Effects of pH Values on Physicochemical Properties and Antioxidant Potential of Whey Protein Isolate-safflower Yellow Complexes

Abstract

Influence of pH values on physicochemical characteristics and antioxidant activities of whey protein isolate (WPI)-safflower yellow (SY) complexes were investigated at 25°C under the aerobic condition for 0, 3 and 6 d. Compared with WPI, WPI-SY had a less particle size at pH 3.5, 6.5, 7.5 and 8.5 from 0 to 6 days. Fluorescence intensity of WPI-SY complexes was appreciably less than WPI, which indicated that the partial tyrosine or tryptophan residues of whey protein had undergone certain changes. Compared with WPI and SY, thermal stability and emulsifying activity of WPI-SY complexes remarkably increased at different pH values. Meanwhile, antioxidant activities of WPI-SY complexes were higher than that of WPI or SY at pH ranged 4.5 to 9.5, kept for 3 and 6 d. Therefore, the interaction of WPI and SY at different pH values, could improve emulsifying activity of WPI and enhance antioxidant activity of SY.

Introduction

The interaction between proteins and polyphenols undergoes their conformation transitions, which might change their functional property and biological activity (Brandelli et al., 2015). Amino acid side chains and polyphenolic aromatic rings form a protein-polyphenol complex by diverse weak forces (primarily hydrophobic), showing that the binding of polyphenols to proteins is primarily a surface phenomenon (Jakobek, 2015). Hydrogen bond is also a kind of interaction between protein and polyphenol, which play a crucial part in maintaining the stability of their complexes (Frazier et al., 2010). Furthermore, in recent years, the interaction between polyphenol and protein has been investigated, using some analyses including fluorescence, light scattering technique, size exclusion chromatography, flow nephelometry and optical microscopy (Von Staszewski et al., 2011). Therefore, previous studies on the interaction of polyphenols with proteins could provide basic data for the development of relevant beverage products containing protein and polyphenol.

Whey proteins are widely used as functional ingredients (Jiang and Brodkorb, 2012). In order to improve and enhance functional properties and physiological functions of whey protein, many methods have been used including its conjugation with carbohydrate (De Souza et al., 2009) or polyphenol (Moreno et al., 2016) via thermal aggregation (Zhang and Zhong, 2010) or enzymatic cross-linking (Jiang et al., 2017) and hydrolysis. For instance, α-lactoalbumin treated by laccase in the presence of ferulic acid had higher surface hydrophobicity and gel strength than α-lactoalbumin (Jiang et al., 2017). Chawla et al. (2009) found that the gamma-irradiation was prone to formation of Maillard reaction products (MRPs) from whey protein and that these MRPs exhibited antioxidant activity. The biologically active peptides obtained from whey protein had physiological effects in vivo, such as antidiabetic, antihypertensive, antimicrobial and antioxidant activities (Brandelli et al., 2015). In addition to above-mentioned methods, the interactions between phenolic compounds and milk proteins were reported. Interaction of flavonoids with proteins has weakened the antioxidant capacity of flavonoids in vitro and in vivo (Xiao et al., 2011). Complexation of tea polyphenols with milk proteins had changed antioxidant activities of tea compounds and secondary structure of milk proteins (Kanakis et al., 2011). Recently, the interaction between proteins and polyphenols are extensively reported, but the effects of pH values on whey protein complexes with polyphenol were little systematically investigated.

Safflower yellow is extracted from the natural plant safflower (Carthamus tinctorius) and has been shown to have numerous pharmacological effects, including anti-inflammation (Zhu et al., 2016) and inhibition of hematoblast conglomeration (Ma et al., 2015). Carthamus tinctorius L has been ratified by China Food and Drug Administration (CFDA) and had beneficial effects during the treatment of cardiovascular and cerebrovascular diseases (Zhu et al., 2016). Safflower yellow mainly consists of hydroxyl safflower yellow A, safflower yellow A and safflower yellow B, as well as other small chemicals (Ma et al., 2016). Safflower yellow contains unique structures of C-glucosylquinochalcone moieties that exist only in Carthamus tinctorius, belonging to the flavonoids family (Mirzajani et al., 2015). Moreover, SY not only has physiological functions, but it is also used as a natural soluble pigment with yellow color in the food industry. However, it is not verified whether SY has stability or keeps its biological activity during the food processing and storage.

