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Effect of covalent modification by (−)-epigallocatechin-3-gallate on physicochemical and functional properties of whey protein isolate
LWT - Food Science and Technology 66 (2016) 305e310
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Effect of covalent modification by ()-epigallocatechin-3-gallate on physicochemical and functional properties of whey protein isolate Zhenbao Jia a, Min Zheng b, Fei Tao c, Wenwei Chen c, Guangrong Huang a, Jiaxin Jiang a, * a
Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, China Jiliang University, Hangzhou 310018, China Department of Basic Medical, Jiangxi Medical College, Shangrao 334000, China c National & Local United Engineering Lab of Quality Controlling Technology and Instrumentation for Marine Food, China Jiliang University, Hangzhou 310018, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2015 Received in revised form 16 October 2015 Accepted 21 October 2015 Available online 23 October 2015
The physicochemical and functional properties of covalently modified whey protein isolate (WPI) by ()-epigallocatechin-3-gallate (EGCG) were investigated. WPI was chemically modified by EGCG under alkaline conditions. The effect of modification on foaming and emulsifying properties was evaluated. The results of SDS-PAGE and size exclusion chromatography indicated that modification by EGCG induced cross-linking on proteins of WPI. Fourier transform infrared spectroscopy (FT-IR) analysis illustrated the incorporation of phenolic groups into the modified WPI and the changes in protein secondary structure. Intrinsic fluorescence spectra revealed that modified WPI had a more compact tertiary structure compared to unmodified WPI. The modified WPI exhibited better foaming and emulsifying properties than unmodified WPI. These results suggest that EGCG modification is a potential method for improving the functional properties of WPI. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Whey protein isolate ()-Epigallocatechin-3-gallate Modification Functional property
1. Introduction Whey proteins and their products are important food ingredients that are extensively utilized in manufacturing of food due to their high nutritional quality and versatile functional properties. WPI, one of the most important whey protein products, is a spraydried powder with high protein content. Functional properties for WPI are particularly important in relation to the texture and structure of processed food. Therefore, increasing interest is directed toward modifying WPI to enhance functionality and thereby add value to the protein. Chemical modification is an effective way to improve the functional properties of WPI. During the past decade, a number of chemical modifications such as phosphorylation, glycation, deamidation, succinylation, and Maillard reaction have been proposed to improve the functional properties of WPI (Li, Enomoto, Ohki, Ohtomo, & Aoki, 2005; Liu & Zhong, 2012; Ma, Forssell, Partanen, Buchert, & Boer, 2011; Morand, Guyomarc'h, Legland, & Famelart, 2012). Phenolic compounds represent the largest group of secondary plant metabolites and are widely distributed in plants. Dietary
* Corresponding author. E-mail address: [email protected] (J. Jiang). http://dx.doi.org/10.1016/j.lwt.2015.10.054 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
phenolic substances have received much attention due to their ability to scavenge reactive oxygen species. Additionally, dietary phenolic compounds are known to interact with proteins in the food matrix. The details of the interactions between target proteins and phenolic compounds are not well understood, but covalent interactions of food proteins with phenolic compounds have been proposed. Phenolic compounds may be oxidized in an alkaline solution to their corresponding quinones (Hurell & Finot, 1984). The electron-deficient quinones represent a species of highly reactive substances that normally react further with nucleophilic amino acid residues in a protein chain (Kroll, Rawel, & Rohn, 2003; Rawel, Rohn, Kruse, & Kroll, 2002). Many food proteins such as whey proteins, myoglobin, lysozyme, bovine serum albumin, and soy proteins could interact with phenolic compounds in this manner. Covalent modification by phenolic compounds produces food protein derivatives that have different physicochemical and conformational properties compared with unmodified proteins. Rawel, Czajka, Rohn and Kroll (2002) reported that soy protein derivatives exhibited different characteristics compared with unmodified soy protein, including isoelectric points, solubility, digestibility, secondary structure, and thermal stability. Ali, Homann, Khalil, Kruse, and Rawel (2013) reported that modification of blactoglobulin with coffee-specific phenolic compounds resulted in
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changed structural properties, and alteration in solubility, surface hydrophobicity, and emulsifying properties were observed. Tea is one of the most widely consumed beverages in the world, and is a rich source of health protective phenolic compounds (Kim et al., 2011; Thielecke & Boschmann, 2009). The predominant bioactive phenolic component of green tea, ()-epigallocatechin-3gallate (EGCG) is known to possess antioxidant, anti-inflammatory, and anti-cancer cell proliferation properties (Katiyar, Afaq, Azizuddin, & Mukhtar, 2001; Lestringant, Guri, Gülseren, Relkin, & Corredig, 2014; Zhong, Chiou, Pan, & Shahidi, 2012). On the basis of previous literature, we hypothesized that EGCG could covalently bind to nucleophilic amino acid residues in whey protein under alkaline conditions, which could lead to conformational changes. It is well known that the functional properties of food proteins are closely related to their physiochemical and structural characteristics. Accordingly, functional properties of whey protein may be altered by modification with EGCG through covalent reaction. However, to our knowledge, no studies have investigated the effects of covalent modification by EGCG on functional properties of WPI, particularly the foaming and emulsifying properties. Therefore, the main objective of the present study was to investigate the effect of covalent modification by EGCG on the physicochemical characteristics of WPI. Furthermore, the foaming and emulsifying properties of modified WPI were evaluated and compared.
TriseCl, pH 6.8, with 2% SDS, 10% glycerol and 0.1% bromophenol blue). Each well in the SDS-PAGE gel was loaded with 10 mg of WPI sample. Middle range unstained protein standard (catalog no. BM525, Sangon Biotech, Shanghai, China) was applied. The gel was stained with Coomassie Blue R-250, destained, and scanned.
2. Material and method
2.6. Intrinsic fluorescence spectroscopy
2.1. Chemicals
The fluorescence spectra were recorded using a Shimadzu RF5301PC spectrofluorometer (Tokyo, Japan) in a 1 cm path length quartz cell. The excitation wavelength was 285 nm. Both the excitation and emission slit widths were set at 5 nm. The concentration of sample solutions prepared in 50 mM phosphate buffer (pH 7.0) was adjusted to 0.3 mg/mL. The emission spectra were collected between 300 and 450 nm.
