a medium mass ratio of GA to OTF (1:20); OGC-H, OTF−GA covalent complex prepared at a high mass ratio of GA to OTF (1:10); OGP-L, OTF−GA physical complex with the same phenolic content as OGC-L; OGP-M, OTF−GA physical complex with the same phenolic content as OGC-M; OGP-H, OTF−GA physical complex with the same phenolic content as OGC-H.

The digestion rate of OGC-H fibrils was the highest among all nanofibrils, and the digestibility of fibrils derived from OTF− GA covalent complexes was higher than that of fibrils prepared from OTF and OTF−GA physical complexes. In all cases, fibrillar structures remained after gastrointestinal digestion. To sum up, fibrils derived from OTF and OTF−GA complexes were resistant to proteolysis, and covalently bound GA could elevate fibril digestibility better than non-covalently bound GA. In summary, bound GA inhibited OTF fibrillation, and more bound GA showed a stronger fibril-inhibitory activity. Covalent bound GA exerted a stronger inhibitory influence on OTF fibrillation than an equal amount of non-covalent bound GA. Building blocks of OTF nanofibrils with bound GA were generally larger than those of OTF nanofibrils. Covalent bound GA shortened OTF nanofibrils significantly, while noncovalent bound GA did not alter the contour length of OTF nanofibrils remarkably. Both covalent and non-covalent bound GA altered the diameter of OTF nanofibrils. Bound GA lowered the isoelectric points of OTF nanofibrils, and covalent bound GA induced greater decreases in the isoelectric points. When compared with an equal amount of non-covalent bound GA, covalent bound GA led to the lower surface hydrophobicity of OTF nanofibrils. The presence of bound GA decreased the viscosity and storage modulus of OTF fibril dispersions. Bound GA endowed fibril dispersions with a strong antioxidant activity. For proteolysis of fibrils, covalent bound GA could elevate the fibril digestion rate better than an equal amount of non-covalent bound GA. The novel study may provide a new approach to modulating the formation and physicochemical properties of protein fibrils according to specific demands.

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AUTHOR INFORMATION

Corresponding Author

Table 7. Digestion Rate of Fibrils Derived from OTF and OTF−GA Complexes after Simulated Intestinal Digestion sample

fibrils; section analysis of nanofibrils; and summary of marked heights of nanofibrils (PDF)

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02630. Schematic formation pathway of OTF−GA covalent complexes; surface hydrophobicity of OTF and OTF− GA complexes; contour length distribution of nanoH

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