Therefore, in this study, we characterized the complexes formed between whey protein isolate and safflower yellow at different pH values and evaluated their performance upon physicochemical properties as well as antioxidant activities.

Materials and Methods

Materials    WPI (93.77% protein content) was purchased from William Mullins, USA. Safflower Yellow (SY) (colors value ≥ 150.0) was bought from the Zhuhai Golden Land Color CO., LTD (Zhuhai, China). Acetonitrile, 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH), and trichloroacetic acid (TCA) were bought from Sigma-Aldrich Co. (St. Louis, USA). The reagents used in the experiments were analytical purity.

Preparation of WPI-SY complexes    Powdered WPI (5% w/v) were dissolved in deionized water and stirred at 25°C for 2 h to ensure complete hydration. The WPI solution was treated at 65°C for 40 min and cooled down. Subsequently, Safflower Yellow (1% w/v) was added into WPI solution and stirred 1 h to make the samples completely dissolved. To prevent microbial growth during sample preparation, sodium azide (0.5% w/v) was also added. The pH value of the WPI-SY solutions was adjusted with 0.1 mol/L NaOH or HCl to 3.5, 4.5, 5.5, 6.5, 7.5, 8.5 and 9.5 respectively. Then, these aqueous systems were mixed with a blender (IKA, T18 digital Ultra-turrax, Germany) at 20 000 rpm for 2 min. Finally, these aqueous systems were stored and analyzed at 25°C under the aerobic condition for 0, 3, and 6 d. WPI or SY was tested as control.

Particle size determination    Droplet size was determined by using the HYL-1076 laser light scattering instrument (Hengyu Instrument Co., Ltd., Dandong, China). The samples were thoroughly mixed within the sampling receptacle to ensure homogeneity in room temperature. Particle size was reported as the size distribution by the median diameter (D₅₀).

Measurement of intrinsic fluorescence spectrum    The intrinsic emission fluorescence spectra of WPI-SY complexes and two control samples were measured by a Fluorphotometer (F4500, Hitachi, Tokyo, Japan). Samples were diluted to a final protein concentration of 0.1 mg/mL, using an aqueous solution with the same pH value of samples. Excitation was set at 280 nm and emission spectrum was set from 300 to 400 nm. Both of excitation and emission slits were set at 5 nm and the scan rate was 240 nm/s.

Determination of emulsification activity and emulsion stability    Emulsification activity was tested according to Agyare et al. (2009) with some modifications. Samples were diluted to a final protein concentration of 1 mg/mL. 1 mL of soybean oil was blended with 3 mL of the diluted sample and then centrifuged at 10 000 rpm. Next, 50 µL of the emulsion at the bottom of the glass container was allowed to stand for 0 minutes and 10 min, added 5 mL of 0.1% SDS solution and mixed thoroughly. The absorbance of the samples was recorded at a wavelength of 500 nm. Emulsification activity (EA) and emulsion stability (ES) were calculated as follows:   

  

where A_500nm is the absorbance at a wavelength of 500 nm, C is the level of the protein (g/mL), ϕ is the volume fraction of the oil phase (ϕ=1/4), A₁₀ is the absorbance of emulsion after it has been left standing for 10 min, A₀ is the absorbance of emulsion before it is left standing and n is the dilution of the sample (n=100).

Determination of thermal stability    Calorimetric analysis was performed using a scanning calorimeter (PE Pyris 6, Perkin Elmer, USA). Calibration was done with zinc and indium as reference materials. Before the determination, all the samples were freeze-dried and then put into aluminum pans. Therefore, each pan was heated up to 125°C at a heating rate of 10°C/min with a constant purging of dry nitrogen (40 mL/min).