WPI was obtained from Hilmar Cheese Co. (Hilmar, CA, USA). According to the product bulletin, the typical composition of the WPI was 89% protein, 1.5% lactose, 0.5% fat, 2.5% ash, and 4.5% moisture. EGCG, KBr, and electrophoresis reagent were purchased from SigmaeAldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Corn oil was purchased from Wumart Stores, Inc. (Hangzhou, China). All other chemicals were of reagent grade and obtained from Mike Chemical Co., Ltd. (Hangzhou, China). 2.2. Preparation of modified WPI with EGCG WPI powder was dispersed in deionized water (35 g into 900 mL), and the pH value of the protein dispersion was adjusted to 9.0 using 0.5 M NaOH. EGCG solution (50 mL of 5 mg/mL) was mixed with the protein dispersion and then the volume was adjusted to 1000 mL with deionized water. After 12 h of reaction time under continuous stirring at 25 C, the pH value of the protein dispersion was adjusted to 6.8 using 0.5 M HCl. To remove the free phenolic compounds in the protein dispersion, an ultrafiltration was performed using a Millipore Pellicon cassette module (Bedford, MA, USA), containing Biomax-5 membrane with a molecular weight cut-off (MWCO) of 5 kDa, with a membrane area of 0.1 m2. The ultrafiltration was operated in the mode of batch ultrafiltration with full recycle of the retentate. Until the final volume reduced to 200 mL, 800 mL of deionized water was added into the retentate. The ultrafiltration was repeated five times, and then the retentate was lyophilized. In addition, a control experiment was performed to obtain the unmodified WPI without EGCG during the same period. 2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed by a slab gel made from 5% stacking gel and 12% separating gel, according to the method of Laemmli (1970). WPI samples were dissolved in loading buffer (50 mM
2.4. Size exclusion chromatography (SEC) Size exclusion chromatography (SEC) experiments were per€ formed using the GE Healthcare AKTA Purifier 100 FPLC system equipped with a monitor UV-900 (Uppsala, Sweden) on a column of Sephacryl S-100 HR (1.6 70 cm). The column was equilibrated and eluted with 100 mM phosphate buffer (pH 7.8) at a flow rate of 1 mL/min. A 500 mL aliquot of WPI solution (5 mg/mL) was loaded on the column. The absorbance at 280 nm was used for detection of protein elution. 2.5. FT-IR spectroscopy Infrared spectra were recorded at room temperature using a Thermo Scientific Nicolet 380 spectrometer (Madison, WI, USA) equipped with a deuterated triglycine sulfate detector. The WPI samples were mixed with KBr and then laminated. The resolution and scanning time were 4 cm1 and 32 times, respectively.
2.7. Determination of foaming properties Foaming properties were determined by using the method described by Aewsiri, Benjakul, and Visessanguan (2009) with some modifications. WPI dispersions (5, 20 and 35 mg/mL) were prepared in graduated test tubes by dispersing WPI powder in 20 mL of 100 mM phosphate buffer (pH 6.8). Whipping treatment was conducted at 12,000 rpm for 2 min at 25 C using the IKA T25 homogenizer (Staufen, Germany). The sample was allowed to stand for 15 min at 25 C. Both foaming capacity (FC) and foaming stability (FS) were calculated from the following equations:
FC ð%Þ ¼
V1 100 V0
FS ð%Þ ¼
V2 100 V1
where, V0 is the liquid volume before whipping, V1 is the initial foam volume after whipping and V2 is the final foam volume after leaving at 25 C for 15 min. 2.8. Emulsion preparation and particle size determination WPI dispersions (10, 20, and 40 mg/mL) were prepared in beakers by dispersing WPI powder in 150.0 mL of 100 mM phosphate buffer (pH 6.8) containing sodium azide (0.1 mg/mL). The emulsion was formed by transferring 50.0 mL of corn oil into the sample dispersion. The mixture was then pre-homogenized with the IKA T25 homogenizer (Staufen, Germany) at 12,000 rpm for 1 min at
Z. Jia et al. / LWT - Food Science and Technology 66 (2016) 305e310
25 C. The crude emulsions were then homogenized with GEA Niro Soavi NS1001L2K high pressure homogenizer (Parma, Italy) for three passes at 40 MPa. After 0 and 28 days of storage at room temperature, a 30 mL aliquot of the emulsion sample was transferred into 50 mL of SDS solution (1 mg/mL) and mixed. Then, the average droplet size was measured with Malvern Nano S90 Zetasizer (Worcestershire, U.K.), and expressed as the volume-surface average droplet size (d32) of emulsion. 2.9. Statistical analysis Determinations of foaming and emulsifying properties were carried out in triplicate and the data was expressed in mean ± standard deviation. Differences between means were assessed using a one-way analysis of variance (ANOVA). Values of P < 0.05 were considered to be statistically significant. Statistical analysis was performed using SPSS statistical software version 13.0 (SPSS Inc, Chicago, USA). 3. Results and discussion 3.1. Profile of modified WPI by EGCG SDS-PAGE run under reducing conditions was used to study change in the protein profile of WPI as a result of modification by EGCG. As shown in Fig. 1, the SDS-PAGE pattern of unmodified WPI showed three main bands attributed to a-lactalbumin (a-La), blactoglobulin (b-Lg), and bovine serum albumin (BSA), respectively (Lane 1). The SDS-PAGE pattern of modified WPI (Lane 2) revealed the formation of protein complexes, demonstrated by the dark stains at the top of the separating gel. Additionally, the formation of a new broad band with an estimated average molecular weight of around 36 kDa was observed at the middle of the separating gel, suggesting that the oligomer of whey proteins was obtained. The intensities of a-La, BSA, and b-Lg bands in modified WPI decreased by comparison to those of unmodified WPI, suggesting that the proteins corresponding to these bands could be involved in the formation of cross-linked products. The SDS-PAGE results
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demonstrated that the reaction by EGCG caused covalent crosslinking of whey proteins. To support this finding, we used size exclusion chromatography (SEC) to analyze the unmodified and modified WPI. Fig. 2 shows the elution profiles of samples. Peaks 1, 2, and 3 in the elution profile of unmodified WPI were attributed to BSA, b-Lg, and a-La, respectively. In the elution profile of modified WPI, a new peak appeared between peak 1 and peak 2, supporting the hypothesis that a crosslinking reaction occurred. Furthermore, the intensities of peaks 2 and 3 decreased and the intensity of peak 1 increased, which indicated that the whey protein components were polymerized to form protein complexes with high molecular weight. The results of SDS-PAGE and SEC illustrated that EGCG induced a cross-linking reaction and caused the change of profile for WPI. Our findings were not in agreement with the results of previous literature. Ishii et al. (2008) investigated the interaction between proteins and EGCG, and showed that EGCG can covalently bind to proteins through autoxidation, generating protein-EGCG conjugates. Meanwhile, Ali et al. (2013) also reported that the interaction between b-Lg and chlorogenic acid under alkaline conditions resulted in the attachment of phenolic derivatives to protein chains, leading to the formation of adducts. We speculated that the characteristics of both the protein and the phenolic compounds played crucial roles in the formation of reaction products. 