The influence of annealing temperature on copper-manganese catalyst towards the catalytic combustion of toluene: The mechanism study

Food Hydrocolloids 98 (2020) 105314

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Impact of covalent or non-covalent bound epigallocatechin-3-gallate (EGCG) on assembly, physicochemical characteristics and digestion of ovotransferrin fibrils Zihao Wei, Qingrong Huang

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Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ, 08901, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Ovotransferrin fibril (−)-Epigallocatechin-3-gallate Covalent binding Non-covalent binding Rheology In vitro digestibility

The objectives of the present study were to investigate impact of covalent or non-covalent bound (−)-epigallocatechin-3-gallate (EGCG) on ovotransferrin (OVT) fibrils. Bound EGCG showed fibril-inhibitory activity in a concentration-dependent manner, and covalent bound EGCG inhibited OVT fibrillation more intensely than an equal amount of non-covalent bound EGCG. Bound EGCG resulted in larger fibril building blocks. Covalent bound EGCG shortened OVT fibrils significantly, and non-covalent bound EGCG induced smaller changes in length of OVT fibrils than covalent bound EGCG. A larger amount of covalent or non-covalent bound EGCG led to shorter OVT fibrils. Covalent bound EGCG did not change thickness of OVT fibrils, while newly emerged thicker fibrils were observed in the presence of non-covalent bound EGCG. Covalent bound EGCG shifted isoelectric point of OVT fibril to lower pHs than non-covalent bound EGCG. Bound EGCG decreased surface hydrophobicity, storage modulus and viscosity of OVT fibrils. OVT fibrils with bound EGCG possessed strong antioxidant capacity. The gastrointestinal digestion result demonstrated that covalent bound EGCG contributed to a higher increase in fibril digestibility than non-covalent bound EGCG.

1. Introduction Amyloid protein fibrils, which possess rod-like structures with high length to diameter ratios, are supramolecular nanostructures resulting from aggregation of proteins or peptides (Jansens et al., 2019; Wei & Huang, 2019a). Rational design of fibrillar protein structures has attracted increasing attention in food science and technology due to intriguing features of amyloid protein fibrils (Jansens et al., 2019; Tang, Wang, & Huang, 2012; Wei, Cheng, & Huang, 2019c; Wei & Huang, 2019b). Although protein fibrils generally have small thickness below 20 nm, protein fibrils have very high mechanical strength, which can be comparable to strength of steel and silk (Smith, Knowles, Dobson, MacPhee, & Welland, 2006). Besides, protein fibrils are relatively stable structures, and they can remain intact in extreme conditions such as low pH and long-term heating (Jansens et al., 2019). In addition, most of protein fibrils have good biocompatibility with no cytotoxicity (Hu et al., 2018; Wei & Huang, 2019a), thus protein fibrils have been regarded as promising building blocks of many natural and artificial functional materials (Knowles & Mezzenga, 2016). Iron-deficiency is a common health problem, and dietary iron supplementation may help to eradicate iron deficiency (Theil, 2004).

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Widespread application of ferritin in food products may act as a natural strategy of iron fortification. Disappointingly, when compared with many other food proteins such as whey protein isolate and zein, comparatively few studies have been conducted about food delivery systems constructed by ferritin, especially ovotransferrin (Wei, Zhu, & Huang, 2019d). Ovotransferrin (OVT) is a ferritin that occupies 12% of egg white proteins, and OVT has many desirable functional properties such as antibacterial and antiviral activity (Giansanti, Leboffe, Pitari, Ippoliti, & Antonini, 2012). In our previous study, iron-bound OVT fibrils with no cytotoxicity are prepared successfully (Wei & Huang, 2019a), and extensive application of iron-bound OVT fibrils in food products may help to minimize dietary iron deficiency. However, although some food protein fibrils such as whey protein fibrils have been systematically studied (Bateman, Ye, & Singh, 2010; Mantovani, Fattori, Michelon, & Cunha, 2016), OVT fibrils have seldom been studied, which has seriously limited their application. Considering that physicochemical properties of fibrils are closely linked with protein type (Jansens et al., 2019), it is essential to study iron-bound OVT fibrils comprehensively. Recent studies demonstrate that OVT fibrils can be employed to construct food-grade delivery vehicles such as organogel-based Pickering emulsions and conventional Pickering emulsions,

Corresponding author. E-mail address: [email protected] (Q. Huang).

https://doi.org/10.1016/j.foodhyd.2019.105314 Received 26 April 2019; Received in revised form 2 August 2019; Accepted 16 August 2019 Available online 17 August 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.

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and these delivery vehicles constructed by OVT fibrils may enhance nutraceutical bioaccessibility (Wei et al., 2019c; Wei & Huang, 2019b, 2019e; Wei, Zhu, Cheng, & Huang, 2019f). Systematic study of OVT fibrils may broaden application of ferritin fibrils in food systems. Mixing of food protein fibrils with other components such as chitosan, phospholipids, calcium ion and lecithin is a feasible approach to modulating formation and physicochemical properties of food protein fibrils (Koo et al., 2018; Liu et al., 2016; Mantovani et al., 2016; Pang, Lin, & Tang, 2017). Since polyphenols can alter physicochemical properties of proteins significantly and protein–polyphenol interactions cannot be excluded during food processing (Le Bourvellec & Renard, 2012), it is necessary to investigate impact of bound polyphenols on formation, physicochemical properties and digestion of protein fibrils. Meanwhile, protein–polyphenol interactions consist of both covalent and non-covalent interactions (Le Bourvellec & Renard, 2012; Wei, Yang, Fan, Yuan, & Gao, 2015). A previous study has demonstrated that covalent and non-covalent protein–polyphenol interactions may result in protein–polyphenol complexes with different physicochemical properties (Wei et al., 2015), suggesting that covalent and non-covalent protein–polyphenol interactions may modify protein materials in different ways. It is expected that covalent and non-covalent bound polyphenols may result in protein fibrils with different physicochemical and functional properties, so it is intriguing to investigate how covalent and non-covalent bound polyphenols exert differential influence on protein fibrils. Because some food components may affect protein fibrils in a concentration-dependent manner (Mantovani et al., 2016), it is essential to study impact of varying amounts of polyphenols on protein fibrils. (−)-Epigallocatechin-3-gallate (EGCG) may be selected as a polyphenol model for the purpose of investigating impact of polyphenols on protein fibrils. EGCG is a major catechin in green tea with molecular weight of 458.37 (Singh, Shankar, & Srivastava, 2011), and EGCG may exist in many food products such as beverages and cakes, implying that interactions of EGCG with many food proteins are possible in a real food industry context. Besides, apart from potentials in treating Parkinson or Alzheimer disease, EGCG can function as antitumor agent or powerful antioxidant (Singh et al., 2011), implying that bound EGCG may possibly endow protein fibrils with many health benefits. Another important reason for selecting EGCG as a polyphenol model is that interactions between EGCG and proteins are relatively well-studied, which facilitates investigating influence of polyphenols on protein fibrils. A previous study shows that both covalent and non-covalent complexations may occur between EGCG and different milk proteins (αlactalbumin, β-lactoglobulin, lactoferrin and sodium caseinate), and protein–EGCG complexations can modulate structural and functional properties of proteins (Wei et al., 2015). The results in the previous study indicates that EGCG is a polyphenol that can modify protein materials effectively, so it is intriguing to study impact of EGCG on protein fibrils. Therefore, the objectives of this study were to investigate impact of varying amount of covalent and non-covalent bound EGCG on formation, physicochemical properties and digestibility of OVT fibrils. Fibril formation was characterized with thioflavin T fluorescence, sodium dodecyl sulfate polyacrylamide gel electrophoresis and atomic force microscopy. The analyzed physicochemical properties of fibrils included zeta potential, surface hydrophobicity as well as rheology, and antioxidant activity of fibrils was also examined. Digestibility of OVT fibrils was studied using an in vitro gastrointestinal model. The novel and comprehensive study could provide deeper insight about impact of polyphenols on protein fibrils.