Antioxidant activity determination

2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity    DPPH radical scavenging activity of WPI-SY, WPI and SY was measured using detection methods of Gülçin et al. (2010) with a slight modification. Samples (250 µL) were added into 1000 µL of 0.1 mol/L DPPH dissolved with ethanol. And this mixture was stirred for 10 s, then put in the dark for 30 min. The absorbance was recorded at 517 nm, with 95% ethanol as the reference. The percentage scavenging ability of free radical DPPH radical scavenging activity was calculated using the following formula:   

Where A₀ is the absorbance of 0.1 mol/L DPPH without sample, A₂ is the absorbance of sample with 0.1 mol/L DPPH and A₁ is the absorbance of sample with ethanol.

Determination of ferrous reducing power    Ferrous reducing power of WPI-SY, WPI and SY was measured using the method of Gülçin et al. (2012) with minor modification. The sample (0.1 mL) was diluted 50-fold with 0.01 mol/L phosphate buffer, and then 0.5 mL of 0.2 mol/L sodium phosphate buffer (pH 6.6) and 0.5 mL of 1% potassium ferricyanide were added. The reaction system was heated in a 50°C bath for 20 min, rapidly cooled, and then 0.5 mL of 10% trichloroacetic acid was added. The mixture was then centrifuged at 3 000 rpm for 10 min. An aliquot of supernatant (0.5 mL) was mixed with 0.5 mL of deionized water and 0.1 mL of 0.1% FeCl₃. The sample was allowed to stand for 10 min. The absorbance was monitored at 700 nm.

Statistical analysis    Three independent preparations of WPI-SY complexes were carried out. Analyses including the determination of median diameter (D₅₀), EAI, ESI, DPPH radical scavenging activity and ferrous reducing power were carried out at triplicate. Results were described as: mean ± standard deviation. Analysis of variance (one way ANOVA) was performed by using the SPSS system software 17.0. The means were compared using Duncan-multiple range test. The significance of difference was set at p < 0.05. For the particle size distribution, molecular weight distribution, intrinsic fluorescence spectrum and DSC analysis, the experiments were repeated in duplicate for each measurement.

Results and Discussion

Analysis of particle sizes    Average particle sizes of WPI-SY complexes and WPI kept at 25°C under the aerobic condition for 0, 3, and 6 d, as a function of pH value, shown in the Fig. 1. WPI-SY complexes and WPI had slightly higher droplet size at pH 3.5 and 4.5 than at pH 5.5, 6.5 and 7.5 (p < 0.05), but exhibited small increase at pH 8.5 and 9.5 at 25°C under the aerobic condition for 0 d. Droplet sizes of WPI-SY and WPI remarkably increased and then decreased as the pH value ranged from 3.5 to 9.5, kept at 25°C for 3 to 6 d (p < 0.05). Liu et al. (2016b) also found that droplet sizes of lactoferrin and lactoferrin-polyphenol conjugates increased first and then decreased with pH values increased. However, compared with WPI, droplet size of WPI-SY significantly decreased after kept at 25°C under aerobic condition for 0, 3 and 6 d, at pH 3.5, 6.5, 7.5 and 8.5, respectively (p < 0.05), which indicated that SY could slow the accumulation of whey proteins and make WPI more stable, due to the aqueous systems contained smaller particle sizes and possessed higher stability to aggregation (Gulseren and Corredig, 2013). Moreover, droplet size of WPI-SY was bigger than that of WPI at pH 4.5, 5.5, and 9.5, respectively. It was reported that preheated whey protein isolate (Pre-HWPI) containing hibiscus extract and tannic acid showed a reduction of the D₄₃ (volume mean diameter) value when the concentration was 0.5% at pH 6.1, while particle sizes of Pre-HWPI containing grape seed extract, continually increased with increasing concentrations of polyphenols at pH 6.1, which indicated that particle sizes of protein-polyphenol complexes mainly depended on pH values and different types of polyphenols (Thongkaew et al., 2014). Therefore, it was obtained that there were significant effects of SY at different pH values on particle sizes of WPI.

Fig. 1.

Average particle size (D₅₀) of WPI and WPI-SY complexes at 25°C under aerobic condition for 0, 3, and 6 days, respectively, as a function of pH values. Error bars indicate the standard deviation from triplicate tests. Different letters indicate significant differences (p < 0.05).