3.2. FT-IR Determining a protein's structure is essential for fully understanding its functional properties. FT-IR is a sensitive tool for detecting conformational changes of protein secondary structure (Jackson & Mantsch, 1995). Fig. 3 shows the FT-IR spectra of unmodified and modified WPI. In the spectrum of unmodified WPI, we observed three strong bands at 3294.2, 1648.0, and 1541.4 cm1, corresponding to the vibrations for the amide A, amide I, and amide Ⅱ of protein, respectively. As compared to the spectrum of unmodified WPI, a new absorption band at 3399.0 cm1 appeared in the EGCG-modified WPI spectrum, attributed to the OeH stretching vibration of the phenolic groups. The new band confirmed the binding of phenolic groups to the protein components in WPI via the covalent bond. Therefore, we speculated that some EGCG derivatives were incorporated into the cross-linked protein as bridging agents. The role of phenolic compounds as bridging agents in laccase-catalyzed cross-linking of protein has been proved in previous reports (Mattinen et al., 2005; Steffensen, Andersen, Degn, & Nielsen, 2008). The amide I band represents the vibration of C]O stretching of the peptide bond, whereas the amide Ⅱ band is primarily due to the vibrations of NeH bending and CeN stretching. Both amide I and amide Ⅱ bands consist of overlapping bands at characteristic frequencies corresponding to different secondary structure elements (e.g., a-helices, b-sheets, turns, and disordered structures). Therefore, the frequencies of amide I and amide Ⅱ bands are highly sensitive to the secondary structure of the polypeptide chain € cker, Ofstad, Sørheim, & Kohler, 2009). There have been (Carton, Bo a large amount of experimental and theoretical studies on these vibrations, especially with regard to their frequencies in relation to the secondary structures of polypeptide chains (Pelton & McLean, 2000; Torii, 2012). As shown in Fig. 3, the frequencies of amide I and amide Ⅱ for modified WPI shifted to 1651.1 and 1538.2 cm1, respectively. This indicated that the secondary structure of WPI was changed after modification. 3.3. Intrinsic fluorescence emission spectrum
Fig. 1. SDS-PAGE patterns of unmodified and modified WPI. Lane 1, Unmodified WPI; Lane 2, Modified WPI; Lanes 3, Molecular weight standard.
The intrinsic fluorescence emission spectrum of protein is
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Fig. 2. SEC elution profiles of unmodified and modified WPI on the column of Sephacryl S-100 HR.
Fig. 3. The FT-IR spectra of unmodified and modified WPI.
usually dominated by tryptophan (Trp) and provides sensitive detection of tertiary conformation of the protein involved. The main proteins in WPI were b-lactoglobulin and a-lactalbumin, which contained two and four Trp residues, respectively. To gain more insight into the conformational changes caused by the reaction of WPI with EGCG, the intrinsic Trp fluorescence assay was performed. Fig. 4 shows the intrinsic emission fluorescence spectra of unmodified and modified WPI with EGCG. The maximum fluorescence emission for modified WPI shifted from 336 nm to 339 nm, and the fluorescence intensity dramatically decreased. The red shift of the emission maximum indicated that the major fluorophore Trp was exposed to a more hydrophilic environment after modification, as a result of perturbation of protein tertiary structure (Kristo, Hazizaj, & Corredig, 2012). The decrease in the fluorescence intensity reflects that the Trp residues are less exposed in the modified WPI as compared to the control. A more compact protein molecule may allow deeper burial of its fluorophores in the protein core than a less compact molecule (Withana-Gamage, Hegedus, Qiu, Mclntosh, & Wanasundara, 2013); therefore, the low
Fig. 4. Intrinsic fluorescence spectra of unmodified and modified WPI.
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309
Table 1 The foaming capacity (FC) and foaming stability (FS) of modified and unmodified WPI at different concentrations. 5 mg/mL
20 mg/mL
FC (%)
FS (%) a
27.2 ± 4.98 50.0 ± 1.32b
Unmodified WPI Modified WPI
35 mg/mL
FC (%) a
FS (%) a
27.0 ± 7.36 25.5 ± 1.68a
157.8 ± 2.42 187.7 ± 14.67b
FC (%) a
14.2 ± 0.58 23.4 ± 4.69b
FS (%) a
159.2 ± 3.48 208.6 ± 1.79b
15.4 ± 1.45a 30.1 ± 2.41b
Results are expressed as the mean ± standard deviation; n ¼ 3. Different letters within the same column are statistically different (P < 0.05).
fluorescence intensity may indirectly indicate a high degree of molecule compactness for modified WPI. Another possible cause for the decrease in the intensity of intrinsic fluorescence may have been the attachment of phenolic moieties to protein, which led to a quenching effect, thereby reducing the emission intensity (Feroz, Mohamad, Bujang, Malek, & Tayyab, 2012). 3.4. Foaming properties Foaming properties, estimated through FC and FS, are presented in Table 1. In the range of 5e35 mg/mL, the FC of modified WPI was significantly higher than those of unmodified WPI (P < 0.05). At the low concentration (5 mg/mL), FS of modified WPI was comparable to that of unmodified WPI (P > 0.05), but at the higher concentrations (25 mg/mL and 35 mg/mL), FS was significantly higher than those of unmodified WPI (P < 0.05). The results revealed that the foaming properties of WPI were greatly improved by EGCG modification. As described above, we found that the WPI was significantly modified by the cross-linking reaction induced by EGCG. The focus of previous studies were on the manipulation of foaming behavior of proteins by means of cross-linking treatment (Foegeding, Luck, & ez, Busti, Ballerini, & Delorenzi, 2010). MoDavis, 2006; Moro, Ba lecular size of food protein was a crucial factor influencing foaming properties. It has been observed that whey protein polymers exhibited higher intrinsic viscosity compared with native whey protein, which generated more stabilized foams by the effective deceleration of drainage rate (Davis & Foegeding, 2004; Vardhanabhuti & Foegeding, 1999). Partanen et al. (2009) reported that the foaming capacity of sodium caseinate was improved due to the cross-linking reaction catalyzed by transglutaminase. Furthermore, the authors indicated that the cross-linking treatment changed the interfacial elasticity and enhanced the foam formation by means of suppressing coalescence and disproportionation. Additionally, it has been demonstrated by Kuan, Bhat, and Karim (2011) that the cross-linking and polymerization of egg white protein improved the foaming properties, because the cross-linked structure effectively enhanced the unfolding of the protein during foam forming and formation of more elastic foam networks at the airewater interfaces. Therefore, the improved foaming properties of modified WPI might be explained by the increased molecular size due to the cross-linking reaction. 3.5. Emulsifying properties In the present study, the original d32 of emulsions (day 0) were