Neova Technologies Inc. (Abbotsford, Canada). According to manufacturer report, OVT had an iron binding activity above 1000 μg Fe/g sample, and OVT had a molecular weight around 76 kDa. The compound (−)-epigallocatechin-3-gallate (EGCG) with a purity of 95% was purchased from DSM Nutritional Products Ltd (Basel, Switzerland). Prestained protein molecular weight marker and pancreatin were purchased from Thermo Fisher Scientific, Inc. (Waltham, USA). Porcine pepsin was bought from Amresco (Solon, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, USA). Water for all experiments was purified through a Milli-Q water purification system. 2.2. Preparation and characterization of covalent and non-covalent OVT–EGCG complexes 2.2.1. Preparation and characterization of covalent OVT–EGCG complexes 2.2.1.1. Preparation of covalent OVT–EGCG complexes. OVT (40 mg) was dispersed in 10 mL of Milli-Q water and stirred overnight at room temperature to ensure complete dissolution. To suppress microbial growth, sodium azide (0.02%, w/v) was added. Different concentrations of EGCG solutions were prepared by dissolving 1, 2 and 4 mg EGCG in 10 mL of Milli-Q water, respectively. EGCG solution (0.1, 0.2 or 0.4 mg/mL) was mixed with the same volume of OVT solution (4 mg/mL) under continuous stirring. The mixture was adjusted to pH 9, and the mixture was stirred with free exposure to air at room temperature for 24 h (Wei et al., 2015). Afterwards, the samples were dialyzed (molecular weight cutoff 3500 Da) against several changes of water to eliminate the free unbound EGCG, followed by lyophilization to acquire covalent OVT–EGCG complexes. To make this paper more concise, the abbreviations cov-OE-S, cov-OE-M and cov-OE-L were used to denote covalent OVT–EGCG complexes obtained by covalent modification of OVT (4 mg/mL) with different concentrations of EGCG (0.1, 0.2 or 0.4 mg/mL), respectively. Modified Lowry method was employed to determine the protein content of covalent OVT–EGCG complexes (Winters & Minchin, 2005). 2.2.1.2. Total phenolic content of covalent OVT–EGCG complexes. The total phenolic content of covalent OVT–EGCG complexes was measured according to the Folin–Ciocalteu method (Gong et al., 2012). EGCG standard curve was used to calculate the total phenolic content, and the total phenol content result was expressed as mg GA equivalents/g dry complex. 2.2.1.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). To confirm successful preparation of covalent OVT–EGCG complexes, SDS-PAGE of OVT and covalent OVT–EGCG complexes was performed in a vertical gel electrophoresis cell (Bio-Rad Laboratories, Hercules, USA) (Laemmli, 1970). The samples (1.5 mg/ mL) were dissolved in 0.062 M Tris–HCl buffer (pH 6.8, 2% SDS, 5% βmercaptoethanol and 1% bromophenol blue), followed by incubation at 95 °C for 5 min. The loading volume of each sample was 10 μL, and the electrophoresis was run at a constant voltage of 100 V. Subsequently, the gel was stained with Coomassie brilliant blue R250. 2.2.1.4. Measurement of free amino groups. The amount of free amino groups in OVT and OVT–EGCG complexes was determined using an OPA (o-phthaldialdehyde) method (Vigo, Malec, Gomez, & Llosa, 1992), and the content of free amino groups was calculated based on L-leucine standard curve. 2.2.2. Preparation and characterization of non-covalent OVT–EGCG complexes 2.2.2.1. Preparation of non-covalent OVT–EGCG complexes. To investigate how covalent and non-covalent bound EGCG exerted differential influence on OVT fibrils, non-covalent OVT–EGCG complexes were prepared as described previously with minor modifications (Wei et al., 2015; Wei & Gao, 2016a, 2016b). To

2. Materials and methods 2.1. Materials Ovotransferrin (OVT) with a purity above 88% was bought from 2

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exclude interference of the EGCG amount while studying how distinct OVT–EGCG interactions affected OVT fibrillation, non-covalent OVT–EGCG complexes with the same phenolic content as corresponding covalent OVT–EGCG complexes were prepared. Specifically, if 1 g of covalent OVT–EGCG complex consisted of M1 g of OVT and M2 g of EGCG, then M2 g of EGCG solution (at pH 5) was gradually added into OVT solution (at pH 5) containing M1 g of OVT. To hold OVT and EGCG together by non-covalent interactions, the resultant mixture (at pH 5) was stirred at room temperature for 24 h, followed by freeze-drying to acquire non-covalent OVT–EGCG complexes. To get rid of EGCG oxidation and possible covalent OVT–EGCG interactions, non-covalent OVT–EGCG complexes were prepared and stored in the dark without exposure to air. To make this paper more concise, the abbreviations non-OE-S, non-OE-M and non-OE-L were used to denote non-covalent OVT–EGCG complexes with the same phenolic content as cov-OE-S, cov-OE-M and cov-OE-L, respectively.