Fluorescence spectra    Effects of pH values on intrinsic fluorescence spectrum of WPI-SY complexes (Fig. 2A), WPI (Fig. 2B) and SY (Fig. 2C) were presented. Fluorescence intensity of WPI was strong due to tryptophan groups presented in its protein and gradually reduced with the pH values from 3.5 to 9.5. However, fluorescence intensity of SY was the weakest and did not show any significant differences at different pH values, since there is little tryptophan groups in SY. Furthermore, the fluorescence intensity of WPI-SY complexes was appreciably less than that of WPI but more than that of SY. WPI-SY complexes had the maximum fluorescence intensity at pH 4.5. Their fluorescence intensity also decreased with the pH value increased from 5.5 to 9.5. This indicated that alkaline condition was more liable to make tryptophan residues of WPI exposed than the acidic condition. Liu et al. (2016c) also reported that fluorescence intensity of lactoferrin was remarkably more than that of lactoferrin-polyphenol complex. Intrinsic tryptophan fluorescence is especially sensitive to the change of the tryptophan local environment, which can indirectly reflect the tertiary structure of a protein. In the folded state, since the tryptophan residue is hydrophobic, it is usually laid in the center of the protein, giving it a high quantum yield and producing strong fluorescence intensity. Otherwise, the folds are opened and the tryptophan residues are exposed to the solvent (hydrophilic), so the fluorescence intensity is reduced (Cao and Xiong, 2015). In this test, WPI in the presence of SY might also be in a partially or fully unfolded state and its tryptophan residues were exposed to a hydrophilic condition. Therefore, compared with WPI, WPI-SY complexes had reduced fluorescence intensities. Almajano et al. (2007) also verified that fluorescence intensity of BSA containing tryptophan group was strong but its fluorescence intensity decreased when mixed with the catechins.

Fig. 2.

The Intrinsic fluorescence spectrum of WPI-SY complexes (A), WPI (B) and SY (C) at 25°C under aerobic condition for 0 days, as a function of pH values. Arrow corresponds to increasing pH value. Excitation wavelength was set at 280 nm and scanning wavelength ranged from 300 nm to 400 nm.

Emulsifying activity and stability measurement    Emulsifying activity (EA) and emulsion stability (ES) are significant indicators to investigate the emulsion performance in food emulsion systems. EA is estimated the relative coverage rate of a protein on an oil bead in emulsion system, while ES is estimated its relative stability. The emulsifying activities of WPI and WPI-SY complexes at different pH values was shown in Fig. 3A. Compared with WPI, WPI-SY complexes exhibited higher emulsifying activity, which indicated that SY increased emulsifying activity of WPI. Liu et al. (2015) also reported that EA of lactoferrin-epigallocatechin gallate complexes increased, compared with lactoferrin. In this test, WPI-SY complexes had smaller particle size than WPI (Fig. 1), but showed higher emulsifying activity. This indicated that the small molecular WPI-SY complexes had greater mobility to adsorb around the oil-water interfaces (O'Sullivan et al., 2015). The average particle size in phosvitin-resveratrol system had an opposite trend with its emulsifying activity (Duan et al., 2016). In comparison, emulsifying activities of WPI-SY complexes and WPI were the smallest at pH 5.5 and 4.5 respectively, and remarkably increased and thereafter decreased with pH value ranged from 5.5 to 9.5 (p < 0.05). Furthermore, emulsifying activities of WPI-SY complexes and of WPI were the maximum at pH 8.5, which may due to the increased electrostatic and steric repulsions between proteins.

Fig. 3.

Emulsifying activity (A) and stability (B) of WPI, SY, and WPI-SY complexes at 25°C under aerobic condition for 0 days, as a function of pH value. Error bars indicate the standard deviation from triplicate tests. Different letters indicate significant differences (p < 0.05).