measured as the indicator of emulsifying ability, and the increases of d32 after 28 days of storage were followed to evaluate the emulsion stability. The results are presented in Table 2. At low concentrations (10 and 20 mg/mL), the modified WPI exhibited significantly lower original d32 values than unmodified WPI (P < 0.05). But as the concentration rose to 40 mg/mL, there was no significant difference between the original d32 values of modified and unmodified WPI (P > 0.05). The results implied that the emulsifying ability of WPI was increased after the modification. At all concentrations assayed, the increases of d32 values of modified WPI emulsions were lower than those of unmodified WPI emulsions after 28 days of storage, indicating EGCG modification improved the emulsion stability of WPI. This finding was consistent with the results in previous literature, which revealed that a-Lapolyphenol covalent complexes had better emulsion stability than a-La (Wang et al., 2015). WPI is considered to be a surface-active agent and can adsorb to the surfaces of oil droplets to form an interfacial film and stabilize the oil/water emulsion against flocculation or coalescence. The emulsifying properties of proteins depend on their abilities to diffuse to the oil/water interface, and then undergo partial unfolding and rearrangement at the interface. Conformational characteristic of food protein is particularly important in relation to the emulsifying properties of protein. According to Jambrak, Lelas, Mason, Kresi c, and Badanjak (2009), the changes of the protein conformational structure could influence surface hydrophobicity and subsequently lead to better adsorption of the oil/water emulsion system. Afizah and Rizvi (2014) stated that the improvement in the emulsifying activity of texturized whey protein concentrate was caused by the increase of exposed aromatic residues, which resulted in the increased affinity of the proteins towards the oilwater interface. They also explained that the aggregated whey protein formed a thicker protein membrane, thus providing better emulsifying stability for the oil droplets. EGCG-induced protein conformational changes were indicated by the results obtained from SDS-PAGE, FT-IR, and intrinsic Trp fluorescence assay. Therefore, the results suggested that the conformational changes on the surface properties of WPI upon EGCG modification led to the improvement of the emulsifying properties. 4. Conclusion In summary, our results indicated the possibility of using EGCG modification to improve the functional properties of WPI. The EGCG modification caused cross-linking and conformational structure changes of proteins in WPI. Furthermore, we found that
Table 2 The average droplet size (d32) of emulsion with modified and unmodified WPI at different concentrations during storage. 0 day
28 days
10 mg/mL Unmodified WPI Modified WPI
20 mg/mL a
689.8 ± 42.31 506.9 ± 31.78b
40 mg/mL a
423.9 ± 30.78 350.2 ± 17.33b
301.3 ± 12.62 288.6 ± 7.17a
10 mg/mL a
20 mg/mL a
804.4 ± 40.84 596.2 ± 26.73b
40 mg/mL a
509.5 ± 31.41 409.4 ± 20.16b
Results are expressed as the mean ± standard deviation; n ¼ 3. Different letters within the same column are statistically different (P < 0.05).
337.2 ± 17.42a 299.4 ± 12.35b
310
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the foaming and emulsifying properties of WPI were enhanced by EGCG modification. This study provides valuable information on the potential application of modified WPI by EGCG. Further research is needed to evaluate the effect of EGCG modification on other functionalities of WPI, in order to develop their applications in food industries. Acknowledgments We gratefully acknowledge the financial support from the National Natural Science Foundation of China (31101319). References Aewsiri, T., Benjakul, S., & Visessanguan, W. (2009). Functional properties of gelatin from cuttlefish (Sepia pharaonis) skin as affected by bleaching using hydrogen peroxide. Food Chemistry, 115, 243e249. Afizah, M. N., & Rizvi, S. S. H. (2014). Functional properties of whey protein concentrate texturized at acidic pH: effect of extrusion temperature. LWT e Food Science and Technology, 57, 290e298. Ali, M., Homann, T., Khalil, M., Kruse, H. P., & Rawel, H. (2013). Milk whey protein modification by coffee-specific phenolics: effect on structural and functional properties. Journal of Agricultural and Food Chemistry, 61, 6911e6920. € cker, U., Ofstad, R., Sørheim, O., & Kohler, A. (2009). Monitoring secCarton, I., Bo ondary structural changes in salted and smoked salmon muscle myofiber proteins by FT-IR microspectroscopy. Journal of Agricultural and Food Chemistry, 57, 3563e3570. Davis, J. P., & Foegeding, E. A. (2004). Foaming and interfacial properties of polymerized whey protein isolate. Journal of Food Science, 69, C404eC410. Feroz, S. R., Mohamad, S. B., Bujang, N., Malek, S. N. A., & Tayyab, S. (2012). Multispectroscopic and molecular modeling approach to investigate the interaction of flavokawain B with human serum albumin. Journal of Agricultural and Food Chemistry, 60, 5899e5908. Foegeding, E. A., Luck, P. J., & Davis, J. P. (2006). Factors determining the physical properties of protein foams. Food Hydrocolloids, 20, 284e292. Hurell, R. F., & Finot, P. A. (1984). Nutritional consequences of the reactions between proteins and oxidized polyphenolic acids. Advances in Experimental Medicine and Biology, 177, 423e435. Ishii, T., Mori, T., Tanaka, T., Mizuno, D., Yamaji, R., Kumazawa, S., et al. (2008). Covalent modification of proteins by green tea polyphenol (e)-epigallocatechin-3-gallate through autoxidation. Free Radical Biology & Medicine, 45, 1384e1394. Jackson, M., & Mantsch, H. H. (1995). The use and misuse of FTIR spectroscopy in the determination of protein structure. Critical Reviews in Biochemistry and Molecular Biology, 30, 95e120. Jambrak, A. R., Lelas, V., Mason, T. J., Kresi c, G., & Badanjak, M. (2009). Physical properties of ultrasound treated soy proteins. Journal of Food Engineering, 93, 386e393. Katiyar, S. K., Afaq, F., Azizuddin, K., & Mukhtar, H. (2001). Inhibition of UVBinduced oxidative stress-mediated phosphorylation of mitogen-activated protein kinase signaling pathways in cultured human epidermal keratinocytes by green tea polyphenol ()-Epigallocatechin-3-gallate. Toxicology and Applied Pharmacology, 176, 110e117. Kim, A., Chiu, A., Barone, M. K., Avino, D., Wang, F., Coleman, C. I., et al. (2011). Green tea catechins decrease total and low-density lipoprotein cholesterol: a systematic review and meta-analysis. Journal of the American Dietetic Association, 111, 1720e1729. Kristo, E., Hazizaj, A., & Corredig, M. (2012). Structural changes imposed on whey proteins by UV irradiation in a continuous UV light reactor. Journal of Agricultural and Food Chemistry, 60, 6204e6209.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effect of covalent modification by ()-epigallocatechin-3-gallate on physicochemical and functional properties of whey protein isolate Zhenbao Jia a, Min Zheng b, Fei Tao c, Wenwei Chen c, Guangrong Huang a, Jiaxin Jiang a, * a
Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, China Jiliang University, Hangzhou 310018, China Department of Basic Medical, Jiangxi Medical College, Shangrao 334000, China c National & Local United Engineering Lab of Quality Controlling Technology and Instrumentation for Marine Food, China Jiliang University, Hangzhou 310018, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2015 Received in revised form 16 October 2015 Accepted 21 October 2015 Available online 23 October 2015
The physicochemical and functional properties of covalently modified whey protein isolate (WPI) by ()-epigallocatechin-3-gallate (EGCG) were investigated. WPI was chemically modified by EGCG under alkaline conditions. The effect of modification on foaming and emulsifying properties was evaluated. The results of SDS-PAGE and size exclusion chromatography indicated that modification by EGCG induced cross-linking on proteins of WPI. Fourier transform infrared spectroscopy (FT-IR) analysis illustrated the incorporation of phenolic groups into the modified WPI and the changes in protein secondary structure. Intrinsic fluorescence spectra revealed that modified WPI had a more compact tertiary structure compared to unmodified WPI. The modified WPI exhibited better foaming and emulsifying properties than unmodified WPI. These results suggest that EGCG modification is a potential method for improving the functional properties of WPI. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Whey protein isolate ()-Epigallocatechin-3-gallate Modification Functional property
1. Introduction Whey proteins and their products are important food ingredients that are extensively utilized in manufacturing of food due to their high nutritional quality and versatile functional properties. WPI, one of the most important whey protein products, is a spraydried powder with high protein content. Functional properties for WPI are particularly important in relation to the texture and structure of processed food. Therefore, increasing interest is directed toward modifying WPI to enhance functionality and thereby add value to the protein. Chemical modification is an effective way to improve the functional properties of WPI. During the past decade, a number of chemical modifications such as phosphorylation, glycation, deamidation, succinylation, and Maillard reaction have been proposed to improve the functional properties of WPI (Li, Enomoto, Ohki, Ohtomo, & Aoki, 2005; Liu & Zhong, 2012; Ma, Forssell, Partanen, Buchert, & Boer, 2011; Morand, Guyomarc'h, Legland, & Famelart, 2012). Phenolic compounds represent the largest group of secondary plant metabolites and are widely distributed in plants. Dietary
* Corresponding author. E-mail address: [email protected] (J. Jiang). http://dx.doi.org/10.1016/j.lwt.2015.10.054 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
phenolic substances have received much attention due to their ability to scavenge reactive oxygen species. Additionally, dietary phenolic compounds are known to interact with proteins in the food matrix. The details of the interactions between target proteins and phenolic compounds are not well understood, but covalent interactions of food proteins with phenolic compounds have been proposed. Phenolic compounds may be oxidized in an alkaline solution to their corresponding quinones (Hurell & Finot, 1984). The electron-deficient quinones represent a species of highly reactive substances that normally react further with nucleophilic amino acid residues in a protein chain (Kroll, Rawel, & Rohn, 2003; Rawel, Rohn, Kruse, & Kroll, 2002). Many food proteins such as whey proteins, myoglobin, lysozyme, bovine serum albumin, and soy proteins could interact with phenolic compounds in this manner. Covalent modification by phenolic compounds produces food protein derivatives that have different physicochemical and conformational properties compared with unmodified proteins. Rawel, Czajka, Rohn and Kroll (2002) reported that soy protein derivatives exhibited different characteristics compared with unmodified soy protein, including isoelectric points, solubility, digestibility, secondary structure, and thermal stability. Ali, Homann, Khalil, Kruse, and Rawel (2013) reported that modification of blactoglobulin with coffee-specific phenolic compounds resulted in
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changed structural properties, and alteration in solubility, surface hydrophobicity, and emulsifying properties were observed. Tea is one of the most widely consumed beverages in the world, and is a rich source of health protective phenolic compounds (Kim et al., 2011; Thielecke & Boschmann, 2009). The predominant bioactive phenolic component of green tea, ()-epigallocatechin-3gallate (EGCG) is known to possess antioxidant, anti-inflammatory, and anti-cancer cell proliferation properties (Katiyar, Afaq, Azizuddin, & Mukhtar, 2001; Lestringant, Guri, Gülseren, Relkin, & Corredig, 2014; Zhong, Chiou, Pan, & Shahidi, 2012). On the basis of previous literature, we hypothesized that EGCG could covalently bind to nucleophilic amino acid residues in whey protein under alkaline conditions, which could lead to conformational changes. It is well known that the functional properties of food proteins are closely related to their physiochemical and structural characteristics. Accordingly, functional properties of whey protein may be altered by modification with EGCG through covalent reaction. However, to our knowledge, no studies have investigated the effects of covalent modification by EGCG on functional properties of WPI, particularly the foaming and emulsifying properties. Therefore, the main objective of the present study was to investigate the effect of covalent modification by EGCG on the physicochemical characteristics of WPI. Furthermore, the foaming and emulsifying properties of modified WPI were evaluated and compared.
TriseCl, pH 6.8, with 2% SDS, 10% glycerol and 0.1% bromophenol blue). Each well in the SDS-PAGE gel was loaded with 10 mg of WPI sample. Middle range unstained protein standard (catalog no. BM525, Sangon Biotech, Shanghai, China) was applied. The gel was stained with Coomassie Blue R-250, destained, and scanned.
2. Material and method
2.6. Intrinsic fluorescence spectroscopy
2.1. Chemicals
The fluorescence spectra were recorded using a Shimadzu RF5301PC spectrofluorometer (Tokyo, Japan) in a 1 cm path length quartz cell. The excitation wavelength was 285 nm. Both the excitation and emission slit widths were set at 5 nm. The concentration of sample solutions prepared in 50 mM phosphate buffer (pH 7.0) was adjusted to 0.3 mg/mL. The emission spectra were collected between 300 and 450 nm.