(pH 2) to a protein concentration of 0.1 mg/mL, and 10 μL of diluted fibrils was spread on a freshly cleaved mica and allowed to adsorb for 2 min at ambient temperature (Wei & Huang, 2019a). The fibrils samples were dried under a nitrogen stream for imaging. NanoScope Analysis Software and FiberApp were employed to analyze AFM data (Usov & Mezzenga, 2015), and contour length distribution of each fibril sample was acquired after analyzing at least 100 individual fibrils.

2.2.2.2. Molecular docking. Interactions between OVT and EGCG in non-covalent OVT–EGCG complexes were studied with the aid of Surflex docking method in SYBYL 8.1 software (Certara, Princeton, USA). The 3D structure of EGCG was optimized at the B3LYP/631 + G(d,p) level of theory using Gaussian 09, and crystal structure of OVT (PDB ID: 1OVT) was prepared by adding hydrogen atoms and removing crystal water. The maximum number of output poses was set as 20, and scoring functions were employed to evaluate the output poses. Ultimately, the optimal pose with the highest C-score and T-score was selected (Cleves & Jain, 2015; Liu et al., 2017).

2.7. Surface hydrophobicity (H0)

2.6. Zeta potential of fibrils Zeta potential of fibrils derived from OVT and OVT–EGCG complexes was measured in the pH range of 2–9. Milli-Q water at preset pH value was used to dilute the fibril samples to a protein concentration of 1 mg/mL, and pH of the diluted fibrils was readjusted with 0.2 M HCl or NaOH. A Zetasizer Nano-ZS90 instrument (Malvern Instruments, Worcestershire, UK) was used to measure zeta potential of the fibril samples at 25 °C.

Surface hydrophobicity of fibrils derived from OVT and OVT–EGCG complexes was measured using ANS (1-anilino-8-naphthalensulfonate) method as described previously (Shen & Tang, 2012). The initial slope of the fluorescence intensity versus fibril concentration was calculated as the index of surface hydrophobicity (Wei & Huang, 2019a). 2.8. Rheological properties of fibrils 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) was applied to measure rheological properties of OVT fibrils and fibrils derived from OVT–EGCG complexes. Fibril dispersions (4 mL) were deposited on the plate of the rheometer and waited for 5 min to allow equilibrium before rheological experiments. Steady-state flow measurements were carried out at 25 ± 0.1 °C, and apparent viscosity of fibrils was recorded as a function of shear rate, varying from 0.01 to 10 s−1. For the dynamic oscillation experiments, the linear viscoelastic regions (LVE) of samples were first determined via the dynamic strain sweep. Dynamic frequency sweep test was carried out at a fixed strain amplitude of 1% (within LVE), and the storage modulus (G′) as well as loss modulus (G″) of fibrils were recorded as a function of frequency, varying from 0.05 to 50 rad/s. The rheology data were obtained and analyzed by Trios Software.

2.3. Fibril formation The optimal fibrillation parameters were acquired in our previous study (Wei & Huang, 2019a). Sodium chloride was added to pH-adjusted Milli-Q water (pH 2) to reach an ionic strength of 150 mM. Afterwards, OVT and OVT–EGCG complexes (400 mg protein equivalent) were dispersed and dissolved in 10 mL of pH-adjusted salt solution (pH 2, 150 mM NaCl), respectively. The final protein concentration of each sample was 40 mg/mL. In order to inhibit microbial growth, sodium azide (0.02%, w/v) was added. For protein fibrillation, the samples were placed into screw-capped vials flushed with nitrogen in the dark, and the vials were heated at 90 °C under magnetic stirring (300 rpm) in an oil bath. After heating at 90 °C for 24 h, the fibril samples were cooled in an ice-water bath immediately and stored in the refrigerator (4 °C) until further analysis. The fibrillation process of OVT and OVT–EGCG complexes was analyzed using characterization tools such as thioflavin T fluorescence, SDS-PAGE and atomic force microscopy.

2.9. DPPH antioxidant activity The ability of scavenging DPPH· (2,2-diphenyl-1-picryl-hydrazylhydrate) was evaluated based on our previous method (Chen et al., 2014), and the antioxidant activity was expressed as nmol Trolox equivalents (TE)/mg sample using Trolox as a standard (Gong et al., 2012).

2.4. Thioflavin T (ThT) fluorescence ThT working solution was freshly prepared based on our previous method (Wei & Huang, 2019a). The tested sample (40 μL) was mixed with 4 mL of ThT working solution and allowed to stand for at least 1 min, followed by immediate measurement to avoid fluorescence quenching. ThT fluorescence was measured on a FluoroMax 3 fluorescence spectrophotometer (Horiba Scientific, Kyoto, Japan). The excitation and emission wavelengths were 440 nm and 482 nm, respectively. The relative ThT fluorescence intensity was acquired via subtracting background signal from ThT working solution.

2.10. In vitro gastrointestinal digestion of fibrils In vitro gastric digestion of fibrils derived from OVT and OVT–EGCG complexes was investigated as described previously (Bateman et al., 2010). The fibril samples were diluted with simulated gastric fluid (pH 1.5, 34 mM NaCl, no enzyme) to reach a final protein concentration of 3 mg/mL. Freshly prepared pepsin solution (10 mg/mL, pH 2) was added into diluted fibril samples, and the final pepsin concentration was fixed as 2 mg/mL. Thereafter, to simulate physiological conditions of in vitro gastric digestion, the samples were incubated at 37 °C and a shaking speed of 50 rpm in VWR 1585 shaking incubator (VWR International, Radnor, USA). Aliquots (160 μL) of the tested samples were drawn after different incubation periods (0–4 h), and 4 mL of ThT working solution was mixed well with the tested samples. Afterwards,

2.5. Morphological observations on fibrils Morphology of fibrils derived from OVT and OVT–EGCG complexes was observed using tapping mode atomic force microscopy (AFM) on a NanoScope IIIA Multimode AFM instrument (Veeco Instruments Inc., Santa Barbara, USA). Fibril samples were diluted with Milli-Q water 3