To a certain extent, the emulsifying stability of protein represented the ability of protein to bind to small oil droplets. Small oil droplets may progressively merge together to form big oil droplets with emulsifying stability of proteins decreased (Sun et al., 2017). From the Fig. 3B, the emulsifying stability of the WPI-SY complexes was higher than that of WPI at only pH 4.5 and 5.5, where the emulsifying stability of WPI was lower than at other higher pH values. Almajano et al. (2007) illustrated that epicatechin and epigallocatechin gallate showed a synergistic increase on emulsion stability of albumin. It was also found that whey protein exhibited the lowest emulsion stability at pH 4.5, and showed increasing trends at more than or less than the isoelectric point. Malik and Saini (2017) also approved that seed or kernel protein isolates exhibited the lowest emulsion activity and stability at the isoelectric point of pH 5. Furthermore, emulsifying stability of WPI-SY complexes increased and then decreased with pH ranged from 3.5 to 9.5, and was the maximum at pH 7.5 (p < 0.05). This might be due to the hydrophilic-lipophilic balance of emulsion affected by the pH values.

Changes of thermal stability    Effects of pH values on calorimetric analysis of WPI-SY complexes (Fig. 4A), WPI (Fig. 4B) and SY (Fig. 4C) at 25°C under the aerobic condition for 0 days, were presented by recording the DSC thermograms. The DSC thermograms revealed endothermic peaks of SY, WPI and WPI-SY, corresponding to the thermal transition temperature at around 60, 60 and 70°C, respectively. Compared with the WPI and SY, thermal transition temperatures of all the WPI-SY complexes with different pH values, severely increased by 10°C. Furthermore, different pH values had some effects on the DSC thermograms of WPI-SY complexes, WPI and SY, but there are no significant regularity. Our results were similar to the report of Liu et al. (2016a), which showed that thermal transition temperature of the Chlorogenic acid-lactoferrin complexes (101.4°C) was significantly higher than that of the lactoferrin (97.1°C), and the degree of the enthalpy change was obviously less. Liu et al. (2015) also reported that the melting endothermic peaks of pure polyphenols disappeared in the DSC thermograms of the complexes, which could be ascribed to the covalent doping between LF and the polyphenols.

Fig. 4.

Calorimetric analysis of WPI-SY complexes (A), WPI (B) and SY (C) at 25°C under aerobic condition for 0 days, as a function of pH value.

Antioxidant properties

DPPH scavenging activity    Influence of pH values on DPPH radical scavenging activity of WPI, SY and WPI-SY complexes at 25°C under the aerobic condition for 0, 3, and 6 d were shown in the Fig. 5A. It was shown that WPI just enhanced the DPPH radical scavenging activity of SY at pH ranged from 5.5 to 7.5 for 0 d. Almajano et al. (2007) reported that interaction of catechins with BSA reduced the rate of ABTS+ scavenging. However, WPI-SY complexes had stronger DPPH radical scavenging activity than SY or WPI at pH ranged from 3.5 to 9.5 (p < 0.05) for 3 to 6 d. It was shown that physicochemical interactions of SY and WPI led to higher DPPH radical scavenging activity. This was probably due to that combinations or interactions of SY molecules might form dimers, trimers and polymers of SY in WPI-SY. Furthermore, WPI had a relatively low DPPH radical scavenging activity and did not show significant differences during the storage time from 3 to 6 d at 25°C. Meanwhile, DPPH radical scavenging activity of SY had significantly weakened when kept at 25°C for 6 d since SY was easily oxidized (Hiramatsu et al., 2009). Compared with WPI or SY, DPPH scavenging capacities of WPI-SY complexes were overwhelmingly improved at pH 3.5, 4.5 and 9.5 with storage time ranged from 0 to 6 d (p < 0.05).

Fig. 5.

DPPH scavenging percentages (A) and ferrous reducing power (B) by WPI, SY and WPI-SY complexes at 25°C under aerobic condition for 0, 3, and 6 days, as a function of pH value.

SY and WPI-SY complexes had a relatively high DPPH radical scavenging activity at pH from 3.5 to 6.5, but showed some reduction at pH ranged from 7.5 to 9.5. Sun et al. (2016) reported that antioxidant activity of sweet potato leaf polyphenols was higher in neutral and weak acid systems, which was similar to our results. Furthermore, antioxidant activity of WPI remained mainly unaffected by the pH of the solution (p > 0.05), except for pH 4.5. Antioxidant activity of WPI decreased at pH 4.5, since WPI had low net charges at pH 4.5, and its electrostatic repulsion was too small to resist any attractive interactions (Liu et al., 2016a).