WPI was obtained from Hilmar Cheese Co. (Hilmar, CA, USA). According to the product bulletin, the typical composition of the WPI was 89% protein, 1.5% lactose, 0.5% fat, 2.5% ash, and 4.5% moisture. EGCG, KBr, and electrophoresis reagent were purchased from SigmaeAldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Corn oil was purchased from Wumart Stores, Inc. (Hangzhou, China). All other chemicals were of reagent grade and obtained from Mike Chemical Co., Ltd. (Hangzhou, China). 2.2. Preparation of modified WPI with EGCG WPI powder was dispersed in deionized water (35 g into 900 mL), and the pH value of the protein dispersion was adjusted to 9.0 using 0.5 M NaOH. EGCG solution (50 mL of 5 mg/mL) was mixed with the protein dispersion and then the volume was adjusted to 1000 mL with deionized water. After 12 h of reaction time under continuous stirring at 25 C, the pH value of the protein dispersion was adjusted to 6.8 using 0.5 M HCl. To remove the free phenolic compounds in the protein dispersion, an ultrafiltration was performed using a Millipore Pellicon cassette module (Bedford, MA, USA), containing Biomax-5 membrane with a molecular weight cut-off (MWCO) of 5 kDa, with a membrane area of 0.1 m2. The ultrafiltration was operated in the mode of batch ultrafiltration with full recycle of the retentate. Until the final volume reduced to 200 mL, 800 mL of deionized water was added into the retentate. The ultrafiltration was repeated five times, and then the retentate was lyophilized. In addition, a control experiment was performed to obtain the unmodified WPI without EGCG during the same period. 2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed by a slab gel made from 5% stacking gel and 12% separating gel, according to the method of Laemmli (1970). WPI samples were dissolved in loading buffer (50 mM
2.4. Size exclusion chromatography (SEC) Size exclusion chromatography (SEC) experiments were per€ formed using the GE Healthcare AKTA Purifier 100 FPLC system equipped with a monitor UV-900 (Uppsala, Sweden) on a column of Sephacryl S-100 HR (1.6 70 cm). The column was equilibrated and eluted with 100 mM phosphate buffer (pH 7.8) at a flow rate of 1 mL/min. A 500 mL aliquot of WPI solution (5 mg/mL) was loaded on the column. The absorbance at 280 nm was used for detection of protein elution. 2.5. FT-IR spectroscopy Infrared spectra were recorded at room temperature using a Thermo Scientific Nicolet 380 spectrometer (Madison, WI, USA) equipped with a deuterated triglycine sulfate detector. The WPI samples were mixed with KBr and then laminated. The resolution and scanning time were 4 cm1 and 32 times, respectively.
2.7. Determination of foaming properties Foaming properties were determined by using the method described by Aewsiri, Benjakul, and Visessanguan (2009) with some modifications. WPI dispersions (5, 20 and 35 mg/mL) were prepared in graduated test tubes by dispersing WPI powder in 20 mL of 100 mM phosphate buffer (pH 6.8). Whipping treatment was conducted at 12,000 rpm for 2 min at 25 C using the IKA T25 homogenizer (Staufen, Germany). The sample was allowed to stand for 15 min at 25 C. Both foaming capacity (FC) and foaming stability (FS) were calculated from the following equations:
FC ð%Þ ¼
V1 100 V0
FS ð%Þ ¼
V2 100 V1
where, V0 is the liquid volume before whipping, V1 is the initial foam volume after whipping and V2 is the final foam volume after leaving at 25 C for 15 min. 2.8. Emulsion preparation and particle size determination WPI dispersions (10, 20, and 40 mg/mL) were prepared in beakers by dispersing WPI powder in 150.0 mL of 100 mM phosphate buffer (pH 6.8) containing sodium azide (0.1 mg/mL). The emulsion was formed by transferring 50.0 mL of corn oil into the sample dispersion. The mixture was then pre-homogenized with the IKA T25 homogenizer (Staufen, Germany) at 12,000 rpm for 1 min at
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25 C. The crude emulsions were then homogenized with GEA Niro Soavi NS1001L2K high pressure homogenizer (Parma, Italy) for three passes at 40 MPa. After 0 and 28 days of storage at room temperature, a 30 mL aliquot of the emulsion sample was transferred into 50 mL of SDS solution (1 mg/mL) and mixed. Then, the average droplet size was measured with Malvern Nano S90 Zetasizer (Worcestershire, U.K.), and expressed as the volume-surface average droplet size (d32) of emulsion. 2.9. Statistical analysis Determinations of foaming and emulsifying properties were carried out in triplicate and the data was expressed in mean ± standard deviation. Differences between means were assessed using a one-way analysis of variance (ANOVA). Values of P < 0.05 were considered to be statistically significant. Statistical analysis was performed using SPSS statistical software version 13.0 (SPSS Inc, Chicago, USA). 3. Results and discussion 3.1. Profile of modified WPI by EGCG SDS-PAGE run under reducing conditions was used to study change in the protein profile of WPI as a result of modification by EGCG. As shown in Fig. 1, the SDS-PAGE pattern of unmodified WPI showed three main bands attributed to a-lactalbumin (a-La), blactoglobulin (b-Lg), and bovine serum albumin (BSA), respectively (Lane 1). The SDS-PAGE pattern of modified WPI (Lane 2) revealed the formation of protein complexes, demonstrated by the dark stains at the top of the separating gel. Additionally, the formation of a new broad band with an estimated average molecular weight of around 36 kDa was observed at the middle of the separating gel, suggesting that the oligomer of whey proteins was obtained. The intensities of a-La, BSA, and b-Lg bands in modified WPI decreased by comparison to those of unmodified WPI, suggesting that the proteins corresponding to these bands could be involved in the formation of cross-linked products. The SDS-PAGE results
307
demonstrated that the reaction by EGCG caused covalent crosslinking of whey proteins. To support this finding, we used size exclusion chromatography (SEC) to analyze the unmodified and modified WPI. Fig. 2 shows the elution profiles of samples. Peaks 1, 2, and 3 in the elution profile of unmodified WPI were attributed to BSA, b-Lg, and a-La, respectively. In the elution profile of modified WPI, a new peak appeared between peak 1 and peak 2, supporting the hypothesis that a crosslinking reaction occurred. Furthermore, the intensities of peaks 2 and 3 decreased and the intensity of peak 1 increased, which indicated that the whey protein components were polymerized to form protein complexes with high molecular weight. The results of SDS-PAGE and SEC illustrated that EGCG induced a cross-linking reaction and caused the change of profile for WPI. Our findings were not in agreement with the results of previous literature. Ishii et al. (2008) investigated the interaction between proteins and EGCG, and showed that EGCG can covalently bind to proteins through autoxidation, generating protein-EGCG conjugates. Meanwhile, Ali et al. (2013) also reported that the interaction between b-Lg and chlorogenic acid under alkaline conditions resulted in the attachment of phenolic derivatives to protein chains, leading to the formation of adducts. We speculated that the characteristics of both the protein and the phenolic compounds played crucial roles in the formation of reaction products. 