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to quantify the remaining amount of fibrils after proteolysis, ThT fluorescence was measured as described in 2.4. Digestibility of fibrils was evaluated using in vitro digestion rate. The in vitro digestion rate of fibril samples was calculated as (I0–I1)/I0, where I0 was the initial ThT fluorescence before digestion, and I1 was the ThT fluorescence at any time points during digestion. The simulated intestinal digestion followed simulated gastric digestion. To initiate simulated intestinal digestion, the pH of the gastric digesta was adjusted to pH 7.0 with 0.1 M NaHCO3, and pancreatin solution (10 mg/mL) was added to reach a final pancreatin concentration of 2 mg/mL. To simulate physiological conditions of in vitro intestinal digestion, the samples were shaken at 37 °C and a shaking speed of 50 rpm for 2 h. When simulated intestinal digestion ended, ThT fluorescence was applied to determine digestion rate of fibrils immediately. 2.11. Statistical analysis All experiments were performed in triplicate. OriginPro 2019 software was applied to perform statistical analysis. Statistical differences (significant if p < 0.05) were determined with the aid of One-way analysis of variance (ANOVA). 3. Results and discussion 3.1. Formation of covalent and non-covalent OVT–EGCG complexes

Fig. 1. SDS-PAGE profiles (10% separating gel) of OVT and covalent OVT–EGCG complexes. Lanes from left to right are protein marker, OVT, covOE-S, cov-OE-M and cov-OE-L.

3.1.1. Formation of covalent OVT–EGCG complexes Protein–polyphenol complexations are classified into covalent and non-covalent ones, which may lead to covalent or non-covalent protein–polyphenol complexes (Rohn, 2014; Wei et al., 2015). Covalent protein–polyphenol complexes can be synthesized in the presence of oxygen at alkaline pH. In this case, polyphenols are oxidized into quinones, and reactive nucleophiles in protein chains may covalently bind to the reactive quinones (Rohn, 2014; Wei et al., 2015). In the current study, covalent OVT–EGCG complexes were also prepared in the presence of oxygen at alkaline pH (pH 9). Since SDS could disrupt non-covalent protein–polyphenol complexations (Kroll, Rawel, & Rohn, 2003), SDS-PAGE was employed to confirm covalent grafting of EGCG onto OVT. As shown in Fig. 1, upon conjugation of OVT with EGCG, an increase of molecular weight was observed in covalent OVT–EGCG complexes, which confirmed successful synthesis of covalent OVT–EGCG complexes (Kroll et al., 2003). Besides, it was observed that molecular weight of covalent OVT–EGCG complexes followed the order: cov-OE-L > cov-OE-M > cov-OE-S, which implied that covalent modification of OVT with a larger amount of EGCG could result in covalent OVT–EGCG complexes with more bound EGCG. Afterwards, the content of covalent bound EGCG in covalent OVT–EGCG complexes was quantified. As depicted in Table 1, total phenolic content of cov-OE-S, cov-OE-M and cov-OE-L was 24.37, 47.08 and 90.68 mg/g sample, which further confirmed that conjugation of OVT with more EGCG led to complexes with more covalent bound EGCG. It was intriguing to explore which reactive nucleophiles of OVT participated in covalent OVT–EGCG binding, so change of nucleophilic groups such as amino groups was determined. As indicated in Table 1, the free amino groups of covalent OVT–EGCG complexes were significantly (p < 0.05) fewer than those of OVT, and the number of free amino groups in covalent OVT–EGCG complexes declined with increasing amounts of EGCG. Since SDS may destroy non-covalent protein interactions during measurements of free amino groups (Kroll et al., 2003), it may be deduced that ε-amino groups of lysine indeed react with EGCG and a larger number of EGCG may react irreversibly with more ε-amino groups in OVT. Based on aforementioned discussion, proposed formation pathway of covalent OVT–EGCG complexes is shown in Fig. S1 (see

Table 1 Total phenolic content and free amino groups of covalent OVT–EGCG complexes. Samples

Total phenolic content (mg/g sample)

Free amino groups (nmol/mg protein)

OVT cov-OE-S cov-OE-M cov-OE-L

0 ± 0a 24.37 ± 0.11b 47.08 ± 0.09c 90.68 ± 0.21d

636.4 521.6 404.6 306.2

± ± ± ±

11.2d 20.4c 13.6b 9.7a

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

supplementary data). First, based on current understandings about EGCG oxidation, EGCG is oxidized into EGCG dimer quinone during the first step (Sang, Yang, Buckley, Ho, & Yang, 2007; Wei et al., 2015). Afterwards, the highly active quinone can react with nucleophilic residues of OVT via Schiff base addition, followed by formation of covalent C–N bonds. 3.1.2. Formation of non-covalent OVT–EGCG complexes Non-covalent protein–polyphenol complexes are constructed via non-covalent protein–polyphenol interactions such as hydrogen bonding and hydrophobic bonding. Generally, non-covalent protein–polyphenol complexes are prepared in the absence of oxygen at acidic pH (Le Bourvellec & Renard, 2012; Wei et al., 2015). In this case, polyphenols remain structurally stable, and polyphenols can only interact with proteins via non-covalent complexations. In the current study, non-covalent OVT–EGCG complexes were also prepared in the absence of oxygen at acidic pH (pH 5). In order to eliminate interference of EGCG content on analyzing how covalent or non-covalent bound EGCG affected OVT fibrils differently, the amount of EGCG in non-covalent OVT–EGCG complexes was the same as corresponding covalent OVT–EGCG complexes. Since Surflex-dock predicted poses precisely in molecular docking study of protein–polyphenol complexes (Cleves & Jain, 2015; Liu et al., 2017), Surflex-dock was applied to study non-covalent OVT–EGCG 4

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Fig. 3. Thioflavin T (ThT) fluorescence of OVT and OVT–EGCG complexes after heating at 90 °C and pH 2.0.