Our results were similar to the report of Xiao et al. (2011), which showed that milk proteins obviously undermined the DPPH radical scavenging activity of polyphenols and DPPH scavenging percentages of polyphenols become larger with increasing affinities of milk protein-polyphenol complexes.

Ferrous reducing power    Effects of pH values on ferrous reducing power of WPI, SY and WPI-SY complexes at 25°C under the aerobic condition for 0, 3, and 6 d were shown in the Fig. 5B. In addition to pH 6.5, the ferrous reducing power of WPI, SY and WPI-SY complexes did not show significant differences at other pH values, which indicated that there was not a powerful relevance between the ferrous reducing powers of three systems and pH values. For instance, the ferrous reducing power of WPI-SY complexes increased by 57.32%, 21.32% than that of WPI and SY, at pH 6.5 for 6 d. Furthermore, the ferrous reducing power of WPI, SY and WPI-SY complexes continually increased at the same pH values (3.5 to 9.5), at 25°C from 0 to 6 d (p < 0.05).

Initially, the immediate ferrous reducing power (Day 0) of the WPI-SY complexes was lower than SY at pH from 3.5 to 9.5 (p < 0.05). Meanwhile, antioxidant activity assays also showed an obvious influence of the various milk proteins on the decrease of in vitro antioxidant activity of polyphenols, probably due to weaker non-covalent bonds (Gallo et al., 2013). However, the ferrous reducing power of WPI-SY complexes was higher than that of WPI and SY at pH from 4.5 to 9.5 when stored at 25°C for 3 and 6 d (p < 0.05). This was in accordance with the result of DPPH radical scavenging activity. It was probably due to prolonged storage contributed to form the new compounds or complexes between degraded products of whey protein and SY, had stronger antioxidant capacities than SY. Furthermore, ferrous reducing power of WPI-SY complexes did not mainly changed with the pH ranged from 3.5 to 9.5 for 0, 3 or 6 days. However, DPPH radical scavenging activity of WPI-SY complexes remarkably decreased with the pH ranged from 3.5 to 9.5 for 3 or 6 d (p < 0.05). This indicated that different pH values could not impact ferrous reducing power of WPI-SY complexes. In the future study, antioxidant capacity and interaction mechanism between WPI and SY need further be clarified.

Conclusions

WPI-SY complexes were prepared at different pH values and kept under an aerobic condition at 25°C for 0, 3, and 6 d. The droplet size of WPI-SY complexes was smaller than that of WPI. The fluorescence intensity of WPI-SY complexes was appreciably less than that of WPI, and decreased with the pH value increased from 3.5 to 9.5. Besides, compared with WPI, emulsifying properties and thermal stability of WPI-SY were improved. SY significantly enhanced antioxidant capacity of WPI at pH ranged from 4.5 to 9.5, kept at 25°C for 3 and 6 d.

Acknowledgements    This study was supported by project for the Natural Science Foundation of Heilongjiang Province of China (No.C2017029), the Academic Research Program of Northeast Agricultural University (No.16XG21) and the Postdoctoral Science Research Foundation from Northeast Agricultural University and Inner Mongolia Mengniu Dairy Group Co. Ltd.