3.2. FT-IR Determining a protein's structure is essential for fully understanding its functional properties. FT-IR is a sensitive tool for detecting conformational changes of protein secondary structure (Jackson & Mantsch, 1995). Fig. 3 shows the FT-IR spectra of unmodified and modified WPI. In the spectrum of unmodified WPI, we observed three strong bands at 3294.2, 1648.0, and 1541.4 cm1, corresponding to the vibrations for the amide A, amide I, and amide Ⅱ of protein, respectively. As compared to the spectrum of unmodified WPI, a new absorption band at 3399.0 cm1 appeared in the EGCG-modified WPI spectrum, attributed to the OeH stretching vibration of the phenolic groups. The new band confirmed the binding of phenolic groups to the protein components in WPI via the covalent bond. Therefore, we speculated that some EGCG derivatives were incorporated into the cross-linked protein as bridging agents. The role of phenolic compounds as bridging agents in laccase-catalyzed cross-linking of protein has been proved in previous reports (Mattinen et al., 2005; Steffensen, Andersen, Degn, & Nielsen, 2008). The amide I band represents the vibration of C]O stretching of the peptide bond, whereas the amide Ⅱ band is primarily due to the vibrations of NeH bending and CeN stretching. Both amide I and amide Ⅱ bands consist of overlapping bands at characteristic frequencies corresponding to different secondary structure elements (e.g., a-helices, b-sheets, turns, and disordered structures). Therefore, the frequencies of amide I and amide Ⅱ bands are highly sensitive to the secondary structure of the polypeptide chain € cker, Ofstad, Sørheim, & Kohler, 2009). There have been (Carton, Bo a large amount of experimental and theoretical studies on these vibrations, especially with regard to their frequencies in relation to the secondary structures of polypeptide chains (Pelton & McLean, 2000; Torii, 2012). As shown in Fig. 3, the frequencies of amide I and amide Ⅱ for modified WPI shifted to 1651.1 and 1538.2 cm1, respectively. This indicated that the secondary structure of WPI was changed after modification. 3.3. Intrinsic fluorescence emission spectrum
Fig. 1. SDS-PAGE patterns of unmodified and modified WPI. Lane 1, Unmodified WPI; Lane 2, Modified WPI; Lanes 3, Molecular weight standard.
The intrinsic fluorescence emission spectrum of protein is
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Fig. 2. SEC elution profiles of unmodified and modified WPI on the column of Sephacryl S-100 HR.
Fig. 3. The FT-IR spectra of unmodified and modified WPI.
usually dominated by tryptophan (Trp) and provides sensitive detection of tertiary conformation of the protein involved. The main proteins in WPI were b-lactoglobulin and a-lactalbumin, which contained two and four Trp residues, respectively. To gain more insight into the conformational changes caused by the reaction of WPI with EGCG, the intrinsic Trp fluorescence assay was performed. Fig. 4 shows the intrinsic emission fluorescence spectra of unmodified and modified WPI with EGCG. The maximum fluorescence emission for modified WPI shifted from 336 nm to 339 nm, and the fluorescence intensity dramatically decreased. The red shift of the emission maximum indicated that the major fluorophore Trp was exposed to a more hydrophilic environment after modification, as a result of perturbation of protein tertiary structure (Kristo, Hazizaj, & Corredig, 2012). The decrease in the fluorescence intensity reflects that the Trp residues are less exposed in the modified WPI as compared to the control. A more compact protein molecule may allow deeper burial of its fluorophores in the protein core than a less compact molecule (Withana-Gamage, Hegedus, Qiu, Mclntosh, & Wanasundara, 2013); therefore, the low
Fig. 4. Intrinsic fluorescence spectra of unmodified and modified WPI.
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309
Table 1 The foaming capacity (FC) and foaming stability (FS) of modified and unmodified WPI at different concentrations. 5 mg/mL
20 mg/mL
FC (%)
FS (%) a
27.2 ± 4.98 50.0 ± 1.32b
Unmodified WPI Modified WPI
35 mg/mL
FC (%) a
FS (%) a
27.0 ± 7.36 25.5 ± 1.68a
157.8 ± 2.42 187.7 ± 14.67b
FC (%) a
14.2 ± 0.58 23.4 ± 4.69b
FS (%) a
159.2 ± 3.48 208.6 ± 1.79b
15.4 ± 1.45a 30.1 ± 2.41b
Results are expressed as the mean ± standard deviation; n ¼ 3. Different letters within the same column are statistically different (P < 0.05).
fluorescence intensity may indirectly indicate a high degree of molecule compactness for modified WPI. Another possible cause for the decrease in the intensity of intrinsic fluorescence may have been the attachment of phenolic moieties to protein, which led to a quenching effect, thereby reducing the emission intensity (Feroz, Mohamad, Bujang, Malek, & Tayyab, 2012). 3.4. Foaming properties Foaming properties, estimated through FC and FS, are presented in Table 1. In the range of 5e35 mg/mL, the FC of modified WPI was significantly higher than those of unmodified WPI (P < 0.05). At the low concentration (5 mg/mL), FS of modified WPI was comparable to that of unmodified WPI (P > 0.05), but at the higher concentrations (25 mg/mL and 35 mg/mL), FS was significantly higher than those of unmodified WPI (P < 0.05). The results revealed that the foaming properties of WPI were greatly improved by EGCG modification. As described above, we found that the WPI was significantly modified by the cross-linking reaction induced by EGCG. The focus of previous studies were on the manipulation of foaming behavior of proteins by means of cross-linking treatment (Foegeding, Luck, & ez, Busti, Ballerini, & Delorenzi, 2010). MoDavis, 2006; Moro, Ba lecular size of food protein was a crucial factor influencing foaming properties. It has been observed that whey protein polymers exhibited higher intrinsic viscosity compared with native whey protein, which generated more stabilized foams by the effective deceleration of drainage rate (Davis & Foegeding, 2004; Vardhanabhuti & Foegeding, 1999). Partanen et al. (2009) reported that the foaming capacity of sodium caseinate was improved due to the cross-linking reaction catalyzed by transglutaminase. Furthermore, the authors indicated that the cross-linking treatment changed the interfacial elasticity and enhanced the foam formation by means of suppressing coalescence and disproportionation. Additionally, it has been demonstrated by Kuan, Bhat, and Karim (2011) that the cross-linking and polymerization of egg white protein improved the foaming properties, because the cross-linked structure effectively enhanced the unfolding of the protein during foam forming and formation of more elastic foam networks at the airewater interfaces. Therefore, the improved foaming properties of modified WPI might be explained by the increased molecular size due to the cross-linking reaction. 3.5. Emulsifying properties In the present study, the original d32 of emulsions (day 0) were