S ≈ non-OE-L > cov-OE-M > cov-OE-L. It was found that covalent bound EGCG exerted stronger inhibition on OVT fibrillation than an equal amount of non-covalent bound EGCG. Besides, bound EGCG showed fibril-inhibitory activity in a concentration-dependent manner, and a larger amount of bound EGCG exerted stronger inhibition on OVT fibrillation. These phenomena may be explained by the following explanations. First, protein fibrils are mainly stabilized by hydrophobic interactions, and disruption of hydrophobic interactions leads to fewer fibrils (Meijer et al., 2007). As shown in Table S2, bound hydrophilic EGCG reduced surface hydrophobicity of OVT, and covalent OVT–EGCG complexes were less hydrophobic than non-covalent OVT–EGCG complexes with an equal amount of bound EGCG. It may be reasonably assumed that the building blocks of fibril derived from covalent OVT–EGCG complexes are less hydrophobic than those of fibrils derived from OVT or non-covalent OVT–EGCG complexes, which contributes to weakened hydrophobic interactions and fewer fibrils. Second, as shown in Table S3, bound EGCG endowed OVT with strong antioxidant activity. It can be reasonably inferred that building blocks of fibril derived from OVT–EGCG complexes have stronger antioxidant capacity than those of OVT fibrils. Since antioxidant ability is positively correlated with anti-fibrillization effects (Shoval, Lichtenberg, & Gazit, 2007), EGCG-bound building blocks with powerful antioxidant capacity should inhibit OVT fibrillation. Third, building blocks of fibrils derived from OVT–EGCG complexes may contain bound EGCG, which brings about steric hindrance during fibrillation. The steric hindrance may prevent aggregation of building blocks and lead to fewer fibrils. Similar anti-fibrillation mechanism is reported in a previous study. In that study, bound saccharides can suppress protein fibrillation via steric hindrance (Zou, Chen, Wang, Wang, & Yang, 2016).

Fig. 2. (a) Surflex docking result of EGCG with OVT. (b) Partial enlarged stereoview of the docked pose of EGCG with OVT (Dotted yellow lines indicate hydrogen bonds). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

binding. Fig. 2 shows preferred OVT–EGCG binding mode. OVT–EGCG binding site was situated in the hydrophobic pocket of OVT, and residues including ARG 246, VAL 247, GLY 321, GLU 323, CYS 405 and LEU 407 were responsible for non-covalent OVT–EGCG binding. Intermolecular hydrogen bonds were also observed in non-covalent OVT–EGCG complexes, which was explained by that hydroxyl groups of EGCG could interact with OVT via hydrogen bonds (Le Bourvellec & Renard, 2012). Thus, non-covalent interactions in non-covalent OVT–EGCG complexes included hydrophobic interactions and hydrogen bondings. To confirm that covalent complexations did not exist in non-covalent OVT–EGCG complexes, the change of reactive nucleophilic groups in OVT was quantified. As indicated in Table S1, the amount of free amino groups remained unchanged upon non-covalent OVT–EGCG complexation, suggesting that only non-covalent complexations existed in non-covalent OVT–EGCG complexes.

3.3. Building blocks of OVT fibrils in the presence of bound EGCG Since SDS-PAGE is an effective method to explore building blocks of protein fibrils (Tang et al., 2012), how bound EGCG affected building blocks of OVT fibrils could be studied with the aid of SDS-PAGE. As depicted in Fig. 4, only SDS-PAGE bands of peptides existed in all fibril dispersions, implying that all fibril samples were mainly composed of peptide fragments. Similar result is reported in a previous study, which shows that peptides are major building blocks of β-conglycinin fibrils (Zou et al., 2016). Fig. 4 shows that major building blocks of OVT fibrils in the absence of EGCG were peptides with molecular weight around 10 kDa. It was also found that average molecular weight of hydrolyzed peptides followed the order: peptides derived from covalent OVT–EGCG complexes > peptides derived from non-covalent OVT–EGCG complexes > peptides derived from OVT, implying that building blocks of fibrils derived from covalent OVT–EGCG complexes were larger than those of fibrils derived from OVT or non-covalent OVT–EGCG

3.2. ThT fluorescence in the presence of bound EGCG Since ThT fluorescence is a sensitive and reliable method to probe the amount of amyloid protein fibrils (Biancalana & Koide, 2010), ThT fluorescence was investigated to understand impact of covalent or noncovalent bound EGCG on OVT fibrils. As depicted in Fig. 3, in the presence of bound EGCG, ThT fluorescence intensity after heating at 24 h followed the order: OVT > non-OE-S > non-OE-M > cov-OE5

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Fig. 4. SDS-PAGE profiles (12% separating gel) of OVT and OVT–EGCG complexes after heating at pH 2.0 and 90 °C for 24 h. Lanes from left to right are marker, heated OVT, cov-OE-S, cov-OE-M, cov-OE-L, non-OE-S, non-OE-M and non-OE-L.

complexes. In the case of fibrils derived from covalent OVT–EGCG complexes, it was worthwhile to note that SDS-PAGE bands with molecular weight over 250 kDa existed, which might be a result of intermolecular crosslinking. Since EGCG quinones are good molecule crosslinkers (Chen, Wang, Zhang, Ren, & Zeng, 2011), EGCG quinones may crosslink the neighboring peptide fragments, which results in the large macromolecules with molecular weight over 250 kDa. Because EGCG-crosslinked peptide aggregates exist in fibrils derived from covalent OVT–EGCG complexes, it is reasonably inferred that EGCGcrosslinked peptide aggregates are also building blocks of fibrils derived from covalent OVT–EGCG complexes. 3.4. Morphology of OVT fibrils in the presence of bound EGCG To understand impact of covalent or non-covalent bound EGCG on length of OVT fibrils, AFM images of OVT fibrils in the absence and presence of bound EGCG were captured and shown in Fig. 5. The contour length distribution of all fibril samples was summarized in Fig. 6. As depicted in Fig. 5, in the case of OVT fibrils, apart from short and flexible fibrils with relatively small contour length and persistence length, long and rigid fibrils with relatively large contour length and persistence length coexisted. As shown in Fig. 6, average contour length of OVT fibrils was 327 nm. The fibrils derived from covalent OVT–EGCG complexes were short with maximum contour length below 200 nm, and average contour length of fibrils derived from cov-OE-S, cov-OE-M and cov-OE-L was 102, 87 and 78 nm, respectively. It was apparent that covalent bound EGCG shortened OVT fibrils significantly. The more the covalent bound EGCG was, the shorter the OVT fibrils were. The following explanation may help to explain this phenomenon. As discussed earlier, building blocks of fibrils derived from covalent OVT–EGCG complexes contain bound EGCG, and the bound EGCG may inhibit linear OVT aggregation via steric hindrance, which results in shorter fibrils. Building blocks of cov-OE-S fibrils have less bound EGCG than those of cov-OE-M fibrils and cov-OE-L fibrils, which may lead to relatively weak steric hindrance and relatively long fibrils. In terms of OVT fibrils with non-covalent bound EGCG, Fig. 5 shows that long and rigid fibrils were still observed, implying that non-covalent bound EGCG induced smaller changes in length of OVT fibrils than covalent bound EGCG. As shown in Fig. 6, average contour length of fibrils derived from non-OE-S, non-OE-M and non-OE-L was 312, 303 and 288 nm, respectively. It was clear that non-covalent bound EGCG shortened OVT fibrils slightly, and a larger amount of non-covalent bound EGCG led to shorter OVT fibrils. The dependence of fibril length on the amount of non-covalent bound EGCG can be explained as