References

•  Agyare,  K. K.,  Addo,  K., and  Xiong,  Y. L. (2009). Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH, temperature and salt. Food Hydrocolloid., 23, 72-81.
•  Almajano,  M. P.,  Delgado,  M. E., and  Gordon,  M. H. (2007). Albumin causes a synergistic increase in the antioxidant activity of green tea catechins in oil-in-water emulsions. Food Chem., 102, 1375-1382.
•  Brandelli,  A.,  Daroit,  D. J., and  Corrêa,  A. P. F. (2015). Whey as a source of peptides with remarkable biological activities. Food Res. Int., 73, 149-161.
•  Cao,  Y. and  Xiong,  Y. L. (2015). Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar protein. Food Chem., 180, 235-243.
•  Chawla,  S. P.,  Chander,  R., and  Sharma,  A. (2009). Antioxidant properties of Maillard reaction products obtained by gamma-irradiation of whey proteins. Food Chem., 116, 122-128.
•  De Souza,  H. K. S.,  Bai,  G.,  Gonçalves,  M. d. P., and  Bastos,  M. (2009). Whey protein isolate-chitosan interactions: A calorimetric and spectroscopy study. Thermochim. Acta, 495, 108-114.
•  Duan,  X.,  Li,  M.,  Ma,  H.,  Xu,  X.,  Jin,  Z., and  Liu,  X. (2016). Physicochemical properties and antioxidant potential of phosvitin-resveratrol complexes in emulsion system. Food Chem., 206, 102-109.
•  Frazier,  R. A.,  Deaville,  E. R.,  Green,  R. J.,  Stringano,  E.,  Willoughby,  I.,  Plant,  J., and  Mueller-Harvey,  I. (2010). Interactions of tea tannins and condensed tannins with proteins. J. Pharm. Biomed. Anal., 51, 490-495.
•  Gülçin,  İ.,  Elmastaş,  M., and  Aboul-Enein,  H. Y. (2012). Antioxidant activity of clove oil-A powerful antioxidant source. Arab. J. Chem., 5, 489-499.
•  Gülçin,  İ.,  Huyut,  Z.,  Elmastaş,  M., and  Aboul-Enein,  H. Y. (2010). Radical scavenging and antioxidant activity of tannic acid. Arab. J. Chem., 3, 43-53.
•  Gallo,  M.,  Vinci,  G.,  Graziani,  G.,  De Simone,  C., and  Ferranti,  P. (2013). The interaction of cocoa polyphenols with milk proteins studied by proteomic techniques. Food Res. Int., 54, 406-415.
•  Gulseren,  I. and  Corredig,  M. (2013). Storage stability and physical characteristics of tea-polyphenol-bearing nanoliposomes prepared with milk fat globule membrane phospholipids. J. Agric. Food Chem., 61, 3242-3251.
•  Hiramatsu,  M.,  Takahashi,  T.,  Komatsu,  M.,  Kido,  T., and  Kasahara,  Y. (2009). Antioxidant and neuroprotective activities of Mogami-benibana (safflower, Carthamus tinctorius Linne). Neurochem. Res, 34, 795-805.
•  Jakobek,  L. (2015). Interactions of polyphenols with carbohydrates, lipids and proteins. Food Chem., 175, 556-567.
•  Jiang,  Z. and  Brodkorb,  A. (2012). Structure and antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model system. Food Chem., 133, 960-968.
•  Jiang,  Z.,  Yuan,  X.,  Yao,  K.,  Li,  X.,  Zhang,  X.,  Mu,  Z.,  Jiang,  L., and  Hou,  J. (2017). Laccase-aided modification: Effects on structure, gel properties and antioxidant activities of α-lactalbumin. LWT-Food Sci. Technol., 80, 355-363.
•  Kanakis,  C. D.,  Hasni,  I.,  Bourassa,  P.,  Tarantilis,  P. A.,  Polissiou,  M. G., and  Tajmir-Riahi,  H. A. (2011). Milk beta-lactoglobulin complexes with tea polyphenols. Food Chem., 127, 1046-1055.
•  Liu,  F.,  Ma,  C.,  McClements,  D. J., and  Gao,  Y. (2016a). Development of polyphenol-protein-polysaccharide ternary complexes as emulsifiers for nutraceutical emulsions: Impact on formation, stability, and bioaccessibility of β-carotene emulsions. Food Hydrocolloid., 61, 578-588.
•  Liu,  F.,  Sun,  C.,  Yang,  W.,  Yuan,  F., and  Gao,  Y. (2015). Structural characterization and functional evaluation of lactoferrin-polyphenol conjugates formed by free-radical graft copolymerization. RSC Adv., 5, 15641-15651.