measured as the indicator of emulsifying ability, and the increases of d32 after 28 days of storage were followed to evaluate the emulsion stability. The results are presented in Table 2. At low concentrations (10 and 20 mg/mL), the modified WPI exhibited significantly lower original d32 values than unmodified WPI (P < 0.05). But as the concentration rose to 40 mg/mL, there was no significant difference between the original d32 values of modified and unmodified WPI (P > 0.05). The results implied that the emulsifying ability of WPI was increased after the modification. At all concentrations assayed, the increases of d32 values of modified WPI emulsions were lower than those of unmodified WPI emulsions after 28 days of storage, indicating EGCG modification improved the emulsion stability of WPI. This finding was consistent with the results in previous literature, which revealed that a-Lapolyphenol covalent complexes had better emulsion stability than a-La (Wang et al., 2015). WPI is considered to be a surface-active agent and can adsorb to the surfaces of oil droplets to form an interfacial film and stabilize the oil/water emulsion against flocculation or coalescence. The emulsifying properties of proteins depend on their abilities to diffuse to the oil/water interface, and then undergo partial unfolding and rearrangement at the interface. Conformational characteristic of food protein is particularly important in relation to the emulsifying properties of protein. According to Jambrak, Lelas, Mason, Kresi c, and Badanjak (2009), the changes of the protein conformational structure could influence surface hydrophobicity and subsequently lead to better adsorption of the oil/water emulsion system. Afizah and Rizvi (2014) stated that the improvement in the emulsifying activity of texturized whey protein concentrate was caused by the increase of exposed aromatic residues, which resulted in the increased affinity of the proteins towards the oilwater interface. They also explained that the aggregated whey protein formed a thicker protein membrane, thus providing better emulsifying stability for the oil droplets. EGCG-induced protein conformational changes were indicated by the results obtained from SDS-PAGE, FT-IR, and intrinsic Trp fluorescence assay. Therefore, the results suggested that the conformational changes on the surface properties of WPI upon EGCG modification led to the improvement of the emulsifying properties. 4. Conclusion In summary, our results indicated the possibility of using EGCG modification to improve the functional properties of WPI. The EGCG modification caused cross-linking and conformational structure changes of proteins in WPI. Furthermore, we found that
Table 2 The average droplet size (d32) of emulsion with modified and unmodified WPI at different concentrations during storage. 0 day
28 days
10 mg/mL Unmodified WPI Modified WPI
20 mg/mL a
689.8 ± 42.31 506.9 ± 31.78b
40 mg/mL a
423.9 ± 30.78 350.2 ± 17.33b
301.3 ± 12.62 288.6 ± 7.17a
10 mg/mL a
20 mg/mL a
804.4 ± 40.84 596.2 ± 26.73b
40 mg/mL a
509.5 ± 31.41 409.4 ± 20.16b
Results are expressed as the mean ± standard deviation; n ¼ 3. Different letters within the same column are statistically different (P < 0.05).
337.2 ± 17.42a 299.4 ± 12.35b
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the foaming and emulsifying properties of WPI were enhanced by EGCG modification. This study provides valuable information on the potential application of modified WPI by EGCG. Further research is needed to evaluate the effect of EGCG modification on other functionalities of WPI, in order to develop their applications in food industries. Acknowledgments We gratefully acknowledge the financial support from the National Natural Science Foundation of China (31101319). References Aewsiri, T., Benjakul, S., & Visessanguan, W. (2009). Functional properties of gelatin from cuttlefish (Sepia pharaonis) skin as affected by bleaching using hydrogen peroxide. Food Chemistry, 115, 243e249. Afizah, M. N., & Rizvi, S. S. H. (2014). Functional properties of whey protein concentrate texturized at acidic pH: effect of extrusion temperature. LWT e Food Science and Technology, 57, 290e298. Ali, M., Homann, T., Khalil, M., Kruse, H. P., & Rawel, H. (2013). Milk whey protein modification by coffee-specific phenolics: effect on structural and functional properties. Journal of Agricultural and Food Chemistry, 61, 6911e6920. € cker, U., Ofstad, R., Sørheim, O., & Kohler, A. (2009). Monitoring secCarton, I., Bo ondary structural changes in salted and smoked salmon muscle myofiber proteins by FT-IR microspectroscopy. Journal of Agricultural and Food Chemistry, 57, 3563e3570. Davis, J. P., & Foegeding, E. A. (2004). Foaming and interfacial properties of polymerized whey protein isolate. Journal of Food Science, 69, C404eC410. Feroz, S. R., Mohamad, S. B., Bujang, N., Malek, S. N. A., & Tayyab, S. (2012). Multispectroscopic and molecular modeling approach to investigate the interaction of flavokawain B with human serum albumin. Journal of Agricultural and Food Chemistry, 60, 5899e5908. Foegeding, E. A., Luck, P. J., & Davis, J. P. (2006). Factors determining the physical properties of protein foams. Food Hydrocolloids, 20, 284e292. Hurell, R. F., & Finot, P. A. (1984). Nutritional consequences of the reactions between proteins and oxidized polyphenolic acids. Advances in Experimental Medicine and Biology, 177, 423e435. Ishii, T., Mori, T., Tanaka, T., Mizuno, D., Yamaji, R., Kumazawa, S., et al. (2008). Covalent modification of proteins by green tea polyphenol (e)-epigallocatechin-3-gallate through autoxidation. Free Radical Biology & Medicine, 45, 1384e1394. Jackson, M., & Mantsch, H. H. (1995). The use and misuse of FTIR spectroscopy in the determination of protein structure. Critical Reviews in Biochemistry and Molecular Biology, 30, 95e120. Jambrak, A. R., Lelas, V., Mason, T. J., Kresi c, G., & Badanjak, M. (2009). Physical properties of ultrasound treated soy proteins. Journal of Food Engineering, 93, 386e393. Katiyar, S. K., Afaq, F., Azizuddin, K., & Mukhtar, H. (2001). Inhibition of UVBinduced oxidative stress-mediated phosphorylation of mitogen-activated protein kinase signaling pathways in cultured human epidermal keratinocytes by green tea polyphenol ()-Epigallocatechin-3-gallate. Toxicology and Applied Pharmacology, 176, 110e117. Kim, A., Chiu, A., Barone, M. K., Avino, D., Wang, F., Coleman, C. I., et al. (2011). Green tea catechins decrease total and low-density lipoprotein cholesterol: a systematic review and meta-analysis. Journal of the American Dietetic Association, 111, 1720e1729. Kristo, E., Hazizaj, A., & Corredig, M. (2012). Structural changes imposed on whey proteins by UV irradiation in a continuous UV light reactor. Journal of Agricultural and Food Chemistry, 60, 6204e6209.
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