Fig. 5. AFM images of fibrils derived from OVT and OVT–EGCG complexes after heating at pH 2.0 and 90 °C for 24 h: (a) OVT fibrils, (b) cov-OE-S fibrils, (c) cov-OE-M fibrils, (d) cov-OE-L fibrils, (e) non-OE-S fibrils, (f) non-OE-M fibrils, (g) non-OE-L fibrils. The scan size is 2 μm × 2 μm, and the z scale is 20 nm. The scale bar represents 200 nm.

follows. Protein fibrils are mainly stabilized by hydrophobic interactions (Meijer et al., 2007), and it may be reasonably inferred that disruption of hydrophobic interactions may lead to shorter fibrils. As discussed earlier, bound hydrophilic EGCG can make building blocks of OVT fibrils less hydrophobic, which may lead to weaker hydrophobic interactions and shorter fibrils. Considering that building blocks of nonOE-L fibrils have more bound hydrophilic EGCG than those of non-OE-S fibrils and non-OE-M fibrils, it is easy to understand that non-OE-L fibril 6

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Fig. 7. Zeta potential of fibrils derived from OVT and OVT–EGCG complexes as a function of pH.

fibrils, while non-covalent bound EGCG altered thickness of OVT fibrils. 3.5. Zeta potential of OVT fibrils with bound EGCG It was possible that bound EGCG could alter surface charge distribution of resultant OVT fibrils, thus influence of covalent or noncovalent bound EGCG on zeta potential of OVT fibrils was studied. As shown in Fig. 7, after non-covalent binding of EGCG to OVT fibrils, isoelectric point (pI) of OVT fibrils decreased from around pH 7.0 to pH 6.1. It was apparent that non-covalent bound EGCG changed distribution of negatively and positively charged groups at the surface of OVT fibrils, which was possibly explained by that non-covalent bound EGCG altered building blocks of OVT fibrils. Fig. 7 depicts that pI of fibril derived from cov-OE-S, cov-OE-M and cov-OE-L was about pH 5.7, pH 5.4 and pH 5.3, respectively. When compared with OVT fibrils with an equal amount of non-covalent bound EGCG, pI of OVT fibrils with covalent bound EGCG shifted to lower pH values. The larger decrease of pI could be explained by blocking of the positively charged amino groups on OVT fibril surfaces (Kroll et al., 2003; Wei et al., 2015). As evidenced in Table 1, covalent modification of OVT with EGCG resulted in blocking of the positively charged amino groups in OVT. It could be reasonably speculated that covalent bound EGCG could also block positively charged amino groups of OVT fibril surfaces, which led to alterations in net surface charge and larger shift of pI to lower pHs. 3.6. Surface hydrophobicity of OVT fibrils with bound EGCG

Fig. 6. Contour length distribution of fibrils derived from OVT and OVT–EGCG complexes: (a) OVT fibrils, (b) cov-OE-S fibrils, (c) cov-OE-M fibrils, (d) cov-OEL fibrils, (e) non-OE-S fibrils, (f) non-OE-M fibrils, (g) non-OE-L fibrils.

It was intriguing to understand how bound EGCG affected distribution and amount of hydrophobic or hydrophilic patches on OVT fibril surfaces, thus surface hydrophobicity of OVT fibrils with bound EGCG was investigated. Table 2 shows that surface hydrophobicity of fibrils followed the order: OVT fibril > non-OE-S fibril > non-OE-M fibril > cov-OE-S fibril > non-OE-L fibril > cov-OE-M fibril > covOE-L fibril. The surface hydrophobicity data implied that bound EGCG decreased surface hydrophobicity of OVT fibrils in a concentrationdependent manner, which could be explained as follows. As evidenced in Table S2, surface hydrophobicity of OVT–EGCG complexes was lower than that of OVT, and it may be reasonably speculated that building blocks of fibrils derived from OVT–EGCG complexes were less hydrophobic than those of OVT fibrils. The less hydrophobic building blocks could contribute to less hydrophobic fibrils derived from OVT–EGCG complexes. Similarly, building blocks of fibrils with more bound EGCG could be less hydrophobic than those of fibrils with less bound EGCG, which contributed to less hydrophobic fibrils. Table 2 also shows that OVT fibrils with covalent bound EGCG were less hydrophobic than OVT fibrils with an equal amount of non-covalent bound EGCG, and the

is the shortest among OVT fibrils with non-covalent bound EGCG. Impact of covalent or non-covalent bound EGCG on thickness of OVT fibrils was also studied. Fig. S2 and Table S4 show that OVT fibrils had a height of 3.0 ± 0.7 or 6.0 ± 0.7 nm. Since protein fibrils are multi-stranded structures composed of many filaments and OVT fibrils have 2-filament or 4-filament multi-stranded structures (Adamcik et al., 2010; Wei & Huang, 2019a), it may be reasonably assumed that average height of each filament in OVT fibrils is around 1.5 nm. As shown in Fig. S2 and Table S4, heights of fibrils derived from covalent OVT–EGCG complexes were centered at 3.0 ± 0.5 or 6.0 ± 0.2 nm, indicating that covalent bound EGCG did not alter thickness of fibrils obviously. In terms of OVT fibrils with non-covalent bound EGCG, it was worthwhile to note that fibrils with thickness of 9.0 ± 0.5 and 12.0 ± 0.7 nm were observed, implying occurrence of 6-filament or 8-filament fibrils in the presence of non-covalent bound EGCG. Overall, covalent bound EGCG did not change the number of multi-stranded filaments in OVT 7

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Table 2 Surface hydrophobicity and DPPH antioxidant activity of fibrils derived from OVT and OVT–EGCG complexes. Samples

Surface hydrophobicity

Antioxidant activity (nmol TE/mg sample)

OVT fibril cov-OE-S fibril cov-OE-M fibril cov-OE-L fibril non-OE-S fibril non-OE-M fibril non-OE-L fibril