•  Liu,  F.,  Wang,  D.,  Sun,  C., and  Gao,  Y. (2016b). Influence of polysaccharides on the physicochemical properties of lactoferrin-polyphenol conjugates coated β-carotene emulsions. Food Hydrocolloid., 52, 661-669.
•  Liu,  F.,  Wang,  D.,  Sun,  C.,  McClements,  D. J., and  Gao,  Y. (2016c). Utilization of interfacial engineering to improve physicochemical stability of beta-carotene emulsions: Multilayer coatings formed using protein and protein-polyphenol conjugates. Food Chem., 205, 129-139.
•  Ma,  Q.,  Ruan,  Y. Y.,  Xu,  H.,  Shi,  X. M.,  Wang,  Z. X., and  Hu,  Y. L. (2015). Safflower yellow reduces lipid peroxidation, neuropathology, tau phosphorylation and ameliorates amyloid beta-induced impairment of learning and memory in rats. Biomed. Pharmacother., 76, 153-164.
•  Ma,  Z.,  Li,  C.,  Qiao,  Y.,  Lu,  C.,  Li,  J.,  Song,  W.,  Sun,  J.,  Zhai,  X.,  Niu,  J.,  Ren,  Q., and  Wen,  A. (2016). Safflower yellow B suppresses HepG2 cell injury induced by oxidative stress through the AKT/Nrf2 pathway. Int. J. Mol. Med., 37, 603-612.
•  Malik,  M. A. and  Saini,  C. S. (2017). Polyphenol removal from sunflower seed and kernel: Effect on functional and rheological properties of protein isolates. Food Hydrocolloid., 63, 705-715.
•  Mirzajani,  F.,  Bernard,  F.,  Zeinali,  S. M., and  Goodarzi,  R. (2015). Identification of hydroxy-safflor yellow A, safflor yellow B, and precarthaminin safflower using LC/ESI-MSMS. J. Food Meas. Charact., 9, 332-336.
•  Moreno,  T.,  de Paz,  E.,  Navarro,  I.,  Rodríguez-Rojo,  S.,  Matías,  A.,  Duarte,  C.,  Sanz-Buenhombre,  M., and  Cocero,  M. J. (2016). Spray Drying Formulation of Polyphenols-Rich Grape Marc Extract: Evaluation of Operating Conditions and Different Natural Carriers. Food Bioprocess Tech., 9, 2046-2058.
•  O'Sullivan,  Jonathan.,  Beevers,  J.,  Park,  Michael.,  Greenwood,  R., and  Norton,  Ian. (2015). Comparative assessment of the effect of ultrasound treatment on protein functionality pre- and post-emulsification. Colloids and Surfaces A: Physicochem. Eng. Aspects., 484, 89-98.
•  Sun,  H.-N.,  Mu,  T.-H., and  Xi,  L. S. (2016). Effect of pH, heat, and light treatments on the antioxidant activity of sweet potato leaf polyphenols. Int. J. Food Prop., 20, 318-332.
•  Sun,  L.,  Sun,  J.,  Thavaraj,  P.,  Yang,  X., and  Guo,  Y. (2017). Effects of thinned young apple polyphenols on the quality of grass carp (Ctenopharyngodon idellus) surimi during cold storage. Food Chem., 224, 372-381.
•  Thongkaew,  C.,  Gibis,  M.,  Hinrichs,  J., and  Weiss,  J. (2014). Polyphenol interactions with whey protein isolate and whey protein isolate-pectin coacervates. Food Hydrocolloid., 41, 103-112.
•  Von Staszewski,  M.,  Jagus,  R. J., and  Pilosof,  A. M. R. (2011). Influence of green tea polyphenols on the colloidal stability and gelation of WPC. Food Hydrocolloid., 25, 1077-1084.
•  Xiao,  J.,  Mao,  F.,  Yang,  F.,  Zhao,  Y.,  Zhang,  C., and  Yamamoto,  K. (2011). Interaction of dietary polyphenols with bovine milk proteins: molecular structure-affinity relationship and influencing bioactivity aspects. Mol. Nutr. Food Res., 55, 1637-45.
•  Zhang,  W. and  Zhong,  Q. (2010). Microemulsions as nanoreactors to produce whey protein nanoparticles with enhanced heat stability by thermal pretreatment. Food Chem., 119, 1318-1325.
•  Zhu,  H.,  Wang,  X.,  Pan,  H.,  Dai,  Y.,  Li,  N.,  Wang,  L.,  Yang,  H., and  Gong,  F. (2016). The Mechanism by Which Safflower Yellow Decreases Body Fat Mass and Improves Insulin Sensitivity in HFD-Induced Obese Mice. Front. Pharmacol., 7, 127.