1021.4 ± 19.2f 306.6 ± 16.4c 195.5 ± 12.8b 98.9 ± 9.6a 491.6 ± 23.4e 392.2 ± 27.3d 269.6 ± 23.2c

149.7 ± 12.2a 443.3 ± 27.9b 898.8 ± 66.6c 1836.1 ± 98.0d 463.0 ± 45.3b 922.5 ± 87.2c 1897.3 ± 79.9d

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

phenomenon could be explained by that covalent bound EGCG introduced a larger amount of hydrophilic hydroxyl groups onto OVT fibril surfaces than non-covalent bound EGCG (Wei et al., 2015). 3.7. Rheological analysis of OVT fibrils with bound EGCG Investigation of rheological properties helps to understand relationship between macroscopic properties and molecular scale properties, and controlling rheological properties is essential for biopolymeric processing and application. Thus, influence of bound EGCG on rheological properties of OVT fibril dispersions was studied. As shown in Fig. 8a, storage modulus of all fibril samples increased with the rise of frequency, and storage modulus was larger than loss modulus in all fibril samples, implying gel-like behavior of all fibril dispersions. It was also observed that storage modulus of all fibrils derived from OVT–EGCG complexes was lower than that of OVT fibrils, suggesting that bound EGCG could lead to gel weakening of fibrils. Fig. 8b shows apparent viscosity of OVT fibrils in the absence and presence of EGCG. All fibril dispersions displayed shear-thinning behavior, and viscosity of all fibrils derived from OVT–EGCG complexes was lower than that of OVT fibrils. Lower storage modulus and viscosity of fibrils derived from OVT–EGCG complexes could be explained by following explanations. First, rheology of fibril dispersion is associated with fibril concentration, and entanglements of fewer fibrils may lead to lower storage modulus and viscosity (Peng et al., 2018). As mentioned in section 3.2, the fibril amount in fibrils derived from OVT–EGCG complexes is smaller than that of OVT fibrils, and the fewer fibrils result in decreasing storage modulus and viscosity. Second, rheological characteristics of fibrils are affected by contour length and persistence length of fibrils. Fibrils with larger contour length and persistence length may form denser entanglement networks, which contribute to higher modulus and viscosity (Loveday, Rao, Creamer, & Singh, 2009). As mentioned in section 3.3, fibrils derived from OVT–EGCG complexes have smaller contour length and persistence length than OVT fibrils, which can lead to lower modulus and viscosity. Third, hydrophobic interactions contribute to fibrillar networks with stronger entanglement, and decreasing hydrophobic interactions may lead to lower storage modulus of fibril dispersions (Semerdzhiev et al., 2018). As mentioned in section 3.6, bound EGCG reduces surface hydrophobicity of OVT fibrils, which may weaken hydrophobic interactions in fibril networks and lead to decrease in storage modulus.

Fig. 8. (a) Storage modulus (G′) and loss modulus (G″) of fibrils derived from OVT and OVT–EGCG complexes as a function of oscillatory frequency. (b) Apparent viscosity of fibrils derived from OVT and OVT–EGCG complexes as a function of shear rate.

antioxidant activity of 149.7 nmol TE/mg sample, and antioxidant capacity of OVT fibrils was attributed to antioxidant activity of their building blocks (OVT peptides) (Kim, Moon, Ahn, Paik, & Park, 2012). Table 2 shows that OVT fibrils with bound EGCG had much stronger antioxidant capacity than OVT fibrils, which was due to introduction of many phenolic hydroxyl groups (Singh et al., 2011). It was also observed that increasing the amount of bound EGCG could enhance antioxidant activity of OVT fibrils. 3.9. Digestibility of OVT fibrils with bound EGCG Because of increasing interest in application of food protein fibrils in functional foods, it is important to know digestibility of food protein fibrils in gastrointestinal tract (Jansens et al., 2019). Digestibility of OVT fibrils with bound EGCG was studied to understand impact of covalent and non-covalent bound EGCG on fibril digestibility. Given that gastric emptying of a solid meal is usually completed between 3 and 4 h (Minekus et al., 2014), the duration of gastric digestion of all fibrils was set as 4 h in the current study. As shown in Table 3, digestion rate of fibril samples after simulated gastric digestion followed the order: cov-OE-L fibril > cov-OE-M fibril > cov-OE-S fibril > nonOE-L fibril > non-OE-M fibril > non-OE-S fibril > OVT fibril. Obviously, digestion rate of OVT fibrils with bound EGCG was greater than that of OVT fibrils, and digestion rate of fibrils derived from covalent

3.8. Antioxidant activity of OVT fibrils with bound EGCG Since oxidative stress may become deleterious and result in numerous diseases (Reynolds, Laurie, Lee Mosley, & Gendelman, 2007), antioxidant activity is a desirable property of food materials. Food protein fibrils are expected to possess strong antioxidant capacity, so antioxidant capacity of OVT fibrils with bound EGCG was studied. As shown in Table 2, OVT fibrils in the absence of bound EGCG had an 8

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possessed much stronger antioxidant capacity. Bound EGCG could improve digestion rate of OVT fibrils, and covalent bound EGCG led to a higher increase in digestibility than non-covalent bound EGCG. The novel findings in this study may have potential implications for rational design of polyphenol-bound protein fibrils with desirable physicochemical properties.

Table 3 Digestion rate of fibrils derived from OVT and OVT–EGCG complexes after simulated gastric digestion and intestinal digestion. Samples

Digestion rate (%) after gastric digestion

OVT fibril cov-OE-S fibril cov-OE-M fibril cov-OE-L fibril non-OE-S fibril non-OE-M fibril non-OE-L fibril

50.3 69.0 75.4 77.3 59.2 65.8 65.6

± ± ± ± ± ± ±

2.8a 1.9d 0.6e 2.7e 0.9b 2.1c 1.8c

Digestion rate (%) after intestinal digestion 83.1 94.3 94.7 95.4 86.6 87.2 87.5

± ± ± ± ± ± ±

1.0a 0.6d 0.9de 0.8e 0.5b 0.6bc 0.3c

Declaration of interests All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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

Acknowledgements

OVT–EGCG complexes was higher than that of fibrils derived from noncovalent OVT–EGCG complexes. This phenomenon may be explained as follows. As mentioned in section 3.3, fibrils derived from OVT–EGCG complexes consist of larger peptides than OVT fibrils, and fibrils derived from covalent OVT–EGCG complexes are composed of larger peptides than fibrils derived from non-covalent OVT–EGCG complexes. It is reasonably speculated that the larger peptide building blocks have more potential pepsin cleavage sites, so larger peptides may be more susceptible to pepsin digestion. Consequently, more peptide building blocks in fibrils derived from covalent OVT–EGCG complexes are decomposed by pepsin digestion, which leads to higher fibril digestion rate than fibrils derived from OVT or non-covalent OVT–EGCG complexes. Simulated intestinal digestion followed gastric digestion, and Table 3 shows digestion rate of fibril samples after intestinal digestion. Although fibril re-formation during simulated gastrointestinal conditions is possible (Jansens et al., 2019), the fibril amount of all fibril samples underwent a further reduction after intestinal digestion. It was observed that OVT fibrils were not fully digested during gastrointestinal digestion, and similar result was found in soy protein fibrils (Lassé et al., 2016). Table 3 also shows that gastrointestinal digestion rate of fibrils followed the order: fibrils derived from covalent OVT–EGCG complexes > fibrils derived from non-covalent OVT–EGCG complexes > OVT fibrils, suggesting that covalent bound EGCG contributed to a higher increase in fibril digestibility than non-covalent bound EGCG. It was noted that some fibrillar structures in fibrils derived from OVT–EGCG complexes remained after gastrointestinal digestion, which could be ascribed to their resistance to proteolytic digestion.

This work was supported by United State Department of Agriculture, National Institute of Food and Agriculture (grant No. 201967017-29176). We acknowledge financial support from the China Scholarship Council for the first author, and we also thank Guizhao Liang from School of Bioengineering in Chongqing University for his help in molecular docking. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.105314. References Adamcik, J., Jung, J. M., Flakowski, J., De Los Rios, P., Dietler, G., & Mezzenga, R. (2010). Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nature Nanotechnology, 5(6), 423–428. Bateman, L., Ye, A., & Singh, H. (2010). In vitro digestion of β-lactoglobulin fibrils formed by heat treatment at low pH. Journal of Agricultural and Food Chemistry, 58(17), 9800–9808. Biancalana, M., & Koide, S. (2010). Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 1804(7), 1405–1412. Chen, Y., Wang, J., Ou, Y., Chen, H., Xiao, S., Liu, G., et al. (2014). Cellular antioxidant activities of polyphenols isolated from Eucalyptus leaves (Eucalyptus grandis×Eucalyptus urophylla GL9). Journal of Functional Foods, 7, 737–745. Chen, R., Wang, J. B., Zhang, X. Q., Ren, J., & Zeng, C. M. (2011). Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins. Archives of Biochemistry and Biophysics, 507(2), 343–349. Cleves, A. E., & Jain, A. N. (2015). Knowledge-guided docking: Accurate prospective prediction of bound configurations of novel ligands using surflex-dock. Journal of Computer-Aided Molecular Design, 29(6), 485–509. Giansanti, F., Leboffe, L., Pitari, G., Ippoliti, R., & Antonini, G. (2012). Physiological roles of ovotransferrin. Biochimica et Biophysica Acta (BBA) - General Subjects, 1820(3), 218–225. Gong, Y., Liu, X., He, W., Xu, H., Yuan, F., & Gao, Y. (2012). Investigation into the antioxidant activity and chemical composition of alcoholic extracts from defatted marigold (Tagetes erecta L.) residue. Fitoterapia, 83(3), 481–489. Hu, B., Shen, Y., Adamcik, J., Fischer, P., Schneider, M., Loessner, M. J., et al. (2018). Polyphenol-binding amyloid fibrils self-assemble into reversible hydrogels with antibacterial activity. ACS Nano, 12(4), 3385–3396. Jansens, K. J. A., Rombouts, I., Grootaert, C., Brijs, K., Van Camp, J., Van der Meeren, P., et al. (2019). Rational design of amyloid‐like fibrillary structures for tailoring food protein techno‐functionality and their potential health implications. Comprehensive Reviews in Food Science and Food Safety, 18, 84–105. Kim, J., Moon, S. H., Ahn, D. U., Paik, H. D., & Park, E. (2012). Antioxidant effects of ovotransferrin and its hydrolysates. Poultry Science, 91(11), 2747–2754. Knowles, T. P. J., & Mezzenga, R. (2016). Amyloid fibrils as building blocks for natural and artificial functional materials. Advanced Materials, 28(31), 6546–6561. Koo, C. K. W., Chung, C., Picard, R., Ogren, T., Mutilangi, W., & McClements, D. J. (2018). Modulation of physical properties of microfluidized whey protein fibrils with chitosan. Food Research International, 113, 149–155. Kroll, J., Rawel, H. M., & Rohn, S. (2003). Reactions of plant phenolics with food proteins and enzymes under special consideration of covalent bonds. Food Science and Technology Research, 9(3), 205–218. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Lassé, M., Ulluwishewa, D., Healy, J., Thompson, D., Miller, A., Roy, N., et al. (2016). Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white. Food Chemistry, 192, 491–498. Le Bourvellec, C., & Renard, C. (2012). Interactions between polyphenols and macromolecules: Quantification methods and mechanisms. Critical Reviews in Food Science

4. Conclusion In conclusion, covalent and non-covalent bound EGCG showed fibril-inhibitory activity in a concentration-dependent manner, and a larger amount of bound EGCG exerted stronger suppression on OVT fibrillation. When compared with an equal amount of non-covalent bound EGCG, covalent bound EGCG inhibited OVT fibrillation more intensely. Presence of bound EGCG resulted in larger building blocks of OVT fibrils. Covalent bound EGCG shortened OVT fibrils significantly, and non-covalent bound EGCG induced smaller changes in length of OVT fibrils than covalent bound EGCG. Increasing amount of covalent or non-covalent bound EGCG could result in shorter OVT fibrils. Interestingly, newly emerged thicker fibrils were observed in fibrils with non-covalent bound EGCG, and covalent bound EGCG did not change thickness of OVT fibrils. Covalent bound EGCG shifted pI of OVT fibril to lower pHs than non-covalent bound EGCG. Bound EGCG decreased surface hydrophobicity of OVT fibrils, and covalent bound EGCG led to a larger decrease in surface hydrophobicity than noncovalent bound EGCG. OVT fibrils with bound EGCG had lower storage modulus and viscosity than OVT fibrils. In comparison with OVT fibrils in the absence of EGCG, OVT fibrils in the presence of bound EGCG 9

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